[Production and characterization of a polyclonal antibody against rabies virus phosphoprotein].
BACKGROUND
The expression of recombinant viral proteins has been a useful tool to study molecular biology and pathogenesis of virus infections. Because commercial specific antibodies to rabies virus phosphoprotein (P) are currently unavailable, these antibodies must be generated de novo in order to study the role of P protein during the infectious process.


OBJECTIVE
A polyclonal antibody was produced and characterized for use against the phosphoprotein (P) of rabies virus. The antibody was raised in rabbits with a recombinant viral phosphoprotein (P) produced in Escherichia coli.


METHODS
Gene P coding for the viral phosphoprotein (P) was amplified by RT-PCR and cloned into the expression vector PinPoint Xa-1 T. The recombinant protein P was expressed in Escherichia coli, purified by affinity chromatography and used to produce a polyclonal antibody anti-P. The antibody anti-P was purified and characterized by immunocytochemistry, immunofluorescence, fluorometric CELL-ELISA and Western blotting.


RESULTS
The recombinant viral phosphoprotein was successfully expressed as a 50 kd biotinylated fusion protein which corresponds to the whole protein P of rabies virus. The polyclonal antibody raised against this recombinant protein P was able to detect with high specificity, protein P in cultures of sensorial neurons infected with rabies virus.


CONCLUSIONS
The P protein obtained from heterologous expression in Escherichia coli became a specific antigen that was used to produce a polyclonal antibody capable of detecting native P protein in rabies virus infected cells.
Castañeda
Nadia Yadira
NY
Instituto de Virología, Universidad El Bosque, Bogotá, D. C, Colombia.
Chaparro-Olaya
Jacqueline
J
Castellanos
Jaime E
JE
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D006801:Humans; D010750:Phosphoproteins / Q000235:genetics / Q000276:immunology*; D011817:Rabbits; D011818:Rabies / Q000517:prevention & control / Q000821:virology; D011820:Rabies virus / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D000956:Antigens, Viral; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins