Trop2病毒样颗粒的制备及纯化研究  

Expression and purification of Trop2 virus like particles

作　　者：王琳[1] 王希[1] 董轲[1] 张惠中[1] 

机构地区：[1]第四军医大学唐都医院临床实验与检验科,陕西西安710038

出　　处：《国际检验医学杂志》2016年第8期1020-1022,1025,共4页International Journal of Laboratory Medicine

基　　金：国家自然科学基金(81201775;81572974);陕西省自然科学基础研究计划(2013JZ010)

摘　　要：目的获得Trop2为靶点的病毒样颗粒(VLPs),为进一步体内诱导实验和疫苗研究提供依据。方法利用分子克隆技术构建Trop2真核表达载体pCAGGs/Trop2和杆状病毒表达载体pFastbac1/Trop2,以pCAGGs/Trop2表达载体转化Hela细胞,用免疫细胞化学方法确定Trop2蛋白表达并锚定于胞膜上;以pFastbac1/Trop2转化大肠杆菌E.coliDH10bac菌株获得重组杆粒Trop2Bacmid,转染Tn5昆虫细胞获得重组病毒Trop2rBV,与本室保存的Gag rBV共感染昆虫细胞得到重组VLPs,透析浓缩后经蔗糖密度梯度离心纯化、蛋白免疫印迹(WB)鉴定,电镜观察病毒形态。结果构建成功的重组杆粒Trop2Bacmid转染Tn5昆虫细胞获得重组病毒Trop2rBV,与Gag rBV共感染昆虫细胞获得重组Trop2VLPs。结论成功制备了Trop2VLPs,为其后续诱导体液和细胞免疫应答研究奠定了基础。Objective To gain the virus like particles（VLPs）based on Trop2 targets,which provided the basis for further in vivo induced experiments and anti-tumor vaccine studies.Methods Using molecular cloning method to constract eukaryotic expression vector of pCAGGs/Trop2 and baculovirus expression vector of pFastbac1/Trop2,expression product of pCAGGs/Trop2 in HeLa cells were intended to be to be anchored to the cell membrane by the methods of Immunohistochemistry;pFastbac1/Trop2 were transformed into E.coliDH10 bac isolates to gain recombinant bacmids,which were transfected into insect cells to express recombinant baculovirus with Gag rBV,then sucrose density gradient centrifugation,Western Blot and electron microscope were performed to purify and identify the Trop2 VLPs.Results Building recombinant bacmids which were transfected into insect cells to express recombinant baculovirus with Gag rBV,gained the recombinant Virus like Particles.Conclusion Trop2 VLPs was successfully prepared,which laid the foundation for the subsequent induction of humoral and cellular immune response.

关 键 词：Trop2 肿瘤疫苗 病毒样颗粒 

分 类 号：R392[医药卫生—免疫学]