人类免疫缺陷病毒Tat融合蛋白后的修饰与生物学活性分析

Construction and expression of the recombinant human immunodeficiency virus Tat gene and analysis on its biological characteristics

目的分析HIV Tat融合后对融合蛋白生物学活性的影响,探讨HIV Tat的生物细胞膜穿透功能和意义。方法以胸腺激酶(TK)基因为报告基因,将不同长度的甘氨酸(Gly)密码子融合在HIV Tat与TK基因之间以及两种基因倒置融合,分别克隆至PBK原核表达载体,大肠埃希菌表达,异丙基-β-D-硫代半乳糖苷(IPTG)诱导,超声波破碎后,经耦联Tat蛋白单克隆抗体的层析柱层析收集。融合蛋白与HepG2细胞共培养,24 h后间接免疫荧光检测。加用更昔洛韦,3 d后锥虫蓝染色计算细胞死亡率,流式细胞仪检测各组细胞凋亡率。结果克隆出含有不同甘氨酸密码子的HIV Tat-Gly(n)-TK(n=0,2,4,6)融合基因,成功表达Tat-TK系列融合蛋白和TK-Tat融合蛋白。间接免疫荧光检测发现Tat-TK系列融合蛋白与Tat蛋白、TK-Tat融合蛋白透过膜效率相似;但流式细胞仪检测表明,在培养基含有更昔洛韦的条件下,Tat-Gly(4)-TK、Tat-Gly(6)-TK、Tat-Gly(2)-TK、Tat-TK融合蛋白、HIV Tat组和TK-Tat致HepG2细胞凋亡率分别为14.77%、4.30%、12.69%、3.00%、1.03%和4.40%。锥虫蓝染色发现细胞死亡率也有类似结果,分别为80.2%、56.7%、65.4%、58.4%、9.1%和57.1%,组间比较差异有统计学意义(P<0.05),但TK-Tat和Tat-TK融合蛋白组之间差异无统计学意义(P=0.24)。结论Tat与TK基因之间融合甘氨酸密码子的数目对融合蛋白中Tat的细胞融合穿透功能不产生影响,而对TK的体外细胞致死作用有较大影响,其中间隔4个甘氨酸密码子对融合蛋白TK体外细胞致死作用的影响最小,同时TK基因与HIV Tat的倒置融合不影响两者生物学功能。

Objective To determine the influence of protein fusion on the biological characteristics of hymidine kinase （TK） and human immunodeficiency virus （HIV） Tat recombinant protein. Methods By utilizing polymerase chain reaction（PCR） technique, different fragments containing two, four or six glycines（Gly） were inserted between the HIV Tat gene and TK, and cloned into PBK vector. After testified by sequencing, the vectorswere transfected into E coll. After induced by isopropyl thiogalactose（IPTG）, bacilli were collected and destructed by ultrasonic, the fusion proteins were determined by monoclonal antibody against HIV protein. HepG2 cells were incubated in DMEM supplement with 10 μg/mL HIV-GIy（n）-TK（n=0, 2, 4, 6） fusion protein, TK-HIV Tat and only HIV Tat. HepG2 cells in different groups were detected by immunofluorescence assay 24 hours after transduction with HIV Tat monoclonal antibody. The rate of apoptosis after cells were incubated withgencilovir（10μg/mL） for 3 days was determined by cell flow cytometry, while survival cell ratio was recorded by trypan blue. The data were analyzed by statistics（t-test）. Results The Tat-Gly（n）-TK （n^0, 2, 4, 6）recombinant genes were constructed and inserted into PBK vectors, which were expressed in E coli and then purified. Cells in different groups, which were incubated with Tat-Gly （n）-TK（n^0, 2, 4, 6） fusion proteins, Tat-TK fusion protein, TK-Tat fusion proteins or only Tat proteins respectively, were detected by immunofluorescence assay. The intensities of fluorescence in different groups were almost same, but the ratios of cell survival or apoptosis were different. The highest ratio of cells apoptosis（14.77%） was in the group that cellular culture medium was mixed with Tat-GIy（4）-TK fusion protein, followed by the groups containing 6, 2 glycines or no TK gene in genes（4.30%, 12.69% and 1.03~, respectively）. There were significant differences between each 2 groups among the all groups（t-test, P^0.05） except between TK-Tat group and Tat-TK group. The order of cells death rates in different groups was similar with that of cell apoptosis rates（the highest rates was in the group that containing Tat-Gly（4）-TK （80.2 % ）, followed by groups that containing Tat-Gly（6）-TK（56.7%）, Tat-Gly（2）-TK（65.4~） or no TK gene（9.1~）. Conclusions Numbers of glycine inserted between HIV Tat and TK does not alter the transcellular function of Tat, but do influence the biological characteristics of downstream protein, that is, cellular lethiferous effects of TK gene. The weakest influence is found when 4 glycines are inserted between TK and Tat. Revers- ing the order of TK and Tat in the fusion protein does not affect the biological functions of these two proteins.