猪链球菌2型Type Ⅱ毒素—抗毒素系统的鉴定与功能研究
发布时间：2019-02-13 21:22
【摘要】：猪链球菌(Streptococcus suis)是引起猪链球菌病的主要病原体,是上呼吸道的条件致病菌,一旦猪发病可引起多种疾病,如脑膜炎,败血症,关节炎,心内膜炎,甚至导致死亡,给世界养猪业带来巨大的经济损失。在33个血清型中,猪链球菌2型(Streptococcus suis 2,S.suis 2)被认为是养猪行业中是最流行的,也是毒力最强的病原菌,而且S.suis 2还是一种严重的人兽共患病原体,可致人脑膜炎和毒素休克综合征。毒素抗毒素系统(toxin-antitoxin system,TAS)是细菌基因组中广泛存在的一类小型遗传元件,最初发现于低拷贝质粒上,用于维持质粒的稳定性,基因组中的TAS改变了细菌的生理框架,使其提高对外界环境的适应能力,促使细菌成为优势菌。本课题通过对预测的7对Type II TAS进行初步的鉴定筛选,探究缺失TAS突变株在毒力方面的影响,取得的主要结果如下:1.RT-PCR验证毒素抗毒素基因共转录提取S.suis 2(SC19菌株)的总RNA,反转录得到cDNA,然后以cDNA为模板,用特异性引物进行PCR扩增,验证毒素抗毒素基因共转录情况,证明毒素抗毒素是由同一操纵子调控转录的。2.毒素和抗毒素功能特性的探究构建成功的重组质粒p BADHis A-toxin(p BADHis A-T)转化至Top10感受态细胞,加入阿拉伯糖诱导毒素表达,绘制生长曲线同时进行活菌计数。结果表明诱导毒素表达时,含有p BADHis A-T1,p BADHis A-T5,p BADHis A-T7,p BADHis A-T9的菌株生长受到明显抑制,诱导毒素表达2h后,其中含有p BADHis A-T1,p BADHis A-T7,p BADHis A-T9的细菌生长趋势已经基本不变,而含有p BADHis A-T5的细菌生长变慢;活菌计数结果表明诱导毒素蛋白1(T1),毒素蛋白7(T7),毒素蛋白9(T9)表达,对细菌有明显的杀菌作用,但细菌并没有被全部杀死,4h后仍然存活的细菌开始缓慢恢复生长,而诱导表达毒素蛋白5(T5)对细菌没有明显的杀菌作用。为进一步确定并验证猪链球菌中存在的毒素抗毒素系统,我们将构建成功的重组质粒p ET30a-antitoxin,和p BADHis A-toxin一起共转化至BL21感受态细胞,加入IPTG和阿拉伯糖,分别诱导抗毒素和毒素表达,绘制细菌的生长曲线,观察抗毒素能否中和毒素,同时对诱导5h后的细菌革兰氏染色,观察细菌在诱导毒素抗毒素蛋白表达情况下的形态。结果表明抗毒素蛋白1(A1),抗毒素蛋白7(A7),抗毒素蛋白9(A9)能够中和相应的毒素蛋白,使细菌生长恢复正常,而只诱导T1,T7,T9表达的细菌,生长明显受到限制,而只诱导T5表达的细菌,生长则恢复到正常水平,说明第5对不是毒素抗毒素系统。革兰氏染色结果表明毒素抗毒素的诱导表达可使E.coli形态发生改变,形成明显的丝状,可能是细菌的一种保护机制。3.毒素抗毒素系统缺失突变株、互补菌株的构建及鉴定根据上述实验结果,对第1对,第7对,第9对毒素抗毒素系统构建相对应的基因缺失突变株。运用温敏性质粒p SET4s构建重组质粒,电转化至SC19感受态细胞,运用温度和壮观霉素抗性筛选得到得到单交换菌株,通过第二次同源重组筛选到基因缺失突变株缺失突变株。运用p SET2构建互补质粒,电转化至相应的毒素抗毒素系统缺失突变株中,以抗性筛选得到互补菌株,最后通过PCR扩增和DNA测序的方法,证明毒素抗毒素系统缺失突变株和互补菌株构建成功。4.毒素抗毒素系统缺失突变株对S.suis 2的影响用SC19和缺失突变株感染小鼠,绘制存活曲线,结果表明突变株对S.suis 2毒力基本不显著。使用红霉素处理野生株和突变株,尝试激活毒素抗毒素系统,对菌株生长曲线进行测定,同时进行活菌计数,结果表明野生株和突变株有一定的差异,但结果不显著。
[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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61
本文编号：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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61
【参考文献】
相关期刊论文 前1条
1 王敏;李明;钟秋;赵岩;饶贤才;黎庶;谭银玲;胡福泉;;高致病性2型猪链球菌毒素-抗毒素系统SezAT的鉴定与活性研究[J];微生物学通报;2012年02期
，本文编号：2421897
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