Purification and characterization of antiviral protein from silkworm fecal matter

Abstract

Antiviral proteins (AVP), present in silkworm fecal matter, show activity against nuclear polyhedrosis virus (NPV) in vitro and in vivo. The extract of silkworm fecal matter prepared in phosphate buffer solution of pH 7.5 was subjected to 50% solid ammonium sulfate precipitation to enrich AVP, then which was dialyzed. The dialysate was applied to the column containing silica gel-G, the column elutes were purified by gelfiltration chromatography. The gelfiltration pattern gave three protein peaks A, B and C. The protein obtained from peak fractions of peak A is found to be active against NPV in vitro. Whereas the proteins were obtained from peak fractions of peaks C and B were not shown activity against NPV in vitro. The peak A fractions were collected and further purified by High Pressure Liquid Chromatography (HPLC) using C4 column. Purified AVP of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) resulted in two protein bands with the molecular mass of 23 KD and 16 KD. Thymol sulphuric acid method of carbohydrate staining indicated that both of these protein bands are glycoproteins. AVP activity is determined in vitro by precipitation reaction. In vivo activity of the AVP is confirmed by conducting the bioassay in silkworms