The expression of the gene includes the corresponding mRNA synthesis (transcription) and protein synthesis (translation), and the protein biosynthesis of the foreign gene in the microorganism depends on the microbial genetic material and the recombinant DNA fragment encoding the target protein. The specific steps are as follows: Step 1, the DNA fragment encoding the protein is isolated from the donor; Step 2, inserting the DNA molecule into the expression vector; Step 3, transfecting the vector into the host; Step 4, culturing Host tissue, gene amplification, mRNA synthesis and peptide synthesis; Step 5, purification of recombinant polypeptide. Polypeptide drug recombination techniques are generally classified into two categories depending on the host cell: (1) eukaryotic expression of the polypeptide drug; (2) prokaryotic expression of the polypeptide drug.
1 eukaryotic expression of peptide synthesis drugs
The eukaryotic expressed polypeptide has the advantages of strong orientation, safe and hygienic product, wide source of raw materials and low cost, and can obtain a polypeptide with high quality, good curative effect and activity and natural polypeptide. At present, eukaryotic host cells mainly include yeast, animal and insect cells, and yeast is most commonly used in eukaryotic expression. Cao et al. successfully expressed chicken b-defensin 6 ( AvBD-6) and decellularized AvBD-6-B bioactive peptides using Pichia pastoris cells, and found that both were strong in the experiment. Bacteriostatic effect. Guo et al. also successfully expressed antibacterial peptide D in Pichia pastoris cells, which has strong antibacterial activity. The yeast expression of antimicrobial peptide D has important guiding significance for the development of antibacterial drugs. Su Caixia et al. previously linked the hepatitis B surface antigen (HBsAg) gene to the S. cerevisiae signal peptide, and optimized the entire sequence to the Hansenula preferred codon, synthesized by PCR technology, and finally successfully expressed the recombination in Hansenula. HBsAg.
2 Prokaryotic expression of peptide synthesis drugs
Host cells used in prokaryotic expression systems include Escherichia coli, Salmonella, Bacillus subtilis, and the like, with E. coli being the most commonly used prokaryotic host cell. By using prokaryotic expression and recombination technology, a large number of bioactive polypeptides can be synthesized in a large amount and efficiently, and the sequence of the polypeptide can be controlled by designing a suitable DNA template, which lays a foundation for gene synthesis of the polypeptide synthesis drug. Li et al. successfully expressed ChickenIFN-y in prokaryotic cell line BL21(DE3) pLysS and demonstrated that ChickenIFN-y has strong antiviral activity by in vitro experiments and is an example of prokaryotic host cell application in peptide synthesis. Lu et al. used a recombinant technique to fuse the hirudin gene with a small ubiquitin-related modification (SUMO), and finally efficiently expressed the hirudin (HV1) subtype in E. coli. The traditional peanut oil extraction process mainly adopts high temperature pressing method and organic solvent extraction method. These two methods cannot be fully utilized due to the severe denaturation of peanut protein at high temperature or residual organic solvent, and Zhang Youwei et al. The technology successfully fermented peanut polypeptides in Bacillus subtilis, which improved the utilization of peanut protein.
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