Ipr1/ PPE68穿梭质粒构建及诱导BALB/c小鼠免疫应答的探讨

摘要

The purpose of this study was to construct an co-expression shuttle plasmid with the intracellular pathogen resistance l(Iprl) gene, the coding sequences of Pro-Pro-Glu 68CPPE68) and OriM, and study the induced immune response. Iprl genes and the coding sequences of PPE68 and OriM were cloned into the MCS sites of the plasmid pBudCE4. 1. After the gene sequences and the protein expression were analyzed by restriction analysis, DNA sequencing and Western-blotting, the BALB/c mice were immunized with plasmid pBIPO. Two weeks after the last immunization, the levels of lgG2a, IL-12 and IFN-y in the serums were detected by ELISA, the quantity of CD4+ and CD8+ T cells were assessed with FCM, and the specific spleen lymphocytes proliferations were evaluated by MTT. At the same time, pathological changes in the lungs and spleens were investigated. The result showed that restriction analysis and DNA sequencing proved that the Iprl gene and PPE68 gene were coincided with theoretical sequences, and the results of Western-blotting showed that Iprl protein and PPE68 protein were expressed successfully. After the mice were immunized with plasmid pBIPO,the levels of lgG2a, IL-12 and IFN-y in the serums and the quantity of CD4+ and CD8+T cells were significantly increase, spleen lymphocyte proliferative responses were no different from BCG group,the lungs and spleens were not obviously pathological changes. This founding suggests that DNA vaccine (pBIPO) are constructed successfully,and it could effectively induce cellular immunity in BALB/c mice. Tthis study lays a foundation for further research about immune protection efficacy of the DNA vaccine.%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.