乙肝病毒X基因(HBVX)慢病毒载体的构建和鉴定.pdfVIP

乙肝病毒X 基因(HBVX)慢病毒载体的构建和鉴定 摘 要： 目的：构建在正常组织来源的肝细胞HL7702 表达的乙型肝炎病毒（HBV ） X 蛋白的基因HBVX 的重组慢病毒载体，研究其在体外的感染效率。 方法:采用PCR 技术从先前构建的载体真核表达载体pcDNA3-X 中扩增出含 HBVX 基因的DNA 片段，用In-Fusion 技术构建表达载体，经PCR 、酶切和单 向测序验证。利用jetPEI 转染试剂三质粒共转染293T 细胞，有限稀释法计算慢 病毒颗粒的滴度。用慢病毒液感染正常组织来源的肝细胞HL7702 ，96 h 后初步 观察慢病毒颗粒的感染情况。Real-time PCR 和Western blot 法检测重组细胞HBV X 基因的表达。 结果:成功构建HBVX 基因的慢病毒表达载体，经293T 细胞包装后，慢病 毒颗粒浓缩后滴度为4x107 7 IU/m1 。用滴度为4x10 IU/m1 的慢病毒感染正常组织 来源的肝细胞HL7702 96h 后，被感染的细胞内可见强弱不等的荧光。Real-time PCR 和Western blot 检测均证实重组肝细胞有HBVX 基因表达。 结论: 成功构建乙肝病毒X 基因(HBVX)慢病毒表达载体，为今后HBVX 基 因对肝细胞的影响的研究提供工具。 关键词: 慢病毒载体 HBVX HL7702 2 Construction and Identification of a Lentiviral Vector Expressing Hepatitis B virus X Gene ABSTRACT Objective To investigate the transfection efficiency of the recombinant HBVX-containing lentiviral vector in the HL7702 cell line from normal tissue . Methods The lentiviral vector was reconstructed by insert the lentiviral vectors with the HBVX gene fragment, which was isolated from pcDNA3-X vector. It was analysised with PCR restriction digestion and bidirectional sequencing.The recombination lentiviral particles were produced by the packaging 293T cell by using the jetPEI method, the culture supernatant was harvested and the titration of them were calculated by the limiting dilution.The HL7702 cells were transduced by the lentiviral particles,after 96h the GFP expression was observed under the fluorescence microscope. The expression level of HBV X in cells were examed by the Real-time PCR and Western blot. Results