Construction and Characterization of a Recombinant Eukaryotic Expression Vector for TFAP2A
Objective Eukaryotic expression vector for transcription factor activating enhancer binding protein 2 alpha(TFAP2A)was constructed and characterized to provide an effective tool for subsequent studies of TFAP2A.Methods The eukaryotic expression vector CV702 plasmid was digested by restriction endonucleas-es BamH Ⅰ and Hind Ⅲ to obtain a linearized vector.The cDNA fragment of TFAP2A was prepared by PCR amplification,and the linearized vector and the cDNA amplification product of TFAP2A were ligated by in v itro cyclization to construct the CV702-TFAP2A recombinant plasmid.The ligation products were transformed into bacteria,and the single clones on the plates were picked for PCR identification,and the positive clones were se-quenced and analyzed.CV702-TFAP2A,a recombinant eukaryotic expression vector,was transfected into breast cancer cells MDA-MB-231,and the expression efficiency of TFAP2A was detected by Western blot.Results The results of colony PCR amplification and sequencing showed that the CV702-TFAP2A recombinant eukary-otic expression vector was successfully constructed;the results of Western blot and Image J grayscale analysis showed that,compared with the CV702 control vector,the relative expression of Flag-TFAP2A protein was higher in MDA-MB-231,which was transfected with the CV702-TFAP2A recombinant plasmid(P<0.05).Conclusion The eukaryotic expression vector of CV702-TFAP2A was successfully constructed,it lays a foun-dation for the follow-up study of the role and molecular mechanism of TFAP2A in triple negative breast cancer.
transcription factor activating enhancer binding protein 2 alphavector constructioneukaryotic ex-pressionbreast cancer
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