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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-HCV core 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-HCV core 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-HCV core 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。

英文摘要:

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 transformed 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 pShuttle- 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.

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