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目的构建Ipr1/PPE68共表达穿梭质粒pBIPO(pBud-Ipr1-PPE68-OriM),探讨其诱导BALB/C小鼠的免疫应答特征。方法将Ipr1基因和PPE68编码序列以及结核分枝杆菌(Mycobacterium tuberculosis,MTB)的复制子OriM分别插入质粒pBudCE4.1的多克隆位点,构建共表达穿梭质粒pBIPO,经酶切测序及Western blotting鉴定后,用其免疫BALB/c小鼠,于末次免疫后2w处死小鼠,ELISA法分别检测小鼠血清中lgG2a、IL-12及IFN-γ的水平,流式细胞仪检测CD4+和CD8+T细胞数量,MTT法检测特异性脾淋巴细胞增殖,同时观察肺脾组织病理学变化。结果酶切测序鉴定PPE68和Ipr1基因序列与理论值相符,Western-blotting鉴定PPE68和Ipr1蛋白表达成功。质粒pBIPO免疫后小鼠血清中的lgG2a、IFN-γ及IL-12水平与CD4+和CD8+T细胞表达均呈现出有意义的提高,脾淋巴细胞增殖与BCG组相比差异无统计学意义,肺脾组织未见病理学改变。结论成功构建DNA疫苗(pBIPO),该疫苗能够有效诱导BALB/c小鼠的细胞免疫应答,为进一步研究其抗MTB的免疫保护作用奠定基础。
Objective To construct a shuttle plasmid pBIPO (pBud-Ipr1-PPE68-OriM) co-expressing Ipr1 / PPE68 and investigate its immune response characteristics in BALB / C mice. Methods The Ipr1 gene and PPE68 coding sequence and OriM, a replicon of Mycobacterium tuberculosis (MTB), were inserted into the multiple cloning site of plasmid pBudCE4.1 to construct the co-expression shuttle plasmid pBIPO. The recombinant plasmid pBIPO was identified by restriction enzyme digestion and Western blotting BALB / c mice were immunized with it and mice were sacrificed 2 weeks after the last immunization. The serum levels of lgG2a, IL-12 and IFN-γ were detected by ELISA. The levels of CD4 + and CD8 + T Cell number and MTT assay were used to detect the proliferation of specific splenic lymphocytes. Pathological changes of lung and spleen were also observed. Results The sequences of PPE68 and Ipr1 were identified by restriction enzyme digestion and the results were consistent with the theoretical values. The expression of PPE68 and Ipr1 protein was confirmed by Western-blotting. The serum levels of lgG2a, IFN-γ and IL-12 in mice after immunization with plasmid pBIPO showed a significant increase in the expression of both CD4 + and CD8 + T cells. There was no significant difference in the proliferation of spleen lymphocytes compared with BCG group, Pulmonary and spleen no pathological changes. Conclusion The recombinant DNA vaccine (pBIPO) was successfully constructed. The vaccine can effectively induce the cellular immune response in BALB / c mice and lay a foundation for further study of its immunoprotection against MTB.