title: [Construction of eukaryotic expression vector of muCD40-IgG1Fc fusion protein and its expression in CHO cells].
abstract: OBJECTIVE
  To construct an eukaryotic expression vector of human muCD40Ig fusion protein,  and to express it stably in Chinese hamster ovary (CHO) cells for obtaining muCD40Ig fusion protein and founding an experimental basis for investigating the soluble muCD40 molecule in vivo.
  
  
  METHODS
  Extracellular domain of human muCD40 gene was amplified by RT-PCR from L929/muCD40-transfected cells,  and the genes encoding the constant regions of human IgG1 were cloned from human splenocytes. The genes were inserted into eukaryotic expression vector pIRES2-EGFP,  respectively. The recombinant vector was transfected into CHO cells by Superfectin. The transfected cells stably secreting muCD40Ig fusion protein was selected with G418 and subcloned. The serum-free culture supernatant of the selected positive clone was subjected to Western blotting  and RT-PCR to confirm the expression of the fusion gene. The affinity of muCD40Ig and L929/CD40L was analyzed by flow cytometry (FCM).
  
  
  RESULTS
  The eukaryotic expression vector pIRES2-EGFP/muCD40Ig was constructed successfully. PCR and Western blotting showed that the transfected CHO cell strain was able to secret muCD40Ig fusion protein stably. FCM demonstrated a good affinity between muCD40Ig and L929/CD40L.
  
  
  CONCLUSIONS
  A transfected CHO cell strain stably expressing muCD40Ig fusion protein has been obtained,  and the muCD40Ig fusion protein can bind to CD40L.
authors: Biotechnology Research Institute, Soochow University, Suzhou 215007, China. eplug1986@126.com
authors: Jian
authors: J
authors: Xu
authors: Qiu-xia
authors: QX
authors: Qu
authors: Yi-bei
authors: YB
authors: Zhu
authors: Xue-guang
authors: XG
authors: Zhang
mesh_terms: D000818:Animals; D023201:CD40 Ligand / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: C499839:CD40Ig protein, recombinant; D011993:Recombinant Fusion Proteins; D023201:CD40 Ligand