猪繁殖与呼吸综合征病毒核衣壳蛋白与糖蛋白的表达纯化及鉴定

Expression,Purification and Identification of Nucleocapsid Protein and Glycoprotein of Porcine Reproductive and Respiratory Syndrome Virus

为获得猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)核衣壳蛋白(N)和糖蛋白(E),试验通过RT-PCR反应获得ORF7和ORF5基因片段,分别克隆至原核表达载体pET-28a(+),转化至大肠杆菌BL21(DE3)中诱导表达,SDS-PAGE分析目的蛋白表达情况,Western blotting检测表达蛋白的免疫活性,采用亲和层析对表达蛋白进行纯化。结果表明,成功表达N和E融合蛋白,Western blotting结果表明表达蛋白具有良好的反应原性,纯化蛋白的纯度分别为89%和90%。融化蛋白的表达为PRRSV的分子生物学功能研究和测定抗体检测方法的建立奠定了基础。

In order to gain nucleocapsid protein （N） and glycoprotein （E） of porcine reproductive and respiratory syndrome virus （PRRSV）, the total RNA was extracted,ORF7 and ORF5 genes were obtained by RT PCR, inserted into pET 28a（ ＋ ） vector, and then transformed into Escherichia coli BL21（DE3）, respectively. The expressed products were identified by SDS PAGE and Western blotting, and purified by affinity chromatography. The results showed that N and E fusion protein were successfully expressed and the proteins had good reactionogenicities by Western blotting analysis. The purities of the purified proteins were 89 % and 90%, respectively. This study could lay foundations for molecular biological function research and es- tablishment of test methods for detection antibodies of PRRSV.