綿羊肺炎支原體檢測方法的建立及黏附蛋白的研究
發(fā)布時間：2018-09-07 06:30
【摘要】：綿羊肺炎支原體(Mycoplasma ovipneumoniae, MO)可感染綿羊和山羊引起傳染性胸膜肺炎,以纖維素滲出性肺炎為主要特征,在很多國家和地區(qū)都報道有該病原的存在。內(nèi)蒙古及周邊地區(qū)是全國的主要養(yǎng)羊區(qū),近年來隨著養(yǎng)殖方式的轉(zhuǎn)變,養(yǎng)殖密度大幅增加,該病在這些地區(qū)分布和流行的趨勢也日益增強,給養(yǎng)羊業(yè)帶來巨大的經(jīng)濟損失。開展該病防控工作的首要任務(wù)是建立準確的診斷方法和研發(fā)有效的疫苗,目前關(guān)于這方面的工作還不很完善,因此,本研究在對華北部分地區(qū)分離的菌株進行了鑒定和分子流行病學(xué)調(diào)查的基礎(chǔ)上,建立了分子水平上的熒光定量PCR檢測方法和血清學(xué)水平上的間接ELISA診斷方法,并預(yù)測了該病原的3個黏附蛋白,對其功能進行了驗證,篩選出了優(yōu)勢抗原表位和功能結(jié)構(gòu)域,以期更深入的了解了該病原的致病特性。本研究對分離自華北大部分地區(qū)的227株綿羊肺炎支原體進行了實驗室傳統(tǒng)的分離和鑒定試驗及分子生物學(xué)鑒定,并分別擴增了16s rDNA、EFTU基因、HSP70基因及16s-23sr DNA。選取其中具有地方代表性的14株菌株,對擴增產(chǎn)物進行了測序,發(fā)現(xiàn)利用EFTU基因構(gòu)建的系統(tǒng)進化樹與依據(jù)16s rDNA構(gòu)建的系統(tǒng)進化樹標準區(qū)別不大,提出EFTU基因可以作為新的分子靶標來分析MO的遺傳進化關(guān)系和種屬鑒別分類,加之其在支原體種間的保守性,建議EFTU基因可以作為MO分子生物學(xué)鑒定的特異性基因。在此基礎(chǔ)上構(gòu)建了基于EFTU基因的MO EvaGreen實時熒光定量PCR檢測方法,通過試驗驗證,該方法在特異性、敏感性和穩(wěn)定性上都優(yōu)于普通PCR檢測方法。根據(jù)本實驗室基因組測序的注釋結(jié)果和生物信息學(xué)軟件分析結(jié)果,將預(yù)測的黏附蛋白P130、P129和P71按保留完整功能結(jié)構(gòu)域和突變位點的要求共截短表達為9段蛋白。利用大腸桿菌表達系統(tǒng)分別表達9段截短蛋白,重組蛋白純化后免疫小鼠進行免疫學(xué)試驗,篩選優(yōu)勢抗原區(qū)。結(jié)果表明,經(jīng)P130-3和P71-3段蛋白免疫可以顯著提高小鼠的細胞和體液免疫指標,在其它的免疫試驗中兩者的效果也比較明顯。建立了綿羊氣管上皮細胞分離鑒定的方法,成功分離到綿羊氣管上皮細胞,并在此基礎(chǔ)上構(gòu)建了MO的感染模型,驗證了其對氣管上皮細胞的黏附能力。應(yīng)用該模型檢測了重組蛋白的黏附能力以及蛋白抗血清對全菌的抑制黏附能力,發(fā)現(xiàn)P130-3和P129-2效果比較明顯,提示其片段上的結(jié)構(gòu)域在菌體黏附過程中起到重要作用。綜合考慮以上試驗結(jié)果,P130-3的檢測性能總體水平比較高,為篩選出的最優(yōu)截短蛋白。用篩選的性能最優(yōu)的蛋白P130-3作為診斷抗原,建立了MO的間接ELISA血清學(xué)檢測方法,并對各反應(yīng)條件參數(shù)進行了優(yōu)化。最后,比較了建立的EvaGreen實時熒光定量PCR方法、P130-3間接ELISA方法、商品化的間接血凝檢測試劑盒以及病原分離培養(yǎng)方法對臨床樣品檢測的敏感性,結(jié)果表明,間接ELISA血清學(xué)檢測方法敏感性最高。
[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.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S858.26
本文編號：2227476
[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.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S858.26
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