HPS自然转化方法的改进及HPS自转运蛋白AT2、AT3的功能研究

发布时间：2018-05-03 05:12

本文选题：副猪嗜血杆菌 + 转化效率　；参考：《华中农业大学》2015年硕士论文

【摘要】：副猪嗜血杆菌(Haemophilus parasuis,HPS)是健康猪上呼吸道的常在菌,在一定条件下侵入机体引发全身性疾病。本研究基于USS序列以及质粒甲基化修饰对现有自然转化方法进行改进,以提高现有自然转化构建缺失株的效率以及扩大转化菌株范围从而利于后续对HPS各基因的功能研究。自转运蛋白可以抑制细胞的吞噬作用以及抵抗补体介导的杀伤,本研究以HPS自转运蛋白AT2、AT3为研究对象,通过构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响。1构建重组质粒根据SH0165全基因组序列设计引物克隆at2、at3、htr A基因上下游同源臂及kanR、gmR抗性盒,通过重叠PCR将抗性表达盒连接到基因上下游同源臂之间,并在同源臂上游同源臂上游及下游同源臂下游添加USS序列得到重组片段。分别将重组片段连接p K18mobsac B和p SHK3质粒载体,得到相应重组质粒。2自然转化方法的改进与评价用p K18mobsac B和p SHK3为载体的重组质粒p K3UK和p S3UK分别自然转化HPS 15种血清型参考菌株及地方分离株SH0165,尝试使用穿梭载体是否能够用于HPS的自然转化并评价其转化效率。随后,将p S3UK电转至HPS菌株,得到体内甲基化的重组质粒p S3UKm,将p S3UKm、p K3UK和p S3UK同时自然转化转化到不同HPS菌株中,评价质粒甲基化对HPS的自然转化效率的影响。3 CF7066Δat2pd、Δat3pd基因缺失株的构建及鉴定将重组质粒依次自然转化到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鉴定,且测序正确。4 at2、at3基因功能研究通过测定CF7066野生菌株及缺失株OD600-时间关系曲线,表明at2、at3的缺失对CF7066的生长趋势没有显著影响。血清抗性试验表明at2、at3缺失后CF7066的抗血清杀伤能力提高。通过荧光定量PCR发现缺失株中cap D、gal U、gal E、omp P2等文献报道HPS与粘附侵入、血清抗性相关的潜在毒力因子出现上调。此外,进行3D4/21对CF7066的吞噬试验发现,at2、at3缺失后CF7066更容易被3D4/21所吞噬。综上,本研究发现并证实H.parasuis-E.coli穿梭载体p SHK3能够用于HPS自然转化构建缺失株,与p K18mobsac B一样,转化效率受基因和菌株影响,但是本研究表明质粒甲基化处理能够显著提高自然转化效率。随后通过构建CF7066△at2pd::ermR、CF7066△at3pd::kanR、CF7066△at2pd::ermR△at3pd::kanR缺失株,初步验证了at2、at3在HPS血清抗性及抗吞噬方面的贡献,为进一步探索HPS抵抗宿主天然免疫的机制提供参考。
[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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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