乙型肝炎病毒全基因组1.3倍体重组腺病毒的构建与表达

Construction of recombinant adenovirus hepatitis B virus1.3 and its expression

摘要:

目的：构建乙型肝炎病毒（hepatitis B virus，HBV）重组腺病毒，为HBV的相关研究提供一种高效率、便捷的实验手段。方法：将HBV1.3倍体的片段构建到pAdTrack-TO4穿梭载体上，再经限制性核酸内切酶PmeⅠ线性化后转入含有腺病毒骨架载体pAd-Easy1的BJ5183感受态中进行重组并鉴定。鉴定正确后的重组腺病毒质粒经PacⅠ线性化后转染入HEK-293细胞进行病毒包装，获取HBV1.3倍体的重组腺病毒（Ad-HBV1.3）。通过荧光显微镜进行感染效率的检测；用ELISA、Western blot方法在蛋白水平检测HBV蛋白表达情况；小鼠尾静脉注射HBV1.3倍体的重组腺病毒，检测经腺病毒感染后的小鼠体内HBV表达情况。结果：在Ad-HBV1.3腺病毒感染后，细胞的感染效率可达到95%以上；ELISA实验检测实验组乙型肝炎s蛋白（hepatitis B virus S protein，HBs）、乙型肝炎e蛋白（hepatitis B virus E protein，HBe）表达量明显高于对照组；Western blot结果显示感染Ad-HBV1.3腺病毒后可有高水平的HBs表达；动物实验显示小鼠在注射Ad-HBV1.3腺病毒后体内HBs在第3天即可开始表达，5 d达到峰值，7 d后逐渐下降。结论：本研究成功构建了可表达HBV的重组腺病毒，其感染效率可高达95%，为HBV相关研究提供一种简单高效的研究手段。

Abstract:

Objective：To construct the recombinant adenovirus vector of hepatitis B virus（HBV） and to provide a more effective and convenient method for HBV-relative research. Methods：HBV 1.3 fold genome was subcloned to shuttle vector pAdTrack-TO4，then pAdTrack-TO4-HBV1.3 was linearized by PmeⅠ and transfected into BJ5183 cells containing pAd-Easy1. The confirmed recombi－nant plasmid with PacⅠ restriction endonuclease was linearized and transfected into HEK-293 cells to generate recombinant aden－oviruses containing HBV1.3 fold genome. Efficiency of the infection was detected by fluorescence microscope and expression of HBV proteins was tested by ELISA and Western blot. Intravenous recombinant adenovirus HBV1.3 was injected into C57BL mouse and changes of HBs expression in mouse were observed. Results：Cellular efficiency of recombinant adenovirus HBV1.3 infection could reach above 95%. Expression of HBs and HBe in experimental group was much higher than that in control group according to ELISA test. High-level HBs expression was detected by Western blot after recombinant adenovirus HBV1.3 infection. Animal experiment showed that after being injected with the recombinant adenovirus HBV1.3，HBs began to express on the 3rd d，peaked on the 5th d and declined gradually on the 7th d. Conclusion：Recombinant adenovirus with high infective efficiency is constructed and can express HBV easily，providing a much more efficiency method for HBV-relative study.