title: [Construction and panning of scFv phage display library against recombinant interleukin 4 receptor].
abstract: Objective To construct the recombinant human interleukin 4 receptor (rhIL-4R) single-chain Fv (scFv) antibody library by phage display technique to obtain the anti-IL-4R scFv clones selected from the library. Methods Total RNA was extracted from splenocytes of the BALB/c mice immunized with rhIL-4R. Complementary DNA fragments of variable heavy (VH) and variable light (VL) chains of the antibodies were prepared by reverse transcription PCR and assembled into scFv by splice overlap extension PCR (SOE-PCR). Both scFv and the pCANTAB5E vector were respectively double-digested with restriction endonuclease Sfi I and Not I, connected with T4 ligase, and then transformed into the competent cells E.coli TG1; it was cultured in medium to obtain the phage scFv antibody library; after three rounds of enrichment and panning, the specific antigen scFv with high affinity was selected for the sequencing. Results After three rounds of panning, we obtained a diversity of approximately 2×10(8) anti-rhIL-4R scFv antibody library. Sequencing analysis of one positive clone showed that the anti-rhIL-4R scFv was 741 bp and coded 247 amino acids. The analysis of VBASE2 database indicated that VH and VL gene sequences of anti-rhIL-4R protein all had three complementarity determining regions and four backbone areas.Conclusion The anti-rhIL-4R scFv was obtained from the scFv antibody library.
authors.affiliation: Guiyang College of Traditional Chinese Medicine, Guiyang 550002, China.
authors.forename: Guangyong
authors.initials: G
authors.lastname: Yang
authors.affiliation: Guiyang College of Traditional Chinese Medicine, Guiyang 550002, China.
authors.forename: Haitao
authors.initials: H
authors.lastname: Guo
authors.affiliation: Guiyang College of Traditional Chinese Medicine, Guiyang 550002, China.
authors.forename: Ximing
authors.initials: X
authors.lastname: Liu
authors.affiliation: Guiyang College of Traditional Chinese Medicine, Guiyang 550002, China. *Corresponding author, E-mail: heguangzhi7711@ sina.com.
authors.forename: Guangzhi
authors.initials: G
authors.lastname: He
authors.affiliation: Guiyang College of Traditional Chinese Medicine, Guiyang 550002, China.
authors.forename: Weiyi
authors.initials: W
authors.lastname: Tian
authors.affiliation: Guiyang College of Traditional Chinese Medicine, Guiyang 550002, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Cai
authors.affiliation: Guiyang College of Traditional Chinese Medicine, Guiyang 550002, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.affiliation: Guiyang College of Traditional Chinese Medicine, Guiyang 550002, China.
authors.forename: Wenjia
authors.initials: W
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D008807:Mice, Inbred BALB C; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D019948:Receptors, Interleukin-4 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D019151:Peptide Library; D019948:Receptors, Interleukin-4; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies