诺如病毒VP1蛋白在马克斯克鲁维酵母中的表达及纯化  

Expression and purification of norovirus VP1 protein in Kluyveromyces marxianus

作　　者：袁军鸿 王晓辉[2] 吴萍萍 冯昌增[2] 周峻岗 褚嘉祐[2] 吕红[4,5] 杨昭庆 YUAN Jun-hong;WANG Xiao-hui;WU Ping-ping;FENG Chang-zeng;ZHOU Jun-gang;CHU Jia-you;LüHong;YANG Zhao-qing(Kunming Medical University,Kunming,Yunnan 650500;Department of Medical Genetics,Institute of Medical Biology,Chinese Academy of Medical Sciences&Peking Union Medical College,Kunming,Yunnan 650118,China;Key Laboratory of Quality Control and Evaluation of Vaccines and Biological Products,State Drug Administration,Chengdu,Sichuan 610097,China;不详)

机构地区：[1]昆明医科大学,云南昆明650500 [2]中国医学科学院,北京协和医学院医学生物学研究所医学遗传学研究室,云南昆明650118 [3]国家药品监督管理局疫苗及生物制品质量控制与评价重点实验室,四川成都610097 [4]复旦大学生命科学学院上海工业菌株工程技术研究中心,上海200438 [5]复旦大学遗传工程国家重点实验室,上海200438

出　　处：《中国病毒病杂志》2022年第1期35-41,共7页Chinese Journal of Viral Diseases

基　　金：云南省高层次卫生健康技术人才培养专项(L-2018003);中国医学科学院医学与健康科技创新工程项目(2016-I2M-1-019,2021-I2M-1-043)。

摘　　要：目的 使用马克斯克鲁维酵母表达系统对GII.17型诺如病毒病毒样颗粒(VLPs)进行表达并对该蛋白纯化方式进行初步探讨。方法 对诺如病毒VP1序列进行密码子优化,使用双酶切构建重组质粒,通过醋酸锂转化法筛选获得重组阳性克隆KM-NOV-GII.17。摇瓶培养72 h后对菌体进行破碎,使用层析柱对目的蛋白进行纯化,并通过电镜观察颗粒形态。结果 成功筛选出高表达量KM-NOV-GII.17,通过Q柱及分子筛串联纯化的方式获得了纯度较高的病毒样颗粒,产量约为0.26 g/L,电镜观察结果显示该VP1序列可自组装为大小均一的病毒样颗粒并且与天然病毒颗粒形态相似。结论 本研究证实了GII.17型诺如病毒VP1蛋白可通过马克斯克鲁维酵母表达系统获得产量高、均一性好的病毒样颗粒,这为诺如病毒基因重组疫苗的研发提供了新的蛋白表达系统。Objective To express and purify virus-like particles(VLPs) of Norovirus GII.17 using Kluyveromyces marxianus expression system. Methods Codon optimization was performed on the VP1 sequence of norovirus, the recombinant plasmid was constructed by double enzyme digestion. Lithium acetate transformation method was used for the screening of positive recombinant clone and KM-NOV-GII.17 clone was obtained. After 72 hours of culture, K.marxianusFim-1 ura3Δ yeast cells were disrupted and the target protein was purified using a chromatographic column. The VLPs were observed under electron microscope. Results KM-NOV-GII.17 clone which produced high levels of VP1 protein was successfully obtained.VP1 protein was purified using Q column along with molecular sieve and the protein yield was as high as 0.26 g/L.Theelectron microscope observation showed that the VP1 protein can self-assemble into uniform size VLPs which were similar with natural virus particles. Conclusion The study shows that norovirus virus-like particles can be produced with high yield and good uniformity by using Kluyveromyces marxianus expression system, which provides a new option for the development of recombinant norovirus vaccine.

关 键 词：诺如病毒 病毒样颗粒 马克斯克鲁维酵母 表达 纯化 

分 类 号：R373.2[医药卫生—病原生物学]