猪圆环病毒2型ORF2基因片段的克隆与原核表达

为在原核表达系统中表达猪圆环病毒2型(PCV2)ORF2基因片段,提取感染PCV2的细胞基因组DNA,PCR扩增ORF2基因片段并克隆至pMD-18T载体上,经PCR、酶切及测序鉴定后,将该基因重组至pET-28a原核表达载体,构建了表达载体pET-28a-ORF2。该重组质粒在大肠杆菌BL21中经1mmol/LIPTG于37℃诱导5h得到最佳表达。表达重组蛋白的分子量为26.0kD,以包涵体形式存在。Western-Blot分析表明,该重组蛋白与PCV2阳性血清发生特异性反应,表明该重组蛋白具有良好的反应原性。

Cloning and Prokaryotic Expression of the ORF2 Gene Fragment of Porcine Circovirus Type 2

The truncated fragment of PCV2 ORF2 gene was amplified by PCR from the PCV2-infected PK-15 cells.The PCR product was cloned using pMD-18T vector and confirmed by restrict enzyme analysis and DNA sequencing.Then,a prokaryotic expression vector pET-28a-ORF2 was constructed.After identification,the recombinant plasmid was transformed into E.coli BL21 strain.The bacteria were induced by 1 mmol/L IPTG for 5h at 37℃.The expression of ORF2 gene was successfully induced.The molecular weight of an induced fusion protein was about 26.0 kD and the expression products mainly existed as inclusion bodies.Western blot assay showed that the recombinant protein reacted with polyclonal antibodies against PCV2,which implies that the ORF2 truncated protein might be suitable as potential antigen for further development of immunoreagents.