论文部分内容阅读
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 monoclonal antibody against MIC8 of N. caninum and to identify it. Methods BALB / c mice were immunized with the recombinant protein pGEX-MIC6 as antigen. The spleen cells were collected and fused with mouse myeloma cells SP2 / 0. The hybridoma cells were screened and then injected intraperitoneally with BALB / c Rat, preparation of ascites, identification of subclasses of mAbs. The ascites were purified by caprylic acid-ammonium sulfate method with protein affinity chromatography. The relative molecular mass of the antibody was analyzed by SDS-PAGE. The antibody titer and specificity were determined by indirect ELISA. The concentration of antibody was determined by BCA protein kit. As a result, two hybridoma cell lines 1A1 and 1H11 that can stably secrete the monoclonal antibody against Neisseria gonorrhoeae MIC6 protein were obtained. The secreted antibodies belong to the subclasses Ig G1 and Ig G2b, respectively. Purified ascites by SDS-PAGE analysis, respectively, the molecular weight of about 50,000 of the heavy chain and light chain of 25 000, the purification effect is better; 1A1 and 1H11 ascites titers were 5.12 × 104 and 1.024 × 105, respectively, at concentrations of 0.935 and 2.010 mg / ml respectively. The two McAbs specifically recognized Neospora and did not cross-react with Toxoplasma gondii. Conclusion The monoclonal antibody against MIC8 against N. caninum is highly specific, which lays the foundation for the further study on the biological function of MIC6 and the establishment of a specific and sensitive method for detection of neosporosis.