猪肺炎支原体P97基因原核表达产物单抗的制备

Preparation of monoclonal antibody to the prokaryotically expressed product of the P97 gene of Mycoplasma hyopneumoniae

目的 纤毛黏附因子P97是猪肺炎支原体特异性的外膜蛋白 ,该蛋白C端的R1区是其主要的抗原决定簇。本研究以大肠杆菌表达的P97黏附因子R1融合蛋白作为免疫原免疫BALB/c鼠制备单克隆抗体。通过酶联免疫吸附实验(ELISA)平行筛选 ,获得两株单克隆抗体杂交瘤细胞株A9-H7、H11-E7。两株杂交瘤细胞经体外连续传代 1个月并冻存复苏后均能分泌高效价的抗融合蛋白抗体 ,并能在小鼠体内形成肿瘤 ,有效诱导产生高效价的腹水抗体。两株单抗腹水的ELISA效价分别为 1× 10 6和 1× 10 5。且均与天然Mhp16

The p97 adhesin of Mycoplasma hyopneumoniae is a membranous protein carrying species-specific antigenic determinants in which the C-terminal R1 region of this protein represents the major immunodominant epitope. In the present study, the p97 adhesin R1 fusion protein expressed on E.coli was used as immunogen to immunize BALB/c mice for the preparation of monoclonal antibody against this specific antigen. Two strains of hybridoma cells, H11-E7 and A9-H7, was obtained through parallel screening with ELISA and Western blotting. These hybridoma cell lines could secrete highly efficient anti-fusion protein antibody even after serial transfers for one month and repeated freezing and thawing. In addition they could induce the development of tumors in mice and produce high titer ascitic antibodies with the ELISA titers of 1×10~6 and 1×10~5 respectively. These monoclonal antibodies also showed strong reactivity with natural Mhp168 strain as demonstrated by Western blotting analysis.