Construction and expression of eukaryotic expression vector of mouse TGFBI gene

Jingyi Niu*, Peng Chen, Ye Wang, Lixin Xie, Yiqiang Wang

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Objective It has been recognized that the mutation of over 30 types of transforming growth factor beta-induced gene (TGFBI) can induce corneal dystrophy, but its molecular mechanism is below understood. The goal of this study was to construct and express mouse TGFBI in eukaryotic expression vector for the further study on the effect of TGFBI in corneal biology and the pathogenesis of corneal dystrophy. Methods The eyeball from 5-week-old BALB/cBy mouse was utilized in this experiment. TGFBI cDNA was obtained by reverse transcription-PCR from total RNA extracted from mouse corneas and cloned into pcDNA3. 1 vector. Recombinant plasmids were identified by restrictive digestion and direct sequencing. Different doses of pcDNA3. 1-TGFBI plasmids were applied for transfection of cultured NIH3T3 cells. NIH3T3 mRNA and protein were harvested from transfected cells for real-time PCR( RT-PCR) analysis and Western blot assay respectively. Results Restriction digestion and sequencing indicated that TGFBI cDNA fragment was successfully inserted into the vector. Transfection of recombinant pcDNA3. 1-TGFBI into NIH3T3 cells resulted in effective expression of TGFBI (about 90% ) , showing the green fluorenscence under the fluorescence microscope. TGFBI mRNA from transfected pcDNA3. 1-TGFBI cells was obviously increased by RT-PCR and TGFBI protein level followed the same pattern by Western blot. Conclusion Recombinant eukaryotic expression vector harboring murine TGFBI cDNA is obtained and efficiently expressed in NIH3T3 cells. The construction might be applied in studying the function and role of TGFBI in pathogenesis of corneal dystrophy.

Original languageEnglish
Pages (from-to)161-165
Number of pages5
JournalChinese Ophthalmic Research
Volume27
Issue number3
Publication statusPublished - Mar 2009
Externally publishedYes

Keywords

  • Corneal dystrophy
  • Eukaryotic expression vector
  • Transforming growth factor beta-induced gene

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