嵌合HEV表位的HBcAg颗粒在毕赤酵母中的分泌表达

发布时间：2018-04-24 23:25

  本文选题：乙肝病毒核心抗原 + 融合蛋白　； 参考：《厦门大学》2008年硕士论文

【摘要】： 毕赤酵母是近年来发展迅速,应用广泛的真核表达系统,能对蛋白进行良好的翻译后修饰,如信号肽加工、蛋白折叠、二硫键形成、部分脂基化和O-连接和N-连接糖基化修饰等。此外,毕赤酵母可以分泌表达外源蛋白,由于其自身胞外分泌的蛋白量很低,与胞内表达外源蛋白的方法相比,没有细胞破碎过程中的细胞内溶物的释放以及所带来的细胞碎片的分离等问题,具有易于分离纯化、下游工艺简单等优点。 戊型肝炎是由戊型肝炎病毒(HEV)引起的一种世界性的危害严重的传染病,主要流行于亚洲、非洲和墨西哥等地区。HEV基因组含3个开放读码框架(ORF),其中ORF2编码660个氨基酸的肽链,为病毒主要结构蛋白。经研究发现,ORF2aa423-438在介导病毒与宿主细胞的结合中起重要作用,是12A10单克隆抗体的识别表位。 乙型肝炎病毒核心抗原(HBcAg)是一种良好的免疫载体,在原核和真核表达系统中均能自我组装成颗粒。当外源表位插入其MIR区时,仍可以形成颗粒,并且外源表位暴露在颗粒的表面,得到充分的展示。本研究将融合12A10表位肽的HBcAg的基因导入毕赤酵母中,构建工程菌株,进行分泌表达,优化了重组菌株的培养条件,并对纯化后的蛋白进行性质分析,验证了融合蛋白上的12A10表位有良好的免疫原性,探讨了HEV表位疫苗的可行性,为酵母分泌表达蛋白颗粒提供了一个很好的范例。 首先,将嵌合有HEV受体相关表位12A10的HBcAg基因克隆到毕赤酵母分泌表达载体pPIC9k,构建重组表达质粒pPIC9k-HBc149-12A10,用内切酶SacI将其线性化后电转入毕赤酵母菌株GS115中。用G418筛选得到高抗性的转化子经过培养和甲醇诱导,培养上清进行SDS-PAGE电泳,与对照GS115/pPIC9k比较,在22kD处有一条差异蛋白带;通过Western Blot鉴定,该条带与单抗12A10有特异性反应。 其次,用MGY/MM、BMG/BMM和BMGY/BMMY三组培养基进行重组菌株的培养,间接法ELISA表明,BMGY/BMMY明显优于其他两组培养基。以BMGY/BMMY培养基为基础,进行诱导温度、诱导甲醇浓度和诱导初始pH值的单因子实验。在以上实验的基础上,通过正交试验进一步优化培养条件,得到最佳诱导条件:初始pH值为7.0,甲醇浓度为1.0%,诱导温度为25℃。培养上清液通过切向流浓缩、更换缓冲液后,进行疏水层析纯化。 最后,CsCl等密度梯度离心测得分泌出来的重组蛋白的密度为1.32g/ml。透射电镜观察显示,纯化的重组蛋白为均一的直径30nm左右的空心颗粒。小鼠免疫实验表明,纯化颗粒免疫8周后鼠血清中的特异性12A10抗体滴度可达到1.6×10~5。与含有12A10表位的p239重组颗粒的免疫原性相比,产生12A10特异性抗体水平有明显的提高,提示重组颗粒较好地呈递了HEV受体相关的非免疫优势表位。 在本研究中,首次构建了能够分泌表达携带有表位多肽的HBcAg蛋白颗粒的毕赤酵母工程菌,为毕赤酵母胞外分泌表达其它大尺度的重组病毒颗粒提供了参考,为研究携带表位多肽的颗粒载体的疫苗提供了范例,为克服酵母胞内表达出现的难以纯化的难题提供了新的途径。
[Abstract]:Pichia pastoris is a kind of eukaryotic expression system which has been developed rapidly in recent years . It can be used for post - translational modification of the protein , such as signal peptide processing , protein folding , disulfide bond formation , partial glycosylation , O - connection and N - linked glycosylation modification .



HEV genome contains three open reading frames ( ORFs ) which encode 660 amino acid peptide chains , which are the main structural proteins of the virus . The results show that ORF2aa423 - 438 plays an important role in mediating the binding of virus to host cell , and is the recognition epitope of monoclonal antibody 12A10 .



The core antigen of hepatitis B virus ( HBV ) is a kind of good immune carrier , which can be self - assembled into particles in both prokaryotic and eukaryotic expression systems . When the exogenous epitope is inserted into its MIR region , it can still form particles , and the exogenous epitope is exposed on the surface of the particles to obtain a sufficient display . The present study provides a good example of the feasibility of the epitope vaccine , and provides a good example for yeast secretion expressing protein particles .



First of all , we cloned into Pichia pastoris secretion expression vector Z9k , and then transformed into Pichia pastoris GS115 by restriction enzyme SacI . The high - resistance transformants were cultured and methanol - induced to culture supernatant for SDS - PAGE electrophoresis . The band was identified by Western Blot .



The results showed that BMGY / BMMY was better than that of other two groups . The optimum conditions were obtained by orthogonal test . The optimal conditions were as follows : initial pH value was 7.0 , methanol concentration was 1.0 % , induction temperature was 25 鈩，

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