MOLECULAR CLONING AND EXPRESSION OF A FRAGMENT OF THE GENE CODIFYING FOR THE PROTEIN ERNS OF CLASSICAL SWINE FEVER VIRUS

摘要

Classical swine fever virus (CSFV), belonging to the genus Pestivirus of the Flaviviridae family, is an enveloped positive stranded RNA virus highly contagious that can cause a fatal disease, characterized by fever, leukopenia and hemorrhage, with substantial economic losses. There is a great demand for a marker vaccine against CSFV. C, Erns, E1, and E2 are the structural proteins of the virus. E2 is the best candidate to be incorporated in vaccine, while Erns becomes an ideal candidate as an antigen in a differential diagnostic test. A synthetic fragment of the Erns gene (codifying for aa 109-160) was subcloned into pET28a vector. The cloning was screened by restriction analysis. The gene was expressed as a his-tag fusion protein in BL21 (DE3) E. coli strain. The recombinant polypeptide formed aggregates of about 7.9; 15.8; 23.7; 31.6; 39.5; 47.5 and 55.3kDa. The protein was not recognized by Western blot using an antibody against the virus. Using an ion metal affinity chromatography procedure, a 90% pure recombinant product was obtained. The potential use of this antigen for detection of CSFV antibodies should be further evaluated.