摘要
目的构建含不同结构域的人端粒酶逆转录酶(hTERT)酵母三杂交系统融合蛋白表达载体并转化酵母。方法采用PCR法从pCLXSN-hTERT质粒中提取不同的hTERTcDNA片段,纯化分离定向克隆到pYESTrp3质粒,构建包含不同hTERT蛋白结构域的编码序列的重组质粒,转化大肠杆菌,经PCR、酶切、测序鉴定,将阳性重组质粒转化酵母菌,进行毒性和自激活检验。结果经过测序验证,重组融合蛋白表达载体pYESTrp3-hTERT构建成功,转化酵母无毒性和自激活性。结论成功构建能用于酵母三杂交系统的融合蛋白表达载体,可于酵母三杂交系统筛选端粒酶抑制剂及hTERT和hTR间的相互作用。
[Objective] To construct different fusion protein expression vectors to yeast three-hybrid system which contain human telomerase catalytic subunit. [Methods] Different fragments of hTERT eDNA were amplified from pCLXSN-hTERT plasmid and cloned into pYESTrp3 prey plasmids respectively. The recombined prey plasmids were transformed into competent cells of Ecoli, Positive ones were identified by specific PCR, enzyme digestion and DNA sequencing analysis. Finally, the successfully recombined plasmids were transfected into yeast L40ura3 and which was tested for toxicity and serf-activation. [Results] Compared with hTERT gene in GenBank, the homology of the recombined expression vector pYESTrp3-hTERT was 99% in nucleotide acid sequence, no toxicity and self-activation were observed. [Conclusions] Fusion protein expression vectors of hTERT were constructed successfully, which might provide the foundation for research on human telomerase reverse transcriptase in the field of anticancer drug screening and the interaction between hTR and hTERT.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2009年第13期1930-1933,共4页
China Journal of Modern Medicine
基金
国家自然科技基金项目(No:306724487)
