重组人血小板CD36基因真核表达载体的构建及蛋白表达  

Construction of eukaryotic expression vector of recombinant human platelet CD36 gene and protein expression

作　　者：付丽辉[1] 张杰 孙春昀 陈麟凤[1] 冯倩[1] 罗圆圆[1] 张晓娟[1] 王可[1] 于洋[1] 汪德清[1] 

机构地区：[1]解放军总医院输血科,解放军临床输血中心,北京100853 [2]北京义翘神州生物技术有限公司

出　　处：《中国输血杂志》2012年第6期557-560,共4页Chinese Journal of Blood Transfusion

摘　　要：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 construct eukaryotic expression vector of recombinant human platelet CD36 gene , and to pu- rified the functional protein of extracellular amino acid residues 30 to 439 segments. Methods The total of RNA was extrac- ted from human liver tissue and the eDNA encoding human platelet CD36 antigen extracellular region （Gly30 ＋ Asn439） residues amplified by RT-PCtL The cDNA was cloned into the prokaryotic expression vector pMD18 and the recombinant vec- tor was transformed into E. eoli DH5α. We screened positive recombinant pMD18-CD36 plasmid. After sequencing,the gene inserted into the transient eukaryotic expression vector pTE2, constructed the pTE2-s-CD36-10 his transient eukaryotic expres- sion vector. Then the recombinant CD36 Gly30 - Asn439 expressed by HEK-293 ceils and was purified with Ni2 ＋ 2NTA chromatography. Results 1.4 kb eDNA was amplified by RT-PCR,sequence analysis of the results was exactly the same as NM_001001547. 2 in Genebank. Plasmid transfected HEK-293 eells, SDS-PAGE confirmed that eells expressed the human CD36 antigen extraeellular protein fragments. Conclusion The CD36 Gly30 - Ash439 can be highly expressed by human embryonic kidney cells（HEK293）. The purified protein should be pave the way for future study.

关 键 词：CD36抗原 血小板 真核载体 蛋白表达 

分 类 号：R457[医药卫生—治疗学] R331.143[医药卫生—临床医学]