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

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19×10^3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Tat蛋白还可分泌至细胞外并具有调控活性。

Eukaryotic expression and activity detection of secretory vector contain tat gene from human immunodeficiency type 1

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 eukaryotic 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 direction .Furthermore ,recombinant plasmids were transfected to 293 cell and expressed protein was detected with Western 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 ,recombinant 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 sequence of tat gene cloned in norientation plasmid was 100% homology with HIV-1 tat gene registered in GenBank .A protein band about 19 ×10^3 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 norientation 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 recombinant Tat protein showed regulation activity not only in cellular but also extra cellular .