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目的 研制抗人端粒酶逆转录酶 (hTERT)蛋白亚基单域重组抗体。方法 以重组His taghTERT融合蛋白为抗原免疫小鼠 ,以逆转录 聚合酶链反应 (RT PCR)扩增小鼠脾细胞重链可变区 ;经克隆噬菌粒载体pCANTAB 5E及转染大肠杆菌建立噬菌体抗体展示文库 ,经过亲和性富集选择阳性克隆 ,并于大肠杆菌 (E .coli.HB2 15 1)中表达为可溶性单域抗体 ;Westernblot确定单域抗体结合特性。结果 以RT PCR扩增His taghTERT免疫小鼠脾细胞RNA ,得到 35 0bp小鼠重链可变区DNA片段 ;通过克隆及转染制备出噬菌体展示抗体文库 ,文库大小是 8× 10 4 ;经过抗原 抗体亲和性筛选 ,得到 4个克隆 ,并表达为可溶性单域抗体 (相对分子质量 16 0 0 0 ) ;WesternBlot分析显示 :其中 2个克隆的可溶性单域抗体可特异性结合His taghTERT融合蛋白 (相对分子质量 16 70 0 ) ,与His tag无关 ;与人癌细胞表达的天然hTERT蛋白 (相对分子质量 12 70 0 0 )分析亦显示其特异性结合能力。DNA序列分析证实可溶性单域抗体为小鼠抗体重链可变区VH基因编码。结论 利用噬菌体展示技术已初步制备出小鼠重链可变区编码的抗hTERT蛋白的特异单域抗体 ,为进一步探索其抑制端粒酶活性及抗肿瘤作用奠定了基础。
Objective To develop a single domain recombinant antibody against human telomerase reverse transcriptase (hTERT) protein subunit. Methods Mice were immunized with recombinant His taghTERT fusion protein and the heavy chain variable region of mouse spleen cells was amplified by reverse transcription polymerase chain reaction (RT PCR). The cloned phagemid vector pCANTAB 5E and E. coli The phage antibody display library was selected for positive clones by affinity enrichment and expressed as a soluble single domain antibody in E. coli (E. coli HB2 15 1); the single domain antibody binding properties were determined by Western blot. Results The spleen cell RNA of mouse immunized with His taghTERT was amplified by RT PCR. The heavy chain variable region DNA fragment of 350 bp was obtained. The library of phage display antibody was prepared by cloning and transfection. The library size was 8 × 10 4. The results of Western Blot analysis showed that the soluble single domain antibodies of two clones could specifically bind to His taghTERT fusion protein (the molecular weight of which was 16000) Relative molecular mass 16 70 0), but not His tag. The analysis of natural hTERT protein (relative molecular mass 12 70 0 0) expressed by human cancer cells also showed its specific binding ability. DNA sequence analysis confirmed that the soluble single domain antibody was the VH domain of mouse antibody heavy chain variable region. Conclusion The phage display technique has been used to preliminarily prepare specific single domain anti-hTERT protein encoded by mouse heavy chain variable region, which lays the foundation for further exploring its anti-telomerase activity and anti-tumor effect.