HIV-1 Tat基因重组原核表达载体的构建及融合蛋白的表达纯化与功能鉴定  

Construction of prokaryotic expression vector carrying HIV Tat gene and the expression,purification and identification of recombinant Tat in E. coli

作　　者：邱会平[1] 程晓东[2] 卢春[2] 

机构地区：[1]南京医科大学基础医学实验教学中心,江苏南京210029 [2]南京医科大学微生物学与免疫学系,江苏南京210029

出　　处：《江苏大学学报（医学版）》2013年第4期308-312,共5页Journal of Jiangsu University：Medicine Edition

摘　　要：目的:在大肠埃希菌中表达人免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)反式激活蛋白(trans activative transcription protein,Tat),为进一步研究可溶性Tat在艾滋病相关卡波氏肉瘤(AIDS-KS)致病过程中的作用提供材料。方法:以本实验室保存的重组质粒pcDNA3.1(+)/Tat101为模板,PCR扩增目的基因Tat。将PCR产物克隆至原核表达载体pET-28a(+)中,构建重组原核表达质粒pET-28a(+)-Tat。将重组质粒转化大肠埃希菌(E.coli)BL21(DE3)感受态细胞,用异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达融合蛋白。表达产物经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹鉴定后,通过镍亲和层析柱纯化。纯化后蛋白采用虫荧光素酶报告实验进行功能验证。结果:限制性内切酶酶切和基因测序证实,成功构建了含有Tat基因的重组原核表达质粒。蛋白质印迹结果显示,His融合蛋白在大肠埃希菌中得到正确表达,纯化后获得了相对分子质量为18×103的融合蛋白。虫荧光素酶报告实验证实,Tat与HIV-1长末端重复序列(long terminal repeat,LTR)具有结合能力。结论:重组质粒pET-28a(+)-Tat能在大肠埃希菌BL21(DE3)中稳定表达Tat蛋白,镍亲和层析柱纯化的His-Tat融合蛋白具有生物学功能。Objective： To construct the prokaryotic expression vector carrying HIV-1 Tat gene and to purify the fusion protein in E.coli.Methods： The DNA fragment of the Tat gene from pcDNA3.1（＋） / Tat101 was cloned into the prokaryotic expression vector pET-28a（＋）,named pET-28a（＋）-Tat.Then the recombinant vector was transformed into E.coli BL21（DE3）.The expression of the histidine-tagged（His-Tag） fusion protein was induced with isopropyl-β-D-thiogalactopyranoside（IPTG）.The fusion protein was detected and purified by Western blot and immobilized Ni2 ＋ absorption chromatographic column,respectively.Results： The prokaryotic expression vector carrying Tat gene was successfully constructed.The expression of the His-Tag fusion protein in E.coli BL21（DE3） was detectable.Finally,the fusion protein with the relative molecular weight of 18 × 103 was gained after purified using the affinity chromatographic column.Conclusion： Recombinant Tat gene can be expressed in E.coli BL21（DE3） and the fusion protein obtained can be functionally active.

关 键 词：TAT基因 原核表达 融合蛋白 人免疫缺陷病毒 卡波氏肉瘤病毒 

分 类 号：R34[医药卫生—基础医学] Q78[生物学—分子生物学]