抗人肝癌单克隆抗体HAb18 Fd及轻链基因的克隆与鉴定  

Cloning and Identification of Fd and Light Chain Genes of MAb HAb18 against Human Hepatoma

作　　者：陈志南[1] 邢金良[1] 杨向民[1] 宋斐[1] 张思河[1] 

机构地区：[1]第四军医大学细胞工程研究中心,西安710032

出　　处：《中国肿瘤生物治疗杂志》2003年第2期105-109,共5页Chinese Journal of Cancer Biotherapy

基　　金：国家"8 63"计划重点课题 (No .2 0 0 1AA2 15 10 1);国家自然科学基金面上项目 (No.3 0 2 0 3 3 0 )资助

摘　　要：目的 :克隆抗人肝癌单抗HAb18Fd及轻链基因 ,并对其可靠性和准确性进行验证。方法 :从分泌单抗HAb18的杂交瘤细胞株中提取总RNA ,利用RT PCR扩增抗体Fd及轻链基因 ,连入 pMD18T载体后 ,挑选阳性克隆进行序列测定并利用相应的软件对序列进行分析。然后将轻链及Fd基因依次克隆到噬菌体展示载体 pComb3中 ,转化大肠杆菌XL1 blue并利用辅助噬菌体M 13K0 7进行挽救 ,收获噬菌体后利用间接ELISA方法检测其抗原特异性。结果 :扩增的HAb18全长轻链和Fd基因大小分别为 6 6 5bp和 6 6 8bp ,序列分析显示 :VH及VL均含有 2个特征性的半胱氨酸 ,CH1属于IgG1亚类 ,CL属于κ亚型。ELISA证实 ,Fab基因表达产物具有与相应抗原特异结合的活性。结论 :成功克隆了肝癌单抗HAb18的Fd及轻链基因 ,为下一步构建多种形式的基因工程抗体奠定了良好的基础。Objective: To clone Fd and light chain genes of monoclonal antibody HAb18 against human hepatoma and verify their accuracy and liability. Methods: Total RNA was extracted from hybridoma cell line secreting MAb HAb18, and Fd and light chain genes were amplified by RT-PCR. After PCR products were ligated into pMD18T vector, positive clones were screened and DNA sequences were tested and analysed by relative softwares. Then, light chain and Fd genes were sequential cloned into phage display vector pComb3. After recombinant vector was transformed into E.coli XL1-blue, recombinant vector was rescued by helper phage M13K07 and the specificity of phages to antigen was detected by indirect ELISA. Results: The size of amplified Fd and light chain genes was separately 665 bp and 668 bp. The results of sequence analysis showed that both VL and VH contained 2 characteristic cystines and CH1 was IgG1 classes and CL was κ. ELISA result identified that expressed Fab antibody could specially bind to corresponding antigen. Conclusion: Fd and light chain genes of MAb HAb18 were successfully cloned, which lay a good foundation for constructing a diversity of engineering antibody.

关 键 词：抗人肝癌单克隆抗体 HAb18Fd 轻链基因 克隆 鉴定 

分 类 号：R735.7[医药卫生—肿瘤] Q786[医药卫生—临床医学]