Expression and Immunogenicity Analysis of the Receptor-binding Region of SARS-CoV Spike  Protein

MA Cui-qin; ZHOU Yu-sen; WANG Yi-jia; GUO Yan; GUAN Jie; WEI Lin; HE Yu-xian

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October 2005

MA Cui-qin, ZHOU Yu-sen, WANG Yi-jia, GUO Yan, GUAN Jie, WEI Lin and HE Yu-xian. Expression and Immunogenicity Analysis of the Receptor-binding Region of SARS-CoV Spike Protein[J]. Virologica Sinica, 2005, 20(5): 472-475.

Citation: MA Cui-qin, ZHOU Yu-sen, WANG Yi-jia, GUO Yan, GUAN Jie, WEI Lin, HE Yu-xian. Expression and Immunogenicity Analysis of the Receptor-binding Region of SARS-CoV Spike Protein .VIROLOGICA SINICA, 2005, 20(5) : 472-475.

SARS-CoV S蛋白受体结合区的重组表达及免疫原性分析

马翠卿 ^1,2 ,
周育森 ^1* ,
王弋嘉 ¹ ,
郭彦 ¹ ,
管洁 ¹ ,
魏林 ² ,
何玉先 ¹

1.
1.军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室北京 2.河北医科大学免疫学教研室河北石家庄

摘要

为了表达SARS-CoV的S蛋白的受体结合区并对其免疫原性进行分析,用PCR方法扩增S蛋白的受体结合区基因片段,克隆至原核表达质粒pET-32a+并在大肠杆菌中表达,应用Western-blot鉴定表达的目的蛋白,而后以该蛋白作为诊断抗原包被酶联板来检测20份SARS病人血清和28份健康人血清,结果原核表达的S蛋白能够和所用的SARS病人血清反应。这提示表达的S重组蛋白具有良好的抗原性。将变性纯化的重组蛋白和复性蛋白分别皮下免疫小鼠,第三次免疫一周后收集抗血清,用ELISA测定抗体和同时测定中和抗体活性。用变性的抗原免疫的小鼠血清均无中和活性;而用复性的蛋白免疫的小鼠产生了中和抗体。实验表明,S蛋白受体结合区无线性中和表位,中和抗体的产生是由构象表位诱导的。提示该蛋白有可能应用于亚单位疫苗的研究。
- 严重急性呼吸窘迫综合征病毒（SARS-CoV）
- , 刺突蛋白（S蛋白）
- , 受体结合区

Expression and Immunogenicity Analysis of the Receptor-binding Region of SARS-CoV Spike Protein

MA Cui-qin ^1,2 ,
ZHOU Yu-sen ^1* ,
WANG Yi-jia ¹ ,
GUO Yan ¹ ,
GUAN Jie ¹ ,
WEI Lin ² ,
HE Yu-xian ¹

Abstract

In order to express the recombinant protein of SARS-CoV S receptor-binding region and to study its immunogenicity,the gene encoding SARS-CoV S receptor-binding region was cloned to pET32a+ and expressed in E.coli.The expression of the recombinant protein was confirmed by Western blot analysis.The S purified protein as diagnostic antigen coated the microtiter plates to detect 20 SARS sera and 28 healthy sera.The results showed that the S purified recombinant protein was able to react with all sera of SARS patients.BALB/c mice were immunized with the denatured and refolding protein respectively.Sera were collected after the third immunization for detection of neutralizing activity against SARS virus.The denatured recombinant S receptor-binding region protein could not induce neutralizing antibodies,whereas refolding recombinant S-RBD could elicit neutralizing antibodies.These results indicated that the receptor-binding region of the S protein could be used for vaccine development.
- Severe acute respiratory syndrome coronavirus（SARS-CoV）
- , Spike protein（S protein）
- , Receptor-binding region

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Expression and Immunogenicity Analysis of the Receptor-binding Region of SARS-CoV Spike Protein

Abstract: In order to express the recombinant protein of SARS-CoV S receptor-binding region and to study its immunogenicity,the gene encoding SARS-CoV S receptor-binding region was cloned to pET32a+ and expressed in E.coli.The expression of the recombinant protein was confirmed by Western blot analysis.The S purified protein as diagnostic antigen coated the microtiter plates to detect 20 SARS sera and 28 healthy sera.The results showed that the S purified recombinant protein was able to react with all sera of SARS patients.BALB/c mice were immunized with the denatured and refolding protein respectively.Sera were collected after the third immunization for detection of neutralizing activity against SARS virus.The denatured recombinant S receptor-binding region protein could not induce neutralizing antibodies,whereas refolding recombinant S-RBD could elicit neutralizing antibodies.These results indicated that the receptor-binding region of the S protein could be used for vaccine development.