Jichu yixue yu linchuang (May 2020)
Construction and functional characterization of human recombinant adenovirus rAd-IL-23R
Abstract
Objective To construct the human recombinant adenovirus expressing interleukin 23 receptor (rAd-IL-23R), and explore the effect of the rAd-IL-23R on the proliferation of human cervical cancer(HeLa) cell line. Methods Using the AdEasyTM system, the pAd-IL-23R was constructed by homologous recombination with the pShuttle-CMV plasmid carried IL-23R and the backbone plasmid pAdeasy-1, which was identified by restriction endonuclease digestion. The linearized recombinant plasmids were packaged and expanded in HEK-293A cells. The viral titer was detected using TCID50. CCK-8 assay was used to analyze the effect of rAd-IL-23R on HeLa cells proliferation. Flow cytometric analysis and Western blot were used to demonstrate the effect of rAd-IL-23R on HeLa cells apoptosis. Results pAd-IL-23R was digested with PacⅠ to yield ~30 kb and 3.0 kb fragments, which could induce HEK-293A cells to produce cytopathic effect and express the IL-23R protein after being transfected into HEK-293A cells. After infecting HeLa cells with increased doses of rAd-IL-23R (0, 1, 2 MOI), the proliferation of HeLa cells was significantly inhibited in a dose-dependent manner (P＜0.05). With the increase of MOI, not only the apoptotic rate of the infected HeLa cells was also increased significantly (P＜0.05), but also the protein expression level of cleaved-caspase 3 was markedly increased. Conclusions rAd-IL-23R was constructed successfully, which may be used to inhibit the proliferation and to promote apoptosis of HeLa cells.
