| It is estimated that there are 350 million carriers of hepatitis B virus (HBV) by the year 2000 according to the WHO. Among them, approximately 5%~ 10% of adults and 80%~90% of children become chronic carriers of HBV. The long term consequence of chronic carriage are cirrhosis, liver failure and hepatocellular carcinoma . Currently, the most effective drugs used for treatment of chronic hepatitis B (CHB) are interferon (IFN) and nucleoside analogues, lamivudine. Interferon- a achieves a short-term outcomes of around 20%~30% loss of HBeAg. The efficacy is even lower in Chinese patients, who are immunotolerant to HBV because of acquisition of the disease during early childhood, than in white patients. Lamivudine, nucleoside analogues, is able to reduce HBV DNA to undetectable levels. However, cessation of treatment usually leads to a rapid relapse of disease, and long-term treatment often results in the selection of lamivudine resistant viral variants due to mutation of YMDD motif of the HBV polymerase gene. These outcomes emphasize the need for novel therapeutic agents and approaches. Specific immunotherapeutic strategies have been proposed as possiblealternatives to the use of IFN or antiviral drugs to break the non-responsiveness of T-cell immunity in chronically infected patients. Among these, specific vaccine therapies with recombinant protein vaccines and gene vaccine and peptide vaccine have been studied with animal models or in human clinical trials.The relatively low efficacy of DNA vaccines in inducing immune responses, especially in large animal species and humans, has impaired their practical use. Despite considerable effort expended on improving DNA vaccine delivery, only minute amounts of antigen(Ag) are available for immune induction following DNA vaccination. Two complementary strategies have been used to improve and modulate the immune response induced by DNA vaccines: (i) supplementing DNA vaccines with plasmids encoding cytokines and (ii) targeting the Ag encoded by DNA vaccine through genetically fusing the Ag to molecules binding cell surface receptors.In this study, we choose HBV preS2+S as targeted antigen. Five recombinant eukaryotic expression plasmids were constructed respectively. Then all of them were transfected into Vero E-6 or SP2/0 cells using LipofectAMINE and the confirmation of the tranciet expression of the fusion proteins by ELISA and IFA respectively. BALB/c mice were immunised with purified recombinant plasmids . According to the references, Bupivacaine was used as priming agents and each plasmid was directly injected into the quadriceps muscle of BALB/c mice with dose of 100ug per mouse. We compared the efficacy of pcDNA3.1 IL-2/Fc as adjuvant on HBV preS2S DNA vaccine-raised immune responses by detected the anti-HBs titers, CTL cytotoxicity level, proliferation of lymphocytes, cytokines level secreted by cultural splenocytes and expression level of CD25. We also compared immune responses induced by pcDNA3.1S2S/IFN a and pcDNA3S2S/Fc plasmids respectively.Contents and conclusions:1. Five recombinant eukaryotic expression plasmids pcDNA3.1S2S,pcDNA3.1S2S/IFN- a ,pcDNA3.1IL-2/Fc,pcDNA3S2S/Fc and pcDNA3IFN- a were constructed by molecular cloning technology respectively. The correct ligation was confirmed by restricted enzyme digestion and sequencing analysis.2. The five recombinant plasmids were transfected into Vero E-6 or SP2/0 cells using LipofectAMINE and detected by ELISA and IFA respectively. The data showed that all the proteins were exactly expressed by erkaryotic cells.3. Groups of BALB/c mice were immunised. Humoral and cellular immune responses were detected respectively.4. Anti-HBs positive serotransformation was occurred in all the pcDNA3.1IL-2/Fc immunised mice at the end of two weeks after immunization. And the CTL cytotoxicity level, proliferation of lymphocytes, level of Thl type cytokines and expression level of CD25 on splenocytes were higher tha...
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