PCR定点诱变改造低效价抗胶质瘤单克隆抗体的初步报告

目的：抗胶质瘤单克隆抗体建株初期效价为1：10^5，经长期传代培养后效价下降至低于1：10^4，为探索其分子机制并将其改造为有活性的单链抗体，为构建靶向基因转移载体奠定基础。方法：通过RT－PCR克隆该单抗可变区基因，发现轻链可变区CDR1区出现一终止密码子，为此利用PCR定点诱变技术将其突变为该亚组相应的氨基酸，进一步构建单链抗体表达载体，并在大肠杆菌表达。结果：改造后的单链抗体能高效表达，Western blot和免疫荧光证实能与相应的膜抗原特异结合。结论：成功地改造并构建单链抗体，可用于胶质瘤靶向基因治疗。

Preliminary report on reforming the lower affinity monoclonal antibody against glioma by PCR site- directed mutagenesis

Objective To explore the molecular mechanism of lower affinity of murine monoclonal antibody against glioma from long passage cultured mouse hybridoma cell line and construct the single chain antibody for the future targeted gene therapy.Methods By means of RT-PCR,320bp light chain variable domain(VL)cDNA was cloned and sequenced.There was a stop code TAA in CDR1 domain of VL,which was changed to the relevant code using PCR site-directed mutation technique that verified by DNA sequencing,then the single-chain Fv (ScFv) was constructed and expressed in E.coli.Results The ScFv can be expressed in E.coli with high efficiency.Western blot and immunofluorescence analysis suggested that it retained its specific tumor binding activity.Conclusion The monoclonal antibody was successfully reformed and constructed into ScFv,the latter of which can be used for the future targeted gene therapy.