重组分泌型人类免疫缺陷病毒Tat蛋白在真核细胞中的表达及活性检测

摘要

目的：构建人类免疫缺陷病毒病毒 tat基因的重组分泌型真核表达载体并在真核细胞中表达，检测表达的重组蛋白活性。方法聚合酶链反应（PCR）从重组质粒pcDNA3．1（＋）/Tat中扩增tat基因，利用 HindⅢ和Bam HⅠ酶切位点分别将tat基因正向、反向插入分泌型载体pSecT ag ，获得正向插入克隆（pSecT at ）和反向插入克隆（pSecTat-AS），经双酶切鉴定连接成功后，利用测序鉴定其序列和插入方向。将构建的重组质粒转染293细胞，利用Western blot法检测Tat蛋白的表达。进而将重组质粒与LTR-CAT报告质粒共转染293细胞和BCBL-1细胞，CAT-酶联免疫吸附试验（ELISA）检测其细胞内CAT表达，从而检测Tat蛋白的活性。最后将转染重组质粒的293细胞与转染LTR-CAT报告质粒的BCBL-1细胞用Transwell培养系统共培养，CAT-ELISA检测分泌型Tat蛋白的旁分泌调控活性。结果 PCR扩增产物在10 g/L琼脂糖凝胶上约300 bp位置出现条带，与预期的324 bp大小相符；经HindⅢ和BamHⅠ分别酶切鉴定，获得2个正向克隆；正向克隆经测序，克隆的 tat基因序列与Gen-Bank中登记的HIV-1 tat基因100％同源。正向克隆质粒转染293细胞后，Western blot法检测观察到约19×103蛋白条带。正向克隆质粒与LTR-CAT报告质粒共转染293细胞和BCBL-1细胞CAT 表达量高于反向克隆质粒转染细胞（P＜0．05）。与反向克隆对照相比，与正向克隆转染的293细胞共培养的BCBL-1细胞CAT表达量更高（P＜0．05）。结论成功构建 HIV-1 tat基因基因分泌型真核表达载体，该载体不但可在真核细胞内表达具有调控活性的T at蛋白，表达的T at蛋白还可分泌至细胞外并具有调控活性。%Objective To construct human immunodeficiency type 1 tat gene recombinant of secretory vector , express in eukaryotic and detect the activity of expressed Tat protein .Methods HIV-1 tat gene was amplified from pcDNA3 .1(+ )/Tat by PCR .Tat DNA fragments was inserted norientation and inverse direction into secretory eu-karyotic expression vector ,pSecTag2B ,with Hind III and BamH I .The norientation and reversing recombinant were termed pSecTat and pSecTat-AS respectively ,and were sequenced to investigate their sequence and inserting direc-tion .Furthermore ,recombinant plasmids were transfected to 293 cell and expressed protein was detected with West-ern blot .Moreover ,the regulating activity of expressed Tat protein was documented by detecting the CAT expression using CAT-ELISA in co-transfected 293 cell and BCBL-1 cells ,in which recombinant plasmids and LTR-CAT plas-mid were co-transfected .Consequently ,in order to investigated the paracrine activity of recombinant Tat protein ,re-combinant plasmids transfected 293 cell and LTR-CAT transfected BCBL-1 cell were co-cultrued in Transwell and expression of CAT in BCBL-1 cell were detected with CAT-ELISA .Results A band about 320 bp as expecting (324 bp) was visible when PCR products were electrophoresis in 1% agarose .Digested with restricted enzyme and se-quenced respectively ,2 norientation positive clone and 1 inverse direction positive clone were determined .The se-quence of tat gene cloned in norientation plasmid was 100% homology with HIV-1 tat gene registered in GenBank .A protein band about 19 × 103 was visible after Western blot was carried out using norientation plasmid transfected 293 cell .CAT expression in pSecTat and LTR-CAT co-transfected cell was higher than that in pSecTat-AS and LTR-CAT co-transfected cell .CAT expression in LTR-CAT transfected BCBL-1 cell ,which was co-cultured with norienta-tion plasmid transfected 293 cell in Transwell ,was higher than inverse direction plasmid control group .Conclusion HIV-1 tat gene was cloned into secretory eukaryotic vector and expressed in eukaryotic cell successfully .The recom-binant Tat protein showed regulation activity not only in cellular but also extra cellular .