SARS-CoV S蛋白的原核表达及其粘膜免疫效应

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．

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

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.