Tang Kie Hie, Yusoff Khatijah, Tan Wen Siang
Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Malaysia.
J Virol Methods. 2009 Aug;159(2):194-9. doi: 10.1016/j.jviromet.2009.03.015. Epub 2009 Mar 26.
Hepatitis B is a major public health problem worldwide which may lead to chronic liver diseases, cirrhosis and hepatocellular carcinoma. An interaction between hepatitis B virus (HBV) envelope protein, particularly the PreS1 region, and a specific cell surface receptor is believed to be the initial step of HBV infection through attachment to hepatocytes. In order to develop a gene delivery system, bacteriophage T7 was modified genetically to display polypeptides of the PreS1 region. A recombinant T7 phage displaying amino acids 60-108 of the PreS1 region (PreS1(60-108)) was demonstrated to be most effective in transfecting HepG2 cells in a dose- and time-dependant manner. The phage genome was recovered from the cell lysate and confirmed by PCR whereas the infectious form of the internalized phage was measured by a plaque-forming assay. The internalized phage exhibited the appearance of green fluorescent dots when examined by immunofluorescence microscopy. Surface modification, particularly by displaying the PreS1(60-108) enhanced phage uptake, resulting in more efficient in vitro gene transfer. The ability of the recombinant phage to transfect HepG2 cells demonstrates the potential of the phage display system as a gene therapy for liver cancer.
乙型肝炎是全球主要的公共卫生问题,可能导致慢性肝病、肝硬化和肝细胞癌。乙肝病毒(HBV)包膜蛋白尤其是前S1区与特定细胞表面受体之间的相互作用被认为是HBV通过附着于肝细胞而感染的起始步骤。为了开发一种基因递送系统,对噬菌体T7进行了基因改造,使其展示前S1区的多肽。一种展示前S1区第60 - 108位氨基酸(PreS1(60 - 108))的重组T7噬菌体被证明在以剂量和时间依赖方式转染HepG2细胞方面最有效。噬菌体基因组从细胞裂解物中回收并通过PCR进行确认,而内化噬菌体的感染形式则通过噬斑形成试验进行测定。通过免疫荧光显微镜检查时,内化的噬菌体呈现绿色荧光点的外观。表面修饰,特别是展示PreS1(60 - 108)可增强噬菌体摄取,从而导致更有效的体外基因转移。重组噬菌体转染HepG2细胞的能力证明了噬菌体展示系统作为肝癌基因治疗方法的潜力。