囊尾蚴病多期复合基因疫苗的构建及免疫效应评价

发布时间：2018-03-09 22:11

本文选题：囊尾蚴病　切入点：六钩蚴　出处：《蚌埠医学院》2011年硕士论文　论文类型：学位论文

【摘要】：目的：(1)构建包含猪带绦虫六钩蚴期和囊尾蚴期特异性疫苗候选分子的囊尾蚴病多期复合基因疫苗。(2)评价该复合基因疫苗的免疫效应。方法：(1)分别提取猪带绦虫六钩蚴和囊尾蚴总RNA，应用RT-PCR方法分别扩增六钩蚴期特异性抗原TSOL18和囊尾蚴期特异性抗原cC1编码基因，另外设计引物应用重叠延伸PCR技术通过一柔性接头（linker）(Gly4Ser)3串联这两个基因，，使TSOL18基因在前，cC1基因在后，即融合基因TSOL18+linker+cC1（简写为TSOL18/L/cC1）。将TSOL18/L/cC1融合基因装载至pMD18-T载体中，并测序验证。提取pMD18-TSOL18/L/cC1质粒DNA，经Xho I和Nhe I双酶切，纯化后的酶切产物TSOL18/L/cC1基因通过T4连接酶与经Xho I和Nhe I双酶切并纯化的pVAX1真核表达质粒连接，构建pVAX1-TSOL18/L/cC1重组质粒，同时构建pVAX1-TSOL18, pVAX1-cC1。将所构建的重组质粒通过阳离子聚合物介导转染293T细胞，设转染pVAX1空载体组和未转染组转染；转染72h后分别收集细胞，提取转染后的细胞总RNA，通过RT-PCR、间接免疫荧光、Western-blot鉴定融合基因在真核细胞中的表达状态；以构建的重组质粒肌肉注射小鼠，以免疫组化检测融合基因在小鼠骨骼肌的真核表达效果。（2）以pVAX1-TSOL18、pVAX1-cC1、pVAX1-TSOL18/L/cC1经肌肉注射途径免疫BALB/c小鼠，以流式细胞术检测免疫后小鼠脾淋巴细胞胞内细胞因子等细胞免疫指标，并以间接ELISA法检测相应的特异性抗体及特异性抗体动态变化等体液免疫指标。结果：(1)构建的pVAX1-TSOL18/L/cC1重组质粒经酶切电泳和测序结果与预期设计的基因序列一致。真核细胞转染后通过RT-PCR检测显示除转染pVAX1空载体组和未转染组外，其余各组均有相应mRNA的表达。真核细胞转染后通过间接免疫荧光、Western-blot检测表明能够在293T细胞中表达出目的蛋白。免疫组化染色后在重组质粒注射部位肌肉组织的肌纤维内显示棕黄色阳性分泌颗粒，并且pVAX1-TSOL18/L/cC1组比pVAX1-TSOL18组棕黄色阳性分泌颗粒多，空载体组与空白对照组骨骼肌组织未见明显的棕黄色颗粒。（2）pVAX1-TSOL18/L/cC1组小鼠血清IgG抗体均显著高于pVAX1-TSOL18、pVAX1-cC1组及PBS组和pVAX1组(P0.05)。 pVAX1-TSOL18/L/cC1组、pVAX1-TSOL18、pVAX1-cC1组分泌IFN-γ的CD4~+T及CD8~+T细胞各亚群比例均显著高于PBS组和pVAX1组(P0.05)， pVAX1-TSOL18/L/cC1组又显著高于pVAX1-TSOL18、pVAX1-cC1组(P0.05)；pVAX1-TSOL18/L/cC1组、pVAX1-TSOL18、pVAX1-cC1组分泌IL-4的CD4~+T细胞亚群比例显著高于PBS组和pVAX1组(P0.05)，而pVAX1-TSOL18/L/cC1组、pVAX1-TSOL18、pVAX1-cC1组与PBS组和pVAX1组组间无显著差异（P＞0.05）；pVAX1-TSOL18、pVAX1-cC1组分泌IL-4的CD8~+T细胞亚群比例显著高于PBS组和pVAX1组(P0.05)，而pVAX1-TSOL18/L/cC1组与与PBS组和pVAX1组组间无显著差异（P＞0.05）。结论：（1）成功构建包含猪带绦虫六钩蚴和囊尾蚴期特异性疫苗候选分子的囊尾蚴病多期复合基因疫苗pVAX1-TSOL18/L/cC1。并且初步揭示了其在体内和体外的表达。（2）囊尾蚴病多期复合基因疫苗pVAX1-TSOL18/L/cC1较单基因疫苗pVAX1-TSOL18、pVAX1-cC1诱导更为全面的免疫应答效应。
[Abstract]:Objective: (1) to construct cysticercosis multiphase complex gene vaccine containing six cysticercus cellulosae and cysticercus stage specific vaccine candidates. (2) evaluate the immune effect of the composite gene vaccine.
Methods: (1) were extracted from Taenia solium oncosphere and cysticercus six total RNA were amplified using RT-PCR method six oncosphere stage specific antigen TSOL18 and cysticercus stage specific antigen cC1 gene encoding, and primers were designed using overlap extension PCR technique through a flexible joint (linker) (Gly4Ser) of the 3 series two genes, the TSOL18 gene in the cC1 gene, after which the fusion gene TSOL18+linker+cC1 (TSOL18/L/cC1). The TSOL18/L/cC1 fusion gene loaded into pMD18-T vector, and pMD18-TSOL18/L/cC1 sequencing. Extraction of plasmid DNA by Xho I and Nhe I double enzyme digestion, pVAX1 eukaryotic purified enzyme digestion products TSOL18/L/cC1 T4 gene by I and Nhe by Xho ligase and I double enzyme digestion and purification of the expression plasmid to construct the recombinant plasmid of pVAX1-TSOL18/L/cC1, and construct pVAX1-TSOL18, recombinant plasmid pVAX1-cC1. constructed by cationic poly Compound mediated transfection of 293T cells with pVAX1 transfected with empty vector transfected group and untransfected group; after transfection of 72h cells were collected, extracted from the transfected cells total RNA by RT-PCR, indirect immunofluorescence, expression of Western-blot fusion gene identification in eukaryotic cells with the recombinant plasmid injected mice; construction and by immunohistochemical detection of fusion gene in skeletal muscle of mice with eukaryotic expression. (2) by pVAX1-TSOL18, pVAX1-cC1, pVAX1-TSOL18/L/cC1 intramuscular immunization of BALB/c mice, with the index of cellular immunity was detected by flow cytometry after immunization of mice spleen lymphocyte intracellular cytokines, and detected by indirect ELISA method corresponding the specific antibody and antibody dynamic changes of humoral immunity.
Results: (1) gene pVAX1-TSOL18/L/cC1 recombinant plasmid by restriction enzyme digestion and sequencing results with the expected design. After transfection of eukaryotic cells was detected by RT-PCR showed that the pVAX1 empty vector group and non transfected group, the expression of other groups have corresponding mRNA. After transfection of eukaryotic cells by indirect immunofluorescence. Western-blot detection showed that can express the target protein in 293T cells. The recombinant plasmid injection site muscle fibers in the muscle tissue after staining immunohistochemistry showed positive brown granules, and the pVAX1-TSOL18/L/cC1 group than in the pVAX1-TSOL18 group Brown positive granules, empty vector group and blank control group in muscle tissue was brown and yellow particles. (2) pVAX1-TSOL18/L/cC1 mice serum IgG antibody were significantly higher than that of pVAX1-TSOL18, pVAX1-cC1 group and PBS group and pVAX1 group (P0.05 pVAX1-TSOL18/L). /cC1 group, pVAX1-TSOL18 group, pVAX1-cC1 CD4~+T and IFN- secretion of CD8~+T cell subsets were significantly higher than that of PBS group and pVAX1 group (P0.05), pVAX1-TSOL18/L/cC1 group was significantly higher than that of pVAX1-TSOL18 group (P0.05, pVAX1-cC1); group pVAX1-TSOL18/L/cC1, pVAX1-TSOL18, CD4~+T cell subsets pVAX1-cC1 secretion of IL-4 was significantly higher than PBS group and pVAX1 group (P0.05), pVAX1-TSOL18/L/cC1 group, pVAX1-TSOL18 group and PBS group, pVAX1-cC1 group and pVAX1 group showed no significant difference (P > 0.05); pVAX1-TSOL18, the proportion of CD8~+T cell subpopulation group pVAX1-cC1 secretion of IL-4 was significantly higher than that of PBS group and pVAX1 group (P0.05), pVAX1-TSOL18/L/cC1 group and PBS group and pVAX1 there was no significant difference between groups (P > 0.05).
Conclusion: (1) the successful construction of cysticercosis contains multiple pVAX1-TSOL18/L/cC1. gene vaccine of Taenia solium oncosphere and cysticercus six stage specific vaccine candidate molecules and reveal its expression in vivo and in vitro. (2) cysticercosis and multiple gene vaccine pVAX1-TSOL18/L/cC1 than single gene pVAX1-TSOL18 vaccine, pVAX1-cC1 effect of immune response induction more comprehensively.

【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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