Purification of Glutathione S-transferase Fusion Proteins of SARS Coronavirus Spike Protein fragment 2 and Preparation of Anti-GST-S2 Polyclonal Antibody

QIN Li; WU Shao-ting; WANG Xi-ming; YUAN Shi-shan; HUANG Da-na; LEI Min-jun; PAN Hui-rong; GAO Shi-tong; ZHANG Ren-li; QU Shen

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June 2005

QIN Li, WU Shao-ting, WANG Xi-ming, YUAN Shi-shan, HUANG Da-na, LEI Min-jun, PAN Hui-rong, GAO Shi-tong, ZHANG Ren-li and QU Shen. Purification of Glutathione S-transferase Fusion Proteins of SARS Coronavirus Spike Protein fragment 2 and Preparation of Anti-GST-S2 Polyclonal Antibody[J]. Virologica Sinica, 2005, 20(3): 217-220.

Citation: QIN Li, WU Shao-ting, WANG Xi-ming, YUAN Shi-shan, HUANG Da-na, LEI Min-jun, PAN Hui-rong, GAO Shi-tong, ZHANG Ren-li, QU Shen. Purification of Glutathione S-transferase Fusion Proteins of SARS Coronavirus Spike Protein fragment 2 and Preparation of Anti-GST-S2 Polyclonal Antibody .VIROLOGICA SINICA, 2005, 20(3) : 217-220.

SARS冠状病毒S蛋白片段2的表达纯化与多克隆抗体的制备

秦莉 ¹ ,
吴少庭 ^2* ,
王西明 ¹ ,
袁仕善 ² ,
黄达娜 ² ,
雷民军 ¹ ,
潘晖榕 ¹ ,
高世同 ² ,
张仁利 ² ,
屈伸 ¹

1.
1.华中科技大学同济医学院生物化学与分子生物学系湖北武汉 2.深圳市疾病预防控制中心分子生物室广东深圳

摘要

采用RTPCR技术从SARS冠状病毒基因组扩增编码S蛋白的S2基因片段(第2170到2814位碱基),克隆到pMD18T载体并测序。用限制性内切酶消化后,S2基因亚克隆至表达载体pGEX4T2,转化大肠杆菌JM109,筛选鉴定阳性菌落。扩增培养含pGEXS2质粒的JM109大肠杆菌,经IPTG诱导,超声破菌,GSHSepharose亲和层析纯化目的蛋白,Westernblot检测SARS患者血清可以识别纯化的蛋白。用此蛋白免疫NIH小鼠,获得了高滴度抗GSTS2抗体的血清,为进一步研究SARS冠状病毒的亚单位疫苗奠定基础。
- SARS
- , 冠状病毒
- , S蛋白
- , 亲和层析
- , 多克隆抗体

Purification of Glutathione S-transferase Fusion Proteins of SARS Coronavirus Spike Protein fragment 2 and Preparation of Anti-GST-S2 Polyclonal Antibody

Abstract

To study the immunogenicity of spike protein of SARS coronavirus, an expressional plasmid was constructed encoding partial sequence of SARS Coronavirus spike protein from 2170 bp to 2814bp, named fragment-2(S2). The gene sequence encoding S2 was amplified by RT-PCR from SARS Coronavirus genome RNA, cloned to pMD18-T vector and subcloned to pGEX-4T-2. E.coli JM109 that contained recombinant expression vector pGEX-S2 was amplified and fusion protein expression was induced by 1mmol/L IPTG at 37℃. Protein was extracted and purified by GSH-Sepharose affinity chromatography. After identified by the serum of SARS patient by Western-blot, the purified protein GST-S2 was used to immunize NIH mice three times at two weeks interval, the immunized mice produced high titer anti-GST-S2 polyclonal antibody. It lies a basis for the future study of subunit vaccine of SARS coronavirus.
- Severe acute respiratory syndrome（SARS）
- , Coronavirus
- , Spike protein
- , Affinity chromatography
- , Polyclonal antibody

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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

Purification of Glutathione S-transferase Fusion Proteins of SARS Coronavirus Spike Protein fragment 2 and Preparation of Anti-GST-S2 Polyclonal Antibody

Abstract: To study the immunogenicity of spike protein of SARS coronavirus, an expressional plasmid was constructed encoding partial sequence of SARS Coronavirus spike protein from 2170 bp to 2814bp, named fragment-2(S2). The gene sequence encoding S2 was amplified by RT-PCR from SARS Coronavirus genome RNA, cloned to pMD18-T vector and subcloned to pGEX-4T-2. E.coli JM109 that contained recombinant expression vector pGEX-S2 was amplified and fusion protein expression was induced by 1mmol/L IPTG at 37℃. Protein was extracted and purified by GSH-Sepharose affinity chromatography. After identified by the serum of SARS patient by Western-blot, the purified protein GST-S2 was used to immunize NIH mice three times at two weeks interval, the immunized mice produced high titer anti-GST-S2 polyclonal antibody. It lies a basis for the future study of subunit vaccine of SARS coronavirus.