【作者】 周平；

【导师】 刘为纹； 房殿春；

【作者基本信息】 第三军医大学 ， 内科学， 2002， 博士

【摘要】 端粒酶与肿瘤的发生有十分密切的联系。研究表明，大多数肿瘤组织端粒酶阳性率达85-90％，除种系细胞、干细胞、淋巴细胞外，正常组织多为阴性。在某些癌前病变中亦发现有端粒酶表达，且其活性随着病变恶性程度的演进而增高。端粒酶与患者肿瘤大小、组织分化、临床分期、预后可能有一定关系，高表达者其预后可能不良。hTERT是人端粒酶催化亚单位，与hTR构成端粒酶的基本核心，以hTR为模板，延长端粒。hTERT mRNA仅存在于永生化或肿瘤细胞中，对端粒酶活性起关键性调控作用。如能发现与hTERT相互作用的端粒相关蛋白将有利于了解其生物学功能、进一步阐述端粒酶的生物学作用及其在永生化或肿瘤细胞中作用的分子机制，可为肿瘤、衰老的发生及防治奠定重要的理论基础。为此，我们采用酵母双杂交技术筛选cDNA文库，以期获得新的端粒相关蛋白。主要研究内容和结果如下： 1．使用缺陷混合氨基酸[色氨酸(Trp)，亮氨酸(Leu)，组氨酸(His)]检测酵母菌株AH109、Y187营养需求。结果显示：AH109、Y187酵母菌株基因表型稳定，在全培养基(YPD)中生长良好，但不能在单缺陷性培养基(-Leu，-Trp，-His)中生长。将AH109、Y187酵母菌株单菌落划线于含有0、2.5、5、7.5、10、12.5、15mM不同3-AT浓度的SD/-His琼脂平板上，30℃培养3～4天。AH109、Y187酵母菌株不能生长。提示AH109、Y187酵母菌株无His渗漏表达，在筛选cDNA文库时无须加入3-AT。采用氯化钙法将酵母双杂交质粒pGBKT7，pGBKT7-53，pGBKT7-Lam，pGADT7，pGADT7-T，Pcl-1等质粒转化入E.coli DH5α中，并经酶切鉴定证实，为cDNA文库筛选提供了基本条件。 2．采用DNA重组技术将hTERT cDNA定向克隆入DNA结合域 第三军医大学博士学位论文 端粒＋以（上白红d码基1习的克隆及功能分折 山NABD）载体pGBKT7的多克隆位点 ECOR、NOt，并经酶切鉴定 证实成功构建了人端粒酶催化亚单位诱饵融合蛋白表达载体pGBKT7－ hhRT。醋酸理法将 pGBKT7－hTERT转入酵母菌株 AH109，涂于 SD／－Trp平 板，30℃培养Zot天，转化株生长良好，显示pGBKT7－hhRT融合蛋白对酵 母细胞生长无毒性作用。p－半乳糖昔酶菌落滤膜影印分析显示pGBKT7－ hhRT融合蛋白无自身激活报告基因的活性。Western blot进一步证实在约 140KD处见一特异条带，显示pGBKT7－hTERT融合蛋白能够在AH109酵母 菌株中有效稳定表达。所构建的端粒酶催化亚单位诱饵融合蛋白表达载体 完全符合筛选CDNA文库的要求，从而为获得与hhRT相互作用的端粒相 关蛋白分子奠定了基础。 3．检测人睾丸cDNA文库转化株Y187效价为4．5f，适 合CDNA文库筛选的要求。采用酵母交配法将pGBKT7－hTERT转化株 AH109与人睾丸cDNA文库转化株Y187交配，转化菌株涂于SD／工eu／－ T叩－His平板，30C培养6～7天。阳性克隆转涂于SD／－Ade／－Leu／－切／－His 平板，30oC培养 8叶0天。获得 Ade＼ Leu＋、Tp＼ His邓性克隆 28个。 日－半乳糖苦酶菌落影印滤膜分析获得Ade＋、His＋、LacZ＋阳性克隆12个。 将12个Ade＼His＼LacZ＋阳性克隆涂于SD／Leu／－切／His平板，30℃ 培养 sed天，反复培养 3次，再次涂于 SD／叭de／工eu／－Tp／爿is平板，30 ℃继续培养8～10天，以弃除非相关质粒。提取12个Ade＋、His＋、LacZ＋ 阳性酵母细胞质粒，与pGBKT7－hTERT、pGBKT7、pGBKT7－Lain、pGADT7 等质粒互换酵母菌株后进行交配实验。p－半乳糖昔酶菌落影印滤膜分析显 示有5个文库质粒具有自身激活报告基因的作用，予以弃除。有7个文库 质粒转化株为真阳性克隆，将真阳性克隆质粒进行测序。其中有2个序列 重复。所测6个真阳性克隆质粒cDNA序列通过Internet利用Blast软件与 GenBank进行同源序列比较，结果获得6个己收录人cDNA序列，它们分 别是：T－STAR，PAWR，Imidazoline receptor candidate，SMARB，LOXL3， HKR3。至今尚未见这一组可能是端粒相关蛋白分于与端粒、端粒酶相关 研究报道，这一新的发现无疑为进一步研究端粒酶全酶的结构、生物学功能 IX z第三军医大学博士学位论文 端2粒相关蛋白聊码基因的克隆及功能分析及其调控机制奠定了良好的实验基础。 4．为获取真正的人端粒相关蛋白编码基因，我们采用哺乳细胞双杂交技术以确证在哺乳细胞内T－STAR与hTERT密切关系。首先，采用DNA重组技术构建DNA－BD载体（pNfhTERT人AD载体（pVP16－TETAR人将盯＊又T山＊A克隆入载体质粒邮的多克隆位点＊＊RI：以k。RI。NOt双酶切鉴定 DNA序列的方向，获取正义重组体 pM上TERT。将T－STAR CDNA定向克隆入载体质粒 PCDNA3?更多还原

【Abstract】 Tumorigenesis is cogently associated with the activation of telomerase. It was exhibited that the telomerase was expressed in 85-90% of human tumors, but not in the majority of normal tissues except for some stem cell ,lymphocyte and germline cells. The expression of telomerase was also identified in some precancerous lesions and its level would be increased with the malignant progression. There might be some relationship between telomerase activity and tumor size, differention, clinical stage and prognosis. Patients would have a poor prognosis if they suffered from tumors with high level of telomerase activity. It was indicated in the in vitro studies that both hTERT( which played a key role in the activation of telomerase)and human telomere RNA(hTR) composed basic core of telomerase. These subunits acted in concert to elongate telomeres by reading from the RNA template sequence carried by the RNA subunit and synthesizing a complementary DNA strand. hTERT mRNA, in contrast to other subunits of telomerase, exists only in immortal or tumor cells. If we could find additional telomere-associated proteins which interact with hTERT, it would be advantageous for us to elucidate the biological function 昦nd molecular mechanism of telomerase in immortal and/or tumor cells. This would establish a sound theoretical basis for the tumor genesis, aging and prevention. Therefore yeast two-hybrid method was employed in the study to screen cDNA libraries with hTERT as bait. The main results are as follows:1. Dropout mixed amino acids [tryptophan(Trp), leucine(Leu), histidine (His)] were employed to detect the nutritional requirement of yeast strains’ Supported by the National Natural Science Foundation of China (No. 39980010)IVAH109, Y187. It was indicated that the gene phenotypes of AH109 and Y187 yeast strains were stable and the strains grew well in the full nutritional supplement(adenine supplemented YPD) but did not in the single dropout supplements(-Leu, -Trp and -His). When the single strain of AH109 and Y187 yeasts was streaked in the SDAHis agar plates with different concentrations of 3-AT (0, 2.5, 5, 7.5, 10, 12.5, 15 mM) and was cultured at 30癈 for 3-4 days. There was no growth of AH 109 and Y187 yeast strains in the culture, which indicated that there was no leak expression of His in AH109 and Y187 and that 3-AT was unnecessary in the screening of cDNA libraries. The plasmids of yeast two hybrid system ( pGBKT7,pGBKT7-53, pGBKT7-Lam, pGADT7, pGADT7-T, Pcl-1) were transformed into the E. coli DH5 a by calcium chlorate and identified by restriction endonuclease. This provide essential conditions for the screening of cDNA libraries.2. hTERT cDNA was directly cloned into the multiclone sites EcoRI, NotI of pGBKT7 vector of DNA binding domain (DNA-BD) by recombinant technique. The recombinant of human telomerase catalytic subunit bait fusion gene, which was named as pGBKT7-hTERT, was identified by restriction endonuclease EcoR I and Not I. pGBKT7-hTERT was transformed into yeast strain AH109 by the method of Li acetate and the transformed strains were streaked onto SDATrp plate and grew well after cultured at 30癈 for 2-4 days. This implied that pGBKT7-hTERT bait fusion protein had no toxic effects on AH 109. It was exhibited in the print analysis of P -galactosidase colony-lift filter assay that there was no autonomous activated reporter gene in pGBKT7-hTERT fusion protein. A specific band was identified in 140KD during electrophoresis by Western blot. This indicated that pGBKT7-hTERT fusion protein could be stably expressed in AH 109 yeast strain. The constructed pGBKT7-hTERT satisfactorily fulfilled the requirement of the screening of cDNA libraries.3. The human testicle cDNA library transformed strain Y187 viability wasdetermined to be 4.5 ~ 5X 107cfu/ml, which was coincided with the screening of cDNA libraries. The mating of pGBKT7-hTERT transformed strain AH 109 with human testicle cDNA library transformed strain Y187 was made by yeast mating method. And the t更多还原