带有人红细胞生成素信号肽序列的IL-1β基因在HepG2细胞中的表达  

The expression of human IL-1β gene containing human erythropoietin signal peptide in HepG2 cells

作　　者：李娜[1] 邸大琳[1] 肖伟玲[1] 付晓燕[1] 梁淑娟[1] 

机构地区：[1]山东省高校免疫学重点实验室,潍坊医学院免疫学教研室,山东潍坊261053

出　　处：《细胞与分子免疫学杂志》2014年第5期493-496,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81272319);山东省科技攻关计划项目(jk47);潍坊医学院前沿探索重点项目(K11TS1007);山东省高校科技计划项目(J12LK02)

摘　　要：目的构建由经典分泌途径和非经典途径表达人IL-1β的慢病毒载体,制备慢病毒后,感染HepG2肝癌细胞,比较肝癌细胞中IL-1β表达的水平。方法分别以表达人IL-1β前体蛋白基因和人红细胞生成素(EPO)信号肽序列与IL-1β成熟蛋白融合基因的pIRES2-EGFP-proIL-1β和pIRES2-EGFP-epoIL-1β质粒为模板,PCR扩增获得人IL-1β全长基因和含有EPO信号肽与IL-1β成熟蛋白融合基因的序列,将其分别克隆入pLenti6/V5慢病毒载体中,构建由非经典途径和经典途径分泌IL-1β的pLenti6/V5-proIL-1β和pLenti6/V5-epoIL-1β慢病毒表达载体。利用三质粒包装体系在HEK293T细胞中包装生产慢病毒,随后感染HepG2肝癌细胞,双抗体夹心ELISA和Western blot法检测细胞质和培养上清中IL-1β的表达水平。结果构建了含有人IL-1β全长基因和含有EPO信号肽与IL-1β成熟蛋白融合基因的pLenti6/V5-proIL-1β和pLenti6/V5-epoIL-1β慢病毒载体,经双酶切和DNA测序证实与基因库中登录的人IL-1β以及EPO信号肽序列一致。利用三质粒系统包装制备经不同途径表达IL-1β的慢病毒,研究证实经由2条不同途径分泌表达人IL-1β的慢病毒感染HepG2细胞后,与转染空载体的对照细胞相比,细胞培养液上清和胞质中IL-1β的水平均显著升高(P<0.01),其中HepG2/epoIL-1β细胞培养液上清中成熟蛋白IL-1β的水平明显高于HepG2/proIL-1β细胞,而胞质中总IL-1β的水平HepG2/proIL-1β高于HepG2/epoIL-1β。结论构建的带有EPO信号肽序列的IL-1β基因的慢病毒载体,在HepG2细胞表达后,分泌成熟IL-1β的水平明显高于非经典途径分泌的表达载体。Objective To construct two lentiviruses secreting human IL-I~ through either classical or nonclassical pathway and analyze their expressions in HepG2 cells after packaging lentMruses and infecting hepatoma carcinoma HepG2 cells. Methods Human full-length IL-1β gene and chimeric gene containing human erythropoietin（EPO） signal peptide and mature IL-1β protein coding sequence were respectively amplified from plRES2-EGFP-prolL-1β and plRES2-EGFP-epolL-1β using PCR. The sequences were subsequently cloned into lentiviral expression vector pLenti6/V5 to construct pLenti6/V5-prolL-1β, which expressed IL-1β through nonclassical pathway, and pLenti6/VS-epolL-1β, which expressed IL-I~ through signal-peptide mediated classical pathway. Lentiviruses expressing human IL-1β through either classical or nonclassical pathway were packaged in HEK293T cells using a three-plasmid packaging system, and then these viruses were used to infect HepG2 cells. The level of IL-1β in both cytoplasm and culture supematant were detected by sandwich ELISA and Westem blotting. Results pLenti6/V5-prolL-1β expressing human full-length IL-1β gene and pLenti6/VS-epolL-1β expressing human EPO signal peptide and mature IL-1β gene were successfully constructed and confirmed through enzymatic assay and DNA sequencing. The lentiviruses expressing IL-1β through different pathways were prepared using a three-plasmid packaging system in HEK293T cells. Compared with the cells infected with control virus, levels of supernatant and cytoplasmic IL-1β in the cells infected with two lentiviruses expressing IL-1β 15 through different pathways were markedly elevated （ P 〈 0.01 ）. However, level of mature IL-113 in supernatant of HepG2/epolL-1β cells was much higher than that of HepG2/prolL-1β cells, while total IL-1β level in cytoplasm of HepG2/prolL-1β cells was significantly higher than that in HepG2/epolL-]15 cells. Conclusion Both classical and nonclassical pathway secretion vectors could express human IL-1β in HepG2 cells, but EP

关 键 词：慢病毒 人IL-1β HEPG2细胞 经典和非经典分泌途径 

分 类 号：R392.11[医药卫生—免疫学] R392-33[医药卫生—基础医学]