抗人膀胱癌单链抗体的构建及表达的研究

Construction and expression of single chain Fv antibody against human bladder carcinoma

目的 为膀胱癌的导向诊断和治疗提供免疫原性更低的单链抗体。 方法 从 1株分泌鼠抗人膀胱癌单克隆抗体的杂交瘤细胞株BDI 1中分离出总RNA ,逆转录cDNA ,并以此为模板用聚合酶链反应技术扩增VH 和VL 基因 ,分别克隆入pUC19质粒 ,进行序列分析。将VH 和VL 插入表达载体pFUW80中 ,并转化大肠埃希菌JM83,诱导表达出抗人膀胱癌单链抗体。用免疫竞争抑制酶联免疫吸附测定法鉴定免疫活性。用固相化的金属离子亲和层析柱纯化抗人膀胱癌单链抗体。 结果VH 全长 36 6bp ,属于小鼠重链可变区Ⅱ亚类 ;VL 全长 32 4bp ,属于小鼠K轻链IV亚类。表达产物可抑制原单抗BDI 1与人膀胱癌细胞结合的活性。纯化的单链抗体是分子量约 2 90 0 0的单一蛋白条带。 结论 抗人膀胱癌单链抗体被成功地构建并表达。

Objective To construct and expre ss single chain Fv antibody against human bladder carcinoma, which was expected to have advantages in targeted diagnosis and therapy with the characteristics o f lower immunogenicity. Methods Hybridoma BD I-1 cell, which secreted a monoclonal antibody against human bladder carcinoma, was used to isolate total RNA. By reverse transcription, the cDNA was synthesiz ed and used as templates for amplifying the immunoglobulin heavy-and light-cha in variable region genes by polymerase chain reaction (PCR). The amplified DNA w as ligated into a sequencing vector pUC19 and sequenced with Sanger′s method. T he V H and V L genes were inserted into expression vector pFUW80. By inducing, the ScFv antibodies were expressed and secreted from Escherichia coli. Bin ding activities against the bladder carcinoma cells were detected by ELISA. The 5× his-tagged ScFv antibodies were purified on IDA-Ni 2+ resin by immobi li zed metal chelate affinity chromatography (IMAC). The purified ScFv antibodies w ere analyzed by SDS-PAGE. Results A full-le ngth of V H and V L genes was 366 and 324 base pairs respectively. Comparing w ith other published sequences, the V H gene was a member of mouse heavy-chain V H subgroup II and originated from rearrangemant of V H, Dsp22 and J H4 ; the V L gene was V K subgroup IV and from V k and J k4 . The ScFv ant ib odies could inhibit 84% of the antigen binding activity of original McAb BDI-1. The purified ScFv antibodies gave a single major band (Mr-29?000) on SDS- PAGE. Conclusion The single chain Fv antibody aga inst human bladder carcinoma was successfully constructed and expressed.