| 摘要: |
| 【目的】制备小鼠抗Luman-N端融合蛋白单克隆抗体。【方法】将GST-Luman-N端载体转化大肠杆菌BL21，用IPTG进行诱导表达，并进行SDS-PAGE电泳，用电洗脱的方法纯化融合蛋白；以纯化的融合蛋白为抗原免疫BALB/C小鼠，取免疫小鼠的脾细胞与SP2/0骨髓瘤细胞进行细胞融合，利用间接ELISA方法筛选阳性孔，采用有限稀释法进行亚克隆，筛选具有抗体分泌活性的单克隆细胞株；应用Western Blot、间接ELISA等方法进行克隆株稳定性、抗体特性的鉴定。【结果】获得1株能稳定分泌抗Luman-N端融合蛋白特异性单克隆抗体的杂交瘤细胞株(2C8)，其腹水ELISA单抗效价为1∶1×107。亚型鉴定和亲和力常数分析表明，2C8产生的单克隆抗体亚型为 IgG2b型，其亲和力常数约为1×107。Western Blot分析证明，该株单抗能与原核表达的Luman-N端融合蛋白特异性结合，与GST-LRF-N端融合蛋白没有特异性结合。染色体分析结果表明，2C8细胞株染色体数目为(53±2)对。【结论】获得了抗Luman-N端融合蛋白的抗体，该抗体具有良好的特异性和结合活性，可用于对Luman-N端蛋白的进一步研究。 |
| 关键词: Luman-N端融合蛋白 间接ELISA 单克隆抗体 |
| DOI： |
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| 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-N-term fusion |
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| Abstract: |
| 【Objective】This study aimed to develop and characterize monoclonal antibodies against Luman-N-term.【Method】E.coli with Luman-N-term fusion gene was induced by IPTG,and identified by SDS-PAGE.The expressed fusion protein was purified by electrical elution.The purified Luman-N-term fusion protein was used to immunize BALB/C mice.The spleen cells and SP2/0 myeloma cells of immunized mice were fused using PEG(4 000),and indirectly ELISA method was used to select positive clones,which were screened by limiting dilution method.【Result】One hybridoma cell (2C8) that can stably secrete anti-Luman-N-term protein monoclonal antibodies were obtained.The monoclonal antibodies were identified:the valence of antibody resist Luman-N-term protein was 1×107 in abdominal dropsy,the subtype of monoclonal antibody was IgG2b,and affinity constant of monoclonal antibody from 2C8 was 1×107.Western Blot indicated that this strain McAb presented high specificity to Luman-N-term protein expressed in the prokaryotic cells and low specificity to GST-LRF-N-term protein.The results of chromosome analysis show that the chromosome number of 2C8 was (53±2).【Conclusion】It is proved that this monoclonal antibody possesses well specificity and associativity,and can be used for the further study on function of Luman. |
| Key words: Luman-N-term fusion protein indirect ELISA monoclonal antibody(McAb) |
