Canine Interferon-γ(CaIFN-γ) cDNA was cloned from spleen T cells of dog by reverse transcription-polymerase chain reaction (RT-PCR). CaIFN-γ cDNA were digested with Hind Ⅲ and NotⅠ, and inserted into pRc/CMV2 exp...Canine Interferon-γ(CaIFN-γ) cDNA was cloned from spleen T cells of dog by reverse transcription-polymerase chain reaction (RT-PCR). CaIFN-γ cDNA were digested with Hind Ⅲ and NotⅠ, and inserted into pRc/CMV2 expression vector. The pRc/CMV2 /CaIFN-γ vector was sequenced, and predicted to produce a signal peptide of 23 amino acids and a mature protein of 143 amino acids with a molecular weight of 19 kD. Two potential N-glycosylation sites are located at positions 16 and 83 of the mature protein. Comparison of the CaIFN-γ protein sequence with that of CaIFN-γ reported from DDBJ/GenBank revealed a homology of 99%. To establish a long time expression system, pRc/CMV2/CaIFN-γ vector was transfected into mouse SP2/0 cell line. The SP2/0 cells culture supernatants was harvested and the antiviral activity was measured following cytopathic-effect inhibition assay using Madin-Darby Canine Kidney (MDCK)-vesicular stomatitis virus(VSV) system. Initial transformants with G418 phenotype produced recombinant CaIFN-γ titers ranging from 2,500 to 5,000 u/mL of culture medium.展开更多
文摘Canine Interferon-γ(CaIFN-γ) cDNA was cloned from spleen T cells of dog by reverse transcription-polymerase chain reaction (RT-PCR). CaIFN-γ cDNA were digested with Hind Ⅲ and NotⅠ, and inserted into pRc/CMV2 expression vector. The pRc/CMV2 /CaIFN-γ vector was sequenced, and predicted to produce a signal peptide of 23 amino acids and a mature protein of 143 amino acids with a molecular weight of 19 kD. Two potential N-glycosylation sites are located at positions 16 and 83 of the mature protein. Comparison of the CaIFN-γ protein sequence with that of CaIFN-γ reported from DDBJ/GenBank revealed a homology of 99%. To establish a long time expression system, pRc/CMV2/CaIFN-γ vector was transfected into mouse SP2/0 cell line. The SP2/0 cells culture supernatants was harvested and the antiviral activity was measured following cytopathic-effect inhibition assay using Madin-Darby Canine Kidney (MDCK)-vesicular stomatitis virus(VSV) system. Initial transformants with G418 phenotype produced recombinant CaIFN-γ titers ranging from 2,500 to 5,000 u/mL of culture medium.
