流产嗜性衣原体MIP蛋白单克隆抗体的制备及特性鉴定

Production and identification of monoclonal antibodiesagainst MIP protein of Chlamydophila abortus

关键词:  流产嗜性衣原体  MIP蛋白  单克隆抗体  鉴定

Key words:  Chlamydophila abortus  MIP protein  Monoclonal antibody  Identification

摘要:

【目的】制备抗流产嗜性衣原体MIP蛋白单克隆抗体,并对其生物学特性进行鉴定。【方法】以纯化的重组MIP蛋白为抗原,免疫Balb/c小鼠后经细胞融合,间接ELISA法筛选阳性细胞克隆并进行有限稀释克隆化,建立分泌抗MIP蛋白单克隆抗体的杂交瘤细胞株。间接ELISA法鉴定单抗抗体效价、相对亲和力、Western Blot鉴定抗MIP蛋白特异性,Mouse单克隆抗体亚类检测试剂盒鉴定类别,鸡胚接种鉴定单抗中和能力。【结果】建立了两株能稳定分泌抗MIP蛋白单克隆抗体的杂交瘤细胞株,分别命名为M1和M3,ELISA法测上清效价为1:1600,染色体鉴定符合杂交瘤细胞的特性,2株单抗免疫球蛋白类型均为IgG1,轻链为κ链,两株单克隆抗体能特异识别MIP重组蛋白,体外中和试验表明,2株单抗能有效中和衣原体感染性。【结论】成功制备了抗MIP单克隆抗体,为MIP蛋白的进一步研究提供了物质基础。

Abstract:

Objective: To produce the monoclonal antibodies (mAbs) against MIP protein of Chlamydophila abortus and identify their biological characteristics. Methods:Balb/c mice were immunized with purified MIP protein. The positive hybridoma cell lines were screened by indirect ELISA after cell fusion and subcloned by limiting dilution. A monoclonal antibody hybridoma cell line which secretes MIP resistant protein was established eventually.The mAbs titers, relative affinity were identified and the specificities to MIP protein were identified by Western Blot , and subtypes of Ig were identifed with mAb subclass detection kit. Chicken embryo inoculation was performed to identify the neutralization activity. Results:Two hybridoma cell lines steadily secreting anti-MIP mAb were obtained and named M1 and M3 and the titer of these mAbs were1:1600 by the way of ELISA. The characteristics of hybridoma was confirmed by Chromosome identification.The subtype of IgG of two strains was IgG1, and the light chain of them was κ chain . The recombinant MIP protein specificity of two strains of the monoclonal was recognized by Western Blot and the neutralization test showed that the mAbs can neutralize chlamydial infection effectively. Conclusion: 2 mAbs anti-MIP have been produced successfully and which can provide some useful date for the study of MIP protein in the future.