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[Construction of a duck hepatitis B virus YMDD mutant and identification of its resistance phenotype].
Fu, Xi-Hua; Liang, Wei-Fang; Wu, Xiao-Dong; Shen, Guo-Jun; He, Hai-Tang; Chen, Jin-Jun; Hou, Jin-Lin.
Afiliação
  • Fu XH; Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. fuxihua80929@yahoo.com.cn
Nan Fang Yi Ke Da Xue Xue Bao ; 31(4): 633-6, 2011 Apr.
Article em Zh | MEDLINE | ID: mdl-21515458
ABSTRACT

OBJECTIVE:

To construct a lamivudine-resistant plasmid containing 1.2 unit genome of duck hepatitis B virus and identify its replication and drug-resistance in avian LMH hepatica cells.

METHODS:

The recombinant plasmid PBS-DHBV1.2 was constructed using the 1.2-genome length DHBV DNA sequence from a dimer DHBV genome with pcDNA3.1 as the template. With site-directed mutagenesis, we obtained PBS-DHBV1.2-M512V plasmids with polymerase gene mutation from PBS-DHBV1.2. Two constructed plasmids were transiently transfected into LMH cells using FuGENETM6 transfection reagent and cultured in the medium containing different concentrations of lamivudine. Southern blot hybridization was performed to detect DHBV replication intermediates.

RESULTS:

PCR amplification, restriction digestion and plasmid sequencing all confirmed successful construction of PBS-DHBV1.2-M512V recombinant plasmid. Southern blot analysis identified the presence of all the expected DHBV replication intermediates in LMH cells. The replication capacity of the mutant plasmid was decreased by 2.7 times compared with that of the wild plasmid. The IC(50) of lamivudine was 37.12∓8.81 ng/ml for the mutant, greater than that of the wild plasmid (10.90∓4.80 ng/ml).

CONCLUSION:

Compared with the wild plasmid, the mutant plasmid has a lower replication capacity and sensitivity to lamivudine in vitro.
Assuntos
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Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Vírus da Hepatite B do Pato / Mutagênese Sítio-Dirigida / Farmacorresistência Viral Tipo de estudo: Diagnostic_studies Idioma: Zh Revista: Nan Fang Yi Ke Da Xue Xue Bao Ano de publicação: 2011 Tipo de documento: Article País de afiliação: China
Buscar no Google
Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Vírus da Hepatite B do Pato / Mutagênese Sítio-Dirigida / Farmacorresistência Viral Tipo de estudo: Diagnostic_studies Idioma: Zh Revista: Nan Fang Yi Ke Da Xue Xue Bao Ano de publicação: 2011 Tipo de documento: Article País de afiliação: China