The Characteristic of the Whole Hepatitis B Virus Gene, Construction of HBV Gene Eukaryotic Cell Expression Vector, and the Efficacy of Adefovir Plus Entecavir or Plus Lamivudine as Rescue Therapies in Chronic Hepatitis B Patients with Entecavir-resistant
|Keywords||Chronic hepatitis B Hepatitis virus Variation Entecavir Resistance Reverse transcriptase Hepatitis B virus Adefovir dipivoxil Lamivudine Salvage therapy Clone Plasmid|
The first part of the ex analysis nucleos (t) ide analogs (NAs) after treatment with entecavir (ETV)-resistant chronic hepatitis B (CHB) patients with hepatitis B virus (HBV entecavir resistant hepatitis B virus reverse transcriptase gene sequence characteristic purpose: ) P-coding region of the reverse transcriptase (RT) gene sequence characteristics. Methods: The subjects selected from April 2006 to February 2011, the First Affiliated Hospital of Suzhou University outpatient or inpatient accept NAs treatment virologic breakthrough or rebound 730 CHB patients, the PCR method in patients with serum HBV RT gene product direct sequencing analysis with Chromas2.0 software HBV RT gene nucleotide and amino acid differences and variation type. Results: A total of ETV resistance variation of 29 cases, including six cases of LAM induced ETV resistance (26.09%) of ETV resistance induced ETV sequential lamivudine (LAM), 23 cases (73.90%). Sequencing results the RT gene ETV resistance mutation rtT184, rtM250 and rtS202G, respectively, for 15 cases (62.07%), 8 cases (27.58%) and 6 cases (26.09%), the type of variation including T184V/A/G/I / S / M, M250I/L/V rtS202G. 29 cases were accompanied by rtM204I / V mutation, which 22 cases with rtL180M of variation. Sequencing showed that the new amino acid substitution A165C, S168C, P170S, S172M, V175I, V183G, V207M / I, V214A, A222T, S223A, I224V, F226T / C, N227T, L229M, T237P, N248H, I254M, V256S etc.. Conclusions: LAM or ETV sequential LAM treatment the induced the ETV resistance mutation, its RT mutation patterns LAM resistance mutation (rtM204I / V with or without associated with rtL180M variation) ETV resistance mutation rtT184, rtM250 or rtS202G the same time. Common types of ETV resistance mutation in RT region rtT184I/G/S, rtS202G, and rtM250L / V. The second part of entecavir or adefovir dipivoxil in combination with lamivudine, adefovir dipivoxil combined with grace salvage therapy En purpose of entecavir drug efficacy studies of patients with chronic hepatitis B: Evaluation of oral adefovir ester (ADV) (10 mg / d) Joint ETV (0.5mg / d) or adefovir dipivoxil combined with LAM (100mg / d) to save the efficacy of the treatment of ETV resistance in CHB patients. Method: April 2006 to February 2011 in the First Affiliated Hospital of Suzhou University outpatient or inpatient accept NAs treatment of CHB patients with viral breakthrough or rebound occurred ETV resistance mutations in 29 patients, depending on the treatment programs were randomly divided for the two groups, A group: ADV10mg / d ETV0.5mg / d, group B: ADV10mg / to d LAM100mg / d, respectively, at baseline, 12 weeks, 24 weeks, 48 ??weeks, 96 weeks to detect liver and kidney function, hepatitis B serum markers and serum HBV DNA level. The decline in group B serum HBV DNA 24 weeks the lt; 2log10copies/ml of 3 patients adjust treatment: ADV10mg / d joint ETV0.5mg / d, to continue to monitor and follow-up. Results: A group of 21 patients, the B group of 8 patients, the mean duration of treatment of 69 weeks (9 to 92 weeks), B group 1 patients died in the course of the first nine weeks of chronic liver failure. 24 weeks of treatment patients with serum HBV DNA from baseline fell by an average 2.87 ± 0.98 and 1.62 ± 1.01 log10copies/ml two groups (p = 0.033); Serum HBV DNA negative rate were 2/19 (10.53%) and 1 / 7 (14.29%), (p = 0.096). The two groups of patients with serum HBV DNA 48 weeks compared to 24 weeks to further decline to 1.0 ± 0.89 and 1.06 ± 1.25 log10copies/ml (p = 0.096); were eight cases and two cases of patients with serum HBV DNA negative (52-82 weeks ), including three cases of HBeAg seroconversion occurred. Treatment of 24 weeks, serum HBV DNA decline in the lt; 2 log10copies/ml patients a total of eight cases (group A, B group 5 cases), the downward trend in 5 cases (A group of 3 cases, 2 cases in group B) patients extend the treatment to Week 48, serum HBV DNA negative rate of 80% (4/5) (group A, B group 2 cases); B group 24 weeks serum HBV DNA downward trend in three patients and adjust treatment After 24 weeks, serum HBV DNA compared with the previous decline further 2.84 ± 0.65 log10copies/ml, 48 weeks, two cases of patients with serum HBV DNA negative. In the course of treatment, in addition to the A group 1 patients had proteinuria, the two groups of patients with no serious adverse events. Conclusion: ADV joint ETV or LAM of ETV resistant CHB patients salvage therapy, but limited efficacy, combined with ETV effective than combined of LAM, can further improve the efficacy of extended course of treatment or adjust treatment according to patient response. Part III entecavir entecavir resistant hepatitis B virus genome amplification and plasmid construction purposes: improvement of HBV gene amplified by polymerase chain reaction (PCR) technology, amplification and analysis of ETV-resistant HBV gene sequences to construct whole genome containing ETV resistance mutations in HBV viral vectors. Method: optimization of sample extraction, PCR amplification protocol (polymerase reaction conditions), improved HBV genome amplification technology; amplification of 19 cases of ETV resistance in CHB patients with peripheral blood HBV genome sequences in GenBank HBV standard Chromas2.0 software analysis, comparison to determine the nucleotide and amino acid differences and variations type. Molecular subcloning technique, will be about 3.2kb, containing ETV-resistant mutations of HBV gene was cloned into the eukaryotic expression vector pcDNA3.1 () ScaI sites, build plasmids pcDNA-HBV3.2. Results: improved PCR gene amplification method successfully 16 cases of ETV resistance in CHB patients 3.2kb HBV DNA samples lowest serum HBV DNA level 3.95 log10copies/ml addition to common RT gene resistance, ETV-resistant HBV genome sequence drug variability, respectively 18.75% (3/16) and 31.25% (5/16) of patients with deletion mutations to occur PreS1 and PreS2; 5 cases (31.25%) the patients PreC District 83 nucleotide G → A substitution ; variation related to the immune cell epitopes overlapping variation 6 11 S area, RT area. The recombinant plasmid pcDNA-HBV3.2 was containing 3.2 kb HBV full-length DNA. Conclusion: Improved PCR method can improve the sensitivity of HBV whole genome amplification, ETV resistant CHB peripheral blood of patients can be successfully amplified the whole genome; ETV-resistant HBV strains in addition to resistance mutation in the RT region, S area, C area deletion variants, S, RT area overlapping variation can occur; build of pcDNA-HBV3.2 plasmid containing ETV-resistant HBV genome.