| Objectives1 To establish a method of HBV phenotypic resistance detection mediated by transient transfection cell model and to evaluate its application.2 To analyze the phenotypic resistance of HBV in-vitro quasispecies and to investigate the regularity between HBV quasispecies population and the occurrence of resistance.Methods1 Construction of transient transfection cell model to detect HBV phenotypic resistanceTwo serum samples containing wild type or rtM204 V mutant HBV strain were selected from CHB patients,after the extraction of HBV DNA from serum, the HBV full-length genome PCR technologies was used to amplify HBV genome. To construct eukaryotic expression plasmid containing the whole genome of HBV,used restriction enzyme Bsp Q I to digest p HY106 plasmid and p EASY-Blunt T vector which had been inserted by HBV genome, then ligated this two compounds and conducted a transform trail.The positive clones was picked and identified to confirm the successful constructs containing full length HBV genome.The two plasmid was extracted and was transiently tranfected into Hep G2 cell, HBV DNA from cell culture supernatant was then harvested and detected by Realtime PCR, the result might confirm the accurate construction of transient transfection cell model. In-vitro drug susceptibility test of the two recombinant vector was performed after transfection, drug treatment within the cell culture wasinitiated and lasted for 4 days.The IC50 was then circulated and used as the evaluation of HBV resistance.2 Phenotypic resistance detection of HBV in-vitro quasispecies populationThe wild type strain was replaced by rt M204 V point mutant through site-directed mutagenesis technologies. Two recombinant vector was then mixed by different proportion as HBV in-vitro quasispecies. Each of the quasispecies group was thus transfected into Hep G2. HBV DNA level and IC50 was analyzed under different drug concentration, and the result might implied the regularity between the quasispecies population and resistance.Results1 Construction of transient transfection cell model to detect HBV phenotypic resistanceLarge amplicon of HBV full-length genome was achieved by PCR, and was used as insert fragments to ligate with PHY106 plasmid. The results indicated that both of the wild type strain or the mutant strain was successfully recombined with p HY106.After transfection in to Hep G2 cell, high copies of HBV DNA was detected, which confirmed a accurate cell model by transient transfection. The in-vitro drug susceptibility test showed the increased IC50 of rt M204 V mutants by 73-fold as compared to the wild type strain, which was identified as phenotypic resistance.2 Phenotypic resistance detection of HBV in-vitro quasispecies populationSite-directed mutagenesis was successfully conducted, and the wild type strain was accurately transformed into the rt M204 V mutant strain.Quasispecies population was formed by the mixture of two recombination plasmid in proportion. Detection of IC50 showed the increased resistance as the proportion of rt M204 V mutant varied from 0 to 100%, which indicated that drug resistance might arise and increase as quasispecies population varied. The results also indicated that there might be a interval of proportion related to the sudden occurrence of phenotypic resistance,which was most likely to be within 25%~30%.Conclusion1 Successfully established a method of HBV phenotypic resistance detection mediated by transient transfection cell model. This method can directly and accurately reflect and quantify the drug resistance, and will open up broad prospects for clinical resistance monitoring and the mechanism research of drug resistance.2 The method of HBV phenotypic resistance detection was successful applied to the resistance analysis of in-vitro quasispecies polulation. The result suggested the proportion of mutants among quasispecies population was related with the occurrence of drug resistance, and strongly implied that the resistance would arise within specific proportion interval of mutants. |
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