Ipr1DNA疫苗诱导BALB/c小鼠免疫应答的探讨

Induction of immune response by Ipr1 DNA vaccine in BALB/c mice

目的构建真核表达质粒pBud-Ipr1-OriM,探讨其诱导BALB/c小鼠的免疫应答,为新型结核病疫苗的研制提供新思路。方法将鼠胞内病原体抗性基因1(Intracellular pathogen resistance,Ipr1)和结核分枝杆菌(Mycobacterium tubercu-losis,MTB)的复制子OriM插入真核表达质粒pBudCE4.1,构建pBud-Ipr1-OriM表达质粒(Ipr1 DNA疫苗);将BALB/c小鼠随机分为4组:生理盐水对照组、BCG组、pBud-OriM组及pBud-Ipr1-OriM组,pBud-OriM组及pBud-Ipr1-OriM组经肌肉注射免疫相应的重组质粒,BCG组经背部皮内注射BCG,每隔2周注射1次,共3次;末次免疫后2周,处死小鼠,分离血清,采用ELISA法检测小鼠血清中IL-12和IFNγ的水平;取脾组织,采用流式细胞术检测CD4+和CD8+T细胞数量,同时观察肺脾组织的病理学变化。结果经酶切、测序鉴定证实,重组真核表达质粒pBud-Ipr1-OriM(Ipr1 DNA疫苗)构建成功;pBud-Ipr1-OriM组免疫小鼠血清中IL-12水平及小鼠脾组织中CD4+和CD8+T细胞数量均高于其他组,肺脾组织未见病理学改变。结论 Ipr1 DNA疫苗能有效诱导BALB/c小鼠的细胞免疫应答,为新型结核病疫苗的研制奠定了基础。

Objective To construct a eukaryotic expression vector pBud-Ipr1-OriM and investigate the immune response induced to provide a new strategy for development of novel vaccine against tuberculosis.Methods Intracellular pathogen resistance 1（Ipr1）gene and the replicon OriM of Mycobacterium tuberculosis（MTB）were inserted into eukaryotic expression vector pBudCE4.1 to construct recombinant plasmid pBud-Ipr1-OriM（Ipr1 DNA vaccine）.BALB / c mice were divided into four groups randomly.The mice in pBud-OriM and pBud-Ipr1-OriM groups were injected i.m.with the corresponding plasmids respectively,while those in control group with physiological saline,and those in BCG group were injected intracutaneously with BCG,every 2 weeks for 3 times.The mice were killed 2 weeks after the last injection,of which the sera were separated and determined for IL-12 and IFNγ levels by ELISA.The spleen tissues of mice were isolated and determined for CD4＋ and CD8＋ T cell counts by flow cytometry and observed for pathological change.Results Restriction analysis and DNA sequencing proved that recombinant plasmid pBud-Ipr1-OriM（Ipr1 DNA vaccine）was constructed correctly.The IL-12 and IFNγ levels in sera as well as CD4＋ and CD8＋ T cell counts in spleen tissues of mice in pBud-Ipr1-OriM group were higher those in other groups.However,no pathological changes were observed in lung or spleen tissue.Conclusion Ipr1 DNA vaccine（pBud-IPR1-OriM）induced cellular immune response in BALB / c mice effectively,which laid a foundation of development of novel vaccine against tuberculosis.