發(fā)布時(shí)間：2018-04-18 23:24

  本文選題：鐵調(diào)控啟動(dòng)子 + 遲鈍愛德華氏菌�。� 參考：《華東理工大學(xué)》2011年碩士論文

【摘要】：重組細(xì)菌載體疫苗作為一種有效的策略可以攜帶抗原蛋白并激發(fā)宿主的產(chǎn)生多重免疫保護(hù)力。目前,利用細(xì)菌載體活疫苗作為遞呈載體是新興現(xiàn)代疫苗的重要方向,接種這種疫苗后,機(jī)體既可產(chǎn)生針對(duì)載體細(xì)菌來(lái)源的病原的免疫保護(hù)力,又可產(chǎn)生針對(duì)攜帶抗原來(lái)源病原的免疫保護(hù)力,能夠達(dá)到同時(shí)預(yù)防多種病原感染的作用。然而,活菌載體的安全性、外源基因表達(dá)穩(wěn)定性等問(wèn)題也是細(xì)菌載體疫苗發(fā)展中不可忽視的問(wèn)題。遲鈍愛德華氏菌(Edwardsiella tarda)是一種重要的革蘭氏陰性魚類病原菌。該病原菌能引起魚類的出血性敗血癥,在本實(shí)驗(yàn)室前期工作中,已經(jīng)從發(fā)病大菱鲆體內(nèi)分離得到一株遲鈍愛德華氏菌EIB202強(qiáng)毒株,并對(duì)其進(jìn)行了全基因組測(cè)序,并構(gòu)建了一系列適用于載體活菌疫苗的缺失減毒菌株。在本研究中,我們篩選了53個(gè)不同細(xì)菌來(lái)源的鐵調(diào)控啟動(dòng)子,并檢測(cè)了它們?cè)隗w外培養(yǎng)時(shí)的轉(zhuǎn)錄活性。將篩選得到的具有較好鐵調(diào)控活性的啟動(dòng)子Pdps和Pdps在巨噬細(xì)胞內(nèi)進(jìn)行胞內(nèi)轉(zhuǎn)錄活性分析,最終選取了能夠在胞內(nèi)高效表達(dá)的啟動(dòng)子Pdps,并在模式動(dòng)物斑馬魚上進(jìn)行了觀察檢測(cè)。實(shí)驗(yàn)證明Pdps在體內(nèi)有一個(gè)轉(zhuǎn)錄增強(qiáng)的趨勢(shì),可以用于體內(nèi)誘導(dǎo)表達(dá)系統(tǒng)的構(gòu)建。為了證明該系統(tǒng)能夠作為成熟的抗原表達(dá)系統(tǒng),我們將來(lái)源于嗜水氣單胞菌(Aeromonas hydrophila) LSA34中的保護(hù)性抗原GAPDH連入含有Pdps的重組質(zhì)粒中,同時(shí),選取遲鈍愛德華氏菌的減毒株WED作為宿主菌,構(gòu)建了細(xì)菌載體疫苗WED/pUTDgap.大菱鲆免疫攻毒實(shí)驗(yàn)證明,WED/pUTDgap針對(duì)對(duì)野生型遲鈍愛德華氏菌和嗜水氣單胞菌均有超過(guò)60%的免疫保護(hù)力。 本研究在遲鈍愛德華氏菌中成功建立了基于鐵誘導(dǎo)啟動(dòng)子的體內(nèi)誘導(dǎo)抗原表達(dá)系統(tǒng),并在遲鈍愛德華氏菌減毒株WED中實(shí)現(xiàn)了嗜水氣單胞菌保護(hù)性抗原蛋白的體內(nèi)表達(dá),成功構(gòu)建了一株具有二價(jià)免疫保護(hù)力的遲鈍愛德華氏菌減毒活疫苗候選株。
[Abstract]:As an effective strategy, recombinant bacterial vector vaccine can carry antigen protein and stimulate multiple immune protection of host.At present, it is an important direction of the new modern vaccine to use the live vaccine of bacterial carrier as the delivery vector. After inoculating this vaccine, the body can not only produce the immune protection ability against the pathogen from which the carrier bacteria come from.It can also produce immune protection against pathogens carrying antigens, and can prevent many kinds of pathogens at the same time.However, the safety of live bacterial vectors and the stability of foreign gene expression are also the problems that can not be ignored in the development of bacterial vector vaccine.Edwardsiella tarda is an important gram-negative fish pathogen.The pathogen can cause hemorrhagic septicemia in fish. In our laboratory, a virulent strain of Euphorbia obtuse (EIB202) was isolated from the infected turbot and sequenced.A series of deletion attenuated strains suitable for vector live vaccine were constructed.In this study, we screened 53 iron regulatory promoters from different bacterial sources and measured their transcriptional activities in vitro.The Pdps and Pdps promoters with better iron regulatory activity were selected for intracellular transcriptional activity analysis in macrophages, and Pdpss, which were highly expressed in the cells, were selected and detected on zebrafish, a model animal.The results show that Pdps has a tendency of transcriptional enhancement in vivo and can be used to construct an in vivo induced expression system.In order to prove that the system can be used as a mature antigen expression system, we linked the protective antigen GAPDH from Aeromonas hydrophila LSA34 to the recombinant plasmid containing Pdps.The vector vaccine WED- pUTDgap. was constructed by selecting the attenuated strain WED of Edwardsiella obtuse as the host strain.The results showed that WED- pUTDgap had more than 60% protection against both wild type Edwardiella obtuse and Aeromonas hydrophila.In this study, we successfully established an in vivo antigen expression system based on iron induced promoters in Edouard dullus, and realized the expression of protective antigen protein of Aeromonas hydrophila in WED, a attenuated strain of E. dullus.A bivalent vaccine candidate for attenuated live vaccine of Edwardsiella dullus was successfully constructed.
【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 黃志斌;劉志軍;廖國(guó)禮;趙明軍;石存斌;;水產(chǎn)養(yǎng)殖動(dòng)物疾病防控與安全用藥[J];廣東飼料;2009年08期

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本文編號(hào)：1770563