【作者】 段鸿元；

【导师】 秦鄂德；

【作者基本信息】 中国人民解放军军事医学科学院 ， 微生物学， 2003， 硕士

【摘要】 登革热是由登革病毒引起的蚊媒病毒病，每年全世界登革热患者达一亿。目前对于该病尚缺乏安全有效的疫苗。登革病毒是单股正链RNA病毒，基因组全长约11Kb，含有单一开放读码框架。登革病毒膜蛋白前体prM和包膜蛋白E及非结构蛋白NS1均含有多种抗原表位，因此这些特异的病毒蛋白为登革新型疫苗的主要靶标。 DNA疫苗可以刺激动物产生体液免疫和细胞免疫。但是通常DNA疫苗的免疫原性较弱。将多种DNA疫苗混合后共同免疫将提供针对多种抗原表位的免疫应答，并有可能提高疫苗的免疫原性。本研究采用RT-PCR法获得了登革3型病毒的prME和NS1基因片段，分别将其插入真核表达载体pcDNA6／V₅·His-B，C，构建了两个真核表达重组质粒。将构建的重组质粒DNA分别转染真核细胞后用免疫荧光法可检测到外源蛋白的表达。然后将两种重组质粒DNA以混合及单独形式分别免疫BALB／C小鼠，免疫的小鼠可产生中和抗体和特异性CTL。在末次免疫后第33天，双质粒重组DNA组与prME重组质粒DNA组的中和抗体水平均可达到1：32。在术次免疫后第41天，当效靶比为40：1时，双质粒重组DNA免疫组的特异性CTL杀伤率为15％，而两个单质粒DNA组分别为10.9％和12.4％。这些结果表明，配伍组重组质粒DNA可同时诱发小鼠体液免疫和细胞免疫，而且产生的细胞免疫应答具有一定的增强效果。 在进一步对所构建的登革3型病毒prME和NS1基因重组质粒DNA及其混合质粒DNA的免疫保护作用进行评价中，由于无合适的小鼠模型，本研究通过采用观察免疫小鼠血清中中和抗体水平的变化进行评估。将不同的重组质粒DNA免疫小鼠后用登革3型病毒进行攻击，分别检测中和抗体的产生情况。在病毒攻击前3天，prME和NS1基因双质粒DNA免疫组与prME基因重组质粒DNA免疫组的中和抗体滴度均为1：4。在攻击病毒后第4和8天，摘要双质粒DNA组的中和抗体滴度分别为1:8和1:4。在攻毒后第15和22天，双质粒DNA组中和抗体上升到1:8和1:16。说明双质粒DNA组诱发的中和抗体在攻毒后可再次升高。而单一的PrME重组质粒DNA组在攻毒后第4天中和抗体水平从攻毒前的1:4上升到1:32。说明单一的PrME重组质粒DNA可在攻毒后使小鼠诱生中和抗体的能力大幅提高。 由于登革3型病毒的PrME基因编码结构蛋白，其重组质粒 DNA免疫小鼠可诱导产生中可抗体，而非结构蛋白NSI基因的重组质粒DNA主要诱导产生细胞免疫。上述结果表明，PrME和NSI双质粒DNA免疫组诱导小鼠产生中和抗体的水平与单一PrME重组质粒DNA的基本一致，而其诱导小鼠产生的细胞免疫反应略高于两个单一重组质粒DNA诱导产生的细胞免疫。本研究为进一步深入探讨登革新型DNA疫苗提供依据。更多还原

【Abstract】 Dengue fever is a kind of mosquito-borne viral diseases caused by dengue viruses(DEN). The figure of dengue fever patients add up to 100 million every year around the world. The effective and safe dengue vaccines are not available yet. The genome of dengue virus is a single-strand positive sense RNA of approximately 11 Kb in length, containing a single open reading frame. The pre-membrane protein prM, envelope protein M and nonstructural protein NS1 of DEN, containing different sorts of antigenic epitopes, have become the major targets of novel dengue vaccines.DNA vaccines can elicit both humoral immune responses and cell-mediated immune responses. However, the immunogenicity of DNA vaccines is weak. The combined vaccines can induce different immune responses targating to various epitopes, thus possibly to improve the immunogenicity of DNA vaccines. PrME gene and NSI gene of dengue type 3 virus were obtained by RT-PCR and were inserted into eukaryonic expression vector pcDNA6AVVHis-B,C to construct two recombinant plasmid DNAs. Then these recombinant plasmid DNAs were transfected into eukaryotic cells, respectively. The foreign proteins were expressed and detected by IFA. To observe the immunogenicity of recombinant plasmid DNAs, two plasmid DNAs were used to immunize BALB/C mice in combined and single form, respectively. As the result, specific cytotoxic T-cell (CTL) response and neutralizing antibodies were produced in corresponding groups. On the 33th day after the latest immunization, the nutralizing antibody level of the group immunized by both combined plasmid DNAs and recombinant plasmid DNA containing prME gene was 1:32. On the 41th day after the latest immunization, when the E/T ratio was 40:1, the percent of specific cytotoxicity of the group immunized with the combinant plasmid DNAs was 15%. While two groupsimmunized with single plasmid DNAs were 10.9% and 12.4%, respectly. These resultsindicated that combined plasmid DNAs were able to induce a wide spectrum of humoral immune response and cell-mediated immune response simutaneously, and the cell-mediated immune response was improved in some degree.Because of lacking ideal mice model, the neutralizing antibody of immunized mice was detected to evaluate the immune protective ability of plasmid DNAs containing prME and NS1 gene of DEN-3. BALB/C mice were challenged with dengue type 3 virus following the immunization by recombined plasmid DNAs. Then the nutralizing antibody was tested. On the 3rd day of pre-challenge, the neutralizing antibodies of both the group immunized with the combined plasmid DNAs and the group immunized with the recombinant containing prME gene were all 1:4. On the 4th and 8th day after the challenge, the nutealizing antibodies of the group immunized by combined plasmid DNAs were 1:8 and 1:4. On the 15th and 21th day post challenge, its level rised to 1:8 and 1:16. This result revealed that another rise of the nutralizing antibody happens after the virus challenge in the group immunized by the combined plasmid DNAs. While, on the 4th day post-challenge, the nutralizing antibody of the group immunized by single recombinant plasmid DNAs containing prME gene has risen from 1:4 of pre-challenge to 1:32 of post-challenge. It suggested that single plasmid containing prME gene could induce humoral immune response and cell-mediated immune response in mice at the same time.The recombinant plasmid DNA containing prME gene which codes a structural protein of DEN can induce neutralizing antibody. While, the recombinant plasmid DNA containing NS1 gene of nonstructural protein mainly elicits cell-mediated immune response. This study reflected that the combined plasmid DNAs had the same ability as the single recombinant plasmid containing prME gene to induce neutralizing antibody. The cell-mediated immune response induced by the combined plasmid DNAs was slightly higher than those induced by the two single recombinant plasmid DNAs. This work laid some basis for the further study of novel dengue vaccine.更多还原