| Objective To construct a recombinant and eukaryotic expression vector pcDNA3.1-preS1-GFP containing both green fluorescent protein(GFP)gene and hepatitis B virus (HBV) preS1gene and to detect expression of this recombinant plasmid in mice sperm.Methods Polymerase chain reaction (PCR)was applied to amplify HBV preS1 gene, plasmid pBR322-HBV being the template. Both the amplified product and GFP containing eukaryotic expression vector pcDNA3.1-GFP underwent double digests with two enzymes, namely EcoR I and Xba I. And T4 ligase was used overnight. Following double digests, sequencing and transfection of sperm with LipofectamineTM2000, expression of GFP was detected with a fluorescence microscope and PCR was applied in detecting the presence of HBV preSl gene.Results Sequencing confirmed that the eukaryotic expression vector pcDNA3.1-preS1-GFP was successfully constructed after recombinant DNA technique, in which preS1 gene was inserted into plasmid pcDNA3.1-GFP, and double digests were done. Fluorescence microscopy revealed green and relatively bright fluorescence in the sperm head after the recombinant plasmid was delivered into mice sperm at some ratio. DNA from transfected sperm was extracted and amplified. And amplified product was 589bp locating in the HBV preSl gene area, as was expected.Conclusion The present study showed recombinant plasmid pcDNA3.1-preS 1-GFP was successfully constructed. This made it possible to establish new modes of HBV vertical transmission in mice. Expression of the recombinant and eukaryotic expression vector pcDNA3.1-preS1-GFP in mice sperm demonstrated that the recombinant plasmid pcDNA3.1-preS1-GFP can be used to produce transgenic mice, with the latter can be seen as a report gene in regard to the presence of HBVpreS1 gene. Thus the present study lay an experimental foundation for the establishment of new modes of HBV vertical transmission in mice and better understanding of the mechanism of HBV vertical transmission. |
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