[Construction of recombinant adenovirus vector co-expressing VEGF165 and SDF-1 genes and its expression in ischemic cerebral tissue of rats].
OBJECTIVE
To construct a recombinant adenoviral vector carrying and co-expressing vascular endothelial growth factor 165 (VEGF165) and stromal cell derived factor 1 (SDF-1) and explore its co-expression in ischemic brain tissue in rats.


METHODS
The VEGF165 and SDF-1 genes were directionally connected with internal ribosome entry site (IRES). And the double gene co-expression recombinant shuttle plasmid pDC316-VEGF165-IRES-SDF-1 was built with homologous recombination. The resultant plasmid pDC316-VEGF165-IRES-SDF-1 and backbone plasmid pBHGlox_E1, 3Cre were transfected into HEK293 cells by liposome and the recombinant adenoviral particles capable of infection were acquired. With the rounds of amplification, the purified adenoviral vector Ad5-VEGF165-IRES-SDF-1 was obtained with a titer of up to 1×10(10) IU/ml. The rat model of middle cerebral artery occlusion (MCAO) was established by intra-luminal suturing. And the viral vectors were transfused into the lateral ventricle by a stereotactic microinjection. The expressions of VEGF165 and SDF-1 in ischemic brain tissue were examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot.


RESULTS
The results of PCR, double enzyme digestion and gene sequencing showed that both the recombinant plasmid and the constructed adenoviral vector were expressed. And the adenoviral vector Ad5-VEGF165-IRES-SDF-1 could mediate a co-expression of VEGF165 and SDF-1 in ischemic cerebral tissue.


CONCLUSIONS
The recombinant adenoviral vector carrying VEGF165 and SDF-1 are successfully constructed. And Ad5-VEGF165-IRES-SDF-1 may mediate a co-expression of VEGF165 and SDF-1 in ischemic cerebral tissue of rats.
Department of Neurosurgery, Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003, China.
Junchen
J
Si
Guojie
G
Hu
Hui
H
Zhu
Shiru
S
Li
Qinghai
Q
Meng
Weicheng
W
Yao
Yugong
Y
Feng
Email: lsfpumc@163.com.
Shifang
S
Li
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002545:Brain Ischemia / Q000235:genetics / Q000378:metabolism*; D054377:Chemokine CXCL12 / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D008297:Male; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
D054377:Chemokine CXCL12; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A