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Antibody diversity of hepatitis B immunoglobulin
ZHOU Shi-quan ZHOU Ji-hang XU Yue-jun ZHUANG Xiao-ling LI Yi-wei LI Shi-bo LIU Xiao-guang*
Acta Anatomica Sinica ›› 2013, Vol. 44 ›› Issue (4) : 479-484.
Antibody diversity of hepatitis B immunoglobulin
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, JHand 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.
Hepatitis B immunoglobulin / Heavy chain / Variable region / RT-PCR / DNA-sequencing / Human
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