CAJ | 학술논문

P65蛋白是猪肺炎支原体的主要免疫优势蛋白，与其他支原体无交叉反应，常作为猪肺炎支原体检测的靶蛋白。本实验应用原核表达的重组 P65蛋白免疫小鼠，用 Mhp全菌蛋白和P65蛋白筛选，制备了1株特异性单克隆抗体3G12。鉴定结果表明，该株单克隆抗体可与 P65蛋白和 Mhp全菌蛋白反应，采用间接 ELISA法测得3G12细胞培养上清与P65蛋白反应抗体的效价为1∶12800，与全菌蛋白反应抗体的效价为1∶3200；3G12腹水与P65蛋白反应的抗体效价为1∶4000000以上，与168全菌蛋白反应的抗体效价为1∶20000以上，细胞经长期体外培养和冻存后复苏能稳定分泌抗体。本研究成功获得到1株针对 P65和Mhp全菌的单克隆抗体，为猪肺炎支原体致病机制和检测方法的进一步研究提供了基础。
P65단백시저폐염지원체적주요면역우세단백，여기타지원체무교차반응，상작위저폐염지원체검측적파단백。본실험응용원핵표체적중조 P65단백면역소서，용 Mhp전균단백화P65단백사선，제비료1주특이성단극륭항체3G12。감정결과표명，해주단극륭항체가여 P65단백화 Mhp전균단백반응，채용간접 ELISA법측득3G12세포배양상청여P65단백반응항체적효개위1∶12800，여전균단백반응항체적효개위1∶3200；3G12복수여P65단백반응적항체효개위1∶4000000이상，여168전균단백반응적항체효개위1∶20000이상，세포경장기체외배양화동존후복소능은정분비항체。본연구성공획득도1주침대 P65화Mhp전균적단극륭항체，위저폐염지원체치병궤제화검측방법적진일보연구제공료기출。
P65 protein, the major immunodominant protein of Mycoplasma hyopneu-moniae (Mhp) exhibiting no cross-reaction with other mycoplasmas, is general y used as a target protein for Mhp detection. In this study, BALB/c mice were immunized with prokaryotical y expressed P65 recombinant protein to prepare monoclonal anti-body. After screening with Mhp whole-cel protein and P65 protein, a specific hy-bridoma cel line, 3G12, was obtained by ELISA. Identification results indicated that the antibody secreted by 3G12 hybridoma cel s could react with P65 protein and Mhp whole-cel protein. According to indirect ELISA assay, 3G12 cel culture super-natant possessed a titer of 1∶12 800 against P65 protein and 1∶3 200 against Mhp whole-cel protein; 3G12 ascites possessed a titer of above 1∶4 000 000 against P65 protein and above 1∶20 000 against Mhp 168 whole-cel protein. After long-term in vitro culture and continuous freezing-thawing, 3G12 cel line could stably secrete antibodies. A monoclonal antibody against P65 protein and Mhp whole-cel protein was successful y obtained in the present study, which provided basis for further in-vestigating the pathogenic mechanism of Mhp and establishing diagnostic methods of Mycoplasmal pneumonia of swine (MPS).