抗人CD3单抗重、轻链可变区基因的体外扩增、克隆和序列分析  

Amplification, Cloning and Sequence Analyses of the V_H and V_K Genes of an Anti-Human CD3 Monoclonal Antibody

作　　者：杨安钢[1] 许辉[1] 吉昌华[1] 韩骅[1] 杨立宏[1] 金伯泉[1] 苏成芝[1] 

机构地区：[1]第四军医大学生物化学教研室

出　　处：《生物化学杂志》1994年第2期213-217,共5页

摘　　要：根据免疫球蛋白重链和轻链可变区基因５’端序列和Ｊ区序列，化学合成适合于体外扩增抗体重、轻链可变区基因的二对引物。从体外培养的ＯＫＴ３杂交瘤细胞中提取总ＲＮＡ，反转录生成ｃＤＮＡ，以ｃＤＮＡ为模板，分别加入合成的重、轻链可变区引物进行ＰＣＲ，扩增出抗体重、轻链可变区基因片段。将扩增产物分别插入ｐＵＣ１９质粒，筛选出阳性克隆，用链终止法进行ＤＮＡ序列测定。所测重链可变区基因全长３５７ｂｐ，编码１１９个氨基酸，轻链可变区基因全长３２１ｂｐ，编码１０７个氨基酸。By comparing the conserved regions at each end of the nucleotide sequences of human and murine germ-line genes encoding V regions and J regions of immunoglobulin heavy chain and K light chain, We designed two sets of primers for the amplification of V_H and V_K genes. In the primers restriction endonucleases EcoRI and SalI sites and the initiation and termination codons were incorporated for forced cloning and expression. Hybridoma cell OKT3, secreting an anti-human CD3 monoclonal antibody, were cultured and the total RNA was extracted. By reverse transcription the cDNAs were synthesized and used as templates for PCR. The PCR was done and the desired V_H and V_K fragments were amplified. The PCR products were then digested with EcoRI and SalI and ligated to pUC19 predigested with the same restriction endonucleases. By screening and identification several recombinants that had been inserted with the target fragments were obtained. The recombinants were sequenced with Sanger's method. It was confirmed by computer-assisted comparative sequence analysis that we have successfully clonetd the full-lenghth and potentially functional V_H and V_K genes of the anti-human CD3 monoclonal antibody OKT3.

关 键 词：免疫球蛋白 可变区 基因 序列分析 

分 类 号：R392.1[医药卫生—免疫学]