本文選題：副豬嗜血桿菌 + 轉(zhuǎn)化效率��； 參考：《華中農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：副豬嗜血桿菌(Haemophilus parasuis,HPS)是健康豬上呼吸道的常在菌,在一定條件下侵入機(jī)體引發(fā)全身性疾病。本研究基于USS序列以及質(zhì)粒甲基化修飾對(duì)現(xiàn)有自然轉(zhuǎn)化方法進(jìn)行改進(jìn),以提高現(xiàn)有自然轉(zhuǎn)化構(gòu)建缺失株的效率以及擴(kuò)大轉(zhuǎn)化菌株范圍從而利于后續(xù)對(duì)HPS各基因的功能研究。自轉(zhuǎn)運(yùn)蛋白可以抑制細(xì)胞的吞噬作用以及抵抗補(bǔ)體介導(dǎo)的殺傷,本研究以HPS自轉(zhuǎn)運(yùn)蛋白AT2、AT3為研究對(duì)象,通過構(gòu)建其基因缺失株研究了AT2、AT3在HPS抵抗宿主天然免疫方面的影響。1構(gòu)建重組質(zhì)粒根據(jù)SH0165全基因組序列設(shè)計(jì)引物克隆at2、at3、htr A基因上下游同源臂及kanR、gmR抗性盒,通過重疊PCR將抗性表達(dá)盒連接到基因上下游同源臂之間,并在同源臂上游同源臂上游及下游同源臂下游添加USS序列得到重組片段。分別將重組片段連接p K18mobsac B和p SHK3質(zhì)粒載體,得到相應(yīng)重組質(zhì)粒。2自然轉(zhuǎn)化方法的改進(jìn)與評(píng)價(jià)用p K18mobsac B和p SHK3為載體的重組質(zhì)粒p K3UK和p S3UK分別自然轉(zhuǎn)化HPS 15種血清型參考菌株及地方分離株SH0165,嘗試使用穿梭載體是否能夠用于HPS的自然轉(zhuǎn)化并評(píng)價(jià)其轉(zhuǎn)化效率。隨后,將p S3UK電轉(zhuǎn)至HPS菌株,得到體內(nèi)甲基化的重組質(zhì)粒p S3UKm,將p S3UKm、p K3UK和p S3UK同時(shí)自然轉(zhuǎn)化轉(zhuǎn)化到不同HPS菌株中,評(píng)價(jià)質(zhì)粒甲基化對(duì)HPS的自然轉(zhuǎn)化效率的影響。3 CF7066Δat2pd、Δat3pd基因缺失株的構(gòu)建及鑒定將重組質(zhì)粒依次自然轉(zhuǎn)化到CF7066菌株,成功得到CF7066缺失株CF7066△at2pd::ermR、CF7066△at3pd::kanR、CF7066△at2pd::ermR△at3pd::kanR。缺失株以PCR、Southern blot以及RT-PCR鑒定,且測序正確。4 at2、at3基因功能研究通過測定CF7066野生菌株及缺失株OD600-時(shí)間關(guān)系曲線,表明at2、at3的缺失對(duì)CF7066的生長趨勢沒有顯著影響。血清抗性試驗(yàn)表明at2、at3缺失后CF7066的抗血清殺傷能力提高。通過熒光定量PCR發(fā)現(xiàn)缺失株中cap D、gal U、gal E、omp P2等文獻(xiàn)報(bào)道HPS與粘附侵入、血清抗性相關(guān)的潛在毒力因子出現(xiàn)上調(diào)。此外,進(jìn)行3D4/21對(duì)CF7066的吞噬試驗(yàn)發(fā)現(xiàn),at2、at3缺失后CF7066更容易被3D4/21所吞噬。綜上,本研究發(fā)現(xiàn)并證實(shí)H.parasuis-E.coli穿梭載體p SHK3能夠用于HPS自然轉(zhuǎn)化構(gòu)建缺失株,與p K18mobsac B一樣,轉(zhuǎn)化效率受基因和菌株影響,但是本研究表明質(zhì)粒甲基化處理能夠顯著提高自然轉(zhuǎn)化效率。隨后通過構(gòu)建CF7066△at2pd::ermR、CF7066△at3pd::kanR、CF7066△at2pd::ermR△at3pd::kanR缺失株,初步驗(yàn)證了at2、at3在HPS血清抗性及抗吞噬方面的貢獻(xiàn),為進(jìn)一步探索HPS抵抗宿主天然免疫的機(jī)制提供參考。
[Abstract]:Haemophilus parasuis (HPS) is a common bacteria in the upper respiratory tract of healthy pigs, which invades the body under certain conditions and causes systemic diseases. Based on the USS sequence and plasmid methylation modification, the existing methods of natural transformation were improved in order to improve the efficiency of the existing natural transformation to construct the missing strain and to expand the range of the transformed strain so as to facilitate the further study on the function of the HPS genes. Self-transporter protein can inhibit phagocytosis of cells and resist complement mediated cytotoxicity. In this study, HPS self-transporter AT2- (AT3) was used as a research object. The effect of AT2N AT3 on the innate immunity of HPS resistant host was studied by constructing its deletion strain. 1 the recombinant plasmid was constructed according to the whole genome sequence of SH0165 to clone the upstream and downstream homologous arms of AT2T3 and kanRgmr-R resistance cassette. The resistance expression cassette was linked to the upstream and downstream homologues of the gene by overlapping PCR, and the USS sequence was added to the upstream and downstream homologues of the homologous arms to obtain the recombinant fragments. The recombinant fragments were ligated into p K18mobsac B and p SHK3 plasmids, respectively. Improvement and Evaluation of Natural Transformation method of corresponding Recombinant plasmid .2; Recombinant plasmids p K3UK and p S3UK using p K18mobsac B and p SHK3 as vectors transformed HPS 15 serotype reference strains and local isolates SH0165, respectively, and attempted to use shuttle Whether the carrier can be used in the natural transformation of HPS and evaluate its conversion efficiency. Then, the recombinant plasmid pS3UKmwas obtained by electroporation of p S3UK into HPS strain. The recombinant plasmid pS3UKm was transformed into different HPS strains at the same time. To evaluate the effect of plasmid methylation on the natural transformation efficiency of HPS, the construction and identification of 3 CF7066 螖 at 2pdand 螖 at3pd gene deletion strain transformed the recombinant plasmid naturally into CF7066 strain in turn. The CF7066 deletion strain CF7066 at2pd1: ermRCF7066 at3kanRCF7066 at2pd::ermR at 3kanRCF7066 was successfully obtained. The deletion strains were identified by PCR Southern blot and RT-PCR and sequenced correctly. The functional analysis of CF7066 wild strain and deletion strain OD600-time showed that the deletion of AT2T3 had no significant effect on the growth trend of CF7066. Serum resistance test showed that the antiserum cytotoxicity of CF7066 was increased after at2 + at3 deletion. Fluorescence quantitative PCR revealed that cap Dendgal Ugal Eomp P2 and other literatures reported that HPS was associated with adhesion invasion, and the potential virulence factors associated with serum resistance were up-regulated. In addition, 3D4/21 phagocytosis test of CF7066 showed that CF7066 was more easily swallowed by 3D4/21 after AT2 + at3 deletion. In conclusion, we found and confirmed that the H.parasuis-E.coli shuttle vector p SHK3 could be used to construct the missing strain of HPS by natural transformation. As with p K18mobsac B, the transformation efficiency was affected by genes and strains. But this study shows that plasmid methylation can significantly improve the efficiency of natural transformation. Then, by constructing the CF7066 at2pd10: ermRRCCF7066 at3pd7: 1 KANRN CF7066 at2pd::ermR at3pd::kanR deletion strain, the contribution of AT2OAT3 to HPS seroresistance and anti-phagocytosis was preliminarily verified, which provided a reference for further exploring the mechanism of HPS resistance to host innate immunity.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.61

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本文編號(hào)：1837112