title: [Construction and identification of a full-length human secretory antibody library in Pichia pastoris].
abstract: OBJECTIVE
  To clone and express functional, recombinant full-length human antibodies in partial N-glycoengineered Pichia pastoris yeast.
  
  
  METHODS
  We constructed double promoter series expressing the constant regions of the light and heavy chain genes using the secretory expression vector pPICZαA; the obtained vector was named pPICZαA-CH;-CL;. Human peripheral blood mononuclear cells (PBMCs) were isolated and RT-PCR was performed using 44 pairs of degenerate primers to generate variable region genes of the heavy and light chains (VH; and VL;). Then, they were inserted into the pPICZαA-CH;-CL; vector. The full-length human antibody library pPICZαA-VH;-CH;-VL;-CL; was transformed into partial N-glycoengineered Pichia pastoris, GS115Y, by electroporation. Sequencing analysis of 20 clones was carried out and the Results were submitted to the IgBLAST-imgt website to determine accuracy and diversity of the antibody library.
  
  
  RESULTS
  The pPICZαA-CH;-CL; vector was sequenced and the expressions of CH; and CL; were determined by immunoblotting. The full-length human antibody library pPICZαA-VH;-CH;-VL;-CL; was constructed with a library capability of 10(5);. The IgBLAST-imgt analysis showed that the antibodies were functional as predicted.
  
  
  CONCLUSIONS
  We successfully constructed a full-length human secretory antibody library in N-glycoengineered Pichia pastoris, which allows the screening of functional antibodies.
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, PLA Academy of Military Medicine, Beijing 100850, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, PLA Academy of Military Medicine, Beijing 100850, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, PLA Academy of Military Medicine, Beijing 100071, China.
authors.forename: Xiaoai
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, PLA Academy of Military Medicine, Beijing 100850, China.
authors.forename: Xiaopan
authors.initials: X
authors.lastname: Yang
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, PLA Academy of Military Medicine, Beijing 100850, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Hua
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, PLA Academy of Military Medicine, Beijing 100850, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Gao
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, PLA Academy of Military Medicine, Beijing 100850, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Fu
authors.affiliation: Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, PLA Academy of Military Medicine, Beijing 100850, China.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Song
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics
chemical_list: D000906:Antibodies