| 摘要: |
| 【目的】制备抗Zhangfei蛋白的单克隆抗体，并鉴定其特性。【方法】用Zhangfei融合蛋白免疫 Balb/c 小鼠，取免疫小鼠脾细胞与SP2/0融合，筛选出阳性克隆，应用有限稀释法筛选抗Zhangfei蛋白的单克隆杂交瘤株；采用腹水诱生法制备单抗腹水，用饱和硫酸铵法纯化腹水抗体；采用间接ELISA、单克隆抗体亚型检测试剂盒、免疫印记技术、曲线拟合方案等分别鉴定单抗效价、亚型、特异性和亲和力等特性。【结果】免疫小鼠的血清抗体效价达1∶5 000以上，细胞融合克隆率为98.96%，阳性克隆率为30.18%，筛选出2株抗Zhangfei蛋白的单克隆杂交瘤细胞，其培养上清效价均达到1∶800，腹水单抗效价分别为1∶105和1∶2×105，亲和常数分别为2.5×106和2.3×106。制备的抗Zhangfei蛋白单克隆抗体的亚型均为IgG1类型。【结论】制备的抗Zhangfei蛋白单克隆抗体具有较高的效价和较强的亲和力，可用于进一步研究Zhangfei蛋白在机体中的生理作用。 |
| 关键词: Zhangfei蛋白 抗体效价 细胞融合 单克隆抗体 特异性 |
| DOI： |
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| 基金项目:国家自然科学基金项目(39970544)；西北农林科技大学青年骨干教师支持计划项目(01140305) |
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| Preparation and identification of monoclonal antibodies against Zhangfei fusion protein |
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ZHANG Yang, TAO Mingliang, LU Baotong,et al
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Government of Muyi Town of Animal Husbandry,Chencang,Baoji,et al
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| Abstract: |
| 【Objective】We prepared the MoAb against Zhangfei Protein and the characteristics of MoAb were identified.【Method】Using Zhangfei Fusion Protein to immunize the Balb/c mouse,we obtained the spleen cells of Balb/c mouse,then combined them with the SP2/0 in fusogenic agent.Indirect ELISA was used to detect masc-clone,limiting dilution assay was used to sieve monoclonal cell line,and induced ascites method was used to prepare the MoAb of Zhangfei Protein.Then saturated ammonium sulfate was used to depurate the antibody and ELISA,Kit,Indirect ELISA and Curve fitting were used to identify the individualities of MoAb.【Result】The valence of antibody in Balb/c mouse reached up to 1∶5×103.Cloning efficiency of cell fusion and masc-cloning efficiency were 98.96% and 30.18%,repectively.We obtained two positive hybridoma cells.The valences of antibody resisting Zhangfei protein were 1∶800 in cell cuiture fluid and 1∶105 or 1∶2×105 in abdominal dropsy.Affinity constants of MoAb from two positive hybridoma cells were 2.5×106 and 2.3×106.The subtypes of the monoclonal antibodies were IgG1.【Conclution】We have prepared the MoAb against Zhangfei protein.The antibodies have high titer and hydro-affinity.It helps future study on Zhangfei protein. |
| Key words: Zhangfei protein antibody titer cell fusion monoclonal antibody individualit |
