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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 analyze the effect of fusion of HIV Tat on the biological activity of fusion protein and explore the function and significance of biological cell membrane penetration of HIV Tat. Methods Thymine kinase (TK) gene was used as a reporter gene, glycine (Gly) codons of different lengths were fused between HIV Tat and TK genes, and the two genes were fused in inversions and cloned into the prokaryotic expression vector PBK. Escherichia coli Expression, induced by isopropyl-β-D-thiogalactopyranoside (IPTG), sonicated, and then purified by column chromatography using a monoclonal antibody conjugated to Tat protein. The fusion protein was co-cultured with HepG2 cells, and indirect immunofluorescence assay was performed 24 hours later. Plus ganciclovir, 3 days after trypan blue staining cell death rate, flow cytometry detection of apoptosis rate in each group. Results The HIV Tat-Gly (n) -TK (n = 0,2,4,6) fusion gene containing different glycine codons was cloned and the Tat-TK fusion protein and TK-Tat fusion protein were successfully expressed. The results of indirect immunofluorescence showed that the efficiency of Tat-TK fusion protein was similar to that of Tat protein and TK-Tat fusion protein. However, the results of flow cytometry showed that Tat-Gly (superscript + 4) The apoptosis rates of HepG2 cells induced by -TK, Tat-Gly (6) -TK, Tat-Gly (2) -TK and Tat-TK fusion protein, HIV Tat and TK-Tat were 14.77% and 4.30% 12.69%, 3.00%, 1.03% and 4.40%. Trypan blue staining showed similar results (80.2%, 56.7%, 65.4%, 58.4%, 9.1% and 57.1%, respectively), with significant difference between the two groups (P <0.05) And Tat-TK fusion protein group was no significant difference (P = 0.24). Conclusion The number of fused glycine codons between Tat and TK gene has no effect on the cell fusion and penetration of Tat in the fusion protein, but has a great effect on the lethal effect of TK in vitro with four glycine codons on the fusion The effect of protein TK on cell death in vitro is minimal, and the inverse fusion of TK gene and HIV Tat does not affect the biological function of both.