狂犬病病毒糖蛋白表达及纯化及其记忆性B细胞结合能力的分析
发布时间：2018-09-04 17:41
【摘要】：表达纯化不同标签、不同大小3个狂犬病病毒糖蛋白,分析其结合功能后,得到具备高亲和力的、可特异性结合记忆性B细胞的狂犬病病毒糖蛋白。本实验通过基因工程的方法,采用不同的原核表达系统分别表达带有不同标签的、全长和膜外区的RVG,纯化蛋白并分析比较其结合功能,荧光标记候选蛋白,结合CD19及CD27的抗体,流式细胞术检测狂犬疫苗免疫后PBMCs中抗狂犬病病毒特异性记忆性B细胞的情况,确认候选蛋白与抗狂犬病毒特异性记忆性B细胞的结合功能。本实验成功构建了3个表达载体pGEX-5X-1-RVG、pET28a-RVG和pET30a-G,优化表达纯化条件成功获得了糖蛋白GST-RVG、His-RVG和His-G。纯化后的GST-RVG、His-RVG和His-G经Western blotting和ELISA鉴定均有抗原特异性;由竞争ELISA法测得3个纯化后糖蛋白与抗狂犬病病毒抗体的亲和力。通过流式细胞术可以检测到狂犬疫苗免疫后阳性志愿者PBMCs中的抗狂犬病病毒特异性记忆性B细胞,从而获得了高亲和力、可用于分选抗原特异性的记忆性B细胞的狂犬病病毒糖蛋白。
[Abstract]:Three rabies virus glycoproteins were expressed and purified with different labels and sizes. After analysis of their binding functions, rabies virus glycoproteins with high affinity and specific binding to memory B cells were obtained. In this study, different prokaryotic expression systems were used to express the purified RVG, proteins with different tags, full length and extracellular region by genetic engineering. The binding functions of the purified proteins were analyzed and compared. The candidate proteins were labeled with fluorescence, and the antibodies against CD19 and CD27 were combined. Flow cytometry was used to detect the specific memory B cells of rabies virus in PBMCs after rabies vaccine immunization, and to confirm the binding function of candidate proteins to specific memory B cells against rabies virus. In this experiment, three expression vectors, pGEX-5X-1-RVG,pET28a-RVG and pET30a-G, were successfully constructed, and the optimized expression and purification conditions were obtained. The glycoprotein GST-RVG,His-RVG and His-G. were obtained successfully. The purified GST-RVG,His-RVG and His-G were identified as antigen-specific by Western blotting and ELISA, and the affinity of three purified glycoproteins to anti-rabies virus antibody was determined by competitive ELISA method. Specific memory B cells against rabies virus were detected by flow cytometry in PBMCs of volunteers immunized with rabies vaccine, which resulted in high affinity. Rabies virus glycoprotein can be used to separate antigen specific memory B cells.
【作者单位】： 中国医学科学院北京协和医学院医学生物学研究所云南省重大传染病疫苗研发重点实验室;
【基金】：中国医学科学院医学与健康科技创新工程重大协同创新项目(No.2016-I2M-1-019)资助~~
【分类号】：R373.9
本文编号：2222880
[Abstract]:Three rabies virus glycoproteins were expressed and purified with different labels and sizes. After analysis of their binding functions, rabies virus glycoproteins with high affinity and specific binding to memory B cells were obtained. In this study, different prokaryotic expression systems were used to express the purified RVG, proteins with different tags, full length and extracellular region by genetic engineering. The binding functions of the purified proteins were analyzed and compared. The candidate proteins were labeled with fluorescence, and the antibodies against CD19 and CD27 were combined. Flow cytometry was used to detect the specific memory B cells of rabies virus in PBMCs after rabies vaccine immunization, and to confirm the binding function of candidate proteins to specific memory B cells against rabies virus. In this experiment, three expression vectors, pGEX-5X-1-RVG,pET28a-RVG and pET30a-G, were successfully constructed, and the optimized expression and purification conditions were obtained. The glycoprotein GST-RVG,His-RVG and His-G. were obtained successfully. The purified GST-RVG,His-RVG and His-G were identified as antigen-specific by Western blotting and ELISA, and the affinity of three purified glycoproteins to anti-rabies virus antibody was determined by competitive ELISA method. Specific memory B cells against rabies virus were detected by flow cytometry in PBMCs of volunteers immunized with rabies vaccine, which resulted in high affinity. Rabies virus glycoprotein can be used to separate antigen specific memory B cells.
【作者单位】： 中国医学科学院北京协和医学院医学生物学研究所云南省重大传染病疫苗研发重点实验室;
【基金】：中国医学科学院医学与健康科技创新工程重大协同创新项目(No.2016-I2M-1-019)资助~~
【分类号】：R373.9
【相似文献】
相关期刊论文 前4条
1 王兴兵,祖东;两类记忆性T细胞的表型、迁移途径及功能的关系[J];国外医学(免疫学分册);2001年05期
2 李卫中;记忆性T细胞的产生与维持[J];国外医学(免疫学分册);2004年06期
3 周钢;记忆性T细胞在移植免疫中的作用[J];国外医学(免疫学分册);2004年05期
4 ;探测EB病毒特异的记忆性T细胞新方法[J];中国肿瘤;1999年06期
相关硕士学位论文 前1条
1 赵德江;离子通道的记忆性对神经元激发参数的影响[D];湘潭大学;2010年
，本文编号：2222880
本文链接：https://www.wllwen.com/xiyixuelunwen/2222880.html
最近更新
教材专著
