本文選題：VP　切入點(diǎn)：生物膜表型　出處：《四川農(nóng)業(yè)大學(xué)》2012年碩士論文

【摘要】：背景：副溶血弧菌(Vibrio parahaemolyticus, VP)是一種革蘭陰性嗜鹽弧菌。廣泛分布于海洋和其他咸水環(huán)境中,多數(shù)海產(chǎn)品均天然攜帶本菌,人們食入未煮熟或烹飪不當(dāng)?shù)漠a(chǎn)品,會(huì)引起胃腸炎或食物中毒,其流行具有季節(jié)性,一般溫暖的月份(5月-10月)易爆發(fā)。該菌最早是從1950年日本大阪的海產(chǎn)品食物中毒事件體檢中篩選出來,此事件中由該菌引發(fā)上的食物中毒占40-60%。近幾年由VP引起急性胃腸炎占世界之首,在我國,特別是一些沿海城市,中毒事件在數(shù)量上超過沙門氏菌,是食源性病原菌之首。其主要癥狀表現(xiàn)為：腹瀉、下腹絞痛、反胃、嘔吐,治療不及時(shí)會(huì)引起敗血病及組織感染,嚴(yán)重者甚至死亡。因此,為降低食源性疾病的風(fēng)險(xiǎn),有必要對副溶血弧菌的致病機(jī)制進(jìn)行深入研究。 副溶血弧菌在生存和感染的過程中都能形成生物膜,同霍亂弧菌(Vibrio cholerae),哈氏弧菌(Vibrio harveyi)及創(chuàng)傷弧菌(Vibrio vulnificus)一樣,生物膜在其適應(yīng)環(huán)境和廣泛傳播過程中發(fā)揮著重要作用。生物膜的形成和消散是一個(gè)動(dòng)態(tài)過程,這過程存在一系列基因表達(dá)的變化,而主要調(diào)控這些基因表達(dá)的就是密度感應(yīng)(Quorum Sensing,QS)系統(tǒng),VP的QS系統(tǒng)調(diào)控子蛋白OpaR與哈氏弧菌的LuxR在氨基酸序列上有96%的同源性,因此二者的QS系統(tǒng)應(yīng)該具有相似的作用機(jī)制。雖然表型實(shí)驗(yàn)及表達(dá)譜分析表明OpaR能調(diào)控VP莢膜多糖基因、胞外多糖基因、c-di-GMP分子代謝及鞭毛基因等生物膜形成相關(guān)基因的表達(dá),但是OpaR對這些基因的轉(zhuǎn)錄調(diào)控機(jī)制并未被闡明,因此有必要做進(jìn)一步的研究。 目的：本研究目的期望建立副溶血弧菌RIMD2210633生物膜表型分析實(shí)驗(yàn)方案和LacZ實(shí)驗(yàn)的技術(shù)平臺(tái)。為進(jìn)一步研究轉(zhuǎn)錄調(diào)控子OpaR的轉(zhuǎn)錄調(diào)控機(jī)制,以及副溶血弧菌后其他調(diào)控子的功能奠定基礎(chǔ)。 方法：(1)生物膜表型實(shí)驗(yàn)方案：生物膜形成過程中可能會(huì)受很多因素的影響,包括溫度、pH、鹽度、營養(yǎng)狀況、培養(yǎng)方式及培養(yǎng)基等,通過對這些因素的比較和分析,建立了3種不同的生物膜表型分析方法(菌落表面褶皺、生物膜基質(zhì)的剛果紅染色、生物膜的結(jié)晶紫染色)。并通過對VP突變株(ΔopaR、ΔtoxR)與野生株(J5421)的生物膜測定分析,來驗(yàn)證這三個(gè)生物膜表型試驗(yàn)各自的技術(shù)平臺(tái)的穩(wěn)定性。 (2)LacZ報(bào)告基因融合實(shí)驗(yàn)技術(shù)：PCR擴(kuò)增靶基因的整個(gè)啟動(dòng)子區(qū)序列,并將其直接克隆入pHRP309質(zhì)粒中,構(gòu)建重組質(zhì)粒。將VPA1513重組質(zhì)粒轉(zhuǎn)入VP野生株(WT)和OpaR突變株(AopaR)中,而后通過比較二者β-半乳糖苷酶活性的差異,確定OpaR對VPA1513的調(diào)控關(guān)系,以檢驗(yàn)實(shí)驗(yàn)平臺(tái)的穩(wěn)定性。 結(jié)果：30℃比37℃更適合VP生長而形成生物膜,在兩種溫度下,200rpm振蕩培養(yǎng)12h就到平臺(tái)期了；VP在營養(yǎng)豐富的HI平板比營養(yǎng)次之的LB、M、APW#3平板的菌落褶皺更明顯；鹽濃度越低(為0.5%)褶皺越明顯；靜置培養(yǎng)更適合褶皺表型,100rpm搖動(dòng)培養(yǎng)方式更適合生物膜形成的定量測定；生物膜的形成在中性和偏堿環(huán)境中的差別不大；培養(yǎng)時(shí)間對剛果紅染色的影響沒有菌落表面褶皺那么明顯,但都有培養(yǎng)時(shí)間越長表型越明顯的趨勢；三種生物膜表型分析試驗(yàn)都證明了OpaR負(fù)調(diào)控和ToxR正調(diào)控生物膜形成。 用載體pHRP309成功構(gòu)建出9個(gè)與VP生物膜或毒力相關(guān)基因的的LacZ(?)重組質(zhì)粒；并通過測定VP突變株AopaR與野生株(J5421)β-半乳糖苷酶活性的差異,證明了OpaR對VPA1513的轉(zhuǎn)錄具有抑制作用。 結(jié)論：成功建立了菌落表面褶皺、生物膜基質(zhì)的剛果紅染色、生物膜的結(jié)晶紫染色三種VP生物膜相關(guān)的表型試驗(yàn)和LacZ報(bào)告基因融合實(shí)驗(yàn)的穩(wěn)定的技術(shù)平臺(tái)。發(fā)現(xiàn)了溫度、鹽濃度、培養(yǎng)時(shí)間、培養(yǎng)方式和營養(yǎng)狀況對生物膜形成有影響,且偏堿環(huán)境不會(huì)影響生物膜的形成。本研究給后續(xù)生物膜表型和其他轉(zhuǎn)錄調(diào)控機(jī)制的研究奠定了基礎(chǔ)。
[Abstract]:Background: Vibrio parahaemolyticus (Vibrio parahaemolyticus VP) is a gram-negative halophilic Vibrio. Widely distributed in marine and other saltwater environment, most of the seafood were carrying the natural bacteria, people undercooked or improper cooking products, can cause gastroenteritis or food poisoning, which is seasonal, general the warm months (May -10 months) explosive. The strain was first screened from medical poisoning incident in Japan in 1950 Osaka seafood food, this incident caused by the bacteria on food poisoning accounted for 40-60%. in recent years caused by VP of acute gastroenteritis accounted for first in the world, in our country, especially in some coastal areas city, poisoning more than Salmonella in quantity, is the first food borne pathogens. The main symptoms are diarrhea, abdominal cramps, nausea, vomiting, not timely treatment will cause septicemia and tissue infection, severe and even Death. Therefore, in order to reduce the risk of food borne diseases, it is necessary to study the pathogenesis of Vibrio parahaemolyticus.
Vibrio parahaemolyticus could form biofilm in the process of survival and infection with Vibrio cholerae (Vibrio, cholerae), Vibrio harveyi (Vibrio harveyi) and Vibrio vulnificus (Vibrio vulnificus), biofilm plays an important role in its adaptation to environment and wide spread process. The formation and dissipation of biofilm is a a dynamic process, this process has a series of changes in the expression of genes, which mainly regulates the expression of these genes is induction density (Quorum Sensing, QS) system, has 96% homology in amino acid sequence of the VP QS system regulator protein OpaR and Vibrio harveyi LuxR, so QS system two. Should have a similar mechanism. Although the expression and spectrum analysis show that OpaR can regulate VP capsular polysaccharide gene phenotype experiments, the extracellular polysaccharide gene, expression of related gene c-di-GMP molecular metabolism and flagellar gene biological membrane, but OpaR The transcriptional regulation mechanism of these genes has not been clarified, so it is necessary to do further research.
Objective: the aim of this study is to establish the experimental program of RIMD2210633 biofilm phenotypic analysis of Vibrio parahaemolyticus and the technological platform of LacZ experiment, in order to further study the transcriptional regulation mechanism of transcriptional regulator OpaR and the function of other regulators of Vibrio parahaemolyticus.
Methods: (1) experimental scheme of biofilm phenotype: biofilm formation may be affected by many factors in the process of pH, including temperature, salinity, nutrient status, culture method and culture medium, through the comparison and analysis of these factors, we establish 3 different biofilm phenotype (colony surface analysis method fold, biofilms stained with Congo red and crystal violet staining) and biofilm. The VP mutant (delta opaR, Delta toxR) and wild-type (J5421) determination of the biological membrane technology platform to verify the stability of the three biofilm phenotype test respectively.
(2) LacZ reporter gene fusion experiments: PCR sequence was amplified by the entire promoter region of target genes, and directly cloned into pHRP309 plasmid, to construct recombinant plasmid VPA1513. The recombinant plasmid was transformed into VP wild strain (WT) and OpaR mutant (AopaR), then the difference of beta galactosidase activity comparison of the two, to determine the regulation between OpaR to VPA1513, to test the stability of the experimental platform.
Results: 30 degrees centigrade than 37 DEG C is more suitable for VP growth and biofilm formation, at the two temperatures, 200rpm 12h oscillating culture to the platform period; VP HI tablet in nutrient rich nutrition than the LB, M, APW#3 were more obvious fold flat; the lower the concentration of salt (0.5%) fold more obvious; static culture is more suitable for the quantitative determination of 100RPM phenotype fold, shaking more suitable for biofilm formation in culture; formed in neutral and alkaline environment is not very different biofilm; effect of culture time on the Congo red staining of the colony surface folds not so obvious, but have a longer phenotype more training obvious; three kinds of biofilm phenotype tests have proved that the OpaR negative regulation and ToxR positive regulation of biofilm formation.
9 recombinant plasmids with VP biofilm or virulence related genes were successfully constructed by vector pHRP309. The difference between VP mutant AopaR and wild J5421 (J5421) beta galactosidase activity was proved by OpaR, which proved that OpaR had inhibitory effect on VPA1513 transcription.
Conclusion: we have successfully established the colony surface folds, Congo red staining of the biofilm matrix, crystal violet staining of the biofilm stable technology platform of three kinds of VP biofilm phenotype correlation test and LacZ reporter gene fusion experiments. It is found that the temperature, salt concentration, culture time, culture and nutrition effect of biofilm the formation, and the alkaline environment will not affect biofilm formation. This study laid the foundation for the subsequent biofilm phenotype and transcriptional regulation.

【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R378

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 齊緒林;林東f ;徐曉剛;樊曉明;吳麗桂;;副溶血弧菌胃腸道感染2247例分析[J];中國臨床醫(yī)學(xué);2008年05期

2 陳珍;覃映雪;鄒文政;徐曉津;邢顏麗;鄢慶枇;;致病性副溶血弧菌生物膜形成特性研究[J];海洋學(xué)報(bào)(中文版);2010年05期

3 劉秀梅,陳艷,王曉英,計(jì)融;1992～2001年食源性疾病暴發(fā)資料分析——國家食源性疾病監(jiān)測網(wǎng)[J];衛(wèi)生研究;2004年06期

4 李志峰,聶軍;副溶血弧菌tlh基因的克隆及序列分析[J];中國人獸共患病雜志;2003年02期

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