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

Liwei Yan; Wei Gong; Wenbing Zhu; Xuemei Zhang; Jingwen XU; Zhongxiang WU; Kongjie LU; Ming Sun; Shaozhong Dong

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

Author: Liwei YAN ¹ ; Wei GONG ¹ ; Wenbing ZHU ¹ ; Xuemei ZHANG ¹ ; Jingwen XU ¹ ; Zhongxiang WU ¹ ; Kongjie LU ¹ ; Ming SUN ¹ ; Shaozhong DONG ¹
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1. Yunnan Provincial Key Laboratory for Development of Vaccines Against Major Infectious Diseases, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming 650118, Yunnan, China.
Publication Type:Journal Article
Keywords: antigen specific memory B cells; flow cytometry; rabies virus glycoprotein
From: Chinese Journal of Biotechnology 2017;33(11):1840-1849
CountryChina
Language:Chinese
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.