鼠抗人血小板CD36分子单克隆抗体的制备及活性分析

发布时间：2019-01-13 09:17

【摘要】：本研究旨在制备具有功能活性的重组人血小板表面CD36抗原的鼠抗人单克隆抗体。提取人肝细胞总RNA,经RT-PCR扩增编码人血小板CD36抗原胞外区(Gly30-Asn439)氨基酸残基cDNA,构建于原核表达载体pMD18并转化大肠杆菌DH5α,筛选获得阳性重组子pMD18-CD36,提取质粒。经序列测定后,将该基因插入到真核细胞瞬时表达载体pTE2上,构建成为pTE2-s-CD36-10 His真核瞬时表达载体。采用lipofectamine 2000转染法,将重组质粒转染至HEK293细胞,表达产物经Ni2+2NTA柱层析纯化。以制备的重组CD36蛋白免疫BALB/c小鼠后,取脾与小鼠骨髓瘤细胞融合,筛选出阳性克隆,行Western blot检测抗体结合活性。结果显示:RT-PCR扩增获得了1.4 kb的片段。经测序,该序列分析结果与GenBank中的NM_001001547.2完全一致。SDS-PAGE证实转染的HEK293细胞表达了人CD36抗原胞外区蛋白片段。鼠单克隆抗体在Western blot中可以识别重组CD36蛋白,灵敏度达到8 ng。结论:成功制备了抗人血小板CD36单克隆抗体,为临床筛选CD36阴性患者及献血员、深入研究人血小板CD36表面抗原对血小板输注无效的影响提供了实验基础。
[Abstract]:The aim of this study was to prepare murine anti-human monoclonal antibodies against CD36 antigen on the surface of recombinant human platelets with functional activity. The total RNA, extracted from human hepatocytes was amplified by RT-PCR and encoded by the amino acid cDNA, encoding the extracellular domain of human platelet CD36 antigen (Gly30-Asn439). The cDNA, was constructed into the prokaryotic expression vector pMD18 and transformed into Escherichia coli DH5 伪, and the pMD18-CD36, extraction plasmid of the positive recombinant plasmid was obtained. After sequencing, the gene was inserted into eukaryotic cell transient expression vector pTE2 and constructed as pTE2-s-CD36-10 His eukaryotic transient expression vector. The recombinant plasmid was transfected into HEK293 cells by lipofectamine 2000 transfection, and the expressed product was purified by Ni2 2NTA column chromatography. After immunizing BALB/c mice with recombinant CD36 protein, the spleen was fused with mouse myeloma cells, and the positive clones were screened out. The antibody binding activity was detected by Western blot. The results showed that 1. 4 kb fragment was obtained by RT-PCR amplification. The sequence analysis was consistent with the NM_001001547.2 in GenBank. SDS-PAGE confirmed that the transfected HEK293 cells expressed the extracellular domain protein fragment of human CD36 antigen. Mouse monoclonal antibody can recognize recombinant CD36 protein in Western blot, and its sensitivity is up to 8 ng.. Conclusion: monoclonal antibodies against human platelet CD36 were successfully prepared, which provided experimental basis for clinical screening of CD36 negative patients and blood donors and further study of the effect of human platelet CD36 surface antigen on platelet transfusion.
【作者单位】：解放军总医院输血科解放军临床输血中心;北京义翘神州生物技术有限公司;
【基金】：国家卫生部课题,编号201002005
【分类号】：R331.143

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