整合缺陷型重组慢病毒载体构建及HCV重组假型慢病毒颗粒的制备与性状分析

Development and Characterization of Integration-deficient Lentiviral Vector and HCV Recombinant Pseudotyped Lentiviral Particles

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 novel hepatitis C virus(HCV) particle vaccine,the traditional three plasmid-base lentiviral system was modificated: the packaging plasmid pHR'CMV Δ R8.2 was transformed into the integration-deficient packaging plasmid pCMVΔR8.2D64E,and confirmed the integration-defective properties in vitro.The reporter gene(GFP) in transfer plasmid was replaced by the HCV non-structural gene NS3.HCV envelope plasmid with different subtypes(from three representative subtypes 1a or 1b or 2a) combined with the above two modified lentiviral plasmids were cotransfected 293FT cells.HCV integration-defective pseudotyped lentiviral particle(IDLVpp) vaccines carrying HCV NS3 were packaged.The HCV IDLVpp were identified by Western blot,and the particle structure was observed by electron microscope.The expression of transgenic HCV NS3 in Huh7 cells infected by HCV IDLVpp was also observed.So a scientific and experimental basis for novel effective HCV vaccines development and applications of HCV IDLVpp vaccines was provided.