副猪嗜血杆菌分子流行病学及hhdA基因克隆表达和免疫保护效力研究

发布时间：2018-12-31 08:28

【摘要】：副猪嗜血杆菌(Haemophilus parasuis,HPS)是猪的革拉斯氏病的病原体,可引起以猪多发性浆膜炎和关节炎等症状。近年来,随着我国规模化养猪场的发展,该病已经成为猪发病率和死亡率增加的重要原因之一。本研究对2012年-2014年期间江西地区HPS临床分离株在已进行琼脂扩散试验方法血清型分型基础上进行ERIC-PCR基因型分析。通过对副猪嗜血杆菌hhd A基因克隆,并在进行HPS hhd A蛋白生物学信息预测的基础上,将HPS hhd A蛋白进行原核表达与鉴定,以及进一步研究其免疫原性,具体如下:1、2012年-2014年江西地区HPS分离株ERIC-PCR基因分型研究以15株副猪嗜血杆菌不同血清型的标准菌株为参照,对2012~2014年江西地区的41株HPS分离株进行基因分型,总共56株菌(41株HPS江西分离株和15株参考菌株)被分为44个基因型,之前无法通过血清分型的8株菌株均得到ERIC-PCR基因分型充分分型,从而弥补了传统血清分型方法不能完全分型的缺陷。2、副猪嗜血杆菌hhd A基因的克隆与生物信息学分析本研究应用PCR技术成功的扩增了副猪嗜血杆菌的hhd A基因序列,获得了长1797bp的hhd A基因,编码599个氨基酸,推导出蛋白质的分子量大小为61.1k Da的亲水性蛋白;该蛋白由内向外和由外向内各有可能一个跨膜区域;通过蛋白质的信号肽预测hhd A蛋白无信号肽,为细胞内蛋白;对hhd A蛋白的二级结构进行了预测和分析,结果显示hhd A含有较多α-螺旋、β-折叠、β转角和无规则卷曲等二级结构,而且有较多柔性区域,呈散在分布。与Gen Bank上公布的ZJ0906株(CP005384)中对应的核苷酸序列同源性为99.9%,氨基酸同源性99.8%,测定表明HPS的hhd A基因十分保守。3、HPS hhd A基因的原核表达及其免疫保护作用的研究将扩增的hhd A基因克隆于原核表达载体p ET-32a(+)中构建重组表达质粒p ET-32a(+)-hhd A,后转化至受体菌E.coli BL21(DE3),IPTG诱导表达,通过SDS-PAGE和Western blot分析重组hhd A蛋白的表达情况和反应原性。重组蛋白经镍柱亲和层析纯化后免疫小鼠,检测其特异性血清Ig G抗体水平及攻毒保护率。结果表明,SDS-PAGE得到约82 KDa与预期分子质量大小一致蛋白;Western blot分析显示,该重组蛋白具有良好反应原性。动物免疫保护试验结果表明,重组hhd A蛋白免疫后能诱导产生高水平的hhd A特异性抗体,并对副猪嗜血杆菌强毒菌株的攻击的提供50%保护率,说明hhd A是副猪嗜血杆菌的一种保护性抗原。
[Abstract]:Haemophilus paraswine (Haemophilus parasuis,HPS) is the pathogen of porcine Graillard disease, which can cause polyserositis and arthritis. In recent years, with the development of large-scale pig farms in China, the disease has become one of the important reasons for the increase of pig morbidity and mortality. In this study, the clinical isolates of HPS from 2012 to 2014 in Jiangxi province were analyzed for ERIC-PCR genotypes on the basis of serotyping of Agar diffusion test method. By cloning the hhd A gene of Haemophilus parais and predicting the biological information of HPS hhd A protein, the prokaryotic expression and identification of HPS hhd A protein were carried out, and the immunogenicity of HPS hhd A protein was further studied. The main results are as follows: 1. According to 15 standard strains of Haemophilus parasuis serotype, 41 HPS isolates in Jiangxi from 2012 to 2014 were genotyped by ERIC-PCR genotyping. A total of 56 strains (41 HPS Jiangxi isolates and 15 reference strains) were classified into 44 genotypes. All 8 strains that could not be genotyped by serum were fully genotyped by ERIC-PCR genotyping. 2. Cloning and bioinformatics analysis of the hhd A gene of Haemophilus parasuis. The hhd A gene sequence of Haemophilus parasuis was successfully amplified by PCR technique. The long 1797bp hhd A gene was obtained, encoding 599 amino acids, and the hydrophilic protein with molecular weight of 61.1 k Da was deduced. There is a transmembrane region from inside to outside and from outside to inside, and the signal peptide of hhd A is predicted to be an intracellular protein by the signal peptide of the protein. The secondary structure of hhd A protein was predicted and analyzed. The results showed that hhd A had more secondary structures, such as 伪 -helix, 尾 -fold, 尾 rotation angle and irregular crimp, and there were more flexible regions and scattered distribution. The homology of nucleotide sequence and amino acid sequence of ZJ0906 strain (CP005384) published on Gen Bank was 99.9 and 99.8 respectively. The results showed that the hhd A gene of HPS was very conserved. Prokaryotic expression of HPS hhd A Gene and its Immunoprotective effect; the amplified hhd A gene was cloned into the prokaryotic expression vector p ET-32a () to construct the recombinant expression plasmid p ET-32a ()-hhd A. The expression of recombinant hhd A protein was analyzed by SDS-PAGE and Western blot. The expression and reactivity of recombinant hhd A protein were analyzed by SDS-PAGE and Western blot. The recombinant protein was purified by nickel column affinity chromatography to immunize mice, and its specific serum Ig G antibody level and protection rate against virus were detected. The results showed that the SDS-PAGE obtained about 82 KDa and the expected molecular weight consistent protein; Western blot analysis showed that the recombinant protein had good reactivity. The results of animal immune protection test showed that recombinant hhd A protein could induce the production of high level of hhd A specific antibody after immunization, and provide 50% protection against the attack of Haemophilus parahaemophilus virulent strain. Hhd A is a protective antigen of Haemophilus parasuis.
【学位授予单位】：江西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.12

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