人端粒相关蛋白T-STAR的克隆及其与端粒酶催化亚单位hTERT的相互作用研究

目的克隆人端粒相关蛋白T-STAR并研究其与端粒酶催化亚单位hTERT在哺乳细胞内的相互作用。方法构建表达载体pGBKT7-hTERT,以pGBKT7-hTERT为诱饵,采用酵母双杂交技术进行cDNA文库筛选;构建pVP16-T-STAR和pMhTERT重组载体,脂质体法将其与报告质粒共转染入胃腺癌细胞株SGC-7901,以pM-53 pVP16-T、pM3-VP16为阳性对照,以pM53 pVP16-CP为阴性对照;以pM-hTERT pVP16、pM pVP16-T-STAR、pM pVP16为交叉阴性对照。ELISA法检测报告基因CAT的表达。结果成功构建真核表达载体pGBKT7-hTERT,将获得的阳性克隆测序,并进行同源性比较证实为是已收录的人cDNA序列T-STAR。pGBKT7-hTERT和pVP16-T-STAR共转染之后,报告基因CAT的表达(A值为0.258)显著高于阴性对照(A值为0.002~0.015)。结论T-STAR与hTERT在胃癌细胞内发生了相互作用,T-STAR可能是人端粒相关蛋白新成员,通过hTER参与端粒酶活性的调控。

Cloning of telomere-associated protein T-STAR and the interaction between T-STAR and hTERT

Objective To clone the telomere-associated protein T-STAR and study the relationship between T-STAR and the telomerase catalyzed subunit hTERT in mammalian cells. Method The expression vector pGBKT7-hTERT was constructed and acted as a decoy in cDNA library screened by yeast two-hybrid technology. Recombinant vectors pVP16-T-STAR and pM-hTERT were constructed and co-transfected with report gene CAT into SGC-7901 cells with liposome. pM-53 pVP16-T and pM3-VP16 were introduced as positive controls, pM-53 pVP16-CP as negative control, and pM-hTERT pVP16, pM pVP16-T-STAR and pM pVP16 as crossing negative controls. The expression of CAT was assayed by ELISA. Results The eukaryotic expression vector pGBKT7-hTERT was successfully constructed. One positive clone achieved by cDNA library screening was sequenced and compared with the isogenous sequences in GenBank by Blast software via Internet. As a result, T-STAR, a recorded cDNA sequence was obtained. The recombinant vectors of pVP16-T-STAR and pM-hTERT were constructed successfully and co-transformed into gastric cancer cells SGC-7901. The OD value of reported gene CAT was 0.258, which was significantly higher than that of the negative and crossing negative controls (0.002-0.015). It revealed that T-STAR interacted with hTERT in the mammalian cells. Conclusions T-STAR interacts with hTERT in the gastric cancer cells. T-STAR may be a new member of telomere-associated protein, and it participates in the regulation of telomerase through hTERT.