绵羊肺炎支原体检测方法的建立及黏附蛋白的研究

发布时间：2018-09-07 06:30

【摘要】：绵羊肺炎支原体(Mycoplasma ovipneumoniae, MO)可感染绵羊和山羊引起传染性胸膜肺炎,以纤维素渗出性肺炎为主要特征,在很多国家和地区都报道有该病原的存在。内蒙古及周边地区是全国的主要养羊区,近年来随着养殖方式的转变,养殖密度大幅增加,该病在这些地区分布和流行的趋势也日益增强,给养羊业带来巨大的经济损失。开展该病防控工作的首要任务是建立准确的诊断方法和研发有效的疫苗,目前关于这方面的工作还不很完善,因此,本研究在对华北部分地区分离的菌株进行了鉴定和分子流行病学调查的基础上,建立了分子水平上的荧光定量PCR检测方法和血清学水平上的间接ELISA诊断方法,并预测了该病原的3个黏附蛋白,对其功能进行了验证,筛选出了优势抗原表位和功能结构域,以期更深入的了解了该病原的致病特性。本研究对分离自华北大部分地区的227株绵羊肺炎支原体进行了实验室传统的分离和鉴定试验及分子生物学鉴定,并分别扩增了16s rDNA、EFTU基因、HSP70基因及16s-23sr DNA。选取其中具有地方代表性的14株菌株,对扩增产物进行了测序,发现利用EFTU基因构建的系统进化树与依据16s rDNA构建的系统进化树标准区别不大,提出EFTU基因可以作为新的分子靶标来分析MO的遗传进化关系和种属鉴别分类,加之其在支原体种间的保守性,建议EFTU基因可以作为MO分子生物学鉴定的特异性基因。在此基础上构建了基于EFTU基因的MO EvaGreen实时荧光定量PCR检测方法,通过试验验证,该方法在特异性、敏感性和稳定性上都优于普通PCR检测方法。根据本实验室基因组测序的注释结果和生物信息学软件分析结果,将预测的黏附蛋白P130、P129和P71按保留完整功能结构域和突变位点的要求共截短表达为9段蛋白。利用大肠杆菌表达系统分别表达9段截短蛋白,重组蛋白纯化后免疫小鼠进行免疫学试验,筛选优势抗原区。结果表明,经P130-3和P71-3段蛋白免疫可以显著提高小鼠的细胞和体液免疫指标,在其它的免疫试验中两者的效果也比较明显。建立了绵羊气管上皮细胞分离鉴定的方法,成功分离到绵羊气管上皮细胞,并在此基础上构建了MO的感染模型,验证了其对气管上皮细胞的黏附能力。应用该模型检测了重组蛋白的黏附能力以及蛋白抗血清对全菌的抑制黏附能力,发现P130-3和P129-2效果比较明显,提示其片段上的结构域在菌体黏附过程中起到重要作用。综合考虑以上试验结果,P130-3的检测性能总体水平比较高,为筛选出的最优截短蛋白。用筛选的性能最优的蛋白P130-3作为诊断抗原,建立了MO的间接ELISA血清学检测方法,并对各反应条件参数进行了优化。最后,比较了建立的EvaGreen实时荧光定量PCR方法、P130-3间接ELISA方法、商品化的间接血凝检测试剂盒以及病原分离培养方法对临床样品检测的敏感性,结果表明,间接ELISA血清学检测方法敏感性最高。
[Abstract]:Mycoplasma pneumoniae (Mycoplasma ovipneumoniae, MO) can infect sheep and goats to cause infectious pleural pneumonia, characterized by cellulose exudative pneumonia, which has been reported in many countries and regions. Inner Mongolia and its surrounding areas are the main sheep breeding areas in China. In recent years with the change of breeding methods the density of breeding has increased significantly. The distribution and epidemic trend of the disease in these areas is increasing day by day which brings great economic losses to the sheep industry. The first task of developing the prevention and control of the disease is to establish accurate diagnostic methods and develop effective vaccines. At present, the work in this field is not perfect, so, On the basis of identification and molecular epidemiology investigation of strains isolated in North China, fluorescence quantitative PCR detection method at molecular level and indirect ELISA diagnostic method at serological level were established. Three adhesion proteins of the pathogen were predicted, their functions were verified, and the dominant antigen epitopes and functional domains were screened out in order to better understand the pathogenicity of the pathogen. In this study, 227 strains of Mycoplasma pneumoniae isolated from most of North China were isolated by traditional laboratory isolation and identification and molecular biological identification, and 16s rDNA,EFTU gene HSP70 and 16s-23sr DNA. were amplified respectively. The amplification products were sequenced from 14 local representative strains. It was found that the phylogenetic tree constructed by EFTU gene was not different from the phylogenetic tree constructed according to 16s rDNA. It is suggested that EFTU gene can be used as a new molecular target to analyze the genetic and evolutionary relationship and species identification of MO, and that EFTU gene can be used as a specific gene for molecular biological identification of MO. On the basis of this, a real-time MO EvaGreen quantitative PCR detection method based on EFTU gene was constructed. The results showed that the method was superior to the conventional PCR method in specificity, sensitivity and stability. According to the results of genome sequencing and bioinformatics software analysis, the predicted adhesion proteins P130, P129 and P71 were cotruncated to 9 segments according to the requirement of preserving complete functional domains and mutation sites. E. coli expression system was used to express 9 truncated proteins respectively. After purification of recombinant proteins, immunological tests were carried out to screen the dominant antigen regions in mice. The results showed that P130-3 and P71-3 protein immunizations could significantly improve the cellular and humoral immunity of mice, and the effects were also obvious in other immunological tests. A method for isolation and identification of sheep tracheal epithelial cells was established and the MO infection model was constructed on the basis of which the adhesion ability of sheep tracheal epithelial cells to tracheal epithelial cells was verified. The adhesion ability of the recombinant protein and the inhibition ability of the antiserum to the whole bacteria were tested by using this model. It was found that the effects of P130-3 and P129-2 were obvious, suggesting that the domain on the fragment played an important role in the process of bacterial adhesion. Considering the above test results, the detection performance of P130-3 is relatively high, which is the best truncated protein. Using P130-3 as diagnostic antigen, indirect ELISA serological detection method for MO was established, and the parameters of reaction conditions were optimized. Finally, the sensitivity of the established EvaGreen real-time fluorescence quantitative PCR method, P130-3 indirect ELISA method, commercial indirect hemagglutination kit and pathogen isolation culture method to clinical samples was compared. Indirect ELISA serological assay has the highest sensitivity.
【学位授予单位】：内蒙古农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S858.26

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