The present study is aimed at studying the gene for TIMP-3,a mammalian tissue inhibitor,by constructing a recombinant eukaryotic cell vector for gene therapy in human breast cancer.We obtained the TIMP-3 gene from the...The present study is aimed at studying the gene for TIMP-3,a mammalian tissue inhibitor,by constructing a recombinant eukaryotic cell vector for gene therapy in human breast cancer.We obtained the TIMP-3 gene from the human placent by RT-PCR.TIMP-3 gene was subcloned into pcDNA3.1 vetor from pMD18T vector by means of gene cloning to construct pcDNA3.1 recombinant vector.Human breast cancer cell line MDA-MB-453 was transfected with pcDNA3.1-TIMP3 recombinant vector using lipofectamine reagent.Then the expression of TIMP-3 and the effect on the metastasis of MDA-MB-453 were examined.The correct construction of pcDNA-TIMP3 was identified by means of restriction enzyme analysis,PCR amplication and nucleotide sequencing.Western blotting showed that the transfected cells were able to express TIMP-3, indicating that our construction of the pcDNA-TIMP3 eukaryotic expression vector was constructed successfully.Our experiments further indicated that the potential of metastasis was significantly reduced for the transfected cell line MDA-MB-453.Cellular & Molecular Immunology.2004;1(4):308-310.展开更多
Lumbrukinase gene from earthworm ( L.bimastus ) was obtained by RT\|PCR. The product, PI 239 , was sequenced and analyzed by biology programs and database. The gene including signal peptide coding sequence was cloned ...Lumbrukinase gene from earthworm ( L.bimastus ) was obtained by RT\|PCR. The product, PI 239 , was sequenced and analyzed by biology programs and database. The gene including signal peptide coding sequence was cloned into an eukaryotic vector and the clones were obtained by transferring into BHK cells. The gene was fused with EGFP gene at C\|terminal to be detected conveniently by its fluorescence. The lumbrukinase gene PI 239 has 852 nucleotides that code for 239 amino acid residues as mature peptide chain. The N terminal of PI 239 shares certain homology with those known lumbrukinase. The enzyme contains relative more acidic amino acid residues, and has homology to serine protease. It belongs to the acidic protein, serine protease. Conformation prediction indicates that its secondary structure mainly consists of β sheet. It has two super secondary structure motifs with the active sites Asp188 and Ser189 in between. The DNA and mRNA of the whole gene could be detected in BHK clones, but no recombinant protein detected. Under cofocal microscope, the cells transferred with fused gene showed fluorescence indicating that the fused protein was expressed. In addition, the cells containing the fused gene died soon while most of the control cells were still alive. It seemed that the protein could be expressed in BHK cells as a cytotoxin, though at a low level.展开更多
文摘The present study is aimed at studying the gene for TIMP-3,a mammalian tissue inhibitor,by constructing a recombinant eukaryotic cell vector for gene therapy in human breast cancer.We obtained the TIMP-3 gene from the human placent by RT-PCR.TIMP-3 gene was subcloned into pcDNA3.1 vetor from pMD18T vector by means of gene cloning to construct pcDNA3.1 recombinant vector.Human breast cancer cell line MDA-MB-453 was transfected with pcDNA3.1-TIMP3 recombinant vector using lipofectamine reagent.Then the expression of TIMP-3 and the effect on the metastasis of MDA-MB-453 were examined.The correct construction of pcDNA-TIMP3 was identified by means of restriction enzyme analysis,PCR amplication and nucleotide sequencing.Western blotting showed that the transfected cells were able to express TIMP-3, indicating that our construction of the pcDNA-TIMP3 eukaryotic expression vector was constructed successfully.Our experiments further indicated that the potential of metastasis was significantly reduced for the transfected cell line MDA-MB-453.Cellular & Molecular Immunology.2004;1(4):308-310.
文摘Lumbrukinase gene from earthworm ( L.bimastus ) was obtained by RT\|PCR. The product, PI 239 , was sequenced and analyzed by biology programs and database. The gene including signal peptide coding sequence was cloned into an eukaryotic vector and the clones were obtained by transferring into BHK cells. The gene was fused with EGFP gene at C\|terminal to be detected conveniently by its fluorescence. The lumbrukinase gene PI 239 has 852 nucleotides that code for 239 amino acid residues as mature peptide chain. The N terminal of PI 239 shares certain homology with those known lumbrukinase. The enzyme contains relative more acidic amino acid residues, and has homology to serine protease. It belongs to the acidic protein, serine protease. Conformation prediction indicates that its secondary structure mainly consists of β sheet. It has two super secondary structure motifs with the active sites Asp188 and Ser189 in between. The DNA and mRNA of the whole gene could be detected in BHK clones, but no recombinant protein detected. Under cofocal microscope, the cells transferred with fused gene showed fluorescence indicating that the fused protein was expressed. In addition, the cells containing the fused gene died soon while most of the control cells were still alive. It seemed that the protein could be expressed in BHK cells as a cytotoxin, though at a low level.
