| Base on DHBV CH4 (GenBank:EU429324) Cap gene which is our laboratory submitted before and staphylococcus aureus nuclease gene (GenBank:CP006630.1) sequence information, the primers of Cap and SNase gene were designed and then them was amplified by PCR, DHBV clinical sample and staphylococcus aureus as temple. Carried out the following research:1. Prokaryotic expression of Staphylococcus aureus SNase gene and its activity detectionThe SNase gene was amplified using PCR assay and then clone to PGM-T. The plasmid was incloned to Prokaryotic expression plasmid pET-32a (+) after sequencing and double enzyme digestion, named pET-32a (+)-SNase, and then transformed into the Escherichia coli Rosetta with IPTG to induce expression of SNase. The expression protein was obtained and it was further detected using TDA discoloration assay. The results showed that we successfully cloned SNase gene 468 bp which was same with expected size. Then we constructed the recombination prokaryotic expression plasmid pET-32a (+)-SNase and transferred into Rosetta. IPTG is using to induce the expression of plasmid. Finally, we successfully got about 35 KDa nuclease protein, In addition, it have SNase activity. Our study provides a new insight into selection of antiviral agent.2. Prokaryotic expression, activity detection and Preparation of Polyclonal Antibodies of fusion protein of DHBV capsid protein and staphylococcus aureus nucleaseBase on DHBV CH4 (Genbank:EU429324) whole Cap gene and staphylococcus aureus nuclease gene (Genbank:CP006630.1) sequence information, the primers of Cap and SNase gene for fusion PCR. DHBV positive serum as temple, Cap gene was amplified using PCR and staphylococcus aureus SNase gene was performed with staphylococcus aureus genome. Cap and SNase gene were fused by PCR. Then fusion gene was ligated into PGM-T vector and detected by enzyme digestion and sequencing, and then subcloned to pET-32a (+) expression vectors. The recombined vector pET-32a (+)-Cap-SNase was transfected into E.coli. Rosetta; In order to enhance the production of the fusion protein, we optimized conditions (temprature, IPTG and time), induced with IPTG and analysised by SDS-PAGE. The prokaryotic expressed fusion protein was purified by Ni-NTA, which was detected by rabbit anti-DHBV Cap antibody; Kunming strain mouse was treated by the fusion protein as antigen to prepare for polyclonal antibody and then detected the activity of fusion protein.The results showed that we successfully cloned and fused Cap-SNase gene 1236 bp gene. The recombined prokaryotic expression vector pET-32a (+)-Cap-SNase was transferred into E.coli. Rosetta. The fusion protein was successfully expressed about 60 KDa. The best optimized condition was 37℃, IPTG (1.0mmol/L),6 h. The expression protein was mainly found in precipitate. The purified protein had nuclease activity. Polyclonal antibody titer was 1:8 and also expressed fusion protein had SNase activity. The study lay the foundation of eukaryotic expression of fusion protein with nuclearase activity, and production polyclone antibody of mouse.3. Establishment and instantaneous expression of DHBV Capsid Targeted Nuclease Expression System15-day duck embryo was used to culture duck embryo liver cell. Eukaryotic expression plasmid pcDNA3.1 (+)-Cap-SNase was transferred into duck embryo liver cell, and then isolated the cell total RNA and protein to detect the mRNA level and protein level by western blotting assay.The results showed that we successfully cultured duck embryo liver cell and detected the mRNA expression of recombined vector from PCR level. Eukaryotic expression plasmid pcDNA3.1 (+)-Cap-SNase was successfully expressed in duck embryo liver cell by western blotting assay with our preparated polyclonal antibody, about 47 KDa. In addition, the fusion protein had nuclearase activity.4. Apply Capsid targeted antiviral inactivation to anti-DHBV researchBased on the pMD18-T-PreS/S vector we constructed before, the real-time PCR standard curve was established and the virus titer was calculated. The vector transformed into duck embryo liver cell, and then infected DHBV and collected the supernatant in different time. Extract the viral genome in the cell supernatant, Real-time PCR was performed to detect the virus titer and PBS, empty vector served as negative control. Statistical analysis of fluorescence quantitative data through SPSS20.0.The results showed that we successfully established standard curve, fluorescence quantitative data analysis results show that the different group and different time DHBV in cell supernatant content were not significant difference (P>0.05)... |
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