title: [Construction and expression of eukaryotic expression vector for intact IgG against human keratin].
abstract: OBJECTIVE
  To construct an eukaryotic expression vector containing the gene of anti-keratin antibody and express it in CHO(dhfr(-)) cells.
  
  
  METHODS
  Both the V(L) and V(H) genes of an anti-keratin Fab from a phage display library were amplified by PCR. Using PCR product as the template, the V(kappa) and V(H) genes with the leader sequences (named L(kappa) and L(H) respectively) were amplified by overlapping PCR. After digested with endonuclease Xba I/BamH I and Xho I/Hind III, L(kappa) and L(H) genes were inserted into pWD digested with Xba I/BamH I and Xho I/Hind III, respectively to construct pWDkappaH. After PCR and sequencing, the expression plasmid pWDkappaH was transfected into CHO (dhfr(-)) cells. The culture supernatant of the transfected cells was collected and assayed for IgG activity.
  
  
  RESULTS
  The eukaryotic expression vector pWDkappaH was constructed successfully and expressed in CHO(dhfr(-)) cells. The expression of intact IgG against keratin was identified by ELISA and RT-PCR.
  
  
  CONCLUSIONS
  The successful expression of intact IgG against keratin lays the foundation for its clinical application.
authors.affiliation: Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Ru-shan
authors.initials: RS
authors.lastname: Xia
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
authors.forename: Jian-yong
authors.initials: JY
authors.lastname: Fan
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Yu-feng
authors.initials: YF
authors.lastname: Liu
mesh_terms: D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D005057:Eukaryotic Cells / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000378:metabolism; D007074:Immunoglobulin G / Q000235:genetics* / Q000378:metabolism; D007633:Keratins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D019151:Peptide Library; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D019151:Peptide Library; D012333:RNA, Messenger; D007633:Keratins