小鼠细小病毒VP2蛋白在昆虫细胞-杆状病毒系统中的表达和免疫原性分析  

Expression and Immunogenicity Analysis of VP2 Protein of Minute Virus of Mice in Insect Cell-baculovirus Expression System

作　　者：马畅 赵迎峰 陈莉 刘彪 赵志刚 恽时锋 MA Chang;ZHAO Yingfeng;CHEN Li;LIU Biao;ZHAO Zhigang;YUN Shifeng(Department of Laboratory Animal,Chinese PLA General Hospital of Eastern Theater Command,Army Education Base of Science and Ethics of Experimental Animal,National Sciene Education Base,Nanjing 210002,China;Department of No.3 Stationed Out-patient,Chinese PLA General Hospital of Eastern Theater Command,Nanjing 210002,China)

机构地区：[1]中国人民解放军东部战区总医院医疗保障中心实验动物室,全军实验动物科普与伦理教育基地,全国科普教育基地,南京210002 [2]中国人民解放军东部战区总医院第三派驻门诊部,南京210002

出　　处：《实验动物科学》2023年第4期28-33,共6页Laboratory Animal Science

基　　金：2019年度军队实验动物专项科研课题(SYDW-2018-11);2021年度东部战区总医院青年课题(YYQN2021099)。

摘　　要：目的利用杆状病毒表达系统合成重组小鼠细小病毒(MVM)VP2蛋白,并对其免疫原性分析。方法将优化后的MVM VP2序列克隆至表达载体pFastBac1,命名为pFastBac1-MVM VP2,再将其转化至DH10Bac感受态大肠杆菌中形成重组杆粒,提取重组杆粒经PCR鉴定正确后转染昆虫细胞Sf9,镜下观察到细胞明显病变后收获重组杆状病毒rBac-MVM VP2。Sf9细胞接重组病毒48和72 h后,用间接免疫荧光(IFA)和蛋白免疫印迹(Western blot)法验证重组蛋白的免疫原性。结果构建的rBac-MVM VP2重组杆状病毒感染Sf9细胞后,可成功表达MVM VP2重组蛋白,经Western blot和IFA检测分析,重组蛋白具有较好的免疫原性且在病毒感染72 h时表达量较高。经蛋白纯化后可得到纯度较高的VP2重组蛋白。结论本研究利用昆虫细胞-杆状病毒系统成功表达MVM VP2蛋白并具有良好的免疫原性,为VP2相关功能的研究和临床诊断方法的建立奠定基础。Objective Recombinant MVM VP2 protein was synthesized by baculovirus expression system and the immunogenicity of the recombinant VP2 protein was analyzed.Method The optimized MVM VP2 gene sequence was cloned into the eukaryotic expression vector pFastBac1,then the recombinant vector pFastBac1-MVM VP2 was transformed into DH10Bac susceptible E.coli to form recombinant plasmid.The recombinant plasmid were transfected into insect cells Sf9.Recombinant baculovirus rBac-MVM VP2 was harvested after significant cellular lesions were observed.Sf9 cells were inoculated with recombinant virus rBac-MVM VP2 for 48 and 72 h.Immunogenicity of the recombinant protein was tested by IFA and Western blot.Result Recombinant baculovirus rBac-MVM VP2 could successfully express recombinant MVM VP2 protein after infecting Sf9 cells.Western blot and IFA analysis showed that the recombinant protein had good immunogenicity and high expression at 72 h of virus infection.After protein purification,a high purity recombinant protein VP2 was obtained.Conclusion In this study,MVM VP2 protein was successfully expressed by insect cell-baculovirus system and the recombinant VP2 protein has good immunogenicity,which lays a foundation for the study of VP2-related functions and the establishment of clinical diagnosis method.

关 键 词：小鼠细小病毒 VP2蛋白 杆状病毒表达 

分 类 号：Q95-3[生物学—动物学]