抗犬新孢子虫MIC6单克隆抗体的制备及鉴定

Preparation and identification of monoclonal antibody against MIC6 of Neospora caninum

目的制备抗犬新孢子虫MIC6单克隆抗体,并进行鉴定。方法用重组蛋白p GEX-MIC6作为抗原免疫BALB/c小鼠,收集脾细胞,与小鼠骨髓瘤细胞SP2/0融合,对融合后的杂交瘤细胞进行筛选后,经腹腔注射BALB/c小鼠,制备腹水,鉴定单抗的亚类。采用辛酸-硫酸铵法结合蛋白亲和层析柱纯化腹水,SDS-PAGE分析抗体的相对分子质量,间接ELISA法检测抗体效价及特异性,BCA蛋白浓度检测试剂盒测定抗体浓度。结果最终获得两株能稳定分泌新孢子虫MIC6蛋白单克隆抗体的杂交瘤细胞株1A1和1H11,分泌的抗体分别属于Ig G1和Ig G2b亚类。纯化后的腹水经SDS-PAGE分析,分别可见相对分子质量约为50 000的重链和25 000的轻链条带,纯化效果较好;1A1和1H11的腹水效价分别为5.12×104和1.024×105,浓度分别为0.935和2.010 mg/ml,两株单抗均能特异性识别新孢子虫,与弓形虫无交叉反应。结论制备的抗犬新孢子虫MIC6单克隆抗体特异性较强,为进一步研究新孢子虫MIC6的生物学功能及建立特异敏感的新孢子虫检测方法奠定了基础。

Objective To prepare and identify the monoclonal antibody（Mc Ab） against MIC6 of Neospora caninum.Methods BALB / c mice were immunized with recombinant protein p GEX-MIC6,of which the spleen cells were collected and fused with mouse myeloma SP2 / 0 cells. The obtained hybridoma cells were screened and injected i.p. to BALB / c mice,of which the ascites was collected and indentified for subclass of Mc Ab. The ascites was purified by caprylic acid-ammonium sulfate method combined with protein affinity chromatography,and the obtained Mc Ab was analyzed for relative molecular mass by SDS-PAGE,for titer and specificity by indirect ELISA,and for concentration by BCA assay kit.Results Two hybridoma cell strains stably secreting Mc Abs against MIC6 of N. caninum,1A1 and 1H11,were obtained,by which the secreted Mc Abs belonged to subclasses Ig G1 and Ig G2 b respectively. Heavy and light chains of Mc Ab,with relative molecular masses of about 50 000 and about 25 000 respectively,were observed on the SDS-PAGE profile of ascites,indicating a satisfactory purification effect. The titers of Mc Abs 1A1 and 1H11 in ascites were 5. 12 × 10^4 and1. 024 × 10^5,while the concentrations were 0. 935 and 2. 010 mg / ml,respectively. Both the Mc Abs recognized N. caninum specifically while showed no cross reaction with T. gondii. Conclusion The prepared Mc Ab against MIC6 of N. caninum showed high specificity,which laid a foundation of further study on biological function and development of specific and sensitive methods for diagnosis of neosporosis.