炭疽杆菌BA2380基因缺失突变株和回复互补株的构建

发布时间：2018-06-26 01:08

本文选题：炭疽杆菌 + Golden　；参考：《吉首大学》2017年硕士论文

【摘要】：炭疽杆菌(Bacillus anthracis)是烈性病原菌,能够引起人畜共患的急性传染病,对全世界人类及畜牧业造成严重威胁。芽胞是炭疽杆菌传播和感染的主要形式,感染后可导致皮肤炭疽、肠道炭疽、肺炭疽等疾病。炭疽杆菌可用作生物武器来储备。故有关炭疽杆菌致病机理的研究一直备受关注。本实验室早期研究结果表明,BA2380碱性丝氨酸蛋白酶与炭疽杆菌的毒力因子和S-层蛋白降解相关,可能参与炭疽杆菌感染机体的致病性,因而有必要对其功能进行深入研究。基于此,我们以炭疽杆菌A16D2为出发菌株,开展了BA2380基因缺失突变株和回复互补株的构建,并研究了两类突变株的部分生理功能。首先,通过PCR扩增获得了BA2380基因上下游同源臂以及抗性基因spc片段,分别连接到T-easy载体,构建了3个供体质粒。利用本实验室改造的“Golden Gate”克隆方法将三个片段同时连入温敏型穿梭载体pKMBK中,构建了基因打靶质粒pK2380usd,将该基因打靶载体导入炭疽杆菌A16D2感受态细胞中,利用同源重组原理,筛选得到炭疽杆菌BA2380基因缺失突变株A16D2△BA2380::spc,并对其进行了验证。接着,利用PCR技术从炭疽芽胞杆菌A16D2株扩增出BA2380基因,通过酶切-连接技术导入大肠杆菌-枯草杆菌穿梭质粒pBE2A中,构建了回复突变质粒pBE2A-BA2380,将该重组质粒电转入BA2380基因缺失株A16D2△BA2380::spc中,筛选获得了回复互补株pBE2A-BA2380/A16D2。最后,以出发菌株A16D2为对照,对BA2380基因缺失突变株A16D2△BA2380::spc和回复互补株pBE2A-BA2380/A16D2的生长特性和芽胞形成情况进行了研究,发现三者在菌株生长和芽胞形成方面没有显著差异,表明BA2380基因缺失对炭疽杆菌生长和芽胞形成没有影响。同时,采用Western blot技术分析了BA2380基因在A16D2、A16D2△BA2380::spc、pBE2A-BA2380/A16D2和pBE2A/A16D2中的表达情况,发现BA2380基因只在出发菌株和回复互补株中表达。结果进一步证明BA2380基因缺失突变株和回复互补株构建成功。本研究工作将为开展炭疽杆菌BA2380基因功能以及与炭疽毒力和S-层蛋白降解的相关性研究奠定基础,为进一步阐明炭疽杆菌致病机理以及炭疽杆菌新疫苗的研究积累科学数据。
[Abstract]:Bacillus anthracis is a potent pathogen, which can cause acute infectious diseases caused by zoonosis and pose a serious threat to human beings and animal husbandry all over the world. Bacillus anthracis is the main form of transmission and infection, after infection can lead to skin anthrax, intestinal anthrax, lung anthrax and other diseases. Bacillus anthracis can be used as a biological weapon to store. Therefore, the study of the pathogenic mechanism of Bacillus anthracis has attracted much attention. The early results of our laboratory indicated that the alkaline serine protease of Bacillus anthracis is related to the virulence factor and the degradation of S- layer protein of Bacillus anthracis, which may be involved in the pathogenicity of Bacillus anthracis infection, so it is necessary to further study its function. Based on this, we used Bacillus anthracis A16D2 as the starting strain to construct the BA2380 gene deletion mutant and the recovery complementary strain, and studied some physiological functions of the two mutants. First, the upstream and downstream homologous arms of BA2380 gene and the spc fragment of resistance gene were obtained by PCR amplification. The fragments were ligated to T-easy vector, and three donor plasmids were constructed. Using the modified "Golden Gate" cloning method in our laboratory, three fragments were simultaneously inserted into the thermo-sensitive shuttle vector pKMBK, and the gene targeting plasmid pK2380usdwas constructed. The gene targeting vector was introduced into the receptive cells of Bacillus anthracis A16D2, and the homologous recombination principle was used. Bacillus anthracis BA2380 gene deletion mutant A16D2 BA2380: SPC was screened and verified. Then, the BA2380 gene was amplified from Bacillus anthracis A16D2 strain by PCR technique, and introduced into the shuttle plasmid pBE2A of Escherichia coli and Bacillus subtilis by enzyme-ligation technique. The recombinant plasmid pBE2A-BA2380 was constructed, and the recombinant plasmid was transferred into BA2380 gene deletion strain A16D2 BA2380: SPC. The response complementary strain pBE2A-BA2380 / A16D2 was obtained. Finally, using the original strain A16D2 as control, the growth characteristics and spores formation of the mutant A16D2 BA2380: 1: SPC and the complementary strain pBE2A-BA2380 / A16D2 were studied. The results showed that there was no significant difference in the growth and spores formation among the three strains. The results showed that the absence of BA2380 gene had no effect on the growth and spores formation of Bacillus anthracis. At the same time, the expression of BA2380 gene in A16D2A16D2 BA2380: BA2380: spcPBE2A2380 / A16D2 and pBE2Ap-A16D2 was analyzed by Western blot technique. It was found that BA2380 gene was expressed only in the starting strain and the complementary response strain. The results further proved that the BA2380 gene deletion mutant and the recovery complementary strain were successfully constructed. This study will lay a foundation for the study of the function of Bacillus anthracis BA2380 gene and its correlation with anthrax virulence and the degradation of S- layer protein. It will also provide scientific data for the further elucidation of the pathogenic mechanism of Bacillus anthracis and the study of new anthrax vaccine.
【学位授予单位】：吉首大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378;S852.616

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