title: Characterization of a single-chain variable fragment specific to Cronobacter spp. from hybridoma based on outer membrane protein A.
abstract: Monoclonal antibody and polyclonal antibody specific to Cronobacter spp. had been reported in previous studies. However, the preparation of single-chain variable fragment (scFv) was faster and convenient. Hence, the aim of this study was to construct a scFv using outer membrane protein A (OmpA) of C. sakazakii as antigen. The protein sequences of OmpA of Cronobacter spp. were analyzed first. The results showed protein OmpA with length of 347 amino acids was conserved in Cronobacter genus (94.83%-100% of protein identity) and was greater than that observed for the other genera tested (8.28-91.64% of protein identity). Then, purified protein OmpA expressed in E. coli was used to prepare hybridoma and to construct scFv further. The scFv was named scFvH81 and analyzed by bioinformatics. The model of scFvH81 built by homologous modeling had a good quality (residues in disallowed regions: 3%) and showed that scFvH81 had a standard pocket-like site. Purified scFvH81 was prepared by denaturation and renaturation of inclusion body and it showed a good specificity and its affinity of Ka=2.39×10(6)M(-1). Therefore, it could be used in the detection and the pathogenesis study of Cronobacter spp.
authors: College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China.
authors: Qiming
authors: Q
authors: Chen
authors: College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China.
authors: Tingting
authors: T
authors: Tao
authors: College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China.
authors: Xiaomei
authors: X
authors: Bie
authors: College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China.
authors: Fengxia
authors: F
authors: Lu
authors: College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China; Department of Public Health, Xuzhou Medical College, 209 Tongshan Road, Xuzhou, Jiangsu 221004, People's Republic of China.
authors: Yuanhong
authors: Y
authors: Li
authors: College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China. Electronic address: fmb@njau.edu.cn.
authors: Zhaoxin
authors: Z
authors: Lu
mesh_terms: D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000737:chemistry / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D003001:Cloning, Molecular; D019295:Computational Biology; D059126:Cronobacter / Q000276:immunology*; D006801:Humans; D006825:Hybridomas; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
chemical_list: D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C078167:OMPA outer membrane proteins
keywords: Bioinformatics analysis; Cronobacter spp.; OmpA; ScFv