Experimental Study Of Antiviral Effects On Hepatitis B Virus With The Recombinant Eucaryotic Plasmid PcDNA3.1-MxA

PART I CONSTRUCTION AND IDENTIFICATION OF THE RECOMBINANT EUCARYOTIC PLASMID PCDNA3.1-MXAObjective: To construct and identify the recombinant eukaryotic plasmid PcDNA3.1-MxA encoding MxA with the eukaryotic plasmid PcDNA3.1.Methods: Recombinant plasmid PAdTrack-MxA which was constructed before and PcDNA3.1 were amplificated in Escherchia coli JM109. Plasmids were double-digested with restrictive endonucleases Not I and Xba I after extracted, and then ligated to construct recombinant eukaryotic plasmid PcDNA3.1-MxA. The recombinant plasmid was selected for Ampicillin resistance. And it was confirmed by digestion with Not I and Xba I , PCR and sequencing. The sequence was homology-analysed compared with the sequence of MxA gene in PAdTrack-MxA and the sequence of MxA gene published in Genebank( NM₀02462) with sofeware DNAssist 2.0.Results: The restrictive endonuclease and PCR analysis confirmed that the recombinant eukaryotic plasmid PcDNA3.1-MxA was constructed successfully. Sequencing analysis revealed that the nucleotide sequence of MxA gene was same to its in PAdTrack-MxA exactly. There were five variations in nucleotide sequence compared with published sequence in Genebank which caused only one amino acid residue replaced from isoleucine to valine. This replaced amino acid residue was located in the non functional area of MxA protein.Conclusions: The recombinant eukaryotic plasmid PcDNA3.1-MxA is constructed successfully. It establishes the foundation for the further study of anti-HBV effects of MxA.PART II EXPERIMENTAL STUDY OF ANTIVIRAL EFFECTS ON HEPATITIS B VIRUS WITH THE RECOMBINANT EUCARYOTIC PLASMID PCDNA3.1-MXAObjective: To investigate the anti-HBV effects in HepG 2.2.15 cells with the recombinant eukaryotic plasmid PcDNA3.1-MxA in vitro. Methods: The recombinant eukaryotic plasmid PcDNA3.1-MxA was transfected into HepG 2.2.15 cells with Lipofection after extracted and confirmed with restrictive endonuclease analysis. According to the neomycin resistance of PcDNA3.1-MxA, HepG2.2.15 cell clone containing PcDNA3.1-MxA was screened out by G418. Two groups was setted up: the treatment group was HepG2.2.15 cells with PcDNA3.1-MxA and the control group was HepG2.2.15 cells without PcDNA3.1-MxA. The levels of MxA mRNA were detected by RT-PCR and the tites of HBsAg and HBeAg were detected by ELISA.Result: HepG2.2.15 cell clone containing PcDNA3.1-MxA was screened out by G418 at 600μg/ml at the 3rd week. The RT-PCR results showed the level of MxA mRNA in the treatment group was much higher than that of the control group. The tites of HBsAg and HBeAg in the treatment group were lower significantly than those in the control group cells(P<0.01).Conclusion: It indicates that MxA protein can inhibit replication and expression of HBV.