Study On A Novel Hepatitis B Virus Multi-epitope Vaccine With Specific SiRNA Expressing Mediated By AAV

According to the latest statistics in2010provided by Ministry of Health, over93millionpeople in China are HBV carrier without symptom, whose infectivity are50-100timesstronger than AIDS, with over1million sufferer per year. Hepatitis B immunity tolerance isthe major failure cause for treatment of hepatitis B. The medicine for clinical treatment ofhepatitis B has such problems as high drug resistance, low tolerance and adverse reaction.Such features as faults of the traditional second traditional vaccine (such as no immuneresponse in some vaccinated people, adverse reaction, null on HBV patients) and unblockedvirus made the nucleic vaccine with prevention and treatment became hot. Although thenewly RNAi technology for treatment of hepatitis B has made progress, the weakorganizational specificity, weak deliver constancy and inefficiency of carrier delivery havebrought the medicine development to bottleneck.This research uses the Type II promoter hAAT to construct liver specificity siRNAexpression unit to target HBV HBsAg gene. The multiple-epitope antigen gene HB isconstructed with the help of artificial selected HBV epitope. The new HBV multiple-epitopevaccine with AAV DNA specify expressing siRNA, which made solid ground for breakingHepatitis B immune tolerance, eliminate the adverse response based on RNAi technologymedicine and improve the expression vaccine efficiency.First, the specificity promoter hAAT terminator U13’box, two copy enhancer APOE andsiHBsAg gene are reconstructed liver specificity expression siHBsAg unit from PCRconstruction. The expression unit is inserted into the AAV-MCS plasmid to makereconstruction plasmid pAAV-siHBsAg, as well as testing siRNA inhibition effect by ELISA.The protein gene, integrated with EGFP which are reconstructed by HBV multiple-epitope, isintegrated into pAAV-siHBsAg plasmid to make pAAV-siHBsAg-HE. The reconstructedplasmid pAAV-siHBsAg-HE, packing plasmid pAAV-RC and auxiliary plasmid pHelper areused to transfect AAV-293cell in order to produce virus rAAV-siHBsAg-HE andpurifying/condensing through anion/cation exchange and centrifugal ultra-filtration. Theconstructed virus rAAV-siHBsAg-HE infect HT1080cell. The reconstruction effect andHB-EGFP expressed in AAV virus are detected by observing green light. The virus titers aretested through flow cytometry detection report for gene EGFP. The ELISA detection is usedto test virus carrier related to gland. Finally, the reconstructed plasmid pAAV-siHBsAg andpAAV-siHBsAg-HE are successfully constructed. EGFP green light observation shows theeffective delivery of HB in rAAV. The AAV virus with high-purity, high enrichment and high titer are got though protein electrophoresis detection and virus electric mirror picture. TheELISA detection showed that the constructed specific expression siRNA plasmid and AAVvirus all have braking effect; the successfully construction and detection of new hepatitis Bmultiple-epitope vaccine with the specificity expression AAV mediate siRNA has providedexperimental base for further research of HBV DNA vaccine.