解剖学报

›› 2012, Vol. 43 ›› Issue (6): 762-766.doi: 10.3969/j.issn.0529-1356-2012.06.008

• 细胞和分子生物学 • 上一篇    下一篇

从兔外周血直接克隆IgG重链可变区 

周世权1; 廖智2; 杨林2; 周吉航1; 刘晓光1*    

  1. 1. 舟山医院细胞分子学实验室，舟山医院中国科学院北京基因组研究所免疫基因组学联合实验室，浙江 舟山 316004; 2. 浙江海洋学院海洋科学学院海洋生物资源及分子工程实验室，浙江 舟山 316000
  • 收稿日期:2011-12-14 修回日期:2012-02-08 出版日期:2012-12-06
  • 通讯作者: 刘晓光

Cloning of IgG heavy chain variable region genes directly from rabbit peripheral blood 

  1. 1. Laboratory of Cell and Molecular Biology, Joint Laboratory of Immunogenomics, BR>Zhoushan Hospital-Beijing Institute of Genomics, Chinese Academy of Sciences, Zhoushan Hospital, Zhejiang Zhoushan 316004, China; 2.Laboratory of Marine Living and Molecular Engineering，College of Marine Science，Zhejiang Ocean University，Zhejiang Zhoushan 316000, China BR>
  • Received:2011-12-14 Revised:2012-02-08 Online:2012-12-06
  • Contact: LIU Xiao-guang

摘要： 目的 从兔外周血总RNA中直接扩增出IgG重链可变区基因。方法 先从IMGT/GENE-DB数据库中获取编码大耳白兔免疫球蛋白（Ig）重链可变区（VH）的3个胚系基因片段VSUB>H/SUB>（IGHV）、D（IGHD）和JSUB>H/SUB>（IGHJ），以及编码γ重链恒定区（CH）基因Cγ（IGHG）的cDNA序列，然后设计嵌套引物，以兔外周血总RNA为模板，进行RT-PCR和Nested-PCR扩增，产物经胶回收后克隆到T载体，随机挑选白色克隆测序，最后将序列用Bioedit软件的Local Blast功能比对出Cγ的基因型，同时提交到IMGT/V-QUEST分析出所属的VH、D和JH胚系基因。结果 获得25个克隆的插入子序列，每个克隆均为兔IgG重链可变区的编码基因，且包含了完整的VSUB>H/SUB>、D、JSUB>H/SUB>胚系基因片段，以及Cγ基因

关键词: 外周血, 免疫球蛋白, 重链, 可变区, 反转录-聚合酶链反应, 序列分析, 兔

Abstract: Objective To amplify IgG heavy chain variable region genes directly from rabbit peripheral blood. Methods Two pairs of PCR primers were designed according to the cDNA sequence of rabbit germline Immunoglobulin（Ig）heavy chain variable region（VH）and constant-region（CH）genes, which were obtained from IMGT/GENE-DB. With the total RNA that was extracted from rabbit peripheral blood as a template, an one-step RT-PCR reaction was done and followed by a nested-PCR. By cloning the PCR products to T vector, DNA sequencing and blasting in Bioedit software and IMGT/V-QUEST network database, the genotype of CH and three VH gene segments, VSUB>H/SUB>（IGHV）, D（IGHD）and JSUB>H/SUB>（IGHJ）, were confirmed, and the specificity and compatibility of the primers were evaluated. Results A total of 25 clones were analyzed. Their DNA sequences were different from each other, and all belonged to rabbit IgG heavy chain coding genes. Their constant regions were encoded by the same allele gene, IGHG*02. Of these 25 clones, there were 2 of 37 IGHV, 8 of 11 IGHD, and 4 of 11 IGHJ functional genes; and they assembled to 18 kinds of VSUB>H/SUB>-D-JSUB>H/SUB> combinations. ［IGHV1S40*01］-［IGHD8-1*01］-［IGHJ4*01］ was the topmost VSUB>H/SUB>-D-JSUB>H/SUB> combinations, which appeared in 4 clones, but some differences existed some differences in sequence. Conclusion We have succeeded in amplifying IgG heavy chain variable region genes rapidly and specifically from rabbit peripheral blood, and the primers, which designed by ourselves, have displayed a relatively good compatibility, but the VH-D-JSUB>H/SUB> combinations with IGHV1S40*01 or IGHV1S45*01 seem to be amplifie

Key words: Peripheral blood, Immunoglobulin, Heavy chain, Variable region, RT-PCR, Sequence analysis, Rabbit

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