结核分枝杆菌特异性抗原基因的克隆及纳米疫苗的初步研究

发布时间：2018-06-29 15:17

本文选题：结核分枝杆菌 + 特异性抗原　；参考：《吉林农业大学》2011年硕士论文

【摘要】：目的：结核病(Tuberculosis,TB)是严重威胁全世界的主要致死疾病之一。尽管人们在出生不久便注射了卡介苗(BCG),但由于BCG存在着免疫缺失,免疫保护力低等缺陷,导致每年因患结核病而死亡的人数在不断地增加,且已成为全球关注的公共卫生和社会问题,因此新型结核疫苗的研制一直是众多研究学者的研究重点,而疫苗研究的关键则在于抗原的选择,目前处于研发阶段的结核抗原主要有ESAT-6、CFP10、Ag85A等。近年来,随着纳米技术在生物、医药等方面的研究,纳米佐剂、纳米载药系统研究的不断成熟又为结核疫苗研究者们带来新的突破,这主要缘于纳米粒子(nanoparticles,NP)具有粒径小、粒径一般在1-100nm之间,可经过人体毛细血管分布至特定组织,因此能够达到药物缓释或控释的作用,可致力于单次免疫疫苗的开发。本研究拟通过扩增特异性抗原基因,构建原核重组质粒与真核重组质粒,一部分将原核重组质粒表达的蛋白对小鼠进行皮下注射免疫,另一部分用无机纳米(SiNP)包裹真核重组质粒对小鼠进行肌肉注射,同时进行纳米质粒与细胞共培养,观察核酸DNA在细胞和肌肉组织中的表达变化,为结核病在纳米缓释疫苗中的研究提供了一定的实验依据。方法：通过PCR扩增ESAT-6、CFP10、MPT51、PpiA蛋白编码的基因,选择CFP10基因构建原核重组质粒pET-30a-CFP-10及表达纯化蛋白,用纯化后的蛋白(40μg/只)免疫小鼠(0、21d),同时构建真核重组质粒pEGFP-N1-CFP10,制备纳米核酸疫苗,一部分与细胞共培养48小时,一部分肌肉注射BALB/c小鼠(50μg/只),于3d后,取肌肉组织,进行冰冻切片,在荧光显微镜下观察蛋白在细胞和肌肉组织中的变化。结果：成功获得原核重组质粒pET-30a-CFP-10及纯化的目的蛋白,并成功将65nm的SiNP与重组真核质粒pEGFP-N1-CFP-10结合,注射小鼠肌肉后,观察到注射肌肉组织中有红色荧光出现(SiNP包裹红色量子点所呈现的红色荧光),证明成功将纳米DNA注入肌肉组织中,‘观察发现红色纳米荧光量子点,却未见质粒表达绿色荧光蛋白。纳米DNA成功导入PK细胞中,并表达绿色荧光蛋白。结论：成功克隆四种特异性抗原基因,表达了CFP10蛋白,构建纳米核酸疫苗SiNP-pEGFP-N1-CFP10并成功在小鼠肌肉中观察,构建CMS-pEGFP-N1成功导入PK细胞并成功表达绿色荧光蛋白。
[Abstract]:Objective: tuberculosis (TB) is one of the major fatal diseases in the world. Although people are injected with BCG shortly after birth, the number of deaths from TB is increasing every year because of deficiencies in BCG, such as lack of immunity and low immune protection. And has become a global public health and social concerns, so the development of new TB vaccine has been the focus of many researchers, and vaccine research is the key to the selection of antigens. At present, the major TB antigens in R & D are ESAT-6 CFP 10, Ag85 A and so on. In recent years, with the research of nanotechnology in biology, medicine and so on, the development of nano-adjuvant and nano-drug carrier system has brought new breakthrough for TB vaccine researchers, which is mainly due to the small particle size of nano-particles (nanoparticlesNP). The particle size is generally between 1-100nm and can be distributed to specific tissues through human capillaries, so it can achieve the function of drug release or controlled release, and can be devoted to the development of single immunization vaccine. The purpose of this study was to construct prokaryotic recombinant plasmid and eukaryotic recombinant plasmid by amplifying specific antigen gene. Some of the proteins expressed by prokaryotic recombinant plasmid were injected subcutaneously into mice. In the other part, mice were injected intramuscularly with inorganic nano-sized (SiNP) encapsulated eukaryotic recombinant plasmids, and then co-cultured with cells to observe the expression of nucleic acid DNA in cells and muscle tissues. It provides a certain experimental basis for the study of TB in nano sustained release vaccine. Methods: the gene encoding ESAT-6 CFP10 MPT51PpiA protein was amplified by PCR. The prokaryotic recombinant plasmid pET-30a-CFP-10 was constructed with CFP10 gene and the purified protein was expressed. Mice were immunized with the purified protein (40 渭 g / mouse) for 0 ~ 21 days, and the eukaryotic recombinant plasmid pEGFP-N1-CFP10 was constructed to prepare nano-nucleic acid vaccine. Some of them were co-cultured with cells for 48 hours, and others were injected intramuscularly into BALB / c mice (50 渭 g / mouse). After 3 days, the muscle tissues were taken and frozen sections were made. The changes of protein in cells and muscle tissues were observed under fluorescence microscope. Results: the prokaryotic recombinant plasmid pET-30a-CFP-10 and the purified target protein were successfully obtained, and the sip of 65nm was successfully combined with the recombinant eukaryotic plasmid pEGFP-N1-CFP-10. Red fluorescence was observed in the injected muscle tissue (red fluorescence presented by SiNP wrapped in red quantum dots), which proved the successful injection of nano-DNA into muscle tissue. However, the plasmid did not express green fluorescent protein. Nano-DNA was successfully introduced into competitive cells and expressed green fluorescent protein. Conclusion: four specific antigen genes were cloned successfully, and CFP10 protein was expressed. SiNP-pEGFP-N1-CFP10 was constructed and observed in mouse muscle successfully. CMS-pEGFP-N1 was successfully introduced into competitive cells and expressed green fluorescent protein.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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