II型猪圆环病毒ORF2基因的原核表达与初步应用  

Prokaryotic Expression and Diagnostic Application of ORF2 of Porcine Circovirus Type Ⅱ

作　　者：吴国平[1] 郭川[1] 刘冰心[2] 梁银龙[2] 苏文金[1] 曹敏杰[2] 

机构地区：[1]厦门大学生命科学学院,福建厦门361005 [2]集美大学生物工程学院,福建厦门361021

出　　处：《厦门大学学报（自然科学版）》2006年第B05期129-133,共5页Journal of Xiamen University：Natural Science

基　　金：福建省科技攻关计划重点项目(2003N083);厦门市科技攻关计划重点项目(3502Z20031054)资助

摘　　要：II型猪圆环病毒(PCV2)是引起断奶仔猪多系统衰竭综合症(PMW S)的重要病原.本研究用PCR以构建的PCV2ORF2重组质粒pBS-T-ORF2为模板,扩增截短的ORF2(tORF2)基因亚克隆至表达载体pGEX-4T-3,构建重组表达质粒pGEX-tORF2并转化大肠杆菌BL21(DE3).经SDS-PAGE及W estern b lot鉴定,成功表达了谷胱甘肽S转移酶(GST)融合的tORF2,重组蛋白分子量约为45ku,表达量约为菌体总蛋白的20%,重组蛋白具有良好的免疫学活性.用tORF2重组蛋白对20份临床猪繁殖与呼吸障碍综合征(PRRS)阳性猪血清进行W estern b lot检测,有4份(20%)血清显示PCV2抗体阳性,说明PCV2与PRRSV存在共同感染现象.本研究初步证明表达的tORF2重组融合蛋白可以应用于临床检测.Porcine circovirus type Ⅱ （PCV2） plays a crucial role in the pathogenesis of post-weaning multisystemic wasting syndrome （PMWS） in swine. The ORF2 gene encoding the capsid protein of PCV2 was amplified by PCR and cloned into plasmid pBS-T to construct recombinant plasmid pBS-T-ORF2, which was used as template to amplify the truncated ORF2 （tORF2）. The tORF2 gene was consequently cloned into the expression vector of pGEX-4T-3 for prokaryotic expression in E. coli. Expression level of the recombinant GST-tagged tORF2 protein reached approximately 20% of the total bacterial proteins and its molecular weight was approximately 45 ku identified by SDS-PAGE and Western blot. The recombinant protein was used as antigen to establish a Western blot diagnostic assay. Twenty PRRS positive swine serum samples were detected by this Western blot assay. The positive rate of these sera was 20% （4/20）, indicating that coinfection of PRRSV and PCV2 exist in swine. Taken together, the recombinant tORF2 protein reporteded in the present study may play a critical role in the detection of PCV2 antibodies immunologically.

关 键 词：Ⅱ型猪圆环病毒 ORP2 原核表达 初步应用 

分 类 号：Q785[生物学—分子生物学]