表达猪瘟病毒E2蛋白重组猪繁殖与呼吸道综合征病毒的研究  

Construction and expression of classical swine fever virus E2 protein by recombinant porcine reproductive and respiratory syndrome virus

作　　者：童武[1,2] 周艳君[1] 徐彦召[1] 姜一峰[1] 王亚欣[1] 张善瑞[1] 朱建平[1] 虞凌雪[1] 孙晶[1] 陈焕春[2] 童光志[1] 

机构地区：[1]中国农业科学院、上海兽医研究所,上海200241 [2]华中农业大学动物医学院,湖北武汉430070

出　　处：《中国预防兽医学报》2012年第7期505-509,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：NSFC-广联合基金项目(U0931003);上海市科技人才计划项目(09XD1405400);国际合作项目(2010DFB33920);国家生猪现代产业技术体系建设项目(NYCYTX-009);中央科研院所公益性基础科研业务费项目(2011JB03)

摘　　要：为构建表达猪瘟病毒(CSFV)E2蛋白重组猪繁殖与呼吸道综合征病毒(PRRSV),本研究首先利用高致病性PRRSV弱毒疫苗HuN4-F112株的感染性分子克隆作为平台,构建了一个在nsp2区有缺失的感染性分子克隆,命名为pHuN4-F112-△480-620。以pHuN4-F112-△480-620作为载体,采用突变PCR的方法将CSFV的主要保护性抗原E2基因1 bp～9 99 bp,1 bp～600 bp,1 bp～330 bp及256 bp～330 bp基因片段分别插到nsp2中aa 480～aa 620位氨基酸缺失编码区域。结果显示,插入完整E2基因或较大E2基因片段的重组PRRSV cDNA质粒均未能拯救出病毒,只有插入较小的E2基因片段(256 bp～330 bp)的重组病毒cDNA质粒成功地拯救出了重组病毒rPRRSV-F112-E2(256-330),拯救的病毒能够在MARC-145细胞上引起明显的细胞病变,而且生长速度明显高于其亲本病毒,间接荧光检测表明该重组病毒能够表达外源基因。To construct recombinant porcine reproductive and respiratory syndrome virus （PRRSV） expressing E protein of classical swine fever virus （CSFV）, the pHuN4-F112-A 480-620 infectious clone with nsp2 region deletion was generated from the infectious clone of attenuated highly pathogenic PRRSV vaccine strain HuN4-F112. Furthermore, the complete E2 gene or tnmcate of E2 genes （1 bp to 999 bp, 1 bp to 600 bp, 1 bp to 330 bp and 256 bp to 330 bp） of CSFV were inserted into the deletion site of pHuN4-Fll2-A480-620 by mutant PCR method, respectively. However, after in vitro transcription followed by transfection of BHK-21 and Marc-145, it was failed to rescue viruses with insertion of longer E2 fragments or complete E2 gene, but only a recombinant PRRSV with an insertion of smaller E2 fragment （256 bp to 330 bp） was rescued which was able to replicate and caused CPE in Marc-145 cells, moreover, the titer of the rescued virus was higher than the parental virus. Expression of inserted E2 epitope in the recombinant PRRSV was identified by indirect immunofluorescence assay with polyclonal antibodies to E2.

关 键 词：猪繁殖与呼吸道综合征病毒 猪瘟病毒 感染性分子克隆 重组病毒 

分 类 号：S852.65[农业科学—基础兽医学]