DEVELOPMENT OF THERAPY FOR HEPATITIS C USING PROTEIN TRANSDUCTION METHOD

虽然干扰素（IFN）治疗可以根除慢性丙型肝炎患者的丙型肝炎病毒（HCV），但到目前为止，根除率仅为30%，这并不令人满意。实现从患者身上根除HCV的最严重障碍是难以清除肝细胞中残留的HCV，或清除HCV感染的肝细胞。为了提供一种从患者体内根除HCV的新方法，我们建立了一个利用人类免疫缺陷病毒TAT蛋白的蛋白转导结构域（ptd）的实验系统。与ptd融合的蛋白注入腹腔后可分布到所有器官。通过安排被HCV基因组NS3结构域编码的HCV蛋白酶识别序列干预的Caspase 3蛋白酶亚基p17和p12，我们建立了一种仅杀死感染HCV的肝细胞的有效方法；丙型肝炎病毒感染的肝细胞具有激活Caspase 3的丙型肝炎病毒蛋白酶，导致肝细胞自杀。将编码pdd - caspase3 p17-NS3裂解位点-p12-NS3裂解位点的质粒pTAT-Casp3-NS3pcs与HCV NS3蛋白酶表达载体结合，诱导HepG2细胞凋亡。另一方面，我们建立了携带HCV NS2-NS5 cDNA的转基因小鼠系。小鼠肝脏中检测到NS2-NS5B基因转录的mRNA。利用这些小鼠，正在进行一项实验，注射从杆状病毒纯化的蛋白，该蛋白转染了pTAT-Casp3-NS3pcs。

Although interferon (IFN) therapy can eradicate hepatitis C virus (HCV) from chronic hepatitis C patients, the eradication rate, to date, is only 30%, which is not satisfactory. The most serious impediment in achieving eradication of HCV from patients lies in the difficulty to clear residual HCV in hepatocytes, or to eliminate HCV-infected hepatocytes.To provide a novel method for eradicating HCV from patients, we conducted to establish an experimental system that utilizes the protein-transducting-domain (ptd) of the TAT protein from human immunodeficiency virus. Fusion proteins with the ptd can be distributed to all organs upon the injection of the proteins into the peritoneal cavity. By arranging the Caspase 3 protease subunits p17 and p12 intervened by the recognition sequences of HCV protease that is coded by the NS3 domain of HCV genome, we produced an efficient method to kill only hepatocytes infected with HCV ; HCV-infected hepatocytes possess the HCV protease that activates the Caspase 3, leading the hepatocytes into suicide.By introducing the plasmid pTAT-Casp3-NS3pcs, which encodes the ptd-Caspase3 p17-NS3 cleavage site-p12-NS3 cleavage site, with an expression vector of HCV NS3 protease, HepG2 cells underwent apoptosis.On the other hand, we established transgenic mouse lines carrying the cDNA for HCV NS2-NS5. mRNA transcribed from the NS2-NS5B genes was detected in the mouse liver. Using these mice, an experiment is underway of injecting the protein purified from baculovirus transfected with pTAT-Casp3-NS3pcs.