بررسي پردازش پلي پروتئين در پاراكوويروس انساني تيپ1 به روش كلونينگ و بيان ژن
Backgrounds and Objections: Human parechovirus is a genus of picornaviridae. All of picornaviruses have a 3C protease which has a key role in virus protein processing and replication. The aim of this study was to analyse polyprotein processing in human Parechovirus type1 by cloning and expression of 3C gene.
Materials and Methods: After preparation of cDNA from human parechovirus type 1(HPEV-1) RNA genome the region of cDNA that was encoded for 3C protein was inserted into plasmid pUBS by Ligase enzyme and recombinant plasmid was prepared. After transformation and replication of this plasmid in E.coli MC 10.22, DNA was isolated by phenol extraction and then expressed in prokaryotic (E.coli BL-21) and In vitro systems under T7 promoter. The results were detected by SDS-PAGE and analyzed.
Results: The products of expression of recombinant plasmids (with out 3C gene) in both prokaryotic and in vitro systems were analyzed. Just one large band same size as primary translation product was observed, but with plasmid including 3C gene, several small bands were detected. These results indicate that human Parechovirus type1 polyprotein processing occurs by 3C protease. 3C protease was checked by anti protease.
Conclusion: Our results showed that HPEV-1 has a processing strategy different from other members of Picornaviruses, and 3C protein seems to be the only virus encoded protease that can catalyze cleavages of all sites in the Parechovirus type1 primary polyprotein.