Introduction to Coagulation Factor Ⅷ

G99

Recombinant mouse anti factor VIII antibody G99 is capable of binding to coagulation factor VIII C2 domain, expressed in Chinese Hamster Ovary cells (CHO).

What is coagulation factor Ⅷ?

 

Human coagulation factor Ⅷ (FⅧ) is an important protein in human plasma, which is the main component of human endogenous coagulation pathway. The low level of FⅧ in blood will lead to different degrees of coagulation dysfunction, that is, hemophilia A (HA).

The structure of human coagulation factor Ⅷ

The FⅧ gene consists of 26 exons and 25 introns, of which 14 and 26 exons are the largest, 3106 bp and 1958 bp, respectively, with a total length of 186 kb, located at the end of the long arm of X chromosome, accounting for about 0.1% of the whole X chromosome. After transcription and cleavage, the FⅧ gene is about 9 kb long and encodes a precursor containing 2351 amino acid residues. After the signal peptide was hydrolyzed in endoplasmic reticulum, a mature FⅧ single chain protein with 2332 amino acid residues and 6 domains was formed. According to the homology of the internal sequence of FⅧ, FⅧ can be divided into three domains: A, B and C. Among them, the A domain can be divided into three types: A1, A2 and A3, each containing about 350 amino acid residues, with 30% homology. A1 and A2 are located in the heavy chain, and A3 in the light chain. The B domain is located in the middle of the molecule, with 908 amino acid residues. The C domain is located in the C end of the light chain, with C1 and C2, each containing about 150 amino acid residues, with 40% homology. The A3 region of FⅧ is located in the phospholipid bilayer of cell membrane and adjacent to C1 and C2 regions; the long axis of C1 region is approximately parallel to the cell membrane plane and almost perpendicular to C2; there are two highly conserved βbarreled domains between A1, A2 and A3 regions with small angles to each other. The whole molecule is arranged as a1-a2-b-a3-c1-c2. Through the complex interaction with various structural proteins and enzymes inside and outside the cell, it affects the protein activity, transmembrane transport and gene expression of FⅧ.

Expression vector and system of recombinant FⅧ

There are three kinds of common vectors: bacterial plasmids, phages and animal and plant viruses. They are self-replicating DNA or RNA molecules that transfer the target gene to the receptor cell. In the construction of recombinant FⅧ vector, these three types have been used, and the application of bacterial plasmids and animal viruses is more common. Virus vectors have been widely used for their advantages of wide host range, large capacity and simple operation. Adenovirus, adeno-associated virus, retrovirus and herpes simplex virus have been reported. In the expression system, prokaryotic expression system is easy to operate, but there are some defects in the expression of eukaryotic recombinant FⅧ protein, such as the wrong link of disulfide bond, the ineffective folding of peptide chain and the low efficiency of glycosylation, so it is not widely used.

Gene therapy

The main pathogenesis of hemophilia A is the inversion and missense mutation of intron 22. With the thorough research on the structure of FⅧ gene and protein by scientists, and the low requirement of individuals on the normal physiological level of FⅧ, many organs can produce active FⅧ. Therefore, hemophilia A is the first choice for somatic gene therapy.

  1. In vitrotreatment

In vitro treatment, the cells of a certain tissue are taken out from the patient’s body first. After in vitro expansion and culture, the vector carrying the recombinant FⅧ gene is transfected into the recipient cells, and the successful transfected cells are selected to be transplanted into the patient’s body, and then the target of treatment is achieved by the expression of FⅧ in these transplanted cells. Because of the insecurity of virus vector, tissue cells such as fibroblasts and myoblasts are used in clinic.

  1. In vivotherapy

In vivo treatment refers to the direct injection of FⅧ expression vector into the patient’s body. At present, the safest treatment is to inject naked DNA without carrier directly into the patient, and to treat by transfection of liver cells in vivo and self -expression of FⅧ, which is an effective treatment in vivo. In addition, through the efficient integration of transposons with mammalian genes, the eukaryotic transposon vector containing FⅧ gene was introduced into the receptor to express f Ⅷ. However, due to the uncertainty of transposon integration gene location, the safety of this method needs to be further verified.

It has to be mentioned that in recent years, with the birth of a new gene editing technology CRISPR/Cas9, scientists only need to design a snippet of CRISPR with dozens of bases, using target specific RNA to bring Cas9 nuclease to specific targets of the genome, which can cut specific gene sites and lead to mutations. This technology has been applied in gene knockout, gene editing related diseases, gene activation and expression and many other fields. CRISPR/Cas9 can also be used as a precise gene cutting tool in eukaryotic transcription regulation. The application of CRISPR/Cas9 in the study of recombinant f Ⅷ can not only simplify the steps of gene editing, but also greatly improve the efficiency of recombination, reduce the residue or deletion of gene fragments caused by cutting, which can improve the success rate of transfection. In the same expression system, CRISPR/Cas9 can correctly fold the expressed recombinant f Ⅷ, thus improving the yield of recombinant FⅧ. CRISPR/Cas9 also has a revolutionary application prospect in the gene therapy of hemophilia A. It is believed that the application of CRISPR/Cas9 in the study of recombinant FⅧ will have profound significance.

Subunit vaccine and its application

Subunit vaccine is made from special immunologically active protein fragments extracted by chemical decomposition or proteolysis from bacteria and viruses. A subunit vaccine is made up of components that present the major protective immunogens of pathogenic bacteria, also known as component vaccines.

What is a subunit vaccine? The vaccine, which does not contain nucleic acid and can induce the body to produce antibodies, is made up by a certain surface structural component (antigen) of microorganisms, called subunit vaccine.

Among the multiple specific antigenic determinants carried by macromolecular antigens, only a small number of antigenic sites contribute to the protective immune response. The special protein structure of bacteria and viruses is extracted by chemical decomposition or controlled proteolysis, and the vaccine prepared by immunologically active fragments is selected as a subunit vaccine.

Subunit vaccine has only a few major surface proteins, avoiding the production of many unrelated antigen-induced antibodies which reducing vaccine side effects and vaccine-related diseases.

The shortcoming of the subunit vaccine is that it is less immunogenic and needs to be combined with an adjuvant to produce a good immune effect. In the 1970s and 1980s, virion subunits and surface antigen (HA and NA) vaccines were developed on the basis of split vaccines. The influenza virus membrane proteins HA and NA were cleaved by appropriate lysing reagents and cleavage conditions, and obtained by appropriate purification methods. Subunit influenza vaccines have very pure antigenic components. In clinical trials in the UK, the subunit influenza vaccines has been demonstrated that the immune effect is the same as that of the split vaccine and can be used in children. It was first approved for use in the UK in 1980 and then extended to other countries.

Subunit vaccine applicationrecombinant subunit vaccine

Recombinant subunit vaccine, also known as genetic engineering subunit vaccine and biosynthetic subunit vaccine, is made from protective antigen gene products, proteins or peptides, expressed in prokaryotic or eukaryotic cells. Traditional vaccines are often made by serial passage or chemical or physical inactivation of certain components. Although they are effective, there is a risk of reversal of virulence. Subunit vaccines prepared by molecular biology techniques are superior in safety to traditional vaccines. Non-recombinant subunit vaccines are vaccines consisting of individual proteins or oligosaccharides, such as bacterial lipopolysaccharides purified from pathogenic microorganisms. Subunit vaccines often require adjuvants or various conjugates to enhance their immunogenicity, and the production of these vaccines usually requires the large-scale cultivation of pathogenic microorganisms, which is costly and has certain risks.

 

Advantages: 1. High safety; 2. High purity, good stability; 3. High yield; 4. For vaccine research that is difficult to culture or potentially carcinogenic, or pathogenic.

Disadvantages: Compared with traditional subunit vaccines, the immune effect is poor.

 

Ways to enhance its immunogenicity:

  1. 1. Adjust the gene combination to express it into a granular structure;
  2. 2. Coagulatein vitro, encapsulated into liposomes or capsule microspheres;
  3. 3.An immunopotentiating compound is added as an adjuvant;

The expression systems for subunit vaccine production mainly include Escherichia coli, Bacillus subtilis, yeast, insect cells, mammalian cells, transgenic plants, and transgenic animals.

The more successful genetic engineering subunit vaccine is the hepatitis B surface antigen vaccine.

Overview of IgG Antibodies and Anti-human IgG Antibodies

What is an IgG antibody?

The IgG antibody is one of the types of mammalian antibodies (also known as immunoglobulins, Ig for short). The antibody-associated immunity against pathogen invasion is mainly provided by four types of antibodies, and is the only one that can provide passive immunity to the fetus through the placenta.IgG antibodies act to activate complement and neutralize multiple toxins in the immune response. IgG antibodies last a long time and are the only antibodies that protect the fetus from the placenta during pregnancy. They also secrete from the mammary gland into the colostrum, giving the newborn the first time to get antibody protection. IgG is a four-chain monomer, accounting for 75% of the total serum Ig. It is the most important antibody component in serum and extracellular fluid. Human IgG can be divided into four sub-categories, depending on the concentration in serum. IgG1, IgG2, IgG3, IgG4. IgG is synthesized three months after birth and is close to the adult level from three to five years old. It is mainly produced by plasma cells in the spleen and lymph nodes. It has a long serum half-life of about 20~23 days. It is the main antibody produced by the re-humoral immune response. It has high affinity, is widely distributed in the body, has important immune effects, and is anti-antibody. The main force of infection, so the clinical use of gamma globulin for immunotherapy should be injected every 2 to 3 weeks is appropriate.

IgG1, IgG3, and IgG4 can cross the placental barrier and play an important role in anti-infective immunity in neonates; IgG1, IgG2, and IgG4 can bind to staphylococcal protein A (SPA) through its Fc segment, thereby purifying antibodies, or For immunodiagnosis; IgG1, IgG3 can efficiently activate complement, and can bind to macrophage and NK cell surface Fc receptors, play a role in opsonization, ADCC, etc.; some autoantibodies and antibodies that cause type II and type III hypersensitivity reactions also belongs to IgG.

IgG antibody function:

It plays an important role in anti-infective immunity, especially in re-immune responses. IgG-type autoantibodies participate in type II and type III hypersensitivity reactions.

  1. Activate the classical pathway of complement, mediating bacteriolysis and cytotoxicity;
  2. Mediating the ADCC effect;
  3. Conditioning phagocytosis;
  4. Combine SPA;
  5. Neutralize toxins and viruses.

What are the techniques for IgG antibodies?

  1. Extraction of globulin

Mostly, ammonium sulfate salting out or sodium sulfate salting out is used. Ammonium sulfate salting has to be precipitated several times, with 40% saturation for the first time, 35% saturation for the second time, and 33% saturation for the third time. The gamma globulin after three extractions is basically an IgG component. The sodium sulphate method is simpler and the gamma globulin can be precipitated with 20%. Although the gamma globulin after salting out is mostly IgG, there are 5% other zone proteins, such as the gamma region heteroprotein. Interference is also caused by the inclusion of other so-called normal IgGs in the IgG component. Therefore, the γ-globulin crudely extracted by the salting-out method can only be used for general experiments or anti-serum with higher antibody titer.

  1. Ion exchange chromatography for IgG extraction

Commonly used ion exchangers are DEAE cellulose or QAE cellulose, which is ideal for QAE-Sephadex, and DE22, 32, 52 are also applicable. Take QAE-Sephadex A25 or A50 acid-treated and equilibrate in 0.05mol/L phosphate buffer pH 7.5-8.6, drain the water, weigh the wet weight Ig in 10ml serum, centrifuge at room temperature for 30min, filter or filter The ion exchanger is removed. The supernatant is treated once more to obtain a relatively pure IgG, even without other miscellaneous proteins. Purification of IgG by this technique is simple and does not damage the antibody, and can be extracted in small amounts or in large quantities.

  1. Affinity chromatography to extract specific IgG

The purified antigen or the crude antigen is cross-linked to Sepharose 4B to prepare an affinity chromatography column. After the antiserum is passed through the column, the unbound heteroprotein is washed away, and then eluted with potassium thiocyanate, and the pure specific IgG antibody is eluted. Because potassium thiocyanate has a destructive effect on the antibody, it should be dialyzed and removed in time. Purified IgG has low content and loses its protective effect. It should be applied in time or lyophilized. It can also be stored at -20 °C, but it is not ideal. Adding triethanolamine or glycerol can protect it.

  1. Preparation of F2 fragment by enzymatic hydrolysis

The point of action of pepsin on IgG is at the C-terminus (232 amino acids) of the disulfide bond connecting the two heavy chains, and as a result, the two Fabs are linked by a disulfide bond, retaining the binding site of the antibody. Compared with IgG, F(ab’)2 is characterized by the removal of the Fc segment, which eliminates the receptor action in cellular immunization experiments; it also causes IgG to lose its primary antigenic properties and is not bound by anti-IgG antibodies; indirect hemagglutination, sensitization of sheep red blood cells with F(ab’)2 is better than IgG.

Anti-human IgG antibody research

In the literature “Experimental study of anti-human IgG antibody combined with mitomycin in the treatment of bladder cancer”, in vitro experimental methods and tumor-bearing nude mice experimental methods were used to observe the combined use of anti-human IgG antibody and mitomycin C (MMC). The biological effects of inhibiting the growth of bladder cancer T24 cells and inducing apoptosis of tumor cells. As a result, it was found that, in addition to B lymphocytes and plasma cells, a variety of epithelial-derived tumor cells and some normal epithelial cells can produce IgG and have a function of promoting tumor cell growth. Anti-human IgG antibody can inhibit the growth of tumor cells, and can also significantly promote the apoptosis of tumor cells. In previous studies, we found that bladder cancer cells can express IgG significantly. Immunohistochemistry, mRNA in situ hybridization, RT-PCR and Western blotting methods also confirmed the presence of IgG in bladder cancer cell lines BIU-87 and T24.

Several protein modification methods (Part One)

The human proteome contains more functional polypeptides than all genes contained in the genome, in part due to simultaneous and post-translational protein modifications. The goal of proteomics research is to obtain a complete picture of the functional proteins present in a particular cell or tissue type, and in healthy or diseased tissues. One of the important areas of proteomics research is the identification of post-translationally modified proteins, their modification sites, the function of modifications, and the interaction of modified proteins in cellular functional networks.

In the past few decades, various methods have been developed for the determination of protein modifications. Here, we highlight the general methods for identifying protein modifications, mass spectrometry; specific methods for identifying phosphorylation, phosphate labeling; methods for identifying ubiquitination; and recognition of histone acetylation and methylation during chromatin remodeling and a method for identifying protein glycosylation.

Mass spectrometry

For the past 20 years, mass spectrometry has become an indispensable tool for determining the type and location of protein modifications. Mass spectrometry can be used for purified proteins or mixtures of proteins, such as cell lysates.

The mass spectrometer produces gas phase ions from a protein sample, separates them according to mass to charge ratio (m/z), and records their abundance. Mass spectrometry can be used for molecular weight determination of polypeptides and proteins, determination of polypeptide amino acid sequences, and detection of post-translational modifications, as well as relative quantification of polypeptides and proteins. This method cannot be used for absolute quantification.

Sample preparation by mass spectrometry, is digested into small molecule polypeptide fragments with restriction endonucleases such as trypsin or lysate. These polypeptide fragments were then evaporated and analyzed to determine their m/z values. Since the cleavage sites are known, a specific amino acid sequence for each peptide can be determined by quality using a computer program. Since the molecular weight and charge of a molecule such as a phosphate group are known, phosphorylation of a specific amino acid in a peptide can also be detected.

A matrix-assisted laser desorption/ionization (MALDI) peptide map or nanoliter mass spectrometer can analyze the digested protein sample. However, the limitation of these methods is that all peptide fragments cannot be completely detected, some peptides are clearly visible, and some peptides are not visible. This is often a major problem for the analysis of complex mixtures. For the analysis of modified peptides and post-translational modifications, the peptides are first separated by reverse phase chromatography, then fractions are collected and analyzed by mass spectrometry (LC/MS). In order to more accurately determine the nature of peptide modification, tandem mass spectrometry (MS/MS) experiments are often used. After the first MS step, the peptide ions are struck with an inert gas, resulting in further fragmentation. These polypeptide fragments are then analyzed in a second MS step. During this process, some of the modified peptides will remain unchanged, and the resulting peptide pattern will be similar to the uninterrupted peptides. Some peptides will be significantly fragmented, and the resulting peptide pattern can be the nature and position of the modified amino acids points provide further information.

In addition to being used to determine the modified state of a single protein, mass spectrometry was also used to determine a broad data set of all proteins with a specific modification such as phosphorylation. This usually involves affinity chromatography prior to mass spectrometry. This technique is used to identify “secondary proteome”; with such modifications, phosphorylation proteome analysis of the activation state of the entire signaling network in cancer can be used.

Mass spectrometry has been used to determine all four modifications described above. However, mass spectrometry is expensive, requires specific equipment, and often requires more quantitative data, so mass spectrometry is often used in conjunction with other biochemical methods to analyze post-translational modifications of proteins.

Phosphorylation

Phosphorylation, or the addition of a phosphate group to a serine, threonine or tyrosine residue, is one of the most common forms of protein modification. In the signal transduction pathway in cells, protein phosphorylation plays an important role and is a reversible, fine-tuned signal. Several in vivo and in vitro methods are used to detect the phosphorylation status of a protein, to detect phosphorylation of a particular amino acid, and to determine whether a particular kinase (or phosphatase) acts on the target protein.

In vitro phosphorylation analysis

The radiometric kinase reaction uses 32P-gamma-ATP and can be used to detect phosphorylation status in vitro. This is also the gold standard for detecting the effects of specific kinases. Purified target protein, kinase, and 32P-gamma-ATP were incubated in reaction buffer. Then, the reaction solution was filtered through a filter, the protein was bound to a filter, the unbound ATP was washed away, and the phosphorylation level (the radioisotope remaining in the filter) was detected by a scintillation counter. Alternatively, the reaction solution was analyzed by SDS-PAGE electrophoresis and observed by X-ray film exposure. This method is semi-quantitative, but the molecular weight of the phosphorylated protein can be determined.

Likewise, the reaction can include a phosphatase, rather than a kinase, to determine if the phosphorylated protein is a substrate for a particular phosphatase.

Radioactive pulse marking

To detect phosphorylation in vivo, radioactive pulse labeling can be used. The cells are grown in the presence of 32P-orthophosphate. A certain antibody is then immunoprecipitated with a specific antibody, and the resulting radioisotope is precipitated by scintillation counter or SDS-PAGE electrophoresis and X-ray film exposure. This method can detect phosphorylation under various physiological conditions. In addition, in combination with protein knockout assays, this method can also be used to analyze whether a particular kinase or phosphatase affects the modified state of the target protein.

Phosphorylation-specific antibody

There are two classes of phosphorylation-specific antibodies. The first class is the universal phosphorylated tyrosine, phosphorylated serine, phosphorylated threonine antibody, which binds to any phosphorylated tyrosine, phosphorylated serine and phosphorylated threonine molecules, and is not related to adjacent amino acid residues. The second class includes antibodies that phosphorylate specific amino acid epitope antibodies.

After the (hypothetical) phosphorylation site is identified by computer or by mass spectrometry, a universal antibody against the phosphorylation site can be purchased from the company by immunoprecipitation to determine if the target protein is phosphorylated at a particular site (eg, a typical cyclin/cdk site). Antibodies prepared for a particular phosphorylated amino acid, such as phosphotyrosine, can also be used. Next, phosphorylated antibodies were prepared against specific epitopes and phosphorylation was detected by simple immunoblotting. In order to analyze a large number of samples, an enzyme-linked immunosorbent assay can be used to bind the target protein to the membrane and then detect it with a phosphorylated specific antibody.

To be continued in Part Two…

Food safety: microbial contamination is a big problem

The theme of “World Health Day” on April 7th is “food safety is the shared responsibility of everyone who grows, processes, transports, stores, sells and consumes food.” This may surprise people who have often been worried about food safety in recent years: has the problem become a worldwide issue?

In a way, it’s Yes.

Earlier, the World Health Organization issued a report on March 31: changes in food production, sales and consumption, as well as environmental changes, emerging pathogens and antimicrobial resistance, have brought challenges to the national food safety system, and increased travel and trade have also increased the likelihood of international spread of contaminated food.

In particular, disease-causing organisms in food are widely spread over long distances through today’s interconnected global food chains, causing the frequency and extent of food-borne illness to escalate and expand, and food contamination at one source is likely to be largely spread, and thus lead to serious health and economic consequences. For this reason, WHO’s new data on food-borne disease hazards highlights the global threat posed by unsafe food and the need for concerted cross-border action throughout the food supply chain.

This also shows us: pay attention to food safety when there is a broader perspective.

In general, food safety mainly involves three aspects: from the perspective of quantity, it is necessary to ensure the balance between supply and demand and meet the demand for food quantity; from the perspective of quality, the nutritional structure of food is required to be reasonable, high quality and health without pollution; from the perspective of development, the acquisition of agricultural products requires attention to the good protection of the ecological environment and the sustainability of resource utilization.

However, it is difficult for the public to know what kind of process they have experienced in reaching the food on the table. From upstream agriculture to midstream food manufacturing, food distribution and catering, and finally to downstream consumers, it involves innovations in various systems, administrative measures, food and other related technologies – all of which constitute a complex food consumption system. Food safety supervision is difficult. One important reason is that the chain from the head to the table is long and has many links. Its regulatory focus has also shifted from the final food testing to the full control of production and operation, establishing a food safety supervision system from farm to table.

However, on overall, food safety issues are complex and severe, but as long as you think about the various aspects of food from raw materials to the table, you can know where the problem may come from: soil, air, and water are sources of various environmental pollution; chemical fertilizers, herbicides, veterinary drugs, nitrites, etc. are sources of cultivation and pollution; packaging pollution, additive abuse, adulteration with non-food substances, and various microbial contamination are all dangerous sources of processed foods. The potential risks of cross-contamination, food spoilage and widespread spread of contaminated food due to the expansion and complication of the food supply chain are also worthy of attention.

It should be pointed out that the public is usually accustomed to arranging food additives, pesticide residues, genetically modified foods, etc. at the forefront of food consumption risks or potential risks of health damage, and the biggest threat to food-borne diseases is not yet given enough attention to the food safety. In fact, for the general population, microbial contamination is the biggest threat to human health in food-borne infectious diseases. Most of these food-borne diseases are caused by bacteria, viruses, worms and fungi, and are usually concealed and difficult to control. Once they occur, they are fulminant. The abuse of antibiotics by the public after the onset of food-borne illness is also very worrying, as it will lead to an increase in the number of resistant bacteria, and in the long run is a threat to food safety.

Lifeasible is a leading partner of microbiology testing in the food industry. Its food microbiology laboratory, which is accredited by the ISO/IEC 17025 general requirements for the competence of testing and calibration laboratories, provides microbiological analysis services in support of your food quality control programs. The microbiological testing portfolio includes analytical services for the detection of pathogenic microbes and spoilage organisms, as well as for the identification and classification of unknown microorganisms. Using the latest technologies, our professional and dedicated teams of microbiologists will help you obtain microbiological testing results with high accuracy, high reliability and short turnaround time.

To cope with and manage food safety risks throughout the supply chain, establish a sound food safety management system, comprehensively improve the level of food safety, and strive to create a rational food consumption environment, we urgently need to look at food safety issues with a broader perspective.

What can be done if cancer turns resistance to KRAS inhibitors?

Recently, KRAS and KRAS-G12C mutant inhibitors, which were previously considered to be “non-drugable” targets, have ignited a development boom. However, KRAS inhibitors are not a “magic bullet” for once and for all. Cancer resistance is a major problem in cancer treatment. It is usual in clinical practice that tumor tissue becomes significantly smaller after targeted therapy, but soon deteriorates again. Can we develop a multi-mechanical combination therapy to eradicate the disease in order to solve the potential drug resistance problem before the tumor develops resistance to the research drug?

After screening 16,019 genes by shRNA screening, researchers at the Francis Crick Institute and the British Cancer Institute found that the KRAS-G12C inhibitor ARS-1620, and the insulin growth factor-1 receptor inhibitor linsitinib, were studied. The combination therapy with mTOR inhibitors can significantly reduce the tumor size of lung adenocarcinoma, and the effect is more durable. The results of this trial were recently published in the journal Science Translational Medicine.

RAS family proteins are mainly divided into three categories: KRAS, HRAS, NRAS, and the KRAS gene mutation rate is more than 80%, which is one of the most common oncogenes. RAS protein regulation includes the MAPK signaling pathway, as well as multiple downstream pathways such as PI3K/AKT/mTOR, which control several key cellular activities, including proliferation, differentiation, survival, and angiogenesis. KRAS mutations occur in more than 90% of pancreatic cancers, 40% of colorectal cancers, and 16% of lung adenocarcinoma cases.

IGF1R is a membrane protein receptor with tyrosine kinase activity, usually overexpressed in lung cancer. It is a key factor for malignant transformation upstream of the MARK and PI3K signaling pathways and is one of the leading causes of acquired resistance to EGFR inhibitors.

Membrane receptor protein IGF-1R regulates downstream MAPK and PI3K/AKT/mTOR signaling pathways

The researchers found that knocking out the MTOR gene made the cells significantly sensitive to KRAS and IGF1R inhibitors. While blocking the three signaling pathways IGF1R, MAPK and PI3K/AKT/mTOR, cancer cells carrying KRAS mutations could not survive. The KRAS-G12C inhibitor ARS-1620 develop resistance and re-grow in a few weeks. The combination of KRAS-G12C inhibitor ARS-1620, IGF1R inhibitor linsitinib, and mTOR inhibitors can significantly reduce the size of mouse and human tumors, and the effect is significantly longer. This combination therapy has the potential to prevent or delay the resistance of KRAS-G12C inhibitors.

Combination therapy under the study, KRAS-G12C inhibitor ARS-1620, IGF1R inhibitor linsitinib, and mTOR inhibitors has significant effect for lung cancer carrying KRAS-G12C mutation. Dr. Julian Downward, one of the authors, believes that the study provides a new approach to improve the efficacy of targeted mutant KRAS proteins. KRAS mutations often lead to greater invasiveness. “This provides a clear direction on how to better use KRAS-G12C inhibitors in the clinic and how to circumvent the possible evolution of cancer when used as a single drug.”

References

  1. 1. Romero-Clavijo. et al. Development of combination therapies to maximize the impact of KRAS-G12C inhibitors in lung cancer. Science Translational Medicine. https://doi.org/10.1126/scitranslmed.aaw7999
  2. 2. Researchers Develop New Class of Lung Cancer Drugs. Retrieved Sep. 19, 2019, from https://www.biospace.com/article/new-class-of-drug-in-combination-shows-promise-for-lung-cancer/
  3. 3. Could a combo treatment boost KRAS inhibitors in lung cancer?. Retrieved Sep. 19, 2019, from https://www.fiercebiotech.com/research/improving-kras-inhibitors-lung-cancers-a-combo
  4. 4. Two Is Better Than One: Combining IGF1R and MEK Blockade as a Promising Novel Treatment Strategy Against KRAS-Mutant Lung Cancer. Retrieved Sep. 19, 2019, from https://cancerdiscovery.aacrjournals.org/content/3/5/491.figures-only

 

A detailed introduction to recombinant protein drugs (Part Three)

  1. 2.Technical methods for expressing host cell construction

(1). Target gene acquisition

1). Primer synthesis technology

Primer refers to a single-stranded DNA molecule that can be covalently bound to a nucleic acid template at the beginning of nucleotide polymerization and function as a replication extension origin. The synthesis of primers uses solid phase DNA synthesis technology. The specific technical process is as follows:

The nucleotide attached to the controlled pore glass is reacted with trichloroacetic acid to remove the protecting group DMT (dimethoxytrityl) to obtain a free 5′-hydroxyl end. Adding a mixture of a phosphite amide monomer and a tetrazolium activator to form a phosphite amide tetrazole active intermediate, which is condensed with a nucleotide free 5′-hydroxyl group in the reaction system, and the nucleotide chain is extended by one base; The subsequent reaction is terminated by the addition of acetic anhydride and methyl imidazole, and the 5′-hydroxyl group which is not involved in the condensation reaction is removed in the final purification stage; the phosphite amide formed in the condensation reaction is converted into a stable phosphate triester by adding a solution of iodine in tetrahydrofuran. The above four steps complete the attachment of a deoxynucleotide to the original nucleotide chain. This process can be repeated to synthesize the desired primer DNA.

2). Gene amplification technology

Generally speaking, it refers to polymerase chain reaction (PCR). When DNA is denatured to a single strand at a high temperature of 95 °C, the primers at 60 °C and the template DNA single strand are refolded by base-pair pairing principle, about 72 °C. The DNA polymerase extends along the direction of phosphoric acid to the five-carbon sugar (5′-3′) to form a complementary strand, which alternates between denaturation, renaturation, and extension temperature, and the DNA strand is continuously replicated and amplified. Now PCR has developed nested PCR, reverse transcription PCR (RT-PCR), in situ PCR (in situ POR), real-time quantitative PCR (RT-PCR), derivative upgrade technology such as digital PCR. There are also new DNA amplification technologies such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA).

3). Reverse transcription PCR to obtain the target protein gene

Earlier, the acquisition of target gene is usually obtained by reverse transcription PCR, which is, tissue samples are obtained from human or animal body, and the specific mRNA extraction kit is generally used to extract messenger RNA (mRNA) in the tissue sample, so that the gene sequence is not an intron containing a gene of a eukaryotic cell. Then, using mRNA as a template, in the presence of appropriate primers, the corresponding DNA single strand is synthesized by reverse transcriptase, which is called cDNA (complementary DNA). After the corresponding RNA is removed by alkali treatment, the DNA is polymerized by using single-stranded cDNA as a template. The enzyme acts to synthesize double-stranded cDNA. At the time of primer design, an appropriate cleavage site is set for subsequent recombination with the vector. Before constructing the expression vector, the cDNA is ligated into a cloning vector, amplified, and then subjected to DNA sequencing, and the codon substitution which is more favorable for expression is selected according to the preference of the degenerate codon of the host cell for sequence optimization.

4). Whole gene chemical synthesis

Currently, as the sequencing of the human genome and other biological genes is completed, more and more functional genes or structural gene sequences are clear. The level of gene synthesis technology is continuously improved, the synthesis time is shortened, and the cost is reduced. On this basis, the full-length gene of the target protein can be obtained by chemical synthesis according to the optimal gene sequence.

The chemical synthesis method is designed into a short gene fragment (<100bp), and the appropriate restriction sites are designed on both sides of the fragment; the full-length primers are synthesized by solid phase DNA synthesis, and each method is obtained by PCR amplification. Fragmented double-stranded DNA; these fragments are then stepwise ligated by DNA ligase, and if necessary, subcloning is used to obtain the complete gene sequence. Each subclone can be identified separately, thus reducing sequence errors. Full-gene chemical synthesis is currently the most accurate and fastest method for obtaining recombinant protein genes.

5). Gene library technology

In molecular biology and genetics, a genome refers to the entire genetic material of an organism, generally referred to as DNA, including genes, non-coding DNA, mitochondria, and DNA in chloroplasts.

A gene library is a collection of all genes in a certain organism. Generally, it is divided into a genomic library and a partial gene library. A genomic library is a collection of clones formed by recombining all genomic DNA of a certain organism to a certain vector and transforming the recipient cells. A partial gene library refers to a collection of clones, such as a cDNA library, in which a part of the gene DNA of a certain organism is ligated to a vector and transformed into a recipient cell.

According to the recombinant vector function of the library, it can also be divided into a clone library and an expression library. The commonly used recombinant vectors include plasmids, phages, cosmids, and bacterial artificial chromosomes (BAC).

The general method for genomic library construction is to first extract and fragment genomic DNA, select a suitable vector and prepare in large quantities, connect the DNA fragment to the vector to form a recombinant vector, and transfect the recombinant vector into the host cell. The cDNA library usually first obtains the total RNA of the cells, and isolates and extracts the mRNA, obtains the cDNA by reverse transcription PCR, and then constructs according to the vector ligation and transformation of the host cells. DNA recombination and its transfected cell technology are described later.

The gene library is usually screened by nucleic acid hybridization method, that is, a probe is formed by labeling a probe with a known sequence, and under certain conditions, the probe can be transferred to the nitrocellulose after being denatured by nucleotide pairing hybridization. The library DNA sequences on the membrane are combined to screen for the gene sequence of interest. For expression libraries, antibody molecules and immunoblotting methods can also be used to bind the protein of interest expressed by the library to determine the gene of interest. The phage vector library can also be diluted in a 96-well plate, and the appropriate PCR buffer system is added to amplify the library gene by PCR, and then the labeled DNA probe is used to bind the target gene, and the dilution is repeated several times to finally select the target gene.

To be continued in Part Four…

 

What are tumor markers and how important are them?

First, what is the tumor marker?

Tumor cells release certain substances, such as hormones, enzymes and antigens, which may be metabolites of tumor cells. These substances may be present in tumor cells or may be present in the body fluids of patients through the immunological properties of these substances to judge and identify the tumor. The metabolism of tumor cells is different from that of normal cells, so there will be some changes in the humoral environment of tumor patients. This change can be judged by detecting tumor markers. In clinical practice, the role of tumor markers is mainly to discover primary tumors. At the same time, it is possible to screen the high-risk population of tumor, and also to judge the therapeutic effect of the tumor and the prediction of the later treatment stage.

Second, does the tumor marker elevated mean cancer present?

It is not necessarily. Although the tumor marker is a metabolite produced by tumor cells, it does not mean that normal cells won’t. Some studies have found that healthy tissues and cells may also produce tumor markers, so it is actually not 100 percent for sure to check tumor by tumor markers.

Moreover, not every tumor patient has obvious tumor markers, so this method can only be used as a reference of the results. If one wants to confirm the diagnosis, you need to go through various inspections before you can determine it.

Third, the significance of tumor markers

  1. Auxiliary support

Tumor markers can be used to assist in the examination of tumors, and for some special cancers, the reference value is relatively high. If there is a problem with the tumor markers, further confirmation by biopsy is needed.

  1. 2.Develop a treatment plan

Cancer patients often need to perform a tumor marker test before making a production plan. This test can reflect the symptoms of the disease and help the doctor to develop a treatment plan.

  1. 3.Monitor the treatment effect

Tumor markers can be used to judge the symptoms during treatment and the therapeutic effect over a period of time. If the tumor markers do not change, in most cases, it means the therapeutic effect is not significant.

  1. 4. Check the recurrence

Tumor markers can also be used to examine tumor recurrence, which can be used as a basis test.

The maximum value of tumor markers should be in their fluctuations. If they fluctuate frequently within a short period of time, attentions should be paid to.

Creative Biolabs provides a comprehensive range of recombinant antibodies of the key tumor for research processes and diagnostic assay developments. The ranges include but not limited to:

 

The knowledge of metastasis, proliferative signaling, metabolism, inflammation, apoptosis is the focus of research. Hence, the identification of tumor markers using antibodies is widely used in cancer diagnostics and research. Tumor markers are proteins or antigens which are used to detect elevated in the body due to the presence of cancer. Elevated quantities of tumor markers in the blood, urine, stool, tumor tissue for cancer diagnosis and prognostic evaluation can be detected by the tumor marker-specific antibodies.

Discovering and detecting cancer cell signaling pathways and the cellular process will help us to find a link between its genotype and phenotype.