猪繁殖与呼吸综合征病毒GP5蛋白糖基化位点缺失突变株的构建  

Construction of porcine reproductive and respiratory syndrome virus with GP5 protein mutants at putative glycosylation sites

作　　者：张辉[1] 王光利[1] 徐大勇[1] 李峰[1] 徐争辉[1] 

机构地区：[1]淮北师范大学生命科学学院,安徽淮北235000

出　　处：《中国兽医科学》2012年第5期511-516,共6页Chinese Veterinary Science

基　　金：安徽省自然科学研究基金项目(KJ2010B192);安徽省优秀青年人才基金项目(2010SQRL082)

摘　　要：以含有猪繁殖与呼吸综合征病毒(PRRSV)VR-2332株全长感染性cDNA克隆的质粒pVR2332为模板,通过融合PCR方法扩增GP5蛋白第34、51位天冬酰胺糖基化位点缺失的突变序列,替换pVR2332质粒中编码GP5蛋白的DNA片段,分别获得重组质粒pVR-N34A、pVR-N51A。在体外转录后转染BHK-21细胞,转染后第48小时收集细胞接种Marc-145细胞,进行突变病毒的拯救。然后通过RT-PCR扩增拯救病毒GP5蛋白的编码序列,序列分析显示构建正确。间接免疫荧光试验证明拯救病毒能够正常表达GP5蛋白。同时,中和试验结果初步表明GP5糖基化位点缺失影响PRRSV与中和抗体的反应。猪繁殖与呼吸综合征病毒GP5蛋白不同糖基化位点缺失突变株的获得将为进一步揭示GP5蛋白糖基化与诱发机体免疫反应之间的关系奠定基础。The plasmid pVR2332 harboring porcine reproductive and respiratory syndrome virus （PRRSV） full-length infectious clone was served as template to generate GP5 mutants, which contained amino acid substitution at putative glycosylation sites （N34, NS1）, by overlap extension PCR. The DNA fragments in the plasmid pVR2332 were replaced by the mutants to obtain the recombinant plasmid pVRN34A and pVR-N51A,respectively. Capped RNA transcripts were generated using the plasmid pVR-N34A or pVR-NS1A as the template in vitro and were then transfected into BHK-21 cells. The mutant virus was rescued by inoculating Marc-145 cells with the supernatant from BHK-21 cells-transfected at hour 48 posttransfection. GP5 mutants were obtained by RT-PCR by using RNA from the rescued viruses as the template,which was confirmed by sequencing analysis. Indirect immunofluorescence assay indicated that the stable expression of GP5 protein was obtained in the rescued PRRSV mutants. Meanwhile, serum neutralization assay showed that deletion of glycosylation sites of PRRSV GP5 effected the ability of the virus to react with neutralizing antibodies. The availability of the PRRSVs with GP5 mutants at different glycosylation sites lays a foundation for further investigating of the relationships between the GP5 protein glycosylation and the immune response induced.

关 键 词：猪繁殖与呼吸综合征病毒 感染性克隆 GP5蛋白 糖基化 

分 类 号：S852.659.6[农业科学—基础兽医学]