hTERT基因片段的克隆、表达和抗hTERT抗体用于端粒酶及hTERT检测的研究

端粒酶是一种重要的肿瘤生物学标志，其活性在生殖细胞、绝大多数肿瘤细胞和体外培养的永生细胞可以测知，但在大多数体细胞中不易测出。人端粒酶由两部分组成，包括hTERC和hTERT，hTERC在正常细胞和肿瘤细胞中均有表达，而hTERT的表达似乎受到严格的调控且和端粒酶活性一致。为了检测肿瘤细胞中端粒酶及hTERT的表达，我们制备了抗hTERT蛋白的特异性多克隆抗体。首先用RT-PCR方法克隆了hTERTcDNA的一个片段，将其连接到GST融合表达载体pGEX-5X-3后在大肠杆菌中融合表达。将纯化的融合蛋白抗原免疫动物，制备抗hTERT蛋白的多克隆抗体。不同的细胞抽提物用该抗体进行了Westernblot分析，结果表明该抗体可特异识别端粒酶阳性细胞株中的hTERT及端粒酶，为端粒酶及hTERT的检测初步提供了一个简单有效的检测手段。

CLONING, EXPRESSION OF A SEGMENT OF hTERT GENE AND DETECTION OF TELOMERASE AND hTERT BY ANTI-hTERT POLYCLONAL ANTIBODY

Telomerase is an important biomarker in cancer cells. It is active in germline cells, most of cancer tissues and cell lines, but not in most somatic tissues. Telomerase is composed of two components, and while hTER is present in normal and tumor cells, expression of hTERT appears to be highly regulated and correlates with telomerase activity. In order to detect the telom-erase enzyme and hTERT protein, anti-hTERT polyclonal antibodies were produced in this study. A segment of hTERT cDNA was amplified by RT-PCR and cloned into the multi-cloning site of the GST gene fusion vector pGEX-5X-3. After the recombinant plasmid was expressed in E. coli BL21, the fusion protein was purified for immunization. Extracts from several cultured cells were analyzed by Western blot, and the results indicated that telomerase enzyme and hTERT protein could be specifically detected by this anti-hTERT antibod'. Thus, a simple and effective method was primarily established for the immunodetection of telomerase enzyme and hTERT protein.