| Antimicrobial peptides are consisted of less than100amino acids with a positivecharge, and has a broad-spectrum resistance as well thermal stability of a class of smallpeptides. Because of their good antibacterial activity, antimicrobial peptides are oftenused to be antimicrobial agents and preservatives. In recent years, some antimicrobialpeptides were found to have the effect of immune adjuvant. In this paper, theantimicrobial peptide Met was synthesised and used as immune adjuvant to avianPasteurella multocida ptfa protein and the effect was evaluated.In this paper, the ptfa gene fragment was amplified by PCR. Then, the genefragment and the vector pET32a were double digested by endonuclease BamHⅠandHindIII. The recombinant plasmid pET32a-ptfa was then obtained by connecting ptfagene fragment and pET32a with T4DNA ligase and transduced into competent cellBL21by chemical method of CaCl2. After that, the expression was induced by IPTGand then the expressed protein was determined through SDS-PAGE electrophoresis.Finally, the ptfa protein was purified by nickel column and concentration was140mg/L.1-day-old non-immunized chicks (purchased from Luoyang Modern Poultry Co.,Ltd.) were randomly divided into six groups, which are ptfa/propolis adjuvant,ptfa/Freund’s adjuvant, ptfa/Freund’s/Met0.2(0.2mg/only), ptfa/Freund’s/Met0.4,ptfa/Freund’s/Met0.6adjuvant and the saline control group. The first immunity test wasperformed on the21stday. The trial groups were injected with corresponding emulsionvaccine while the contol group was injected with the normal saline. After that, thevenous blood samples were collected on post-immune day7,14,21,28,35,42andthen the serum was separated from the samples after being placed under room temperature. The serum thereafter was stored at-20℃. The second immunity test wascarried out on42ndday and the peripheral blood samples were collected for MTT teston post-immune day7,14,21. After second immunity test, lymphocyte were analysedby MTT assays and Venous serum was analysed by ELISA assays so that the results ofELISA and MTT could be used for evaluating the immune influence of Met for the ptfaprotein in chicken’s body.Protein ptfa was successfully expressed and purified in this study. The results ofELISA tests showed that the antibody levels of each group reached the highest point inthe second week of the second injection and all the antibody levels of treatment groupswere higher than that of the control group in the same period, and the difference wassignificant (P <0.05). In the first three weeks of the first injection, the antibody levelsof each group got increased.The Met0.4group had the highest antibody level and theMet0.6group was the second highest one. There was no difference between propolisand the saline control group. After the second injection, the antibody levels of Met0.2and Freund’s adjuvant group were similar. The antibody levels of Met0.4and Met0.6group however were significantly higher than that of the other three groups. Met0.4group’s antibody level reached its maximum in the second week and the antibody titerwas1：51200, which means that Met is effective for chicks’ humoral immunity as anadjuvant and the best dosage is0.4mg. The results of MTT assay showed that in thesecond week after the second injection each treatment group reached the maximumlevel and all of them were obviously higher than that of control group. The differencesbetween Met0.4or Met0.6group and Freund’s adjuvant group were very significant (P<0.01). Additionally, Met0.4was more effective in promoting lymphocytetransformation compared to Met0.2and Met0.6so that we can deduce thatantimicrobial peptides Met can effectively promote the lymphocyte transformation andit can promote cellular immune response in chicks to some extent. In this study, weused ptfa protein as antigens and antimicrobial peptides as adjucants to demonstratethat antimicrobial peptides Met had the good effect as immune adjuvants, which laidthe foundation for the research of modern immune adjuvants. |
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