猪圆环病毒2型 ORF4基因的原核表达及多克隆抗体的制备

摘要

Porcine circovirus 2 ( PCV2 ) is the primary causative factor of postweaning multisystemic wasting syn-drome ( PMWS) in swine.The first three open reading frames have been described while ORF 4 is the fourth ORF in PCV2 genome and its function is still unknown .In this study, ORF4 gene was cloned into the vector of pGEX-4T-1 to construct recombinant plasmid pGEX-4T-ORF4.The recombinant plasmids confirmed by sequencing were trans-formed into BL21 competent strain and induced by IPTG .The purified GST-ORF4 protein was subcutaneously injec-ted into the rabbits for preparation of the anti-GST-ORF4 antibody .Blood was collected four weeks later and the titer of polyclonal antibody was assessed by indirect enzyme-linked immune sorbent assay ( ELISA ) .The results showed that the GST-ORF4 protein was successfully expressed by SDS-PAGE analysis and could be specifically detected with resulting polyclonal antibodies against GST-ORF4 protein by Western blot .The ELISA result indicated that the titer of the antibody was over 1∶12 800 and the antibody can be used for further functional study of ORF 4 which may facil-itate understanding the pathogenesis of PCV 2 and interaction between PCV 2 and host .%猪圆环病毒2型（PCV2）是引起断奶仔猪多系统衰竭综合征（PMWS）的主要病原，ORF4是PCV2中继ORF1，ORF2，ORF3之后的第四大开放阅读框。本研究以提取的PCV2基因组为模板经PCR扩增获得ORF4基因（386～565 nt），并克隆到pGEX-4T-1表达载体上，构建pGEX -4T-ORF4表达载体。经PCR和测序鉴定后，将重组质粒转化受体菌BL21诱导表达并进行目的蛋白GST-ORF4纯化，然后用纯化的蛋白免疫新西兰兔，在免疫期间采血并用间接ELISA对多抗血清进行效价测定。 SDS-PAGE电泳检测结果表明，重组质粒成功表达了目的蛋白。 ELISA检测结果显示抗体效价达到1∶12800以上。 Western blot分析表明ORF4融合蛋白具有很好的免疫原性，获得的抗体可以和大肠杆菌表达的GST-ORF4融合蛋白特异性反应。