A Porcine Bactericidal Permeability Increase Protein N-terminal Gene Cloning And Prokaryotic Expression Of Antimicrobial Activity

With the emergence of a large number of contemporary society, the overuse of antibiotics, drug residues phenomenon has become more and more serious. Bactericidal permeability-increasing protein （BPI） in its capacity as a new substance that has a natural defense function appears. Which is characterized by a broad spectrum of antibacterial, and biological characteristics of anti-tumor, anti-parasitic and other aspects. And has not prone to bacterial resistance characteristics have been people hope in the future to replace antibiotics.Swine bactericidal permeability-increasing protein （BPI） as an antimicrobial active substance from a pig neutrophils isolated. Among them, the swine bactericidal permeability-increasing protein （BPI） N terminal gene generally from 200 to 240 amino acids.Most of the Gram-negative bacteria from swine bactericidal permeability-increasing protein （BPI） is very sensitive to the N-terminal gene, and, BPI-N gene to a small number of Gram-positive bacteria also have some antibacterial activity. In this study, using genetic engineering techniques to swine bactericidal permeability-increasing protein （BPI） N terminal gene cloning and expression studies of antibacterial activity. After sterilization so as swine permeability-increasing protein （BPI） of the actual production and application of the foundation.1, BPI-N terminal gene amplificationAccording to the NCBI published swine bactericidal permeability-increasing protein （BPI） gene sequence, using software to analyze results of the swine bactericidal permeability-increasing protein （BPI） gene sequences of the N-terminus. Reuse DNAstaer, Primer 5 and Oligo software designed two swine bactericidal permeability-increasing protein primers （BPI） N terminal gene of P1, P2. And manually adding enterokinase cleavage site, to avoid being wrapped histidine tag without having antibacterial activity. The extracted RNA pig liver was amplified. Insert it into pMD-19T vector and sequenced. The obtained sequences with published swine bactericidal permeability-increasing protein （BPI） N terminal gene sequence （GenBank ID:NM₀01159307.1） are identical.2, BPI-N terminal gene in E. coli pET-32a cloning, expressionBy using Primer 5 software selected two BamH I and Xho I restriction sites. After use and BamH I and Xho I, respectively, containing the gene of BPI-N recombinant plasmid pMD 19-T-BPI-N and the expression vector pET-32a was double-digested recovery, purification. Overnight connection to construct prokaryotic expression plasmid, which was named pET-32a-BPI-N. The recombinant plasmid pET-32a-BPI-N was transformed into DH5@ the screened simultaneously identified by PCR and restriction enzyme digestion. After successful identification, extraction recombinant plasmid pET-32a-BPI-N. The recombinant plasmid pET-32a-BPI-N was transformed into E. coli BL21 （DE3）, and by IPTG （concentration of approximately 1M） induction. To express antimicrobial peptides BPI-N protein. SDS-PAGE and by optimizing the identification of expression conditions showed pET-32a-BPI-N has gained highly expressed in E. coli, SDS-PAGE showed that the molecular weight of the expression product is about 47.0KD. And expressed in inclusion bodies were. To determine the activity of the fusion protein. The fusion protein nickel ion affinity chromatography. Thereby obtaining high purity fusion protein. After enterokinase cleavage, after removing the histidine tag, to obtain a molecular weight of about 26.0KD about BPI-N terminal protein.3, swine bactericidal/permeability-increasing protein N-terminal （BPI-N） antibacterial activityAfter cutting the purified porcine source bactericidal/permeability-increasing protein N-terminal （BPI-N） to study specific antibacterial activity, minimum inhibitory concentration, and its stability. The results showed that:Swine bactericidal/ permeability-increasing protein N-terminal （BPI-N） for most Gram-negative bacteria have antibacterial activity. A small number of Gram-positive bacteria also have antibacterial activity. UV irradiation, PH value, heat treatment for swine bactericidal/ permeability-increasing protein N-terminal （BPI-N） antimicrobial activity to a certain extent, but the effect is very weak, indicating swine bactericidal/ permeability-increasing protein N end （BPI-N） has a strong stability.