In order to detect the protein delivery mediated by the PTD (protein transduction domain) of TAT Protein, a expression vector, named pT7460-GFP, was constructed by insert the PTD DNA Sequence, followed by a GFP (green...In order to detect the protein delivery mediated by the PTD (protein transduction domain) of TAT Protein, a expression vector, named pT7460-GFP, was constructed by insert the PTD DNA Sequence, followed by a GFP (green fluorescent protein) gene fused in-frame, into the pT7450 vector. The TAT-GFP fusion protein was expressed in the E.coli ER2566. Most of the fusion protein was presented in the inclusion body. The protein was purified by Ni 2+ affinity chromatography under denature conditions, then by a Sepharose Q column to remove urea. The soluble denatured protein was added directly to medium containing the Myeloma Cell SP2/0. It came out that the fusion protein could be detected delivered into the cells under fluorescent microscope in a short time.展开更多
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In order to detect the protein delivery mediated by the PTD (protein transduction domain) of TAT Protein, a expression vector, named pT7460-GFP, was constructed by insert the PTD DNA Sequence, followed by a GFP (green fluorescent protein) gene fused in-frame, into the pT7450 vector. The TAT-GFP fusion protein was expressed in the E.coli ER2566. Most of the fusion protein was presented in the inclusion body. The protein was purified by Ni 2+ affinity chromatography under denature conditions, then by a Sepharose Q column to remove urea. The soluble denatured protein was added directly to medium containing the Myeloma Cell SP2/0. It came out that the fusion protein could be detected delivered into the cells under fluorescent microscope in a short time.
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.
