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…