抗人膀胱癌单克隆抗体重链可变区基因的克隆及原核表达载体的构建

Cloning and sequence analysis of the heavy chain variable region genes of monoclonal antibody against human bladder carcinoma and constructing prokaryotic expression vector

目的克隆抗人膀胱癌单克隆抗体重链可变区基因(VH),并构建其原核表达载体。方法从能分泌抗人膀胱癌单克隆抗体的杂交瘤细胞BDI-1中提取总RNA,通过RT-PCR扩增出VHcDNA,用HindⅢ和XhoⅠ酶切纯化的RT-PCR产物和原核表达载体pET28a(+),在T4DNA连接酶作用下室温连接。重组质粒经酶切鉴定,阳性克隆测序并进行序列分析。结果扩增出VHcDNA片段,大小约为370bp,重组质粒的酶切鉴定结果与预期一致。VH基因序列长度为366bp,编码122个氨基酸。VH基因属于鼠免疫球蛋白重链Ⅱ亚类。结论成功克隆出抗人膀胱癌单克隆抗体重链可变区基因,并成功构建其原核表达载体。

Objective To clone and sequence the heavy chain variable region genes of monoclonal antibody against human bladder carcinoma and construct its prokaryotic expression vector. Methods Total RNA was isolated from hybridoma BDI-1 cells secreting a monoclonal antibody against human bladder carcinoma. The cDNA of VH was amplified by RT-PCR. The purified PCR products and the prokaryotic expression vector pET28a（＋）, after having been digested with Hind Ⅲ and Xho Ⅰ , were ligated in the presence of T4 DNA ligase. The recombinant plasmids were identified via sequential digestion with Hind Ⅲ and Xho Ⅰ. The positive clones were sequenced for analysis. Results An expected fragment of approximately 370 base pair（bp） in length was amplified. Enzymatic digestion showed that the length was in consistence with the expected. The VH gene was 366 bp in length,encoding 122 amino acid residues,belonging to the mouse Ig kappa heavy chain subgroup Ⅱ. Conclusion The VH gene of monoclonal antibody against human bladder carcinoma was successfully cloned, and the prokaryotic expression vector was also successfully constructed.