摘要目的 构建含有人微管蛋白辅助因子A(TBCA)的真核表达载体并观察其对肾癌细胞株786-0增殖的影响.方法 构建真核表达载体pcDNA3.0-Flag-TBCA和pCMV-TBCA-IRES-EGFP,并进行酶切、测序检验、脂质体转染细胞后利用Western blot法检测蛋白表达.以四唑氮化合物(MTS)法绘制细胞生长曲线.结果 成功构建了真核表达载体pcDNA3.0-Flag-TBCA和pCMV-TBCA-IRES-EGFP.转染后TBCA在细胞中表达明显升高(P＜0.05).但在转染后第3、4、5、6天,重组质粒组吸光度值分别为0.52±0.04、0.85 ±0.04、1.20±0.08、1.61 ±0.11;与对照组比较无统计学意义(P＞0.05).结论 TBCA的过表达载体构建成功,但在786-0细胞中过表达TBCA后对并不能对细胞增殖产生明显影响.

abstractsObjective To construct eukaryotic expression vector of human tubulin cofactor A (TBCA) and to study the effect of it on proliferation of renal cell carcinoma cell line 786-0.Methods The eukaryotic expression vectors of human tubulin cofactor A,pCMV-IRES-EGFP and pcDNA3.0-Flag,were constructed and transfected into 786-0 cells by liposome mediate method.TBCA protein level in 786-0 cells was detected by Western blotting.The effects of TBCA on proliferation of 786-0 cells were analyzed by 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method.Results The eukaryotic expression vectors of human tubulin cofactor A,pCMV-IRES-EGFP and pcDNA3.0-Flag,were constructed correctly,which was confirmed by restriction endonuclease digestion and DNA sequencing analysis.Western blotting indicated that TBCA was highly expression in transfected 786-0 cells (P ＜ 0.05).At 3,4,5 and 6 d after transfection,the recombinant plasmid group absorbance values were 0.52 ± 0.04,0.85 ± 0.04,1.20 ± 0.08,1.61 ± 0.11 ; no statistical significance compared with the control group (P ＞ 0.05).The colony forming efficiency in the recombinant plasmid group was higher than other groups.Conclusion The eukaryotic expression vectors of TBCA was constructed correctly and TBCA should not promote the proliferation of renal cell carcinoma cells.