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目的建立HBV表型耐药分析方法 ,体外培养慢性乙型肝炎患者血清HBV分离株并系统分析其对拉米夫定(LAM)的敏感性。方法从1例LAM耐药慢性乙型肝炎患者血清中提取HBV DNA,PCR扩增RT基因,克隆到pGEM-Teasy载体中,随机挑选10个克隆进行DNA序列测定,并分析RT区LAM耐药相关的突变位点。用XhoI和NcoI双酶切pGEM-Teasy-RT及pTriEx-HBV(C),构建1.1倍HBV野生株和LAM耐药突变株的重组载体pTriEx-wRT和pTriEx-mRT,转染人肝癌细胞系HepG2细胞。转染60h后加入不同浓度(0、0.01、0.1、1、10、100μmol/L)的LAM,药物连续作用5d后,抽提病毒核心颗粒HBV DNA,通过实时荧光PCR及Southern blotting检测不同药物浓度作用下HBV DNA的复制水平。结果成功建立体外HBV表型耐药分析方法。野生未突变株重组载体pTriEx-wRT转染HepG2细胞后,随着LAM浓度的升高,HBV DNA水平明显下降,而突变株重组载体pTriEx-mRT转染HepG2细胞后,HBV DNA水平无明显下降,IC50值与野生株相比增加了2500倍。结论建立的HBV表型耐药分析方法对监测HBV耐药性具有可行性。
OBJECTIVE: To establish a method for the analysis of drug resistance in HBV phenotype. Serum HBV isolates from patients with chronic hepatitis B were cultured in vitro and their susceptibility to lamivudine (LAM) was systematically analyzed. Methods HBV DNA was extracted from the serum of a patient with LAM-resistant chronic hepatitis B virus. The RT gene was amplified by PCR and cloned into pGEM-Teasy vector. Ten clones were randomly selected for DNA sequencing and RT-PCR analysis of LAM resistance Of the mutation sites. PGEM-Teasy-RT and pTriEx-HBV (C) were double digested with XhoI and NcoI to construct recombinant plasmids pTriEx-wRT and pTriEx-mRT which were 1.1 times of HBV wild-type and LAM-resistant mutants and transfected into human hepatoma cell line HepG2 cell. After 60h of transfection, LAM with different concentrations (0, 0.01, 0.1, 1, 10 and 100μmol / L) were added into the LAM. After five days of continuous treatment, the HBV DNA of the core particles was extracted and detected by real-time fluorescence PCR and Southern blotting The level of HBV DNA replication. Results The HBV resistance assay was successfully established in vitro. After transfected with pTriEx-wRT, the level of HBV DNA in HepG2 cells was significantly decreased after the recombinant plasmid pTriEx-mRT was transfected into HepG2 cells. However, the level of HBV DNA was not significantly decreased after HepG2 cells were transfected with pTriEx-mRT. The IC50 value increased 2500 fold compared to wild-type strain. Conclusion The established method of HBV phenotype analysis is feasible to monitor HBV resistance.