本文選題：支原體 + 單克隆抗體��； 參考：《中國協(xié)和醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 肺支原體、溶神經(jīng)支原體和關(guān)節(jié)炎支原體是嚙齒類實驗動物的主要致病菌，也是清潔級實驗動物必須排除的病原微生物。我國實驗動物支原體感染的實驗室檢測方法以分離培養(yǎng)和檢測血清抗體為主，前者操作繁瑣、結(jié)果受到操作者技術(shù)水平的影響，并且檢測所需時間長；后者為間接判斷動物的感染、不適用于某些免疫缺陷或免疫修飾而導(dǎo)致產(chǎn)生抗體障礙的動物的檢測。為此，本研究通過制備抗實驗嚙齒類動物支原體單克隆抗體，進而建立以檢測支原體抗原為對象的雙抗體夾心ELISA方法，旨在能夠快速、敏感、直接判斷動物感染。 首先，肺支原體mAb的制備與鑒定。肺支原體抗原譜較廣，以19612全菌蛋白為免疫原，3種嚙齒類動物易感支原體全蛋白為篩選抗原，制備出9種單抗細(xì)胞株，，與先前的豬鼻單抗、三種支原體共同抗原的單抗進行了系統(tǒng)的鑒定，包括亞類鑒定、相對分子質(zhì)量、特異性及敏感性等。 其次，選擇最佳配對抗體，建立雙抗夾心ELISA法。我們將不同來源的單抗進行配對組合，最終選擇12A9作為捕獲抗體，酶標(biāo)E5作為檢測抗體用作雙抗體夾心ELISA的檢測試劑，對嚙齒類實驗動物易感的三種支原體模式菌株的檢測靈敏度達到ng級水平，診斷特異性和敏感性均較好。 最后，該雙抗體夾心ELISA法應(yīng)用于實際檢測。在對120只感染動物的實際檢測中，雙抗體夾心ELISA的陽性檢出率較高，與分離培養(yǎng)的檢測結(jié)果一致性分別為92.6％(M53)、91.7％(19611)、95.2％(19612)、92.9％(19988)。 本研究為將來實驗動物支原體抗原的實際檢測打下了基礎(chǔ)，為其他實驗動物微生物監(jiān)測方法的改良提供了實驗依據(jù)。 支原體是一類缺乏細(xì)胞壁的原核微生物，能引起鼠呼吸系統(tǒng)支原體病(MRM)和鼠生殖系統(tǒng)支原體病(MGM)，其膜蛋白是支原體最重要的表面抗原物質(zhì)。除無膽甾原體外，目前用于支原體的培養(yǎng)基大都含動物血清，以此為其生長繁殖提供脂質(zhì)前體，而脂類成分的抗原是支原體的主要抗原。由于不同動物的血清成份存在差異，故其所培養(yǎng)的支原體的抗原性可能亦有差異。為此，我們將肺支原體用含馬血清和豬血清的培養(yǎng)基培養(yǎng)，觀察不同的血清環(huán)境和傳代次數(shù)對肺支原體致病性和抗原性的影響。 在致病性研究中，我們利用肺支原體模式菌株19612和M53感染實驗動物，結(jié)果19612感染實驗大鼠、小鼠均失敗，后通過感染實驗鼠離體氣管粘膜細(xì)胞，發(fā)現(xiàn)經(jīng)多次傳代的肺支原體模式株19612失去黏附性，無法黏附到動物氣管粘膜細(xì)胞上，喪失致病性。 在抗原性研究中，免疫印跡結(jié)果提示：一，用不同培養(yǎng)基培養(yǎng)的同一株肺支原體，相對分子質(zhì)量33×10~3處的蛋白表達量不同；二，相對分子質(zhì)量33×10~3處的蛋白也是血清中的抗原成分；三，不同肺支原體株之間的抗原條帶存在差異。 本研究考察了與肺原體致病性和抗原性有關(guān)的一些因素，這些為今后診斷試劑的研制提供了參考，具有重要意義。
[Abstract]:Mycoplasma pneumoniae, Mycoplasma dissolve mycoplasma and Mycoplasma arthritis are the main pathogenic bacteria in the rodent experiment animals. It is also the pathogenic microorganism that must be eliminated by the clean laboratory animals. The laboratory test method of mycoplasma infection in our country is mainly to separate and train and detect the serum antibody, the former is complicated and the result is the operator technique. The effect of the level is long and the time required is detected. The latter is used to indirectly determine the infection of the animal, which is not suitable for the detection of an animal with antibody barrier caused by some immunodeficiency or immune modification. To this end, the study was made to prepare monoclonal anti body of Mycoplasma Mycoplasma, and then to establish the detection of Mycoplasma antigen. The double antibody sandwich ELISA method is designed to be quick, sensitive and direct for animal infection.
First, the preparation and identification of Mycoplasma pneumoniae mAb. The antigen spectrum of Mycoplasma pneumoniae is wide, 19612 total bacteria protein is used as immunogen, and the total protein of Mycoplasma Mycoplasma in 3 rodents is selected as the screening antigen and 9 kinds of monoclonal antibody cell lines are prepared. The monoclonal antibody of the previous McAb and three kinds of Mycoplasma common antigen is systematically identified, including subclass identification and phase. Molecular weight, specificity, sensitivity, and so on.
Secondly, we selected the best paired antibody and established the double anti sandwich ELISA method. We paired the McAbs from different sources, finally selected 12A9 as the capture antibody, and the enzyme labeled E5 was used as the detection antibody for the double antibody sandwich ELISA, and the sensitivity of the three mycoplasma strains susceptible to the rodent test animals was ng The diagnostic specificity and sensitivity were good at the level level.
Finally, the double antibody sandwich ELISA method was applied to the actual detection. In the actual detection of 120 infected animals, the positive detection rate of the double antibody sandwich ELISA was higher, and the consistency with the isolated culture detection results was 92.6% (M53), 91.7% (19611), 95.2% (19612), 92.9% (19988).
This study laid the foundation for the practical detection of Mycoplasma antigens in laboratory animals in the future, and provided experimental evidence for the improvement of microbiological monitoring methods in other laboratory animals.
Mycoplasma is a type of prokaryotic microorganism lacking cell wall, which can cause Mycoplasma Mycoplasma (MRM) and Mycoplasma disease (MGM) in rat's respiratory system, and its membrane protein is the most important surface antigen of Mycoplasma. In addition to no steroids, most of the culture medium used for mycoplasma is contained in animal serum to provide lipid for its growth and reproduction. The antigen of the lipid component is the main antigen of Mycoplasma. The antigenicity of Mycoplasma may be different because of the difference in the serum composition of different animals. For this reason, we culture Mycoplasma pneumoniae with the culture medium containing horse serum and pig serum, and observe the different serum environment and the number of passages to Mycoplasma pneumoniae. The effects of disease and antigenicity.
In the pathogenicity study, we used Mycoplasma pneumoniae model strain 19612 and M53 to infect experimental animals. Results 19612 infected experimental rats and mice failed. After infection of the isolated tracheal mucosa cells of the experimental rats, the Mycoplasma pneumoniae model 19612 lost adhesion and was unable to adhere to the tracheal mucosa cells of the animal's trachea. Pathogenicity.
In the study of antigenicity, the results of immunoblotting suggested that the protein expression of the same strain of Mycoplasma pneumoniae cultured in different medium was different in the relative molecular mass of 33 x 10~3; two, the protein in the relative molecular mass of 33 x 10~3 was also the antigen component in the serum; three, the antigen bands of different mycoplasma strains were different.
In this study, some factors related to pathogenicity and antigenicity of mycoplasma pneumonia were investigated, which provide a reference for the development of diagnostic reagents in the future.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392;R375

【引證文獻】

相關(guān)期刊論文 前1條

1 任澤民;姜勇;巴曉亮;胡長敏;陳穎鈺;彭清潔;陳煥春;郭愛珍;;牛支原體單克隆抗體的制備與鑒定[J];中國畜牧獸醫(yī);2012年07期

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