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Experimental study on establishing eukaryotic expression vector pcDNA3.1(+) TPA and its expressing activity in human
Hong-bo Deng1, Yu Yang, Jin-fu Yang
1Department of Gerontology, Second Xiangya Hospital, Central South University, Changsha 410011, China.
Objective:
To establish a cell line stably expressing the tissue plasminogen activator (TPA) in human skin fibroblasts so as to develop the function analysis and gene therapy of TPA in ischemic heart diseases.
Methods:
Eukaryotic expression vector pcDNA3.1(+) TPA was constructed and transferred into human skin fibroblasts. After G418 selection, the exogenous expression and activity of TPA were observed subsequently by reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and chromogenic substrate assay.
Results:
Eukaryotic expression vector pcDNA3.1(+) TPA was expressed effectively in human skin fibroblasts. Quantitative ELISA showed that the expression of TPA protein of the experiment group was much higher than that of the control group (643.5 ng/10(6) cells per 24 hours vs. 19.2 ng/10(6) cells per 24 hours). And the chromogenic substrate assay showed that the exogenous TPA activity of the experimental group was also much higher than that of the control group (122.6 U/10(6) cells per 24 hours vs. 5.8 U/10(6) cells per 24 hours).
Conclusion:
The exogenous TPA gene can be expressed effectively after pcDNA3.1(+)TPA was transferred into human skin fibroblasts, suggesting that the cell model will become an important tool in the further study of TPA function and gene therapy in ischemic heart diseases.
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