【摘要】：表達(dá)純化不同標(biāo)簽、不同大小3個狂犬病病毒糖蛋白,分析其結(jié)合功能后,得到具備高親和力的、可特異性結(jié)合記憶性B細(xì)胞的狂犬病病毒糖蛋白。本實驗通過基因工程的方法,采用不同的原核表達(dá)系統(tǒng)分別表達(dá)帶有不同標(biāo)簽的、全長和膜外區(qū)的RVG,純化蛋白并分析比較其結(jié)合功能,熒光標(biāo)記候選蛋白,結(jié)合CD19及CD27的抗體,流式細(xì)胞術(shù)檢測狂犬疫苗免疫后PBMCs中抗狂犬病病毒特異性記憶性B細(xì)胞的情況,確認(rèn)候選蛋白與抗狂犬病毒特異性記憶性B細(xì)胞的結(jié)合功能。本實驗成功構(gòu)建了3個表達(dá)載體pGEX-5X-1-RVG、pET28a-RVG和pET30a-G,優(yōu)化表達(dá)純化條件成功獲得了糖蛋白GST-RVG、His-RVG和His-G。純化后的GST-RVG、His-RVG和His-G經(jīng)Western blotting和ELISA鑒定均有抗原特異性;由競爭ELISA法測得3個純化后糖蛋白與抗狂犬病病毒抗體的親和力。通過流式細(xì)胞術(shù)可以檢測到狂犬疫苗免疫后陽性志愿者PBMCs中的抗狂犬病病毒特異性記憶性B細(xì)胞,從而獲得了高親和力、可用于分選抗原特異性的記憶性B細(xì)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.
【作者單位】： 中國醫(yī)學(xué)科學(xué)院北京協(xié)和醫(yī)學(xué)院醫(yī)學(xué)生物學(xué)研究所云南省重大傳染病疫苗研發(fā)重點實驗室;
【基金】：中國醫(yī)學(xué)科學(xué)院醫(yī)學(xué)與健康科技創(chuàng)新工程重大協(xié)同創(chuàng)新項目(No.2016-I2M-1-019)資助~~
【分類號】：R373.9

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