丙型肝炎病毒核心蛋白在大肠杆菌和毕赤酵母中的表达

发布时间：2018-04-08 16:35

  本文选题：丙型肝炎病毒　切入点：Core蛋白　出处：《西北大学》2006年硕士论文

【摘要】：丙型肝炎病毒(Hepatitis C virus,HCV)感染是导致慢性肝炎、肝硬化和肝癌的重要原因之一。目前尚无有效的疫苗及治疗药物,对HCV感染的早期诊断是控制HCV传播的重要手段。 HCV感染的临床诊断途径有:检测血清HCV RNA、HCV抗体及HCV抗原。目前PCR检测HCV RNA是HCV诊断最灵敏、特异的方法,但其操作复杂,易造成污染,且价格昂贵,不适于大规模的筛查应用。HCV患者血清中HCV抗原水平很低,常规免疫学方法难以获得阳性结果。故HCV诊断最常用的方法是利用HCV特异性蛋白质抗原来筛查血清中HCV抗体。 HCV核心蛋白(Core蛋白)高度保守且有很强的免疫原性,可诱发高水平的特异性体液免疫及细胞免疫,是HCV诊断试剂中必不可少的组分之一。近年来Core蛋白先后在大肠杆菌中获得了表达,但是大肠杆菌不具有真核生物的基因表达调控机制和蛋白质的加工修饰能力,表达的蛋白往往形成的包涵体,需要经过变性、复性等处理才能应用。与大肠杆菌相比,毕赤酵母是单细胞真核生物,既具有生长快的特点,又能有效克服大肠杆菌系统缺乏蛋白翻译后加工、修饰的不足,表达外源蛋白可分泌到胞外,便于产品的分离纯化。因此本研究在大肠杆菌系统中表达Core蛋白并构建其毕赤酵母表达系统,初步对两个系统表达的Core蛋白进行对比。 将克隆有Core基因的原核表达载体pBVIL1-Core转化大肠杆菌HB101,温度诱导表达Core蛋白,进行SDS-PAGE及Western-Blot分析;同时通过PCR方法扩增Core基因,克隆入表达载体pPICZaA,筛选阳性克隆pPICZaA-Core。将经过酶切鉴定和测序正确的阳性克隆电转入毕赤酵母GS115中,挑取酵母转化单菌落,接种于BMGY培养基,30℃、250r/min培养至OD_(600)=2.0时室温1,500r/min离心5min,用BMMY培养基重悬菌体使OD_(600)=1.0,重新放入摇床进行诱导。每24h补加甲醇至终浓度为1.0%,连续诱导72h,诱导后上清进行Western-Blot分析。 结果显示pBVIL1-Core的表达产物经SDS-PAGE分析出现一条约31KD的带,与预期融合蛋白的分子量相符,表达蛋白存在于包涵体中且表达量占菌体总蛋白的20%,Western-blot显示诱导后菌体在相应位置出现特异性杂交带,表明
[Abstract]:Hepatitis C virus (HCV) infection is one of the important causes of chronic hepatitis, liver cirrhosis and liver cancer.At present, there are no effective vaccines and therapeutic drugs. The early diagnosis of HCV infection is an important means to control the spread of HCV.The methods of clinical diagnosis of HCV infection were as follows: detection of serum HCV RNA antibody and HCV antigen.At present, PCR detection of HCV RNA is the most sensitive and specific method for the diagnosis of HCV, but its operation is complex, easy to cause pollution, and expensive, so it is not suitable for large-scale screening application. The level of HCV antigen in serum of patients with HCV is very low.It is difficult to obtain positive results by routine immunological methods.Therefore, the most commonly used method for the diagnosis of HCV is to screen HCV antibody in serum by HCV specific protein.HCV core protein is highly conserved and has strong immunogenicity, which can induce high level of specific humoral immunity and cellular immunity. It is an essential component of HCV diagnostic reagents.In recent years, Core protein has been expressed in E. coli, but E. coli does not have eukaryotic gene expression regulation mechanism and protein processing modification ability, the expressed protein often forms inclusion body, which needs to be denatured.Renaturation and other treatment can be applied.Compared with Escherichia coli, Pichia pastoris is a single cell eukaryote, which has the characteristics of fast growth and can effectively overcome the deficiency of protein translation and modification in Escherichia coli system. The expression of exogenous protein can be secreted out of the cell.It is easy to separate and purify the product.In this study, Core protein was expressed in Escherichia coli and Pichia pastoris expression system was constructed, and the two Core proteins were compared.The prokaryotic expression vector pBVIL1-Core with Core gene was transformed into Escherichia coli HB101, and the Core protein was expressed by temperature induction. The Core gene was amplified by PCR method and cloned into the expression vector pPICZaA to screen the positive clone pPICZaA-Core.The positive clones identified by enzyme digestion and sequenced were transferred into Pichia pastoris GS115.At room temperature, 1500r/min was centrifuged for 5 mins at room temperature when inoculated on BMGY medium at 30 鈩，

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