The Generation Of The Transgenic Mice Harbouring Hepatitis B Virus (adr Subtype) PreS2-S Gene

Human hepatitis B virus is the prototype for a family of viruses, referred to as hepadnaviridae. It has at least 4 subtypes, ayw, adr, ayr, and adw, among which subtype adr is the most prevailing in China. The complete genomic DNA of subtype adr has been cloned in China. It is only 3.2kbp in length and different from the other 3 subtypes in DNA and protein sequence. Human hepatitis B virus encodes 4 overlapping reading frames. Among them, the ORF which codes the envelope protein is divided by two initiation codons into L, M, S protein.L protein includes gene preSl, preS2 and S, M protein includes gene preS2 and S, S protein is coded by gene S. The functions of M protein are not clear. Due to the usefulness of transgenic model to identify the functions of genes, the transgenic mouse system of M protein will be an ideal experimental model to study the functions of M protein, which could provide new insight into it.In the present study, we used preS2-S gene (adr subtype) expression vector pcDNA3-S2-S, containing CMV promoter and preS2-S gene open reading frame by recombination DNA technique. It was identified by restriction endonucleases digestion and confined by DNA direct sequencing. The sequence of preS2-S gene was coincident with the reported preS2-S gene sequence. After restriction enzyme BglII digestion, the coding elements were micro injected into male pronuclei of mice zygotes. Following microinjection, the embyros were transferred to oviducts of psedopregnant females. 23 pups were born and survived. 5 of them were verified to integrate the preS2-S gene in their genomic DNA by multiplex PCR assay, named C57-TgN(preS2-S/adr)SMMU. With immunohistochemistry, M protein was detected mainly in hepatocytes cytoplasm of C57-TgN(preS2-S/adr)SMMU transgenic mice. Then the transgenic mice were mated to normal mice to establish transgenic mice strains. Now one of them had F2 offsprings.These results suggest that preS2-S gene could be steadily inherited to the subsequent generations and express M protein. This transgenic mouse line is useful for studying the functions of the M protein and may be prospective to develop the therapeutic HBV DNA vaccine based on theprS2-S gene.