[Construction of recombinant adenovirus for the angiogenic inhibitor, vasostatin, and its expression in vitro].
OBJECTIVE
To construct a recombinant replication-deficient adenovirus for the angiogenic inhibitor, vasostatin and assay its expression in vitro.


METHODS
The cDNA for vasostatin was got by PCR amplification, then it was cloned into T vector and confirmed by enzymatic analysis and direct sequencing. Subsequently the cDNA was subcloned into the shuttle plasmid, and co-transformed into 293 cells with backbone plasmid. The resulting recombinant andenovirus was confirmed by PCR and western blot. The activity was assayed by inhibition of HUVEC growth.


RESULTS
The PCR product was about 590 bp in length, and sequencing result was identical to that reported. The construction of recombinant replication-deficient adenovirus was confirmed by PCR. Western blot analysis showed the expression of vasostatin in vitro. The supernatant from transduced HeLa cells inhibited the growth of HUVEC specifically.


CONCLUSIONS
The recombinant adenovirus for vasostatin efficiently mediated the expression of the protein in vitro.
Key Laboratory of Biotherapy of Human Diseases of Ministry of Education, Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China.
Zhen-yu
ZY
Ding
Li
L
Yang
Ling
L
Tian
Fen
F
Liu
Fei
F
Xiao
Yu-quan
YQ
We
D000256:Adenoviridae / Q000235:genetics*; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D037282:Calreticulin; D002455:Cell Division; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000166:cytology; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D012261:Ribonucleoproteins / Q000096:biosynthesis / Q000235:genetics*
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D018076:DNA, Complementary; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; C121986:vasostatin