title: [Construction and expression of recombinant human serum albumin-EPO fusion protein].
abstract: OBJECTIVE To construct the recombinant plasmid pCI-HLE encoding human serum album-EPO (HSA-EPO) fusion protein and to express it in CHO cell.
  
  
  METHODS
  The cDNA encoding human serum album and EPO were amplified by PCR, and then spliced with the synsitic DNA fragment encoding GS (GGGGS), by overlap PCR extension to form LEPO. After BamH I digestion, the HSA and LEPO was ligated to generate the fusion HSA-EPO gene and was then cloned into the expression vector pCI-neo to generate the recombinant plasmid pCI-HLE. The plasmid pCI-HLE was transfected into CHO cell by liposome protocol. Then, the recombinant cells were screened by G418 and identified by PCR and Western blot. Expression of fusion protein was evaluated by Enzyme Linked Immunosorbent Assay (ELISA).
  
  
  RESULTS
  Restrictive enzymes digestion and DNA sequencing revealed that HSA-EPO fusion gene was cloned into expression vector pCI-neo successfully. PCR and Western blot analysis confirmed that the fusion gene was integrated in the genome of CHO cells and expressed successfully. The HSA-EPO production varied from 86 Iu/(mL x 10(6) x 72 h) to 637 IU/(mLx 10(6) x 72 h).
  
  
  CONCLUSIONS
  The results confirmed that HSA-EPO fusion gene can be expressed in the CHO cells, with EPO immunogenicity, which could serve as foundation for the development of long-lasting recombinant HSA-EPO protein.
authors.affiliation: Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu 610041, China.
authors.forename: Ying-Chun
authors.initials: YC
authors.lastname: Huang
authors.forename: Xing-Hua
authors.initials: XH
authors.lastname: Gou
authors.forename: Lei
authors.initials: L
authors.lastname: Han
authors.forename: De-Hua
authors.initials: DH
authors.lastname: Li
authors.forename: Lan-Ying
authors.initials: LY
authors.lastname: Zhao
authors.forename: Qia-Qing
authors.initials: QQ
authors.lastname: Wu
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004921:Erythropoietin / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012709:Serum Albumin / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D004921:Erythropoietin