猪繁殖呼吸综合征病毒的NSP7蛋白密码子优化和表达及其免疫学活性鉴定

Codon optimization expression of PRRSV NSP7 protein and immunological activity identification

目的:提高蓝耳病毒NSP7基因的表达水平和促进其可溶性表达,验证重组蛋白的免疫学活性,为猪繁殖呼吸综合征血清学鉴定奠定基础。方法:通过同源性分析软件分析28株NSP7基因的保守性,抗原表位预测网站预测NSP7蛋白的抗原表位,可溶性表达预测网站分析NSP7基因可溶性表达概率,随后挑选保守性高的NSP7基因进行密码子优化并合成,最后利用SDS-PAGE探究重组蛋白表达产物存在的形式和表达的最佳条件,Western blot和ELISA探究重组蛋白的免疫学活性。结果:蓝耳病毒NSP7基因较保守,抗原表位较多,经过密码子优化后以可溶性形式表达; SDS-PAGE分析表明重组蛋白的相对分子质量约为49 k D,最佳表达条件为34℃0. 8 mmol/L IPTG 7 h; Western blot和ELISA结果显示纯化的蛋白具有免疫学活性。结论:重组蛋白以可溶性形式表达且具有免疫学活性,为PRRSV血清学鉴定奠定基础。

Objective: To improve the expression level of PRRSV NSP7 gene,promote its soluble expression,and verify the immunological activity of recombinant protein,so as to lay a foundation for serological identification of PRRSV.Methods: The conservatism of 28 strains NSP7 genes was analyzed by homologous analysis software.Epitope prediction website predicts the epitope of NSP7 protein.The probability of soluble expression of NSP7 gene was analyzed by the soluble expression prediction website.Subsequently,the highly conservative NSP7 gene was selected for codon optimization and synthesis.Finally,SDS-PAGE was used to investigate the form of expression product and best condition for expression of recombinant protein,Western blot and ELISA were used to explore the immunological activity of recombinant protein.Results: The PRRSV NSP7 gene was more conservative and had more epitope and the expressed form of PRRSV NSP7 gene was soluble after codon optimization;SDS-PAGE analysis showed that the relative molecular mass of the recombinant protein was about 49 k D,and the optimum expression conditions was 34℃ 0.8 mmol/L IPTG 7 h.The results of Western blot and ELISA showed that the recombinant protein possesses immunologic activity.Conclusion: Recombinant proteins are expressed in soluble form and possesses immunological activity,which lays the foundation for PRRSV serological identification.