本文選題：霍亂弧菌 + OxyR抗氧化調(diào)節(jié)子　； 參考：《南京農(nóng)業(yè)大學(xué)》2012年碩士論文

【摘要】：霍亂是一種古老且流行廣泛的急性腸道感染疾病,臨床表現(xiàn)為大量米泔樣大便,嚴(yán)重時(shí)候?qū)е虏∪诵菘松踔了劳觥８锾m氏陰性細(xì)菌霍亂弧菌是引起霍亂的病原體,其致病能力主要由霍亂毒素和毒素共調(diào)節(jié)菌毛所介導(dǎo)。 LysR型轉(zhuǎn)錄調(diào)控因子是廣泛存在于原核生物中的一類DNA結(jié)合蛋白家族,該家族蛋白雖然結(jié)構(gòu)高度保守,但調(diào)控范圍涉及生物代謝、群體感應(yīng)、毒素產(chǎn)生、固氮和游動(dòng)性等多方面�？寡趸{(diào)節(jié)子OxyR是LysR型轉(zhuǎn)錄調(diào)控蛋白家族的成員之一,大小約34KDa,是菌株抵抗氧化損傷作用的最重要的防御屏障。對(duì)于OxyR調(diào)節(jié)子的研究在大腸桿菌、綠膿桿菌等細(xì)菌中進(jìn)行得比較廣泛和深入,而在霍亂弧菌中尚未有研究報(bào)道,因此本實(shí)驗(yàn)以霍亂弧菌OxyR調(diào)節(jié)子作為研究對(duì)象。 采用框內(nèi)敲除方法構(gòu)建霍亂弧菌oxyR突變株,發(fā)現(xiàn)有氧條件下培養(yǎng)時(shí)該突變株具有嚴(yán)重的生長(zhǎng)缺陷,而霍亂弧菌野生株的培養(yǎng)物上清液可以彌補(bǔ)這種生長(zhǎng)缺陷,使oxyR突變株恢復(fù)正常生長(zhǎng)；繼而通過一系列實(shí)驗(yàn)證明上清液中發(fā)揮作用的的活性物質(zhì)是與抗氧化相關(guān)的蛋白質(zhì),同時(shí)檢測(cè)到牛肝過氧化氫酶也具有彌補(bǔ)oxyR突變株生長(zhǎng)缺陷的活性,推測(cè)有氧條件下霍亂弧菌oxyR突變株的生長(zhǎng)缺陷是由外界環(huán)境中的氧分壓所導(dǎo)致,上清液中發(fā)揮作用的的活性物質(zhì)可能是與抗氧化相關(guān)的酶類。 構(gòu)建霍亂弧菌中幾種抗氧化功能基因(katG、katB、vc2637-8)的缺失突變株,檢測(cè)突變株上清液是否能夠幫助.oxyR突變株恢復(fù)正常生長(zhǎng),結(jié)果發(fā)現(xiàn)KatG、KatB蛋白是存在于上清液中的主要活性物質(zhì)；利用鎳柱純化帶有His標(biāo)簽的KatG、KatB融合蛋白并添加到oxyR突變株的培養(yǎng)液中,證實(shí)了二者確實(shí)具有彌補(bǔ)oxyR突變株生長(zhǎng)缺陷的活性；以lux作為報(bào)告基因構(gòu)建目的基因表達(dá)分析的檢測(cè)菌株,通過冷光檢測(cè)發(fā)現(xiàn)OxyR對(duì)katG、katB的表達(dá)不具有調(diào)控作用,因此證明了為何oxyR突變株上清液具有與野生株上清液相同的彌補(bǔ)oxyR突變株生長(zhǎng)缺陷的功能；通過在霍亂弧菌體內(nèi)超量表達(dá)帶有Flag標(biāo)簽的KatG、KatB融合蛋白,以霍亂弧菌確定的胞內(nèi)蛋白HapR作為對(duì)照,通過Western blotting檢測(cè)證實(shí)這兩個(gè)蛋白均是通過細(xì)胞裂解方式釋放到上清液中從而發(fā)揮作用的。 論文進(jìn)一步研究了霍亂弧菌oxyR、katG、katB、vc2637-8等抗氧化基因缺失株的生理功能。通過檢測(cè)生長(zhǎng)情況,發(fā)現(xiàn)除oxyR缺失導(dǎo)致菌株嚴(yán)重的生長(zhǎng)缺陷之外,katG-katB-vc2637-8三重基因缺失突變株的生長(zhǎng)也會(huì)受到一定程度的抑制；通過檢測(cè)突變株對(duì)H202的敏感性,發(fā)現(xiàn)抗氧化基因缺失會(huì)導(dǎo)致菌株對(duì)H2O2敏感性提高；通過檢測(cè)突變株的定殖能力,發(fā)現(xiàn)抗氧化基因的缺失不影響霍亂弧菌在乳鼠腸道的正常生長(zhǎng)與定殖。 綜上所述,在體外條件下,OxyR調(diào)節(jié)子對(duì)于霍亂弧菌抗氧化能力整體水平的調(diào)控十分重要,是保證菌株在有氧條件下正常生長(zhǎng)的關(guān)鍵。在后面的研究中,有必要對(duì)霍亂弧菌的OxyR抗氧化調(diào)控系統(tǒng)進(jìn)行更深入的研究,篩選受OxyR蛋白調(diào)控的功能基因。
[Abstract]:cholera is an ancient and widespread acute intestinal infection disease characterized by a large amount of rice wash - like stool , a severe time leading to shock or even death in the patient . Gram - negative bacteria cholera is a pathogen causing cholera , which is primarily mediated by cholera toxin and toxin co - regulation .

Anti - oxidation regulator OxyR is one of the most important defense barriers against oxidative damage . The research of OxyR regulator is one of the most important defense barriers against oxidative damage . The research of OxyR regulator has not been reported in E . coli and Pseudomonas aeruginosa .

The oxyR mutant strain was constructed by the method of in - frame knock - out method , and the mutant strain was found to have serious growth defects under the condition of aerobic condition , and the supernatant of the culture supernatant of the wild strain of Vibrio cholera can make up for the growth defect , so that the oxyR mutant strain can be restored to normal growth ;
A series of experiments prove that the active substance which plays a role in the supernatant is a protein related to oxidation resistance , and also has the activity of compensating for the growth defect of the oxyR mutant strain , and the growth defect of the oxyR mutant strain under aerobic conditions is caused by the oxygen partial pressure in the external environment , and the active substance in the supernatant fluid may be an enzyme which is related to oxidation resistance .

The deletion mutant of several antioxidant genes ( katG , katB , vc2637 - 8 ) was constructed . The results showed that KatG and KatB proteins were the main active substances in the supernatant .
KatG , KatB fusion protein with His tag was purified by nickel column and added to the culture solution of oxyR mutant strain .
The expression of katG and katB was not regulated by cold light detection , and it was proved that the supernatant of oxyR mutant had the same function as the supernatant of wild strain to make up the growth defect of oxyR mutant .
The KatG , KatB fusion protein with Flag tag was expressed in the body of Vibrio cholera , and the intracellular protein HapR determined by Vibrio cholera was used as a control , and the two proteins were detected by Western blotting to confirm that both proteins were released into the supernatant by cell lysis .

In this paper , the physiological function of the anti - oxidation gene deletion strain , such as oxyR , katG , katB , vc2637 - 8 and so on , was further studied . By detecting the growth conditions , it was found that the growth of katG - katB - vc2637 - 8 triple gene deletion mutant was inhibited by some extent .
By detecting the sensitivity of mutant strain H202 , it was found that the deletion of anti - oxidation gene could increase the sensitivity of strain to H2O2 .
By detecting the colonizing ability of mutant strain , it was found that the deletion of anti - oxidation gene did not affect the normal growth and colonization of cholera in the intestinal tract .

In conclusion , under the condition of in vitro , the regulation of OxyR regulator is very important for the whole level of anti - oxidation ability of Vibrio , and it is the key to guarantee the normal growth of the strain under aerobic conditions . In the latter study , it is necessary to study the OxyR anti - oxidation regulatory system of Vibrio cholin more deeply and screen the functional gene regulated by OxyR protein .

【學(xué)位授予單位】：南京農(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

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

1 汪保衛(wèi);施慶珊;歐陽友生;陳儀本;;細(xì)菌抗氧化系統(tǒng)-oxyR調(diào)節(jié)子研究進(jìn)展[J];微生物學(xué)報(bào);2008年11期

相關(guān)博士學(xué)位論文 前1條

1 王鵬;中慢生天山根瘤菌胞外多糖在共生過程中的功能研究[D];南京農(nóng)業(yè)大學(xué);2010年

相關(guān)碩士學(xué)位論文 前2條

1 陳蘇舒;霍亂弧菌LysR/MFS調(diào)控體系的功能研究[D];南京農(nóng)業(yè)大學(xué);2011年

2 沈晶;龜裂鏈霉菌（Streptomyces rimosus）M4018中rex基因的克隆、表達(dá)和生物功能初步研究[D];華東理工大學(xué);2010年

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本文編號(hào)：1835243