人可溶性肿瘤坏死因子受体1基因原核表达及活性鉴定
人可溶性肿瘤病坏死因子受体1;原核表达;免疫印迹;细胞毒性作用;,傅蕾,彭仕芳,谭德明,刘洪波,傅蕾,通讯作者:,Cloning,prokaryoticexpressionandactivityidentificationofhumansol
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傅蕾, 彭仕芳, 谭德明, 刘洪波, 中南大学湘雅医院感染病科 湖南省长沙市 410008
傅蕾, 2005年中南大学医学博士, 主治医师, 主要从事肝功能衰竭临床与基础的研究.
湖南省自然科学基金资助项目, No. 05JJ30178
通讯作者: 谭德明, 410008, 湖南省长沙市湘雅路87号, 中南大学湘雅医院感染病科. dmt2008@yahoo.com.cn
电话: 0731-4327221 传真: 0731-4327281
收稿日期: 2007-01-10 接受日期: 2007-03-06
Cloning, prokaryotic expression and activity identification of human soluble tumor necrosis factor receptor 1 gene in Escherichia Coli JM109
Lei Fu, Shi-Fang Peng, De-Ming Tan, Hong-Bo Liu
Lei Fu, Shi-Fang Peng, De-Ming Tan, Hong-Bo Liu,Department of infectious Diseases, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
Supported bythe Natural Science Foundation of Hunan Province, No. 05JJ30178
Correspondence to:De-Ming Tan, Department of Infectious Diseases, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China. dmt2008@yahoo.com.cn
Received:2007-01-10 Accepted:2007-03-06
Abstract
AIM: To construct a prokaryotic expression plasmid of human soluble tumor necrosis factor receptor 1 (sTNFR1) gene, induce the expression of sTNFR1-maltose binding protein (MBP) fusion protein and investigate the bioactivity of expression products.
METHODS: The total RNA was extracted from HeLa cells and used as a template to amplify human sTNFR1 gene by reverse transcription-polymerase chain reaction (RT-PCR). The PCR products were cloned into T vector and sub-cloned into plasmid pMAL-c2x, a prokaryotic expression plasmid. The recombinant plasmid was transferred into Escherichia Coli JM109 and induced by isopropyl-β-D-thiogalactopyranoside (IPTG) to express fusion protein sTNFR1-MBP. sTNFR1-MBP was purified by amylose resin affinity chromatography and identified by sequencing and Western blot analysis. The bioactivity of sTNFR1-MBP was estimated by MTT assay.
RESULTS: A 558-bp fragment of human sTNFR1 gene had been amplified by RT-PCR and successfully cloned into vector pMAL-c2x as recombinant vector pMAL-c2x-sTNFR1, which was confirmed by DNA sequencing ......
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