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A Study On Establishment Of Hepatitis B Virus (HBV)Mouse Models

Posted on:2014-03-21Degree:MasterType:Thesis
Country:ChinaCandidate:H J YangFull Text:PDF
GTID:2284330467985119Subject:Pathogen Biology
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Aim1. To establish and identify a mouse model supporting HBV persistence2. To establish an inducible HBV expression system based on Cre-loxP recombination and its corresponding inducible transgenic mouse modelMethod1. Establishment of a mouse model supporting HBV persistence(1) Identification of the plasmid HBV1.3-56:the plasmid HBV1.3-56containing1.3copies HBV genome was constructed based on HBV genome with serotype adw and genotype B as template and pUC19as vector. Identified by restriction analysis, the plasmid was transfected by Huh7cell line. HBsAg and HBeAg in cell culture supernatant were tested after72h by ELISA; HBV DNA and RNA were detected by Southern Blot and Northern Blot.(2) Establishment of a mouse model supporting HBV persistence:the plasmid HBV1.3-56was delivered by hydrodynamic injection into the liver of BALB/c mouse via tail vein. Collect serum of the treated mouse every week after injection till the17th week and HBsAg and HBeAg were tested by ELISA; HBV DNA and RNA in treated mouse liver were detected by Southern Blot and Northern Blot.2. Construction of an inducible HBV expression system and establishment of the corresponding transgenic mouse model(1) Construction of an inducible HBV expression system:based on Cre-loxP recombination system and mechanism of intron excision in post-transcription, we insert an exogenous gene, including five different polyA signal sequences and DsRed gene promoted by EnhancerⅡ/Cp, which were flanked by IoxP sites and intron sites into S gene region of1.3copies of HBV genome (serotype adw, genotype B) and then constructed the plasmid pHBV-intron-REP (HnR). Cre recombinase was expressed by pShuttle-Cre plasmid and mediated the recombination of the sequence between two loxP sites. Therefore, HBV expressed in a controlled manner.(2) Identified by restriction analysis, the inducible HBV expression system was proved in vitro and in vivo. In three groups including inducible group (HnR/pShuttle-Cre), un-induced group (HnR/pShuttle) and positive control group (HBV1.3-56), plasmids were transfected into Huh7cell line or delivered into the mice’s liver. ELISA, Southern Blot and Northern Blot were carried on separately to test the HBV expression on protein, DNA and RNA levels.(3) Establishment of HnR transgenic mice:HnR plasmid was digested by KpnI/PciI into the linear DNA fragment. HnR transgenic founder mice were created through microinjection of the DNA fragment into C57/CBA pronucleus and PCR identification. After mating with wildtype C57BL/6for several generation and with positive transgenic mice in the same brood, the HnR transgenic mice were established。Results1. Establishment of a mouse model supporting HBV persistence(1) Identification of HBV1.3-56:after restriction analysis of HBV1.3-56, the bands were the same as we expected. After transfected into Huh7cell line, HBsAg and HBeAg were positive; Southern Blot showed corresponding replicative DNA intermediates and Northern Blot showed viral transcripts.(2) Identification of the mouse model supporting HBV persistence:After hydrodynamic injection of plasmid HBV1.3-56into BALB/c mice, HBsAg and HBeAg were secreted and kept at a detectable level for almost4months despite of declining as days increasing. The level of HBeAg was coincident with but slightly lower than that of HBsAg. Viral replication and transcription could be detected in the first19days after injection. However, when RNA transcription decreased, DNA replication was robust.2. Construction of inducible HBV expression system and establishment of inducible transgemc mouse(1) Identification of inducible HBV expression system:after restriction analysis of HnR plasmid and pShuttle-Cre plasmid, the results were the same as we expected. In vitro, the red fluorescent protein could be observed in un-induced group (HnR/pShuttle) which was different from the observation in inducible group; HBsAg could be induced to secrete in indubible group as in positive group and did not express in un-induced group. As the insertion cannot stop HBeAg expression, HBeAg can be detected in three groups; HBV replicative DNA intermediats and transcripts could be detected in inducible group and positive group; RT-PCR showed recombination in inducible group. In vivo, HBsAg, viral replicative DNA and RNA transcripts could be detected in inducible group and positive group but not in un-induced group; HBeAg secreted in all three groups; RT-PCR showed recombination in inducible group. All of these results indicated the inducible HBV expression system worked.(2) Establishment of HnR transgenic mice:After HnR transgenic founder mice were crossed with wildtype C57BL/6and mice in the same brood were crossed with each other, we obtained HnR transgenic mice.Conclusion1. We established a mouse model supporting HBV persistence by hydrodynamic injection. HBeAg and HBsAg can persist for at least4months; viral replicative DNA intermediates and RNA transcripts can be detected at the first19days. These results were different from the previous reports which means this model can be well studied in the field of HBV chronic infection.2. We constructed successfully an inducible HBV expression system. Identification in vitro and in vivo all suggested the HBsAg, viral replicative DNA intermediates and RNA transcripts could be induced in a controlled manner. Establishment of inducible HBV transgenic mice came over the limitation of conventional transgenic mice which mice were tolerant to the transgenic products. We can control HBV expression spatially and timely in animal models.
Keywords/Search Tags:Hepatitis B virus, hydrodynamic injection, animal model, transgenicmice
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