摘要
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。
AIM： To express the nucleocapsid （N） protein of SARS coronavirus （SARS-CoV） in E. coil and construct its DNA vaccine. METHODS： The prokaryotic expression vector pQEN containing N gene was constructed and transformed into the E. coil. The recombinant N protein was then expressed and purified by Ni^2＋-NTA affinity resin. In addition, the N gene was cloned into the eukaryotic expression plasmid pSecTagB and the eukaryotic recombinant expression vector pSecN was obtained. The DNA vaccine pSecN was injected to immunize the BALB/c mice to produce the antiserum against N protein of SARS-CoV. Subsequently, the reactivity of the antiserum with recombinant N protein and SARS-CoV particles was assayed by ELISA. RESULTS： Recombinant N protein reacted strongly and specifically with the sera from immunized mice and SARS patients. Similarly, the sera of immunized mice could also react specifically with SARS-CoV particles. CONCLUSION： The recombinant N protein could be used as a good antigen to detect SARS. The DNA vaccine pSecN could also efficiently induce the production of IgG against N protein of SARS-CoV, which offered clues to the development of a potential DNA vaccine.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2005年第6期731-733,737,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
广州市荔湾区科技攻关项目(No.20032104056)
广州市科技攻关重大项目(No.2003Z1-E0011)
