Immunodetection of Canine Parvovirus (CPV) in clinical samples by polyclonal antisera against CPV-VP2 protein expressed in Esherichia coli as an antigen.
The entire virion protein 2 (VP2) gene of Canine Parvovirus (CPV) was amplified by polymerase chain reaction (PCR) and engineered to be expressed by a bacterial expression vector pET-28a, under the control of the IPTG-inducible T7lac promoter. SDS-PAGE gel revealed that VP2 expressed as a 67kDa, and found mainly in the pellet of the bacterial lysates, suggesting that cytoplasmic expression is not preferred. The recombinant protein VP2 fused with His-tag was purified from Esherichia coli using Ni-NTA resin under denaturing conditions. SDS-PAGE analysis also showed the high expression of several lower molecular weight (LMW) bands. Western blot analysis showed that polyclonal antisera produced by rabbit against E. coli-VP2 protein reacted specifically with the purified VP2 protein as well as two other LMW bands. Some of the resulting LMW products failed to keep their antigenic site in the N-terminal region of the VP2. The degradation of recombinant VP2 protein in E. coli could be due to the action of host proteases. The immunodetection ability of the polyclonal antisera was compared with that of a commercial monoclonal antibody to test numerous clinical specimens by immuno-dot blot assays. There were distinctive differences in the degree of immunodetection ability of polyclonal antisera and monoclonal antibody to react with CPV antigens. The reaction time of polyclonal antisera was much faster in visual color appearance than that of monoclonal antibody during NBT/BCIP staining. The result from diagnostic PCR assay confirmed the presence of CPV in 44 out of 46 specimens collected, consistent with polyclonal antisera-positive result. Therefore, the polyclonal antisera can be used for CPV detection in the faeces of diarrhoeic dogs, which was found to be more rapid, sensitive, broad but less specific than the monoclonal antibody.
Park
Jae Sung
JS
Department of Biology & Medicinal Science, Pai Chai University, Daejeon 302-735, Republic of Korea.
Choi
Bong Kum
BK
Vijayachandran
Lakshmi Sumitra
LS
Ayyappan
Vasudevan
V
Chong
Chom-Kyu
CK
Lee
Ki-Sung
KS
Kim
Sei Chang
SC
Choi
Chang Won
CW
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology*; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology*; D004283:Dog Diseases / Q000821:virology; D004285:Dogs; D004926:Escherichia coli / Q000235:genetics; D005243:Feces / Q000821:virology; D005822:Genetic Vectors; D007106:Immune Sera; D007118:Immunoassay / Q000379:methods*; D010322:Parvoviridae Infections / Q000662:veterinary / Q000821:virology; D017993:Parvovirus, Canine / Q000276:immunology / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000276:immunology