抗人CD14新克隆ZCH-7-2F9单抗轻重链可变区基因克隆与序列分析  

Cloning and sequencing of VL and VH genes from a novel clone ZCH-7-2F9 of anti-hCD14 monoclonal antibody

作　　者：宁铂涛[1] 汤永民[1] 曹江[1] 沈红强[1] 钱柏芹[1] 

机构地区：[1]浙江大学医学院附属儿童医院血液肿瘤科,浙江杭州310003

出　　处：《浙江大学学报（医学版）》2007年第4期348-354,共7页Journal of Zhejiang University(Medical Sciences)

基　　金：国家自然科学基金(30170391);浙江省自然科学基金(Y206009);浙江大学医学院中青年科技启动基金(542918)

摘　　要：目的:获取抗人CD14单克隆抗体(单抗)ZCH-7-2F9(简称2F9)轻链可变区(VL)和重链可变区(VH)基因,为2F9单链抗体(ScFv2F9)的构建奠定基础。方法:复苏2F9和NS-1细胞株,并亚克隆2F9细胞株,从其最佳克隆93A10提取总RNA,RQ1 RNase-Free DNase处理污染的DNA,与NS-1细胞进行同步对照,通过RT-PCR,扩增获得2F9单抗VL基因和VH基因,分别克隆入pGEM○R-T Easy载体,然后转化DH5α细菌,酶切鉴定并纯化阳性重组子,测序后进行在线核苷酸序列分析。结果:2F9单抗VL基因全长321 bp,编码107个氨基酸,归属于小鼠Ig的Vκ基因,氨基酸序列分析结果显示,VL含有明确的4个框架区(FR)和3个抗原决定簇互补区(CDR),在第23位和第88位为半胱氨酸,是与抗体二硫键形成有关的两种特征性氨基酸;其VH基因全长360 bp,编码120个氨基酸,归属于小鼠Ig的VH基因,氨基酸序列分析结果显示,VH含有明确的4个框架区和3个抗原决定簇互补区,在第22位和第96位为半胱氨酸,是与抗体二硫键形成有关的两种特征性氨基酸。结论:经核苷酸序列分析证明所克隆的基因分别是2F9单抗的VL和VH基因,为2F9基因工程抗体研究奠定了坚实的基础。Objective： To acquire the genes of light chain variable region （VL） and heavy chain variable region （VH） of a novel clone ZCH-7-2F9（2F9） of anti-hCD14 for construction of anti -hCD14 single chain antibody（ScFv）. Methods： From the mouse hybridoma cell line 2F9 and its fusion partner murine myeloma cell line NS-1 ,total RNA was prepared. The VL and VH genes were amplified by RT-PCR with family specific primer pairs ,respectively. The PCR products were cloned into pGEM-T Easy vectors,then transfected into DH5α and the positive recombinants were identified and purified. After sequencing with automatic DNA sequencer the sequences were analyzed online. Results： VL gene of the new clone of CD14 monoclonal antibody （McAb） 2F9 consisted of 321 bps encoding a peptide of 107 amino acid residues,and VH gene of the 2F9 antibody contained 360 bps encoding a peptide of 120 amino acid residues. According to IMMUNOGENETICS online analysis by IMGT/V-QUEST,the VL and VH genes belonged to mouse IGKV and mouse IGHV subgroups,respectively. On the position 23/88 of the light chain and 22/96 of the heavy chain genes there were cysteines,which play the key role in forming

关 键 词：抗体 单克隆 抗原 CD14 序列分析 免疫球蛋白可变区/遗传学 基因 

分 类 号：R392[医药卫生—免疫学] R733.7[医药卫生—基础医学]