基于脂蛋白P48的牛支原体快速检测方法的建立

发布时间：2018-03-17 07:15

本文选题：牛支原体　切入点：多克隆抗体　出处：《甘肃农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：牛支原体(Mycoplasma bovis)是一种引起牛呼吸道疾病的病原体,通常可以引起肺炎、乳腺炎、关节炎、角膜结膜炎等,成为阻碍养牛业发展的一大重要隐患。随着养牛业的不断发展,牛支原体病的流行范围越来越广,发生频率也越来越高,加之我国对于本病的研究起步较晚,并且国际上尚无公认的特异性诊断方法,以及目前对于该病致病机制的研究尚未达成共识,从而为该病的预防控制带来了不便。基于此,本研究对牛支原体P48基因进行扩增,并构建原核表达载体进行原核表达,成功制备出P48蛋白单克隆抗体,对P48蛋白相关功能进行了研究,从而为该病原的防控、免疫学功能及致病机制的研究奠定了基础。同时本研究还建立了牛支原体与无乳支原体等病原的鉴别诊断方法,对牛支原体感染临床快速、准确的诊断具有较高的实用价值。1.牛支原体P48蛋白的原核表达纯化及多克隆抗体的制备参照GeneBank中牛支原体P48株基因序列设计特异性引物,将编码区中的五处终止密码子TGA等义突变为TGG,将扩增获得的P48基因克隆至表达载体pET-28a中,构建原核表达质粒pET-P48,表达质粒转化大肠杆菌BL21(DE3),并在IPTG诱导下成功获得融合蛋白表达产物rMbP48,大小约为51ku。将纯化后的表达产物(His-P48)免疫新西兰大白兔制备多克隆抗体,并通过western blot验证其免疫原性,结果表明P48抗血清能和纯化后的抗原His-P48发生特异性反应。2.牛支原体P48蛋白亚细胞初步定位为了检测P48蛋白的分布情况,分别提取牛支原体全菌蛋白、膜蛋白、胞浆蛋白,用制备好的P48抗血清进行western blot检测,结果表明,P48蛋白在牛支原体的膜蛋白和胞浆蛋白中均有分布,但主要以膜上为主。分别用膜蛋白和胞浆蛋白作为抗原包被后,以制备好的P48抗血清为一抗进行ELISA检测,同时用荧光抗体检测脂蛋白P48在牛支原体中的分布情况,结果表明P48蛋白主要存在于牛支原体的膜分离相。全菌免疫荧光试验进一步证实脂蛋白P48位于牛支原体的膜表面。3.牛支原体P48蛋白单克隆抗体的制备将纯化后的表达产物(His-P48)免疫BALB/C小鼠制备得到单克隆抗体,成功获得两株能够稳定、持续分泌单克隆抗体的杂交瘤细胞,细胞上清效价可达1:2000左右,单克隆抗体效价可达1:640 000,与可以引起牛呼吸道疾病的其他病原无交叉反应,具有稳定性高、特异性强等特点。4.牛支原体PCR检测方法的建立根据P48基因序列特点,设计了一对特异性引物对牛支原体、无乳支原体、丝状支原体、滑液支原体、鸡毒支原体等基因进行扩增,并进行了敏感性分析,同时对临洮、武威等地的牛支原体病料及疑似支原体感染病料进行相关处理后进行PCR扩增,结果表明该引物具有很高的特异性和敏感性,除牛支原体外其他物种基因均无目的条带出现,对牛支原体新鲜临床病料进行PCR扩增后有清晰的目的条带出现,说明该诊断方法对于牛支原体的鉴别诊断具有较高的实用价值。
[Abstract]:Mycoplasma bovis (Mycoplasma bovis) is a kind of bovine respiratory disease caused by the pathogen, usually can cause pneumonia, mastitis, arthritis, keratoconjunctivitis, become one of the important problems that hinder the development of the cattle industry. With the continuous development of the cattle industry, popular more and more widely Mycoplasma disease of cattle, the frequency of occurrence and the more high, coupled with our research on the disease started late, specific diagnosis and there is no international recognized, as well as the current research about the pathogenic mechanism of this disease has not yet reached a consensus, so as to bring inconvenience for the disease prevention and control. Based on this, the study of bovine mycoplasma P48 gene was amplified, and the construction of the original the eukaryotic expression vector of prokaryotic expression, preparation of monoclonal antibodies against P48 protein, the function of P48 protein were studied, so as to control the pathogen, immunological function and pathogenic mechanism research To lay the foundation. At the same time this study also established a method for differential diagnosis of bovine mycoplasma and Mycoplasma agalactiae pathogens, bovine mycoplasma infection clinical rapid, accurate diagnosis has high practical value of.1. bovine mycoplasma P48 prokaryotic expression and purification of protein and preparation of polyclonal antibody to GeneBank in Mycoplasma bovis strain P48 gene sequence design specific primers, five encoding region of the termination codon TGA missense mutation in TGG, the amplification of the P48 gene cloned into expression vector pET-28a, to construct the prokaryotic expression plasmid pET-P48, the recombinant plasmid was transformed into E.coli BL21 (DE3), and induced by IPTG fusion protein successfully expressed rMbP48 the size is about 51ku., the purified expression products (His-P48) to immunize New Zealand rabbits to prepare polyclonal antibody, and its immunogenicity was verified by Western blot, and the results show that the P48 antiserum The purified His-P48 antigen specific reaction.2. Mycoplasma bovis P48 protein subcellular distribution of preliminary positioning to the detection of P48 protein, bovine mycoplasma whole bacterial proteins were extracted, membrane protein, Cytoplasmic Protein, P48 antiserum prepared by Western blot test, the results show that the distribution of membrane protein and cytosolic protein P48 protein in Mycoplasma bovis, but mainly in the membrane. With membrane protein and cytosolic protein as antigen, anti ELISA was detected by P48 antiserum was prepared for use at the same time, fluorescent antibody detection of apolipoprotein P48 in Mycoplasma bovis in distribution, results show that the membrane separation P48 protein mainly exists in the whole bacteria Mycoplasma bovis. Immunofluorescence assay further confirmed the lipoprotein P48 of Mycoplasma bovis in bovine mycoplasma.3. membrane surface preparation of monoclonal antibodies against P48 protein purified expression products (H Is-P48) BALB/C mice were immunized with the prepared monoclonal antibody, successfully obtained two strains of hybridoma cells stably, continuously secrete monoclonal antibody, cell supernatant titer was about 1:2000, the monoclonal antibody titer was 1:640 000, and can cause the pathogen of bovine respiratory disease and no cross reaction with high stability, specificity and other characteristics.4. PCR method for detection of Mycoplasma bovis is established according to P48 gene sequence, a pair of specific primers was designed for bovine mycoplasma pneumoniae, Mycoplasma agalactiae, Mycoplasma mycoides, Mycoplasma synoviae, such as poison chicken Mycoplasma gene was amplified, and sensitivity analysis is carried out at the same time, to Lintao, Wuwei and other places of cattle Mycoplasma disease and suspected mycoplasma infection the disease is expected to conduct correlation processing for PCR amplification. The results show that the primer with high specificity and sensitivity, in addition to the bovine mycoplasma species gene was he No target strip appeared. A clear target band appeared after PCR amplification of new Mycoplasma bovis clinical material, indicating that this diagnostic method has high practical value for the differential diagnosis of Mycoplasma bovis.

【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.62

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