Real-time analysis of the interaction of a multiple-epitope peptide with antibodies against classical swine fever virus using surface plasmon resonance.
The E2 envelope glycoprotein is the major immunodominant protein of classical swine fever virus (CSFV), and can induce neutralizing antibodies and protective immune responses in infected swine. We developed a tandem-repeat multiple-epitope recombinant protein that contains two copies of each of the regions of E2 spanned by residues 693-704, 770-780, and 826-843, coupled by two copies of the region spanned by residues 1446-1460 of the CSFV nonstructural protein NS2-3. The chemically synthesized gene was expressed in Escherichia coli as a fusion with glutathione S-8 (GST), named GST-BT21. After it was purified with Glutathione Sepharose 4B, we used Western blotting to characterize the construct and surface plasmon resonance to analyze its affinity and specific interaction with CSFV-positive serum. Purified GST-BT21 protein displayed excellent immunoreactivity with antiserum against CSFV (Tian et al., 2012), and surface plasmon resonance confirmed the specific affinity between BT21, but not GST, and antibodies in serum from animals infected with CSFV. Surface plasmon resonance is a sensitive and precise method for epitope evaluation, and it can be used to characterize the immunogenicity and functions of recombinant proteins.
Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu 730046, China.
Xiangmin
X
Hou
Hong
H
Tian
Jinyan
J
Wu
Jiang
J
Tao
Yan
Y
Chen
Shuanghui
S
Yin
Keshan
K
Zhang
Youjun
Y
Shang
Xiangtao
X
Liu
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology*; D015153:Blotting, Western; D006691:Classical Swine Fever / Q000276:immunology / Q000821:virology; D006692:Classical Swine Fever Virus / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D003199:Computer Systems; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000378:metabolism; D005814:Genes, Viral / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D055767:Immobilized Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods*; D013499:Surface Properties; D013552:Swine / Q000276:immunology / Q000821:virology
D000914:Antibodies, Viral; D000939:Epitopes; D055767:Immobilized Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins