猪源A组轮状病毒Vp4基因工程乳酸菌的制备及其免疫原性分析

Preparation and immunogenicity analysis of recombinant Lactobacillus expressing VP4 of A group porcine rotavirus

为了解猪轮状病毒(RV)Vp4基因工程乳酸菌口服免疫小鼠是否能产生有效的免疫保护作用,本研究应用电转化技术将猪源A组RV重组质粒pW425et-Vp4转化至嗜酸乳杆菌中,重组菌经SDS-PAGE分析可见约87ku的重组蛋白。Western blot分析表明该蛋白能与猪RV多克隆抗体反应。大量培养pW425et-Vp4乳酸菌,经离心加入灭菌脱脂乳液给BALB/c小鼠灌服;应用ELISA方法检测血清抗体水平,试剂盒检测肠黏膜SIgA水平,最后进行小鼠免疫保护性试验。结果表明pW425et-Vp4乳酸菌能增强小鼠的黏膜免疫反应,对猪PV强毒株的攻击具有一定的保护作用,为下一步在猪体内进行试验奠定基础。

In order to identify if recombinaut Lactobacillus expressing group A Rotavirus Vp4 of could produce effective immunoprotection after orally immunizing to BALB/c mice, pW425et-Vp4 recombinant plasmid of Porcine Rotavirus was transformed into Lactobacillus acidophilus, positive clone were identified by restriction enzyme digestions and PCR amplification, The recombinant protein was of 87 ku by SDS-PAGE analysis, and could react to polyclonal antibody against RV by western blot analysis. The recombinant Lactobacillus with adjuvantation of sterilized nonfat milk was prepared and orally immunized to BALB/c mice. IgG in serum were examined by ELISA and SIgA in intestines mucosa were examined by SIgA kit. The result indicated the recombinant Lactobacillus could enhance the mucosa immune response of mice and protect against the challenge of porcine rotavirus; and might establish the groundwork for the the recombinant Lactobacillus test in vivo of swine.