两株登革2型病毒NS1基因真核重组质粒免疫BALB／c小鼠产生特异性抗体水平的比较  

The comparison of specific antibodies levels in the BALB/c mice immnnized with the pcDNA3.1 recombinant plasmids of NS1 gene partial sequence from different dengue type 2 virus strains

作　　者：任丽娟[1] 左丽[1] 朱海东[1] 胡方芳[1] 

机构地区：[1]贵阳医学院免疫学教研室贵州省疾病预防控制中心,550004

出　　处：《中华微生物学和免疫学杂志》2011年第4期350-355,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助（30560148）;贵州省优秀教育科技人才省长基金（2009）82号

摘　　要：目的利用登革2型病毒（denguetype2virus，DENV2）M株和NC,C株NSl基因部分别pcDNA3．1重组质粒初次和加强免疫BALB／c小鼠，观察免疫小鼠体液免疫应答的差异。方法分别构建两株DENV2NSl基因部分序列（1—413bp）的pcDNA3．1真核重组质粒和pET28a（＋）质粒，进行原核蛋白的表达、鉴定、纯化和定量；并用pcDNA3．1重组质粒免疫BALB／c小鼠，初次免疫及第7天、14天分别加强免疫1次，共免疫3次。收集初次免疫后第7、14、28和56天外周血标本，间接ELISA法测定小鼠血清特异性IgM／IgG类抗体水平，细胞病变抑制法检测特异保护性抗体水平。结果构建了pET28a（＋）-NS1m／pET28a（＋）-NS1原核表达重组质粒，SDS-PAGE分析表明，NS1基因部分序列获得表达，其相对分子质量均约22．3×10^3；Westernblot表明该目的蛋白可与抗His标签单克隆抗体结合；经Ni柱亲和层析法得到纯度达92％的表达蛋白，对C6／36细胞有毒性，并可用于ELASA检测。不同DENV2毒株NS1基因部分序列的pcDNA3．1重组质粒初次和加强免疫BALB／c小鼠诱导特异性IgM、IgG类和中和抗体的产生存在差异，M株重组质粒加强免疫小鼠后特异性抗体效价水平较高并持续较长时间。结论DENV2两毒株NS1基因部分序列重组质粒免疫小鼠后诱生的特异性抗体类别、水平存在差异。Objective To compare the humoral immune response of BALB/c mice immunized by recombinant plasmids PeDNA3.1-M-NS1 and PeDNA3.1-N-NS1. Methods Dengue type 2 virus （DENV2） NSI gene were constructed two partial sequences（ 1-413 bp） of the pcDNA3. 1 eukaryotic plasmids and pET28a （ ＋ ） plasmid for prokaryotic expression, identification, purification and quantification. The BALB/ e mice were immunized by poDNA3. 1-M-NS1, pcDNA3. 1-N-NS1 recombinant plasmids with adjuvant. Each animal received a primary inoculation and two boosts at 1-week intervals. Then the blood samples of BALB/c mice were collected from different experiment groups at day 7, 14, 28 and 56, respectively after first immunization. The specific IgM/IgG antibodies for NS1 protein in serum were confirmed by indirect ELISA. And then the activities of the specific protective antibody were determined by cytopathic effect inhi- bition（CPEI）. Results Construction of the pET28a （ ＋ ） -NSlm/pET28a （ ＋ ）-NSln prokaryotic expres- sion plasmid, SDS-PAGE analysis showed that, NS1 gene partial sequence was expressed, both the relative molecular weight of about 22.3 x 103 ; Western blot showed that the protein can bind anti-His tag monoclonal antibody; by Ni affinity chromatography with a purity of 92% protein, on the C6/36 cell toxicity, and can be used ELASA detection. The results showed that the levels of specific IgM/IgG antibody and neutralizing anti- body activities were increased in pcDNA3.1-M-NS1 booster immunization group than other groups. The re- suit had been observed longer duration of antibody level in pcDNA3.1-M-NS1 booster immunization group. Conclusion Humoral immune response were significantly different between PeDNA3.1-M-NS1 and pcDNA3.1-N-NS1 recombinant plasmid immunized mice groups.

关 键 词：登革2型病毒 NS1基因 重组质粒 BALB/C小鼠 体液免疫应答 

分 类 号：R3[医药卫生—基础医学]