摘要
目的 :HER2 /neu过度表达见于多种恶性肿瘤。作为肿瘤抗原 ,p185 erbB2 是一个理想的肿瘤治疗靶点。本实验在既往工作基础上 ,构建嵌合型抗p185 erbB2 单克隆抗体 (单抗 )的真核表达载体 ,并在CHO dhfr-细胞中表达 ,从而降低鼠源性抗体的免疫原性 ,为进一步的应用研究奠定基础。方法 :RT -PCR扩增p185 erbB2 单抗 1 2的轻、重链可变区基因 ,插入嵌合抗体真核表达载体pWSD2中 ,脂质体转染CHO dhfr-细胞。经RT -PCR、间接ELISA、Westernblot检测人 -鼠嵌合抗体的表达水平 ,用ELISA、免疫沉淀方法对嵌合抗体的亲和力和特异性进行初步鉴定。此外 ,采用MTT比色法检测嵌合抗体对高表达p185 erbB2 的乳腺癌细胞系SKBR3的生长抑制作用。结果 :用RT -PCR、间接ELISA、Westernblot方法证明人 -鼠嵌合抗体在CHO dhfr-细胞中表达 ;ELISA检测提示嵌合抗体与高表达p185 erbB2的细胞反应阳性 ,与低表达p185 erbB2 的细胞反应阴性 ;免疫沉淀结果表明嵌合抗体可与p185 erbB2 分子特异性结合 ;MTT比色法检测表明嵌合抗体对过表达p185 erbB2 的乳腺癌细胞SKBR3的生长有抑制作用。结论 :CHO dhfr-细胞表达的人 -鼠嵌合抗体可与p185 erbB2 特异性结合 ,并可抑制高表达p185 erbB2 的肿瘤细胞生长 ,表明抗人p185 erbB2 人
Objective: Overexpression of the HER2/neu oncogene is a frequent molecular event in multiple human cancers. Being a cancer antigen, p185 erbB2 is an ideal target for immunotherapy. In order to decrease the immunogenicity of mouse anti-p185 erbB2 monoclonal antibody in human cancer therapy, we constructed the eukaryotic expression vector of anti-p185 erbB2 chimeric monoclonal antibody and verified expression of the chimeric antibody in CHO-dhfr - cell. Methods: The variable regions of light chain and heavy chain were amplified with RT-PCR and inserted into the chimeric antibody vector pWSD2. After CHO-dhfr - cells were transfected with recombination plasmid by lipofectAMINE, the chimeric antibody expressing level was identified with RT-PCR, indirect-ELISA, and Western blot. The specificity of the anti-p185 erbB2 chimeric antibody was testified with ELISA assay and immunoprecipitation. Moreover, the effects of chimeric antibody on the proliferation of breast cancer cell line SKBR3, which is overexpressing p185 erbB2 , were measured with MTT assay in vitro. Results: The anti-p185 erbB2 chimeric antibody eukaryotic expression vector was constructed successfully and the expression of the chimeric antibody in CHO-dhfr - was verified by RT-PCR, indirect-ELISA, and Western blot. ELISA assay showed that chimeric antibody reacted with cells overexpressing p185 erbB2 specifically, but did not react with that non-overexpressing p185 erbB2 . Immunoprecipitation test confirmed that the chimeric antibody could bind to p185 erbB2 specifically. The MTT assay demonstrated that the chimeric antibody could inhibit the growth of SKBR3 cells overexpressing p185 erbB2 . Conclusion: The anti-p185 erbB2 mouse/human chimeric antibody that was expressed in CHO-dhfr - cells can bind to p185 erbB2 specifically and inhibit proliferation of SKBR3 cells overexpressing p185 erbB2 . It has a potential application in biotherapy of cancer.
出处
《北京大学学报（医学版）》
CAS
CSCD
北大核心
2004年第5期491-495,共5页
Journal of Peking University:Health Sciences
基金
国家自然科学基金 ( 3 9970 83 7)
教育部教育振兴行动计划特殊专项 ( 985工程 )资助~~
