Establishment Of A Replication-competent HBV Mouse Model And A Dox-inducible And Liver-specific UPA Transgenic Mice

Objective1.To establish and identify a replication-competent HBV mouse model by hydrodynamic-based transfection and investigate its value in HBV vaccine evaluation.2.To develop dox-inducible and liver-specific uPA transgenic mice.Method1.Establishment and application of a replication-competent HBV mouse model.(1) The identification of pAAV-HBV1.3 plasmid: A replication-competent HBV DNA,pAAV-HBV1.3,contained inverted terminal repeat elements (ITR) of adeno-associated virus (AAV) and 1.3 copies HBV genome (ayw subtype),was kindly provided by Professor Wenjie Tan from National Institute for Viral Disease Control and Provention.After enzyme digestion and DNA sequence analysis, the pAAV-HBV1.3 plasmid was transfected into Huh7 cells using Lipofectamine 2000(Invitrogen Co,CA).HBsAg and HBeAg were detected in cell culture supernatant 72 h post-transfection.(2) The establishment of a mouse model with acute HBV infection: 12 micrograms of pAAV-HBV1.3 plasmid was transfected into C57BL/6 mice via the tail vein in a volume of PBS using a hydrodynamic in vivo transfection procedure.These mice were sacrificed on day 10, 30 and 60 after plasmid injection.The serum specimens were assayed for the expression of HBsAg, HBeAg and the quantity of HBV DNA by ELISA and Real-Time PCR respectively.The liver of each mouse was divided into two parts, one part was preserved in formaldehyde solution for HE and immunohistochemical analysis and the other was lysed for Real-Time PCR assay.(3) Development of the replication-competent HBV mouse: The mouse was injected intraperitoneally triple with 0.2 milliliter dexamethasone(50mg/Kg) every two days before pAAV-HBV1.3 plasmid transfection. The serum specimens were assayed for HBsAg,HBeAg and HBV DNA at the indicated times after injection.(4) Identification of the replication-competent HBV mouse using commercially available vaccine: C57BL/6 mice were immunized 2 times at 14 days interval by intramuscular injection with commercially available vaccine(An Zai Shi). 20 micrograms pAAV-HBV1.3 plasmid in 1.6 millilitres PBS buffer solution was injected hydrodynamically into the tail veins of C57BL/6 mouse after the protective antibody(HBsAb)appeared. The serum were assayed for the expression of HBsAg,HBeAg and HBV DNA at indicated time by commercial ELISA kit.(5) Application of the replication-competent HBV mouse in vaccine evaluation: A candidate vaccine AdS contained HBV preS1-S-preS2 gene was provided by Director Shi-chun Lu from Section of Hepatobiliary Surgery in Beijing You-An Hospital. C57BL/6 mice were immunized 5 times at 14 days interval by intramuscular injection with commercially available vaccine and AdS respectively. 20 micrograms pAAV-HBV1.3 plasmid in 1.6 millilitres PBS buffer solution was injected hydrodynamically into the tail veins of C57BL/6 mouse after the HBsAb appeared. At different specified time, the serum were collected and assayed for the expression of HBsAg and HBeAg by commercial ELISA kit and virus load by Real-Time PCR.2.Generation of a dox-inducible and liver-specific uPA transgenic mice(1) Construction of pTet-on Albumin: pTet-on Albumin was constructed by cloning albumin promoter and enhancer sequence into the pTet-on plasmid.After testing the transcriptional activity of the albumin promoter in Huh7 cells, the pTet-on-Albumin was linearized and injected into superovulated pronuclear zygotes to produce transgenic mice.The transgenic founders were identified by PCR and the positive ones were crossed with C57BL/6 mice. Expression of rtTA protein in tissues of F1 generation was detected by RT-PCR and Western blot.(2)Construction of pTRE2–uPA : pTRE2–uPA was constructed by cloning mouse uPA cDNA fragment and exon 11 sequence of uPA into the vector pTRE2. The biological activity of uPA were detected by cotransfection of pTet-on and pTRE2 -uPA into Huh7 and followed by the induction with doxycycline(Dox) 24 hours later. Another 36 hours later, the cells and supernatant were collected respectively and the expression and biological activity of uPA were detected. pTRE2 -uPA was linearized and injected into superovulated pronuclear zygotes to produce transgenic mice.The transgenic founders were identified by PCR and the positive ones were crossed with C57BL/6 mice. The F1 generation was detected by PCR.(3) Generation of a Dox-inducible and liver-specific uPA transgenic mice : The pTet- on-Albumin transgenic mice crossed with the pTRE2 -uPA transgenic mice.The pTet- on-Albumin–uPA double transgenic mice were detected by PCR and were maintained on regular drinking water until they reach 4 weeks old.These mice were sacrificed after Dox- induced for 12 weeks. The serum specimens were assayed for ALT and the liver specimens were assayed for HE and immunohistochemical analysis.Results1,Establishment and application of a replication-competent HBV mouse model.(1) The identification of pAAV-HBV1.3: pAAV-HBV1.3 was identified by BamHⅠand EcoRⅠdigestion respectively,the expected 7007bp,150bp,912bp,766bp and 10,187bp fragments could be observed.DNA sequence analysis shows accordance with that of the NCBI database.Compared with control group,HBsAg and HBeAg could be detected on 72 hours post-transfection with the pAAV-HBV1.3. The optical density(OD) value was 0.309( HBsAg) and 1.279 (HBeAg) respectively.(2) The identification of a mouse model with acute HBV infection: The HBsAg and HBeAg were positive in serum and liver of experimental mice at day 10 after transfection, then they became negative at day 30 and 60. Meanwhile the viral load in serum and liver in experimental group were significantly higher than that in control group at day 10, 30 and 60 after transfected and the peak point was on day 10. Immunohistochemical analysis showed that the HBsAg and HBcAg were expressed in hepatocytes at day 10.(3) The prolong expression of HBV antigen in the developed replication-competent HBV mouse model: The serum HBsAg and HBeAg of the four groups immunosuppressive mice were significant difference(P<0.01,P<0.05). The HBV antigens expression were longest in the IP Dex 8 weeks group. HBsAg and HBeAg in immunosuppressed mouse model were positive until 8 weeks.In conclusion,by suppressing the immune status of mice injected with dexamethasone, the replication of the virus and expression of HBV antigens were extended longer than that in normal adult mice.(4) The replication-competent HBV mouse model was effective in vaccine evaluation: The HBsAb was detected in commercially available vaccine (An Zai Shi) group after the second immunization. The HBsAg and HBeAg were negative after the transfection of pAAV-HBV1.3 in An Zai Shi group, and positive in the NS group.(5) The evaluation of a candidate vaccine using the replication-competent HBV mouse model: The HBsAb was detected in An Zai Shi group after the second immunization.The difference between the other two groups was significantly (P<0.01,P<0.01). The HBsAb was detected in AdS group after the fouth immunization and there were significant difference when compared with NS group(P<0.01). After transfection with the pAAV- HBV1.3 plasmid, HBsAg and HBeAg were negative in An Zai Shi group. However, NS group's HBsAg and HBeAg endurance time were significantly longer than AdS group's (P<0.05,P<0.05). The viral load in AdS group and NS group were significantly higher than that in An Zai Shi group(P<0.05 ,P<0.05).There were significant difference between AdS group and NS group after transfection 20 days(P<0.05).2,Generation of Dox-inducible and liver-specific uPA transgenic mice(1) The constructin of pTet-on-Albumin: pTet-on-Albumin was identified by BamHⅠd igestion, the expected 3026bp,3026bp,2681bp and 2000bp fragment can be observed. Cotransfection with pTRE2-EGFP and Dox-induced for 24 hours,the expression of green fluorescent could be observed, which confirmed the transcriptional activity of albumin promoter . The genomic DNA of transgenic offsprings were isolated and identified by PCR assay. The rtTA mRNA and protein were only detected in the live tissues of offspring after Dox-induced.(2) The constructin of pTRE2- uPA : pTRE2- uPA was identified by SmaⅠ/ HindⅢdigestion, the expected 3549bp,1027bp,960bp and 158bp fragments can be observed. Cotransfection with pTet-on and Dox -induced for 36 hours, the expression and biological activity of uPA could be detected. After microinjection,the genomic DNA of transgenic offsprings were isolated and identified by PCR assay.(3) Generation of Dox-inducible and liver-specific uPA transgenic mice: The pTet- on-Albumin transgenic mice crossed with the pTRE2 -uPA transgenic mice.The double transgenic mice were identified by PCR. HE results showed multiple spotty necrosis in liver tissues in positive mice after Dox induced. Immunohistochemical analysis showed the uPA expression in hepatocytes.The serum ALT level for the double transgenic mice in induced state was higher than that when non-induced. Conclusion1.An acute HBV-replicating mouse model was developed successfully by hydrodynamic-based transfection. By suppressing the immune status of mice injected with dexamethasone, the expression of HBV antigens was extended longer than that in normal adult mice.2.The commercially available vaccine was used for model evaluation, the results show that the model can be used for vaccine evaluation effectively. Then the model was used to evaluate a new vaccine,the candidate AdS DNA vaccine have better immune effect.3.The inducible double transgenic mouse was breeding for 5 generations successfully,which set the foundation for .the study of the chimeric mouse model.