本文選題：菌種鑒定 + 蛋白質(zhì)組��； 參考：《江蘇大學(xué)》2017年碩士論文

【摘要】：醫(yī)院周?chē)h(huán)境的細(xì)菌污染是當(dāng)前醫(yī)療衛(wèi)生行業(yè)關(guān)注的重要問(wèn)題。本研究選取醫(yī)院分離的兩株對(duì)家蠶具有致死性的細(xì)菌為研究對(duì)象,進(jìn)行菌種鑒定,比較蛋白質(zhì)組差異,對(duì)致病基因進(jìn)行PCR鑒定,非溶血性腸毒素進(jìn)行了原核表達(dá),構(gòu)建穿梭質(zhì)粒。主要的研究?jī)?nèi)容及結(jié)果如下:(1)兩種致病性不同的細(xì)菌的菌種鑒定對(duì)從醫(yī)院分離的具有不同致病性的兩株細(xì)菌,通過(guò)表型、生理生化特征反應(yīng)以及16S rRNA基因序列構(gòu)建系統(tǒng)發(fā)育樹(shù)分析。鑒定結(jié)果顯示細(xì)菌65-5為枯草芽孢桿菌(Bacillus subtilis),細(xì)菌41-1為廣義的蠟樣芽孢桿菌(Bacilluscereus sensu lato)。(2)兩株細(xì)菌的蛋白質(zhì)組比較研究通過(guò)二維電泳串聯(lián)質(zhì)譜的方法,比較兩種細(xì)菌的蛋白質(zhì)組差異。兩種細(xì)菌蛋白質(zhì)圖譜中的蛋白點(diǎn)存在較大的差異。選取圖譜上的蛋白點(diǎn),進(jìn)行質(zhì)譜鑒定,進(jìn)一步確定強(qiáng)致病菌41-1為蠟樣芽孢桿菌。通過(guò)蛋白質(zhì)組研究細(xì)菌分泌的蛋白,蠟樣桿菌上清的蛋白中包含有鞭毛蛋白和金屬蛋白酶。(3)PCR鑒定蠟樣芽孢桿菌中致病基因41-1菌株通過(guò)菌種鑒定確定為蠟樣芽孢桿菌。蠟樣芽孢桿菌是條件致病菌,能產(chǎn)生多種致病性毒素。利用PCR檢測(cè)鑒定出來(lái)的蠟樣芽孢桿菌存在的毒素基因,確定該菌株包含非溶血性腸毒素基因nhe A、nhe B、nhe C,不包含溶血性腸毒素基因hbl A、hbl C、hbl D,細(xì)胞毒素K基因cyt K和腸毒素Cereulide基因ces。并獲得nhe A、nhe B、nhe C三種致病基因的克隆。(4)對(duì)三種nhe A、nhe B、nhe C基因的原核表達(dá)將克隆獲得的三種致病基因nhe A、nhe B、nhe C基因,分別構(gòu)建成大腸桿菌表達(dá)載體 pET-30a(+)-nhe A、pET-30a(+)-nheB 以及 pET-30a(+)-nhe C,三種非溶血性腸毒素亞基成功在大腸桿菌中表達(dá),NheB亞基在原核表達(dá)時(shí)易于形成多聚體,質(zhì)譜鑒定結(jié)果顯示三種蛋白正確表達(dá)。用割膠純化的三種非溶血性腸毒素蛋白作為抗原免疫新西蘭大白兔,獲得三種目的蛋白的多克隆抗血清。用原核表達(dá)相應(yīng)的重組蛋白驗(yàn)證多克隆抗體的效果,確定三種多克隆抗體成功制備。用制備的多克隆抗體對(duì)蠟樣芽孢桿菌上清中的蛋白進(jìn)行了檢測(cè),非溶血性腸毒素亞基Nhe A和Nhe B被特異性的鑒定,而未鑒定到非溶血性腸毒素Nhe C亞基。(5)穿梭質(zhì)粒的構(gòu)建為了研究三種非溶血性腸毒素亞基的功能和相互作用之間的關(guān)系。利用基于A(yíng)cMNPV bacmid表達(dá)系統(tǒng),將三種非溶血性腸毒素基因克隆到桿狀病毒的穿梭載體上,獲得對(duì)應(yīng)的穿梭質(zhì)粒。將穿梭質(zhì)粒轉(zhuǎn)化到Bacmid的感受態(tài)細(xì)胞發(fā)生轉(zhuǎn)座,成功構(gòu)造 pACnhe C-nhe-nhe B bacmid,pACnhe C-nhe A bacmid,pACnhe C-nhe B bacmid。
[Abstract]:Bacterial pollution in the surrounding environment of hospitals is an important issue concerned by the medical and health industry.In this study, two strains of bacteria that are lethal to silkworm (Bombyx mori) were selected as the research object. The strains were identified, the proteome difference was compared, the pathogenic gene was identified by PCR, and the non-hemolytic enterotoxin was expressed in prokaryotic.The shuttle plasmid was constructed.The main contents and results of this study are as follows: (1) the identification of two strains of bacteria with different pathogenicity to two strains of bacteria with different pathogenicity isolated from hospitals, through phenotypes,Physiological and biochemical responses and phylogenetic tree analysis of 16s rRNA gene sequence.The results showed that bacteria 65-5 were Bacillus subtilisus and bacteria 41-1 were generalized Bacillus cereus sensu lato.2.The proteome differences between the two strains were compared by two-dimensional electrophoresis tandem mass spectrometry.The protein spots in the protein map of the two bacteria differ greatly.The protein spots on the map were selected and identified by mass spectrometry, and the strong pathogenic bacteria 41-1 was further identified as Bacillus cereus.The protein secreted by bacteria was studied by proteome. The protein of supernatant of Bacillus cereus contained flagellin and metalloproteinase. The pathogenic gene 41-1 of Bacillus cereus was identified as Bacillus cereus by PCR.Bacillus cereus is a conditional pathogen that produces a variety of pathogenic toxins.The toxin gene of Bacillus cereus was identified by PCR detection. It was determined that the strain contained the non-hemolytic enterotoxin gene nhe Anhe Bnhe C, not the hemolytic enterotoxin gene hbl nhe hbl ChblD, the cytotoxin K gene cyt K and the enterotoxin Cereulide gene Ces.The prokaryotic expression of three pathogenicity genes, nhe Anhe Bnhe Bnhe C, was obtained from the prokaryotic expression of three kinds of nhe Anhe Bnhe C genes, and the three pathogenicity genes nhe Anhe Bnhe C gene were cloned, and the three pathogenicity genes nhe Anhe Bnhe C gene were cloned.E. coli expression vectors pET-30a (pET-30a) and pET-30a (pET-30a) and pET-30a (pET-30a) were constructed respectively. Three non-hemolytic enterotoxin subunits were successfully expressed in E. coli.The results of mass spectrometry showed that the three proteins were correctly expressed.Three kinds of non-hemolytic enterotoxin proteins purified by tapping were used as antigens to immunize New Zealand white rabbits and polyclonal antisera of three kinds of target proteins were obtained.The effect of polyclonal antibody was verified by prokaryotic expression of corresponding recombinant protein, and three polyclonal antibodies were successfully prepared.The protein in the supernatant of Bacillus cereus was detected by polyclonal antibody. The non-hemolytic enterotoxin subunits Nhe A and Nhe B were specifically identified.In order to study the relationship between the function and interaction of the three non-hemolytic enterotoxin subunits, the shuttle plasmid of Nhe C subunit of non-hemolytic enterotoxin was not identified.Based on AcMNPV bacmid expression system, three non-hemolytic enterotoxin genes were cloned into the shuttle vector of baculovirus and the corresponding shuttle plasmid was obtained.The shuttle plasmid was transformed into the competent cells of Bacmid, and pACnhe C-nhe-nhe B bacmidhe C-nhe A bacmidhe C-nhe C-nhe B was successfully constructed.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R446.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 杜美紅;趙瑞雪;李靜雯;;基質(zhì)輔助激光解吸電離飛行時(shí)間質(zhì)譜分析技術(shù)在微生物檢測(cè)與鑒定中的應(yīng)用[J];食品安全質(zhì)量檢測(cè)學(xué)報(bào);2017年02期

2 宗凱;周莉質(zhì);李云飛;陳雪嬌;孫娟娟;鄭海松;余曉峰;;基于MALDI-TOF-MS質(zhì)譜技術(shù)對(duì)蜂蜜中芽孢桿菌的鑒定與分型[J];安徽農(nóng)業(yè)科學(xué);2016年08期

3 王艷萍;夏云;;應(yīng)用16S rRNA基因序列分析技術(shù)鑒定臨床非典型細(xì)菌的研究[J];中國(guó)病原生物學(xué)雜志;2013年11期

4 頓玉慧;趙更峰;鄭啟偉;;蠟樣芽孢桿菌致吐毒素的研究現(xiàn)狀[J];食品科學(xué);2009年09期

5 劉國(guó)紅;劉波;林乃銓;林營(yíng)志;;芽孢桿菌的系統(tǒng)進(jìn)化及其屬分類(lèi)學(xué)特征[J];福建農(nóng)業(yè)學(xué)報(bào);2008年04期

6 儀淑敏;李遠(yuǎn)釗;張培正;梁浩;朱英蓮;;蠟樣芽孢桿菌在營(yíng)養(yǎng)肉湯和維也納香腸中的生長(zhǎng)模型及控制[J];食品工業(yè)科技;2007年09期

相關(guān)博士學(xué)位論文 前1條

1 崔一芳;蠟樣芽孢桿菌毒素特征及耐藥基因tet(45)的可移動(dòng)性研究[D];中國(guó)農(nóng)業(yè)大學(xué);2016年

，

本文編號(hào)：1751781