HCV core 1b亚型和HA共表达重组腺病毒载体的构建与鉴定

Construction and Identification of Recombinant Adenovirus Vector Co-expressing HCV Core 1b and HA

目的:构建HCV core 1b亚型和HA共表达的腺病毒载体并予以鉴定。方法:合成HCV核心蛋白1b亚型的核苷酸,连接入pcmv5-HA质粒中。用XhoⅠ单酶切,后用Klenow酶补平,再用HindⅢ单酶切下HA-HCV core 1b片段,连入pShuttle-cmv穿梭质粒中,构建穿梭质粒pShuttle-CMV-HA core 1b.将pShuttle-CMV-HA core 1b转化至含有AdEasy-1的BJ5183感受态细菌中进行同源重组。PacⅠ酶切线性化重组质粒pAd-HA-HCV core 1b并转染AD293细胞进行病毒包装和扩增。用RT-PCR检测HA-HCVcore 1b的mRNA的表达水平,并用Western blot检测融合表达蛋白HA-HCV core 1b的表达水平。结果:穿梭质粒pShuttle-CMV-HA-HCV core 1b经PCR和测序证实构建成功。重组腺病毒载体经AD293细胞包装后,可观察到CPE现象。用获得的重组腺病毒载体感染AD293细胞,经RT-PCR检测,在mRNA水平上有表达;经Western blot检测,HCV core 1b亚型腺病毒载体(Ad-HA-HCV core 1b)感染组与未感染腺病毒载体组及空白对照组相比,只有Ad-HA-HCV core 1b感染组有融合蛋白HA-HCVcore 1b的表达。结论:通过分子克隆体外重组技术,成功构建了HCV core 1b亚型和HA共表达的重组腺病毒载体Ad-HA-HCVcore 1b。为进一步研究丙型肝炎病毒1b亚型引起丙肝感染中胰岛素抵抗的作用机制提供了方法。

Objective： To construct and identify the recombinant adenovirus vector of co-expression HCV core 1b and HA.Methods： The HCV gene was cloned and intested into a pcmv5-HA plasmid,then was connect into pShuttle-CMV plasmid and trans-formed the plasmid into E.coli DH5α.The recombinant plasmid pShuttle-CMV-HA-HCV core 1b was screened and transformed into BJ5183 to perform homologous recombination with pAdEasy-1.A recombinant adenovirus vector was acquired.Then it was transfected into AD293 cells to prepare the replication deficient adenovirus Ad-HA-HCV core 1b.AD293 cells was infected with Ad-HA-HCV core 1b and the expression of HA-HCV core 1b was detected by RT-PCR and western blot.Results：（1） The recombinant plasmid pShut-tle-CMV-HA-HCV core 1b were constructed successfully.（2）After recombination of pShuttle-CMV-HA-HCV core 1b and pAdEasy-1,the product was digested by restriction endonuclease PacI.In gel electrophoresis,they performed as the 3.0 kb segment which was as the same segment as prospection.（3） Ad-HA-HCV core 1b could infect AD293 cells and the extrinsic genes can be expressed in cells.Conclusion： The recombinant adenoviral vector Ad-HA-HCV core 1b was constructed successfully.