抗耐药结核多效基因工程疫苗的构建及免疫效果分析

发布时间：2018-06-20 04:26

本文选题：DNA疫苗 + 结核分枝杆菌　；参考：《遵义医学院》2017年硕士论文

【摘要】：目的:构建抗耐药结核重组质粒DNA疫苗p IEMKMAGP,并初步评价其免疫效果,为临床耐药结核病的治疗提供新的选择。方法:(1)利用DNA重组技术,将结核分枝杆菌表面抗原MPT64、Ag85B基因编码区和野生型kat G基因编码区,以及穿孔素PRF1、颗粒溶素GNLY基因编码区插入到真核表达载体p IRES2-EGFP中,构建抗结核多基因真核串联表达载体p IEMKMAGP(MPT64-Ag85B/kat G/GNLY-PRF1);(2)利用电转化的方法,将质粒p IEMKMAGP转化耻垢分枝杆菌制备重组耻垢分枝杆菌菌苗;(3)利用脂质体转染技术,将质粒p IEMKMAGP转染293T细胞,应用荧光定量PCR技术检测目的基因表达水平,应用Western blot技术检测目的蛋白表达情况;(4)质粒p IEMKMAGP免疫接种C57BL/6小鼠和SD大鼠,ELISA试剂盒检测IL-12和IFN-γ细胞因子表达水平。结果:(1)成功构建了抗耐药结核多效基因工程DNA疫苗p IEMKMAGP(MPT64-Ag85B/kat G/GNLY-PRF1);(2)成功制备重组耻垢分枝杆菌菌苗;(3)目的基因MPT64/Ag85B、kat G、GNLY/PRF1在293T细胞中成功表达,融合蛋白MPT64/Ag85B、融合蛋白GNLY/PRF1成功表达,且融合蛋白MPT64/Ag85B可分泌表达;(4)目的基因MPT64/Ag85B、kat G、GNLY/PRF1在C57BL/6小鼠组织器官m RNA水平上有一定表达,但蛋白水平上未检测到目的蛋白的表达;(5)质粒p IEMKMAGP免疫接种SD大鼠中外周血血清中的IL-12和IFN-γ细胞因子表达水平明显升高。结论:(1)抗耐药结核真核表达质粒p IEMKMAGP(MPT64-Ag85B/kat G/GNLY-PRF1)作为DNA疫苗能够在细胞水平上有效表达,有望为进一步开发新型的抗结核疫苗提供了实验依据。(2)本研究构建的抗耐药结核DNA疫苗p IEMKMAGP(MPT64-Ag85B/kat G/GNLY-PRF1)可以引起IL-12和IFN-γ细胞因子水平的升高。
[Abstract]:Objective: to construct the recombinant plasmid DNA vaccine pIEMKMAGPand evaluate its immunological effect and provide a new choice for the treatment of drug-resistant tuberculosis. Methods using DNA recombination technique, the coding region of MPT64-Ag85B gene and wild-type kat G gene, as well as perforin PRF1, granulysin GNLY gene coding region, were inserted into eukaryotic expression vector pIRES2-EGFP. Construction of anti-tuberculosis polygene eukaryotic tandem expression vector pIEMKMAGP-MPT64-Ag85B / kat G / GNLY-PRF1 / 2) using electroporation method, plasmid pIEMKMAGP was transformed into Mycobacterium smearicum to prepare recombinant Mycobacterium smeitum vaccine, and the plasmid pIEMKMAGP was transfected into 293T cells by liposome transfection technique, and the recombinant plasmid pIEMKMAGP was transfected into 293T cells by liposome transfection. The expression levels of IL-12 and IFN- 纬 cytokines in C57BL / 6 mice and SD rats immunized with pIEMKMAGP were detected by fluorescence quantitative polymerase chain reaction (FQ-PCR) and Western blot. Results: (1) successfully constructed the multifunctional DNA vaccine pIEMKMAGPN MPT64-Ag85Br / Kat G / GNLY-PRF1 / 2) the target gene MPT64 / Ag85BY / PRF1 was successfully expressed in 293T cells, and the fusion protein MPT64 / Ag85Ag85B was successfully expressed in GNLYP PRF1, and the fusion protein GNLYP / PRF1 was successfully expressed in 293T cells. And the fusion protein MPT64 / Ag85B can secrete the expression of nong4.) the target gene MPT64 / Ag85BNY / PRF1 is expressed at the level of mRNAs in C57BL / 6 mouse tissues and organs. However, the expression of IL-12 and IFN- 纬 cytokines in peripheral blood of SD rats immunized with pIEMKMAGP was not detected at the protein level. Conclusion the eukaryotic expression plasmid pIEMKMAGPN MPT64-Ag85B / kat G / GNLY-PRF1 can be effectively expressed as a DNA vaccine. It is expected to provide an experimental basis for the further development of new anti-TB vaccine. (2) the DNA vaccine against drug-resistant tuberculosis, named as IEMKMAGPG, MPT64-Ag85Br / Kat G / GNLY-PRF1, can increase the levels of IL-12 and IFN- 纬 cytokines.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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