Purification, partial characterization and molecular cloning of the novel antiviral protein RC28.
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 28.251kDa as measured by MALDI-TOF mass spectrography. Our preparation of RC28 inhibited HSV-1 replication in vitro with an IC(50) value of 0.078mg/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.
Gong
Meng
M
Ministry of Health, West China Hospital, Sichuan University, Chengdu, China.
Piraino
Frank
F
Yan
Naihong
N
Zhang
Jing
J
Xia
Minjie
M
Ma
Jia
J
Cheng
Jingqiu
J
Liu
Xuyang
X
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D001483:Base Sequence; D001487:Basidiomycota / Q000737:chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D018259:Herpesvirus 1, Human / Q000187:drug effects; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000998:Antiviral Agents; D017931:DNA Primers; D005656:Fungal Proteins; C542559:RC28 protein, Rozites caperata; D011994:Recombinant Proteins