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目的:在大肠杆菌中表达SARS冠状病毒核衣壳N蛋白,并构建其DNA疫苗。方法:构建含N基因的原核表达载体pQEN,并在大肠杆菌M15中表达N蛋白。采用Ni2+亲和层析法纯化目的蛋白。将N基因克隆入真核表达载体pSecTagB中,构建真核重组质粒pSecN。以其免疫小鼠制备抗血清,并用ELISA法检测其与大肠杆菌中表达的重组N蛋白及天然全病毒N蛋白的反应性。结果:重组N蛋白能与DNA疫苗免疫的小鼠血清以及SARS患者血清发生特异性反应;SARS-CoV病毒颗粒也可与DNA疫苗免疫的小鼠血清发生特异性反应。结论:重组N蛋白保留了病毒的一些特异性抗原表位,可作为用ELISA法检测SARS-CoV的抗原。构建的DNA疫苗可在小鼠体内产生高效价的抗SARS病毒N蛋白的特异性抗体,从而为该疫苗的开发奠定了基础。
OBJECTIVE: To express SARS-CoV nucleocapsid N protein in Escherichia coli and construct its DNA vaccine. Methods: The prokaryotic expression vector pQEN containing N gene was constructed and N protein was expressed in Escherichia coli M15. The target protein was purified by Ni2 + affinity chromatography. The N gene was cloned into eukaryotic expression vector pSecTagB to construct eukaryotic recombinant plasmid pSecN. The antiserum was prepared from the immunized mice, and its reactivity with recombinant N protein expressed in E. coli and natural whole virus N protein was detected by ELISA. Results: The recombinant N protein reacted specifically with the serum of mice immunized with DNA vaccine and the serum of SARS patients. The SARS-CoV virus particles could also react specifically with the serum of mice immunized with DNA vaccine. Conclusion: The recombinant N protein retains some specific epitopes of the virus and can be used as an antigen to detect SARS-CoV by ELISA. The constructed DNA vaccine can produce high titer anti-SARS virus N protein-specific antibody in mice, which lays a foundation for the development of the vaccine.