【作者】 程希远；

【导师】 吴曙光；

【作者基本信息】 第一军医大学 ， 药理学， 2002， 硕士

【摘要】 端粒是真核细胞染色体末端的特殊结构，由呈串联排列的重复亚单位DNA片段和端粒结合蛋白构成，保护染色体免于降解、末端重合、重排、和丢失，维持染色体的稳定性和完整性。人端粒的重复亚单位序列为TTAGGG，长约5-20kb。体细胞每传代一次，端粒缩短50-200bp，缩短至一定程度时(2-4kb)细胞衰老或死亡。 与正常细胞相比肿瘤衍生细胞系和生殖细胞的端粒并不随细胞分裂进行性缩短，提示细胞内存在将端粒片段加至端粒末端的酶。Greider和Blackburn验证了端粒酶的存在，之后证实端粒酶是含RNA(端粒合成的模板)的核糖核蛋白，为RNA依赖性的DNA聚合酶，具有反转录活性。端粒酶可能在肿瘤无限增殖的维持中起重要作用。尽管现在端粒酶的激活机制还不甚明了，但可以确定端粒酶的激活能持续合成端粒并加到染色体末端，阻止其缩短，在癌症的发生、发展过程中起关键作用。它已成为一种新的肿瘤标记物及抗癌靶点 近来人端粒酶组分RNA和相关蛋白(TP1和hTRT)的分离、研究及其全基因的成功克隆，为围绕端粒酶而进行的机制研究、端粒酶结构研究、端粒酶抑制剂研究、及其与临床诊断和治疗的相关研究等等提供了可靠的基因材料。现已在体外证实端粒酶的RNA组分和IP1结合后表现出端粒酶活性，但其基因的表达与端粒酶的活性并不……致。体外重配实验证实hTRT具有端粒酶活性，并发现hTRT水平与细胞端粒酶活性成正相关。目前研究结果认为，hTRT是端粒酶催化亚基，是其活性调节的限速亚单位，与端粒酶活性密切相关，端粒酶的激活可能与hTRT的表达、调控有关。hTRT属于逆转录酶家族的成员，它的逆转录功能区包括7个小功能区(reverse transcriptase motif)和1个序列保守的特异性结构域(Tmotif)，它们是端粒酶发挥作用的必要条件，一直伴随在肿瘤的发生、发展过程中，是个非常好的生物靶点。目前端粒酶活性抑制剂主要集中在阻断端粒酶RNA的模板作用和诱导细胞分化两方面。前者有反义寡核苷酸、肽酶等，这些抑制剂的研究虽然取得可喜的进展，但由于肿瘤细胞中RNA的转录量增加并不伴随有端粒酶活 第一军医大学硕士学位论文性的增加，使得这类抑制剂抑制RNA时并不总能抑制端粒酶活性因而有一定的局限性：后。者具有代表性的如维甲酸和们m；“‘’．这类药能明显抑制白血病端粒酶的活件，们对实体瘤细胞不能发挥作用。端粒酶蛋白组分基冈的成功克隆为端粒酶抑制剂的研究提供了新的思路。山于端粒酶蛋臼在组织细胞I－．l含虽甚微，很难提取纯化，若是通过体外表达出端粒酶逆转录功能区基囚，可以方便、大泉获得端粒酶逆转求功能区基因的蛋白质。获得这个研究端粒酶的有效－卜具之后，我们可以进行肽库筛选研究端粒酶结构、可能影响端粒酶活性的表位及小肽，从中筛选出端粒酶的抑制剂；还可制备端粒酶逆转录功能区的抗体，用于端粒酶活性的检测，为端粒酶调节机制的研究和临床诊断、治疗肿瘤提供新的简使方法。 为此，我们用基囚工程的方法表达端粒酶催化亚基。设讣一对5’端有酶切位点仇砒1和3’端为Hi（1厂1的引物，以巾。K－MI订为模板可扩增出长为 um…的基因片段，此片段包括端粒酶作为逆转录酶发挥逆转录功能所需的 8个功能区门”motif“和 7个小功能区（sin＊11。n。V”＊。将经pC【（j）增的片段克隆至带有6个连续组氨酸的原核表达载体祉－2引。，u‘lG诱导表达后，用伽st。rn十lot检测鉴定确定是否为目的蛋白。随后分析目的蛋白的存在形式，是以分泌形式还是包含体形式存在。若是包涵体形式存在用尿素裂解后用余属毅合层析方法纯化日的蛋白并对之进行复性，以期获得可溶的日的蛋白，并用此蛋白免疫新西兰白兔制备多克隆抗体，【lISA的方法检测多克隆抗体的效价。 我们成功地表达出带有6个组氨酸的融合蛋白，表达量经扫描显示为20％，井验证它是以包涵体形式存在的。经过摸索确定8＼1的尿素为最适溶解包if4体的变性剂浓度，在变件条件－厂用朵）aiM合）2析杜迪过N i与组氮酸的鳌合作用，获得纯度较高的目的蛋白。接有对变性状态下的蛋白质进行梯度透析最后得到可溶的日的蛋肉。用此蛋白制各的多克隆抗体经o。ISA检测最适稀释度为卜100）00。更多还原

【Abstract】 Linear eukaryotic chromosome are capped by tandem repeats of simple sequence,G-rich and oriented 5’ -3’ toward the chromosome ends. These termini,called telomere,protects the chromosome ends from degradation,recombination,and complete replication of the chromosome ends . For telomere shorten with each round of cell division,these will lead to cell senescence. Human telomeric sequence is estimated as 5-20 kb .which consists of regular repeat (ITAGGG).Telomerase losts 50-200 bp at every somatic cell division. Eventually,if unckecked,progressive telomere erosion leads to cellular death.Normal cultured cells have limited capacity of replication because of telomere shorten,whereas in tumor cells and other immortal cells can maintain telomere length,which indicates telomere sequence is re-added onto chromosome end. It is now clearly that the enzyme telomerase activated can add sequence to the end of telomere. Telomerase is a sepecial RNA dependent DNA polymerase,which maintains telomere length and prevents cell senescence,and contains telomerase RNA component and protein components.. It appears to be selectively expressed in tumors versus normal cells,and this enzyme is considered as a good biomarker for cancer,whereas telomerase activation mechanism has not been clearly interpreted.Telomerase RNA component and Telomerase protein components,has been isolated .cloned and sequenced from human,which highly propels telomerase studies in the fields of telomerase activation mechanism,structure,inhibitorand others. Telomerase -associated Protein (TP1) shows specifically interact with telomerase RXA subunit,and these components are not required for in vitrotelomerase activity. The key protein component is telomerase reverse transcriptase (hTRT) catalytic subunit.a limiting factor for its activity,which contains seven reverse transcriptase motifs and sepcific region with conserved sequence termed 1<T motif" . T motif is distinguished between telomerase reverse transcriptase and reverse transcriptase families and required for telomerase activity. Furthermore,hTRT activity is detectable from tumor early-stage to tumor late-stage in clinical studies. These results have lead to a rapid increase in interest in telomerase reverse transcriptase as a highly selective anti-can -cer target.Recently,telomerase inhibitor mainly focuses on blocking the RNA template and inducing cell differentiation. By blocking tumor cell RNA,antisense oligonucleotides can down-regulate telomerase activity,but it is not idea inhibitor for telomerase RNA is not related to telomerase activity. Retinoic acid is a typical kind of differentiation-inducing agents did not directly inhibit telomerase activity,suggesting that the inhibition of telomerase activity is in response to induction of differentiation,but it cannot down-regulate entity tumor telomerase activity. Now,we can present a new strategies to achieve efficient telomerase inhibitor targeting telomerase reverse transcriptase catalytic subunit after telomerase protein compnent gene cloned and sequenced. By genetic engineering method,we express and purify a large amount of bacterial recombinant protein which is encoded by human telomerase reverse trascriptase functional region gene. This bacterial expression system and the purification method is simple and efficient,will be useful not only for studies to determine the molecular functions andstructure of telomerase but also for development of strategies of drug design targeting human telomerase reverse trascriptase such as phage display peptide library selection and preparation of polyclonal anti-human telomerase resever transcriptase func -tional region antibody to examine telomerase activity.our purpose is to clone a 1. 3kb gene fragment of telomerase catalytic subunit gene which contains seven reverse transcriptase motifs and specific region with conserved sequence termed "T motif" and was amplified by PCR into expression vector pET28b. The recombinant plasmid was induced by 1PTG for 4h and produced a 52KD recombinant protein.Amou更多还原