How Important Is the Role of the Lentiviral Vector?


Abstract: Transgenic technology makes it possible for humans to change the trait phenotype of organisms according to their subjective wishes. The type and quality of vectors are directly related to the efficiency of subsequent transgenes. Therefore, the construction of expression vectors has become one of the key links in transgenic research. Lentivirus, also known as retrovirus, belongs to the family of retroviruses and is an RNA virus. Since this virus contains reverse transcriptase, it is called retrovirus. Lentivirus in the host cell, using RNA as a template, synthesizing cDNA under the action of self-reverse transcriptase, and then synthesizing double-stranded DNA using cDNA as a template, and cyclizing and integrating the chromosome of the host cell by viral integrase Expressed on the long term. The lentiviral vector (LV) is constructed by the lentivirus to remove part of the gene based on its genome and is constructed with the desired gene and marker of interest. Compared with other vectors, the lentiviral vector has a large capacity to carry the gene fragment. The dyeing efficiency is high, it can infect dividing cells and non-dividing cells, and the target gene can be stably expressed in the host cell for a long time and has good safety. It has become an ideal carrier for transferring the target gene. This article will focus on thelentiviral vectorconstruction and their applications in genetics.

Keywords: lentiviral vector, construction, application

The development of lentiviral vectors

In the early days of the development of lentiviral vectors in the early 1990s, HIV-1-based lentiviral vector research was mainly used for the study of the biological characteristics of HIV and the treatment of AIDS. The expression vector is mainly constructed for target cells expressing CD4, and most of the packaging systems retain all HIV genes as a control, and a series of viral genomes such as mutation or deletions of a certain sequence are modified. The purpose was to observe the effects on viral packaging, replication, infection, etc., and to study the genome composition of lentivirus HIV-1. This laid a solid foundation for the subsequent research of lentiviral expression vectors.

In the mid-1990s, the three-plasmid system of Didier Trono of the University of Geneva and Luigi Naldini and Inder Verma of the Sack Biological Research Institute of California marked the birth of the first generation of the lentiviral vector multi-plasmid system. The system consists of a vector plasmid, a packaging plasmid, and an envelope plasmid. The vector plasmid is responsible for loading the foreign gene, deleting genes such as gag, pol, env, but retaining 5′ and 3′, LTR, Psi sites, and Rev Response elements, thus improving the carrier loading while ensuring its efficiency. On the basis of this, Zufferey et al designed the second generation lentiviral vector, and deleted three auxiliary genes of vif, vpr and nef on the packaging plasmid, and obtained the infection of non-dividing cells, mononuclear macrophages and nerves. Cell-capable lentiviral vector and did not affect viral yield. Since these helper genes can express virulence viral proteins, their deletion not only increases the safety of lentiviral vectors but also increases the stability of the packaging cell line. The third generation of lentiviral vectors subsequently has higher biosafety. The vector plasmid contains only the cis-acting site and does not express any HIV protein. The packaging plasmid contains only three HIV functional genes, gag, pol and rev, and all other helper genes are deleted.

Construction of lentiviral vector

Ø The capacity and characteristics of lentiviral vectors

Due to the restriction of the size of the inserted gene in the packaging capacity of the viral capsid, the possible loading capacity of the target gene in the vector should be considered first when constructing the vector. The length of the HIV proviral genome is about 9.8 kb, and the length of the plasmid vector can be subtracted. The maximum length of the fragment of interest is loaded, which is the biggest disadvantage of lentiviral vectors compared to micromanipulation. At the same time, the packaging systems for the 2nd and 3rd generation lentiviral vectors are not universal because the second generation vector plasmid is Tat-dependent and the third generation packaging system does not contain the Tat gene.

Ø Optimization of lentiviral vectors

The expression efficiency can be increased by inserting a regulatory sequence. For example, by inserting the woodchuck hepatitis virus post-transcriptional regulatory element into the three untranslated region of the target gene, the expression level of the target gene can be increased by 5 to 8 times.

Ø Double gene transfer vector design


In the transgenic research, the strategy of double gene co-transfer is often used. The most common method for constructing lentiviral double gene transfer vector is to design bicistronics using internal ribosome entry site (IRES). The disadvantage of this strategy is downstream. The gene expression is low, and even the expression of downstream genes is not detected at all, and the use of two transcription units in tandem on the lentiviral vector effectively overcomes this shortcoming, enabling the downstream gene to be efficiently expressed.

Application of lentiviral vector

Treating HIV infection

With the surge in AIDS patients worldwide, the way to treat AIDS as soon as possible has become the most important issue. Lentiviral vectors have many advantages in the treatment of HIV-1 infection. First, some lentiviral vectors from HIV-1 and HIV-2 compete with viral RNA for packaging and competition for viral proteins for viral replication, inhibiting their replication, thereby controlling HIV infection. Second, lentiviral vectors can stably transfect dividing cells and non-dividing cells, especially those associated with HIV-1 replication and immune recovery, such as dendritic cells, T cells, and the like. Third, lentiviral vectors can be designed to express genes with therapeutic anti-HIV-1, specifically controlling the various stages of viral replication.

Treatment of tumor

For a long time, the treatment of tumors has been based on surgery, radiotherapy and drugs. Tumor gene therapy is a new treatment method after these traditional treatment methods. The lentiviral vector can safely and effectively implant the tumor gene into the human body for long-term effective expression. The use of lentiviral vectors increases the sensitivity of tumor cells to traditional chemotherapeutic drugs. RNAi technology is also used in tumor gene therapy. The occurrence of cancer is mainly due to the mutation and abnormal activation of proto-oncogenes. Lentivirus can specifically reduce the expression of RNAi against proto-oncogenes in various cells, thereby inhibiting canceration. Cell proliferation; shRNA capable of efficiently transmitting tumor-associated genes, silencing after transcription, inhibiting the expression of the target gene, reducing its promotion in tumor development and tumor invasion, thereby playing a therapeutic role.

Treatment of transplant rejection

In recent years, organ transplantation has been widely used to treat severe organ failure, but how to reduce transplant rejection and graft-versus-host disease (GVHD) after organ transplantation has become the most urgent problem to be solved. Activation, differentiation and proliferation of T lymphocytes play a key role in the immune response to transplantation. Genetic modification of T lymphocytes to reduce rejection and GVHD is one of the important directions in the field of transplantation. Li Zhenyu et al. observed the expression of the lentiviral vector in mouse lymphocytes by constructing a lentiviral three-plasmid system containing the green fluorescent protein gene, and found that the lentiviral vector has high infection efficiency on mouse T lymphocytes and can be rapidly stabilized. Transfer foreign genes to T cells.

References

[1] SUN Ke-ning, ZHU Hua-bin, LIN Feng, Wang Dong, HAO Hai-sheng, DU Wei-hua, ZHAO Xue-ming. Development on Lentiviral Vector Construction and Transgentic Animals by Lentiviral Vector.

[2] Buchschacher G L J, Panganiban A T. Human immunodeficiency virus vectors for inducible expression of foreign genes [J]. J  Virol, 1992, 66(5):2731-2740

The current situation and development trend of cancer vaccine

The cancer vaccine uses the patient’s own tumor cells, tumor-specific antigens and other immune-regulatory cells to treat and prevent cancer. The difference between the cancer vaccine and the mechanism of action of the pathogenic vaccine is that the former mainly achieves therapeutic purposes by stimulating the body’s specific immune response to cancer antigens. Thecancer vaccine is mainly obtained from the host and most macromolecules, is a normal autoantigen present in normal cells, has the specificity of the immune system, and can accurately identify cancer cell antigens from the host cells. The development of cancer vaccines is based on the premise that tumor cells are fundamentally different from normal cells, and that the immune system is able to identify and impart (by immunization), these distinctions to the goal of identifying malignant cells and regulating tumor rejection.

1. Pancreatic cancer vaccine

Pancreatic cancer is the fourth most common malignant tumor in developed countries, with a 5-year survival rate of only 7%. Due to the special anatomical location and physiological characteristics of pancreatic cancer, the onset of pancreatic cancer is insidious, there is no obvious symptoms in the early stage of the disease, the diagnosis is difficult, and it is highly invasive, so the mortality rate is very high.Diagnosis of pancreatic cancer can detect cancer antigen 19-9 levels. Cancer antigen 19-9 can be used as pancreatic cancer. An auxiliary diagnostic indicator for malignant tumors such as gallbladder cancer. In the embryonic stage, the pancreas, gallbladder, liver, intestine and other tissues have such antigens, and the normal human tissue content is very low; in the digestive tract malignant tumors, especially in the serum of patients with gallbladder cancer and pancreatic cancer, the content of cancer antigen 19-9 is significantly increased. However, the early diagnosis is of little value, mainly as a monitoring indicator and an indicator of recurrence. In addition, differential diagnosis of digestive tract diseases (such as pancreatic cancer and pancreatitis, gastric cancer and gastric ulcer) also has certain value.

Pancreatic cancer is highly immunosuppressive, and tumor-associated reactive T cell infiltration in the microenvironment is associated with a good prognosis for pancreatic cancer. Studies have shown that pancreatic cancer has multiple mechanisms to evade surveillance by the immune system, such as recruitment of negative regulatory T cells, secretion of transforming growth factor beta (TGF-beta) and interleukin 10 to suppress the immune response of the immune system, and down-regulation Expression of major histocompatibility complex I (MHC I), and the like. Recent studies have shown that necrotic apoptosis in pancreatic cancer induces chemokine ligand and macrophage-induced C-type lectin receptor signaling, promotes macrophage-induced adaptive immunosuppression, and accelerates pancreatic cancer progression.

Breaking through the immunosuppression of pancreatic cancer, enhancing the recognition of tumor-associated antigen (TAA) and activation of tumor-specific T cell responses are the core issues in the treatment of pancreatic cancer tumor vaccines. In recent years, with the development of omics, more and more relatively specific TAAs have been discovered. For example, exon sequencing can be specifically focused on a certain gene coding part, so most of the oncogene changes can be identified.There are an average of 63 genes in pancreatic cancer. These genes are mainly concentrated in 12 core signaling pathways such as Kras signal, TGF-β signal, SHH signaling pathway, etc. These changes are important for tumor tissue growth and differentiation, suggesting pancreas Cancer is the result of multiple gene mutations, and it also provides a basis for the target of pancreatic cancer tumor vaccines.The pancreatic cancer genotypes are divided into four subtypes: squamous, pancreatic progenitor, immunogenic and aberrantly differentiated endocrine exocrine (ADEX). The immunogenic gene program is associated with B cell signaling pathway, antigen presentation, CD4+ T cells, CD8+ T cells, and Toll-like receptor signaling pathways, and its acquired immune signaling pathways such as CTLA-4 and PD1 are up-regulated, indicating specificity. There may be breakthroughs in the study of tumor vaccines for immunogenic genotypes, suggesting that the treatment of pancreatic cancer needs to be personalized and precise.

1.2. Pancreatic cancer vaccine type

1.2.1. Peptide/gene vaccine

1.2.1.1. KRAS peptide vaccine

The KRAS gene belongs to a member of the Ras gene family and plays an important role in regulating cell proliferation and differentiation. More than 90% of pancreatic cancers have KRAS mutations and are among the earliest genetic alterations in precancerous lesions. When the KRAS gene is mutated, the conformational change of the Ras protein promotes invasion and metastasis of pancreatic cancer through its downstream RAF/MEK/ERK protein kinase cascade.

1.2.1.2. Telomerase vaccine

Telomerase maintains chromosome stability and plays an important role in cell senescence and carcinogenesis. When telomerase is activated, tumor cells are prevented from telomere-mediated cell death. Telomerase is a key molecule in inducing cell carcinogenesis. In general, telomerase is activated in more than 85% of tumor cells.

1.2.1.3. Vascular endothelial growth factor receptor 2 protein vaccine

Vascular endothelial growth factor (VEGF) signaling pathway plays a crucial role in tumor angiogenesis, in which VEGF receptor 2 (VEGFR2) mediates vascular endothelial proliferation and chemotaxis. Cells, which increase the permeability of blood vessels, are the main functional receptors of VEGF. In pancreatic cancer, VEGF/VEGFR2 is closely related to tumor growth and infiltration by regulating angiogenesis.

1.2.1.4. Mucin vaccine

Mucin1 (MUC1) is a high molecular weight type I transmembrane glycoprotein, which is normally expressed in the proximal luminal or glandular surface of epithelial cells in various tissues and organs, but in 90% of patients with pancreatic cancer. Overexpression. MUC1 interferes with cell-cell and cell-matrix linkages and plays a role in tumor signal transduction, invasion, and distant metastasis.

1.2.1.5. WT1 epitope peptide vaccine

Wilm’s tumor protein, (WT1) is a type of TAA, which is expressed in solid tumors such as lung cancer, breast cancer, thyroid cancer, and pancreatic cancer, in addition to high expression in various types of leukemia.

1.2 .2. Cell vaccine

1.2.2.1. Tumor cell vaccine

Injection of a radiation-irradiated tumor cell culture vaccine is the most primitive tumor vaccine, which utilizes all relevant tumor antigen expression expressed by tumor cells to produce a specific anti-tumor immune response. GVAX is an allogeneic whole-cell vaccine derived from two tumor cells and genetically engineered to express granulocyte colony-stimulating factor (GM-CSF). Α-galactosyl (α-GAL) is not synthesized in normal human cells, and serum contains a large amount of anti-α-GAL antibody, but tumor cells can synthesize α-GAL, so α-GAL can be used as an antigen. Induction of an anti-tumor response. Algenpantucel-L is an allogeneic tumor vaccine produced by NEWLink, which is produced using two human pancreatic ductal cancer cells genetically modified to express α-GAL.

1.2.2.2. Dendritic cell vaccine

Dendritic cell (DC) is a professional antigen presenting cell (APC), which can efficiently present and activate MHC I and MHC II to CD8+ and CD4+ T cells. Stimulating memory T cells and memory B cells produce specific anti-tumor responses and play an important role in primary and secondary immune responses against tumors.

1.2.3. Vaccine combination therapy

1.2.3.1. Immunological checkpoint treatment and vaccine combination therapy

Immunological checkpoints are key to maintaining immune tolerance to chronic antigen exposure and preventing tissue damage. T cell activation is dependent on the interaction between co-stimulatory, co-inhibitory receptors and their ligand complexes. Usually, co-stimulatory receptors have CD40, CD28, OX40 and 4-1BB, while inhibitory receptors have CTLA-4, PD-1, B7 family receptors and their ligands CD80, CD86, PD-L1 and PD-L2. . Immunological checkpoint therapy is an antibody based on the inhibitory receptors CTLA-4, PD-1 and its ligands.

1.2.3.2. CAR-T Cell Therapy

The chimeric antigen receptor (CAR) is a type of genetically engineered transmembrane fusion receptor that binds to primitive cell surface antigens and transmits specific T cell activation signals. CAR-T cells (CAR-T) are a type of T-cells that are genetically engineered to encode tumor-specific antigen receptor genes, which allow T cells to express related antigen receptors and restore T cells. Immune surveillance can identify tumor surface antigens, so that a large number of tumor-associated antigens released by tumor cell rupture are presented, which can trigger the recognition and complete killing effect of the body’s immune system on tumors, which can be regarded as a special kind of cell vaccine. The key to developing CAR-T cell therapy is to select the appropriate targeting antigen and immune receptor.

2.Colon cancer vaccine

Colon cancer is one of the high-grade malignant tumors of the digestive system. The incidence rate is the third in the world for malignant tumors and has risen to the second place in economically developed areas. The emergence of coloncancervaccine will definitely bring new hope to the treatment of colon cancer.

Tumor vaccines use tumor cells or tumor antigens to induce the body to produce immune responses against tumor cells, inhibit their growth, and prevent recurrence and metastasis. Tumor antigens have been found on the surface of spontaneous tumors and human tumor cells in animals. Tumor antigens are generally classified, and two anti-tumor antigen classification methods including tumor-specific antigen (TSA) and tumor-associated antigen (TAA) are introduced. TSA is only present on the surface of tumor cells and is an antigen unique to a certain tumor cell. TAA is unique to non-tumor cells and is an antigen that can be expressed by normal cells. However, when cells are cancerous, their content is significantly increased, and such antigens only show quantitative changes without

Strict tumor specificity, embryonic antigen is a typical representative of it. Embryonic antigen refers to the normal component produced by embryonic cells during embryonic development. It is reduced in the late stage of embryonic development, gradually disappears after birth or remains extremely small, and such antigens regenerate when the cells become cancerous. At present, there are two kinds of embryo antigens that are the most intensive: 1) Alpha-fetoprotein (AFP): a glycoprotein synthesized by fetal liver cells, which inhibits maternal immune rejection. Adults are almost undetectable, and hepatocellular carcinoma is abundantly expressed when it is cancerous. 2) Carcinoembryonic antigen (CEA): It is an antigen that loosely binds to the cell membrane and is easily detached, such as carcinoembryonic antigen produced by intestinal cancer cells. AFP and CEA are weakly immune, as they have emerged during the embryonic period, and the body’s immune system has been immune to it and does not produce an immune response. However, AFP and CEA can be used as tumor markers to detect the early diagnosis of primary liver cancer and colon cancer by detecting the levels of AFP and CEA in the serum of patients.

At present, the main research tumor vaccines include the following:

2.1. Inactivate tumor cell vaccine

The tumor cell vaccine extracts tumor cells from the tumor tissues of the body, and inactivates the tumor cells that have lost the tumorigenicity but still maintains their immunogenicity, thereby inducing the body’s active immune response. In theory, such vaccines can provide tumor antigens, including TSA and TAA, to induce the body to produce an anti-tumor immune response.

2.2. Dendritic cell vaccine

Dendritic cells (DC) vaccine (referred to as DC vaccine) is the most active and fruitful biotherapeutic topic in research today. DC can be used as an important component of tumor immunotherapy. The mechanism of DC vaccine for malignant tumors is as follows: 1) Dendritic cells are special antigen-presenting cells, which help the immune system recognize tumor cells; 2) DCs carrying tumor antigens will antigen The information is presented to and activated by T cells, which induces the body to produce a large number of T lymphocytes with specific cytotoxic functions, which have specific killing effects on tumor cells. Dendritic cell therapy is a very cutting-edge new technology, and the application of DC vaccine has brought good news to the treatment of cancer patients. Numerous studies have shown that DC vaccines are safe, easy to handle, and immunosuppressive for a range of tumor types. The safety of the DC vaccine is also very good, and there have been no reports of serious adverse reactions. The successful development of dendritic cell vaccine treatment has brought new hopes to countless tumor patients and opened up a new way for the treatment of cancer.

2.3. Protein or polysaccharide vaccine

Such vaccines are obtained by mixing or linking tumor-associated proteins or polysaccharides and adjuvant molecules into the human body to induce humoral or cellular immunity, thereby achieving the purpose of killing tumor cells. The reason why tumors cannot be recognized by the immune system is mainly due to the weak immunogenicity of tumors. Therefore, the use of immunoadjuvants to enhance the immunogenicity of tumors is a hallmark of early tumor vaccines. The protein or polysaccharide vaccine is composed of an adjuvant such as Corynebacterium, alum, BCG, Freund’s complete adjuvant, etc. in the lysate of autologous or allogeneic tumor cells or tumor cells. Its mechanism of action may be related to the activation of antigen presenting cells (APC) by the inflammatory response at the injection site, the production of cytokines and the accumulation of B and T cells around the antigen.

2.4. Gene vaccine

Gene therapy is a hot research area in current medicine and biology. Gene vaccines, also known as nucleic acid vaccines or DNA vaccines, are often referred to as “naked” DNA vaccines. It contains no peptide, protein or viral vector, but consists of an antigen-encoding gene derived from the pathogen and plasmid DNA as its carrier. The birth of the genetic vaccine has revolutionized the treatment of colon cancer patients. : It is easy to operate and can be easily controlled by increasing or decreasing the amount of DNA injected. It does not require complex processes such as separation and purification of proteins. One or two weeks after DNA vaccine injection, an immune response is produced. After 14 days, the expression in the muscle is reached. The peak, then gradually decline, remains at low levels for months or even 1 year. In recent years, many researchers have actively developed tumor-related gene vaccines. The preliminary experiments have also proved that genetic vaccines have good curative effect and high immune performance, which makes people have a strong interest and expectation for the development of genetic vaccines.

The use of tumor vaccines can cause specific immune responses, thereby inhibiting tumor growth. Although tumor vaccines use the patient’s own tumor cells, tumor-specific antigens and other immune-regulatory cells to treat and prevent tumors, opening up a modern way to safely and effectively treat tumors, but at present, there are some difficulties in the development of tumor vaccines, such as further regulation. Enhance immune effector cells, etc. Once these problems are resolved, the tumor vaccine can be used on a large scale in the clinic. In addition, due to the complex composition of human tumors and the heterogeneous expression of tumor antigens, it may be necessary to immunize with a variety of tumor antigens in order to induce an effective immune response in patients.

Reference

[1] Keilholz U, Weber J, Finke J H, et al. Immunologic monitoring of cancer vaccine therapy: results of a workshop sponsored by the Society for Biological Therapy.[J]. Journal of Immunotherapy, 2002, 25(2):97.

[2] Maron D F. Cancer Vaccine.[J]. Scientific American, 2017, 317(5):16-16.

[3] Li X, Min M, Du N, et al. Chitin, Chitosan, and Glycated Chitosan Regulate Immune Responses: The Novel Adjuvants for Cancer Vaccine[J]. Clinical & Developmental Immunology, 2015, 2013(7378):387023.

[4] Shindo Y, Hazama S, Nakamura Y, et al. miR-196b, miR-378a and miR-486 are predictive biomarkers for the efficacy of vaccine treatment in colorectal cancer[J]. Oncology Letters, 2017, 14(2):1355-1362.

[5] Berry J, Vreeland T, Trappey A, et al. Cancer vaccines in colon and rectal cancer over the last decade: lessons learned and future directions[J]. Expert Review of Clinical Immunology, 2017, 13(3):1.

Notoginseng folium saponins is a traditional Chinese medicine

Notoginseng folium saponins is a traditional Chinese medicine that has the effect of shortening sleep time, prolonging sleep time, reducing the number of awakenings, improving headache, dizziness, palpitations, fatigue, and treating neurasthenia.

Function

In addition to the special effects of treating bruises, Notoginseng folium has the effect of nourishing and strengthening.  Modern pharmacological studies have shown that Notoginseng folium has obvious preventive and therapeutic effects on hyperlipidemia, hyperviscosity, hypertension, and arrhythmia.

The main pharmacological effects of the main active constituents of Panax notoginseng, Panax notoginseng, are calming, soothing, analgesic and lipid-lowering.

The role of the central nervous system: only 100-200mg / kg dose of notoginsenoside can significantly reduce the spontaneous activity of mice, enhance the sedative and hypnotic effects of thiopental, pentobarbital sodium, etc., and can fight the central excitement. The excitatory effect caused by caffeine indicates that this product has significant central inhibition.  Panax notoginseng saponins can improve the blood supply of the brain, nourish and regulate the nerves, restore the coordination functions of the nervous system, thereby restoring physiological sleep to treat neurasthenia, and strengthen the inhibition of the cerebral cortex and make the cortex  The rise of excitability is reduced and it is resistant to anxiety and can be used to treat generalized anxiety disorder.

Panax notoginseng saponins are better for shortening sleep time, prolonging sleep time, reducing the number of awakenings, and improving headache, dizziness, palpitations and fatigue.  The total effective rate of treatment of neurasthenia was significantly higher than that of the gastrodin control group.

Effect of lowering blood fat: Panax notoginseng saponins can significantly reduce serum total cholesterol (TC) and serum triglyceride (TG) levels in rats with high-fat models, which are related to the ginseng diol contained therein.  The type of saponin is related.  According to reports, TC decreased by an average of 22.4%, TG decreased by an average of 37.7%, and high-density lipoprotein increased by an average of 18.8%.  Using self-control method, taking 100 mg of esculin in patients diagnosed with hyperlipidemia, 3 times a day, 60 days for a course of treatment, the total effective rate was 81.6%.

Content ratio

Panax notoginseng leaves usually refers to the dried stems of the stems and leaves of the aboveground parts of Panax notoginseng. It has high medicinal value. The main pharmacological effects of the extracted total saponins of Panax notoginseng are calming and soothing, analgesic and lipid-lowering.  notoginseng has the characteristics of convenient eating, moistening mouth and thirst quenching. The main function is similar to that of Sanqi stem. It is also raw and cooked.  Rawnotoginseng can lower blood pressure, lower blood sugar, lower blood fat, etc.; notoginseng can also be soaked like drinking tea, drink a few cups a day!  The monomeric saponins contained in Panax notoginseng are mainly 20(s)-protopanaxadiol saponins, and contain almost no original ginseng triol saponins, which is the biggest difference between Panax notoginseng saponins and Panax notoginseng saponins.  The total saponin content of Panax notoginseng leaves is 4% to 6%, mainly containing ginsenoside Rb3, Rb1

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A Highly Sensitive Method for Protein Product

Simple Introduction

First of all, let’s start with its definition: Peptide mapping is the main method for the analysis and identification of protein products and preparations. In addition, there are some other core techniques such as peptide mass mapping and peptide mapping ms.

 

The following are functions of Peptide mapping:

To confirm that a protein primary structure (amino acid sequence) including the N and C terminals.

  • To provide protein loci and the proportion of modified groups, such as glycosylation, acetylation, sulphates a00nd phosphorylation.
  • To provide qualitative and quantitative information on protein degradation products
  • To get information on protein oxidation and deamination directly.

According to these useful features above, peptide mapping has already gained in popularity in biological field. After getting numerous data from peptide mapping, the next step is deeper analysis.

 

A sample graph of peptide mapping

Peptide Mapping Analysis

Based on graphs of peptide mapping and also peptide mass mapping, scientists can get a lot of useful information. According to the size of molecular weight of proteins, peptides and amino acid composition characteristics, the use of strong specificity of proteolytic enzyme [is commonly endopeptidase] on the special peptide chain site will peptide fragment into smaller fragments, through the separation of a certain form characteristic fingerprint detection methods. eolytic enzyme [is commonly endopeptidase] on the special peptide chain site will peptide fragment into smaller fragments, through th

The picture below shows the steps of peptide mapping method:

Step 1:

Protein digestion: Immobilized trypsin provides rapid and simple protein digestion with high reproducibility, high sensitivity and excellent data quality in a format that is compatible with automated operations.

Step 2:

Peptide separation: this technique provides a complete set of chromatographic tools for all development applications requiring peptide separation and analysis

Step 3:

Biological mass spectrometer: It provides a fast and easy to solve multicomponent analysis method, Used for sequence determination, structural analysis, molecular weight determination and component content determination of polypeptides. What’s more, it has the characteristics of high sensitivity, strong selectivity and good accuracy.

Step 4:

Peptide analysis software: Integrated software can save time and identify more materials. Simple software workflows can guide biotherapy, characterization pathways, and provide comprehensive coverage, including peptide sequence validation and identification of all variants and modifications.

 

So peptide mapping analysis is quite effective and important in some specific aspect of research. Let’s take an example here:

Study on quality control of impurities and harmful substances in Genetic Recombinant Drug is a crucial project., in particular, when the recombinants are used in the production of genetically engineered drugs undergo mutations. Probably, they would bring some mutations into drugs. Besides strengthen control of original material and process of production, peptide mapping analysis is necessary method to ensure safety and consistency.

 

As a result, peptide mapping analysis has given rise to a number of new things. For instance, HPCE, A new electrical technology for separation which emerged in 1980s. Because of its relatively high resolution, HPCE has started a wider road for structural analysis and quality control of protein drugs.

 

At present, in addition to the routine analysis of amino acid sequence of some small peptides, the peptide graph analysis is one of the important conventional indicators to control the consistency of most gene engineering products. All in all, technologies on peptide mapping have already penetrated into every corner of our everyday life.

 

New antibody screening technology – protein chip

What is an autoantibody?

Autoantibodies are antibodies that target tissues, organs, cells, and cellular components. The growth, development and survival of the human body have the maintenance of a complete autoimmune tolerance mechanism. The normal immune response has a protective defense effect, that is, it does not react to its own tissues and components. Once the integrity of self-tolerance is destroyed, the body regards its own tissues and components as “foreign substances”, and an autoimmune reaction occurs to produce autoantibodies. Normal human blood may have low titers of autoantibodies, but no disease occurs. However, if the titer of autoantibodies exceeds a certain level, it may cause damage to the body and induce disease.

There are many kinds of autoimmune diseases antibodies , the most important of which are antinuclear antibodies. In addition, anticardiolipin antibodies, neutrophil cytoplasmic antibodies, anti-mitochondrial antibodies, anti-erythrocyte antibodies, anti-platelet antibodies, anti-endothelial cells antibodies, anti-neurovirus antibodies, rheumatoid factor, anti-thyroglobulin antibodies, anti-insulin bodies Antibodies and the like are also autoantibodies.

Reasons for autoantibody production:

Antibodies are generally produced by the immune system by foreign proteins or other substances (especially pathogenic bacteria) that enter the body and are used in immune reactions to eliminate harmful foreign substances. Usually, the immune system can recognize and ignore the body’s own cells, and does not produce antibodies to it; at the same time, the immune system does not overreact to substances (such as food) that are not threatened in the environment. However, under certain circumstances, the immune system recognizes the body’s own substances and treats them as foreign invaders, thereby producing antibodies (ie, autoantibodies) against these substances, triggering autoimmunity. These autoantibodies attack the cells, tissues, and organs of the body, causing an inflammatory reaction and causing damage to the body.

The production of autoantibodies may be due to the presence of some of the same molecular structures between pathogenic antigens (bacteria, viruses, etc.) and their own components: an immune response that cross-reacts with autoantigens; or some infectious agents that cause autoantigens Denatured, the immune system produces autoantibodies to these exposed new antigens.

The pathogenic effect of autoantibodies is still unclear. Whether it is the “cause” or “consequence” of autoimmune diseases has different opinions. For patients with high titers of autoantibodies, those without clinical symptoms may not need treatment, but should go to the hospital regularly. Review and review.

 

In tumors, inflammation, autoimmune diseases (such as lupus erythematosus, genital warts, Crohn’s disease, multiple sclerosis), neurodegenerative diseases, infectious diseases, etc., a large number of autoantibodies are produced and accumulated in patients. Some autoantibodies have emerged in the early stages of a specific disease, even before the onset of symptoms of the disease, providing a reliable disease biomarker for the early diagnosis; some autoantibodies are the body’s own protection against disease. Sexual antibodies, which provide new ideas for the treatment of the disease, as data from the world-renowned pharmaceutical giants show that 60% of the profits of large pharmaceutical companies have come from drugs that belong to antibodies. So how do you discover these potential autoantibodies?

Methods for screening for autoantibodies:

At present, the most suitable method for screening autoantibodies is the protein chip method. A protein chip often has thousands of protein spots, which can screen one autoantibody that can interact with these proteins at one time, and then pass fluorescent These autoantibodies can be found by incubation of the marker against the anti-Human IgG secondary antibody and fluorescence detection.

The principle is simple, but it is very difficult to do, why? This has to say about the binding process of antibodies to antigens. In short, the corresponding antibody recognizes a specific epitope on the antigen and then binds it. The epitopes are divided into two types, linear epitopes and non-continuous epitopes. A linear epitope consists of a contiguous sequence of amino acids. An antibody that recognizes a linear epitope recognizes this amino acid sequence and produces an antigen-antibody binding reaction; a non-contiguous epitope is composed of a discontinuous amino acid, which is correct by the antigenic protein. After folding, they are close together and recognized by the corresponding antibody, producing an antigen-antibody binding reaction.

 

In the body, most autoantibodies are non-continuous epitopes that recognize antigens. Correct identification and screening of these autoantibodies requires that the proteins on the protein chip be full-length, correctly folded, and biologically functional. However, the traditional protein chip can only guarantee that the protein synthesized on the chip is full-length (some can not be guaranteed), can not guarantee the correct folding of the protein, and can not guarantee the normal biological function of the corresponding protein. Other problems affecting the screening of autoantibodies by traditional protein chips include high CV values (>30%), poor reproducibility, low resolution, high background signal, and inability to distinguish autoantibodies with low expression levels. Screening for autoantibodies with such protein chips is like fishing with a large network full of loopholes. Screening of autoantibodies with such protein chips has brought great resistance to researchers and companies in the research of antibody screen. Sengenics’ ImmunomeTM Protein Chip Research Platform, invented by Professor Jonathan Blackburn at the University of Cambridge in 1996, is a collaboration between Oxford and Cambridge. It is the only one in the world that is fully-length, correctly folded and functionally validated. Protein chip platform. Autoantibodies that recognize non-contiguous epitopes can be screened for advantages that cannot be replaced by other protein chip products.

The ImmunomeTM Protein Microarray Research Platform contains 1631 full-length, correctly folded and functionally validated human proteins, covering cancer antigens, transcription factors, kinases, signaling pathway molecules, etc., to meet the research needs of users in different directions. Daban’s low autoantibodies provide strong technical support as biomarkers. Compared to traditional protein chip products in the screening of autoantibody applications, Sengenics ImmunomeTM protein chip products can be described as “Skynet is restored, not leaking”, full-length, correct folding, functional verification, wide coverage, low coverage CV low background signal, high-resolution full-generation protein chip platform technology helps researchers and business users to “capture big fish” in the field of autoantibody research, and return home.

Researchers correct genetic mutation

UCLA researchers led by Dr. Donald Kohn have created a method for modifying blood stem cells to reverse the genetic mutation that causes a life-threatening autoimmune syndrome called IPEX. The gene therapy, which was tested in mice, is similar to the technique Kohn has used to cure patients with another immune disease, severe combined immune deficiency, or SCID, also known as bubble baby disease.

The work is described in a study published in the journal Cell Stem Cell.

IPEX is caused by a mutation that prevents a gene called FoxP3 from making a protein needed for blood stem cells to produce immune cells called regulatory T cells. Regulatory T cells keep the body’s immune system in check; without them, the immune system attacks the body’s own tissues and organs, which is known as autoimmunity.

The approach adds a normal copy of the FoxP3 gene to blood stem cells, which can produce all types of blood cells. In the study, the approach corrected the genetic mutation in mice with a version of IPEX that’s similar to the human version of the disease, and it restored proper immune regulation.

To get the normal copy of the FoxP3 gene to the proper place within the blood stem cells, the researchers used a tool called a viral vector — a specially modified virus that can carry genetic information to a cell’s nucleus without causing a viral infection. The UCLA team engineered the viral vector used in the study so that the gene is turned on only in regulatory T cells, but not in other types of cells.

“It’s exciting to see how our gene therapy techniques can be used for multiple immune conditions,” said Kohn, a professor of pediatrics and microbiology, immunology and molecular genetics at the David Geffen School of Medicine at UCLA and member of the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA. “This is the first time we’ve tested a technique that targets an autoimmune disorder, and the findings could help us better understand or lead to novel treatments for other autoimmune conditions such as multiple sclerosis or lupus.”

The name IPEX stands for immune dysregulation, polyendocrinopathy, enteropathy, X-linked. The syndrome can affect the intestines, skin and hormone-producing glands such as the pancreas and thyroid, as well as other parts of the body. It is typically diagnosed within the first year of life and can be life-threatening in early childhood. IPEX can be treated with a bone marrow transplant, but finding a matched bone marrow donor can be difficult, and the transplant procedure is often risky because people with IPEX can be very sick.

In the new study, the UCLA researchers used viral vectors to deliver normal copies of the FoxP3 gene to the genome of the mice’s blood stem cells so that they produced functional regulatory T cells. All of the mice in the study were virtually free of IPEX symptoms shortly after the treatment.

“It’s incredibly important that we only create regulatory T cells that have the non-mutated FoxP3 gene,” said Katelyn Masiuk, a student in the ULCA physician-scientist degree program and the study’s first author. “We found that if the FoxP3 protein is turned on in blood stem cells, the whole blood system functions abnormally. We realized that we needed a vector that only made FoxP3 in the regulatory T cells made from the blood stem cells, but not in the blood stem cells themselves or other types of blood cells they make.”

The researchers also put their IPEX-targeting vector into human blood stem cells and then transfused those cells into mice without immune systems. The human blood stem cells were able to produce regulatory T cells that turned on the vector.

Kohn, who also is a member of the UCLA Children’s Discovery and Innovation Institute and the UCLA Jonsson Comprehensive Cancer Center, said the results are promising and the researchers hope to test the approach in human patients.

Kohn said that to treat humans with IPEX, blood stem cells would be removed from the bone marrow of patients with IPEX. Then, the FoxP3 mutation would be corrected in a lab using the IPEX-targeting vector. The patients would receive a transplant of their own corrected blood stem cells, which would produce a continuous life-long supply of regulatory T cells.

Kohn is also the principal investigator in a clinical trial that is testing the use of patients’ own genetically corrected blood stem cells to treat sickle cell disease, the most common inherited blood disorder in the U.S. And in another study led by Kohn, a similar technique has cured 40 babies with SCID.

Kohn, Masiuk, Dr. Roger Hollis (a study co-author and member of Kohn’s lab) and Dr. Maria Grazia Roncarolo of Stanford University are inventors of the FoxP3 vector, for which a patent application has been filed by the UCLA Technology Development Group on behalf of the Regents of the University of California.

The FoxP3 vector for IPEX is not yet available in clinical trials and has not been approved by the FDA for use in humans.

The research was funded by the UCLA Molecular Biology Institute’s Whitcome Predoctoral Training Program and the T32 Medical Scientist Training Program, a program of the National Institute of General Medical Sciences.

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Introduction to a protein engineering technique—mutation

Positional mutation is a protein engineering technique that substitutes, inserts or deletes specific nucleotides in known DNA sequences based on the known structure and function of proteins to produce mutant protein (enzyme) molecules with novel traits. The technology is widely used in the biological and medical fields. Position mutation technology has the characteristics of high mutation rate, simple and easy to perform, and good repeatability. As a research method, localization mutation technology is also widely used to study the relationship between protein structure and function, so as to elucidate the regulation mechanism of genes, the etiology and mechanism of diseases.

 

 

Introduction

The “small change” of protein molecules based on natural protein structure refers to the modification, substitution or deletion of a few residues of proteins of known structure. This is the most widely used method in protein engineering, and can be mainly divided into proteins. Two types of modification and gene location mutation. Gene-localized mutation refers to the transformation of protein molecules at the genetic level, that is, the method of site-directed mutagenesis, the insertion, deletion, substitution and reorganization of nucleotide codons of genes encoding proteins, and then the mutated genes are carried out. The protein expresses and analyzes the functional activity of the expressed protein, and the result provides a new design for protein molecular engineering.

 

Design goals and solutions for location mutation

The common design goals of localization mutations are to improve the heat and acid stability of proteins, increase activity, reduce side effects, improve specificity, and conduct structural-functional studies through protein engineering. Hartley is equal to 1986 to complete a design goal and solution that we want, and still has important reference value. The stability of protein is an important prerequisite for the normal biological activity of proteins. Therefore, improving the stability of proteins has become one of the important goals of protein design and transformation.

 

Type of mutation

There are many ways to change the nucleotide sequence of a gene, such as chemical synthesis of genes, direct modification of genes, and cassette mutation technology. Depending on the manner in which the gene is mutated, it can also be classified into three categories: insertion of one or more amino acid residues; deletion of one or more amino acid residues; replacement or substitution of one or more amino acid residues. In order to achieve the purpose of gene location mutation, in vitro recombinant DNA technology or PCR method is often used.

 

Site-directed mutation

The amino acids in a protein are determined by the triplet codon in the gene. By changing one or two bases, the amino acid species can be changed to produce a new protein. It is usually the amino acid that changes a position in the functional region to study the structure, stability or catalytic properties of the protein. The work of point mutation is the main body of current protein engineering research. So far, many kinds of proteins such as subtilisin, T4 lysozyme, dihydrofolate reductase, trypsin and ribonuclease have been modified. For example, replacing Asn117 of a tissue-type plasminogen activator (t-PA) with Glu117, thereby removing an original glycosylation site; since the original sugar chain can promote t -PA is cleared from plasma, so point mutations can reduce plasma clearance of t-PA and prolong plasma half-life.

 

Box mutation

In 1985, Wells proposed a genetic modification technique for a box-type mutation that can produce 20 different amino acid mutants at one site, and can perform “saturation” analysis of important amino acids in protein molecules. Using the localization mutation, two original vectors and endonuclease cleavage points not present on the gene are added on both sides of the amino acid code to be modified, and the endonuclease is used to digest the gene, and then the synthesized double-stranded DNA fragment with different changes is substituted for digestion. part. A variety of mutant genes can be obtained in such a single treatment.

 

Procedure for locating mutations

The protein molecular design program for gene localization mutation follows the procedure in the design principle, but the gene location mutation has its own particularity, and its specific procedure is as follows.

  1. Establish a structural model of the protein under study

Establishing a three-dimensional structural model of a protein is critical to establishing a mutation site or region and predicting the structure and function of the mutated protein. The structure can be determined by X-ray crystallography, two-dimensional nuclear magnetic resonance, or the like, or a structural model can be established based on the structure of the analog or other structural prediction methods.

  1. Identify locations that have a significant impact on the required properties
  2. Predict the structure of the mutant
  3. Construct mutants. Mutant protein
  4. Examination of mutant proteins

 

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Introduction to plant tissue culture

Tissue culture techniques of higher plants refer to techniques for isolating one or several individual cells or a part of a plant body for cultivation.

Generally speaking, we refer to a generalized tissue culture in which a part of a plant body (i.e., an explant) is isolated by aseptic processing, inoculated on a medium, and cultured under artificially controlled conditions to produce a complete plant.

Image: plant cell culture

Physiological basis of plant cell culture

Cell totipotency: Each cell of a plant carries a complete set of genomes and has the potential to develop into a full plant.

Plant growth regulating substances play a key role in the differentiation and determination of plant cell tissues. It includes: auxins, cytokinins, gibberellins, abscisic acid, ethylene, and the like.

Auxins are mainly used for the formation of callus, the production of somatic embryos and the rooting of test-tube seedlings. Commonly used auxins are 2,4-D, NAA (naphthaleneacetic acid), IBA (indolebutyric acid), IAA (indole acetic acid) and the like. Its effect is 2,4-D>NAA>IBA>IAA.

Cytokinins promote cell division and differentiation, delay tissue aging, and promote bud production. Commonly used cytokinins are Zip, KT (clopidogrel), 6-BA (6-benzylaminoadenine), ZT (zeatin) and others. The order from strong to weak is Zip>KT>6-BA>ZT.

Gibberellin promotes the growth of differentiated shoots and breaks the dormancy of seeds. Commonly used gibberellin is GA3.

Types of plant cell culture

Tissue culture can be divided into tissue or callus culture, organ culture, plant culture, cell and protoplast culture according to the culture target.

  1. Tissue or callus culture is a tissue culture in a narrow sense, which is to cultivate various parts of the plant, such as shoot tip meristem, formation layer, xylem, phloem, epidermal tissue, endosperm tissue and thin wall tissue, etc.; or culture of callus produced by plant organ culture, both of which are induced to form plants by re-differentiation.
  2. Organ culture, namely the culture of isolated organs, depend on the crop and needs, may include isolation of shoot tips, stem segments, root tips, leaves, leaf primordia, cotyledons, petals, stamens, pistils, ovules, embryos, ovary, or fruits culture of explants.
  3. Plant culture is the cultivation of intact plant material, such as seedlings and larger plants.
  4. Cell culture is a culture of ex vivo single cells or pollen single cells or small cell clusters which can maintain good dispersibility by liquid shaking culture of callus or the like.
  5. Protoplast culture is the cultivation of protoplasts that remove cell walls by

enzymatic and physical methods.

Characteristics of plant cell culture

Tissue culture is a new technology developed in this century. Due to the advancement of science and technology, especially the application of exogenous hormones, tissue culture not only provides theoretically reliable experimental evidence for related disciplines, but also becomes a kind of a new method for large-scale, batch-scale production of seedlings.

The reason why plant tissue culture has developed so rapidly is that it has such a wide range of applications due to the following characteristics:

  1. Culture conditions can be artificially controlled.

The plant material used in tissue culture is completely grown under artificially supplied culture medium and microclimate environment. It is free from the adverse effects of four seasons, day and night changes and severe weather in nature, and the conditions are uniform, which is very beneficial to plant growth. It is convenient for stable annual production.

  1. Short growth cycle and high reproductive rate.

Due to artificially controlled culture conditions of plant tissue culture, it provides different culture conditions according to various requirements of parts of plants, so the growth is faster. In addition, the plants are also relatively small, often 20-30d for a cycle. Therefore, although plant tissue culture requires certain equipment and energy consumption, since plant materials can be produced in a geometrical order, the overall cost is low, and high-quality seedlings or virus-free seedlings of uniform specifications can be provided in time.

  1. Convenient management, which is conducive to factory production and automation control

Plant tissue culture is conducted under certain conditions of temperature, light, humidity, nutrition, hormones, etc. in a certain place and environment, which is highly conducive to high intensification and high-density factory production, and is also conducive to automatic control of production.

It is the development direction of future agricultural factory cultivation. Compared with pot cultivation and field cultivation, it saves a series of complicated labors such as cultivating and weeding, watering and fertilizing, and controlling pests and diseases, which can greatly save manpower, material resources and land needed for field planting.

Lifeasible offers a complete range of high-quality plant tissue culture products that facilitate the development of new plant traits and large-scale production, including Amino Acids, Antibiotics, Auxins etc.

Detailed introduction of protein structure

Protein is mainly composed of chemical elements such as carbon, hydrogen, oxygen and nitrogen. It is an important biological macromolecule. All proteins are multimers formed by the connection of 20 different amino acids. After Forming proteins, these amino acids are also called as a residue.

 

The boundaries between proteins and peptides are not very clear. Some people believe that the number of residues required for a functionally acting domain is called a polypeptide or peptide if the number of residues is less than 40. To function biologically, proteins need to be properly folded into a specific configuration, mainly through a large number of non-covalent interactions (such as hydrogen bonds, ionic bonds, van der Waals forces and hydrophobic interactions); in addition, in some proteins (especially in the case of secreted proteins), disulfide bonds also play a key role. In order to understand the mechanism of action of proteins at the molecular level, it is often necessary to determine the three-dimensional structure of a protein. Structural biology has been developed by studying protein structure, using techniques including X-ray crystallography, nuclear magnetic resonance, etc. to resolve protein structures.

 

A certain number of residues are necessary to exert a certain biochemical function; 40-50 residues are usually the lower limit of the size of a functional domain. Protein size can range from such a lower limit up to thousands of residues. The current estimated average length of proteins differs between different species, typically about 200-380 residues, while eukaryotes have an average protein length of about 55% longer than prokaryotes. Larger protein aggregates can be formed by many protein subunits; for example, by the polymerization of thousands of actin molecules to form protein fibers.

 

Discovery history

In 1959, Perutz and Kendrew analyzed the structure of hemoglobin and myoglobin, solved the three-dimensional structure, and won the 1962 Nobel Prize in Chemistry.

Pauling discovered the basic structure of the protein. Based on the X-ray diffraction data, Crick and Watson proposed a model of the three-dimensional structure of DNA. Received the 1962 Nobel Prize in Physiology or Medicine. After the 1950s, Hauptmann and Karle established a purely mathematical theory for the direct determination of crystal structures using X-ray analysis, which has epoch-making significance in crystal research, especially in the study of macromolecular biological substances such as hormones, antibiotics, and proteins. And the molecular structure of new drugs played an important role. They were awarded the 1985 Nobel Prize in Chemistry.

 

Structure type

Protein molecules are covalent polypeptide chains formed by the condensation of amino acids end-to-end, but natural protein molecules are not loose random polypeptide chains. Each natural protein has its own unique spatial structure or three-dimensional structure, which is often referred to as the conformation of the protein, ie the structure of the protein.

The molecular structure of a protein can be divided into four levels to describe its different aspects:

Primary structure: A linear amino acid sequence that makes up a protein polypeptide chain.

Secondary structure: a stable structure formed by hydrogen bonds between C=O and N-H groups between different amino acids, mainly α-helix and β-sheet.

Tertiary structure: The three-dimensional structure of a protein molecule formed by the arrangement of multiple secondary structural elements in three dimensions.

Quaternary structure: used to describe a protein complex molecule that is functionally formed by interactions between different polypeptide chains (subunits).

In addition to these structural levels, proteins can be transformed in multiple similar structures to perform their biological functions. For functional structural changes, these tertiary or quaternary structures are usually described in a chemical conformation, and the corresponding structural transformation is referred to as a conformational change.

 

 

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What is Adjuvant Selection?

Adjuvant Selection is the main method for finding molecular genetic markers, vaccines like bacterial vaccines, cancer vaccines, RNA vaccines, MAS breeding and so on.

Major methods for finding molecular genetic markers

DNA markers are divided into two types: type Ⅰ markers, mainly are some single genes, and used to compare the homologous loci varieties of relative distance and chain and linear correlation; Ⅱ type markers, mainly high polymorphism, information content rich DNA fragments, is one of the most commonly used microsatellite marker. Through Adjuvant Optimization, more and more kinds of molecular markers were introduced, including restricted fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and Microsatellites. At present, there are 2,505 markers in the world’s pig research, including 1,391 microsatellite markers. Ⅰ type 873, type Ⅱ mark 1632.

The main methods to search for molecular genetic markers are candidate gene method and genome scanning method.

  1. Candidate gene method as a candidate gene for a trait, it is usually some genes whose biological functions and nucleic acid sequences are known, and they are involved in the growth and development process of the trait. These genes may be structural genes, regulatory genes or genes that affect the expression of traits in biochemical metabolic pathways. Candidate gene method research should follow certain steps, such as candidate gene selection primer design, gene specific fragment amplification, polymorphic locus search and so on. Candidate gene search USES genes that are thought to have a direct physiological function for a trait to find QTLS. In addition, genes found in other species that control some traits can be studied as candidate genes for pigs. For example, the h-fabp gene affects the backfat thickness and intramuscular fat content of pigs (Gerbens et al., 1999). 2000; 2001); The melanocorticoid receptor 4 (MC4R) gene was significantly correlated with the intake, backfat thickness and growth rate of pigs (Kim et al., 1999; 2000).
  2. Genome scanning: all genetic information is stored on the 19 pairs of chromosomes of the pig. Reference families were established, such as meishan European and American pig species, wild boar big white pig, and their hybrid offspring were used to find QTL through genetic markers. The most effective design is the genotype analysis of F2 generation isolation population. Figure 4-2 is a simple schematic diagram of single genetic marker and linkage QTL analysis. Alleles of genetic markers and their linkage QTL in F1 generation were heterozygous. In the F2 generation isolation population, the ratio of the three possible genotypes per seat should be 1:2:1, when the average performance of marker genotypes is compared, the existence of linkage QTL can be analyzed.

Anderson (1994), such as reported with a wild boar by the results of the large white building reference group, using the 105 DNA markers in the genetic map, the separation of F2 generation 200 pigs linkage analysis research found that on chromosome 4 seat back fat and control the growth rate, the average genetic effect 24 g/d and 5 mm, respectively, the equivalent of F2 DaiQun total phenotypic variation of 12% and 18%. Daily weight gain can differ by more than 50g between two extreme homozygous genotypes, resulting in a 10kg weight difference at market time in pigs.

(iii) MAS breeding

In pig breeding selection, it is difficult to determine the sex efficiency of low heritability (e.g., reproductive traits), high cost of measurement (e.g., disease resistance), phenotypic values (e.g., lean meat rate) or limited sexual performance (e.g., milk production) early in development. It is estimated that the selection of the marker before the determination of the offspring can increase the selectivity response by 10%~15%. The MAS of compatriots who choose to combine can be increased by about 40%. Combining multiple genetic markers and trait information, the selectivity response can be increased by 50%~200%. Using marker selection in cross breeding can predict and make full use of heterosis. Molecular genetic markers can also be applied to early selection and screening and detection of large populations to select populations with desired genotypes.

For example, there is little progress in the improvement of pig litter, a low genetic trait, by traditional methods. Rothschild et al. found in 1994 that the estrogen receptor (ESR) gene was one of the main genes responsible for the litter size of pigs, which could control the total litter size of 1.5 pigs and the live litter size of 1 pig in the meishan synthetic line of China. In the Chinese two-flower face hybrid population, the agricultural university of China not only confirmed the results of Rothschild et al., but also found another main gene locus controlling the number of piglets – FSH, which can control the total number of piglets and the number of live piglets by 2.0.

Although MAS can improve the effectiveness of selection and the annual amount of genetic improvement, its effectiveness is also affected by many factors. In addition to the heritability of traits, the intensity of selection, and the size of the selected population, the determinants are the linkage between genetic markers and QTLS. Zhang (1992) pointed out that each QTL could be specifically detected by using genetic markers closely linked to QTL, and the final selection of genetic markers would be equivalent to the selection of QTL itself. Therefore, genetic markers closely related to QTL must be obtained in order to improve MAS efficiency. Resources at present, through the establishment of the pig family, has some related to the growth, reproduction and carcass, meat quality of QTL mapping in some microsatellite nearby, such as on chromosome 3 microsatellite Sw2427 – Sw251 area and daily gain of pigs, on chromosome 4 S0101 – S0107 area and back fat belly fat, 7 chromosome S0064 S0066 regional composition and has a strong correlation between birth weight and body. It can be predicted that with the discovery of more genetic markers closely linked to QTL, MAS will be applied more effectively in practical breeding.