Construction And Identification Of A Recombinant Adenovirus Vector Containing HIL-24△103 Gene And Its Biological Effects To A549 Cell

Objects: To construct a recombinant adenovirus vector containing hIL-24Δ103 gene for investigation of the functions of hIL-24Δ103 gene to A549 cell and the intracellular signal pathways of interleukin 24.Methods: Polymerase chain reaction (PCR) technique was used to amplify the 104-206 coding region of IL-24 with Pgex-IL-24 containing IL-24 of human as template;the gene was cloned into pMD18-T vector and then sub-cloned into the shuttle vector pAdTrack-CMV, and subsequently co-transformed into BJ5183 cells with an adenoviral backbone plasmid pAdEasy-1. The recombinant adenovirus plasmid then transformed into HEK293 cells for amplification of Ad-hIL-24Δ103 virus. The inhibitory effect of hIL-24Δ103 to A549 cells was detected by MTT assay;Apoptosis was detected by Hoechst 33258 stain,flow cytometry;The expression of PKR and eIF-2αwas detected by Western blot.Results: The hIL-24Δ103 gene was amplified with polymerase chain reaction (PCR) technique by using plasmid Pgex-IL-24 as template to obtain the targeted gene;the PCR product was cloned into pMD18-T vector and digested with Kpn I and Xho I together, the fragment subsequently sub-cloned into pAdTrack-CMV to construct the shuttle vector. The exact recombinant pAd-Track-CMV- hIL-24Δ103 was linearized with Pme I, and homologous recombined with pAdEasy-1. Identification using Pac I digestion obtaining a 23kb fragment and a 4.5kb small characteristic fragment, which suggested the successful construction of recombinant adenovirus vector. The packaging, identification and expression of recombinant adenovirus vector: The recombinant adenovirus vector was transformed into HEK293 cells using liposome to obtain hIL-24Δ103 adenovirus. The adenovirus was then amplified in 293 cells and the titer was 2×1010pfu/mL. PCR and Western blotting detected the expression of hIL-24Δ103. The adenovirus was then provided to A549 cells to measure the biological effects of hIL-24Δ103. MTT assay detected that the cell viability decreased after treatment of Ad-hIL-24Δ103, and the inhibitory effect was dose-dependent. Hoechst 33258 staining and flow cytometry analysis detected cell apoptosis after Ad-hIL-24Δ103 treatment. Western blot analysis demonstrated that PKR as well as its downstream target eIF-2αwas upregulated and phosphorylated in Ad-hIL-24Δ103 infected A549 cells, suggesting that PKR might play an important role in IL-24Δ103-induced apoptosis and celluar growth inhibition.Conclusion: The Ad-hIL-24Δ103 adenovirus was constructed and IL-24Δ103 expression inhibits cellular proliferation and induces apoptosis in A549 cells.