Purification, partial characterization and molecular cloning of the novel antiviral protein RC28

Abstract

An antiviral protein, RC28, with anti-herpesvirus activity was purified from a PBS extract of Rozites caperata ( Cortinarious caperata) by acetone precipitation followed by gel filtration and ion exchange chromatography. The molecular weight of this protein was 28.251 kDa as measured by MALDI-TOF mass spectrography. Our preparation of RC28 inhibited HSV-1 replication in vitro with an IC 50 value of 0.078 mg/ml and a therapeutic index >32. The first 30 amino acid residues of RC28 were sequenced by Edman degradation to be MLTYRGKLNWYNYAVNEGFTLILPGXELKV. Based on that sequence, two degenerate primers were designed, the RC28 cDNA fragment was cloned by 3′-RACE, and the rest of the amino acid sequence was inferred from the cDNA sequence. The full-length peptide chain of RC28 has 235 amino acid residues, and was modified after its translation naturally. A search of the literature showed that this sequence has not been reported before and does not belong to any known protein family. A preliminary expression system was also constructed by inserting the cDNA into the PQE-30 vector, and transformed into Escherichia coli.