基础医学与临床 ›› 2020, Vol. 40 ›› Issue (5): 662-667.

• 研究论文 • 上一篇    下一篇

人重组腺病毒rAd-IL-23R的构建与功能鉴定

苑晓梅, 刘力*   

  1. 中国医学科学院基础医学研究所 北京协和医学院基础学院 病原生物学系, 北京 100005
  • 收稿日期:2020-02-14 修回日期:2020-03-18 出版日期:2020-05-05 发布日期:2020-04-30
  • 通讯作者: *lliu8@263.net
  • 基金资助:
    中国医学科学院创新工程(2017-I2M-3-007)

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

YUAN Xiao-mei, LIU Li*   

  1. Department of Etiology, Institute of Basic Medical Sciences CAMS, School of Basic Medicine PUMC, Beijing 100005, China
  • Received:2020-02-14 Revised:2020-03-18 Online:2020-05-05 Published:2020-04-30
  • Contact: *lliu8@263.net

摘要: 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。

关键词: 重组腺病毒, IL-23R, 宫颈癌, 增殖, 凋亡

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.

Key words: recombinant adenovirus, IL-23R, cervical cancer, proliferation, apoptosis

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