Construction of human eukaryotic expression plasmid vascular endothelial growth factor 165 and its expression in transfected vascular smooth muscles.
BACKGROUND
The highly specific vascular endothelial growth factor (VEGF) induces the growth of vascular endothelial cell. This study was to construct the eukaryotic expression plasmid of vascular endothelial growth factor165 (VEGF165) and observe its expression in vascular smooth muscles (VSMCs).


METHODS
The primers were designed and synthesized according to the gene sequences of human VEGF165. The VEGF165 gene was obtained from umbilic artery tissue by the method of RT-PCR, then it was cloned to eukaryotic expression plasmid pBudCE4.1 by recombination strategy. The eukaryotic expression plasmid named pBudCE4.1/VEGF165 was identified by restriction enzyme digestion, and was sequenced. The pBudCE4.1/VEGF165 was transfected into VSMCs by using lipofection. The VEGF165 expression of mRNA and protein was detected by RT-PCR and Western blot respectively.


RESULTS
VEGF165 was shown about 576bp by RT-PCR. Sequencing revealed the amplified VEGF165 gene was identical with that in the GeneBank. Restrictive enzyme (Hind III, Bam HI) digestion analysis showed that recombinant expression plasmid pBudCE4.1/tVEGF165 had been constructed successfully. The expression of VEGF165 at mRNA and protein levels in the transformed VSMCs had been demonstrated by RT-PCR and Western blot.


CONCLUSIONS
The recombinant eukaryotic expression plasmid pBudCE4.1/VEGF165 has been successfully constructed and expressed in transformed VSMCs. The present study has laid a foundation for VEGF165 gene therapy of vascular stenosis in the transplant organ.
Organ Transplant Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China. wzjxdfpxy@zju.edu.cn
Zhong-Jun
ZJ
Wu
Xiao-Hong
XH
Yang
Shu-Sen
SS
Zheng
Su-Fen
SF
Yang
De
D
Shi
D015153:Blotting, Western; D015870:Gene Expression; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000502:physiology*; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
D012333:RNA, Messenger; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A