綿羊肺炎支原體檢測(cè)方法的建立及黏附蛋白的研究
發(fā)布時(shí)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(bào)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(shì)也日益增強(qiáng),給養(yǎng)羊業(yè)帶來巨大的經(jīng)濟(jì)損失。開展該病防控工作的首要任務(wù)是建立準(zhǔn)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(duì)華北部分地區(qū)分離的菌株進(jìn)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(cè)方法和血清學(xué)水平上的間接ELISA診斷方法,并預(yù)測(cè)了該病原的3個(gè)黏附蛋白,對(duì)其功能進(jìn)行了驗(yàn)證,篩選出了優(yōu)勢(shì)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(duì)分離自華北大部分地區(qū)的227株綿羊肺炎支原體進(jìn)行了實(shí)驗(yàn)室傳統(tǒng)的分離和鑒定試驗(yàn)及分子生物學(xué)鑒定,并分別擴(kuò)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(duì)擴(kuò)增產(chǎn)物進(jìn)行了測(cè)序,發(fā)現(xiàn)利用EFTU基因構(gòu)建的系統(tǒng)進(jìn)化樹與依據(jù)16s rDNA構(gòu)建的系統(tǒng)進(jìn)化樹標(biāo)準(zhǔn)區(qū)別不大,提出EFTU基因可以作為新的分子靶標(biāo)來分析MO的遺傳進(jìn)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(shí)時(shí)熒光定量PCR檢測(cè)方法,通過試驗(yàn)驗(yàn)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(cè)方法。根據(jù)本實(shí)驗(yàn)室基因組測(cè)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(cè)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(diǎn)的要求共截短表達(dá)為9段蛋白。利用大腸桿菌表達(dá)系統(tǒng)分別表達(dá)9段截短蛋白,重組蛋白純化后免疫小鼠進(jìn)行免疫學(xué)試驗(yàn),篩選優(yōu)勢(shì)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(xì)胞和體液免疫指標(biāo),在其它的免疫試驗(yàn)中兩者的效果也比較明顯。建立了綿羊氣管上皮細(xì)胞分離鑒定的方法,成功分離到綿羊氣管上皮細(xì)胞,并在此基礎(chǔ)上構(gòu)建了MO的感染模型,驗(yàn)證了其對(duì)氣管上皮細(xì)胞的黏附能力。應(yīng)用該模型檢測(cè)了重組蛋白的黏附能力以及蛋白抗血清對(duì)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(yàn)結(jié)果,P130-3的檢測(cè)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(cè)方法,并對(duì)各反應(yīng)條件參數(shù)進(jìn)行了優(yōu)化。最后,比較了建立的EvaGreen實(shí)時(shí)熒光定量PCR方法、P130-3間接ELISA方法、商品化的間接血凝檢測(cè)試劑盒以及病原分離培養(yǎng)方法對(duì)臨床樣品檢測(cè)的敏感性,結(jié)果表明,間接ELISA血清學(xué)檢測(cè)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(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S858.26
本文編號(hào)：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(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S858.26
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