解剖学报

解剖学报 ›› 2013, Vol. 44 ›› Issue (4 ): 479-484.doi: 10.3969/j.issn.0529-1356.2013.04.007

• 细胞和分子生物学 • 上一篇    下一篇

乙肝免疫球蛋白抗体的多样性

周世权1 周吉航1 徐岳军2 庄晓玲2 李翊卫2 李世波3 刘晓光1 *   

  1. 1. 舟山医院细胞分子生物学实验室，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 316021; 2. 舟山医院检验中心，浙江 舟山 316021; 3. 舟山医院传染科，浙江 舟山 316021
  • 收稿日期:2012-12-26 修回日期:2013-03-05 出版日期:2013-08-06 发布日期:2013-09-04
  • 通讯作者: 周世权 E-mail:zhoushiquan@hotmail.com
  • 基金资助:

    浙江省自然科学基金项目（Y2101355）;国家973计划基金资助项目

Antibody diversity of hepatitis B immunoglobulin

ZHOU Shi-quan1 ZHOU Ji-hang1 XU Yue-jun2 ZHUANG Xiao-ling2 LI Yi-wei2 LI Shi-bo3 LIU Xiao-guang 1*   

  1. 1.Laboratory of Immunogenomics, Zhoushan HospitalBeijing Institute of Genomics,Chinese Academy  of Sciences, Laboratory of Cell and Molecular Biology, Zhoushan Hospital, Zhejiang 316021, China; 2. Clinical Laboratory of Zhoushan Hospital, Zhejiang  Zhoushan 316021, China; 3. Infection Department of Zhoushan Hospital, Zhejiang Zhoushan 316021, China
  • Received:2012-12-26 Revised:2013-03-05 Online:2013-08-06 Published:2013-09-04

摘要：

目的 基于重链可变区的编码基因序列对乙肝免疫球蛋白（HBIG）中的IgG抗体进行分类。方法 借用文献提供的引物，以HBsAb强阳性健康个体外周血中的总RNA为模板进行RT-PCR和Nested-PCR扩增，通过将产物克隆到T载体、随机挑选测序，分析并统计出插入子的VH-JH-Cγ组
合方式。结果 共获得56个有效克隆，全部为人VH3-D-JH-Cγ序列，可分成49种序列，其中5种序列有2或3个序列完全相同的克隆。4个Cγ功能基因也全部出现，其中IGHG2频率最高（28次）；23个VH3功能基因有11个出现，其中频率最高的是IGHV3-23（29次）；23个D功能基因有16个出
现，其中频率最高的是IGHD1-26（8次）；6个JH 功能基因则全部出现，其中频率最高的是IGHJ4（33次）。VH3-D-JH组合有33种，频率最高的是IGHV3-23/IGHD1-26/IGHJ4（8次），其中5种与IGHG2拼接，但这5种VH3-D-JH-Cγ2序列仍有明显的差异。结论 成功地从HBsAb强阳性健康个体外周血中克隆到由VH3亚家族参与重排的IgG重链可变区序列；初步证实可变区序列不仅在VH3-D-JH-Cγ组合方式上具有极大的多样性，而且在序列上也具有明显的差异性；基于可变区测序对IgG抗体或B细胞进行分类是可行的，但需大大提高挑取克隆的数量或改用新一代大规模测序技术。

关键词: 乙肝免疫球蛋白, 重链, 可变区, 反转录-聚合酶链反应, DNA测序, 人

Abstract:

Objective To classify IgG antibodies in hepatitis B immunoglobulin(HBIG) based on DNA-sequencing. Methods The total RNA was extracted as template from the peripheral blood of a healthy individual with high titer of HBsAb(equivalent to HBIG donor), and a two-step RT-PCR and Nested-PCR reaction was conducted sequentially with two sets of compatible primers, which obtained from a literature and specialized for amplifing all IgG heavy chain variable region(VH) genes that begin with VH3 subgroup. Then, the products were cloned to T-vector, and all white colonies were chosen for DNA-sequencing. Finally, the sequence ofevery insert was submitted to IMGT/V-QUEST network database for comfirming the genotype of VH (IGHV), D(IGHD) and JH(IGHJ), or blasted in Bioedit software for confirming the genotype of Cγ. Results A total of 56 clones containing insert were chosen and verified by DNA sequencing, and all comprised of VH3, D, JHand Cγ germline genes. These 56 sequences were divided into 49 kinds, in which 5 kinds came from 2 or 3 clones. Of 49 kinds of sequences, there appeared all 4 Cγ functional genes(IGHG2 topped with 28 clones), 11 of 23 VH3 functional genes(IGHV3-23 topped with 29 clones), 16 of 23 D functional genes(IGHD1-26 topped with 8 clones) and all 6 JH functional genes(IGHJ4 topped with 33 clones). Irrespective of constant-regions, there were 33 kinds of VH3-D-JH combinations. The［IGHV3-23]-［IGHD1-26］-［IGHJ4 combination was the topmost one, which appeared in 8 clones. Although 5 of which were linked to the same constant-region gene, IGHG2, their variable region sequences still had significant differences. Conclusion We succeeded in amplifying IgG heavy chain variable region genes rapidly and specifically from human peripheral blood with high titer of HBsAb, and found that it is practical to categorize antibody in HBIG or B cells in blood based on variable region DNA-sequencing, but the antibody diversity is so vast that it needs to magnify the number of clones al least several scales.

Key words: Hepatitis B immunoglobulin, Heavy chain, Variable region, RT-PCR, DNA-sequencing, Human