作为基因转移载体的乙肝病毒衣壳的制备及其功能的初步鉴定

Construction of a liver targeting gene transfer vector using hepatitis B virus envelope particles and assessment of its function

目的构建具有肝脏靶向性的乙型肝炎病毒衣壳基因转移载体。方法采用PEG8000病毒浓缩法提取HepG2.2.15细胞上清液中的乙型肝炎病毒,用β-丙内酯法制备乙型肝炎病毒衣壳,用它包裹5.3kb的绿色荧光蛋白质粒以测试其装载能力,并用ELISA法、PCR法、SDS-聚丙烯酰胺凝胶电泳、透射电镜等对它进行定量、定性研究,最后将其转染HepG2细胞,通过荧光显微镜观察绿色荧光蛋白表达情况,通过流式细胞仪检测细胞发光率。结果制备的乙型肝炎病毒衣壳载体保留病毒衣壳的表面蛋白HBsAg+preS1+preS2,无病毒DNA;该载体对绿色荧光蛋白质粒较高装载容量,装载绿色荧光蛋白质粒后在肝癌细胞中有很高的转移效率,并能实现高表达。结论采用PEG8000病毒浓缩法、β-丙内酯法可从HepG2·2·15细胞上清液中制备乙型肝炎病毒衣壳基因转移载体,该载体具有良好的生物学功能。

Objective： To construct a liver targeting gene transfer vector using hepatitis B virus envelope particles. Methods： Hepatitis B viruses were obtained from the supematant of HepG 2.2. I5 cells by a PEG8000 system and were inactivated by β-propiolactone to prepare hepatitis B virus envelope. The hepatitis B virus envelope was used to pack 5.3 kb pIRES2-EGFP to assess their packing ability. Subsequently, the products were studied with ELISA, PCR, SDSPAGE, and electron microscopy. Finally, the product was used to transfect HepG2 cells and the green fluorescent protein （GFP） expression was observed under a fluorescent microscope. The rate of GFP positive cells was determined by flow cytometer. Results ： The acquired hepatitis B virus envelope retained the surface protein HBsAg ＋ pre S1 ＋ pre S2, but with no virus DNA. The prepared envelope had high packing ability for GFP and the packed GFP had a high transfection rate in HepG2 cell. Conclusion： Hepatitis B virus envelope has been successfully obtained from the supernatant of HepG 2. 2. 15 cells with a PEG8000 system and β-propiolactone.