霍乱弧菌oxyR突变株生长缺陷机制的研究

发布时间：2018-05-02 19:49

本文选题：霍乱弧菌 + OxyR抗氧化调节子　；参考：《南京农业大学》2012年硕士论文

【摘要】：霍乱是一种古老且流行广泛的急性肠道感染疾病,临床表现为大量米泔样大便,严重时候导致病人休克甚至死亡。革兰氏阴性细菌霍乱弧菌是引起霍乱的病原体,其致病能力主要由霍乱毒素和毒素共调节菌毛所介导。 LysR型转录调控因子是广泛存在于原核生物中的一类DNA结合蛋白家族,该家族蛋白虽然结构高度保守,但调控范围涉及生物代谢、群体感应、毒素产生、固氮和游动性等多方面。抗氧化调节子OxyR是LysR型转录调控蛋白家族的成员之一,大小约34KDa,是菌株抵抗氧化损伤作用的最重要的防御屏障。对于OxyR调节子的研究在大肠杆菌、绿脓杆菌等细菌中进行得比较广泛和深入,而在霍乱弧菌中尚未有研究报道,因此本实验以霍乱弧菌OxyR调节子作为研究对象。采用框内敲除方法构建霍乱弧菌oxyR突变株,发现有氧条件下培养时该突变株具有严重的生长缺陷,而霍乱弧菌野生株的培养物上清液可以弥补这种生长缺陷,使oxyR突变株恢复正常生长；继而通过一系列实验证明上清液中发挥作用的的活性物质是与抗氧化相关的蛋白质,同时检测到牛肝过氧化氢酶也具有弥补oxyR突变株生长缺陷的活性,推测有氧条件下霍乱弧菌oxyR突变株的生长缺陷是由外界环境中的氧分压所导致,上清液中发挥作用的的活性物质可能是与抗氧化相关的酶类。构建霍乱弧菌中几种抗氧化功能基因(katG、katB、vc2637-8)的缺失突变株,检测突变株上清液是否能够帮助.oxyR突变株恢复正常生长,结果发现KatG、KatB蛋白是存在于上清液中的主要活性物质；利用镍柱纯化带有His标签的KatG、KatB融合蛋白并添加到oxyR突变株的培养液中,证实了二者确实具有弥补oxyR突变株生长缺陷的活性；以lux作为报告基因构建目的基因表达分析的检测菌株,通过冷光检测发现OxyR对katG、katB的表达不具有调控作用,因此证明了为何oxyR突变株上清液具有与野生株上清液相同的弥补oxyR突变株生长缺陷的功能；通过在霍乱弧菌体内超量表达带有Flag标签的KatG、KatB融合蛋白,以霍乱弧菌确定的胞内蛋白HapR作为对照,通过Western blotting检测证实这两个蛋白均是通过细胞裂解方式释放到上清液中从而发挥作用的。论文进一步研究了霍乱弧菌oxyR、katG、katB、vc2637-8等抗氧化基因缺失株的生理功能。通过检测生长情况,发现除oxyR缺失导致菌株严重的生长缺陷之外,katG-katB-vc2637-8三重基因缺失突变株的生长也会受到一定程度的抑制；通过检测突变株对H202的敏感性,发现抗氧化基因缺失会导致菌株对H2O2敏感性提高；通过检测突变株的定殖能力,发现抗氧化基因的缺失不影响霍乱弧菌在乳鼠肠道的正常生长与定殖。综上所述,在体外条件下,OxyR调节子对于霍乱弧菌抗氧化能力整体水平的调控十分重要,是保证菌株在有氧条件下正常生长的关键。在后面的研究中,有必要对霍乱弧菌的OxyR抗氧化调控系统进行更深入的研究,筛选受OxyR蛋白调控的功能基因。
[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 .

【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

【共引文献】