title: Antisera preparation and epitope mapping of a recombinant protein comprising three peptide fragments of the cystic fibrosis transmembrane conductance regulator.
abstract: Antibodies targeting a single epitope of the cystic fibrosis transmembrane conductance regulator (CFTR) have been reported to influence the validity of immunological analyses; however, autoimmune mechanisms associated with CFTR epitopes are not well understood. In this study, antiserum raised against a multi-epitope recombinant protein composed of three peptide fragments of CFTR (r-CFTR-3P) was prepared and B cell epitope mapping of the protein was carried out using biosynthetic peptides. The r-CFTR-3P gene was cloned into the pSY621 expression plasmid and the protein was expressed in the BL21 strain of Escherichia coli. The rabbit r-CFTR-3P antiserum recognized the native CFTR antigen extracted from human sperm and the GST188 fusion peptides CFTR(25-36), CFTR(103-117), and CFTR(1387-1480) spanning different regions of CFTR. Four novel r-CFTR-3P B cell epitopes were identified: (29)RQRLEL(34), (104)RIIASY(109), (111)PDN(113), and (1447)VKLF(1450) of CFTR. Other proteins from various species shared sequence homology with the identified epitopes based on NCBI BLAST alignment. This study provides new tools for detecting CFTR protein and insight into the characteristics of minimal B cell epitopes of CFTR and associated immunological mechanisms.
authors.affiliation: Unit of Reproductive Physiology, Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang 310013, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Li
authors.affiliation: National Population and Family Planning Key Laboratory of Contraceptive Drugs & Devices, Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China.
authors.forename: Haiping
authors.initials: H
authors.lastname: Tang
authors.affiliation: National Population and Family Planning Key Laboratory of Contraceptive Drugs & Devices, Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China.
authors.forename: Wanxiang
authors.initials: W
authors.lastname: Xu
authors.affiliation: Unit of Reproductive Physiology, Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang 310013, China.
authors.forename: Aijun
authors.initials: A
authors.lastname: Chen
authors.affiliation: Unit of Reproductive Physiology, Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang 310013, China.
authors.forename: Qixian
authors.initials: Q
authors.lastname: Shi
authors.affiliation: National Population and Family Planning Key Laboratory of Contraceptive Drugs & Devices, Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China.
authors.forename: Zhida
authors.initials: Z
authors.lastname: Sun
authors.affiliation: National Population and Family Planning Key Laboratory of Contraceptive Drugs & Devices, Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China.
authors.forename: Liyan
authors.initials: L
authors.lastname: Wang
authors.affiliation: Unit of Reproductive Physiology, Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang 310013, China. Electronic address: niya99@126.com.
authors.forename: Ya
authors.initials: Y
authors.lastname: Ni
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D018604:Epitope Mapping; D018985:Epitopes, B-Lymphocyte / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D013094:Spermatozoa / Q000737:chemistry
chemical_list: D000906:Antibodies; D018985:Epitopes, B-Lymphocyte; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
keywords: Antibody; B cell epitope; CFTR; Epitope mapping; Polyclonal antiserum; Protein expression