Acta Anatomica Sinica

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

Previous Articles     Next Articles

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

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