Bovine interferon-tau expression inEscherichia coliand identification of its biological activities

Abstract

AbstractThe bovine interferon-tau (bIFN-τ) gene, with signal sequence, was obtained through polymerase chain reaction (PCR) from bovine early embryos and subcloned into a pGEM-T vector. After being verified, the fragments, with or without signal sequence, were inserted into the expression vector pET-30a(+). Two recombinant plasmids were induced to express the recombinant proteins by isopropyl β-d-1-thiogalactopyranoside. The results showed that the bIFN-τ gene could be obtained from five bovine blastocysts by PCR without extraction of genomic DNA. It had 99% homology with nucleotides and 97% with amino acids in the GenBank sequence (accession number: XM_593584). The products of recombinant bIFN-τ, minus signal sequence, expressed in pET-30a(+) were analysed by SDS-PAGE. A new 20 kDa protein was detected and its molecular weight was as expected. The antiviral activity of recombinant bIFN-τ was 1×104IU/mg using a standard cytopathic reduction assay. Marked morphological changes were induced by recombinant bIFN-τ in bovine endometrial epithelial cells. The cell volume was larger than that of controls and a lot of vesicles appeared in the cytoplasm after 24 h culture in the presence of 2.9 μg/ml recombinant bIFN-τ. In conclusion, a purified recombinant, biologically active bIFN-τ was obtained in this experiment.