摘要
目的:探索一种针对慢性乙型肝炎的特异性长效基因治疗手段。方法:改造乙型肝炎病毒(hepatitis Bvirus,HBV)为基因治疗载体,敲除其所有的开放阅读框(open reading frames,ORFs),同时保留包装信号序列,并且插入增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)作为报告基因。同时构建辅助细胞系,为上述病毒载体(假病毒)提供包装所需要的病毒蛋白。以该假病毒载体转染辅助细胞系,以酶联免疫方法检测细胞培养上清中乙型肝炎病毒抗原,并在荧光显微镜下观察EGFP的表达,用透射电镜观察培养上清中的病毒颗粒,同时用实时定量PCR的方法对分泌到细胞培养上清中的假病毒进行了定量检测。结果与结论:以假病毒载体转染辅助细胞后,电镜观察到培养上清中乙肝病毒的丹氏粒(Dane particle)。实时定量PCR分析结果显示,细胞分泌到培养上清的假病毒含量达103拷贝/μl。ELISA结果显示该上清中同时表达了HBV表面抗原和e抗原,通过荧光显微镜观察发现外源基因绿色荧光蛋白获得有效表达。
Objective：To develop a liver-specific gene delivery system for one-shot gene therapy of chronic hepatitis B virus infection. Methods： A genetically modified hepatitis B virus （the pseudovirus vector） was used as a liver-specific gene delivery viral vector. The pseudovirus vector was constructed by sequentially knocking out the viral genes （ X, C, P, and S） using PCR-based site-directed mutagenesis, and an enhanced green fluorescent protein gene （EGFP） was inserted into this pseudovirus as a reporter. Meanwhile host cell lines （helpers） expressing all the wide type HBV proteins were developed for the production of the pseudovirus. After the helpers were transfected with the pseudovirus vector, the conditioned media were analyzed for the expression of HBsAg and HBeAg by ELISA, and the pseudovirons by transmission electron microscopy and real-time PCR. The expression of EGFP was also examined by fluorescence microscopy. Results and Conclusion：After transfection with the pseudovirus, helper cells secreted the HBV pseudovirons （confirmed by real-time PCR and transmission electron microscopy）, HBsAg and HBeAg （by ELISA ）. The heterogeneous EGFP was expressed in the transfected helper cells.
出处
《军事医学科学院院刊》
CSCD
北大核心
2007年第4期338-342,共5页
Bulletin of the Academy of Military Medical Sciences
