| Hepatitis A(HA), which accounts for both endemic and epidemic hepatitis world wide, is caused by the hepatitis A virus(HAV). People in china infected with HAV had a total of 9.7 hundred millions, and infectious rate had exceeded 80%. HA largely harmed to people. At present there had not special therapy to HA which were mostly prevented by attenuated vaccine and inactivated vaccine. Attenuated HA vaccine is cheaper one, but it has potential hazard after injection. Inactivated vaccine has great and longer immunity, but it is expensive. The vaccines are not popularized in poor region. It was very important to develop a new HA vaccine, which is easily immunized, low charge, and good immune effect. DNA vaccine was born in 1990s. It is the third vaccine after attenuate vaccine inactive vaccine and subunit vaccine. It has great advantage. People pay more attention about it. DNA vaccine is composed with protective antigen gene and plasmid vector. Protective antigen gene can be a single gene or a group of genes cooperate with each other. It also can be linked together. HAV is a positive-strand RNA virus with a genome length of approximately 7,450 nucleotides. both (11-25aa)and VP3(101-121aa) capsid proteins are very important to induce antibody. In our study, a multi-epitopes antigen gene of HAV was fused with C gene of HBV to enhance its immunity. Then CVPX was expressed in E.coli and CHO. This research is the foundation for the further study for HAV DNA vaccine. Hybrid gene CVPX whose DNA fragement was about 561bp, was constructed by overlapping PCR with template of C gene of HBcAg and every PCR circulation product. Specific primers were planned by us. Considering the special structure of two conformational epitopes, we inserted several flexible amino acids into two epitopes. A specific DNA fragment about 561bp was recovered. Ligation of the DNA with the vector pMD18-T was done and then transformed into competent host strain Escherichia coli DH5α. Plasmids were isolated from recombinant clones by alkaline lysis, and PCR identification, restriction analysis and DNA sequencing were carried out to verify the recombinant plasmids pMD18-T-CVPX. All results showed that the CVPX DNA was the exact fragment. In this study, the recombinant plasmid pMD18-T-CVPX,pMD18-T-C were cleavaged with EcoRI ,XhoI and inserted into the expression vector pET-28a and subsequently subjected to restriction endonuclease analysis and sequencing, the result indicated that the prokaryotic expression vector pET-28a-CVPX ,pET-28a-C were constructed successfully. After the expression plasmid were extracted and transformed into expression hosts BL21(DE3) of E.coli, the transformed hosts were induced by IPTG, by SDS-PAGE analysis of host protein, the expression of the objective gene were 24KDa and 20.3KDa. The host was induced by 1mmol/L IPTG and taken out 1mml every two hours after induced 1 hour to study on the best expression condition. The result shows 5 hour after host induced is the best choice. Western Blot analysis indicate the protein of CVPX expression could react to HAV antisera. specificly. CVPX and C genes were sub-cloned into the eukaryotic expression...
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