ABSTRACT
@#Objective To explore the effects of 2,3,5,4'-tetrahydroxy stilbene-2-β-D-glycoside(TSG)on the over-expression and aggregation of α-synuclein in vitro.Methods TSG in different concentrations was incubated with α-synuclein transgenic COS-7 cells for 24 h.The cell viability was measured by MTT method.The expression of α-synuclein protein was determined by immunocytochemistry and Western blotting method.Results Incubation of TSG at the range of 12.5～200.0 μmol/L with α-synuclein transgenic COS-7 cells for 24 h did not influence cell viability,but a dose-dependently inhibition for the over-expression of α-synuclein protein could be observed in the tests of immunocytochemistry and Western blotting.Conclusion TSG can inhibit the over-expression of α-synuclein protein in COS-7 cells in vitro.
ABSTRACT
Objective:To construct the recombinant eukaryotic expression vector pcDNA3.1(+)/hFasL and investigate its transfection and transient expression in COS-7 cells.Methods:The full-length cDNA of hFasL was obtained from the plasmid vector pBluescript II KS(+)/hFasL cut with Xba I and then subcloned into eukaryotic expression vector pcDNA3.1(+).The recombinant plasmid pcDNA3.1(+)/hFasL was identified with restriction enzyme digestion and sequence analysis.It was then transfected into the COS-7 cells by Lipofectin Reagent and its expression was determined by immunocytochemical staining.Results:Restriction enzyme digestion analysis with XbaⅠ?DraⅡ?HindⅢ and DNA sequencing indicated the correct construction of the recombinant plasmid;Immunocytochemical staining showed the expession of hFasL on COS-7 cells.Conclusion:The construction and expression of the eukaryotic expression plasmid pcDNA3.1(+)/hFasL have been achieved successfully.
ABSTRACT
Cdc42 is a member of the Rho family of small GTP-ase and plays an important role in intracellular signaling pathways regulating cell morphology, motility and stimulation of DNA synthesis. We have isolated cDNA encoding Cdc42 from a rat brain cDNA library using PCR-cloning strategy. The sequence of isolated gene revealed an open reading frame of 576 nucleotides with the potential to encode a protein of 191 amino acids with a predicted molecular weight of 21 kD. The resulting sequence was incorporated into the GenBank with accession number, AF205635. Sequence analysis revealed that overall cDNA sequence identity is 96% with human G25K and 52% with rat Chp, a homologue of the GTPase human Cdc42Hs, and having one nucleotide difference from the mouse Cdc42. However, putative protein sequence was identical to the mouse and human brain Cdc42Hs. On expression of the cDNA in COS-7 cells, a protein molecular weight of 21 kD was detected in immunoblotting using anti-human Cdc42 antibodies. Therefore, these results suggest that the cDNA we are reporting is most likely the rat homologue of the GTPase human Cdc42.
Subject(s)
Humans , Rats , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Comparative Study , Cross Reactions , DNA, Complementary/genetics , Molecular Sequence Data , Sequence Analysis, DNA , Sequence Homology, Amino Acid , cdc42 GTP-Binding Protein/immunology , cdc42 GTP-Binding Protein/geneticsABSTRACT
In order to improve the expression efficiency of recombinant IL~6 in eukaryotic cells, we constructed a new vector pcDIIL-6 to express IL-6 in eukaryotic cells on the basis of an eukaryotic vector pcDNA3IL-6 by introducin g the first intron of human?-globin gene into the downstream of CMV promoter in the pcDNASIL - 6. The IL-6 level expressed by pcDIIL - 6 is 7. 5 times higher than that of pcDNA3IL - 6. On the other hand , we studied the influence of pAdVAntage vector co - transfection on the expression level of IL-6 in eukaryotic cells and found the level of IL-6 is 3. 6 times higher than that of pcDNA3IL - 6. The results we got are beneficial to express cytokines with high efficiency in eukaryotic cells.
ABSTRACT
Objective:To express human dentin sialoprotein (hDSP) gene in COS-7 cells. Methods:hDSP gene was subcloned into mammalian expression vector pcDNA3. The recombined plasmids were transfected into COS-7 cells using lipofectamune PLUS TM kit for transient expression. Western blot analysis and immunohistochemical staining were used to examine the gene products. Results:The constructed vectors were confirmed by digestion with restriction enzyme. An immuno-reaction positive band with relative molecular mass of 60 000 was found by Western blot analysis in culture supernatant and cytoplasms of COS-7 cells. Immunohistochemical staining showed strong positive particles in the cytoplasms. Conclution:hDSP gene can be expressed in COS-7 cells.
ABSTRACT
Objective:To clone HLA A *0201 cDNA and express it transiently on COS 7 cells.Methods:HLA A cDNA isolated from the HLA A *0201 positive lymphocytes by using RT PCR was cloned into pBluescript II SK vector directionally.After the sequence was confirmed,the cDNA was inserted into mammalian expression vector pcDNA3.The recombinant vector was transfected into COS 7 cells by cation liposome.The transient expression on the cells was measured by flow cyteometer.Results:The cDNA was identical with HLA A *0201 cDNA published on Genebank.Determined by flow cytemeter,the expressing rate was recorded for 57%.Conclusion:We have cloned HLA A *0201 successfully and expressed it transiently on COS 7 cell,which would be potentially useful in research on killing tumor restricted by HLA A2.