The Research Of Bovine Antimicrobial Peptides Bac7,Bac5 And β-defense

Bovine antimicrobial peptides bac7,bac5 are linear cation micromolecular polypeptides and bovineβ- defense(LAP) belongs to the defense element class, which play an important role in both innate and acquired immunity. In the present study, based on the gene sequences encoding mature bovine antimicrobial peptides bac7 and bac5 as registered in Gen Bank, the fragment of the fusion gene Bac7-Bac5 was synthesized and cloned into the prokaryotic expression vector pET32 (a+) to construct a recombinant expression vector, pET-B7-B5. The recombinant construct was then transformed into E coli BL21 (DE3) to over-express the recombinant protein B7-B5 (rB7-B5). The expressed rB7-B5 was localized in inclusion bodies and accounted for 36.6% of total bacterial protein. The molecular weight of rB7-B5 was 33kD, a figure consistent with the predicted value. Following purification by Ni affinity chromatography and stepwise renaturation by dialysis, rB7-B5 showed sound antimicrobial activity in porcine actinobacillus pleuropneumoniae and antibiotics-resistant E. coli. The present study provides basis for the research and development of novel antibacterial preparations.In the present study, based on the gene sequences encoding mature bovine antimicrobial peptides bac7,bac5 andβ- defense(LAP) as registered in Gen Bank, The fragment of the fusion gene Bac7-Bac5-βdefense was synthesized and cloned into the pFASTHTb vector of BAC-TO-BACTM recombinant baculovirus expression system to construct a recombinant expression vector, The plasmid pFASTBac-B7-B5-Bβwas then transformed into DH10BACTM competent cell. High molecular weight DNA, the recombinant bacmid, was prepared from the overnight cultures of selected E. coli DH10BACTM colonies. By transfecting Sf9 cells with the recombinant bacmid the recombinant baculovirus from the superenatant was obtained, which could further infect Sf9 cells obtained and express the target protein. The expression of rB7-B5-Bβwas confirmed by 12% SDS-PAGE and accounted for 10.6% of total bacterial protein,the molecular weight of rB7-B5-Bβwas 20kD, a figure consistent with the predicted value. rB7-B5-Bβshowed antimicrobial activity in E. coli. The present study provides basis for the research and development of novel antibacterial preparations.