title: [Stable expression of human anti-IL-33 scFv-IgG1Fc fusion protein in CHO k1 cells].
abstract: OBJECTIVE
  To construct two different eukaryotic expression vectors of human anti-interleukin 33 (IL-33) single-chain antibody fragment (scFv-Fc) to transfect Chinese hamster ovary (CHO) k1 cells and select the stably and high-level expressed cell lines to improve the expression level of the fusion protein.
  
  
  METHODS
  The previously constructed recombinant plasmid pcDNA3.1/SP-scFv-Fc was digested to obtain SP-scFv-Fc fragments, and the fragments were inserted into the plasmid PMH3(EN) to construct recombinant plasmid PMH3(EN)/SP-scFv-Fc. The plasmids PMH3(EN)/SP-scFv-Fc and pcDNA3.1/SP-scFv-Fc were separately transfected into CHO k1 cells. The transcription and translation level of the SP-scFv-Fc were detected by reverse transcription PCR (RT-PCR) and Western blotting, respectively. The stably and high-level expressed cell lines were screened by Dot blotting. The expression level and binding activity of the expressed scFv-Fc were measured by ELISA.
  
  
  RESULTS
  The recombinant plasmid PMH3(EN)/SP-scFv-Fc was successfully constructed and the size of the inserted SP-scFv-Fc was about 1560 bp. The RT-PCR results showed that the SP-scFv-Fc was successfully transfected into CHO k1 cells. The scFv-Fc proteins could be secreted into the cultural supernatant and specifically bind to human IL-33 and anti human IgG1 Fc antibody. The expression level of scFv-Fc in plasmid PMH3(EN) was higher than that in plasmid pcDNA3.1. After four rounds of screening, the stably and high-level expressed cell strains were obtained. The expression level of the scFv-Fc was about 10 mg/L. The competitive ELISA results showed that the expressed scFv-Fc fusion proteins could inhibit the binding of IL-33 to ST2.
  
  
  CONCLUSIONS
  The anti-IL-33 scFv-Fc proteins were highly expressed in CHO k1 cells.
authors.affiliation: School of Basic Medic Science, Sichuan Medical University, Luzhou 646000, China.
authors.forename: Yingchun
authors.initials: Y
authors.lastname: Ye
authors.affiliation: School of Basic Medic Science, Sichuan Medical University, Luzhou 646000, China.
authors.forename: Siji
authors.initials: S
authors.lastname: Nian
authors.affiliation: School of Basic Medic Science, Sichuan Medical University, Luzhou 646000, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Wang
authors.affiliation: School of Basic Medic Science, Sichuan Medical University, Luzhou 646000, China.
authors.forename: Tong
authors.initials: T
authors.lastname: Wu
authors.affiliation: School of Basic Medic Science, Sichuan Medical University, Luzhou 646000, China.
authors.forename: Wenfeng
authors.initials: W
authors.lastname: Xu
authors.affiliation: School of Basic Medic Science, Sichuan Medical University, Luzhou 646000, China. *Corresponding author, E-mail: yuanqing_ok@hotmail.com.
authors.forename: Qing
authors.initials: Q
authors.lastname: Yuan
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics*; D000067596:Interleukin-33 / Q000276:immunology*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000494:pharmacology; D057127:Single-Chain Antibodies / Q000235:genetics*
chemical_list: D007074:Immunoglobulin G; D000067596:Interleukin-33; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies