猪圆环病毒2型ORF2基因的优化表达及活性检测

Prokaryotic expression of modified ORF2 gene of porcine circovirus type 2 and immunogenicity of the expressed protein

通过软件分析后,截去猪圆环病毒2型ORF2基因中影响蛋白表达的保守基因,并对其余的基因进行了密码子优化。把改造后的ORF2基因连接到pGEX-4T-1载体上并转入表达宿主菌大肠杆菌BL21(DE3)进行IPTG诱导表达。结果显示,重组菌可以表达出分子质量为50ku的重组蛋白,在0.2mmol/L的IPTG诱导5h情况下表达效果最好,表达的蛋白以包涵体形式存在于菌体中。表达产物经琼脂糖凝胶纯化可得到较纯的蛋白。经Western-blot分析,所纯化的蛋白能刺激小鼠产生相应抗体,并能与PCV2阳性血清进行特异性免疫印迹反应。试验结果证实表达的蛋白具有良好的抗原性。

The conserved fragment that influences expression of porcine circovirus type 2(PCV2)ORF2gene was removed,and the rest part of ORF2gene was modified.Then the optimized ORF2gene was cloned into pGEX-4T-1expression vector,and the recombinant plasmid was transformed into Escherichia coli BL21(DE3).The transformed bacteria were induced with IPTG and a recombinant protein of 50ku was produced.The result showed that 0.2mmol/L IPTG and 5hof inducting time were the best conditions for this protein production and more pure proteins would be produced after agarose gel purification.The purified protein could stimulate mice to produce antibodies and react with a PCV2positive serum in Westernblot,which showed that the expressed protein had a strong antigenicity.