SARS病毒S蛋白的原核表达与DNA疫苗的构建

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—CoV S蛋白抗血清。然后用纯化的重组蛋白rSa和rSb建立的SARS—CoV S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—CoV S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SARS DNA疫苗的研制提供一定的借鉴作用。

Prokaryotic expression of SARS coronavirus spike protein and construction of its DNA vaccine]

OBJECTIVE: To study the immunological characteristics of the spike (S) protein of SARS coronavirus (SARS-CoV) and analyze the feasibility of using this protein as the component for SARS vaccine development. METHODS: The two truncated fragments of S gene were separately cloned into the prokaryotic expression vector pET-15b and expressed in E.coli. The resulting recombinant proteins, rS(a) and rS(b), were purified by affinity chromatography. The full-length S gene was cloned into the eukaryotic expression plasmid pSecTagB to prepare recombinant plasmid pSecS as the DNA vaccine to immunize BALB/c mice for inducing the secretion of anti-SARS-CoV protein. The immunological effect of anti-SARS-CoV antibody was tested with purified rS(a) and rS(b) proteins by enzyme-linked immunosorbent assay (ELISA). RESULTS: Both the truncated recombinant proteins were expressed in soluble forms and reacted specifically with the sera from immunized pSecS mice and clinically diagnosed SARS patients. The prokaryotically expressed recombinant truncated S protein had similar antigenicity with SARS-CoV S protein. CONCLUSION: The recombinant protein could be used as an antigen for detecting the serum of SARS CoV-infected patients. The SARS-CoV S gene vaccine could induce the production of specific antibody, which offers clues for the research of SARS DNA vaccine.