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  本文選題：PPE68　切入點：結(jié)核分枝桿菌　出處：《重慶醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的： 構(gòu)建帶有結(jié)核分枝桿菌PPE68基因和分枝桿菌復(fù)制子OriM基因的穿梭質(zhì)粒pBudCE4.1/PPE68/OriM，將其電轉(zhuǎn)入BCG，構(gòu)建PPE68重組BCG，用其免疫BALB／c小鼠，，與BCG和DNA疫苗比較，探討其誘導(dǎo)BALB／c小鼠體內(nèi)免疫應(yīng)答特征。 方法： 以pZM03為模板，用PCR法擴增出分枝桿菌復(fù)制子OriM基因，將其亞克隆入已含PPE68基因的帶有雙啟動子的真核共表達載體pBudCE4.1的NheI位點中，經(jīng)過酶切測序鑒定后，得到穿梭質(zhì)粒pBudCE4.1/PPE68/OriM；將PPE68基因轉(zhuǎn)染入RAW264.7（小鼠巨噬細胞株），通過RT-PCR和Western-blot檢測PPE68的表達。用鑒定正確的穿梭質(zhì)粒pBudCE4.1/PPE68/OriM電轉(zhuǎn)入BCG中，制備PPE68重組BCG，菌落PCR法進行鑒定，擴增。用鑒定正確的PPE68重組BCG皮內(nèi)免疫BALB／c小鼠，同時設(shè)DNA疫苗組、空載體組、BCG、OriM重組BCG和生理鹽水組對照組。于末次免疫后2周，分別檢測小鼠體內(nèi)IgG2a、IL-12、IL-4以及IFN-γ水平；特異性蛋白刺激后淋巴細胞增殖狀態(tài)；CD4+T淋巴細胞和CD8+T淋巴細胞數(shù)及CD4+/CD8+比值；觀察小鼠肺脾器官荷菌量、組織病理改變情況和免疫組織化學(xué)結(jié)果。 結(jié)果： RT-PCR、Western-blot法檢測PPE68基因在巨噬細胞內(nèi)的表達。成功地構(gòu)建了PPE68重組卡介苗，PPE68重組末次免疫后2周PPE68重組BCG組較BCG組血清IgG2a水平明顯升高(P0．05)；IFN-γ和IL-12水平高于生理鹽水組（P0．05），但低于BCG組和DNA疫苗組(P0．05)，其中，DNA疫苗組IFN-γ和IL-12水平顯著升高(P0．05);各組產(chǎn)生IL-4水平無顯著性差異；特異性脾淋巴細胞增殖明顯高于BCG組（P0．05）；與BCG組和DNA疫苗組相比，PPE68重組BCG組小鼠CD4+T細胞和CD8+T細胞百分比增加，CD4+／CD8+T細胞比值降低；免疫組化法檢測到小鼠肺組織中PPE68的表達；肺組織輕度充血程度與BCG組相當，余未見明顯病理改變、脾肺無菌落生長。 結(jié)論： 成功構(gòu)建PPE68重組BCG疫苗，該疫苗可誘導(dǎo)BALB／c小鼠體內(nèi)IgG2a水平升高，IL-12和IFN-γ含量增加，脾淋巴細胞的增殖明顯，CD4+T細胞和CD8+T細胞明顯增加，提高機體Th1型免疫效應(yīng)，為進一步研究其抗MTB的免疫保護作用奠定了基礎(chǔ)。
[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- 緯 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 緯 and IL-12 in BCG group were significantly higher than those in normal saline group. The levels of IFN- 緯 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- 緯, 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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392.11

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