肠道病毒71型病毒样颗粒在汉逊酵母中的表达  

Expression of virus-like particles of enterovirus 71 in Hansenula polymorpha

作　　者：顾美荣[1] 宋琳琳[2] 许珊珊[2] 付作申 林福玉[2] 张现臣[2] 魏文进[2] 贾士儒[1] 

机构地区：[1]天津科技大学生物工程学院,300457 [2]北京民海生物科技有限公司研发中心,102600

出　　处：《中华微生物学和免疫学杂志》2013年第8期604-609,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家高技术研究与发展计划（863计划）（2012AA02A403）

摘　　要：目的应用汉逊酵母表达系统进行肠道病毒71型（EV71）病毒样颗粒（VLP）的表达。方法将经过汉逊酵母密码子优化的人EV71的P1和3CD基因片段克隆到汉逊酵母表达载体PMV上，获得重组表达质粒PMV．P1-3CD，转化汉逊酵母宿主菌AU0501，PCR方法及稳定传代培养筛选整合P1和3CD基因的重组菌株。重组菌种接种在含有1％甲醇的培养基中进行诱导表达，对表达产物进行SDS．PAGE、Western blot检测。挑选优胜表达菌株进行30L发酵罐发酵培养，发酵产物经过粗略纯化后进行电镜分析。结果筛选获得EV71重组表达菌株；SDS-PAGE检测结果显示在相对分子质量（M，）为26×10^3、33×10^3、35×10^3处有明显的VP3、VP1、VP0蛋白条带，M大小与预期的目的蛋白大小一致；Western blot检测结果显示表达产物与EV71-VP1单克隆抗体在犯为33×10^3处有较为明显的VPl反应条带，表达产物具有良好的免疫反应性；表达菌株发酵表达量可达200mg／L，电镜分析可见24～30nin的VLP，且颗粒结构完好。结论应用汉逊酵母表达系统成功表达了EV71VLP，为今后研制EV71VLP疫苗奠定基础。Objective To express virus-like particles （VLP） of enterovirns 71 （EV71） in Han- senula polymorpha. Methods The coding sequences of P1 and 3CD genes of EV71 were optimized accord- ing to codon usage bias of Hansenula polymorpha for achieving high expression, and then cloned into the ex- pression vector PMV of Hansenula polymorpha. The recombinant expression vector PMV-P1-3CD was trans- formed into Hansenula polymorpha AU0501. The transformants were stably cultured in selective medium （Yeast Nitrogen Base） and screened for strains with positive P1 and 3CD genes by PCR. Then an induced cultivation on the recombinant strains were performed in a medium supplemented with methanol to a final concentration of 1.0% and the expressed products were analyzed by SDS-PAGE and Western blot assays to select high expression strains. The high expression strains were cultured in 30 L fermentor and its fermenta- tion products were analyzed by electronic microscope after purification. Results EVT1 recombinant expres- sion strains were successfully constructed. The results of SDS-PAGE showed that the expressed products had obvious VP3, VP1, VP0 protein bands with molecular weights of 26x103, 33x103 and 35x103, respective- ly, which were consistent with the expected molecular weight of the fusion proteins. Western blot demonstra- ted that the expressed products could be specifically recognized by the polyclonal antibody against EVT1-VP1 at 33x103, indicating its high immunoreactivity. ELISA confirmed that the expression level of EV71 fermen- tation products was reached to 200 mg/L. Electronic microscope analysis showed that the VLP of recombi- nant EV71 were 24-30 nm in diameter with normal structure. Conclusion The virus-like particles of human enterovirus 71 are successfully expressed in Hansenula polymorpha, which provides a foundation for the fur- ther development of EV71 VLP vaccine.

关 键 词：肠道病毒71型 汉逊酵母 病毒样颗粒 表达 

分 类 号：R373[医药卫生—病原生物学]