抗氯霉素抗体可变区基因的克隆与序列分析  

CLONING AND SEQUENCE ANALYSIS OF VARIABLE GENES OF MONOCLONAL ANTIBODY AGAINST CHLORAMPHENICOL

作　　者：李黄金[1] 赵林[1] 陈伟[1] 刘家劲[1] 李秋[1] 郭志云[1] 林冬霞[1] 田素娟[1] 黄树林[1] 

机构地区：[1]广东药学院生命科学与生物制药学院生物技术系,广州510006

出　　处：《现代预防医学》2009年第24期4655-4658,共4页Modern Preventive Medicine

基　　金：广东省科技攻关计划资助项目(2007A020300007-8;2006B20501005);广东药学院科研启动基金(2006SMK06)

摘　　要：[目的]克隆并分析抗氯霉素单克隆抗体(抗CAPmAb)的轻、重链可变区基因VL和VH。[方法]从分泌抗CAPmAb的杂交瘤细胞株4D10中提取总RNA,利用RT-PCR技术和一系列简并引物扩增VL和VH基因,扩增产物克隆于pMD18-T后测序,利用VBASE2数据库进行比对分析。[结果]有多对引物扩增到VL或VH基因,但同一基因的不同引物扩增到的序列相同。VL和VH基因长度分别为342bp和357bp,分别编码114个和119个氨基酸残基,编码产物均具有4个FR区、3个CDR区和2个特征性半胱氨酸残基,符合典型的KABAT结构特点,分别属于IGKV1亚群和IGH-V3609VH8家族。所克隆序列已被GenBank收录,序列号分别为FJ477893和FJ477894。[结论]成功克隆到抗CAPmAb的VL和VH基因,为进一步构建氯霉素残留检测用基因工程抗体打下了基础。[Objective] To clone and sequence variable genes of light chain（VL）and heavy chain（VH）of monoclon-al antibody against chloramphenicol（anti-CAP mAb）.[Methods] The RNAs was extracted from hybridoma cell line 3E4 se-creting anti-CAP mAb,from which VL and VH genes were amplified by RT-PCR with a set of primers.The PCR products were cloned into pMD18-T,and then sequenced and analyzed by using VBASE2,and VBASE2 was used to compare.[Results] VL and VH genes were amplified by PCRs with several degenerate primers.The cloned VL and VH sequences turned out to be 342 bp and 357 bp long,both sequences were identical to KABAT＇s typical V segment,which were containing four FRs,three CDRs and two characteristic point of cysteine residues,and using a V segment of the IGKV1 subgroup and Igh-V3609 VH8 family,respectively.Cloned VL and VH sequences had been released in GenBank,where accession numbers were FJ477893 and FJ477894,respectively.[Conclusion] The VL and VH genes of anti-CAP mAb were successfully cloned,which laid a good foundation for the preparation of recombinant antibody anti-CAP.

关 键 词：抗氯霉素单克隆抗体 轻链可变区基因 重链可变区基因 克隆 序列分析 

分 类 号：R392.11[医药卫生—免疫学] R392-33[医药卫生—基础医学]