猪繁殖与呼吸综合征病毒ORF7基因的表达和重组蛋白的纯化及免疫学活性分析

猪繁殖与呼吸综合征病毒（PRRSV）为不分节段、聚腺苷酸化、有囊膜的单股正链RNA病毒。其基因组长约15kb，含有8个开放的阅读框架（ORFs）。PRRSV基因组的顺序依次为：5’-ORFla-ORFlb-ORF（2—6）．ORFT-3’。鲁韦韦等分析表明，PRRSV的ORF7基因保守性较好，变异较小。此外，ORF7编码的核衣壳蛋白（N蛋白）具有良好的免疫原性及反应原性，机体产生的PRRSV抗体主要是针对N蛋白的。因此，N蛋白常可作为诊断抗原来检测PRRSV。

Expression of Porcine Reproductive and Respiratory Syndrome Virus ORF7 Gene and Purification and Immunological Activity Analysis of the Recombinant Protein

Objective] The aim of this study was to realize efficient expression of the porcine reproductive and respiratory syndrome virus (PRRSV) ORF7 gene in genetic engineering bacteria and analyze the immunological activity of the recombinant protein after purification. Method] The constructed recombinant expression vector pET-ORF7 was transformed into Escherichia coliBL21 (DE3) and induced by IPTG under the optimal condition. After analysis of SDS-PAGE and Western Blot, the expression products were purified by Ni-NTA His·Bind Resin chromatographic column under denaturing condition and renatured by gradient dialysis. Subsequently, the immunological activity of the renatured recombinant protein was detected by Western Blot and indirect ELISA. Result] The recombinant plasmid pET-ORF7 expressed in E.coli successfully, and the fusion protein was in the form of inclusion body. By SDS-PAGE detection, the molecular weight of the expression protein was approximate 33 kD, according with the expectation. Analysis by Bandscan software showed that the expressed fusion protein was about 50% of total bacterial protein of BL21(DE3). Western Blot and indirect ELISA detection showed that the renatured protein could react with PRRSV positive serum specifically, indicating its good immunological activity. Conclusion] This study lays a foundation for the preparation of PRRSV monoclonal antibody and diagnostic kit.