PPE68重组BCG诱导机体免疫应答的探讨

发布时间：2018-03-27 17:14

本文选题：PPE68　切入点：结核分枝杆菌　出处：《重庆医科大学》2012年硕士论文

【摘要】：目的：构建带有结核分枝杆菌PPE68基因和分枝杆菌复制子OriM基因的穿梭质粒pBudCE4.1/PPE68/OriM，将其电转入BCG，构建PPE68重组BCG，用其免疫BALB／c小鼠，，与BCG和DNA疫苗比较，探讨其诱导BALB／c小鼠体内免疫应答特征。方法：以pZM03为模板，用PCR法扩增出分枝杆菌复制子OriM基因，将其亚克隆入已含PPE68基因的带有双启动子的真核共表达载体pBudCE4.1的NheI位点中，经过酶切测序鉴定后，得到穿梭质粒pBudCE4.1/PPE68/OriM；将PPE68基因转染入RAW264.7（小鼠巨噬细胞株），通过RT-PCR和Western-blot检测PPE68的表达。用鉴定正确的穿梭质粒pBudCE4.1/PPE68/OriM电转入BCG中，制备PPE68重组BCG，菌落PCR法进行鉴定，扩增。用鉴定正确的PPE68重组BCG皮内免疫BALB／c小鼠，同时设DNA疫苗组、空载体组、BCG、OriM重组BCG和生理盐水组对照组。于末次免疫后2周，分别检测小鼠体内IgG2a、IL-12、IL-4以及IFN-γ水平；特异性蛋白刺激后淋巴细胞增殖状态；CD4+T淋巴细胞和CD8+T淋巴细胞数及CD4+/CD8+比值；观察小鼠肺脾器官荷菌量、组织病理改变情况和免疫组织化学结果。结果： RT-PCR、Western-blot法检测PPE68基因在巨噬细胞内的表达。成功地构建了PPE68重组卡介苗，PPE68重组末次免疫后2周PPE68重组BCG组较BCG组血清IgG2a水平明显升高(P0．05)；IFN-γ和IL-12水平高于生理盐水组（P0．05），但低于BCG组和DNA疫苗组(P0．05)，其中，DNA疫苗组IFN-γ和IL-12水平显著升高(P0．05);各组产生IL-4水平无显著性差异；特异性脾淋巴细胞增殖明显高于BCG组（P0．05）；与BCG组和DNA疫苗组相比，PPE68重组BCG组小鼠CD4+T细胞和CD8+T细胞百分比增加，CD4+／CD8+T细胞比值降低；免疫组化法检测到小鼠肺组织中PPE68的表达；肺组织轻度充血程度与BCG组相当，余未见明显病理改变、脾肺无菌落生长。结论：成功构建PPE68重组BCG疫苗，该疫苗可诱导BALB／c小鼠体内IgG2a水平升高，IL-12和IFN-γ含量增加，脾淋巴细胞的增殖明显，CD4+T细胞和CD8+T细胞明显增加，提高机体Th1型免疫效应，为进一步研究其抗MTB的免疫保护作用奠定了基础。
[Abstract]:Objective:. The shuttle plasmid pBudCE4.1 / PPE68 / OriMwith the PPE68 gene of Mycobacterium tuberculosis and the OriM gene of Mycobacterium replicas was constructed. The recombinant PPE68 was transfected into BCGs, and the BALB/c mice were immunized with PPE68. Compared with BCG and DNA vaccines, the characteristics of in vivo immune response induced by pBudCE4.1 / PPE68 / OriMwere studied. Methods:. Using pZM03 as template, the OriM gene of Mycobacterium replicas was amplified by PCR and subcloned into the NheI site of eukaryotic coexpression vector pBudCE4.1 with double promoter containing PPE68 gene. The shuttle plasmid pBudCE4.1 / PPE68 / OriM was obtained, the PPE68 gene was transfected into RAW264.7 (mouse macrophage cell line), and the expression of PPE68 was detected by RT-PCR and Western-blot. The recombinant PPE68 BCGs were prepared by electroporation into BCG with identified shuttle plasmid pBudCE4.1/PPE68/OriM, and identified by colony PCR method. Amplification. Intradermal immunization of PPE68 recombinant BCG was used to immunize BALB/c mice. DNA vaccine group, blank vector group, BCG OriM recombinant BCG group and normal saline group were used to detect the levels of IL-12 IL-4 and IFN- 纬 in mice at 2 weeks after the last immunization. The number of CD 4 T lymphocytes and CD8 T lymphocytes and the ratio of CD4 / CD 8 were observed after stimulation of specific protein, and the amount of murine lung and spleen organs, histopathological changes and immunohistochemical results were observed. Results:. The expression of PPE68 gene in macrophages was detected by RT-PCR Western-blot. The level of serum IgG2a in PPE68 recombinant BCG group was significantly higher than that in BCG group two weeks after the last immunization of PPE68 recombinant BCG vaccine PPE68, but the levels of IgG2a 纬 and IL-12 in BCG group were significantly higher than those in normal saline group. The levels of IFN- 纬 and IL-12 in DNA vaccine group were significantly higher than those in BCG group and DNA vaccine group, but there was no significant difference in IL-4 production among each group. Compared with BCG group and DNA vaccine group, the percentage of CD4 T cells and CD8 T cells in PPE68 recombinant BCG group decreased, and the ratio of CD 4 / CD 8 T cells in lung tissue was detected by immunohistochemistry. The degree of mild hyperemia in lung tissue was similar to that in BCG group. Conclusion:. PPE68 recombinant BCG vaccine was successfully constructed, which could induce the increase of IgG2a level in BALB/c mice and increase the contents of IL-12 and IFN- 纬, and the proliferation of splenic lymphocytes, CD4 T cells and CD8 T cells. It lays a foundation for further study on the immune protection of MTB.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.11

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