Expression of human endostatin gene in SW1990 cell line mediated by recombinant adenovirus

Objective To construct a human endostatin adenovirus vector and observe the ex- pression and bioactivity of endostatin in SW1990 cell line after recombinant adenovirus infection.Methods Human endostain cDNA was cloned into shuttle vector pShuttle-CMV,and the resultant plasmid was linearized and subsequently cotransformed into E.coli BJ5183 with an adenoviral backbone plasmid pAdEasy-1.Recombinants were then selected and the linearized recombinant plasmid was transfected into 293 cell line to produce recombinant adenovirus.Endostatin recombinant adenovirus was confirmed by PCR and Western blot analysis.Recombinant adenovirus Ad-Lac Z constructed as the same steps was used to infect SW1990 cells at different MOI to find its optimal MOI.SW1990 cells were infected by endostatin adenovirus at the optimal MOI,and the endostatin protein concentration and bioactivity in supernatant were detected every day within a week.Results A human endostatin recombinant adenovirus vector was constructed successfully and could infect SW1990 cell line with high efficiency.MOI=100 was the opti- mal multiplicity of infection.During 1-7 days after transfection,endostatin protein concentration in the su- pematant of SW1990 was (1.6±0.3),(8.5±0.6),(54.3±4.4),(256.9±25.8),(596.6±38.7),(321.7±21.2) and (132.6±7.6)μg/L respectively.This endostatin protein significantly in- hibited the proliferation and migration of human umbilical vein endothelial cells (P0.01).Conclusion Recombinant adenovirus can mediate efficient and effective expression of human endostatin in SW1990 cells.