本文選題：炭疽桿菌 + Golden��； 參考：《吉首大學(xué)》2017年碩士論文

【摘要】：炭疽桿菌(Bacillus anthracis)是烈性病原菌,能夠引起人畜共患的急性傳染病,對(duì)全世界人類及畜牧業(yè)造成嚴(yán)重威脅。芽胞是炭疽桿菌傳播和感染的主要形式,感染后可導(dǎo)致皮膚炭疽、腸道炭疽、肺炭疽等疾病。炭疽桿菌可用作生物武器來儲(chǔ)備。故有關(guān)炭疽桿菌致病機(jī)理的研究一直備受關(guān)注。本實(shí)驗(yàn)室早期研究結(jié)果表明,BA2380堿性絲氨酸蛋白酶與炭疽桿菌的毒力因子和S-層蛋白降解相關(guān),可能參與炭疽桿菌感染機(jī)體的致病性,因而有必要對(duì)其功能進(jìn)行深入研究�；诖�,我們以炭疽桿菌A16D2為出發(fā)菌株,開展了BA2380基因缺失突變株和回復(fù)互補(bǔ)株的構(gòu)建,并研究了兩類突變株的部分生理功能。首先,通過PCR擴(kuò)增獲得了BA2380基因上下游同源臂以及抗性基因spc片段,分別連接到T-easy載體,構(gòu)建了3個(gè)供體質(zhì)粒。利用本實(shí)驗(yàn)室改造的“Golden Gate”克隆方法將三個(gè)片段同時(shí)連入溫敏型穿梭載體pKMBK中,構(gòu)建了基因打靶質(zhì)粒pK2380usd,將該基因打靶載體導(dǎo)入炭疽桿菌A16D2感受態(tài)細(xì)胞中,利用同源重組原理,篩選得到炭疽桿菌BA2380基因缺失突變株A16D2△BA2380::spc,并對(duì)其進(jìn)行了驗(yàn)證。接著,利用PCR技術(shù)從炭疽芽胞桿菌A16D2株擴(kuò)增出BA2380基因,通過酶切-連接技術(shù)導(dǎo)入大腸桿菌-枯草桿菌穿梭質(zhì)粒pBE2A中,構(gòu)建了回復(fù)突變質(zhì)粒pBE2A-BA2380,將該重組質(zhì)粒電轉(zhuǎn)入BA2380基因缺失株A16D2△BA2380::spc中,篩選獲得了回復(fù)互補(bǔ)株pBE2A-BA2380/A16D2。最后,以出發(fā)菌株A16D2為對(duì)照,對(duì)BA2380基因缺失突變株A16D2△BA2380::spc和回復(fù)互補(bǔ)株pBE2A-BA2380/A16D2的生長(zhǎng)特性和芽胞形成情況進(jìn)行了研究,發(fā)現(xiàn)三者在菌株生長(zhǎng)和芽胞形成方面沒有顯著差異,表明BA2380基因缺失對(duì)炭疽桿菌生長(zhǎng)和芽胞形成沒有影響。同時(shí),采用Western blot技術(shù)分析了BA2380基因在A16D2、A16D2△BA2380::spc、pBE2A-BA2380/A16D2和pBE2A/A16D2中的表達(dá)情況,發(fā)現(xiàn)BA2380基因只在出發(fā)菌株和回復(fù)互補(bǔ)株中表達(dá)。結(jié)果進(jìn)一步證明BA2380基因缺失突變株和回復(fù)互補(bǔ)株構(gòu)建成功。本研究工作將為開展炭疽桿菌BA2380基因功能以及與炭疽毒力和S-層蛋白降解的相關(guān)性研究奠定基礎(chǔ),為進(jìn)一步闡明炭疽桿菌致病機(jī)理以及炭疽桿菌新疫苗的研究積累科學(xué)數(shù)據(jù)。
[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.
【學(xué)位授予單位】：吉首大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R378;S852.616

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 王玉飛;曲R，

本文編號(hào)：2068356