ABSTRACT
OBJECTIVE: To investigate the effects of plasmid-based siRNA targeting to oncogene c-myc on c-myc/ c-Myc expressions and cells proliferation in MCF-7 breast cancer cells. METHODS: siRNA eukaryotic expression plasmid p-Mat01-1 targeting to the sequence 589-609 of oncogene c-myc and its mismatch plasmid p-Mis09-1 were constructed, and transiently transfected MCF-7 cells using Lipo2000. Semi-quantitative RT-PCR and Western blot were used to analyze the expressions of c-myc/c-Myc in MCF-7 cells, and cells proliferation was detected by MTT assay. RESULTS: p-Mat01-1 inhibited the expressions of c-myc mRNA (24 h: P < 0.01) and c-Myc protein (5 d. P < 0.01) in MCF-7 cells as compared with pEGFP-C1 and p-Mis09-1 controls, and suppressed the proliferation of MCF-7 cells significantly (3 d: P < 0.05, 5, 7 d: P < 0. 01). CONCLUSION: Plasmid-based siRNA targeting to oncogene c-myc could inhibit the expressions of c-myc/c-Myc in MCF-7 breast cancer cells efficiently, suggesting that the downregulation of c-myc/c-Myc could suppress the proliferation of MCF-7 cells in vitro.
Subject(s)
Cell Proliferation , Proto-Oncogene Proteins c-myc/genetics , RNA Interference , RNA, Small Interfering/genetics , Base Sequence , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Female , Humans , Molecular Sequence Data , Plasmids/genetics , Proto-Oncogene Proteins c-myc/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
OBJECTIVE: To construct anti-human AFP single chain fragment variable (ScFv) gene, transform it into BL-21 (DE3) E. coli for expression, and identify its bioactivity. METHODS: VH and VL genes of anti-human AFP monoclonal antibody were cloned by RT-PCR from hybridoma. The ScFv gene was spliced by sequence overlap extending (SOE) PCR, and then it was ligated into pGEM-T vector to be identified by endonuclease digestion, PCR and sequencing. ScFv gene was cloned into pET32 (a+) vector and transformed into BL-21 (DE3) E. coli. The positive clones were screened out by IPTG induction, and the ScFv antibody was purified to be identified by SDS-PAGE and competitive inhibition ELISA test. RESULTS: The VH DNA consisted of 339 bases, coming from the mouse IgG gamma chain. The VL DNA consisted of 312 bases, coming from the mouse IgG kappa chain. The VH and VL genes were spliced by 45 bases coding a (G4S)3 flexible linker. The ScFv gene consisted of 696 bases. The ScFv antibody expressed by BL-21 (DE3) fused with TrxA tag protein and formed inclusions. The relative molecular mass of TrxA-ScFv fusion protein is about 40 x 10(3) and that of ScFv is about 24 x 10(3). The ScFv antibody has excellent activity tested by competitive inhibition ELISA, the TrxA-ScFv could inhibit about 41% of the McAb to bind antigen and ScFv could inhibit about 53%. CONCLUSION: We have successfully constructed an anti-human AFP ScFv gene with 696 bases; it can express in BL-21 with high activity.
