论文部分内容阅读
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。
Expression and purification of different tags, different sizes of three rabies virus glycoprotein, analysis of its binding function, with high affinity, can specifically bind to memory B cells of rabies virus glycoprotein. In this experiment, different prokaryotic expression systems were used to express RVG with different tags, full-length and extracellular regions, respectively. The purified proteins were analyzed and their binding functions were compared. Fluorescently labeled candidate proteins, CD19 and CD27 Antibodies and flow cytometry were used to detect anti-rabies virus-specific memory B cells in PBMCs after rabies vaccine immunization to confirm the binding function between candidate proteins and anti-rabies virus-specific memory B cells. Three expression vectors pGEX-5X-1-RVG, pET28a-RVG and pET30a-G were successfully constructed in this study. The glycoproteins GST-RVG, His-RVG and His-G were successfully obtained by optimizing the expression and purification conditions. The purified GST-RVG, His-RVG and His-G were identified by Western blotting and ELISA respectively. The affinity of the three purified glycoproteins to anti-rabies virus was measured by competitive ELISA. Rabies virus-specific memory B cells in PBMCs of rabies vaccinated post-rabies vaccine-immunized rabbits can be detected by flow cytometry to obtain high affinity rabies virus glycoproteins that can be used to sort antigen-specific memory B cells protein.