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DKK1 eukaryotic expression plasmid and expression product identification.

G Y Bao1, K Y Lu2, S F Cui2

  • 1Department of Clinical Laboratory, Yangzhou First People's Hospital, Yangzhou, Jiangsu, China baogy_fml@163.com.

Genetics and Molecular Research : GMR
|July 1, 2015
PubMed
Summary
This summary is machine-generated.

Researchers successfully cloned the dickkopf 1 (DKK1) gene and expressed DKK1 protein in human cells. This advancement aids in studying DKK1

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Area of Science:

  • Molecular Biology
  • Cancer Research
  • Protein Expression

Background:

  • Dickkopf 1 (DKK1) is implicated in tumorigenesis.
  • Understanding DKK1's role requires reliable methods for its production and study.

Purpose of the Study:

  • To construct a eukaryotic expression plasmid for human dickkopf 1 (DKK1).
  • To express, purify, and verify the DKK1 protein.
  • To establish a foundation for studying DKK1's biological activity in tumorigenesis.

Main Methods:

  • Extraction of mRNA from cervical cancer tissue.
  • Reverse transcription-polymerase chain reaction (RT-PCR) to amplify the DKK1 gene.
  • Construction of the recombinant plasmid pCMV-HA2/DKK1.
  • Transfection into Free-Style 293-F cells using liposome vectors.
  • Western blotting for DKK1 protein detection.

Main Results:

  • The amplification product confirmed the expected size of the DKK1 gene fragment.
  • Restriction enzyme digestion and sequencing verified the recombinant plasmid as pCMV-HA2/DKK1.
  • Western blotting successfully detected the expressed DKK1 protein using an anti-DKK1 antibody.

Conclusions:

  • The successful cloning of the DKK1 gene and expression of DKK1 protein were achieved.
  • This methodology provides a valuable tool for investigating the biological functions of DKK1 in cancer development.