發(fā)布時間：2018-04-09 08:35

  本文選題：綿羊肺炎支原體　切入點：EF-Tu　出處：《中國農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：綿羊肺炎支原體(Mycoplasma ovipneumoniae, M. ovipneumoniae)是一種可引起綿羊、山羊、大角羊和野生小反芻動物慢性肺炎的主要病原,主要導(dǎo)致患病動物體重下降、痙攣性咳嗽、貧血等癥狀,病程可持續(xù)數(shù)月至數(shù)年。目前我國已從四川、新疆、寧夏、河北等多個省份的羊群中分離到該病原,且近些年該病在世界各地均有發(fā)生,對羊養(yǎng)殖業(yè)造成極大的經(jīng)濟(jì)損失。免疫預(yù)防及血清學(xué)診斷是控制動物傳染病的有效手段,因此疫苗研究篩選具有良好免疫原性蛋白及建立血清學(xué)檢測方法對預(yù)防控制本病極為重要。本研究選取綿羊肺炎支原體細(xì)菌延伸因子Tu (elongation factor Tu, EF-Tu)蛋白和熱休克70蛋白(heat shock protein 70, HSP70),對其進(jìn)行免疫原性分析,并建立了綿羊肺炎支原體血清抗體ELISA檢測方法,以期為綿羊肺炎支原體病的防治提供一些研究基礎(chǔ)。本文首先對綿羊肺炎支原體臨床分離株Mo-1的elongation factor Tu, EF-Tu) EF-Tu和HSP70蛋白進(jìn)行了表達(dá)和純化。然后,通過免疫印跡試驗證明EF-Tu和HSP70兩種蛋白均存在于由綿羊肺炎支原體膜蛋白的TritonX-114提取物中。將原核表達(dá)的重組蛋白rEF-Tu(recombinant EF-Tu)和rHSP70 (recombinant HSP70)分別免疫BALB/c小鼠,進(jìn)行免疫原性分析。結(jié)果表明,與綿羊肺炎支原體全細(xì)胞提取物(Mo extracts)免疫對照組比較,上述兩種重組蛋白均能刺激小鼠產(chǎn)生較高的IgG水平,且IgG1和IgG2a水平顯著高于Mo extracts對照組。rEF-Tu和rHSP70免疫組小鼠血清中Thl型(IFN-γ、TNF-α、IL-12p70)和Th2型(IL-4、IL-5、IL-6)細(xì)胞因子水平顯著高于Mo extracts對照組。ELISPOT試驗進(jìn)一步證實, rHSP70比rEF-Tu和Mo extracts能誘導(dǎo)更多的特異性分泌IFN-γ的淋巴細(xì)胞。由此說明,rEF-Tu和rHSP70蛋白可使小鼠同時產(chǎn)生較強(qiáng)體液免疫應(yīng)答及細(xì)胞免疫應(yīng)答。另外,體外生長抑制試驗表明,抗rHSP70、抗rEF-Tu和抗Moextracts小鼠血清均可有效抑制固體培養(yǎng)基上綿羊肺炎支原體的生長,其中rHSP70組抑菌效果優(yōu)于rEF-Tu組和Mo extracts組。本文利用菊糖提純豚鼠血清中的補(bǔ)體C3b成分,免疫小鼠后篩選得到可用于建立補(bǔ)體結(jié)合酶聯(lián)免疫吸附試驗方法(complement fixation ELISA, CF-ELISA)的抗豚鼠補(bǔ)體C3b單克隆抗體。經(jīng)鑒定,所獲得的單克隆抗體具有高的親和力及特異性,可特異識別豚鼠補(bǔ)體C3bα'鏈。在此基礎(chǔ)上,選擇免疫原性較優(yōu)且具有高度保守性的rHSP70作為包被抗原,建立了Mo-rHSP70-CF-ELISA和Mo-rHSP70-iELISA方法,兩者均具有良好的敏感性和特異性。用Mo-rHSP70-CF-ELISA、Mo-rHSP70-iELISA以及文獻(xiàn)報道的以全菌抗原為包被抗原的綿羊肺炎支原體間接ELISA方法(Mo-iELISA)對361份采集自羊場的臨床血清樣品進(jìn)行了檢測。結(jié)果表明,Mo-rHSP70-CF-ELISA和Mo-rHSP70-iELISA方法的Kappa系數(shù)為0.7640,兩種方法一致性良好。Mo-rHSP70-CF-ELISA和Mo-rHSP70-iELISA分別與Mo-iELISA比較,Kappa系數(shù)分別為0.8150和0.8649,一致性良好。綜上所述,綿羊肺炎支原體EF-Tu和HSP70蛋白可誘導(dǎo)較強(qiáng)的體液免疫反應(yīng)和細(xì)胞免疫反應(yīng),具有良好的免疫原性,有可能成為綿羊肺炎支原體疫苗的候選抗原。另外,我們以Mo-rHSP70作為包被抗原建立的Mo-rHSP70-CF-ELISA和Mo-rHSP70-iELISA方法具有較高的靈敏度及特異性,可用于綿羊肺炎支原體血清抗體的檢測.。
[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.

【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S852.62

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