酵母重组表达PCV2病毒样颗粒的纯化工艺  

The Purification Process of Recombinant Porcine Circovirus Type 2 Virus-like Particles in Yeast

作　　者：谢晋 杨德强 周峻岗[1,3] 李翊峰 吕红 XIE Jin;YANG Deqiang;ZHOU Jungang;LI Yifeng;LüHong(State Key Laboratory of Genetic Engineering, Fudan University, Shanghai 200438, China;WuXi Biologics Co., Ltd., Wuxi, Jiangsu 214092, China;Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, Shanghai 200438, China;WuXi Biologics(Shanghai)Co.,Ltd.,Shanghai 200131,China)

机构地区：[1]复旦大学遗传工程国家重点实验室,上海200438 [2]无锡药明生物技术有限公司,江苏无锡214092 [3]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 [4]上海药明生物技术有限公司,上海200131

出　　处：《复旦学报（自然科学版）》2020年第6期685-694,共10页Journal of Fudan University：Natural Science

基　　金：上海市科学技术委员会项目(18391901800,19DZ2282100)。

摘　　要：猪圆环病毒(PCV)是一种引起断奶仔猪多系统衰竭综合征、呼吸道综合征及繁殖障碍的病毒,已经成为危害养猪业最主要的病原之一,而接种猪圆环病毒2型(PCV2)疫苗是控制猪圆环病毒相关疾病最有效的措施.利用马克斯克鲁维酵母(Kluyveromyces marxianus,KM)表达猪圆环病毒PCV2 Cap的产量可达2g/L,高于其他表达系统的产量,并且重组表达的Cap蛋白在胞内能高效自组装成病毒样颗粒(Virus-Like Particles,VLPs).酵母细胞破碎之后样品中含有大量的微小的细胞碎片,高速离心澄清难以分离,影响了VLPs的得率、纯度以及澄清度.本研究考察了超声波破碎、玻璃珠研磨破碎以及高压均质破碎等不同破碎方法,发现1300bar高压均质法对重组表达PCV Cap蛋白的马克斯克鲁维酵母破碎效果最佳.在此基础上,检测添加有机溶剂、表面活性剂和化学絮凝剂的处理效果,发现添加1%(质量分数)Triton X-100对酵母破碎菌液具有65%以上的浊度降低率,处理后样品可直接用于离子交换层析,得率大于92%,HPLC纯度大于85%.Porcine circovirus(PCV)is a virus that causes post weaning multisystemic wasting syndrome(PMWS),respiratory syndrome and reproductive disorders in weaned pigs.It has become one of the most harmful pathogens that destroy the porcine industry.PCV2 vaccine is the most effective way to prevent porcine circovirus-related diseases.The yield of PCV2 Cap expressed by Kluyveromyces marxianus is up to 2g/L,which is higher than that of other expression systems,and the recombinant Cap protein can assemble into virus-like particles(VLPs)in cells efficiently.For intracellular expression of VLPs in yeast,disruption of yeast cells created a large number of cell fragments,including proteins,glycoproteins,polysaccharides,polyphosphate lipids and nucleic acids.Large cell fragments can be effectively separated by centrifugation,but small cell fragments suspended in the supernatant caused a high turbidity,which might block the chromatography column,increased the difficulty of cleaning the column and decreased the service life of the packing media.In this study,the Ultrasonication,glass bead grinding and high-pressure homogenization were used to disrupt the cells of K.marxianus PCV2 strain,and results indicated that high pressure homogenization at 1300bar for twice was the optimal condition.After screening of different organic solvents,surfactants and chemical reagents,it was found that more than 65%of the turbidity decreased after clarified with 1%(mass fraction)Triton X-100,the purity and the recovery rates of PCV2 VLPs can reached above 92%and 85%,respectively.

关 键 词：马克斯克鲁维酵母 猪圆环病毒 病毒样颗粒 浊度 纯化 

分 类 号：Q819[生物学—生物工程]