一组端粒相关蛋白分子的克隆

Cloning of a group of telomere-associated proteins

目的:进一步了解端粒酶的生物学功能及其在永生化或肿瘤细胞中作用的分子机制,筛选新的端粒相关蛋白分子.方法:基于酵母双杂交技术原理,采用DNA重组技术构建诱饵融合蛋白表达载体,筛选cDNA文库,β-半乳糖苷酶滤膜影印分析确证阳性克隆,对阳性克隆质粒测序并在GenBank进行cDNA序列同源性比较.结果:AH109、Y187酵母菌基因表型稳定,无His渗漏表达.成功构建端粒酶催化亚单位诱饵融合蛋白表达载体pGBKT7-hTERT,其表达的融合蛋白对AH109酵母细胞生长无毒性且不具有激活自身报告基因(LacZ)的作用.从cDNA文库中筛选到Ade+、Leu+、Trp+和His+阳性克隆28个,β-半乳糖苷酶菌落影印滤膜分析筛选到Ade+、His+和LacZ+阳性克隆12个,其中有5个文库质粒具有自身激活报告基因的作用,予以弃除.7个阳性克隆质粒测序发现有2个为重复序列,cDNA序列同源性比较最终获得6个已收录人cDNA序列,他们分别为T-STAR、PAWR、I-1、SMARCB1、LOXL3和HKR3.结论:获取新的端粒相关蛋白可了解端粒酶全酶的结构及其在肿瘤、细胞永生化的生物学功能及分子机制,为肿瘤、衰老的发生及防治奠定重要的理论基础.

AIM： To elucidate the biological function and molecular mechanism of telomerase in immortal and/or tumor cells. METHODS： Based on the principle of the yeast two-hybrid technology, the recombinant of human telomerase catalytic subunit bait fusion gene was constructed by DNA recombination technique, which was employed to screen eDNA libraries. The positive clones were confirmed by β-galactosidase colony-lift filter assay and furthermore the obtained plasmids cDNA sequences were compared with the isogenous sequences in GenBank. RESULTS： The gene phenotypes of AH109 and Y187 yeast strains were stable and there was no leak expression of His. The recombinant of human telomerase catalytic subunit bait fusion gene, which was named as pGBKT7-hTERT, was constructed, and its bait fusion protein had no toxic effects on AH109 yeast cells and no activation effect on the autonomous reporter gene LacZ. Twenty-eight Ade＋, Leu＋, Trp＋, and His＋ clones were screened from the cDNA libraries and 12 Ade＋, His＋ and LacZ＋ clones were obtained by the print analysis of β-galactosidase colony-lift filter assay. Of the 12 positive clones, 5 library plasmids possessed the effect of autonomously activating the reporter gene, so they were removed. There were 7 true positive clones among the library plasmids, 2 of which were found repeated after sequencing. The six obtained plasrnids cDNA sequences were compared with the isogenous sequences in GenBank by Blast software via Internet. Finally, 6 recorded cDNA sequences were obtained, including T-STAR, PAWR, I-1, SMARCB1, LOXL3 and HKR3. CONCLUSION： The 6 obtained telomereassociated proteins may help us understand the structure of telomerase holoenzyme and the biological function and molecular mechanism of telomerase in tumor and/or immortal cells, providing a theoretical basis for the tumor genesis, aging and prevention.