[Construction and expression of recombinant adenovirus vector carrying human endostatin, K5 and endostatin-K5 gene].
OBJECTIVE
To construct the recombinant adenoviral vectors expressing human endostatin, K5 and endostatin-K5 gene respectively, and study their bioactivity in vitro.


METHODS
Human endostatin, K5 and endostatin-K5 gene were amplified by PCR, which were then subcloned into shuttle vector pAd5-CMV-H1H2-MCS-6His by enzyme and ligation respectively. The positive recombinant plasmids linearized by Pac I were cotransfected into HEK 293 cells with the Pac I linearized adenoviral backbone plasmid using calcium phosphate precipitation method. The recombinant viruses were purified by CsCl density gradient centrifugation. The protein expression at different time points (24 h, 48 h and 72 h) was determined by Western blot. The inhibitory effect of the protein on ECV-304 growth was detected by MTT.


RESULTS
The recombinant adenoviral vectors expressing human endostatin, K5 and endostatin-K5 gene were successfully constructed in HEK293 cells. Protein expression was detected by Western blot. The level of protein expression was increased with the prolonged incubation of the infected HeLa cells. Three kinds of the protein expressed by the recombinant adenoviral vectors showed obvious inhibitory effect on ECV-304 cell growth.


CONCLUSIONS
The protein expressed by adenoviral vectors carrying endostatin, K5 and endostatin-K5 gene has an obvious inhibitory effect on ECV-304 cell growth.
Li
Hui-jin
HJ
Laboratory of Gene Therapy, College of Life Sciences, Shaanxi Normal University, Xi'an 710062, China. lihuijin@stu.snnu.edu.cn
Zhang
Wei-feng
WF
Wang
Dong-yang
DY
Li
Rong-hua
RH
Qi
Zong-li
ZL
Zhao
Jun-li
JL
Xia
Hai-bin
HB
D000256:Adenoviridae / Q000235:genetics*; D003001:Cloning, Molecular; D043169:Endostatins / Q000235:genetics*; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D018082:Kringles / Q000235:genetics*; D011995:Recombination, Genetic
D043169:Endostatins