HIV-1基质蛋白P17的原核表达与纯化  

Overexpression of HIV-1 P17 protein in Escherichia coli and its purification

作　　者：李钟洙[1] 金宁一[2] 王宏伟[2] 郭志儒[2] 李萍[2] 杨翰仪[3] 殷震[2] 

机构地区：[1]吉林大学第二医院皮肤科,吉林 长春 130041 [2]解放军农牧大学基因工程实验室 [3]吉林大学基础医学院

出　　处：《白求恩医科大学学报》2001年第1期19-21,共3页Journal of Norman Bethune University of Medical Science

摘　　要：目的 :HIV- 1p17基因在大肠杆菌中高效表达并纯化目的蛋白。方法 :1PCR法扩增的 HIV-1p17基因片段克隆至 p ET2 8c质粒 ;2重组质粒分别在大肠杆菌 BL 2 1、BL 2 1(DE3)、BL 2 1(DE3) plys S及 HMS174 (DE3)中表达 ;3用 Ni- NTA金属树脂层析法纯化目的蛋白 ;4用酶联免疫法和免疫印迹法检测纯化蛋白的特异性。结果 :重组质粒在 BL (DE3)中表达量最高 ,目的蛋白表达量占全菌体裂解物的32 % ,纯化后其纯度达 95% ,纯化蛋白可与 p17Mc Ab和 HIV- 1阳性血清发生特异反应。结论 :带有HIV- 1p17片段的重组质粒 p ET2 8c在大肠杆菌 BL2 1(DE3)中获得高效表达 ,蛋白表达量可满足其免疫、生物活性的进一步研究 ;用 Ni- NTA金属树脂层析法纯化蛋白 ,方法简便 ,蛋白丢失少 ,纯度高 ;纯化的重组蛋白 HIV- 1p17可用来临床早期诊断 HIV- 1感染者 ,同时可预测患者的临床进程。? Objective:To highly express HIV 1p17 in E.coli and purify the protein.Methods:①Recombinant plasmid was constructed by inserting HIV 1p17 gene amplified by PCR into plasmid vector,pET28c;②The recombinant plasmid was expressed in BL21,BL21(DE3),BL21(DE3) plysS and HMS174(DE3) of E.coli separately;③The target protein were purified with Ni NTA resin;④The purified protein was detected by western blot and ELISA.Results:The expression of the P17 protein in BL21(DE3) represented up to 32% of total protein in E.coli ,which was the most amounts compared with other kinds of E.coli .The purity of the purified protein reached 95%.The purified protein was recognized by HIV 1P17McAb as well as by HIV 1 positive serum.Conclusion:The recombinant plasmid is highly expressed in BL21(DE3) of E.coli that can be proceeded to the immunocompetence and the bioactivity research.The method of Ni NTA resin is simple with low protein losing and high purity.And the purified p17 can be employed in early detection of HIV 1 infection and prediction of the clinical progression. 〔

关 键 词：基因表达 纯化 HIV-1 p17蛋白 酶联免疫法 免疫印迹法 

分 类 号：R373[医药卫生—病原生物学] R394[医药卫生—基础医学]