Preparation method and application of novel antimicrobial peptide Misgurin mutant
A mutant and antimicrobial peptide technology, applied in the preparation of recombinant novel antimicrobial peptide Misgurin mutants, the field of novel antimicrobial peptide Misgurin mutants, can solve the problem of no commercialized new antimicrobial peptide Misgurin mutants, no mention of recombinant protein antimicrobial activity, etc. problem, to achieve the effect of good clinical use, improve the protection rate of chicks, and high expression
Inactive Publication Date: 2012-06-27
上海市农业科学院畜牧兽医研究所
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Problems solved by technology
In 2002, Gong Jiewan et al. concatenated multiple copies of the Misgurin gene in the same direction, cloned it into the expression plasmid pPIC9K, and electrotransformed it into Pichia pastoris strain GS115, and screened out recombinants that integrated multiple copies of the foreign gene. The antibacterial activity of the recombinant protein was not mentioned in the paper (Jiewan Gong. The construction of gene expression vectors for mae and misgurin and their expression in Pichia pastoris. Jinan University, Ph.D. dissertation, 2002.48-57.)
At present, there is no report on the successful use of genetic engineering technology to produce active antimicrobial peptide Misgurin, and there is no report on the commercialization of new antimicrobial peptide Misgurin mutants
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Embodiment Construction
[0022] 1. Design a Misgurin mutant containing glutamine, and artificially synthesize the Misgurin mutant gene
[0023] After intensive and extensive research, the inventors designed a mutant of misgurin containing glutamine to enhance its antibacterial activity on the basis of referring to the amino acid sequence of misgurin in GenBank (Gene Bank P81474). At the same time, the Misgurin mutant gene was designed with yeast preferred codons, and an α signal peptide Kex2 cleavage site was added to its N-terminus to ensure that the secreted and expressed antimicrobial peptide had a natural N-terminus, and XhoI and XbaI restriction sites were introduced at both ends point for easy cloning into the vector pPICZα-A.
[0024] 2. Construct a secretory yeast expression vector (such as figure 1 shown)
[0025]The recovered product containing the Misgurin mutant gene digested by XhoI and XbaI was mixed with the expression vector pPICZαA recovered by the same enzyme digestion in a certain...
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The invention belongs to the technical field of biology and relates to a preparation method and application of a novel antimicrobial peptide Misgurin mutant. The technical scheme is as follows: introducing glutamine at the C tail end of the amino acid sequence of natural Misgurin so as to design a novel Misgurin mutant; selecting Pichia yeast biased codons so as to artificially synthesize a complete gene of the novel Misgurin mutant; constructing a Pichia yeast secretion type expression vector pPICZaA-M; transforming a host cell SMD1168 by a vector electroporation method; selecting to obtain high-efficiency expression strains; and under proper culture conditions, ensuring 100 microgram of recombinant novel Misgurin mutant is contained in per milliliter of expression supernatant. The obtained product has a good nematocidal activity on staphylococcus aureus, pig salmonella and pig pathogenic Escherichia coli K88, K99 and 987P, and has an obvious effect on prevention and treatment of clinical bacterial diseases.
Description
technical field [0001] The invention belongs to the high-tech research field of biotechnology, and relates to a novel antimicrobial peptide Misgurin mutant, more specifically to a preparation method of a recombinant novel antimicrobial peptide Misgurin mutant and its application in controlling and preventing bacterial diseases. Background technique [0002] Antibacterial peptides (ABP) are cationic small peptides produced by organisms with strong antibacterial effects, and are important components of biological innate immunity. The mechanism of action of antimicrobial peptides is different from that of traditional antibiotics, and they are the most promising substitutes for antibiotics. Compared with traditional antibiotics, antimicrobial peptides have a unique antibacterial mechanism, which is not easy to cause drug-resistant mutations in bacteria. Antimicrobial peptides can also specifically inhibit fungi, viruses, parasites and tumors, and their biological activities are...
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IPC IPC(8): C12N15/11C12N15/81C07K14/435A61K38/17A61P31/04
Inventor 缪德年樊生超姚惠娟文豪
Owner 上海市农业科学院畜牧兽医研究所