构建卡介苗突变株的初步研究

发布时间：2018-05-17 16:31

本文选题：卡介苗 + ERP基因　；参考：《石河子大学》2006年硕士论文

【摘要】： 结核病是人类主要的传染性疾病之一,全球每年新发结核病人数超过800万,死亡人数约300万,全世界结核杆菌感染人数超过1/3。近年来HIV的广泛流行及多重耐药结核菌株的出现使结核病的发病率和死亡率呈上升趋势。世界卫生组织于1993年宣布“全球结核病紧急状态”,并指出应优先发展有效的结核病疫苗。卡介苗是目前全球唯一一种用于预防结核病的疫苗,它是牛分支杆菌的减毒活菌苗株。卡介苗能够有效预防严重的儿童结核病,但对成人结核病的保护效力在不同的地区人群中有显著差异(0-80%),因而亟需一种更为有效的结核病疫苗。目前正在研究中的新型疫苗包括亚单位疫苗、DNA疫苗、减毒活疫苗、重组卡介苗等。由于卡介苗具有广泛使用的安全性(全球已有超过30亿人接种了卡介苗),对卡介苗进行改造是开发新疫苗的一种策略。ERP基因是结核杆菌中的一个重要的毒力基因,研究表明,它不仅存在于有毒力的结核杆菌中,也存在于卡介苗中,免疫功能缺陷者(如HIV患者)接种卡介苗可能引起严重的播散性结核病。目的本研究利用基因敲除技术和电穿孔技术构建卡介苗ERP基因缺失突变株,为初步探讨卡介苗突变株的功能奠定了基础,并为研制新型结核病疫苗进行了有益的探索。方法对卡介苗菌进行体外培养,用改进的方法手抽卡介苗菌基因组DNA,PCR扩增ERP基因两侧的两个800bp左右的片段,经纯化后连接T载体,通过蓝白斑筛选及菌落PCR鉴定后挑选阳性克隆送测序,测序正确的两个片段分别连接到PKO载体预定位点,构建卡介苗ERP基因置换型打靶载体,经抗生素,菌落PCR、质粒PCR及酶切鉴定后挑选阳性克隆再次测序,验证载体构建成功与否。将构建好的打靶载体电穿孔卡介苗,通过抗生素及PCR鉴定出卡介苗ERP基因缺失突变株。结果1.提取卡介苗菌基因组DNA。2.扩增出两个目的片段。3.将两个目的片段插入到PKO载体的预定位点,构建卡介苗ERP基因置换型打靶载体。4.将打靶载体电穿孔入卡介苗,筛选并鉴定卡介苗ERP基因缺失突变株。结论构建了卡介苗ERP基因置换型打靶载体,并构建筛选卡介苗ERP基因缺失突变株。
[Abstract]:Tuberculosis is one of the major infectious diseases in human beings. Worldwide, the number of new TB cases is more than 8 million every year, the death toll is about 3 million, and the number of tuberculosis bacilli infection in the world is more than one third. In recent years, the prevalence of HIV and the emergence of multidrug resistant tuberculous bacilli have increased the incidence and mortality of tuberculosis. The World Health Organization (WHO) declared a global TB emergency in 1993 and stated that priority should be given to the development of effective TB vaccines. BCG is the only vaccine used to prevent tuberculosis in the world. It is a live attenuated strain of Mycobacterium bovis. BCG vaccine can effectively prevent severe tuberculosis in children, but the protective effect of BCG vaccine on adult tuberculosis is significantly different among different populations in different regions. Therefore, a more effective TB vaccine is urgently needed. New vaccines are currently under study, including subunit DNA vaccine, live attenuated vaccine, recombinant BCG vaccine and so on. Because the BCG vaccine has been widely used safely (more than 3 billion people have been vaccinated with BCG vaccine in the world, the modification of BCG vaccine is a strategy for developing new vaccine. ERP gene is an important virulence gene in Mycobacterium tuberculosis. It exists not only in noxious Mycobacterium tuberculosis, but also in BCG vaccine. Vaccination of BCG in patients with immune deficiency (such as HIV patients) may cause serious disseminated tuberculosis. Objective to construct BCG ERP mutant by gene knockout technique and electroporation technique, and to establish a foundation for the function of BCG mutant strain, and to explore the development of new TB vaccine. Methods BCG was cultured in vitro. Two 800bp fragments were amplified from the genomic DNA of BCG by hand extraction, and then ligated into T vector after purification. The positive clones were selected and sequenced by blue-white spot screening and colony PCR identification. The two correct fragments were sequenced and linked to the predetermined site of the PKO vector respectively. The BCG ERP gene replacement targeting vector was constructed, and antibiotics were used to construct the BCG ERP gene replacement target vector. The positive clones were selected and sequenced after colony PCR, plasmid PCR and restriction endonuclease digestion to verify the success of the construction of the vector. The target vector electroporation BCG was constructed and the ERP gene deletion mutant of BCG vaccine was identified by antibiotics and PCR. Result 1. The genomic DNA of Bacillus Calmette-Guerin (BCG) was extracted. Two target fragments. Two target fragments were inserted into the predetermined site of PKO vector to construct BCG ERP gene replacement targeting vector. 4. The target vector was electroporated into the BCG vaccine to screen and identify the BCG ERP gene deletion mutant. Conclusion BCG ERP gene replacement targeting vector was constructed and BCG ERP gene deletion mutant was constructed.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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