抗人端粒酶蛋白亚基单域抗体制备和鉴定

Single domain antibody to human telomerase catalytic subunit:preparation and characterization

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 recombinant single domain antibody against hTERT, human telomerase catalytic subunit. Methods A previously prepared His-tagged hTERT fusion protein was used as the antigen,and the variable regions in heavy chain (VH) of immunized mice were RT-PCR amplified and cloned into the pCANTAB 5E, a phagemid vector. By transfection, the display library of mouse VH was developed. The candidate clones were selected by affinity panning, and soluble VH were obtained after expression in E.coli, HB2151. The resultant single VH antibodies were characterized on their binding potentials by western blotting. Results An about 350 bp VH fragment was amplified from spleen cells of mice immunized by His-tagged hTERT and expressed by phage displayed as VH library. The size of the library was 8×10 4. After three rounds of affinity panning, 4 independent clones were chosen and consequently expressed as soluble single domain antibodies (Mr=16 000). In Western blot analysis, the single domain antibody from 2 of 4 clones proved to react with the His-tagged hTERT fusion protein (Mr=167 000) without dependence of His-tags and also detect the native hTERT (Mr=127 000) extracted from the human HeLa cancer cell line. DNA sequencing showed both of the single domain antibodies were encoded by the heavy chain variable region of the mouse. Conclusions The single domain antibodies developed were hTERT recognizable and hTERT specific, thus providing a basis for application of recombinant single domain antibody in inhibition of telomerase activity and anticancer therapy.