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| 颗粒酶B的表达纯化和鉴定 | |
| 引用本文： | 李先茂,曾位森,张亚历,张振书,赖卓胜,刘晓晴.颗粒酶B的表达纯化和鉴定[J].第四军医大学学报,2002,23(17):1556-1558. |
| 作者姓名： | 李先茂 曾位森 张亚历 张振书 赖卓胜 刘晓晴 |
| 作者单位： | 1. 第一军医大学南方医院消化病研究所,广东广州,510515 2. 第一军医大学细胞生物教研室,广东广州,510515 3. 西藏军区直属门诊部,860000 |
| 基金项目： | 国家自然科学基金 (3 9870 72 3 )，广东省组织工程团队项目，南方医院院长基金 (10 72 0 ) |
| 摘 要： | 目的：克隆颗粒酶B(GraB)，构建GraB的原核表达载体，从而建立其原核表达体系，获得高效表达的重组GraB。方法：抽提人外周血淋巴细胞总RNA,进行RT-PCR克隆GraB cDNA，构建GraB原核表达载体，用限制性酶切和DNA测序进行鉴定；异丙基-1-硫代-β-呋喃半乳糖苷（IPTG）诱导表达，通过聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表达产物。表达产物亲和层析纯化并用免疫印迹法鉴定，以GraB底物测定其活性。结果：表达产物以可溶性分子形式存在于菌体中，具有良好的抗原性和特异性，并能作用于GraB特异性底物。结论：建立了GraB原核表达系统。 |
| 关 键 词： | 颗粒酶B 基因克隆 原核表达 鉴定 |
| 文章编号： | 1000-2790(2002)17-1556-03 |
| 修稿时间： | 2002年4月9日 |
Cloning and purification of human GraB in E.coli |
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| LI Xian Mao ,ZENG Wei Sheng ,ZHANG Ya Li ,ZHANG Zhen Shu ,LAI Zhuo Sheng ,LIU Xiao Qing Instititue of Gastroenterology,Nanfang Hospital.Cloning and purification of human GraB in E.coli[J].Journal of the Fourth Military Medical University,2002,23(17):1556-1558. | |
| Authors: | LI Xian Mao ZENG Wei Sheng ZHANG Ya Li ZHANG Zhen Shu LAI Zhuo Sheng LIU Xiao Qing Instititue of Gastroenterology Nanfang Hospital |
| Institution: | LI Xian Mao 1,ZENG Wei Sheng 2,ZHANG Ya Li 1,ZHANG Zhen Shu 1,LAI Zhuo Sheng 1,LIU Xiao Qing 3 1Instititue of Gastroenterology,Nanfang Hospital,2Department of Cellular Biology,First Military Medical University,Guangzhou 5 |
| Abstract: | AIM To develop an inducible system for expression of GraB in E.coli . METHODS GraB cDNA was amplified by RT PCR via extracting lymphocyte total RNA. After identification by DNA sequence analysis, the gene was inserted into E. coli expression vector pTrcHis2A. The prokaryo tic expression plasmid pTrcHis2A /granzyme B was constructed and transformed into Top10F. RESULTS After 8 h of IPTG induction, the granzyme B was expressed by 15% of total proteins by SDS PAGE. Western blot assay proved the expressed protein to be of good antigenicity and high specificity. The recombinant GraB purified by affinity chromatography has effect on GraB substrate. CONCLUSION The human granzyme B was expressived successfully in E.coli . |
| Keywords: | granzyme B gene cloning gene expression |
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