【作者】 苗佩宏；

【导师】 徐江平； 庞建新；

【作者基本信息】 第一军医大学 ， 药理学， 2004， 硕士

【摘要】 端粒是真核细胞线性染色体末端特殊的DNA蛋白质结构，其长度的维持即正常的端粒功能依赖于端粒酶的激活，由于端粒酶的表达与肿瘤的密切相关使得端粒和端粒酶成为很有潜能的研究靶点。因此，有可能通过对端粒及端粒酶的结构和功能的探索寻找到理想的抗肿瘤以及衰老相关性疾病的有效治疗方法。 目前，组成人端粒酶复合物的3个主要成分已被鉴定，其中最重要的一种组分是人端粒酶催化亚基(hTERT)，研究表明：hTERT是人端粒酶复合物的核心成分之一，其基因表达状态是细胞调控端粒酶活性的主要环节。hTERT在大部分恶性肿瘤细胞中表达，在正常组织中不表达，hTERT的表达与端粒酶活性密切相关，是端粒酶的催化亚基和酶活性的限速决定因子。hTERT属于逆转录酶家族的成员，包含7个保守的逆转录酶基序(RT motifs)和一个端粒酶特异的基序(T motif)，它们是端粒酶发挥作用的必要条件。这些结果提示hTERT在细胞中的表达是决定端粒酶活性的主要因素，因此研究hTERT基因表达的调控机制并设计针对hTERT的小分子抑制剂是揭示端粒酶为开发靶点的一个关键步骤，可能对衰老、肿瘤发生等重大生物学问题的解决产生深远的影响。 噬菌体表面展示技术是一种对多肽功能非常有效的筛选技术，它将外源蛋白分子或多肽的基因克隆到丝状噬菌体基因组中，与噬菌体外膜蛋白融合表达，展示在噬菌体颗粒的表面。由于外源蛋白或多肽的基因型和表型统一在同一噬菌体颗粒内，因此，通过表型筛选就可以获得它的编码基因，所以说，噬菌体展示技术为筛选小分子活性物质提供了重要的技术支持。 本研究通过端粒酶逆转录功能区融合蛋白的表达，抗端粒酶催化亚基抗体的制备，并以此纯化抗体为靶分子筛选噬菌体线性十二肽库，从而获得模拟端粒酶逆转录功能区表位的短肽序列，为研究开发端粒酶逆转录酶的小分子抑制剂奠定基础，包括以下三个方面：一、用基因工程的方法表达出包含所有基序的端粒酶催化亚基: 自行设计引物，以pLPC一hTERT为模板扩增出长为1 3 1 obP的基因片段，此片段包括端粒酶发挥逆转录功能所需的全部8个基序。将PCR扩增的此片段克隆至带有6个连续组氨酸标签的原核表达载体pET一32a中，IPTG诱导表达后，用SDS一PAGE和W七stem一Blot检测确定表达出端粒酶逆转录功能区融合蛋白。以SM尿素溶解以包涵体形式存在的目的蛋白，然后用金属鳌合层析柱通过Ni与组氨酸的鳌合作用纯化融合蛋白并对之进行复性，获得具有生物活性的hTERT蛋白。二、制备鼠抗hTERT多克隆抗体: 用端粒酶逆转录功能区融合蛋白常规免疫昆明鼠制备鼠抗hTERT多克隆抗体，经SPG柱纯化后，Dot一ELISA鉴定此抗体与hTERT融合蛋白的结合，ELISA检测多克隆抗体的效价。结果表明制备的抗体能够特异地与hTERT蛋白结合，获得的鼠抗hTERT多抗血清效价达到l:64000，符合下一步作为靶分子筛选肤库的要求。三、模拟端粒酶逆转录功能区表位的筛选: 以纯化的鼠抗hTERT多抗和正常鼠IgG为靶，差减筛选噬菌体线性12肤库，获得模拟hTERT表位的短肤序列，所得阳性克隆通过夹心ELISA、抗hTERT多抗特异性阻断实验、竞争抑制实验表明能够模拟hTERT的表位，搜索FASTA，BLAsT数据库，所获得的11个不同序列富含组氨酸(最高达41.6%)及亲水氨基酸(最高达91.67%)，但它们没有共同的保守基序，且与己知序列无相似性，与hTERT序列亦无同源性，可能模拟的是hTERT的非线性表位，也为今后研制针对hTERT的小分抑制剂提供了实验依据。更多还原

【Abstract】 The extreme ends of linear eukaryotic chromosomes contain specialized DNA-protein structures called telomere which cap the ends of chromosomes, preventing chromosomal degradation and fusion, and allowing for continuous cell proliferation. Telomerase is a unique RNA-dependent DNA polymerase with specialized reverse transcriptase characterization that uses a short templating sequence which contained within its RNA subunit to direct synthesis of telomeric repeats by the catalytic protein component, TERT (telomerase reverse transcriptase).Human telomerase reverse transcriptase , hTERT , has been identified as a catalytic subunit required for telomere elongation , it is the rate-limiting factor for telomerase activity both biologically and enzymatically . The correlation between positive for high telomerase activity and human tumors has led to the hypothesis that tumor growth requires reactivation of telomerase. Most normal human somatic cells do not have detectable telomerase activity and lack expression of hTERT, whereas most immortalized cells and approximately 85% human cancers demonstrate up-regulated telomerase activity and express hTERT. Recent studies reveal that mRNA of the human telomerase catalytic subunit protein, hTERT, has been shown to correlate strongly with telomerase activity. During human tumorigenesis, telomerase becomes reactivated by transcriptional up-regulation of hTERT . One critical function served by hTERT reactivation during cancer progression is to avert the adverse consequences of telomere shortening and loss of chromosomal capping function.Taken together , these findings indicate that the expression of hTERT as the rate-limiting step in telomerase activity and bring the study of hTERT gene expression to the forefront of telomerase regulation research, making ita valuable target in cancer diagnosis. Therefore, Further understanding of the mechanisms involved in hTERT regulation is necessary of great importance and hTERT become a promising target for new strategies of drug development for anti-cancer as well as the treatment of other age- related diseases.Phage-displayed peptide libraries emerge as a proven method for the exploring of novel biologically active molecules and offer an attractive approach for the identification of discontinuous and linear and even nonproteinaceous peptide ligands for various targets. Most peptides selected by phage display bind at sites that coincide with natural ligand binding sites, and consequently act as agonists or antagonists of protein-protein interaction. A short, active peptide could then be used as a leading compound for the rational design of small, non-peptide orally available therapeutic molecules.The study can be divided into following three parts: Part 1 Expression and purification of telomerase reverse transcriptaseClone 1.3kb foreign DNA insert of telomerase catalytic subunit gene which contains all 8 motifs and was amplified by PCR into expression vector of pET-32a. The recombinant plasmid was induced by IPTG for 4h and produced 59KD recombinant protein which appeared in the form of inclusion body. This inclusion body was dissolved in 8mol/L urea and purified by affinity chromatograph using Ni-NTA resin under denaturing conditions. Purified hTERT protein was identified by SDS-PAGE and Western-Blot. Results revealed hTERT functional region recombinant protein was expressed and purified correctly.Part 2 Prepariation of mouse multi-clonal antibody target hTERTImmuned normal mice using purified hTERT protein by standard method to obtain polyclonal anti-hTERT antibody. Dot-ELISA and ELISA identified that polyclonal antibody responsed specifically to recombinant hTERT protein in a concertation correlated manner, the titering of this antibody serum achieved 1:64000. After purification of this polyclonalantibody through SPG column according the protocol and the antibody with such characteristics can be used as target in the three-round screening of phage display peptide library.Part 3 Screening and identification of hTERT e更多还原