霍亂弧菌調(diào)控aphB的基因篩選及功能研究及銅綠假單胞菌對霍亂弧菌抑制作用研究
發(fā)布時間：2018-07-28 19:08
【摘要】：霍亂是一種急性腹瀉疾病,由革蘭氏陰性細(xì)菌霍亂弧菌(Vibrio cholerae)引起。通常是血清群01群的古典型和埃爾托生物型霍亂弧菌致病,臨床表現(xiàn)為大量米泔樣大便,嚴(yán)重時候?qū)е虏∪水a(chǎn)生霍亂肌無力并且迅速脫水。如果迅速和適當(dāng)?shù)闹委煕]有跟進(jìn),可導(dǎo)致病人死亡。 毒素協(xié)同調(diào)節(jié)菌毛(the toxin-coregulated pilus, TCP)是霍亂感染中的重要毒力因子,能協(xié)助霍亂弧菌定殖于小腸�；魜y弧菌產(chǎn)生的霍亂毒素(cholera toxin, CT)則導(dǎo)致病人劇烈腹瀉。負(fù)責(zé)合成TCP的基因以操縱子的方式位于霍亂弧菌基因組中一個大毒力島上,被稱做TCP-ACF元件或霍亂毒力島(vibrio pathogenicit island,VPI).CT毒素是由ctxA和ctxB兩個基因編碼,它們位于另一個遺傳元件原性噬菌體CTXΦ上。而位于霍亂毒力島上的ToxT是AraC型調(diào)控子,能直接激活tcp,ctxAB和其他輔助定殖因子的表達(dá)。ToxT的表達(dá)則是依賴于兩組基因的合作,包括大染色體上的跨膜調(diào)控蛋白編碼基因toxRS和霍亂毒力島上的tcpPH。而AphB與AphA協(xié)同作用可以激活tcpPH的表達(dá),同時也是導(dǎo)致兩大霍亂致病型古典型和埃爾托生物型毒力基因表達(dá)差異的原因。 霍亂弧菌有三套平行的群體感應(yīng)系統(tǒng),共同調(diào)控霍亂毒力基因表達(dá)。研究證明,霍亂弧菌群體感應(yīng)系統(tǒng)通過關(guān)鍵調(diào)控蛋白HapR負(fù)調(diào)控毒力基因的表達(dá).HapR通過直接作用于aphA的啟動子區(qū)調(diào)控毒力級聯(lián)系統(tǒng),降低毒力的表達(dá)。但是HapR并不影響aphB的表達(dá)。為了研究aphB的表達(dá)調(diào)控機(jī)理,我們利用轉(zhuǎn)座子插入突變建庫的方法進(jìn)行篩選。我們將aphB啟動子區(qū)克隆到兩個報告質(zhì)粒pBBRLux和pKP302上,并將其導(dǎo)入埃爾托生物型霍亂弧菌C6706(lacZ-)中,以此作為出發(fā)菌株。利用出發(fā)菌株與轉(zhuǎn)座子pSC123接合建庫,通過檢測aphB啟動子的表達(dá)水平來篩選影響aphB表達(dá)的突變株。在本研究中,我們篩選到兩株突變株T1和T2能影響aphB的表達(dá)。運(yùn)用隨機(jī)擴(kuò)增PCR方法檢測轉(zhuǎn)座子插入位點(diǎn),通過測序比對分析后發(fā)現(xiàn)T1中轉(zhuǎn)座子插入在vc1585讀碼框內(nèi),而T2中轉(zhuǎn)座子則插入在距vc1602基因末端7bp處。通過分別構(gòu)建框內(nèi)缺失株,我們確定vc1585能夠影響aphB的表達(dá),而vc1602及vc1601單缺失并不影響aphB的表達(dá)。 在另一個實(shí)驗(yàn)中我們擬篩選能夠抑制霍亂弧菌C6706生長的細(xì)菌。經(jīng)過篩選我們發(fā)現(xiàn)了十株細(xì)菌能顯著抑制霍亂弧菌的生長。經(jīng)API20E試紙條鑒定,發(fā)現(xiàn)十株菌均為銅綠假單胞菌(Pseudomonas aeruginosa)。通過與銅綠假單胞菌標(biāo)準(zhǔn)菌株ATCC27853對比發(fā)現(xiàn)可能是綠膿素(Pyocyanin, PCN)在抑菌過程中起主要作用。
[Abstract]:Cholera is an acute diarrhea disease caused by Vibrio cholerae (Vibrio cholerae). Vibrio cholerae, usually an ancient and biologic serotype of serogroup 01, is clinically characterized by a large amount of hogwash stool, which in severe cases leads to cholera myasthenia and rapid dehydration. If prompt and appropriate treatment is not followed up, the patient may die. Toxin coregulatory pili (the toxin-coregulated pilus, TCP) is an important virulence factor in cholera infection, which can help Vibrio cholerae colonize the small intestine. The cholera toxin (cholera toxin, CT) produced by Vibrio cholerae causes severe diarrhea. The genes responsible for the synthesis of TCP are located in a virulent island in the Vibrio cholerae genome in the form of manipulators, known as TCP-ACF elements or (vibrio pathogenicit islandVPI) .CT toxins are encoded by two genes, ctxA and ctxB. They are located on another genetic element, CTX 桅. ToxT, located on cholera virulence island, is a AraC type regulator, which can directly activate the expression of tcpnctxAB and other auxiliary colonizing factors. The expression of ToxT depends on the cooperation of two groups of genes. The transmembrane regulatory protein encoding gene toxRS and the cholera virulence island tcpPH. The synergistic action of AphB and AphA can activate the expression of tcpPH, and it is also the reason for the difference of virulence gene expression between the two major pathogenic types of cholera, paleotypic and Elto. Vibrio cholerae has three sets of parallel population sensing systems to regulate cholera virulence gene expression. The results showed that the virulence cascade system of Vibrio cholerae could reduce the expression of virulence genes by directly acting on the promoter region of aphA through the negative regulation of virulence gene expression of the key regulatory protein HapR by the population sensing system of Vibrio cholerae (Vibrio cholerae). But HapR does not affect the expression of aphB. In order to study the expression regulation mechanism of aphB, we used transposon insertion mutation to construct library. The aphB promoter region was cloned into two reporter plasmids pBBRLux and pKP302 and introduced into Vibrio cholerae C6706 (lacZ-). The library was constructed by the conjugation of the original strain and transposon pSC123, and the expression level of aphB promoter was detected to screen the mutants that affected the expression of aphB. In this study, we selected two mutant strains T 1 and T 2 to influence the expression of aphB. The insertion site of transposon was detected by random amplified PCR. The results showed that T1 transposon was inserted into vc1585 reading frame, while T2 transposon was inserted at the end of vc1602 gene 7bp. We determined that vc1585 could affect the expression of aphB, while vc1602 and vc1601 single deletion did not affect the expression of aphB. In another experiment, we intended to screen bacteria that could inhibit the growth of Vibrio cholerae C 6706. After screening, we found that ten strains of bacteria could significantly inhibit the growth of Vibrio cholerae. Ten strains of Pseudomonas aeruginosa (Pseudomonas aeruginosa). Were identified by API20E test strip. Compared with the standard Pseudomonas aeruginosa strain ATCC27853, it was found that the pyocyanin (Pyocyanin, PCN) may play a major role in the inhibition of Pseudomonas aeruginosa.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R378
[Abstract]:Cholera is an acute diarrhea disease caused by Vibrio cholerae (Vibrio cholerae). Vibrio cholerae, usually an ancient and biologic serotype of serogroup 01, is clinically characterized by a large amount of hogwash stool, which in severe cases leads to cholera myasthenia and rapid dehydration. If prompt and appropriate treatment is not followed up, the patient may die. Toxin coregulatory pili (the toxin-coregulated pilus, TCP) is an important virulence factor in cholera infection, which can help Vibrio cholerae colonize the small intestine. The cholera toxin (cholera toxin, CT) produced by Vibrio cholerae causes severe diarrhea. The genes responsible for the synthesis of TCP are located in a virulent island in the Vibrio cholerae genome in the form of manipulators, known as TCP-ACF elements or (vibrio pathogenicit islandVPI) .CT toxins are encoded by two genes, ctxA and ctxB. They are located on another genetic element, CTX 桅. ToxT, located on cholera virulence island, is a AraC type regulator, which can directly activate the expression of tcpnctxAB and other auxiliary colonizing factors. The expression of ToxT depends on the cooperation of two groups of genes. The transmembrane regulatory protein encoding gene toxRS and the cholera virulence island tcpPH. The synergistic action of AphB and AphA can activate the expression of tcpPH, and it is also the reason for the difference of virulence gene expression between the two major pathogenic types of cholera, paleotypic and Elto. Vibrio cholerae has three sets of parallel population sensing systems to regulate cholera virulence gene expression. The results showed that the virulence cascade system of Vibrio cholerae could reduce the expression of virulence genes by directly acting on the promoter region of aphA through the negative regulation of virulence gene expression of the key regulatory protein HapR by the population sensing system of Vibrio cholerae (Vibrio cholerae). But HapR does not affect the expression of aphB. In order to study the expression regulation mechanism of aphB, we used transposon insertion mutation to construct library. The aphB promoter region was cloned into two reporter plasmids pBBRLux and pKP302 and introduced into Vibrio cholerae C6706 (lacZ-). The library was constructed by the conjugation of the original strain and transposon pSC123, and the expression level of aphB promoter was detected to screen the mutants that affected the expression of aphB. In this study, we selected two mutant strains T 1 and T 2 to influence the expression of aphB. The insertion site of transposon was detected by random amplified PCR. The results showed that T1 transposon was inserted into vc1585 reading frame, while T2 transposon was inserted at the end of vc1602 gene 7bp. We determined that vc1585 could affect the expression of aphB, while vc1602 and vc1601 single deletion did not affect the expression of aphB. In another experiment, we intended to screen bacteria that could inhibit the growth of Vibrio cholerae C 6706. After screening, we found that ten strains of bacteria could significantly inhibit the growth of Vibrio cholerae. Ten strains of Pseudomonas aeruginosa (Pseudomonas aeruginosa). Were identified by API20E test strip. Compared with the standard Pseudomonas aeruginosa strain ATCC27853, it was found that the pyocyanin (Pyocyanin, PCN) may play a major role in the inhibition of Pseudomonas aeruginosa.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R378
【共引文獻(xiàn)】
相關(guān)期刊論文 前1條
1 田輝;;腸道致病菌群體感應(yīng)研究進(jìn)展[J];世界華人消化雜志;2007年08期
相關(guān)博士學(xué)位論文 前2條
1 史曉，
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