| In the anti-hepatitis b virus (anti-HBV) infection treatment, nucleos(t)ide analogues (NA) are widely used currently, which can inhibit the reverse transcriptase activity of HBV and are able to control effectively the illness development of the chronic HBV patients. NA have the advantage of less side effect and inhibition of HBV’s rapid replication. But, NA can play a direct role in covalently-closed circular DNA (ccc DNA), the HBV sufferers have to receive long-tern drug treatment. Due to the lack of strict3’-5’proofreading function of the reverse transcriptase during replication, it will cause HBV easy to variate. Under the pressure of choosing drugs, the resistance mutation of the virus strains are ble to be amplified selectively, which may directly induce RT area genes to produce adaptability resistance mutations and cause the decline of the sensitivity of the advantage virus strains for the drugs so as to reduce the curative effect for the disease. Therefore, the resistance to the virus becomes a key problem during the treatment of anti-HBV.The analysis of in viro resistant phenotype is the current commonly-used resistance research methods. Based on the transfection from HBV DNA recombinant plasmid vector to the human hepatocellular carcinoma cells (HHCC), HBV biological characteristics, anti-HBV drugs screening and immune therapy evaluation are studied. Of which, HBV instantaneous replication express cell model is the common model in identifying the clinical separation of the mutant viruses resistant phenotype. Because of the multifarious vector construction process, the long time-consuming, low level of in vitro viral genome replication and unstable in expression, the urgent clinical need phenotypes resistance analysis model of cells hasn’t been established yet at present in our country. Just because the common genetic type of HBV cell models with Chinese characteristics built up in the laboratories are fewer; and the number of those established is very limited, lacking the representativeness of all kinds of HBV gene variants sequence. All these can no longer meet the need of study on various kind of HBV mutant types which have or may have important clinical and biological significance for the lack of source of patients with significant HBV mutant strain cell models. In term of the above-mentioned problems, the study is conducted to satisfy the need of the increasing studies of the new antiviral drug screening, immune therapy and genetic therapy evaluations.The study tend to build up a stable and practical in vitro phenotypes resistance analysis model based on Chinese common HBV genetic type to evaluate virus resistance level and direct the clinical rational drug use and individualized treatment.Objective:To construct a1.1ploid genome length HBV recombination in vitro resistance expression vector based on common Chinese HBV gene types. And based on this, to build a simple, rapid and accurate HBV in vitro resistant phenotype analysis model applied to clinical practice.Methods:(1) Chronic HBV patients in Chinse Mainland are selected, and their serum HBV DNA will be extacted, full-length cloned and sequence identified.(2) The DNA of clear sequence HBV C genotype will be used as model after cyclization. Then two primer of the specific enzymes cutting sites are designed and added and amplified into two DNA fragments of1.1ploid genome length HBV genome by polymerase chain reaction (PCR). And the enzymes cutting sites combine with vector to form1.1ploid genome length HBV recombinant plasmid vector.(3) The identified recombinant vector is applied to transfecting Huh7cells, then the culture supernatant HbsAg and HbeAg expressions in the transfected cell culture, the HBV DNA replication level and the replicative intermediate are detected.(4) Drug sensitive test will be performed on the Huh7cells transfected with recombinant vector by adding different concentrations of antiviral drug (Lamivudine) in48hours. The drug works continously for5days, then the replication and expression level of the virus under different concentrations of drug are monitoredResults:(1)1.1ploid length HBV genome recombinant plasmid vector of Chinese HBV C genotype has sucessfully been constructed. (2) After the in vitro transfection of hepatocarcinoma cells, HBsAg、HbeAg can be detected in the culture supernatant; higher HBV DNA replication level (107～109copies/ml) by fluorescence quantitative PCR detection system; and the virus replication intermediates can be found with Southern blot.(3) The recombinant expression vector will be applied to phenotype resistance analysis. With the increase of the antivirus drug concentrations, the replication and expression levels of HBV will decrease obviously.Conclusions:(1) The successfully constructed1.1ploid length HBV genome recombinant plasmid vector based on the common Chinese HBV genotype can be mediated a high level of HBV replication and gene expression in the in vitro cultured cells.(2) The established HBV resistant phenotype analysis model may be applied to evaluating the virus resistance level. |
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