南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (01): 33-36.

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SARS病毒S蛋白的原核表达与DNA疫苗的构建

李建娜1, 向开军2, 周荣2,3, 黄春华2, 丁勇强2, 曾其毅3, 钟秋平1   

  1. 1. 华南热带农业大学, 海南儋州571737;
    2. 广州华银基因科技有限公司, 广东广州510150;
    3. 广州市儿童医院, 广东广州510120
  • 出版日期:2005-01-20 发布日期:2005-01-20
  • 基金资助:
    收稿日期:2004-9-14。
    基金项目:广州市科技攻关重大项目(2003Z1-10011);广州市荔湾区科技攻关项目(20032104056)
    作者简介:李建娜(1979- ),女,在读硕士,研究方向为应用微生物,E-mail:christina0125@163.com
    通讯作者:钟秋平,华南热带农业大学工学院,E-mail:qiupingzhong@scuta.edu.cn,电话:0898-23309279

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

LI Jian-na1, XIANG Kai-jun2, ZHOU Rong2,3, HUANG Chun-hua2, DING Yong-qiang2, ZENG Qi-yi3, ZHONG Qiu-ping1   

  1. 1. 华南热带农业大学, 海南儋州571737;
    2. 广州华银基因科技有限公司, 广东广州510150;
    3. 广州市儿童医院, 广东广州510120
  • Online:2005-01-20 Published:2005-01-20

摘要: 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。

Abstract: 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, rSa and rSb, 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 rSa and rSb 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.

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