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登革热在全球范围内广泛流行,但是目前为止却仍然没有疫苗上市,疫苗的开发迫在眉睫。抗体依赖增强感染效应是登革病毒疫苗开发中遇到的一个瓶颈问题。研究表明登革病毒的包膜蛋白Ⅲ区能够介导中和抗体产生,且诱导产生较少的交叉抗体或无交叉抗体,能够大大减弱抗体依赖增强感染效应,因而是登革热重组蛋白疫苗的首选靶标。通过酵母密码子优化后合成同时包含4种血清型登革病毒包膜蛋白Ⅲ区的四价联合DV EDⅢ蛋白序列,随后构建酵母表达质粒,并获得酵母表达菌株,经诱导后四联DV EDⅢ蛋白获得高效表达。通过Western blot、ELISA检测及蛋白质免疫原性鉴定,结果表明登革病毒四联DV EDⅢ蛋白表达质粒构建成功,重组蛋白在毕赤酵母获得高效表达,免疫小鼠后能够介导产生较高水平的血清效价。这表明已获得了能引起有效免疫反应的四型登革病毒EDⅢ蛋白,为登革病毒疫苗的研究提供了良好的基础。
Dengue is widely prevalent in the world, but so far no vaccines have yet been released and the development of vaccines is imminent. Antibody dependence enhances the infection effect is a bottleneck encountered in dengue virus vaccine development. Studies have shown that dengue virus envelope protein Ⅲ region can mediate neutralizing antibody production, and induced less cross-antibody or no cross-antibody, can greatly reduce the antibody dependence to enhance the infection effect, which is the preferred target of dengue recombinant protein vaccine . After optimization by yeast codon, tetravalent joint DV EDⅢ protein sequence containing the 4 serotypes of dengue virus envelope protein III was synthesized, then the yeast expression plasmid was constructed and the yeast expression strain was obtained. After induction, the quadruple DV EDⅢ protein Efficient expression. The results of Western blot, ELISA and protein immunogenicity identification showed that the expression plasmid of dengue virus quadruple DV EDⅢ protein was successfully constructed. The recombinant protein was highly expressed in Pichia pastoris and could induce higher levels of Serum titer. This indicates that we have obtained the ED III protein of type IV dengue virus that can induce an effective immune response, which provides a good foundation for the study of dengue virus vaccine.