Expression of Coxsackievirus B4 proteins VP0 and 2C in Escherichia coli and generation of virus protein recognizing antisera.
Coxsackievirus B4 (CBV-4) capsid protein VP0 and non-structural 2C protein were expressed and purified using a glutathione-S-transferase (GST) fusion protein expression system. We used a full-size CBV-4 cDNA as a template to amplify the genes by polymerase chain reaction (PCR). The genes were cloned into expression vector pGEX-2T and expressed as a fusion protein with GST. The GST-fusion proteins (GST-2C and GST-VP0) were purified in denatured and native forms and used to generate antibodies in rabbits. The antisera raised against GST-VP0 fusion protein recognized the corresponding structural proteins (VP0, VP2 and VP4) from purified CBV-4 preparations and infected cell lysates. In addition, cross-reactivity with CAV-9 and CBV-5 capsid proteins was observed. Anti-GST-2C antisera precipitated viral 2C protein in CBV-4-infected GMK cells, showing that the antibodies recognize the corresponding natural antigen.
Härkönen
T
T
Enterovirus Laboratory, National Public Health Institute, Helsinki, Finland. taina.harkonen@ktl.fi
Hovi
T
T
Roivainen
M
M
D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis / Q000276:immunology*; D002213:Capsid / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D036022:Capsid Proteins; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D029822:Enterovirus B, Human / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D005982:Glutathione Transferase; D006801:Humans; D015151:Immunoblotting; D010957:Plasmids; D011233:Precipitin Tests; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D000914:Antibodies, Viral; D036022:Capsid Proteins; D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D005982:Glutathione Transferase; C076023:2C protein, viral