[Complete HBV DNA clone and sequence from serum samples of severe hepatitis B patients].
OBJECTIVE
To study the association between hepatitis B virus (HBV) mutants and the pathogenesis of severe hepatitis B by full-length HBV genome.


METHODS
Serum samples from 10 severe hepatitis B patients were collected in our hospital. Serum HBV DNAs were extracted using DNA mini Kit, and amplified by LA Taq DNA polymerase to yield full-length HBV DNA. PCR products were isolated and cloned into vector pUCm-T, then transfected into DH-5 alpha cells. Positive clones were selected and checked by digestion, and full-length HBV DNAs were sequenced.


RESULTS
4 cases were cloned into vector pUCm-T successfully and completed the full-length sequencing. Among them, 3 cases had a G to A mutation at nucleotide 1896 in pre-C region and 1 had a double mutation of T1762-A1764 in the core promoter region. Some amino acid changes occurred within the known CTL, B or T cell epitopes of the PrS2 and C regions.


CONCLUSIONS
This method could serve to study the relationship between HBV genome and the pathogenesis of severe hepatitis B.
Wu
Wei
W
Department of Infectious Diseases, First Hospital of Zhejiang University, Hangzhou 310003, China.
Li
Lan-juan
LJ
Chen
Yu
Y
Li
Jun
J
Qian
Xiang
X
Cheng
Dong-qing
DQ
D000328:Adult; D001402:B-Lymphocytes / Q000378:metabolism; D003001:Cloning, Molecular; D000939:Epitopes / Q000235:genetics; D016679:Genome, Viral / Q000235:genetics; D006509:Hepatitis B / Q000209:etiology* / Q000821:virology; D006514:Hepatitis B Surface Antigens / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D011498:Protein Precursors / Q000235:genetics*; D017421:Sequence Analysis; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism
D000939:Epitopes; D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; C050807:presurface protein 2, hepatitis B surface antigen