猪伪狂犬病病毒变异株TK/gE/gI三基因缺失突变株在小鼠体内的免疫原性

Efficacy evaluation of a TK/gE/gI-gene-deleted pseudorabies virus variant

应用PCR方法扩增了猪伪狂犬病病毒(PRV)NY株TK基因两侧序列,克隆至pUC-19载体,同时插入绿色荧光蛋白标记基因,构建转移质粒pUC-TKLRE,并与PRV变异株gE/gI双基因缺失突变株rPRV NY-gE-/gI-的基因组DNA共转染ST细胞,通过蚀斑纯化,获得表达荧光蛋白的PRV三基因缺失突变株。分别用PRV三基因缺失突变株、rPRV NY-gE-/gI-、PRV商品活疫苗Bartha-K61株、DMEM细胞培养液免疫6周龄雌性小鼠,2周后对小鼠进行第2次免疫,且首免后6周用PRV强毒NY株对小鼠进行攻毒试验。结果显示,成功拯救PRV三基因缺失突变株rPRV NY-gE-/gI-/TK--EGFP+,且对小鼠是安全的。间接ELISA试验和病毒中和试验证实rPRV NY-gE-/gI-/TK--EGFP+使小鼠机体产生了较高滴度的PRV特异性抗体,小鼠外周血T淋巴细胞亚群的测定证明,其诱导小鼠机体产生了细胞免疫应答。攻毒试验结果显示,rPRV NY-gE-/gI-/TK--EGFP+对PRV强毒NY株的攻击具有一定的抵抗力。本试验获得的PRV三基因缺失病毒rPRV NY-gE-/gI-/TK--EGFP+有望成为一种防控当前PR流行的疫苗株。

In this study,the flanking sequences of TK genes were PCR amplified from PRV NY strain as homologous arms and cloned into pUC-19 plasmid.As a reporter,the enhanced green fluorescent protein(EGFP)was inserted into the recombinant plasmid to obtain PRV transfer plasmid pUC-TKLRE.Then the pUC-TKLRE plasmid and genome DNA of rPRV NY-gE-/gI-were further co-transfected into ST cells,and a gE/gI/TK triple deletion mutant with green fluorescent protein gene(named rPRV NY-gE-/gI-/TK--EGFP+) was picked up during plaque purification in ST cells.6-week-old female mice were immunized with rPRV NY-gE-/gI-/TK--EGFP+,rPRV NY-gE-/gI-,commercial activated vaccine Bartha-K61,and DMEM culture solution.The injections were repeated after 2 weeks in all of the groups,and then challenged with the virulent PRV NY strain at 6 weeks after the first immunization.The results showed that the rPRV NY-gE-/gI-/TK--EGFP+ was successfully generated,and had ideal safety to mice.The indirect ELISA and the neutralization test displayed that the rPRV NY-gE-/gI-/TK--EGFP+ could induce higher PRV-specific antibody,and the recombinant virus elicited significant cellular immune response towards the determination of T lymphocyte subsets.The challenge test showed that rPRV NY-gE-/gI-/TK--EGFP+-immunized group could protect the mice against the virulent PRV NY challenge to some extent.In conclusion,the rPRV NY-gE-/gI-/TK--EGFP+might be a promising candidate vaccine for the control of the currently epidemic PR in China.