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利用PCV2鄂州株的基因序列设计特异性引物,分别在上、下游引物中加入Sac I和Hind III酶切位点,扩增出PCV2鄂州株的ORF2基因;将该片段克隆至p ET-28a原核表达载体,经PCR、酶切鉴定,成功构建了阳性重组表达质粒p ET-ORF2。将其转入大肠杆菌Rosetta中,经IPTG诱导,SDS-PAGE电泳分析确定重组蛋白大小为34.5 k D,主要以包涵体的形式表达。优化后确定最佳诱导条件为:37℃,至OD值0.4~0.6时加入终浓度为1 mmol/L的IPTG,诱导5 h。表达产物经His-tag镍柱纯化后进行Western-blot分析,结果表明表达的重组蛋白能够与PCV2标准阳性血清发生特异性反应,具有良好的反应原性。
The specific primers were designed according to the gene sequence of Ezhou strain of PCV2. The restriction sites of Sac I and Hind III were added to the upstream and downstream primers to amplify the ORF2 gene of PCV2 Ezhou strain. The fragment was cloned into p ET-28a pronucleus The recombinant plasmid p ET-ORF2 was successfully constructed by PCR and restriction enzyme digestion. The recombinant protein was transformed into E.coli Rosetta and induced by IPTG. SDS-PAGE analysis indicated that the recombinant protein was 34.5 kD in size and expressed mainly in inclusion bodies. Optimized conditions to determine the best induction: 37 ℃, OD value of 0.4 to 0.6 when added to a final concentration of 1 mmol / L of IPTG induced 5 h. The expressed product was purified by His-tag nickel column and analyzed by Western-blot. The results showed that the expressed recombinant protein could react specifically with PCV2 standard positive serum and had good reactionogenicity.