表达PEDV S和PoRV VP7蛋白的重组伪狂犬毒株构建

【背景】猪流行性腹泻、猪轮状病毒病与猪伪狂犬病是严重危害全球养猪业的3种重要传染病，混合感染往往导致猪场更严重的损失。【目的】利用同源重组技术构建共表达猪流行性腹泻病毒(Porcine epidemic diarrhea virus，PEDV) S蛋白和猪轮状病毒(Rotavirus，PoRV) VP7蛋白的猪伪狂犬三联基因工程疫苗株，并研究其部分生物学特性。【方法】通过序列比对、蛋白结构分析筛选s基因的475?804 aa和vp7基因的17?339 aa作为毒株构建的目的片段，依次构建了pMD-S、pMD-VP7、pMD-VP7.S克隆载体和pEGFP-VP7.S转移载体。将质粒pEGFP-VP7.S和PRV XJ亲本株同源重组，空斑纯化得到重组毒株PRV (CM)，对其稳定性和增殖特性进行研究。【结果】构建了共表达S蛋白和VP7蛋白的伪狂犬基因工程病毒，连续传代20次，均能检测到vp7和s基因，而gE基因阴性；Western blotting证实2种外源基因在重组病毒中均能实现良好的表达；测定亲本毒株和重组毒株的TCID50分别是10?7.59/0.1 mL和10?7.25/0.1 mL。【结论】获得了伪狂犬基因工程重组弱毒株PRV (CM)，外源基因稳定存在，毒力基因稳定缺失，增殖特性差异不大，为PRV、PEDV和PoRV基因工程三联苗研究奠定了基础。

Construction of recombinant pseudorabies virus strain expressing PEDV S and PoRV VP7 protein

Backgroud] Porcine epidemic diarrhea, porcine rotavirus disease and pseudorabies are three important infectious diseases that seriously endanger the global pig industry. Mixed infections often lead to more serious losses on the farm. Objective] A homologous recombination technique was used to construct a triple genetic engineering attenuated strain of pig pseudorabies co-expressing s gene and vp7 gene. Methods] Through the sequence alignment and protein structure analysis of PEDV s and PoRV vp7 genes, 475?804 aa of s gene and 17?339 aa of vp7 gene were selected as candidate regions for the construction of strains. Three cloning vectors of pMD-S, pMD-VP7 and pMD-VP7.S and one transfer vector of pEGFP-VP7.S were constructed, respectively. Plaque purification was employed to obtain recombinant strain. Results] A genetically engineered pseudorabies strain co-expressing S protein and VP7 protein was constructed. The strain was serially passaged for 20 generations, and both vp7 and s genes were detected without gE gene. Western blotting experiments confirmed foreign gene expression by recombinant virus. The TCID50 of the parent strain and the recombinant strain was determined by the Reed-Muench method to be 10?7.59/0.1 mL and 10?7.25/0.1 mL, respectively. Conclusion] Recombinant pseudorabies strain PRV (CM) was obtained, which stably carried the two foreign genes, but with the deletion of virulence genes. The recombinant strain owned a similar proliferation characteristic with the parent strain. The study laid the foundation for further study on development of PRV, PEDV and PoRV triple genetic engineering vaccine.