端粒酶抑制剂筛选模型诱饵载体的构建与转化

Construction and transformation of bait fusion vector used in high throughput screening assay model for telomerase inhibitors

目的构建含不同人端粒酶RNA基因片段的诱饵融合表达载体并转化酵母,用于酵母三杂交方法筛选端粒酶抑制剂。方法采用RT-PCR方法从肿瘤细胞中扩增人端粒酶RNA(hTR)基因后,分别扩增hTR基因假结合体区CR4-CR5及H/ACA和CR7区hTR基因片段,纯化分离定向克隆到pRH3诱饵质粒,构建包含不同hTR目的基因片段的重组质粒,转化大肠杆菌,经PCR、酶切、测序鉴定后,将阳性重组质粒转化酵母菌,进行毒性自激活检验。结果测序结果证实,含不同hTR基因片段诱饵重组质粒pRH3—hTR构建成功,转化酵母后无毒性和自激活性。结论成功构建了酵母三杂交系统诱饵载体,可于酵母三杂交系统筛选端粒酶抑制剂及在酵母中研究hTERT和hTR间的相互作用。

Objective To construct bait fusion vectors of yeasffor three-hybridization system carrying different human telomerase RNA genes regions （hTR） and transform yeas. Methods The hTR cDNA was analyzed after being amplified using RT-PCR from the tumor cells.Different fragments of hTR cDNA carrying pseudoknot,CR4-CR5 ,BoxH/ACA, CR7 regions were amplified and cloned to pRH3 prey plasmids. The recombinant prey plasmids were transformed into competent cells of E. coli DH5α.The positive recombinant clones were identified by specific PCR, enzyme digestion and DNA sequence analysis. The successfully recombined plasmids were finally introduced into yeast L40ura3 and tested for toxicity and self- activation. Results The sequencing results indicate that different hTR gene fragments of pRH-hTR recombinant vectors was 100% homogeneciry with hTR gene （NO. 7012） published by Genebank, No toxicity and self-activation was observed. Conclusions Human telomerase RNA bait vectors were constructed successfully, which may provide the foundation for research on human telomerase reverse transcriptase in the field of anticancer drug screening and the interaction between hTR and hTERT.