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

Objective To construct eukaryotic expression vector of recombinant human platelet CD36 gene,and to purified the functional protein of extracellular amino acid residues 30 to 439 segments.Methods The total of RNA was extracted from human liver tissue and the cDNA encoding human platelet CD36 antigen extracellular region(Gly30 ~ Asn439) residues amplified by RT-PCR.The cDNA was cloned into the prokaryotic expression vector pMD18 and the recombinant vector was transformed into E.coli 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 expression vector.Then the recombinant CD36 Gly30 ~ Asn439 expressed by HEK-293 cells and was purified with Ni2 +2NTA chromatography.Results 1.4 kb cDNA was amplified by RT-PCR,sequence analysis of the results was exactly the same as NM_001001547.2 in Genebank.Plasmid transfected HEK-293 cells,SDS-PAGE confirmed that cells expressed the human CD36 antigen extracellular protein fragments.Conclusion The CD36 Gly30 ~ Asn439 can be highly expressed by human embryonic kidney cells(HEK293).The purified protein should be pave the way for future study.