Prokaryotic expression of truncated VP7 of bluetongue virus (BTV) and reactivity of the purified recombinant protein with all BTV type-specific sera.
Purification of bluetongue virus (BTV) group-specific VP7 protein, expressed in prokaryotic system as histidine-tagged fusion protein is described in the present study. The major antigenic portion of VP7 gene of BTV 23 was amplified from the extracted RNA by reverse transcription polymerase chain reaction and cloned. The recombinant expression construct (pET-VP7) was identified by the polymerase chain reaction and sequencing analysis. Expression of histidine-tagged fusion truncated VP7 protein with a molecular mass of 36 kDa was determined by Western blot analysis using anti-His antibody. The expressed VP7 was purified to near homogeneity by chromatography on nickel-agarose column as judged by sodium dodesyl sulfate-polyacrylamide gel electrophoresis analysis. The purified VP7 protein was recognized by antibody to BTV in Western blot analysis. The capability of the recombinant VP7 protein to differentiate hyperimmune serum of rabbit to BTV from normal rabbit serum was evident in the enzyme-linked immunosorbent assay (ELISA). The purified VP7 reacted well with the 24 BTV serotype-specific sera obtained from OIE Reference Laboratory on bluetongue. Our results indicated that the expressed VP7 protein could be used as antigen for development of antibody-capture ELISA for detection BTV group-specific antibodies. This recombinant protein may also be used as antigen in competitive ELISA format.
Pathak
K B
KB
Division of Virology, Indian Veterinary Research Institute, Mukteswar Campus, District Nainital 263 138, Uttarakhand, India.
Biswas
S K
SK
Tembhurne
P A
PA
Hosamani
M
M
Bhanuprakash
V
V
Prasad
Gaya
G
Singh
R K
RK
Rasool
T J
TJ
Mondal
B
B
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood / Q000378:metabolism; D001819:Bluetongue / Q000175:diagnosis; D001820:Bluetongue virus / Q000235:genetics*; D002460:Cell Line; D002846:Chromatography, Affinity; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012756:Sheep; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*
D000914:Antibodies, Viral; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; C066862:VP7 protein, orbivirus