豬鏈球菌2型Type Ⅱ毒素—抗毒素系統(tǒng)的鑒定與功能研究
發(fā)布時間：2019-02-13 21:22
【摘要】：豬鏈球菌(Streptococcus suis)是引起豬鏈球菌病的主要病原體,是上呼吸道的條件致病菌,一旦豬發(fā)病可引起多種疾病,如腦膜炎,敗血癥,關(guān)節(jié)炎,心內(nèi)膜炎,甚至導(dǎo)致死亡,給世界養(yǎng)豬業(yè)帶來巨大的經(jīng)濟(jì)損失。在33個血清型中,豬鏈球菌2型(Streptococcus suis 2,S.suis 2)被認(rèn)為是養(yǎng)豬行業(yè)中是最流行的,也是毒力最強(qiáng)的病原菌,而且S.suis 2還是一種嚴(yán)重的人獸共患病原體,可致人腦膜炎和毒素休克綜合征。毒素抗毒素系統(tǒng)(toxin-antitoxin system,TAS)是細(xì)菌基因組中廣泛存在的一類小型遺傳元件,最初發(fā)現(xiàn)于低拷貝質(zhì)粒上,用于維持質(zhì)粒的穩(wěn)定性,基因組中的TAS改變了細(xì)菌的生理框架,使其提高對外界環(huán)境的適應(yīng)能力,促使細(xì)菌成為優(yōu)勢菌。本課題通過對預(yù)測的7對Type II TAS進(jìn)行初步的鑒定篩選,探究缺失TAS突變株在毒力方面的影響,取得的主要結(jié)果如下:1.RT-PCR驗(yàn)證毒素抗毒素基因共轉(zhuǎn)錄提取S.suis 2(SC19菌株)的總RNA,反轉(zhuǎn)錄得到cDNA,然后以cDNA為模板,用特異性引物進(jìn)行PCR擴(kuò)增,驗(yàn)證毒素抗毒素基因共轉(zhuǎn)錄情況,證明毒素抗毒素是由同一操縱子調(diào)控轉(zhuǎn)錄的。2.毒素和抗毒素功能特性的探究構(gòu)建成功的重組質(zhì)粒p BADHis A-toxin(p BADHis A-T)轉(zhuǎn)化至Top10感受態(tài)細(xì)胞,加入阿拉伯糖誘導(dǎo)毒素表達(dá),繪制生長曲線同時進(jìn)行活菌計數(shù)。結(jié)果表明誘導(dǎo)毒素表達(dá)時,含有p BADHis A-T1,p BADHis A-T5,p BADHis A-T7,p BADHis A-T9的菌株生長受到明顯抑制,誘導(dǎo)毒素表達(dá)2h后,其中含有p BADHis A-T1,p BADHis A-T7,p BADHis A-T9的細(xì)菌生長趨勢已經(jīng)基本不變,而含有p BADHis A-T5的細(xì)菌生長變慢;活菌計數(shù)結(jié)果表明誘導(dǎo)毒素蛋白1(T1),毒素蛋白7(T7),毒素蛋白9(T9)表達(dá),對細(xì)菌有明顯的殺菌作用,但細(xì)菌并沒有被全部殺死,4h后仍然存活的細(xì)菌開始緩慢恢復(fù)生長,而誘導(dǎo)表達(dá)毒素蛋白5(T5)對細(xì)菌沒有明顯的殺菌作用。為進(jìn)一步確定并驗(yàn)證豬鏈球菌中存在的毒素抗毒素系統(tǒng),我們將構(gòu)建成功的重組質(zhì)粒p ET30a-antitoxin,和p BADHis A-toxin一起共轉(zhuǎn)化至BL21感受態(tài)細(xì)胞,加入IPTG和阿拉伯糖,分別誘導(dǎo)抗毒素和毒素表達(dá),繪制細(xì)菌的生長曲線,觀察抗毒素能否中和毒素,同時對誘導(dǎo)5h后的細(xì)菌革蘭氏染色,觀察細(xì)菌在誘導(dǎo)毒素抗毒素蛋白表達(dá)情況下的形態(tài)。結(jié)果表明抗毒素蛋白1(A1),抗毒素蛋白7(A7),抗毒素蛋白9(A9)能夠中和相應(yīng)的毒素蛋白,使細(xì)菌生長恢復(fù)正常,而只誘導(dǎo)T1,T7,T9表達(dá)的細(xì)菌,生長明顯受到限制,而只誘導(dǎo)T5表達(dá)的細(xì)菌,生長則恢復(fù)到正常水平,說明第5對不是毒素抗毒素系統(tǒng)。革蘭氏染色結(jié)果表明毒素抗毒素的誘導(dǎo)表達(dá)可使E.coli形態(tài)發(fā)生改變,形成明顯的絲狀,可能是細(xì)菌的一種保護(hù)機(jī)制。3.毒素抗毒素系統(tǒng)缺失突變株、互補(bǔ)菌株的構(gòu)建及鑒定根據(jù)上述實(shí)驗(yàn)結(jié)果,對第1對,第7對,第9對毒素抗毒素系統(tǒng)構(gòu)建相對應(yīng)的基因缺失突變株。運(yùn)用溫敏性質(zhì)粒p SET4s構(gòu)建重組質(zhì)粒,電轉(zhuǎn)化至SC19感受態(tài)細(xì)胞,運(yùn)用溫度和壯觀霉素抗性篩選得到得到單交換菌株,通過第二次同源重組篩選到基因缺失突變株缺失突變株。運(yùn)用p SET2構(gòu)建互補(bǔ)質(zhì)粒,電轉(zhuǎn)化至相應(yīng)的毒素抗毒素系統(tǒng)缺失突變株中,以抗性篩選得到互補(bǔ)菌株,最后通過PCR擴(kuò)增和DNA測序的方法,證明毒素抗毒素系統(tǒng)缺失突變株和互補(bǔ)菌株構(gòu)建成功。4.毒素抗毒素系統(tǒng)缺失突變株對S.suis 2的影響用SC19和缺失突變株感染小鼠,繪制存活曲線,結(jié)果表明突變株對S.suis 2毒力基本不顯著。使用紅霉素處理野生株和突變株,嘗試激活毒素抗毒素系統(tǒng),對菌株生長曲線進(jìn)行測定,同時進(jìn)行活菌計數(shù),結(jié)果表明野生株和突變株有一定的差異,但結(jié)果不顯著。
[Abstract]:Streptococus suis is the main pathogen that causes the swine streptococcosis, is the condition pathogenic bacteria of the upper respiratory tract, and once the disease of the pig can cause various diseases, such as meningitis, septicemia, arthritis, endocarditis, and even death, it brings great economic loss to the pig industry in the world. In 33 serotypes, Streptococcus suis 2, S. suis 2, is considered to be the most popular and most virulent pathogen in the pig industry, and S. suis 2 or a serious human animal has a pathogen, which can cause human meningitis and toxin shock syndrome. The toxin-antitoxin system (TAS) is a class of small genetic elements that are widely present in the bacterial genome, initially found on a low-copy plasmid for maintaining the stability of the plasmid, the TAS in the genome changes the physiological framework of the bacteria, and the bacteria are caused to be the dominant bacteria. The main results were as follows: 1. The total RNA of S. suis 2 (SC19 strain) was extracted by RT-PCR and the cDNA was obtained by reverse transcription, and then the cDNA was used as a template. PCR amplification was carried out with specific primers to verify the co-transcription of the toxin anti-toxin gene, and it was proved that the antitoxin of the toxin was regulated by the same operon. A successful recombinant plasmid pBADHis A-t (p BADHis A-T) was transformed into a Top10 competent cell, and the expression of the arabinose-induced toxin was added, and the growth curve was plotted to count the live bacteria. The results showed that the growth of the strains containing p BADHis A-T1, p BADHis A-T5, p BADHis A-T7 and p BADHis A-T9 was significantly inhibited when the toxin was induced to express, and the growth trend of the bacteria containing p BADHis A-T1, p BADHis A-T7 and p BADHis A-T9 was almost unchanged. and the bacterial growth of the p-BADHis A-T5 is slow, the result of the living bacteria is that the expression of the toxin protein 1 (T1), the toxin protein 7 (T7) and the toxin protein 9 (T9) is induced, and the induced expression of the toxin protein 5 (T5) has no obvious bactericidal effect on the bacteria. in order to further determine and verify the toxin anti-toxin system existing in the streptococcus suis, we will construct a successful recombinant plasmid pET30a-antiloxin, and p BADHis A-teoxin to co-convert to the BL21 competent cell, add IPTG and arabinose, respectively induce the expression of the anti-toxin and the toxin, and draw the growth curve of the bacteria, It is observed that the anti-toxin can neutralize the toxin, and at the same time, the bacterial Gram-positive staining after 5h is induced, and the morphology of the bacteria in the expression of the toxin-resistant protein of the toxin is observed. The results showed that the anti-toxin protein 1 (A1), the anti-toxin protein 7 (A7) and the anti-toxin protein 9 (A9) can neutralize the corresponding toxin protein, so that the growth of the bacteria is normal, and only the bacteria expressing T1, T7 and T9 are induced, the growth is obviously restricted, and only the bacteria that are expressed in T5 are induced, The growth is restored to a normal level, indicating that the fifth pair is not a toxin antitoxin system. Gram-staining showed that the induced expression of the toxin-resistant toxin could change the morphology of E. coli, which could be a protective mechanism of bacteria. A corresponding gene deletion mutant was constructed in the first pair, the seventh pair, and the ninth to the toxin-resistant system according to the above-mentioned experimental results by the construction and identification of the toxin-antitoxin system deletion mutant and the complementary strain. The recombinant plasmid was constructed by using the temperature-sensitive granule, pSET4s, and then transformed into the competent cells of the SC19, and the single-exchange strain was obtained by the temperature and the resistance of the spectinomycin, and the mutant strain was deleted by the second homologous recombination to the deletion mutant of the gene. A complementary plasmid was constructed by using p-SET2, and then transformed into a corresponding toxin-antitoxin system deletion mutant strain, and the complementary strain was obtained by resistance screening, and finally, the method of PCR amplification and DNA sequencing was used to prove that the toxin-antitoxin system deletion mutant and the complementary strain were successfully constructed. The effect of the toxin anti-toxin system on S. suis 2 was infected with SC19 and the deletion mutant strain, and the survival curves were drawn. The results showed that the virulence of the mutant strain to S. suis 2 was not significant. The wild strain and the mutant strain were treated with erythromycin, the toxin-resistant system was tried to activate the toxin-antitoxin system, the growth curve of the strain was measured, and the living bacteria were counted. The results showed that the wild strain and the mutant had a certain difference, but the result was not significant.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.61
本文編號：2421897
[Abstract]:Streptococus suis is the main pathogen that causes the swine streptococcosis, is the condition pathogenic bacteria of the upper respiratory tract, and once the disease of the pig can cause various diseases, such as meningitis, septicemia, arthritis, endocarditis, and even death, it brings great economic loss to the pig industry in the world. In 33 serotypes, Streptococcus suis 2, S. suis 2, is considered to be the most popular and most virulent pathogen in the pig industry, and S. suis 2 or a serious human animal has a pathogen, which can cause human meningitis and toxin shock syndrome. The toxin-antitoxin system (TAS) is a class of small genetic elements that are widely present in the bacterial genome, initially found on a low-copy plasmid for maintaining the stability of the plasmid, the TAS in the genome changes the physiological framework of the bacteria, and the bacteria are caused to be the dominant bacteria. The main results were as follows: 1. The total RNA of S. suis 2 (SC19 strain) was extracted by RT-PCR and the cDNA was obtained by reverse transcription, and then the cDNA was used as a template. PCR amplification was carried out with specific primers to verify the co-transcription of the toxin anti-toxin gene, and it was proved that the antitoxin of the toxin was regulated by the same operon. A successful recombinant plasmid pBADHis A-t (p BADHis A-T) was transformed into a Top10 competent cell, and the expression of the arabinose-induced toxin was added, and the growth curve was plotted to count the live bacteria. The results showed that the growth of the strains containing p BADHis A-T1, p BADHis A-T5, p BADHis A-T7 and p BADHis A-T9 was significantly inhibited when the toxin was induced to express, and the growth trend of the bacteria containing p BADHis A-T1, p BADHis A-T7 and p BADHis A-T9 was almost unchanged. and the bacterial growth of the p-BADHis A-T5 is slow, the result of the living bacteria is that the expression of the toxin protein 1 (T1), the toxin protein 7 (T7) and the toxin protein 9 (T9) is induced, and the induced expression of the toxin protein 5 (T5) has no obvious bactericidal effect on the bacteria. in order to further determine and verify the toxin anti-toxin system existing in the streptococcus suis, we will construct a successful recombinant plasmid pET30a-antiloxin, and p BADHis A-teoxin to co-convert to the BL21 competent cell, add IPTG and arabinose, respectively induce the expression of the anti-toxin and the toxin, and draw the growth curve of the bacteria, It is observed that the anti-toxin can neutralize the toxin, and at the same time, the bacterial Gram-positive staining after 5h is induced, and the morphology of the bacteria in the expression of the toxin-resistant protein of the toxin is observed. The results showed that the anti-toxin protein 1 (A1), the anti-toxin protein 7 (A7) and the anti-toxin protein 9 (A9) can neutralize the corresponding toxin protein, so that the growth of the bacteria is normal, and only the bacteria expressing T1, T7 and T9 are induced, the growth is obviously restricted, and only the bacteria that are expressed in T5 are induced, The growth is restored to a normal level, indicating that the fifth pair is not a toxin antitoxin system. Gram-staining showed that the induced expression of the toxin-resistant toxin could change the morphology of E. coli, which could be a protective mechanism of bacteria. A corresponding gene deletion mutant was constructed in the first pair, the seventh pair, and the ninth to the toxin-resistant system according to the above-mentioned experimental results by the construction and identification of the toxin-antitoxin system deletion mutant and the complementary strain. The recombinant plasmid was constructed by using the temperature-sensitive granule, pSET4s, and then transformed into the competent cells of the SC19, and the single-exchange strain was obtained by the temperature and the resistance of the spectinomycin, and the mutant strain was deleted by the second homologous recombination to the deletion mutant of the gene. A complementary plasmid was constructed by using p-SET2, and then transformed into a corresponding toxin-antitoxin system deletion mutant strain, and the complementary strain was obtained by resistance screening, and finally, the method of PCR amplification and DNA sequencing was used to prove that the toxin-antitoxin system deletion mutant and the complementary strain were successfully constructed. The effect of the toxin anti-toxin system on S. suis 2 was infected with SC19 and the deletion mutant strain, and the survival curves were drawn. The results showed that the virulence of the mutant strain to S. suis 2 was not significant. The wild strain and the mutant strain were treated with erythromycin, the toxin-resistant system was tried to activate the toxin-antitoxin system, the growth curve of the strain was measured, and the living bacteria were counted. The results showed that the wild strain and the mutant had a certain difference, but the result was not significant.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.61
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 王敏;李明;鐘秋;趙巖;饒賢才;黎庶;譚銀玲;胡福泉;;高致病性2型豬鏈球菌毒素-抗毒素系統(tǒng)SezAT的鑒定與活性研究[J];微生物學(xué)通報;2012年02期
，本文編號：2421897
本文鏈接：http://sikaile.net/yixuelunwen/dongwuyixue/2421897.html
最近更新
教材專著
