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目的制备具有功能活性的重组人血小板表面CD36抗原的纯化表达蛋白胞外区30～439氨基酸残基段。方法提取人肝细胞组织总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(invitrogen)转染法,将重组质粒转染HEK-293细胞,表达产物经Ni2+2NTA柱层析纯化。结果 RT-PCR扩增获得了1.4 kb的片段。invitrogen测序,该序列分析结果与Genebank中的NM_001001547.2完全一致。SDS-PAGE证实转染的HEK-293细胞表达了人CD36抗原胞外区蛋白片段。结论利用构建的真核载体pTE2-s-CD36转染人胚肾细胞(HEK293)可高效表达CD36 Gly30～Asn439,得到纯化蛋白,为人血小板CD36表面抗原对血小板输注无效影响的深入研究奠定基础。
Objective To prepare the purified extracellular domain of 30 ~ 439 amino acid residues of CD36 antigen on recombinant human platelet with functional activity. Methods The total RNA was extracted from human hepatocytes. The cDNA encoding the amino acid residues of the extracellular domain (Gly30 ~ Asn439) of human CD36 antigen was amplified by RT-PCR and was then cloned into prokaryotic expression vector pMD18 and transformed into E. coli DH5α. The positive recombinant pMD18-CD36, plasmid was extracted. After sequencing, the gene was inserted into the transient expression vector pTE2 of eukaryotic cell to construct the eukaryotic transient expression vector pTE2-s-CD36-10 his. The recombinant plasmid was transfected into HEK-293 cells by lipofectamine 2000 (invitrogen), and the expressed product was purified by Ni2 + 2NTA column chromatography. Results A 1.4 kb fragment was obtained by RT-PCR. Invitrogen sequencing, the sequence analysis results and Genebank in NM_001001547.2 exactly the same. SDS-PAGE confirmed that the transfected HEK-293 cells expressed the extracellular domain of human CD36 antigen. Conclusion The recombinant eukaryotic vector pTE2-s-CD36 was transfected into human embryonic kidney cells (HEK293) to express CD36 Gly30 ~ Asn439 efficiently, which provided a basis for the further study on the effect of platelet CD36 surface antigen on the ineffectiveness of platelet transfusion.