绵羊肺炎支原体EF-Tu和HSP70蛋白免疫原性分析及补体ELISA方法的建立

发布时间：2018-04-09 08:35

本文选题：绵羊肺炎支原体　切入点：EF-Tu　出处：《中国农业大学》2016年博士论文

【摘要】：绵羊肺炎支原体(Mycoplasma ovipneumoniae, M. ovipneumoniae)是一种可引起绵羊、山羊、大角羊和野生小反刍动物慢性肺炎的主要病原,主要导致患病动物体重下降、痉挛性咳嗽、贫血等症状,病程可持续数月至数年。目前我国已从四川、新疆、宁夏、河北等多个省份的羊群中分离到该病原,且近些年该病在世界各地均有发生,对羊养殖业造成极大的经济损失。免疫预防及血清学诊断是控制动物传染病的有效手段,因此疫苗研究筛选具有良好免疫原性蛋白及建立血清学检测方法对预防控制本病极为重要。本研究选取绵羊肺炎支原体细菌延伸因子Tu (elongation factor Tu, EF-Tu)蛋白和热休克70蛋白(heat shock protein 70, HSP70),对其进行免疫原性分析,并建立了绵羊肺炎支原体血清抗体ELISA检测方法,以期为绵羊肺炎支原体病的防治提供一些研究基础。本文首先对绵羊肺炎支原体临床分离株Mo-1的elongation factor Tu, EF-Tu) EF-Tu和HSP70蛋白进行了表达和纯化。然后,通过免疫印迹试验证明EF-Tu和HSP70两种蛋白均存在于由绵羊肺炎支原体膜蛋白的TritonX-114提取物中。将原核表达的重组蛋白rEF-Tu(recombinant EF-Tu)和rHSP70 (recombinant HSP70)分别免疫BALB/c小鼠,进行免疫原性分析。结果表明,与绵羊肺炎支原体全细胞提取物(Mo extracts)免疫对照组比较,上述两种重组蛋白均能刺激小鼠产生较高的IgG水平,且IgG1和IgG2a水平显著高于Mo extracts对照组。rEF-Tu和rHSP70免疫组小鼠血清中Thl型(IFN-γ、TNF-α、IL-12p70)和Th2型(IL-4、IL-5、IL-6)细胞因子水平显著高于Mo extracts对照组。ELISPOT试验进一步证实, rHSP70比rEF-Tu和Mo extracts能诱导更多的特异性分泌IFN-γ的淋巴细胞。由此说明,rEF-Tu和rHSP70蛋白可使小鼠同时产生较强体液免疫应答及细胞免疫应答。另外,体外生长抑制试验表明,抗rHSP70、抗rEF-Tu和抗Moextracts小鼠血清均可有效抑制固体培养基上绵羊肺炎支原体的生长,其中rHSP70组抑菌效果优于rEF-Tu组和Mo extracts组。本文利用菊糖提纯豚鼠血清中的补体C3b成分,免疫小鼠后筛选得到可用于建立补体结合酶联免疫吸附试验方法(complement fixation ELISA, CF-ELISA)的抗豚鼠补体C3b单克隆抗体。经鉴定,所获得的单克隆抗体具有高的亲和力及特异性,可特异识别豚鼠补体C3bα'链。在此基础上,选择免疫原性较优且具有高度保守性的rHSP70作为包被抗原,建立了Mo-rHSP70-CF-ELISA和Mo-rHSP70-iELISA方法,两者均具有良好的敏感性和特异性。用Mo-rHSP70-CF-ELISA、Mo-rHSP70-iELISA以及文献报道的以全菌抗原为包被抗原的绵羊肺炎支原体间接ELISA方法(Mo-iELISA)对361份采集自羊场的临床血清样品进行了检测。结果表明,Mo-rHSP70-CF-ELISA和Mo-rHSP70-iELISA方法的Kappa系数为0.7640,两种方法一致性良好。Mo-rHSP70-CF-ELISA和Mo-rHSP70-iELISA分别与Mo-iELISA比较,Kappa系数分别为0.8150和0.8649,一致性良好。综上所述,绵羊肺炎支原体EF-Tu和HSP70蛋白可诱导较强的体液免疫反应和细胞免疫反应,具有良好的免疫原性,有可能成为绵羊肺炎支原体疫苗的候选抗原。另外,我们以Mo-rHSP70作为包被抗原建立的Mo-rHSP70-CF-ELISA和Mo-rHSP70-iELISA方法具有较高的灵敏度及特异性,可用于绵羊肺炎支原体血清抗体的检测.。
[Abstract]:Mycoplasma pneumoniae (Mycoplasma ovipneumoniae, M. ovipneumoniae) is a main pathogen caused by sheep, goats, wild bighorn sheep and small ruminant animal chronic pneumonia, caused the sick animal weight loss, spasmodic cough, anemia and other symptoms, the course may be continued for several months to several years. At present, China has from Sichuan, Xinjiang Ningxia, isolated the pathogen in Hebei and other provinces of the flock, and in recent years, the disease occurs throughout the world, causing great economic losses to the sheep industry. Immune prevention and serological diagnosis is an effective means to control the animal infectious disease, so the vaccine has good immunogenicity of screening protein and establishment of serological detection method of prevention and control of this disease is very important. This research selects Mycoplasma pneumoniae bacterial elongation factor Tu (elongation factor Tu, EF-Tu) protein and heat shock protein 70 (heat shock Protein 70, HSP70), analyze the immunogenicity of the sheep and the establishment of mycoplasma pneumonia serum antibody detection ELISA method, in order to provide some basis for the study of prevention and treatment of Mycoplasma pneumoniae disease. Firstly, Mycoplasma pneumoniae clinical isolates of Mo-1 elongation factor Tu, EF-Tu EF-Tu) and HSP70 protein and expression and purification of TritonX-114. Then, the extract of Western blot test showed that EF-Tu and HSP70 two proteins were found in the membrane protein of Mycoplasma pneumoniae. The prokaryotic expression of the recombinant protein rEF-Tu (recombinant EF-Tu) and rHSP70 (recombinant HSP70) BALB/c mice were immunized with analysis of immunogenicity. The results showed that with the sheep whole cell extracts of Mycoplasma pneumoniae (Mo extracts) immune control group, these two recombinant proteins can stimulate mice to produce higher levels of IgG and IgG1, and Ig The level of G2a was significantly higher than that of control group.REF-Tu Mo extracts type Thl and rHSP70 in the serum of immunized mice (IFN- gamma, alpha TNF-, IL-12p70) and Th2 (IL-4, IL-5, IL-6) cytokine levels were significantly higher than that of Mo extracts control group.ELISPOT test confirmed that rHSP70 rEF-Tu and Mo extracts than the more specific secretion of IFN- gamma induced lymphocyte. Therefore, rEF-Tu and rHSP70 protein in the mice also produce strong humoral immune response and cellular immune response. In addition, the in vitro growth inhibition test showed that anti rHSP70, anti rEF-Tu and anti Moextracts serum could effectively inhibit the growth of solid culture of Mycoplasma ovipneumoniae based on the inhibitory effect of rHSP70 was better than rEF-Tu Mo group and extracts group. The purification of inulin in guinea pig serum complement C3b component, after immunization of mice obtained can be used for the establishment of complement binding ELISA Test method (complement fixation ELISA, CF-ELISA) anti guinea pig complement C3b monoclonal antibody. After identification, with high affinity and specificity of the McAbs, specific guinea pig complement C3b 'alpha chain. On this basis, select the better immunogenicity and is highly conserved rHSP70 as antigen the establishment of Mo-rHSP70-CF-ELISA and Mo-rHSP70-iELISA, both of them have good sensitivity and specificity. Mo-rHSP70-CF-ELISA, Mo-rHSP70-iELISA and reported to the whole cell antigen coated sheep mycoplasma pneumonia indirect ELISA antigen (Mo-iELISA) of the 361 samples collected were detected from clinical serum samples of sheep. The results show that the coefficient of Kappa Mo-rHSP70-CF-ELISA and Mo-rHSP70-iELISA was 0.7640, two methods.Mo-rHSP70-CF-ELISA and Mo-rHSP70-iELISA were consistent with Mo-iELIS A comparison, Kappa coefficients were 0.8150 and 0.8649, good consistency. In summary, Mycoplasma pneumoniae EF-Tu and HSP70 protein can induce humoral and cellular immune responses in strong, has good immunogenicity, may be a candidate antigen of Mycoplasma pneumoniae vaccine. In addition, we Mo-rHSP70-CF-ELISA and Mo-rHSP70-iELISA method for coating antigen to establish Mo-rHSP70 as a high sensitivity and specificity, can be used for detection of Mycoplasma pneumoniae antibodies.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.62

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