（一）实验动物支原体感染双抗体夹心ELISA方法的建立（二）人工培养环境对肺支原体致病性和抗原性的影响

发布时间：2018-07-10 05:10

本文选题：支原体 + 单克隆抗体　；参考：《中国协和医科大学》2007年硕士论文

【摘要】： 肺支原体、溶神经支原体和关节炎支原体是啮齿类实验动物的主要致病菌，也是清洁级实验动物必须排除的病原微生物。我国实验动物支原体感染的实验室检测方法以分离培养和检测血清抗体为主，前者操作繁琐、结果受到操作者技术水平的影响，并且检测所需时间长；后者为间接判断动物的感染、不适用于某些免疫缺陷或免疫修饰而导致产生抗体障碍的动物的检测。为此，本研究通过制备抗实验啮齿类动物支原体单克隆抗体，进而建立以检测支原体抗原为对象的双抗体夹心ELISA方法，旨在能够快速、敏感、直接判断动物感染。首先，肺支原体mAb的制备与鉴定。肺支原体抗原谱较广，以19612全菌蛋白为免疫原，3种啮齿类动物易感支原体全蛋白为筛选抗原，制备出9种单抗细胞株，，与先前的猪鼻单抗、三种支原体共同抗原的单抗进行了系统的鉴定，包括亚类鉴定、相对分子质量、特异性及敏感性等。其次，选择最佳配对抗体，建立双抗夹心ELISA法。我们将不同来源的单抗进行配对组合，最终选择12A9作为捕获抗体，酶标E5作为检测抗体用作双抗体夹心ELISA的检测试剂，对啮齿类实验动物易感的三种支原体模式菌株的检测灵敏度达到ng级水平，诊断特异性和敏感性均较好。最后，该双抗体夹心ELISA法应用于实际检测。在对120只感染动物的实际检测中，双抗体夹心ELISA的阳性检出率较高，与分离培养的检测结果一致性分别为92.6％(M53)、91.7％(19611)、95.2％(19612)、92.9％(19988)。本研究为将来实验动物支原体抗原的实际检测打下了基础，为其他实验动物微生物监测方法的改良提供了实验依据。支原体是一类缺乏细胞壁的原核微生物，能引起鼠呼吸系统支原体病(MRM)和鼠生殖系统支原体病(MGM)，其膜蛋白是支原体最重要的表面抗原物质。除无胆甾原体外，目前用于支原体的培养基大都含动物血清，以此为其生长繁殖提供脂质前体，而脂类成分的抗原是支原体的主要抗原。由于不同动物的血清成份存在差异，故其所培养的支原体的抗原性可能亦有差异。为此，我们将肺支原体用含马血清和猪血清的培养基培养，观察不同的血清环境和传代次数对肺支原体致病性和抗原性的影响。在致病性研究中，我们利用肺支原体模式菌株19612和M53感染实验动物，结果19612感染实验大鼠、小鼠均失败，后通过感染实验鼠离体气管粘膜细胞，发现经多次传代的肺支原体模式株19612失去黏附性，无法黏附到动物气管粘膜细胞上，丧失致病性。在抗原性研究中，免疫印迹结果提示：一，用不同培养基培养的同一株肺支原体，相对分子质量33×10~3处的蛋白表达量不同；二，相对分子质量33×10~3处的蛋白也是血清中的抗原成分；三，不同肺支原体株之间的抗原条带存在差异。本研究考察了与肺原体致病性和抗原性有关的一些因素，这些为今后诊断试剂的研制提供了参考，具有重要意义。
[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.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392;R375

【引证文献】

（一）实验动物支原体感染双抗体夹心ELISA方法的建立 （二）人工培养环境对肺支原体致病性和抗原性的影响

（一）实验动物支原体感染双抗体夹心ELISA方法的建立（二）人工培养环境对肺支原体致病性和抗原性的影响