用RLM-RACE法克隆抗CD20单克隆抗体可变区基因及其信号肽基因  

Cloning of variable region and signal peptide genes of anti-CD20 monoclonal antibody by RLM-RACE

作　　者：王玉刚[1] 冯健男[1] 沈倍奋[1] 

机构地区：[1]军事医学科学院基础医学研究所分子免疫室,北京100850

出　　处：《细胞与分子免疫学杂志》2005年第4期466-469,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金重大项目(No.30490240)

摘　　要：目的:寻找一种可同时钓取抗CD20mAbVL、VH基因及其信号肽基因的方法。方法:使用Trizol提取杂交瘤细胞1-28的总RNA,分别采用传统的快速扩增5′cDNA末端(traditionalrapidamplificationof5′cDNAend,T5′RACE)和RNA连接酶介导的快速扩增5′cDNA末端(RNAligasemediatedrapidamplificationof5′cDNAend,RLMRACE)的方法钓取目的基因。将其克隆到pGEMTEasy载体上,测序后,与Kabat数据库和GenBank中相应的序列进行比对。结果:采用RLMRACE法可同时钓取到抗CD20mAbVL、VH基因及其信号肽基因,而用T5′RACE法仅能获得VL基因及其信号肽基因。结论:RLMRACE法是钓取抗体V区基因和信号肽基因的好方法。AIM: To seek a good method for simultaneous cloning V_L, V_H and signal peptide genes of anti-CD20 monoclonal antibody (mAb) from hybridoma cells 1-28. METHODS: Total RNA was prepared from hybridoma cells 1-28 by Trizol. Two methods were used to clone the target genes. One was traditional rapid amplification of 5′ cDNA end (T-5′ RACE), and the other was RNA ligase-mediated rapid amplification of 5′ cDNA end (RLM-RACE). The PCR products were then cloned into pGEM -T Easy vector, sequenced and analyzed with Blast method using Kabat and GenBank Databases. RESULTS: The target genes of L chain and H chain were both successfully cloned by RLM-RACE, while only V_L gene and its signal peptide genes were obtained by T-5′ RACE. CONCLUSION: RLM-RACE is a good method for cloning antibody’s V gene and signal peptide genes.

关 键 词：RACE Ⅴ区基因 信号肽基因 单克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]