Research Article

DKK1 eukaryotic expression plasmid and expression product identification

Published: June 11, 2015
Genet. Mol. Res. 14 (2) : 6312-6318 DOI: 10.4238/2015.June.11.5

Abstract

We constructed the human dickkopf 1 (DKK1) eukaryotic expression plasmid and expressed, purified, and identified its expression product. We extracted cancer cells from cervical cancer tissue, followed by extraction of mRNA. Reverse transcription-polymerase chain reaction was conducted to obtain DKK1 gene fragments. Using these fragments, we prepared the recombinant plasmid pCMV-HA2/DKK1. The recombinant plasmid was restriction enzyme-digested and sequenced, and using liposome vectors, was transiently transfected into Free-Style 293-F cells (serum-free medium). DKK1 protein was detected by western blotting. The amplification product showed the expected size. Restriction enzyme digestion and sequence analysis showed that the recombinant plasmid was PCMV-HA2/DKK1. The expression product was verified properly by western blotting using an anti-DKKI antibody. The successful cloning of the DKKI gene and expression of DKKI protein will be useful for studying the biological activity of tumorigenesis.

We constructed the human dickkopf 1 (DKK1) eukaryotic expression plasmid and expressed, purified, and identified its expression product. We extracted cancer cells from cervical cancer tissue, followed by extraction of mRNA. Reverse transcription-polymerase chain reaction was conducted to obtain DKK1 gene fragments. Using these fragments, we prepared the recombinant plasmid pCMV-HA2/DKK1. The recombinant plasmid was restriction enzyme-digested and sequenced, and using liposome vectors, was transiently transfected into Free-Style 293-F cells (serum-free medium). DKK1 protein was detected by western blotting. The amplification product showed the expected size. Restriction enzyme digestion and sequence analysis showed that the recombinant plasmid was PCMV-HA2/DKK1. The expression product was verified properly by western blotting using an anti-DKKI antibody. The successful cloning of the DKKI gene and expression of DKKI protein will be useful for studying the biological activity of tumorigenesis.

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