表达猪轮状病毒VP4基因重组乳酸球菌表达载体构建及免疫原性分析

Construction of Recombinant Lactococcus Lactis Expressing VP4 of Porcine Rotavirus and Analysis of Immunogenicity

【目的】构建猪轮状病毒主要保护性抗原VP4基因的重组乳酸乳球菌口服疫苗,为猪轮状病毒的防治提供有效方法。【方法】将猪轮状病毒免疫保护性抗原基因VP4主要结构功能区定向插入乳酸菌表面表达载体pNZ8112,电转化乳酸乳球菌NZ9000,构建了pNZ8112/NZ9000乳酸乳球菌表面表达系统。通过SDS-PAGE、Western-blot和免疫荧光方法鉴定,以此活菌系统口服免疫6～8周龄Balb/c小鼠,测定了免疫动物的局部体液免疫和系统免疫应答。【结果】证明插入的外源基因在菌体表面得到了正确表达,表达猪轮状病毒免疫保护性抗原的重组乳酸乳球菌免疫动物后,分别在粪便、泪液和阴道洗液中检测到了特异性分泌型IgA抗体和在血液中检测到了IgG抗体的存在;体外细胞中和试验结果表明,血清抗体对猪轮状病毒的中和效价为1﹕40。【结论】表达猪轮状病毒免疫保护性抗原的重组乳酸乳球菌口服免疫动物能够产生良好的局部和系统体液免疫应答而且具有免疫中和效力。

【Objective】 The main protective antigen gene segment VP4 of porcine rotavirus was inserted into the surface display vector pNZ8112 of Lactococcus. 【Method】 It was electrotransformed into Lactococcus lactis NZ9000,and the surface display system pNZ8112/NZ9000 was construced. To prove the expression of the exogenous gene,the expression product was detected by SDS-PAGE,Western-blot and immunofluorescence.The reconbinant strain was orally administered to the 6-8 week Balb/c mice. 【Result】 The regional humoral immunity and systematic immunity were detected in the immunized mice.The results indicate that specific sIgA and IgG can be found in fecal pellet,ophthalmic wash,vaginal wash and in the serum after administration.The result of neutralization test indicate the titer of serum is 1：40. 【Conclusion】 All the results indicate that VP4 expressed in the recombinant strain can elicit regional immunity and systematic immunity.