Cloning and Expression of Nucleocapsid Protein Gene of SARS Associated Coronavirus

ZHU Han—ping; LU Qun—ying; LU Yi—yu; YAO Ping—ping; XU Fang; GE Qong; WENG Jing—qing; YAN Ju—ying; GONG Li—ming; SHI W en; ZHAO Zhi—ya; ZHU Zhi—yong

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October 2003

ZHU Han—ping, LU Qun—ying, LU Yi—yu, YAO Ping—ping, XU Fang, GE Qong, WENG Jing—qing, YAN Ju—ying, GONG Li—ming, SHI W en, ZHAO Zhi—ya and ZHU Zhi—yong. Cloning and Expression of Nucleocapsid Protein Gene of SARS Associated Coronavirus[J]. Virologica Sinica, 2003, 18(5): 451-453.

Citation: ZHU Han—ping, LU Qun—ying, LU Yi—yu, YAO Ping—ping, XU Fang, GE Qong, WENG Jing—qing, YAN Ju—ying, GONG Li—ming, SHI W en, ZHAO Zhi—ya, ZHU Zhi—yong. Cloning and Expression of Nucleocapsid Protein Gene of SARS Associated Coronavirus .VIROLOGICA SINICA, 2003, 18(5) : 451-453.

SARS病毒核蛋白基因的克隆及其表达研究

1.
浙江省疾病预防控制中心病毒研究所，浙江杭州，310009

摘要

摘要：从一例输入性传染性非典型性肺炎病人血清中提取病毒RNA，通过RT-PCR方法扩增出SARS病毒核蛋白基因片段，克隆入质粒载体pUCm—T后，进行核苷酸序列的测定及分析，与已公布的SARS病毒基因序列进行比较，证实为SARS冠状病毒核蛋白基因。为了解该病毒核蛋白的抗原特性，将核蛋白基因插入表达载体，构建重组质粒pET28a—SN，转导大肠杆菌BL21(DE3)后，加IPTG诱导表达。产物经SDS—PAGE电泳分析，表达出相对分子量约为50kDa的蛋白，占整个菌体的45％左右。Western—blot分析表明，表达产物仅与SARS阳性病人血清起反应，而与正常血清不起反应。间接ELISA免疫检测，抗原滴度达l：12500。表明表达的核蛋白为SARS特异性抗原，这为SARS病毒的诊断试剂的研制提供了方便而安全的抗原来源。
- SARS病毒(SARS—associated coronavirus
- , SARS—CoV)
- , 核蛋白基因：序列分析
- , 表达：抗原性

Cloning and Expression of Nucleocapsid Protein Gene of SARS Associated Coronavirus

1.
Institutefor Virus Disease Research，Zhefiang Centerfor Disease Prevention and Control,Hangzhou 310009，China

Abstract

Abstract：Nucleocapsid gene of SARS·-associated coronavirus(SARS·-CoV)was obtained by reverse tran scription and polymerase chain reaction from a pateint sufered from severe acute respiratory syndrome(SARS)from Beijing，subsequently cloned into pUCm—T vector．The sequence of positive recombinants was determined by the method of dideoxy chain termination，which revealed that the nucleocapsid gene segment is 1 269 nucleotide in length with an open reading frame encoding a protein of 422 am ino acids．Nucleocapsid gene was subcloned into the prokaryotic vector pET28a．Th e recombinant plasmid pET28a—SN was transformed into host cell BL21(DE3)．After inducing by IPTG~ about 50kD protein was expressed and the expression level was about 45％．W estern—blot an alysis demonstrated that the recombinant proteins reacted with SARS positive sera but not with norm al sera tested． SARS nucleocapsid protein expressed by the E．coli system ofer a safe source of specific antigen for diagnostic purposes．
- Key word：SARS-associated coronavirus(SARS-CoV)
- , Nucleocapsid gene
- , Sequence analysis
- , Protein expression

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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

Cloning and Expression of Nucleocapsid Protein Gene of SARS Associated Coronavirus

1. Institutefor Virus Disease Research，Zhefiang Centerfor Disease Prevention and Control,Hangzhou 310009，China

Abstract: Abstract：Nucleocapsid gene of SARS·-associated coronavirus(SARS·-CoV)was obtained by reverse tran scription and polymerase chain reaction from a pateint sufered from severe acute respiratory syndrome(SARS)from Beijing，subsequently cloned into pUCm—T vector．The sequence of positive recombinants was determined by the method of dideoxy chain termination，which revealed that the nucleocapsid gene segment is 1 269 nucleotide in length with an open reading frame encoding a protein of 422 am ino acids．Nucleocapsid gene was subcloned into the prokaryotic vector pET28a．Th e recombinant plasmid pET28a—SN was transformed into host cell BL21(DE3)．After inducing by IPTG~ about 50kD protein was expressed and the expression level was about 45％．W estern—blot an alysis demonstrated that the recombinant proteins reacted with SARS positive sera but not with norm al sera tested． SARS nucleocapsid protein expressed by the E．coli system ofer a safe source of specific antigen for diagnostic purposes．