Construction of recombinant adenovirus vector expressing hIL-17F and functional study of expressed IL-17 F / 中华微生物学和免疫学杂志

ABSTRACT

Objective To construct a recombinant adenovirus vector ( Ad-hIL-17F) expressing human interleukin 17F (hIL-17F) and to investigate the effects of expressed hIL-17F on angiogenesis. Methods The hIL-17F fragments was amplified by PCR using pUCm-T/hIL-17F plasmids as templates and then cloned into pAdTrack-CMV transfer vector to form pAdTrack-CMV-hIL-17F.The pAdTrack-CMV-hIL-17F transfer vector was linearized with PmeI digestion and then transformed into competent BJ 5183 with pAdEasy-1 backbone vector for homologous recombination .Then it was linearized with PacI digestion and transfected into human embryonic kidney 293 (QBI-293A) cells to construct Ad-hIL-17F.RT-PCR analysis and indirect immunofluorescent assay (IFA) were performed to determine the expressions of hIL-17F.MTT assay was used to detect the inhibitory effects on cell growth of ECV 304 .The expressions of VEGF and Ang-1 in 293 A and ECV304 cells were measured by ELISA .The effects of Ad-hIL-17 F on the expressions of VEGF in 293A cells were analyzed by Real-Time PCR.Results The sequencing result verified that hIL-17F gene fragment was correctly inserted in the vector .The expressions of hIL-17F gene at mRNA and pro-tein levels were confirmed by RT-PCR and IFA.Ad-hIL-17F could significantly inhibit the growth of ECV304 cells and down-regulate the expressions of VEGF and Ang-1 in 293A and ECV304 cells.Conclu-sion Ad-hIL17F expressing hIL-17F was successfully constructed .The expressed hIL-17F could inhibit the angiogenesis through down-regulating the expressions of VEGF and Ang-1.