title: [Construction and expression of recombinant adenovirus vector carrying human endostatin, K5 and endostatin-K5 gene].
abstract: 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.
authors.affiliation: Laboratory of Gene Therapy, College of Life Sciences, Shaanxi Normal University, Xi'an 710062, China. lihuijin@stu.snnu.edu.cn
authors.forename: Hui-jin
authors.initials: HJ
authors.lastname: Li
authors.forename: Wei-feng
authors.initials: WF
authors.lastname: Zhang
authors.forename: Dong-yang
authors.initials: DY
authors.lastname: Wang
authors.forename: Rong-hua
authors.initials: RH
authors.lastname: Li
authors.forename: Zong-li
authors.initials: ZL
authors.lastname: Qi
authors.forename: Jun-li
authors.initials: JL
authors.lastname: Zhao
authors.forename: Hai-bin
authors.initials: HB
authors.lastname: Xia
mesh_terms: 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
chemical_list: D043169:Endostatins