丙型肝炎病毒非结构蛋白NS2的核酸适体对病毒生活周期的影响
发布时间：2019-01-01 16:47
【摘要】：世界上大约有3%(1.7亿)的人感染了丙型肝炎病毒(HCV)，HCV可引起严重的肝脏疾病，如慢性肝炎、肝硬化乃至肝癌。由于缺少针对丙肝的疫苗，且目前的治疗方法有一定的局限性，研究新型、高效的抗HCV药物的重任迫在眉睫。NS2蛋白能够影响子代病毒颗粒装配，也能通过干扰宿主细胞的生理过程来促进病毒的复制。以NS2蛋白作为药物研究的靶点，具有巨大的潜力。核酸适体是一类新型的识别分子,具有亲和力强、选择性高、稳定性好、易于修饰等优点，，广泛用于生物检测和新药开发等方面。在HCV编码的非结构蛋白中，针对NS2蛋白的药物尚未见报道。本文以NS2蛋白为靶标，筛选出特异结合的核酸适体，并研究了适体对病毒生活周期的影响，取得了以下结果： （1）采用普通PCR方法成功扩增出HCV NS2基因，并将目的基因成功构建到pET28b原核表达载体中。 （2）将重组质粒pET28b-NS2转到BL21感受态细胞中进行蛋白大量表达，发现此蛋白可溶性低。采用超速离心法，并加入膜表面活性剂—月桂酸，可获得可溶性强的目的蛋白，用镍柱进一步纯化得到蛋白。 （3）以纯化的NS2蛋白为靶标，经过5轮筛选，得到特异结合的ssNDAaptamers。从中选择三条适体（NS2-1、NS2-2、NS2-3）分析发现，这3种核酸适体能够特异结合到NS2蛋白上，不会与对照蛋白HCV NS5B和LacZ发生非特异性结合。 （4）用荧光定量PCR和免疫荧光法检测转染核酸适体后细胞中的病毒复制和释放能力变化，发现核酸适体以一种呈剂量依赖型的方式抑制病毒复制，此外核酸适体NS2-2、NS2-3还能够抑制病毒颗粒的释放。结果表明针对NS2蛋白的核酸适体能够抑制病毒的复制和释放，有望研究成一种新型的靶向治疗HCV的药物。
[Abstract]:About 3% (170 million) of people in the world are infected with hepatitis C virus (HCV), which can cause serious liver diseases, such as chronic hepatitis, liver cirrhosis and even liver cancer. Due to the lack of a vaccine against hepatitis C and the limitations of current treatment methods, it is urgent to study new and efficient antiviral drugs. NS2 protein can affect the assembly of viral particles in offspring. Virus replication can also be promoted by interfering with the physiological processes of host cells. Using NS2 protein as the target of drug research has great potential. Aptamer is a new class of recognition molecules with high affinity, high selectivity, good stability and easy modification. It is widely used in biological detection and new drug development. Among the non-structural proteins encoded by HCV, drugs targeting NS2 proteins have not been reported. In this paper, NS2 protein was used as the target to screen out the specific binding aptamers, and the effects of aptamers on virus life cycle were studied. The following results were obtained: (1) the HCV NS2 gene was successfully amplified by ordinary PCR method. The target gene was successfully constructed into pET28b prokaryotic expression vector. (2) the recombinant plasmid pET28b-NS2 was transfered into BL21 receptive cells to express the protein in large quantities, and it was found that the protein was low soluble. The soluble target protein was obtained by ultracentrifugation and membrane surfactant-lauric acid. The protein was further purified by nickel column. (3) the purified NS2 protein was used as the target, and the specific binding ssNDAaptamers. was obtained after five rounds of screening. Analysis of three aptamers (NS2-1,NS2-2,NS2-3) showed that the three aptamers could specifically bind to NS2 protein and could not bind to HCV NS5B and LacZ. (4) the ability of virus replication and release after transfection of nucleic acid aptamer was detected by fluorescence quantitative PCR and immunofluorescence method. It was found that the aptamer inhibited virus replication in a dose-dependent manner, and the aptamer NS2-2, inhibited virus replication in a dose-dependent manner. NS2-3 can also inhibit the release of virus particles. The results showed that the aptamer targeting NS2 protein could inhibit the replication and release of the virus, and it was expected to be a novel drug targeting the treatment of HCV.
【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373
本文编号：2397844
[Abstract]:About 3% (170 million) of people in the world are infected with hepatitis C virus (HCV), which can cause serious liver diseases, such as chronic hepatitis, liver cirrhosis and even liver cancer. Due to the lack of a vaccine against hepatitis C and the limitations of current treatment methods, it is urgent to study new and efficient antiviral drugs. NS2 protein can affect the assembly of viral particles in offspring. Virus replication can also be promoted by interfering with the physiological processes of host cells. Using NS2 protein as the target of drug research has great potential. Aptamer is a new class of recognition molecules with high affinity, high selectivity, good stability and easy modification. It is widely used in biological detection and new drug development. Among the non-structural proteins encoded by HCV, drugs targeting NS2 proteins have not been reported. In this paper, NS2 protein was used as the target to screen out the specific binding aptamers, and the effects of aptamers on virus life cycle were studied. The following results were obtained: (1) the HCV NS2 gene was successfully amplified by ordinary PCR method. The target gene was successfully constructed into pET28b prokaryotic expression vector. (2) the recombinant plasmid pET28b-NS2 was transfered into BL21 receptive cells to express the protein in large quantities, and it was found that the protein was low soluble. The soluble target protein was obtained by ultracentrifugation and membrane surfactant-lauric acid. The protein was further purified by nickel column. (3) the purified NS2 protein was used as the target, and the specific binding ssNDAaptamers. was obtained after five rounds of screening. Analysis of three aptamers (NS2-1,NS2-2,NS2-3) showed that the three aptamers could specifically bind to NS2 protein and could not bind to HCV NS5B and LacZ. (4) the ability of virus replication and release after transfection of nucleic acid aptamer was detected by fluorescence quantitative PCR and immunofluorescence method. It was found that the aptamer inhibited virus replication in a dose-dependent manner, and the aptamer NS2-2, inhibited virus replication in a dose-dependent manner. NS2-3 can also inhibit the release of virus particles. The results showed that the aptamer targeting NS2 protein could inhibit the replication and release of the virus, and it was expected to be a novel drug targeting the treatment of HCV.
【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373
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