猪圆环病毒2型Cap蛋白的原核表达及其抗原性分析

Prokaryotic expression and antigenic analysis of the capsid protein porcine circovirus type 2

根据GenBank中登录的PCV-2序列,设计1对特异性引物,PCR扩增PCV-2去除核定位信号(nuclear localization signal,NLS)的Cap蛋白基因,经Bam HI和Xho I双酶切后将其插入到表达载体pEGX-4T-1多克隆位点,并转化到表达菌株Rossetta(DM3)中,采用IPTG进行诱导表达,采用SDS-PAGE薄层灰度分析表达情况,收集菌体,使用GST-Protein Purification Kit亲和层析纯化方法对表达的GST-Cap融合蛋白蛋白进行纯化,SDS-PAGE电泳检测纯化效果,Western blot分析纯化后的重组Cap蛋白(rCap)免疫学活性。结果表明,成功克隆了大小为579 bp去除核定位信号的ORF2基因,成功诱导表达出预期大小45.3 ku相一致的rCap蛋白,表达量占总菌体的25%,纯化后的rCap蛋白SDS-PAGE电泳分析纯度达到90%以上,Western blot分析表明纯化的rCap蛋白能与PCV-2阳性血清发生特异性反应,具有良好的免疫学活性。

According to the published ORF2 genes sequence of PCV-2 in GenBank, one pair of PCR primers were designed, and the nuclear localization signal （NLS）-defected capsid protein gene（d Cap）of porcine circovirus type 2 （PCVo2）was expressed. Then the PCR product was cloned into pEGX-4T-lvector by Barn HI and Xho I digestion and a recombinant plasmid named pEGX-4T-1-ORF2 was obtained, which was induced by IPTG; The expressive product was purified by the GST-Protein Purification Kit. The purification effect and the specificity of the purified recombinant capsid proteins were detected respectively by SDS-PAGE and Western blot assay. The results demonstrated that ORF2 gene was successfully cloned, which was 579 bp; the size of rCap protein was 45.3 ku, which was in line with the expected size; there is only one purpose band of 45.3 ku in size with above 90% purity. The results show that the recombinant Cap have been well purified and could react with the polyclonal antibody against PCV-2, and possess good antigenicity.