title: [Preparation and activity analysis of mouse anti human platelet CD36 monoclonal antibody].
abstract: 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-Asn439 expressed by HEK-293 cells was purified with Ni(2+) 2NTA chromatography. The results showed that 1.4 kb cDNA was amplified by RT-PCR, sequencing of the cDNA indicated the sequence 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.
authors.affiliation: Department of Blood Transfusion, Chinese PLA General Hospital, Beijing, China.
authors.forename: Lin-Feng
authors.initials: LF
authors.lastname: Chen
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Ja-Hui
authors.initials: JH
authors.lastname: Yang
authors.forename: Yuan-Yuan
authors.initials: YY
authors.lastname: Luo
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zhuang
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.forename: Qian
authors.initials: Q
authors.lastname: Feng
authors.forename: De-Qing
authors.initials: DQ
authors.lastname: Wang
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000302:isolation & purification; D001792:Blood Platelets / Q000276:immunology*; D015153:Blotting, Western; D018955:CD36 Antigens / Q000235:genetics* / Q000276:immunology; D018076:DNA, Complementary; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D010957:Plasmids; D017713:Platelet Transfusion; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000911:Antibodies, Monoclonal; D018955:CD36 Antigens; D018076:DNA, Complementary