Characterization and epitope mapping of monoclonal antibodies recognizing N-terminus of Rep of porcine circovirus type 2.
Two genotypes of porcine circovirus (PCV) have been described, the non-pathogenic PCV1 and the pathogenic PCV2 in pigs. PCV-ORF1 encodes Rep and Rep' proteins which have identical N-terminal sequence (RepN) within each PCV strain, but RepN has only 88% similarity between PCV1 and PCV2. Purified RepN of PCV2 was used as an immunogen to produce monoclonal antibodies (mAbs). 11 mAbs were screened out and established, and they were divided into two groups according to Western blot and IFA results. One group, including 1C1, bound only PCV2-RepN, while the other, including 3D10, had cross reactivity with RepN of both PCV1 and PCV2. Epitope mapping indicated that 1C1 and 3D10 recognized the linear epitopes L(39)FDYFIVG(46) and K(99)EGNLLIE(106) in PCV2-RepN, respectively. Protein sequence alignment of RepN indicated L(39)FDYFIVG(46) is conserved in all PCV2 in NCBI database, whereas the PCV1 has amino acid substitutions V(41)C(42) in this region. mAb 3D10 could recognize all PCV because all natural mutations in its epitope did not affect its binding. The information about characteristics and epitope of monoclonal antibodies may be useful for the development of diagnostic methods for PCV2 and for analyzing the function of Rep and Rep' of PCV.
Meng
Tao
T
Animal Health Biotechnology, Temasek Life Sciences Laboratory, National University of Singapore, 117604 Singapore, Singapore.
Jia
Qiang
Q
Liu
Sen
S
Karuppannan
Anbu K
AK
Chang
Chih-Cheng
CC
Kwang
Jimmy
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D002460:Cell Line; D018173:Circoviridae Infections / Q000175:diagnosis / Q000662:veterinary / Q000821:virology; D017925:Circovirus / Q000201:enzymology / Q000276:immunology* / Q000302:isolation & purification; D003429:Cross Reactions; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000276:immunology*; D018604:Epitope Mapping; D019084:Fluorescent Antibody Technique, Indirect; D016056:Immunodominant Epitopes / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D013553:Swine Diseases / Q000175:diagnosis / Q000821:virology; D020079:Terminal Repeat Sequences; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D016056:Immunodominant Epitopes; D011994:Recombinant Proteins; D014764:Viral Proteins; C023140:DNA replicase; D004259:DNA-Directed DNA Polymerase