| 摘要: |
| 【目的】制备抗Luman募集因子N端（Luman-recruiting factor N terminal,LRF-N）蛋白的单克隆抗体。【方法】PCR扩增LRF-N端的194 bp序列，构建LRF-N重组质粒pGEX-LRF-N，转染BL21(DE3)菌，诱导表达并纯化GST-LRF-N融合蛋白，以其作为抗原免疫6～8周龄雌性BALB/c小鼠，免疫4次，每次间隔10 d，第4次加强免疫3 d后采取小鼠脾脏，分离脾细胞，与SP2/0细胞融合，用ELISA和Western blot进行阳性细胞株的筛选和鉴定，对获得的杂交瘤细胞的细胞核型、分泌稳定性以及所分泌的单克隆抗体进行鉴定。【结果】pGEX-LRF-N在大肠杆菌BL21(DE3)中得到高效表达，GST-LRF-N蛋白分子质量约为33 ku，与预期结果一致。试验共获得3株稳定分泌抗LRF-N抗体的杂交瘤细胞株，分别命名为2AC9、2AG10和3G7。单抗2AC9、2AG10、3G7 能够特异性地识别GST-LRF-N蛋白，亚型鉴定结果显示，其重链分别为IgG2a、IgM和IgG1。【结论】获得的抗LRF N抗体的杂交瘤细胞株2AC9、2AG10、3G7单抗特异性良好，能够稳定分泌抗体，可用于小鼠LRF蛋白生物学功能及活性的进一步研究。 |
| 关键词: Luman募集因子 pEGX-LRF-N GST-LRF N重组蛋白 单克隆抗体 |
| DOI： |
| 分类号: |
| 基金项目:高等学校博士点学科专项科研基金项目(20110204120010) |
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| Preparation and identification of monoclonal antibodies against Luman-recruiting factor N-terminal |
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LI Xiao, LAN Xiangli, XU Xiaobin
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College of Veterinary Medicine,Northwest A&F University
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| Abstract: |
| 【Objective】This study was to develop and characterize monoclonal antibodies against Luman-recruiting factor N terminal(LRF-N).【Method】PCR was used to amplified 194 bp fragment of LRF gene N-terminal and a recombinant plasmid pGEX-LRF-N was constructed.Then,the GST-LRF-N recombinant protein was expressed and purified before being used to immunize the female BALB/c Mus musculus (with age of 6-8 weeks) once every 10 days for a total of 4 times.The spleens were extracted 3 days after the fourth immunization,and splenocytes were fused with SP2/0 cells.The selection and identification of positive samples and the identification of karyotypes,secretion stabilities and the obtained 3 monoclonal antibodies against GST-LRF-N (MAb,named as 2AC9,2AG10,and 3G7,respectively) were finally conducted by indirect ELISA and Western blot assays.【Result】Recombinant plasmid pGEX-LRF-N was expressed efficiently in the Escherichia coliBL21(DE3),and the molecular weight of its expressive GST-LRF-N protein was 33 ku,Mouse positive antiserum could recognize GST-LRF-N protein.The MAbs 2AC9,2AG10,and 3G7 could specifically recognize pGEX-LRF-N,from pGEX-Luman protein,pGEX-Atac2 protein and pGEX-4T-1 protein.The results of isotype analysis showed that MAbs 2AC9，2AG10，and 3G7 could be categorized to IgG2a,IgM,and IgG1 subclasses,respectively.【Conclusion】The obtained MAbs from 2AC9,2AG10，and 3G7 hybridoma cells have good monospecificities and can stably secrete antibodies,thus can be used for further research of LRF protein in mouse tissues. |
| Key words: Luman recruiting factor pGEX-LRF-N GST-LRF-N recombinant protein monoclonal antibody |
