内含肽介导的鼠多瘤病毒样颗粒生产表达和纯化

Intein-Mediated Expression and Purification of Murine Polyomavirus Virus-Like Particles

鼠多瘤病毒样颗粒(VLPs)可由结构蛋白VP1自组装而成,可用于疫苗开发、基因治疗、药物传递和生物材料等方面.针对目前VP1生产中需要凝血酶去除谷胱甘肽S-转移酶(GST)标签,生产成本较高、操作复杂的问题,利用内含肽(intein)自剪切的特征,将内含肽插入到VP1与GST标签之间进行融合表达,表达产物经亲和色谱纯化,并通过改变柱内pH值、温度诱导内含肽剪切,使VP1与GST-intein分离从而获得VP1蛋白,产量为4,mg/(L培养基).纯化的VP1可自组装成与天然鼠多瘤病毒样颗粒一致的VLPs.结果表明,内含肽介导鼠多瘤病毒样颗粒生产纯化流程简单易行,且无需凝血酶参与,大幅降低成本,为VLPs的高效制备奠定了基础.

Virus-like particles（VLPs）of murine polyomavirus can be manufactured through in vitro self-assembly of structural protein VP1. VLPs have been utilized in the development of vaccines,gene therapy,drug delivery, biomaterials,etc. However,removing of GST-tag by thrombin in VP1 purification has the problems of high cost and complicated procedures. Therefore,the self-cleavage feature of intein was utilized by inserting the gene segment of intein between VP1 and GST. Then,affinity chromatography was used in the purification process and temperature and pH value were changed to induce the self-cleavage of intein. Thus,GST-intein tag and VP1 were separated. The production of VP1 was 4 mg/（L culture）. Purified VP1 was assembled in vitro into VLPs,which were consistent with the natural VLPs of murine polyomavirus. Hence,the intein-mediated expression and purification process of murine polyomavirus VLPs are successfully developed,which is helpful for lower-cost,higher-efficiency production of VLPs without the involvement of thrombin.