title: [Cloning and expression of human sPD-1-Fc in Cos-7 cells].
abstract: OBJECTIVE
  To investigate human soluble PD-1 (sPD-1) biological activity sPD-1 gene be cloned and expressed in eucaryote cells.
  
  
  METHODS
  sPD-1 gene was amplified from human PBMC mRNA by RT-PCR and cloned into eucaryotic expression vector pSG5-Fc. And the positive recombinant plasmid pSG5-Fc -sPD-1 was screened by enzyme digestion and sequencing.The correct sequencing of the recombinant plasmid pSG5-Fc-sPD-1 was transfected into COS-7 cells. The expressed recombinant protein in the supernatant was concentrated with protein A-agarose and analyzed by Western blot. The binding activity to PD-L1 which was expressed with prokaryotic cells was detected with Co-IP.
  
  
  RESULTS
  The human sPD-1 fragment was obtained through RT-PCR. The plasmid pSG5-Fc-sPD-1 was constructed by double enzyme digestion and ligation of vector pSG5-Fc and fragment sPD-1. Sequenced sPD-1 gene was coincident with the theoretical sequence. sPD-1-Fc fusion protein in the supernatant was expressed in COS-7 cells and identified by Western blot. The activity of recombinant fusion protein sPD-1-Fc bound to PD-L1 had been detected with Co-IP.
  
  
  CONCLUSIONS
  The human sPD-1 has been cloned and expressed in eucaryote cells successfully. The sPD-1-Fc fusion protein can be effective in binding to PD-L1, which can be used for further research in the function and clinical application of sPD-1.
authors.affiliation: Institute of Molecular Biology, Three Gorges University, Yichang 443002, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.forename: Chao-qi
authors.initials: CQ
authors.lastname: Liu
authors.forename: Jian-zhi
authors.initials: JZ
authors.lastname: Wang
authors.forename: Ji-jing
authors.initials: JJ
authors.lastname: Shi
authors.forename: Bai-rui
authors.initials: BR
authors.lastname: Lv
authors.forename: Xiao-lin
authors.initials: XL
authors.lastname: Qin
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D002999:Clone Cells; D003001:Cloning, Molecular / Q000379:methods*; D005057:Eukaryotic Cells / Q000166:cytology / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D061026:Programmed Cell Death 1 Receptor; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D015703:Antigens, CD; D051017:Apoptosis Regulatory Proteins; C105992:PDCD1 protein, human; D061026:Programmed Cell Death 1 Receptor; D011993:Recombinant Fusion Proteins