JOURNAL ARTICLE

[Construction of a plasmid vector of fused protein genes driven by human insulin-like growth factor II P3 promoter]

Hong-ke Zhou, Dong-hua Yang, Shao-hui Tang, Wei Huang, Xiao-hua Lu
Zhonghua Yi Xue za Zhi [Chinese medical journal] 2006 January 10, 86 (2): 106-10
16620715

OBJECTIVE: To construct a shuttle plasmid vector of fused herpes simplex virus thymidine kinase (HSV-tk) gene and enhanced green fluorescent protein (EGFP) gene driven by human insulin-like growth factor II (IGF-II) P3 promoter, and investigate the special killing effect of the HSV-tk/ganciclovir (GCV) system on hepatocellular carcinoma (HCC) cells.

METHODS: An adenovirus shuttle plasmid, pDC316-tkEGFP-CMV containing fused genes tkEGFP and an adenovirus shuttle plasmid pDC316-tkEGFP-P3 driven by IGF-II P3 promoter were constructed by techniques of gene recombination and screening, and identified by restriction digestion and sequencing analysis. Human hepatocellular carcinoma cells HepG2 and human cervical carcinoma cells HeLa were cultured and transfected with these 2 recombinant shuttle plasmids. RT-PCR was used to detect the mRNA expression of EGFP and HSV/tk. GCV of the final concentrations of 0, 1, 10, and 100 microg/ml respectively was added into the culture fluid of the HepG2 cells transfected with pDC316-tkEGFP-CMV or pDC316-tkEGFP-P3, and MTT method was used to detect the cell inhibition rate.

RESULTS: Digestion and sequencing analysis showed that the recombinant plasmid pDC316-tkEGFP-P3 accorded with the design. Fluorescent microscopy showed that EGFP was expressed only in the HepG2 cells, but not in the HeLa cells. RT-PCR showed that mRNA expression of EGFP and HSV/tk could be seen in both HepG2 and HeLa cells transfected with pDC316-tkEGFP-CMV or pDC316-tkEGFP-P3, however, only in the pDC316-tkEGFP-P3 transfected HepG2 cells, but not in the HeLa cells transfected with pDC316-tkEGFP-P3. MTT assay showed that GCV dose-dependently inhibited the 2 cancer cells, the inhibition rates of GCV of the final concentrations of 1, 10, and 100 microg/ml were 24.1% +/- 1.9%, 45.1% +/- 1.7%, and 69.4% +/- 3.6% in the HepG2 cells, and 25.1% +/- 1.6%, 49.3% +/- 1.1%, and 72.2% +/- 2.9% in the HeLa cells. However, the inhibition rates of the pDC316-tkEGFP-P3-transfected HepG2 cells by GCV of the final concentrations of 1, 10, and 100 microg/ml wee 19.8% +/- 1.3%, 36.2% +/- 2.0% and 48.7% +/- 1.9% respectively, all significantly lower than those of the pDC316-tkEGFP-CMV-transfected HepG2 cells (all P < 0.01), and no significant cell inhibition was found in the HeLa cells transfected with pDC316-tkEGFP-CMV.

CONCLUSION: A shuttle plasmid vector containing the tkEGFP fusion protein gene driven by IGF-II P3 promoter has been constructed successfully and its specific expression in HepG2 cells provides a sound basis for targeted gene therapy for HCC.

Full Text Links

Find Full Text Links for this Article

Discussion

You are not logged in. Sign Up or Log In to join the discussion.

Related Papers

Remove bar
Read by QxMD icon Read
16620715
×

Save your favorite articles in one place with a free QxMD account.

×

Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"