【摘要】 参照GenBank中猪圆环病毒Ⅱ型(Porcine circovirus type 2,PCV2)的ORF2基因序列,设计合成1对引物,对PCV-2ZS株ORF2基因上含主要抗原位点的片段(ORF2-ME)进行PCR扩增,并克隆到pMD18-T Simple Vector中,进行序列测定。将重组质粒pMD-ORF2-ME的EcoR Ⅴ和Xhol Ⅰ双酶切产物插入原核表达载体pET-32a(+),构建原核表达载体pET-ORF-ME。测序分析表明,克隆的ORF2-ME与GenBank上公布的PCV2毒株核苷酸序列同源性为99%~100%,推导的氨基酸序列同源性介于91%~100%之间。原核表达载体导入BL21(DE3)后用IPTG进行诱导表达,收集菌液进行SDS-PAGE和Western Blotting分析,结果表明ORF2-ME在BL21(DE3)中成功表达,所表达融合蛋白的相对分子质量约为40000,并能被PCV2阳性血清所识别,这就为PCV2感染诊断和抗体检测提供了依据。更多还原

【Abstract】 A pair of specific primers was designed according to the sequences published in the GenBank of ORF2 gene of PCV2 to amplify the ORF2 truncated gene,which including the major epitope (ME),from the PCV2 ZS strain.The fragment was cloned into pMD 18-T Simple Vector and then sequenced.The recombinant plasmid pMD-ORF2-ME was digested by EcoRⅤ and XholⅠ and the truncated gene was cloned into the prokaryotic expressing vector pET-32a(+),i.e.the recombinant plasmid named pET-ORF-ME was constructed.The sequencing results indicated that the ORF2 truncated gene′s nucleotide sequecse of PCV2 ZS strain shared 99%-100% homology with other PCV2 strains that had been published in GenBank with the identity of the deduced amino acid to be between 91%-100%.The recombinant plasmid pET-ORF-ME was transformed into BL21(DE3)competent cell of E .coli and induced by IPTG .SDS-PAGE analysis showed that the ORF2-ME had been expressed with the molecular weight of the expressed fusion protein to be about 40 000.Western Blotting assay indicated that the recombinant protein could react with polyclonal antibody against PCV2,sharing a good antigencity.The results suggest that the ORF2 truncated protein might be suitable as potential antigen for routine serodiagnosis and epidemiological surveys of PCV2 infection.更多还原