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人CD154-GST融合蛋白基因在大肠杆菌中的表达
引用本文:张春艳,李树浓,宁波,张志方,曹开源. 人CD154-GST融合蛋白基因在大肠杆菌中的表达[J]. 中国病理生理杂志, 2000, 16(8): 673-677
作者姓名:张春艳  李树浓  宁波  张志方  曹开源
作者单位:1. 中山医科大学免疫学教研室, 广东 广州 510089;
2. 中山医科大学病理生理教研室, 广东 广州 510089;
3. 中山医科大学生理教研室, 广东 广州 510089
摘    要:目的:为制备重组人CD154-谷胱甘肽巯基转移酶(GST)融合蛋白(hCD154-GST),用于人CD154单克隆抗体研制。方法:根据人CD154基因序列设计合成特异性引物,RT-PCR扩增人CD154基因,并插入融合蛋白原核表达载体pGEX-4T-1中,得到重组表达质粒pGEX-4T-1/CD154;用此重组质粒转化大肠杆菌BL21细胞,转化菌落经BamHⅠ、EcoRⅠ酶切鉴定。IPTG诱导大肠杆菌表达人CD154蛋白,SDS-PAGE电泳鉴定表达产物。结果:从人外周血淋巴细胞扩增出820bp的hCD154cDNA;将其克隆至pGEX-4T-1质粒中,经双酶切鉴定及DNA序列分析证实含有目的基因;IPTG诱导后的大肠杆菌经SDS-PAGE电泳鉴定出现明显的55kD蛋白带。结论:成功构建了人CD154-GST原核表达质粒,并在大肠杆菌中表达出人CD154-GST融合蛋白,为人CD154单克隆抗体的研制及进一步抗排斥反应的研究打下了基础。

关 键 词:人CD154  融合蛋白  基因表达  克隆   分子  
收稿时间:1999-12-27

Cloning of hCD154 gene from human activated peripheral blood mononuclear cell and expression of hCD154-GST fusion protein in prokaryote
ZHANG Chun-yan,LI Shu-nong,NING Bo,ZHANG Zhi-fang,CAO Kai-yuan. Cloning of hCD154 gene from human activated peripheral blood mononuclear cell and expression of hCD154-GST fusion protein in prokaryote[J]. Chinese Journal of Pathophysiology, 2000, 16(8): 673-677
Authors:ZHANG Chun-yan  LI Shu-nong  NING Bo  ZHANG Zhi-fang  CAO Kai-yuan
Affiliation:1. Department of Immunology, Sun Yat-sen University of Medical Sciences, Guangzhou 510089, China;
2. Department of Pathphysiology, Sun Yat-sen University of Medical Sciences, Guangzhou 510089, China;
3. Department of Physiology, Sun Yat-sen University of Medical Sciences, Guangzhou 510089, China
Abstract:AIM:To express recombinant hCD154-GST fusion protein, to prepare anti-hCD154 monoclonal antibody, and to investigate the effect of anti-hCD154 monoclonal antibody on graft rejection. METHODS AND RESULTS: Total RNA was prepared from human peripheral blood mononuclear cell (PBMC) activated with 10ng/mL PMA and 1 μg/mL PHA for 8h, the total RNA was reversetranscribed to cDNA. The entire coding region and a part of the 3'non-coding regions were amplified by PCR using a pair of primers designed and synthesized according to the sequence of human CD154 gene from gene bank. The amplified product, a 820bp DNA fragment was cloned into pGEX-4T-1 plasmid expressing glutathione S-transferase(GST). The cloned insert was identified by double digestion of the cloned pGEX-4T-1 plasmid with retriction enzymes BamHⅠand EcoRⅠ.The fusion protein expression plasmid of PGEX-4T-1/hCD154 was constructed, then transformed to E coli BL21. The human CD154-GST fusion protein expression was induced by IPTG in BL21. The expression of recombinant 26kD GST and 55kD human CD154-GST fusion protein were confirmed by SDS-PAGE. CONCLUSION: We have express the recombinant human CD154-GST fusion protein. The expressed hCD154-GST fusion protein will be used to prepare anti-hCD154 monoclonal antibody, to investigate the role of anti-CD154 monoclonal antibody on graft rejection.
Keywords:Human CD154  Fusion protein  Gene expression  Cloning   molecular
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