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目的探讨SARS冠状病毒的刺突(spike,S)蛋白的免疫学特性,以及S蛋白作为SARS-CoV病毒疫苗组分的可行性。方法将S蛋白基因分段克隆入原核表达载体pET-15b,并在大肠杆菌中表达,经过亲和层析得到纯化的重组蛋白rSa和rSb;将全长S基因克隆入真核分泌表达载体pSecTagB,得到重组DNA疫苗pSecS,免疫小鼠,得到SAS-CoVS蛋白抗血清。然后用纯化的重组蛋白rSa和rSb建立的SARS-CoVS抗体ELISA检测技术研究所构建的S-DNA疫苗的免疫效果。结果分段的重组蛋白rSa和rSb在大肠杆菌中均以可溶性形式得到高效表达,并能与SARS确诊病人血清以及pSecS免疫鼠血清发生特异性抗原抗体反应,原核表达的重组分段S蛋白具有SARS-CoVS蛋白相似的抗原性。结论原核表达的两段重组S蛋白有可能作为抗原组分用于临床SARS-CoV检测中;所构建的SARS-CoV的S基因核酸疫苗能在小鼠体内产生特异性抗体,这为进一步SARSDNA疫苗的研制提供一定的借鉴作用。
Objective To investigate the immunological characteristics of spike protein (SSP) of SARS coronavirus and the feasibility of using S protein as vaccine component of SARS-CoV virus. Methods The S protein gene was cloned into the prokaryotic expression vector pET-15b by fragment and expressed in E. coli. The purified recombinant proteins rSa and rSb were obtained by affinity chromatography. The full-length S gene was cloned into eukaryotic expression vector pSecTagB , Get recombinant DNA vaccine pSecS, mice were immunized to obtain SAS-CoVS protein antiserum. The immune effect of the constructed S-DNA vaccine was then investigated using the ELISA technique of SARS-CoVS established by the purified recombinant proteins rSa and rSb. Results The segmented recombinant proteins rSa and rSb were efficiently expressed in soluble form in Escherichia coli and could react with the serum of patients with SARS and the serum of pSecS immunized mice to produce specific antigen and antibody. The expressed prokaryotic recombinant protein S has SARS -CoVS protein similar antigenicity. CONCLUSION: Two recombinant S protein expressed in prokaryotic cells may be used as an antigen component in the clinical SARS-CoV detection. The constructed S gene nucleic acid vaccine against SARS-CoV can generate specific antibodies in mice, which is a potential vaccine for further SARS DNA vaccine Provide some reference for the development of.