Prokaryotic Expression And Antimicrobial Activity Of Porcine β-defensin-1

Defensin are arginine and cysteine residue rich cationic endogenous peptides in animals and plants,which feature multi-biological activities, anti-pathogenic microorganisms such as bacteria andenveloped virus, but do not introduce bacterial resistance. Moreover, defensin also hasimmunoregulatory function, and thus defensin is considered as a new type of antibiotics, which has awide application prospect. This study constructed Porcine β-defensin-1(Porcine β-defensin-1, PBD-1)prokaryotic expression plasmid, expressed and purified recombinant PBD-1, and then analysed theanti-microbial activity of recombinant PBD-1in vitro and vivo so as to to lay the foundations for furtherdeveloping new therapeutic agents.In the study, the genome sequence of PBD-1with a length of247bp was obtained by RT-PCR anda recombinant cloning vector pGM-T-PBD-1was constructed. Moreover, we also constructed arecombinant expressing vector PET-32a-PBD-1, which was successfully expressed24.5kDarecombinant PBD-1in E.coli BL21cells. Furthermore, we successfully got the pure PBD-1using6×His-tagged proteins purification kit.In vitro, recombinant PBD-1had anti-microbial activities aganist E. coli, Salmonella,Streptococcus, Staphylococcus and Pasteurella. The anti-bacterial activities increased with theconcentration of recombinant PBD-1. In additionm, the recombinant PBD-1was able to inhibited theproliferation of the virus in the corresponding cells and prevented cytopathic effect (CPE) when therecombinant PBD-1combined prior with porcine epidemic diarrhea virus (Porcine epidemic diarrheavirus, PEDV) and avian influenza virus (Avian influenza virus, AIV)(H9N2) in vitro for1hour,respectively. However, the recombinant PBD-1faild to inhibit CPE caused by PEDV and showedinhibiting effect on AIV (H9N2) after PEDV or AIV (H9N2) infected relevant cells.The results in vivo showed that the recombinant PBD-1with a similar treatment effect to that ofceftriaxone significantly decreased the morbidity, mortality and gross pathological degree of chickensinfected by Escherichia coli (E. coli) O141strain. There was a superior treatment effect whencombination of recombinant PBD-1and ceftriaxone was used for treatment.One week after infection, the average body weights both the group of recombinant PBD-1treatment and the group of combination of recombinant PBD-1and ceftriaxone were significantlyhigher than that of the non-treatment group (P<0.05).But the body weights between the group ofceftriaxone treatment and the group of non-treated were not significant difference (P>0.05). In conclusion, the results indicated that the recombinant PBD-1had extensive anti-microbialactivities. However, different inhibition effects displayed among various bacteria and virus species. Therecombinant PBD-1had obvious therapeutic effect on the sick chickens infected by E. coli O141strainand was more effective when combination with antibiotics.