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目的 克隆抗人膀胱癌单克隆抗体轻链可变区基因 (VL) ,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VLcDNA。用HindⅢ和XhoⅠ酶切纯化的RT -PCR产物和原核表达载体pET2 8a(+) ,在T4 DNA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VLcDNA片段 ,大小约为 340bp ,重组质粒的酶切鉴定结果与预期一致。VL 基因序列长度为 32 4bp ,编码 10 8个氨基酸。VL 基因属于鼠免疫球蛋白κ轻链Ⅳ亚类。结论 成功克隆出抗人膀胱癌单克隆抗体轻链可变区基因 ,并成功构建其原核表达载体。
Objective To clone the light chain variable region (VL) of monoclonal antibody against human bladder cancer and construct its prokaryotic expression vector. METHODS: Total RNA was extracted from BDI-1 cells secreting anti-human bladder cancer monoclonal antibody and VL cDNA was amplified by RT-PCR. The purified RT-PCR product and prokaryotic expression vector pET2 8a (+) were digested with HindIII and XhoI, ligated at room temperature under the action of T4 DNA ligase, and the recombinant plasmids were identified by restriction enzyme digestion. The positive clones were sequenced and sequenced. The results of amplification of VLcDNA fragment, the size of about 340bp, recombinant plasmid digestion identification results with the expected. The length of VL gene sequence is 32 4bp, encoding 108 amino acids. The VL gene belongs to the murine immunoglobulin κ light chain Ⅳ subclass. Conclusion The light chain variable region gene of monoclonal antibody against human bladder cancer was successfully cloned and its prokaryotic expression vector was successfully constructed.