从兔外周血直接克隆IgG重链可变区  

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

作　　者：周世权[1,2] 廖智[3] 杨林[3] 周吉航[1,2] 刘晓光[1,2] 

机构地区：[1]舟山医院细胞分子学实验室 [2]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 [3]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

出　　处：《解剖学报》2012年第6期762-766,共5页Acta Anatomica Sinica

基　　金：浙江省科技计划面上社会发展资助项目(2009C33128)

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VH(IGHV)、D(IGHD)和JH(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VH、D、JH胚系基因片段,以及Cγ基因第1个外显子(CH1)序列的5’端部分,但没有任何2个克隆的序列完全相同。37个IGHV功能基因出现了2个;11个IGHD功能基因出现了8个;11个IGHJ功能基因出现了4个。VH-D-JH组合方式共出现18种,即使VH-D-JH组合方式相同,序列仍具有明显的差异。结论从兔外周血总RNA中直接扩增出了IgG重链可变区,自行设计的引物显示出了高度的特异性和良好的兼并性。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 eDNA 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, Vs （IGHV）, D （IGHD）and J_H（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 V_H-D-J_H combinations. [ IGHV1S40 ＊ 01 ] -[ IGHD8-1 ＊ 01 ] -[ IGHJ4 ＊ 01 ] was the topmost V_H-D-J_H 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 Vs-D-Ja combinations with IGHV1 S40 ＊ 01 or IGHV1 S45 ＊ 01 seem to be amplified preferentially.

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 

分 类 号：Q291[生物学—细胞生物学] R392.13[医药卫生—免疫学]