摘要
根据GenBank数据库中猪圆环病毒Ⅱ型的基因组序列设计引物,采用PCR技术从病料基因组DNA中扩增出PCVⅡ河南地方株ORF4基因,全长180 bp,编码59个氨基酸.将该基因克隆至载体pGEX-4T-3中形成pGEX-4T-3-ORF4表达载体.经PCR、酶切和测序鉴定后,转化表达菌株BL21(DE3)诱导表达.SDS-PAGE结果显示:ORF4能够在大肠杆菌中表达,产物的分子量约为32 kD,且以包涵体形式存在.Western Blot检测结果显示,纯化后的ORF4蛋白能够与鼠抗6×His标签单克隆抗体发生特异性反应,为进一步研究PCVⅡORF4蛋白的特性与功能奠定了基础.
According to the genomic sequence of porcine circovirus typeⅡ （PCVⅡ） from GenBank,a pair of specific primers were designed.The full-length PCVⅡORF4 gene of Henan strain was amplified from pathological samples suspected PCVⅡ infection by PCR,which was 180 bp in length and encoded 59 amino acids.The ORF4 gene was cloned into the vector of pGEX-4T-3 to construct recombinant plasmid pGEX-4T-3-ORF4.The recombinant plasmid confirmed by sequencing was transformed into BL21 （DE3） and induced by IPTG.SDS-PAGE analysis showed that the ORF4 gene could express in E.coli BL21 （DE3） and the expression prudct of ORF4 gene was about 32 kD recombinant protein, mainly in form of inclusion bodies.The results of western blot showed that the purified ORF4 protein could be specifically reacted with 6 ＆#215;His monclonal antibody.Cloning and prokaryotic expression of ORF4 gene were benefit to research for function and characteristics of ORF4 of PCVⅡ.
出处
《河南科技学院学报（自然科学版）》
2014年第4期52-55,61,共5页
Journal of Henan Institute of Science and Technology(Natural Science Edition)
基金
河南省高等学校青年骨干教师资助计划项目(2011GGJS-192)
