SARS-CoV S蛋白的原核表达及其粘膜免疫效应  

Prokaryotic Expression of the SARS-CoV Spike Protein and Preliminary Studies on Its Mucosal Immune Response

作　　者：罗君毅[1] 盖伟伟[1] 雷磊[1] 涂浩波[1] 鄢慧民[1] 

机构地区：[1]武汉大学生命科学学院/病毒学国家重点实验室,湖北武汉430072

出　　处：《武汉大学学报（理学版）》2006年第6期733-738,共6页Journal of Wuhan University:Natural Science Edition

基　　金：湖北省科技攻关项目(2003AA305B03);武汉大学SARS科技攻关特别基金资助项目

摘　　要：SARS冠状病毒（SARS-CoV）的S（Spike）蛋白是病毒表面最主要的膜蛋白，在病毒的感染过程中起重要作用，是冠状病毒的主要抗原．以SARS冠状病毒的cDNA为模板，通过PCR得到S△1（151～1200bp）、S△2（1201～2250bp）和S△3（2251～3150bp）3段基因序列，克隆到表达载体pET-32a，在大肠杆菌BL21中诱导表达得到包涵体蛋白，经Western blot检测正确后，将此作为抗原以滴鼻和腹腔注射两种小同的途径免疫BALB／c雌性小鼠，3次免疫之后采集小鼠的血清和肺洗液，经酶联免疫吸附实验（enzyme—linked immunosorbent assay，ELISA）检测其中的抗体IgG和IgA．结果表明S蛋白通过滴鼻途径既能刺激产生一定的血清IgG，更能诱导肺粘膜产生特异IgA抗体．进一步比较分析发现，诱导粘膜免疫的效应区域主要位于S△2（401～750aa）蛋白区段．The spike （S） protein is a major membrane protein of the SARS-CoV, which plays an important role in receptor interaction, immune recognition and inducing neutralizing antibody. In this study, three nonoverlapping fragments （151- 1 200 bp, 1 201-2 250 bp, and 2 251-3 150 bp） of the S gene were amplified by PCR, cloned in pET- 32a and expressed in Escherichia coli. Recombinant proteins were confirmed by western blotting and were used as antigens for immunization of BALB/c mice. Immunizations were performed three times intranasally or intraperitoneally. Serum and lung extracts of immunized mice were collected and tested by enzyme-linked immunosorbent assays （ELISA） for S protein specific IgG or IgA antibodies. The results indicated that the S protein could stimulate IgA antibodies in the lung mucosal tissue as well as IgG antibodies in the serum by intranasal immunization. Further more, we found the predominant antigenic regions of the S protein for mucosal immune response mainly located in amino acid residues from 401 to 750.

关 键 词：SARS-COV S蛋白 粘膜免疫效应 滴鼻免疫 

分 类 号：Q93[生物学—微生物学]