猪繁殖与呼吸综合征病毒糖基化囊膜蛋白5原核表达载体的构建及免疫原性分析

为分析猪繁殖与呼吸综合征病毒(PRRSV)糖基化囊膜蛋白5（GP5）的免疫原性，本研究通过提取PRRSV分离株（GenBank登录号：HQ701732.1）RNA和RT-PCR扩增得到开放阅读框5（ORF5）基因。根据ORF5的基因序列，设计2对引物，经PCR扩增分别获得不含信号肽和跨膜功能区的2段基因片段。利用酶切位点，将2段基因连接到原核表达载体pET-28a（+）上，获得重组表达质粒pET28a-GP5。将重组质粒导入BL21（DE3）感受态细胞，经IPTG诱导获得表达。经Western blotting鉴定，重组蛋白可被PRRSV阳性血清识别。将纯化的重组蛋白免疫BALB/c小鼠，ELISA方法检测，小鼠能产生针对蛋白的血清抗体。因此，该重组PRRSV GP5蛋白具有良好的生物学活性，为进一步研究GP5蛋白的结构和功能奠定基础。

Prokaryotic Expression Vector Construction and Immunogenicity Analysis of Glycosylated Envelope Protein 5 from Porcine Reproductive and Respiratory Syndrome Virus

In order to analyze the immunogenicity of glycosylated envelope protein 5 (GP5) from porcine reproductive and respiratory syndrome virus (PRRSV), ORF5 gene fragment was amplified by RT-PCR from the PRRSV (GenBank: HQ701732.1). Based on the ORF5 gene sequence, two pairs of primers were used to amplify two gene fragments, excluding the signal peptide sequence and transmembrane regions. The two gene fragments were cloned into the prokaryotic expression vector pET-28a（+）, and then the recombinant plasmid was transformed into Escherichia coli. The results of Western blotting and ELISA respectively showed that the expression of GP5 could be recognized by positive serum antibody of PRRSV, and corresponding antibodies of GP5 protein could produce in the immunized BALB/c mice. Therefore, the recombinant GP5 protein had good biological activity, and could provide fundamental data for the further study on the structure and function of GP5 protein of PRRSV.