HBcAg展示技术表达含有礁珊瑚荧光蛋白的病毒样颗粒

发布时间：2018-10-23 10:03

【摘要】：乙肝核心抗原(HBcAg)具有天然形成病毒样颗粒的能力,同时这种结构能使机体产生特异性的体液免疫和细胞免疫作用,故成为了良好的免疫载体蛋白。本实验用含有231个氨基酸的大分子礁珊瑚荧光蛋白(ZsGreen)作为的目标抗原,以具备颗粒装配区的乙肝核心抗原蛋白为抗原递呈的载体,通过基因克隆技术将表达ZsGreen蛋白的基因插入HBcAg中位于颗粒表面的主要免疫优势抗原决定簇c/e1表位区,并将这部分融合基因与载体pET28a进行连接,构建重组表达载体。重组表达载体转化至大肠杆菌BL21(DE3),经过对表达条件及发酵工艺条件的优化后,重组融合蛋白(Zscore)在大肠杆菌中的表达量达到20%以上。表达菌体经过破璧,离心澄清,硫酸铵沉淀,阳离子及分子筛层析等方法的分离纯化,最终获得颗粒纯度大于80%的重组融合蛋白。重组融合蛋白经过系统的结构分析及免疫学研究,证实:(1)HBcAg在融合大分子蛋白ZsGreen后其产物仍能形成病毒样颗粒(VLP)的结构,(2)且展示在重组蛋白颗粒表面融合表达的ZsGreen能激发更高的免疫原性。这一系列的证据表明乙肝核心抗原展示表达外源蛋白的平台具备疫苗表达载体的能力,为进一步的研究开发打下基础。
[Abstract]:Hepatitis B core antigen (HBcAg) has the ability of forming virus-like particles naturally, and this structure can make the body produce specific humoral and cellular immunity, so it has become a good immune carrier protein. In this experiment, the fluorescent protein (ZsGreen) of reef coral containing 231 amino acids was used as the target antigen, and the hepatitis B core antigen protein with granular assembly region was used as the antigen presenting carrier. The gene expressing ZsGreen protein was inserted into the c/e1 epitope of the main immune dominant antigen determinant on the surface of particles in HBcAg by gene cloning technique. The fusion gene was ligated with the vector pET28a to construct the recombinant expression vector. The recombinant expression vector was transformed into Escherichia coli BL21 (DE3). After optimizing the expression conditions and fermentation conditions, the recombinant fusion protein (Zscore) was expressed more than 20% in Escherichia coli. The recombinant fusion protein with particle purity of more than 80% was obtained by the separation and purification of the expressed bacteria through the separation and purification of the expressed bacteria by centrifugation, ammonium sulfate precipitation, cationic and molecular sieve chromatography and so on. The recombinant fusion protein was studied by systematic structural analysis and immunological analysis. The results showed that: (1) HBcAg could still form the structure of virus-like (VLP) after fusion of macromolecular protein ZsGreen, and (2) ZsGreen expressed on the surface of recombinant protein particles could stimulate higher immunogenicity. This series of evidences indicate that hepatitis B core antigen display the ability to express foreign protein on the platform of vaccine expression vector and lay the foundation for further research and development.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

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