本文關(guān)鍵詞：金黃色葡萄球菌轉(zhuǎn)座子突變文庫的構(gòu)建與腸毒素A相關(guān)基因的研究　出處：《西北農(nóng)林科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 金黃色葡萄球菌 腸毒素A 質(zhì)粒 轉(zhuǎn)座子

【摘要】：金黃色葡萄球菌(Staphylococcus aureus,S.aureus)簡稱金葡菌,是一種重要的食源性致病菌,其引起食物中毒的主要致病因子是腸毒素。由于進(jìn)食被金黃色葡萄球菌及其產(chǎn)生的腸毒素所污染的食物而引起的食物中毒被稱為金黃色葡萄球菌食物中毒(Staphylococcal food poisoning,SFP),一般在短時(shí)間內(nèi)就會(huì)有相應(yīng)的癥狀發(fā)作,包括惡心、嘔吐、腹部絞痛或腹瀉等。由于金黃色葡萄球菌無處不在,廣泛分布于空氣、灰塵和食品加工設(shè)備表面等,使得食品極易產(chǎn)生交叉污染,所以盡管嚴(yán)格控制食品加工的各個(gè)過程,還是會(huì)受到不同程度的污染。其中,食源性動(dòng)物和食品加工者的手或呼吸道分泌物被視為食品污染金葡菌的主要來源。在常見的金葡菌食物中毒事件中,大部分是由SEA引起的,其次依次是SED、SEC、SEB和SEE。轉(zhuǎn)座子(transposon)是細(xì)菌和許多原核與真核生物基因組中一段特異的具有轉(zhuǎn)位特性的獨(dú)立的DNA序列,可以在細(xì)菌的染色體、質(zhì)�；蚴删w之間水平轉(zhuǎn)移。研究質(zhì)粒消除和Tn917轉(zhuǎn)座子的隨機(jī)插入對(duì)食源性金黃色葡萄球菌腸毒素A表達(dá)的影響,是本課題的主要研究內(nèi)容。本研究對(duì)48株不同來源并攜帶腸毒素sea基因的金葡菌先進(jìn)行質(zhì)粒消除,再對(duì)消除前后菌株進(jìn)行18種腸毒素基因和29種耐藥基因的PCR檢測(cè),以及12種臨床常用抗生素的藥敏檢測(cè)。隨后將質(zhì)粒消除后金葡菌通過隨機(jī)插入轉(zhuǎn)座子Tn917對(duì)其染色體進(jìn)行誘變,產(chǎn)生變異體,并建立轉(zhuǎn)座子文庫。最后通過ELISA方法篩選腸毒素A表達(dá)異常的菌株,同時(shí)檢測(cè)生物膜形成能力以及產(chǎn)溶血毒素異常的突變株,并與原始菌株比較其差異性。這為闡明腸毒素A分子機(jī)制和金黃色葡萄球菌引起食物中毒的防控提供了理論依據(jù)。主要研究結(jié)果如下:(1)對(duì)48株不同來源并攜帶腸毒素sea基因的金葡菌先進(jìn)行質(zhì)粒消除,再對(duì)消除前后菌株進(jìn)行18種腸毒素基因和29種耐藥基因的PCR檢測(cè),以及12種臨床常用抗生素的藥敏檢測(cè)。結(jié)果表明,質(zhì)粒消除對(duì)腸毒素基因see,耐藥基因tet O和tetK,以及抗生素AMP的影響較為顯著,對(duì)ERY有影響但不顯著,說明耐藥基因tetO和tetK存在細(xì)菌質(zhì)粒上,以及AMP和ERY的基因也可能在質(zhì)粒上。金葡菌的腸毒素基因和耐藥基因位點(diǎn)比較復(fù)雜,同種基因在不同菌株間存在差異,說明其具有無規(guī)律性。此外,耐藥基因型和耐藥表型之間也存在一定差異。(2)將含有轉(zhuǎn)座子Tn917的溫敏穿梭質(zhì)粒轉(zhuǎn)入菌株Y1和Y2中,并在ERY和42℃高溫條件下進(jìn)行誘導(dǎo)轉(zhuǎn)座。在初步建立轉(zhuǎn)座子文庫中,我們隨機(jī)挑取200個(gè)單菌落進(jìn)行抗性篩選,最終得到2株復(fù)制子融合菌株,其余均為成功發(fā)生轉(zhuǎn)座作用突變株。(3)對(duì)轉(zhuǎn)座子突變株進(jìn)行了SEA產(chǎn)量、生物膜和溶血毒素的測(cè)定,最終篩選出了2株不產(chǎn)SEA和溶血毒素的菌株,1株生物膜形成能力較弱的菌株。
[Abstract]:Staphylococcus aureus (Staphylococcus, aureus, S.aureus) S.aureus, is one of the important food borne pathogens, the main pathogenic factor causing food poisoning is caused by enterotoxin. Contaminated by Staphylococcus aureus enterotoxin food and the food poisoning is called staphylococcal food (Staphylococcal food poisoning, SFP poisoning), usually in a short period of time there will be corresponding to the onset of symptoms, including nausea, vomiting, abdominal cramps and diarrhea. Due to Staphylococcus aureus is widely distributed in the air, dust and food processing equipment such as surface, making the food easy to produce cross contamination, so although each process strict control of food processing, will be subject to different degrees of pollution. Among them, animal and food processors hand or respiratory secretions are regarded as food. The main source of infection of Staphylococcus aureus. In common staphylococcal food poisoning incidents, most of which is caused by SEA, followed by SED, SEC, SEB and SEE. transposon (transposon) are DNA sequences of bacteria and many prokaryotic and eukaryotic genomes has a specific translocation features alone the can in the bacterial chromosome, horizontal transfer between plasmids and phages. Study on plasmid elimination and Tn917 transposon random insertion effect on the expression of foodborne Staphylococcus aureus enterotoxin A, is the main content of the research. The study of 48 different strains and carrying enterotoxin sea gene of Staphylococcus aureus first detection of plasmid elimination, 18 enterotoxin genes and 29 kinds of resistance genes to eliminate the strains before and after PCR, and the susceptibility of 12 antibiotics in clinical detection. Then the plasmid elimination of postburn Staphylococcus aureus T by random transposon insertion The n917 mutation, the chromosome generated variants, and the establishment of the transposon library. Finally, using ELISA method to screen the abnormal expression of enterotoxin A strains, the simultaneous detection of biofilm formation ability and hemolytic toxin abnormal mutant, and compared with the original strain of the differences. This provides the theoretical basis for clarifying the molecular mechanism and enterotoxin A Staphylococcus aureus caused food poisoning prevention and control. The main results are as follows: (1) of 48 strains of different sources and carrying the enterotoxin sea gene of Staphylococcus aureus by plasmid elimination, detection of 18 kinds of enterotoxin genes and 29 kinds of resistance genes to eliminate the strains before and after PCR, and the drug sensitivity of 12 kinds of commonly used antibiotics in clinic the detection. The results showed that the plasmid elimination of enterotoxin genes see, O and tetK resistance genes Tet, AMP and the effects of antibiotics on ERY effect is significant, but not significant, the drug resistance gene TetO and tetK are bacterial plasmids, and genes AMP and ERY may also be in the plasmid. The enterotoxin genes and drug resistance gene of Staphylococcus aureus is more complex, there are differences between the different strains in the same gene, indicating that it has no regularity. In addition, there are some differences between the resistant genotype and drug resistance phenotype. (2) containing a temperature sensitive transposon Tn917 shuttle plasmid was transformed into strain Y1 and Y2, and induced by transposition in ERY and 42 DEG C under high temperature conditions. In the initial establishment of the transposon library, we randomly selected 200 single colonies screened 2 strains, the final replicon fusion strains, the rest are the success of transposition. The mutant. (3) the transposon mutant of SEA production, determination of biofilm and hemolytic toxin, screened 2 strains producing SEA and hemolytic toxin, 1 strains of biofilm formation ability of strains.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TS201.3

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李瓊瓊;范一靈;宋明輝;施春雷;楊美成;;食源性金黃色葡萄球菌腸毒素及其檢測(cè)方法[J];食品安全質(zhì)量檢測(cè)學(xué)報(bào);2016年02期

2 朱淑英;胡元瑋;張兵;吳國平;;82株不同來源金黃色葡萄球菌腸毒素的分型檢測(cè)分析[J];中國衛(wèi)生檢驗(yàn)雜志;2015年01期

3 姜北;黎庶;張驍鵬;于俊媛;袁文常;胡啟文;饒賢才;胡曉梅;;金黃色葡萄球菌轉(zhuǎn)座文庫構(gòu)建與持留突變株研究[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2014年09期

4 徐振波;劉曉晨;李琳;李冰;;金黃色葡萄球菌腸毒素在食源性微生物中的研究進(jìn)展[J];現(xiàn)代食品科技;2013年09期

5 劉偉;王菊光;孫曉華;謝利軍;紀(jì)黎黎;;日常食品及食物中毒樣本中金黃色葡萄球菌腸毒素的分型檢測(cè)分析[J];中國預(yù)防醫(yī)學(xué)雜志;2013年08期

6 吳可可;吳躍進(jìn);陳慧燕;;食源性金黃色葡萄球菌耐藥基因和腸毒素基因定位[J];中國衛(wèi)生檢驗(yàn)雜志;2013年02期

7 張小娟;張榮光;;影響電穿孔法轉(zhuǎn)化效率的因素[J];醫(yī)學(xué)綜述;2011年05期

8 王飛;劉代成;楊宏軍;何洪彬;王長法;高運(yùn)東;仲躋峰;;應(yīng)用雙重PCR方法對(duì)奶牛乳腺炎金葡菌溶血毒素進(jìn)行分型[J];家畜生態(tài)學(xué)報(bào);2010年06期

9 馬艷平;劉永生;張杰;丁耀忠;楊生海;;轉(zhuǎn)座子應(yīng)用的研究進(jìn)展[J];江西農(nóng)業(yè)學(xué)報(bào);2009年05期

10 李欣;楊坤寧;劉志勇;陳紅兵;;電轉(zhuǎn)化法制備高轉(zhuǎn)化效率的E．coli感受態(tài)細(xì)胞研究[J];食品與生物技術(shù)學(xué)報(bào);2007年06期

相關(guān)碩士學(xué)位論文 前4條

1 楊沁南;雞肉中大腸桿菌生物膜的污染情況及石榴皮單寧對(duì)大腸桿菌生物膜的干預(yù)作用[D];西北農(nóng)林科技大學(xué);2016年

2 李洪恩;甘草次酸降低α-溶血素的表達(dá)以及其對(duì)金黃色葡萄球菌體內(nèi)外致病性影響的研究[D];吉林大學(xué);2013年

3 馬瑜丹;單核細(xì)胞增生李斯特菌菌膜形成突變株的篩選[D];上海交通大學(xué);2008年

4 范芳華;金葡菌腸毒素A基因及其突變體表達(dá)產(chǎn)物生物學(xué)活性研究[D];浙江大學(xué);2007年

，

本文編號(hào)：1385791