河北地区猪圆环病毒二型ORF2基因片段的克隆与原核表达

Cloning and Expressing of the Fragment of Porcine Circovirus2 ORF2 Gene

以PCV2 PK-15细胞毒提取的DNA为模板,用PCR扩增PCV2ORF2基因的后部约600 bp片段;对PCR产物回收纯化后与载体pMD19-T进行连接、转化,经酶切和序列分析后亚克隆到原核表达载体pET-32a中,构建重组表达质粒pET-32a-ORF2,转入大肠埃希氏菌BL21(DE3)中,以IPTG诱导表达,进行SDS-PAGE电泳和Western-Blotting分析。结果表明,重组表达质粒在大肠中所表达的融合蛋白相对分子量为40 kDa,Western-blotting分析表明该蛋白具有PCV2抗原性。

The behind fragment of PCV20RF2 gene was amplified from the extracted DNA of PCV2 in inoculated in PK-15 cells;The PCR product was connected with pMD19-T vector, which was confirmed by sequence analysis and restrict enzyme identification, and then was cloned into pET-32aVector, Plasmid containing the right insertion was digested with enzymes to confirm its identity and retransformed the recombinant plasmid into E. coli BI221 strain. The bacteria were induced by IPTG and the lysates were analysed directly by SDS-PAGE . Results were as follows. A fusion protain approximately 40 kDa in molecular weight was observed on the SDS-PAGE. Western-blotting confirmed that had antigenic activity of PCV2.