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Ipr1-PPE68重组BCG诱导BALB-C小鼠免疫应答的探讨
Immune Response of BALB/C Mice Induced by Ipr1/PPE68 Recombinant BCG
张壮苗1,2,杨春1,2*,徐蕾1,2,何永林1,2,王静娴1,2,董志玲1,2,杨静1,2
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作者单位:(1. 重庆医科大学病原生物学教研室,重庆400016; 2. 重庆医科大学分子医学与肿瘤研究中心,重庆400016)
中文关键字:结核分枝杆菌;Ipr1/PPE68重组BCG;免疫应答
英文关键字:Mycobacterium tuberculosis (MTB); Ipr1/PPE68-rBCG; immune response
中文摘要:目的 构建Ipr1/PPE68重组卡介苗(Recombinant BCGrBCG),探讨其诱导BALB/C小鼠免疫应答的效果。方法 Ipr1PPE68基因及结核分枝杆菌(Mycobacterium tuberculosisMTB)复制子OriM分别插入pBudCE4.1的多克隆位点,构建共表达穿梭质粒pBIPO,将其电转入BCG,构建Ipr1/PPE68-rBCG并将rBCG免疫BALB/C小鼠,检测小鼠血清中IgG2aIL-12IFN-γIL-4的水平、特异性脾淋巴细胞增殖和CD4+CD8+T细胞数量,同时观察脾、肺荷菌量及脾、肺组织病理学变化。结果 酶切测序及菌落PCR鉴定Ipr1PPE68以及OriM基因序列与理论值相符Western-blotting结果显示Ipr1PPE68蛋白成功表达。Ipr1/PPE68-rBCG免疫小鼠后,血清中的IgG2aIL-12水平及脾淋巴细胞增殖情况明显高于对照组,但与BCG组相比没有显著意义;IFN-γ水平显著低于BCG组,与对照组相比无显著性差异;各组别IL-4的水平差异均不明显。肺荷菌实验未见菌落生长,肺脾组织未见病理学改变。结论 成功构建Ipr1/PPE68-rBCG,该重组BCG能诱导BALB/C小鼠的细胞免疫应答。
英文摘要:Objective  To construct the recombinant BCG with intracellular pathogen resistance 1 (Ipr1) gene and coding region of Pentose-5-phosphate-3-epimerase 68 (PPE68), and to study the relevant immune response of rBCG immunized BALB/C mice. Methods  Ipr1 and PPE68 genes and OriM were cloned into the MCS sites of plasmid pBudCE4.1, respectively. Then the recombinant plasmid pBIPO was verified by enzyme restriction, DNA sequencing and Western-blotting. Subsequently, pBIPO was electro-transferred into BCG and positive colonies were analyzed by PCR. BALB/C mice were then immunized with Ipr1/PPE68-rBCG for two weeks, and the productions of IgG2a, IL-12, IFN-γ and IL-4 in the sera were detected by ELISA, the quantity of CD4+ and CD8+ T cells were assessed by FACS, specific spleen lymphocytes proliferations were evaluated by MTT. At the mean time, numbers of residual Mycobacterium tuberculosis and pathological changes in different organs of BALB/C mice were investigated. Results  Enzyme restriction, DNA sequencing and Western-blotting confirmed that the Ipr1/PPE68-rBCG was successfully constructed. Compared with control group, Ipr1/PPE68-rBCG immunized mice showed a significantly increased production of IgG2a and IL-12 in the sera, and enhanced spleen lymphocyte proliferative responses. However, no significant difference was observed between Ipr1/PPE68-rBCG group and BCG group. Although the productions of IFN-γ in the sera were significantly lower than BCG group, no significant difference was observed between Ipr1/PPE68-rBCG group and control group. All the group showed an equal production of IL-4. No residual Mycobacterium tuberculosis was found, and there were no obvious pathological changes in the lung and spleen. Conclusion  Ipr1/PPE68-rBCG were successfully constructed which could induce effective cellular immunity in BALB/C mice.
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