CAJ | 학술논문

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.
근거GenBank수거고중저원배병독Ⅱ형적기인조서렬설계인물,채용PCR기술종병료기인조DNA중확증출PCVⅡ하남지방주ORF4기인,전장180 bp,편마59개안기산.장해기인극륭지재체pGEX-4T-3중형성pGEX-4T-3-ORF4표체재체.경PCR、매절화측서감정후,전화표체균주BL21（DE3）유도표체.SDS-PAGE결과현시：ORF4능구재대장간균중표체,산물적분자량약위32 kD,차이포함체형식존재.Western Blot검측결과현시,순화후적ORF4단백능구여서항6×His표첨단극륭항체발생특이성반응,위진일보연구PCVⅡORF4단백적특성여공능전정료기출.
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 ×His monclonal antibody.Cloning and prokaryotic expression of ORF4 gene were benefit to research for function and characteristics of ORF4 of PCVⅡ.