鼠抗人血小板CD36分子单克隆抗体的制备及活性分析

Preparation and Activity Analysis of Mouse Anti Human Platelet CD36 Monoclonal Antibody

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。

This study was purposed to prepare eukaryotic expression vector of recombinant human platelet CD36 gene. The total RNA was extracted from human liver tissue and the cDNA encoding human platelet CD36 antigen extracellular region （ Gly30 - Asn439） was amplified by RT-PCR. The cDNA was cloned into the prokaryotic expression vector pMD18 and the recombinant vector was transformed into E. coli DH5α. The positive recombinant pMD18-CD36 plasmid was screened. After sequencing, this combinant vector was inserted into the transient eukaryotic expression vector pTE2, the pTE2-s-CD36-10 His transient eukaryotic expression vector was constructed. The recombinant CD36 Gly30 - Ash439 expressed by HEK-293 cells was purifid with Ni2 ＋ 2NTA chromatography. The results showed that 1.4 kb cDNA was amplified by RT-PCR, sequencing of the cDNA indicated the sequance was exactly the same to that in Genbank NM_001001547.2. The HEK293 cells with the plasmid were transfected, and SDS-PAGE confirmed that the transfect HEK293 cells expressed the human CD36 antigen extracellular protein fragments. Western-blot showed that the monoclonal antibody could recognize the recombinant CD36 with the sensitivity of 8 ng. It is concluded that the CD36 Gly30-Asn439 can be highly expressed by human embryonic kidney cells （ HEK293 ）, and the monoclonal antibody with biological activity has been obtained, which provide the basis for further study on platelet transfusion refractoriness.