PCV3河南株Cap基因的克隆、测序及其在大肠杆菌中的表达

Cloning,sequencing and expression of Cap gene of Henan PCV3 strain in Escherichia coli

为研究猪圆环病毒3型（PCV3）Cap基因在大肠杆菌中的表达产物是否具有免疫原性,根据GenBank中发表的PCV3基因组序列,设计1对特异性引物,PCR扩增PCV3 Cap基因,克隆到pMD18-T载体。测序结果表明获得了Cap基因,其大小为543bp。并将Cap亚克隆到原核表达载体pET-28a中,再转化入大肠杆菌Rosetta（DE3）中,获得重组菌株。获得的重组菌37℃、0.6mmol/L IPTG诱导6h,进行SDS-PAGE及Western blot分析。重组菌破碎后从沉淀中获取1条约24 600的新蛋白带,可与小鼠抗His单克隆抗体发生特异性反应。有研究表明PCV3与PCV2不具有免疫交叉保护作用,因此本试验表达的具有良好的抗原性PCV3 Cap蛋白,为PCV3新型亚单位疫苗和ELISA诊断试剂盒的研制奠定了基础。

To make sure whether the expression product of Cap gene of porcine circovirus type 3 （PCV3）in Escherichia coli has immunogenicity,a pair of specific primer was designed and synthe- sized according to the nucleotide sequence of the PCV3 gene published in GenBank. PCV3 Cap gene was amplified by P1/P2 and PCR from total DNA extracted from clinical samples,PCR prod- uct was cloned into pMD18-T vector for sequencing. The result revealed that the nucleotide se- quence of PCV3 Cap gene consisted of 543 bp. A prokaryotic plasmid of pET28a-SUMO-PBD1 was obtained by subeloning the encoding region of the mature peptide of PCV3 Cap gene into pET28a-SUMO vector and transformed Escherichia coli Rosetta （DE3）competent cell. The ex- pressed products were analyzed by SDS-PAGE electrophoresis and Western blot test after induced by addition of IPTG to a final concentration of 0.6 mm/L for 6 h at 37℃. The expressed protein was 24 600 its can specifically react to the anti-his monoclonal antibody. Studies showed that there is not cross-protection between PCV2 and PCV3. So the expressed PCV3 Cap protein has an anti- genieity,which laid the foundation for the development of new type of PCV3 subunit vaccine and ELISA diagnostic kit.