嗜水气单胞菌菌蜕系统的构建及其免疫效果研究

Establishment of Aeromonas hydrophila ghosts system and its immune effect

【目的】探讨鱼源嗜水气单胞菌菌蜕系统的可行性和应用性。【方法】以PhiX174基因组DNA为模板,对LysisE基因进行PCR扩增,并将纯化的PCR产物与原核表达载体pBV220双酶切后连接,构建溶菌质粒pBV220-LysisE。将pBV220-LysisE转入嗜水气单胞菌LN0925株中,构建LN0925(pBV220-LysisE)菌蜕疫苗(AHGs),进而通过溶菌动力学过程检测、电镜下细菌形态观察和动物免疫保护试验等评价所制备的菌蜕疫苗。【结果】PCR扩增成功获得长度为276bp的噬菌体LysisE基因;成功构建pBV220-LysisE重组质粒及AHGs。在42℃诱导60min后,LN0925(pBV220-LysisE)菌株开始出现溶菌现象,至3h后溶菌基本结束;溶菌至210min时,其裂解效率达到99.99%。菌液浓度对菌蜕裂解效率的影响试验表明,不同浓度质粒pBV220-LysisE均可以高效诱导嗜水气单胞菌LN0925株裂解。电镜观察发现,AHGs形成明显的溶菌孔道,整体细胞形态完好,且内容物流失。动物免疫保护试验结果表明,AHGs疫苗能明显提高鲤鱼的血清抗体水平,在免疫后5-6周血清抗体凝集效价达到1∶256,从第7周开始呈下降趋势;AHGs和甲醛灭活疫苗(FKC)的相对保护率分别为77.78%和55.56%。【结论】AHGs能够有效激活鱼体的免疫系统并产生免疫保护,且较FKC具有更好的免疫保护效果。

[Objective]This study aimed to study thefeasibility and application of BGs in Aeromonas hydrophilaisolated from fish.[Method]ThelysisE gene in the bacteriophage PhiX174 was cloned by PCR amplification and the PCR products were then digestedby double enzyme and ligated to the prokaryotic expression vector pBV220.The lysis plasmid pBV220-LysisE was constructed and then transformed to Aeromonas hydrophila LN0925 strain to prepare A.hydrophilaghosts（AHGs）.The lysis curves,morphology observation and animal immunoprotection test of AHGs were performed to evaluate the effects of AHGs prepared.[Result]The LysisEgene with size of 276 bp was cloned into pBV220 vector and the recombinant plasmid pBV220-LysisE was constructed and successfully transformed to LN0925 strain to prepare AHGs.LN0925（pBV220-LysisE）lysed at 60 min after induced at 42℃and the lysis process continued for3 h.The lysis efficiency of AHGs induction was 99.99%.Effects of initial culture concentrations on lysis efficiency indicated that pBV220-LysisE could highly induce lysis of LN0925 strain.The morphology oflysed bacteria was observed by scanning electron microscope and transmission electron microscope,and it was found that AHGs formed obvious lysis tunnels with entire cell structure.Animal protective immunity test was carried out to evaluate the immune effect of AHGs on common carp（Cyprinus carpio）.The results suggested that AHGs could obviously improve the level of antibody in serum and the agglutination titer reached 1∶256after 5-6weeks by immunization with vaccines.Challenge test showed that the relative percent survival（RPS）of AHGs and Formalin-killed vaccine（FKC）were 77.78% and 55.56%,respectively.[Conclusion]As a novel type of vaccine system,AHGs could active the immune system of fish and provide a better protective effects against A.hydrophilainfection in common carp when compared to FKC.