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为提高慢病毒载体应用的安全性及改进HCV新型颗粒疫苗的制备,首先对传统的慢病毒三质粒系统进行改造:将包装质粒pHR’CMVΔR8.2改造成整合缺陷型的包装质粒pHR’CMVΔR8.2D64E,并在体外感染实验验证其整合缺陷性;将转移质粒中的报告基因GFP替换成HCV的NS3基因,选用表达3种亚型(1a、1b、2a)HCV包膜E1E2的包膜质粒,用改造后的三质粒系统制备了3种亚型(1a,1b,2a)的假型丙型肝炎病毒(HCV)整合缺陷型慢病毒颗粒,并用Western blot方法确定了颗粒上包膜蛋白的表达,通过电镜可观察到浓缩后的HCV慢病毒颗粒结构,HCV慢病毒颗粒感染Huh7细胞后可观察到转基因NS3的表达。为安全高效的慢病毒载体的应用及HCV假型颗粒疫苗研发提供了新的技术路线。
In order to improve the safety of lentiviral vector and improve the preparation of new HCV particle vaccine, we firstly modified the traditional lentivirus three plasmid system: the packaging plasmid pHR’CMVΔR8.2 was transformed into the defective packaging plasmid pHR’CMVΔR8. 2D64E was used to test its integration defect in vitro. The reporter gene GFP in the transfer plasmid was replaced by NS3 gene of HCV, and the envelope plasmids expressing HCV envelope E1E2 in three subtypes (1a, 1b, 2a) Three pseudotyped hepatitis C virus (HCV) -conjugated lentivirus particles of three subtypes (1a, 1b, 2a) were prepared using the modified three-plasmid system and the expression of the envelope protein on the granule was determined by Western blot , The condensed lentiviral particle structure was observed by electron microscopy, and the expression of transgenic NS3 was observed after infected with HCV lentivirus particles Huh7 cells. It provides a new technical route for the safe and efficient application of lentiviral vector and the development of HCV pseudotyped particle vaccine.