Molecular Resistense Viration And Vitro Expression Of HBV RT Fregment Origirated From Chronic HBV Infected Patients

ObjectiveThis issue through the infection of hepatitis B virus-positive patients carried by the Pregion of the HBV genomic DNA from large sample surveys, in order to find variants ofsingle or multiple righteousness, and thus infer that the correlation of anti-HBV drugs,and HBV reverse transcriptase mutation, for the discovery of HBV resistance locus andclinical anti-HBV drug offers new material and theoretical reference; select the typicalvariation of HBV of gDNA samples, constructed the recombinant clones of the reversetranscriptase activity in the area of transfected cells by indirect immunofluorescence detectthe expression of activity, ready to build its permanent expression cell lines, as well as thestudy of HBV mutation and drug resistance lay the foundation.Method1.Clinical samples were collectedSelect from April2011to December2011in Tai’an88hospitals with nucleos (t) forTreatment of Chronic Hepatitis B (Chronic Hepatitis B CHB) patients with HBeAg,whichever is the whole blood and serum was separated,-80°C labeled frozen.2.Extraction of DNA in the serum of chronic hepatitis B HBeAgUsing BIOMIGA blood genomic DNA extraction kit from a saved serum separation,purification of HBV of gDNA. 3.HBV DNA gene was amplifiedLearn from the HBV S gene fragment was amplified by PCR positive for nucleic acidsamples, two pairs of primers mutually overlapping HBV DNA was amplified by PCR,two full-length gene (divided into f1, f2, of two parts).4. HBV DNA in gene cloning and sequencing of the full-lengthF1, f2will be amplified PCR products were prokaryotic vector pMD18T vecterconnected, respectively, named the T-f1, T-f2, the advantage of resistant colonies grownon solid culture dish, the initial specific detection positive colony after the cloning andsequencing, splicing and sequencing results, than the right, correction, analysis of theHBV genome nucleotide differences, variation type.5.Construction of recombinant plasmid pcDNA-RTSelect one cases of HBV by RT District occurred classic variation of the sample, thesample T-f1,T-f2, template design containing EcoR I and BamH I, restriction sites point ofthe primers amplified recycling RT fragment, and cloning vector of the pMD18T-thesimple vecter connection, verified by sequencing, the RT fragment digested recycling andsubcloned into pcDNA3.1(-) eukaryotic expression recombinant plasmid pc-RT.6. Screening the concentration of CHO cells sensitive to neomycinUse200ug/ml,400ug/ml600ug/ml,700ug/ml,800ug/ml,1000ug/ml neomycinconcentration gradient effect in CHO cells were observed to choose the cells all diedwithin10-14days minimum neomycin concentration.7.HBV RT transient expression in CHO cells.The recombinant plasmid pc-RT in the positive liposome-mediated transfection ofCHO cells with neomycin screening concentration of the medium after6hours,24hoursafter detection of the expression of the RT region in CHO cells.8. IFA and Westernblot analysis the expressionThe IFA experimental results show that under a fluorescence microscope, positivecells in more than70%. Western Blot experimental results of HBV RT region protein wassuccessfully expressed in CHO cells. Result1. Full length cDNA sequencing of HBV DNAIn the P region gene reverse transcriptase district, a few to be concerned about thevariation of the (two cases of variation and above),may be caused by chronic hepatitis B(CHB) patients with drug-resistant mutation sites.2. CHO cells to determine the concentration of neomycin-sensitiveScreening by the concentration gradient delivery times diluted,and finally todetermine the best screening concentration of CHO cells to neomycin for700ug/ml.3. Indirect immunofluorescence assay and Western blot assay of HBV RTdistrict transient expression resultsThe IFA experimental results show that under a fluorescence microscope, positivecells in more than70%. Western Blot experimental results of HBV RT region protein wassuccessfully expressed in CHO cells.Conclusion1. Chronic hepatitis B patients with nucleoside (acid) drugs during the treatment, Pemergence of several to be concerned about the variation of the classic five cases ofsamples that have been reported of HBV by RT variation,there are six amino acid differentvariation and confirmed the preliminary findings of HBV resistance potential variation.2. The success of HBV by RT subcloned into pcDNA3.1(-) eukaryotic expressionrecombinant plasmid pc-RT.3. Successfully expressed in eukaryotic cells of HBV by RT for building the of HBVRT section of the permanent expression cell lines and to further investigate the molecularmechanisms and screening of new treatments to experimental basis.