[Construction of co-expression plasmid containing tPA and VEGF165 genes and its expression in VSMCs].
OBJECTIVE
To construct the eukaryotic co-expression plasmid of tissue-plasminogen activator (tPA) genes and vascular endothelia growth factor165 (VEGF165) and observe its expression in vascular smooth muscle cells (VSMCs).


METHODS
The tPA and VEGF165 genes were cloned from hominal heart tissue with RT-PCR, and then the tPA and VEGF165 genes were cloned into eukaryotic expression plasmid pBudCE4.1 to construct the eukaryotic co-expression plasmid pBudCE4.1/tPA-VEGF165. The pBudCE4.1/tPA-VEGF165 was transfected into VSMCs via lipofection mediation. The expression levels of tPA and VEGF165 mRNAs in the transformed VSMCs were detected by RT-PCR and the expression levels of tPA and VEGF165 proteins were detected by ELISA.


RESULTS
The size of RT-PCR product of tPA and VEGF165 genes was 1.9 kb and 576 bp respectively. Restrictive enzyme digestion analysis showed that recombinant co-expression plasmid pBudCE4.1/tPA-VEGF165 had been constructed successfully. The expression of tPA and VEGF165 at mRNA and protein levels in the transformed VSMCs and cells culture supernatant were detected respectively by RT-PCR and ELISA.


CONCLUSIONS
The recombinant eukaryotic co-expression plasmid pBudCE4.1/tPA-VEGF165 has been successfully constructed. The tPA and VEGF165 are expressed in transformed VSMCs.
Wu
Zhong-jun
ZJ
The Organ Transplant Center, The 1st Affiliated Hospital, Zhejiang University, Hangzhou 310003, China.
Shi
De
D
Luo
Xu-dong
XD
Li
Yu
Y
Xie
Hai-yang
HY
Zheng
Shu-sen
SS
D002478:Cells, Cultured; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics*
D012333:RNA, Messenger; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D010959:Tissue Plasminogen Activator