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

作　　者：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] 

机构地区：[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

出　　处：《四川动物》2013年第4期595-600,共6页Sichuan Journal of Zoology

基　　金：国家自然科学基金(编号:30901280)

摘　　要：目的构建Ipr1/PPE68重组卡介苗(Recombinant BCG,rBCG),探讨其诱导BALB/C小鼠免疫应答的效果。方法将Ipr1和PPE68基因及结核分枝杆菌(mycobacterium tuberculosis,MTB)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IgG2a、IL-12、IFN-γ及IL-4的水平、特异性脾淋巴细胞增殖和CD4+和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Ipr1和PPE68以及OriM基因序列与理论值相符,Western-blotting结果显示Ipr1和PPE68蛋白成功表达。Ipr1/PPE68-rBCG免疫小鼠后,血清中的IgG2a和IL-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。脾、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。Objective To construct the recombinant BCG with intracellular pathogen resistance 1 （ Iprl ） 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 Iprl and PPE68 genes and OriM were cloned into the MCS sites of plasmid pBudCE4.1, respec- tively. 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 Iprl/PPE68-rBCG for two weeks, and the productions of IgG2a, IL-12, IFN-＇,/ 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 pro- liferations were evaluated by MTF. At the mean time, numbers of residual Mycobacterium tuberculosis and pathological chan- ges in different organs of BALB/C mice were investigated. Results Enzyme restriction, DNA sequencing and Western- blotting confirmed that the Iprl/PPE68-rBCG was successfully constructed. Compared with control group, Iprl/PPE68-rB- CG immunized mice showed a significantly increased production of IgG2a and IL-12 in the sera, and enhanced spleen lym- phocyte proliferative responses. However, no significant difference was observed between Iprl/PPE68-rBCG group and BCG group. Although the productions of IFN-3, in the sera were significantly lower than BCG group, no significant difference was observed between Iprl/PPE68-rBCG group and control group. All the group showed an equal production of IL-4. No residu- al Mycobacterium tuberculosis was found, and there were no obvious pathological changes in the lung and spleen. Conclu- sion Iprl/PPE68-rBCG were successfully constructed which could induce effective cellular immunity in BALB/C mice.

关 键 词：结核分枝杆菌 Ipr1/PPE68重组BCG 免疫应答 

分 类 号：R392[医药卫生—免疫学]