Experiment On Expression Of HBV Core Gene In Eukaryotic Cell

Objectives1. To research HepG2 cell infected with HBV and How HBV replicated and the expression of HBcAg.2. To constrret eukaryotes expression vector of HBV.,ore gene with its regulatory sequence.3. To research the expression of HBV core gene in HepG2 and the role of upper regulatory sequence.4. To research the integration mechanism of HBV DR area.Methods1. Human Hepatoma(HepG2) Cells was infected with HBV that was in the sera positive for HBsAg, HBeAg and anti-HBc in vitro and the HBV DNA concentration in this sera was 1.5 X 1010copies/ml which was tested by fluorescence quantification, then HBeAg in the cell culture supernatant was tested by ELISA, HBcAg by immunol histochemistry(SABC), HBV cccDNA by PCR, HBc mRNA by RT-PCR.2. HBV core gene with upper regulatory sequence was amplified by recombinant PCR, after it was double digested, then was linked to pcDNA3.1(+) vector whose CMV promoter was damaged using T4 DNA Ligase, the pcDNA3.1(+)-HBV X-C eukaryotes expression vector was constructed.3. Transfect pcDNA3.1(+)-HBV X-C eukaryotes expression vector into HepG2 cell with LipofectamineTM2000, the transfected cell lines were selected by G418, HBcAg and HBxAg were tested by immunol histochemistry(SABC), HBeAg by ELISA, HBx mRNA and HBc mRNA by RT-PCR.4. Extract total DNA from HepG2 whose genome was integrated with HBV X-C DNA sequence, then X-C, X and C DNA sequence were amplified by PCR.Results1. The infected HepG2 cells were washed 8 times using PBS, then after culturing 12h, HBeAg cound be detected by ELISA in the cell culture supernatant, but the concentration of HBeAg falled down quickly, and HBcAg coundn't be detected by immunol histochemistry(SABC), HBc mRNA coundn't be detected by RT-PCR, and also HBV cccDNA had not been detected by PCR all the time.2. The recombinant PCR method for amplifying HBV X-C sequence was set up and the pcDNA3.1(+)-HBV X-C eukaryotes expression vector was constructed Successfully.3. The recombinant pcDNA3.1(+)-HBV X-C eukaryotes expression vector cound express out HBcAg in HepG2 cells, but HBeAg had not been detected by ELISA in the cell culture supernatant.4. The total DNA of HepG2 cells was extracted from positive cell line, it cound only detect X gene and C gene by PCR, but the whole X-C DNA sequence had not been detected.Conclusions1. Whether HepG2 cells can be infected with HBV that was in the sera positive for HBsAg, HBeAg and anti-HBc in vitro should made a further research.2. We had set up a method of amplifying HBV X-C DNA sequence which has two gaps in this area by recombinant PCR, constructed pcDNA3.1(+)-HBV X-C eukaryotes expression vector successfully, laid the foundation for a future research about expression regulation of HBV core gene.3. Under the regulation of the upper regulatory sequence, HBV core gene can be expressed successfully in HepG2 cells, but the secretion of HBeAg may be also regulated by other factors except gene regulation.4. X gene and C gene can be integrated into HepG2 genome separately, but X-C DNA sequence can not be integrated into HepG2 genome as a whole, which may related to the replication and integration of HBV DR area.