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

Expression and purification of rabies virus glycoprotein and analysis of its specific binding capacity to memory B cells

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:

We aimed to express and purify three rabies virus glycoproteins with different tags and sizes. After analyzing their binding function, we wish to obtain a rabies virus glycoprotein with higher affinity and ability to specifically bind memory B cells. Experiments were carried out to express full length, as well as the ectodomain RVG by gene engineering method. Combined with the antibody of CD19 and CD27, the candidate protein labeling with fluorescence was used to analyze its binding function. Flow cytometry was used to detect the anti-rabies virus specific memory B cells in PBMCs, and confirm the binding ability between the candidate proteins and anti-rabies virus-specific memory B cells. We successfully constructed three expression vectors pGEX-5X-1-RVG, pET28a-RVG and pET30a-G. Three glycoproteins GST-RVG, His-RVG and His-G were obtained by optimized expression and purification conditions. The antigen specificity of purified GST-RVG, His-RVG and His-G were identified by Western blotting and ELISA. The affinity of these three purified glycoproteins to anti-rabies virus antibody were detected by competitive ELISA. Anti-rabies virus specific memory B cells in positive PBMCs gained from people who had ever been injected with the vaccine can be detected by flow cytometry. Thus, we got a recombinant rabies virus glycoprotein that had high-affinity and could sort antigen specific memory B cells.