登革病毒四型联合重组包膜蛋白Ⅲ区的表达和免疫原性鉴定  

Expression and Immunological Analysis of Recombinant Tetravalent Envelope Domain Ⅲ Protein of Dengue Virus

作　　者：邱丽娟[1,2] 赵玉娇[1,2] 李多[1,2] 黄新伟[1,2] 王晓丹[1,2] 席珏敏[1,2] 潘玥[1,2] 陈俊英[1,2] 孙强明[1,2] 

机构地区：[1]北京协和医学院/中国医学科学院医学生物学研究所,昆明650118 [2]云南省重大传染病疫苗研发重点实验室,昆明650118

出　　处：《中国生物工程杂志》2016年第1期1-6,共6页China Biotechnology

基　　金：国家"十二五"重大新药创制科技重大专项(2012ZX09104-302);国家自然科学基金面上项目(81171946);云南省科技厅社会发展科技计划(2011CA016);云南省自然科学基金(2009ZC187M;2012FB188)资助项目

摘　　要：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。Four serotypes of dengue virus（ DENV1-4） circulate globally,causing more human illness than any other arthropod-borne virus.So far,licensed vaccine against dengue is not yet available.The greatest risk factor for severe dengue is a previous infection with a different DENV serotype.The theory for why sequential heterotypic infections increases risk of severe disease is ＂antibody-dependent enhancement＂（ ADE）.Previous research determinants that the predominant immune response is against the E protein which is comprised of three structurally distinct domains（ DI,DⅡ,DⅢ）.EDⅢ might exclusively generate potent neutralizing antibodies with little or no cross-reactivity,and therefore of reduced ability to promote ADE.This suggested that EDⅢ is more likely to be an attractive vaccine.Here,a tetravalent recombinant dengue domain Ⅲ protein gene sequence were yeast expression optimized and synthesized,then expression plasmids were constructed and transducted into pichia pastoris to selected an high-efficiency expression strain.EDⅢ protein were obtained by inducing expression and purification followed by identification using SDS-PAGE,Western blot.The result showed that EDⅢ expression plasmid was successfully constructed and highly expressed in pichia pastoris.Mice immunized with tetravalent DENV DⅢ generated higher levels of serum titer.In conclusion,a tetravalent recombinant dengue domain Ⅲ protein with high purity were obtained.It lays a good foundation for the development of dengue vaccine.

关 键 词：登革病毒 包膜EDⅢ蛋白 疫苗 重组蛋白 

分 类 号：Q939[生物学—微生物学]