抗HCV包膜蛋白E1/E2高中和活性单链抗体的筛选及抗病毒效果评价

Hepatitis C virus (HCV) infection is one of the leading causes of chronic liver disease, which is associated with a high risk of cirrhosis and hepatocellular carcinoma. Currently there was no available vaccine to prevent HCV infection. The standard therapy for chronic infection with pregylated alpha interferon (IFN-α) and ribavirin is only effective in 40%-60% of cases. Although new effective HCV antiviral regimens which were approved recently can cure the vast majority of patients, but too expensive. In former study, HCVpp and HCVcc have been obtained and Single Chain Fragment Variable (ScFv) gene has been screened from HCV NS5B with ribosome display technology. We will investigated the humor immune responses in HCV patients by neutralization assay with HCVpp. PBMC from patients with higher titers neutralization antibodies will be isolated, VH and VL gene will be amplified by PCR from the cDNA library inverse transcription from total RNA of PBMC. ScFv gene library of HCV E1/E2 will be obtained by ribosome display technology. The ScFv gene will be identified and expressed in coli. After obtaining ScFv protein, the ScFv inhibitory activity to HCVpp infected Huh-7.5 cells will be analyzed. And HCVcc replication in Huh7.5 will be determined after treating with the ScFv. These study would be provided new strategy and reliable experiment results for development of HCV antibody medicine.

丙型肝炎病毒（HCV）感染严重威胁人类健康，极易慢性化和发展为肝硬化及肝癌，迄今疫苗研究进展缓慢。重组干扰素联合利巴韦林抗病毒治疗效果不尽人意，新型蛋白酶抑制剂显示较好的疗效，但价格昂贵，迫切需要研制更加经济和安全有效的新型抗病毒药物。本研究在建立HCVpp、HCVcc系统及利用核糖体展示库技术筛选抗HCV NS5B蛋白单链抗体（ScFv）工作基础上，通过HCVpp测定中和抗体，选择持续产生高中和抗体滴度的HCV患者，分离其PBMC，扩增全套重链可变区（VH）和轻链可变区（VL）的基因，构建抗包膜蛋白的核糖体展示的ScFv基因库，筛选针对包膜蛋白E1/E2的ScFv；对所筛选到的ScFv进行表达和鉴定；观察抗E1/E2的ScFv对HCVpp感染HuH-7.5细胞的阻断作用，以及对HCVcc复制的抑制作用。该研究结果可为研发具有高中和活性的新型HCV抗体药物提供可靠的实验依据。

丙型肝炎病毒（HCV）感染后极易慢性化和发展为肝硬化及肝癌，迄今尚无预防疫苗。重组干扰素联合利巴韦林抗病毒治疗效果不尽人意，DAA药物显示较好的疗效，但价格昂贵，迫切需要研制更加经济和安全有效的新型抗病毒药物。本课题利用核糖体展示库技术，通过HCVpp测定中和抗体，选择持续产生高中和抗体滴度的HCV患者，分离其PBMC，扩增全套重链可变区（VH）和轻链可变区（VL）的基因，构建抗包膜蛋白的核糖体展示的ScFv基因库，筛选针对包膜蛋白E2核心区的ScFv；筛选到抗包膜蛋白的的ScFv，对ScFv进行表达和鉴定；并观察抗E1/E2的ScFv对HCVpp感染HuH-7.5细胞的阻断作用，以及对HCVcc复制的抑制作用。结果筛选到的ScFv能有效抑制HCVpp和HCVcc感染HuH-7.5。该研究结果可为研发具有高中和活性的新型HCV抗体药物提供可靠的实验依据。课题已发表相关论文，正准备申请专利。