禽病原性大腸桿菌腸菌素相關(guān)基因entE、entS、tolC突變株的構(gòu)建及其致病性評價
發(fā)布時間：2019-05-05 09:40
【摘要】：禽致病性大腸桿菌(APEC)屬于腸道外致病性大腸桿菌(ExPEC)的成員,能通過呼吸道和生殖道等多種途徑感染并引起禽類各種腸道外疾病。目前,該病已成為危害養(yǎng)禽業(yè)的重要細菌病之一,給養(yǎng)禽業(yè)造成了重大經(jīng)濟損失,尤其是那些養(yǎng)禽業(yè)發(fā)達的國家,例如,巴西、中國、美國。對于大腸桿菌來說鐵離子是一種必需成分,但動物體內(nèi)游離的鐵離子濃度很低,不能滿足細菌的生存要求,因此大腸桿菌進化出多種攝鐵機制從宿主體內(nèi)競爭鐵離子,腸菌素鐵攝取系統(tǒng)就是其中一種。鐵離子對于大腸桿菌在宿主體內(nèi)生存和發(fā)揮毒力起著關(guān)鍵作用,這使得鐵攝取系統(tǒng)如鐵結(jié)合性復(fù)合物能夠成為發(fā)展新型藥物的靶位點。本研究旨在研究腸菌素相關(guān)基因entE、entS、tolC與APEC致病性之間的關(guān)系,為揭示APEC的致病機理及進一步構(gòu)建APEC弱毒疫苗株奠定基礎(chǔ)。運用λRed重組系統(tǒng)構(gòu)建E058△entE、E058△entS、E058△tolC、E058△entE△entS、 E058△entE△entS△tolC基因缺失突變株,同時將帶有相應(yīng)啟動子的tolC基因完整閱讀框插入低拷貝載體pACYC184中,通過電轉(zhuǎn)化突變株來篩選獲得回復(fù)株。RT-PCR結(jié)果表明entE和tolC基因均不能轉(zhuǎn)錄,而其上下游基因的轉(zhuǎn)錄不受影響。1日齡SPF雞致死性試驗結(jié)果表明,與野生株相比,E058△tolC及E058△entE△entS△tolC突變株的致病力顯著降低,而E058△entE、E058△entS、E058△entE△entS突變株致弱不明顯。35日齡SPF雞體內(nèi)定居與持續(xù)試驗結(jié)果表明,E058△tolC和E058△entE△entS△tolC突變株在各臟器的定居與持續(xù)能力均顯著低于野生株E058(P0.0001),同時,E058△entE△entS△tolC突變株較E058Ato/C突變株的定居與持續(xù)能力顯著降低。生長曲線的測定結(jié)果顯示,在LB培養(yǎng)基中,各突變株生長速度與野生株相似,并未表現(xiàn)出明顯差異,但在貧鐵培養(yǎng)基中,E058△entE△entS△tolC三基因突變株的生長速度顯著低于野生株,而其它突變株生長速度較野生株均沒有表現(xiàn)出顯著差異。血清殺菌試驗顯示各突變株和野生株沒有表現(xiàn)出顯著差異。體外競爭試驗結(jié)果顯示,在LB培養(yǎng)基中,除E058△entS外,突變株均表現(xiàn)為輕度致弱,而在貧鐵培養(yǎng)基中,三基因突變株E058△entE△entS△tolC表現(xiàn)為中度致弱。HD-11細胞吞噬試驗結(jié)果顯示,與突變株E058Ato/C相比,突變株E058△entE△entS△tolC對HD-11細胞侵襲率明顯增加,差異顯著。這些結(jié)果表明,參與腸菌素外膜輸出基因tolC與APEC E058菌株的致病性高度相關(guān),而參與腸菌素合成基因entE和內(nèi)膜運輸基因entS不影響APEC的致病性。并且三基因缺失株E058△entE△entS△tolC的生長速度在貧鐵條件下受到抑制,在雞的感染模型中,這種生長方面的缺陷或許會在體內(nèi)有所放大,引起致病性下降。
[Abstract]:Avian pathogenic Escherichia coli (APEC) is a member of extraintestinal pathogenic Escherichia coli (ExPEC). It can be infected by respiratory tract and reproductive tract and can cause all kinds of extraintestinal diseases in poultry. At present, the disease has become one of the most important bacterial diseases in poultry industry, causing great economic losses to poultry industry, especially in countries with advanced poultry industry, such as Brazil, China and the United States. Iron ion is an essential component for Escherichia coli, but the concentration of free iron ion in animals is very low and cannot meet the survival requirements of bacteria. Therefore, E. coli has evolved a variety of iron uptake mechanisms to compete for iron ions from the host body. The enterocin iron uptake system is one of them. Iron ions play a key role in the survival and virulence of Escherichia coli in host, which makes iron uptake system, such as iron-binding complexes, become the target sites for the development of new drugs. The aim of this study was to study the relationship between enterocin-related gene entE,entS,tolC and the pathogenicity of APEC, and to lay a foundation for revealing the pathogenesis of APEC and constructing the attenuated vaccine strain of APEC. The E058 entE,E058 entS,E058 tolC,E058 entE entS,E058 entE entS tolC gene deletion mutant was constructed by 位 Red recombination system, and the complete reading frame of the tolC gene with corresponding promoter was inserted into the low copy vector pACYC184. The results of RT-PCR showed that both entE and tolC genes could not be transcribed, but the transcription of the upstream and downstream genes was not affected. One-day-old SPF chickens showed that compared with wild strains, the results of lethal test showed that the two genes could not be transcribed and the transcription of the upstream and downstream genes was not affected. The pathogenicity of E058 tolC and E058 entE entS tolC mutants was significantly decreased, but that of E058 entE,E058 entS,E058 entE entS mutants was not obvious. 35-day-old SPF chickens were settled in vivo and continuously tested. The colonization and sustainability of E058 tolC and E058 entE entS tolC mutants in various organs were significantly lower than those of wild E058 (P0.0001), and the colonization and sustainability of E058 entE entS tolC mutants were significantly lower than those of E058Ato/C mutants. The results of growth curve showed that the growth rate of mutants in LB medium was similar to that of wild plants, but there was no significant difference in the growth rate of mutants, but in iron-poor medium, the growth rates of mutants were similar to those of wild plants. The growth rate of E058 entE entS tolC trigene mutants was significantly lower than that of wild ones, while the growth rates of other mutants were not significantly different from those of wild ones. Serum bactericidal test showed that there was no significant difference among the mutants and wild strains. The results of in vitro competition test showed that all mutants showed mild weakening in LB medium except E058 entS, but in iron-poor medium, the mutants showed mild weakening in the medium with the exception of E058 entS. The results of phagocytosis test of HD-11 cells showed that the invasiveness of mutant E058 entE entS tolC to HD-11 cells was significantly higher than that of mutant E058 entE entS tolC (P < 0.05), and the difference was significant. These results indicated that the exportation gene tolC of enterocin was highly correlated with the pathogenicity of APEC E058, while entE and entS did not affect the pathogenicity of APEC. The growth rate of the three gene deletion strain E058 entE entS tolC was inhibited under the condition of iron deficiency. In the chicken infection model, the growth deficiency might be magnified in vivo, resulting in a decrease in pathogenicity.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61
本文編號：2469464
[Abstract]:Avian pathogenic Escherichia coli (APEC) is a member of extraintestinal pathogenic Escherichia coli (ExPEC). It can be infected by respiratory tract and reproductive tract and can cause all kinds of extraintestinal diseases in poultry. At present, the disease has become one of the most important bacterial diseases in poultry industry, causing great economic losses to poultry industry, especially in countries with advanced poultry industry, such as Brazil, China and the United States. Iron ion is an essential component for Escherichia coli, but the concentration of free iron ion in animals is very low and cannot meet the survival requirements of bacteria. Therefore, E. coli has evolved a variety of iron uptake mechanisms to compete for iron ions from the host body. The enterocin iron uptake system is one of them. Iron ions play a key role in the survival and virulence of Escherichia coli in host, which makes iron uptake system, such as iron-binding complexes, become the target sites for the development of new drugs. The aim of this study was to study the relationship between enterocin-related gene entE,entS,tolC and the pathogenicity of APEC, and to lay a foundation for revealing the pathogenesis of APEC and constructing the attenuated vaccine strain of APEC. The E058 entE,E058 entS,E058 tolC,E058 entE entS,E058 entE entS tolC gene deletion mutant was constructed by 位 Red recombination system, and the complete reading frame of the tolC gene with corresponding promoter was inserted into the low copy vector pACYC184. The results of RT-PCR showed that both entE and tolC genes could not be transcribed, but the transcription of the upstream and downstream genes was not affected. One-day-old SPF chickens showed that compared with wild strains, the results of lethal test showed that the two genes could not be transcribed and the transcription of the upstream and downstream genes was not affected. The pathogenicity of E058 tolC and E058 entE entS tolC mutants was significantly decreased, but that of E058 entE,E058 entS,E058 entE entS mutants was not obvious. 35-day-old SPF chickens were settled in vivo and continuously tested. The colonization and sustainability of E058 tolC and E058 entE entS tolC mutants in various organs were significantly lower than those of wild E058 (P0.0001), and the colonization and sustainability of E058 entE entS tolC mutants were significantly lower than those of E058Ato/C mutants. The results of growth curve showed that the growth rate of mutants in LB medium was similar to that of wild plants, but there was no significant difference in the growth rate of mutants, but in iron-poor medium, the growth rates of mutants were similar to those of wild plants. The growth rate of E058 entE entS tolC trigene mutants was significantly lower than that of wild ones, while the growth rates of other mutants were not significantly different from those of wild ones. Serum bactericidal test showed that there was no significant difference among the mutants and wild strains. The results of in vitro competition test showed that all mutants showed mild weakening in LB medium except E058 entS, but in iron-poor medium, the mutants showed mild weakening in the medium with the exception of E058 entS. The results of phagocytosis test of HD-11 cells showed that the invasiveness of mutant E058 entE entS tolC to HD-11 cells was significantly higher than that of mutant E058 entE entS tolC (P < 0.05), and the difference was significant. These results indicated that the exportation gene tolC of enterocin was highly correlated with the pathogenicity of APEC E058, while entE and entS did not affect the pathogenicity of APEC. The growth rate of the three gene deletion strain E058 entE entS tolC was inhibited under the condition of iron deficiency. In the chicken infection model, the growth deficiency might be magnified in vivo, resulting in a decrease in pathogenicity.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61
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