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Through PCR amplification, 5’ flanking region and partial open reading frame(ORF) of gene of Nile tilapia(Oreochromis niloticus) was cloned by PCR amplification. Sequence analysis showed that no difference was found in known functional regions. This study was to construct and identify the mammalian expression vector of pEGFP-β-actin and to detect whether it could express in HEK 293T cell line. pEGFP-β-actin was transfected into HEK 293T cells with Lipofectamine 2000. The results showed that correct construction of recombinant pEGFP-β-actin has been shown by restriction enzyme digestion. The expression of gene in HEK 293T cells could be observed under microfluoroscope. pEGFP-β-actin could repress EGFP protein in HEK 293T cells. The results showed that β-actin gene promoter possessed effective transcription activities in eukaryotic cells. The work laid foundations for further study on the gene engineering and autotransgenic tilapia.
Through PCR amplification, 5 ’flanking region and partial open reading frame (ORF) of gene of Nile tilapia (Oreochromis niloticus) was cloned by PCR amplification. Sequence analysis showed that no difference was found in known functional regions. This study was to construct and identify the mammalian expression vector of pEGFP-β-actin and to detect whether it could express in HEK 293T cell line. pEGFP-β-actin was transfected into HEK 293T cells with Lipofectamine 2000. The results showed that correct construction of recombinant pEGFP-β -actin has been shown by restriction enzyme digestion. The expression of gene in HEK 293T cells could be observed under microfluoroscope. pEGFP-β-actin could repress EGFP protein in HEK 293T cells. The results showed that β-actin gene promoter possessed effective transcription activities in eukaryotic cells. The work laid foundations for further study on the gene engineering and autotransgenic tilapia.