猴源GII．17型诺如病毒基因组P结构域及其变体的原核表达、纯化和抗原性鉴定  

Expression, Purification and Antigenicity of P Domain and Its Variant of Monkey GII.17 Norovirus Genome

作　　者：刘波[1] 李超[1] 陶玉芬[1] 李昕潼 刘建生[1] 和占龙[1] 刘红旗[1] 

机构地区：[1]中国医学科学院/北京协和医学院医学生物学研究所,昆明650118

出　　处：《实验动物与比较医学》2016年第5期334-339,共6页Laboratory Animal and Comparative Medicine

基　　金：云南省应用基础研究计划项目（2013FZ143）;医学生物学研究所重点项目（2014IMB03ZD）;云南省创新团队（2015HC027）

摘　　要：目的 表达、纯化和鉴定猴源GII.17型诺如病毒（NoV）基因组P结构域及其变体蛋白.方法 基于本实验室获得的猴源GII.17型NoV基因组P结构域的核苷酸序列,按大肠杆菌密码子使用的偏好性优化P结构域的核苷酸序列,人工合成相应的基因.以此基因为模板,通过PCR方法扩增得到P结构域的变体基因.采用无缝连接的方法将P结构域基因及其变体克隆到原核表达载体,进行蛋白诱导表达和纯化.最后,通过免疫印迹法和ELISA法分析表达蛋白的抗原性.结果 测序结果表明,P结构域及其变体成功地克隆至原核表达载体.SDS-PAGE分析显示,P结构域在原核细胞的超声裂解上清和沉淀中都有表达,而其变体主要表达于上清.免疫印迹和ELISA结果显示,表达纯化得到的P结构域及其变体蛋白具有很好的抗原性.表达的蛋白免疫小鼠制备的相应抗体ELSIA效价可达到1∶32000.结论 成功地表达了猴源GII.17 NoV基因组P结构域及其变体蛋白,并且通过免疫小鼠得到了相应的抗体,为后期检测试剂盒的研发、病毒鉴定和病毒受体结合实验奠定了基础.Objective To express P domain and its variant of monkey GII.17 norovirus genome, purify protein and analyze antigenicity. Methods The gene of P domain was synthesized, following designation based on the nucleotide sequence that was obtained from monkey GII. 17 norovirus genome and bias of E. coli. PCR with these specific primers was performed to amplify P domain and make its variant, followed by cloning into prokaryotic expression vector via in-Fusion ligation. Protein expression was induced by IPTG, purified through GST affinity column and analyzed by Coomassie staining, immunoblot and ELISA assay. Results Sequencing results showed that P domain and its variant were successfully cloned into the expression vector. Analysis of SDS-PAGE revealed that P domain was observed in both the supernatant and pellet, and its variant was mostly expressed in the supernatant. The expressed and purified proteins were further confirmed by immunoblot analysis via the anti-GST antibody. The results of immunoblot analysis and ELISA assay indicated the good antigenicity of the proteins. The ELISA titers of antibodies induced by these proteins were 1 ; 32000. Conclusion The P domain and its variant of monkey GII.17 norovirus genome were successfully expressed. The antibodies were obtained after immunization of mice with these two proteins. The findings of this study pave the way for the development of viral detection kit, viral identification and assay of virus-receptor binding.

关 键 词：猴 诺如病毒基因组 P结构域 蛋白表达 抗原性 

分 类 号：R373[医药卫生—病原生物学]