【摘要】： 目的構(gòu)建日本血吸蟲多價膜錨定表達(dá)疫苗pIRES-Sj97-Sj14-Sj26,并研究其免疫原性。 方法采用分子克隆技術(shù),構(gòu)建日本血吸蟲脂肪酸結(jié)合蛋白(Sj14)、GST(Sj26)和副肌球蛋白(Sj97)的跨膜共表達(dá)質(zhì)粒pIRES-Sj97-Sj14-Sj26,轉(zhuǎn)染Hela細(xì)胞,通過RT-PCR法檢測Sj14、Sj26和Sj97mRNA的表達(dá),間接免疫熒光檢測Sj26蛋白的表達(dá)。用此疫苗免疫BALB/c小鼠,同時設(shè)置生理鹽水對照組、空白質(zhì)粒對照組、pIRES-Sj26組和pIRES-Sj14-Sj26組。用ELISA法檢測免疫小鼠血清中的總IgG抗體和小鼠脾細(xì)胞分泌IFN-γ的水平。以MTT法檢測淋巴細(xì)胞的增殖情況。并以FCM分析脾細(xì)胞亞群。 結(jié)果經(jīng)瞬時轉(zhuǎn)染Hela細(xì)胞證明質(zhì)粒pIRES-Sj97-Sj14-Sj26能在體外進(jìn)行表達(dá)。免疫小鼠后ELISA法檢測抗體結(jié)果表明pIRES-Sj97-Sj14-Sj26能夠較各對照組誘導(dǎo)產(chǎn)生高水平的IgG抗體(P0.01,P0.05).該組的脾淋巴細(xì)胞刺激指數(shù)較各對照組也有顯著增高(P0.01),IFN-γ的水平與空白對照組和空載體組有顯著差異(P0.01). CD4+和CD8+T細(xì)胞含量百分比有明顯增高(P0.05)。 結(jié)論pIRES-Sj97-Sj14-Sj26疫苗具有較強(qiáng)的免疫原性,能明顯增強(qiáng)BALB/c小鼠的免疫應(yīng)答反應(yīng).為該疫苗抗感染效應(yīng)的研究奠定了基礎(chǔ),并為日本血吸蟲病的防治尋求一種新的多基因組合疫苗的免疫策略。
[Abstract]:Objective to construct and study the immunogenicity of the multivalent membrane anchored pIRES-Sj97-Sj14-Sj26, vaccine of Schistosoma japonicum. Methods the transmembrane coexpression plasmid pIRES-Sj97-Sj14-Sj26, of fatty acid binding protein (Sj26) and accessory myosin (Sj97) of Schistosoma japonicum was constructed by molecular cloning technique. The expression of Sj14,Sj26 and Sj97mRNA was detected by RT-PCR method, and the expression of Sj26 protein was detected by indirect immunofluorescence. BALB/c mice were immunized with this vaccine, and the control group of normal saline, the control group of blank plasmid pIRES-Sj26 and the group of pIRES-Sj14-Sj26 were set up at the same time. The total IgG antibody in serum of immunized mice and the level of IFN- 緯 secreted by spleen cells of mice were detected by ELISA method. The proliferation of lymphocytes was detected by MTT method. Spleen cell subsets were analyzed by FCM. Results transient transfection of Hela showed that plasmid pIRES-Sj97-Sj14-Sj26 could be expressed in vitro. After immunizing mice, the results of ELISA assay showed that pIRES-Sj97-Sj14-Sj26 could produce higher level of IgG antibody than that of control group (P0.01P 0.05). The level of IFN- 緯 was significantly higher in this group than that in the control group (P0.01), and there was a significant difference between the control group and the blank carrier group (P0.01). The percentage of CD4 and CD8 T cells was significantly increased (P0.05). Conclusion pIRES-Sj97-Sj14-Sj26 vaccine has strong immunogenicity and can obviously enhance the immune response of BALB/c mice. It lays a foundation for the study of the anti-infective effect of the vaccine and seeks a new immunization strategy for the prevention and control of schistosomiasis japonicum.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392

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