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目的构建人端粒酶RNA组分(h TR)的真核表达载体,并对其生物学功能进行初步检测。方法以人胚肾293T细胞RNA逆转录得到的c DNA为模板,采用PCR技术扩增出h TR编码序列,将其插入到p CDNA 3.0载体中;将重组质粒与空载体分别转染293T细胞,实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)检测其表达情况,在Hep G2细胞中构建此基因的稳定细胞系并用qRT-PCR检测其效果,通过RNA结合免疫共沉淀(RIP)实验验证其和人端粒酶逆转录酶(h TERT)及角化不良蛋白(dyskerin)的相互作用,通过端粒酶重复序列扩增技术(telomerase repeat amplification protocol,TRAP)检测过表达h TR后Hep G2细胞的端粒酶活性。结果双酶切和测序结果表明,p CDNA3.0-h TR的真核表达载体构建成功;转染293T细胞用qRT-PCR证明成功表达;qRT-PCR证实Hep G2 p CDNA3.0-h TR稳定细胞系筛选成功;RIP实验证实了h TR与h TERT和角化不良蛋白之间存在相互作用;TRAP实验发现过表达h TR并不影响Hep G2细胞的端粒酶活性。结论成功构建了p CDNA 3.0-h TR真核表达载体,为进一步研究以针对h TR为靶标的癌症治疗奠定了基础。
Objective To construct an eukaryotic expression vector of human telomerase RNA (hTR) and to detect its biological function. Methods The c DNA obtained by RT - PCR of human embryonic kidney 293T cells was used as a template. The h TR coding sequence was amplified by PCR and inserted into pCDNA 3.0 vector. 293T cells were transfected with recombinant plasmid and empty vector, respectively. The expression of the gene was detected by real-time quantitative PCR (qRT-PCR). The stable cell line of this gene was constructed in Hep G2 cells and the effect of the gene was detected by qRT-PCR. By RNA interference co-immunoprecipitation (RIP) The interaction between human telomerase reverse transcriptase (h TERT) and dyskerin was verified by RT-PCR. The expression of Hep was detected by telomerase repeat amplification protocol (TRAP) G2 cell telomerase activity. Results The results of double enzyme digestion and sequencing showed that the eukaryotic expression vector of pCDNA3.0-h TR was successfully constructed. The transfected 293T cells were successfully proved by qRT-PCR. The qRT-PCR confirmed the stability of Hep G2p CDNA3.0-h TR The results of RIP showed that there was interaction between h TR and h TERT and dyskeratin. TRAP showed that h TR did not affect the telomerase activity of Hep G2 cells. Conclusion The eukaryotic expression vector of pCDNA 3.0-h TR was successfully constructed and laid the foundation for further research on the treatment of cancer targeting h TR.