[Analysis and recombinant construction of HBV the reverse transcriptase gene with drug-resistant mutations from 40 patients with chronic hepatitis B].
OBJECTIVE
To analyze genetic mutation associated with drug resistance in the reverse transcriptase (RT) domain of HBV from 40 patients with chronic hepatitis B, and to construct mutant RT gene recombinant vectors for drug-resistant phenotypic analysis.


METHODS
HBV DNA was extracted from sera of the 40 patients receiving anti-HBV nucleot (5) ide analogue. The complete RT domain-encoding gene was amplified by nested PCR, and then cloned into pGEM-T-easy vector. Three to Five clones were randomly selected for DNA sequencing. Data were analyzed by UNASTAR software. The pTriEx-HBV (C) 1.1 expression vectors were constructed by replacing the 1250-hp Xho I/Nco I fragments containing complete RT domain from individual patients samples.


RESULTS
All samples were detected with drug-resistant mutations associated with lamivudine, adefovir, and entacavir singly or in combination. Ninety-six mutant RT genes were cloned into pGEM-T-easy vector, from which 40 major mutant RT genes were replaced into pTriEx-HBV (C) 1.1 expression vectors. The construction was confinned to be successful by verifying mutation existence using DNA sequencing, and detectable HBsAg and HBeAg in the cell supernatant after transfecting recombinant expression vectors into Huh7 cells.


CONCLUSIONS
The analysis of drug-resistant mutation and the construction of mutant-recombinant expression vectors were successfully implemented using the samples frum clinical patients. The work lays a foundation for drug-resistant phenotypic analysis of HBV mutants.
Su
He-ling
HL
Biotechnology college, Guilin Medical School, 541004 Guilin, China.
Liu
Yan
Y
Liu
Wen
W
Zhong
Yan-wei
YW
Chen
Xue-yuan
XY
Wang
Chun-mei
CM
Liu
Yong-ming
YM
Xu
Dong-ping
DP
D000328:Adult; D000998:Antiviral Agents / Q000494:pharmacology / Q000627:therapeutic use; D002460:Cell Line; D003001:Cloning, Molecular; D024882:Drug Resistance, Viral; D005260:Female; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006515:Hepatitis B virus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D019694:Hepatitis B, Chronic / Q000188:drug therapy / Q000821:virology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D055815:Young Adult
D000998:Antiviral Agents; D012194:RNA-Directed DNA Polymerase