人重组腺病毒rAd-IL-23R的构建与功能鉴定

目的构建表达白介素23受体的人重组腺病毒(rAd-IL-23R),并探究该病毒对人子宫颈癌(HeLa)细胞系增殖的影响。方法采用AdEasyTM系统,将带有IL-23R的穿梭载体与腺病毒骨架载体进行同源重组,构建pAd-IL-23R,酶切消化鉴定,线性化的病毒质粒在HEK-293A细胞中进行病毒包装和扩增,TCID50法测定病毒滴度。CCK-8法检测rAd-IL-23R对HeLa细胞增殖的影响,流式细胞计量术分析和Western blot检测rAd-IL-23R对HeLa细胞凋亡的影响。结果pAd-IL-23R经PacⅠ酶切消化为约30 kb和3 kb的核酸片段,可诱导HEK-293A细胞产生细胞病变,并成功表达IL-23R蛋白;用不同滴度的rAd-IL-23R(0、1和2 MOI)感染HeLa细胞后,rAd-IL-23R对细胞增殖的抑制具有明显的剂量效应(P<0.05),随着MOI增加,细胞凋亡率显著增加(P<0.05),同时cleaved-caspase 3的蛋白表达水平增高。结论成功构建rAd-IL-23R,该重组腺病毒可抑制HeLa细胞增殖并促进凋亡发生。

Construction and functional characterization of human recombinant adenovirus rAd-IL-23R

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.MethodsUsing 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 PacⅠto 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 ofcleaved-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.