利用His-杆状病毒表达系统制备HPV16L1VLP  

Functional HPV16L1 VLP can be efficiently expressed by His-tag baculovirus expression system

作　　者：陈宏伟[1] 郑瑾[1] 杨筱凤[1] 王静[1] 来宝长[1] 司履生[1] 王一理[1] 

机构地区：[1]西安交通大学生命科学与技术学院,陕西西安710061

出　　处：《西北大学学报（自然科学版）》2005年第1期67-70,共4页Journal of Northwest University（Natural Science Edition）

基　　金：国家"863"高科技研究发展计划资助项目(2001AA215221)

摘　　要：目的　改进现有的昆虫细胞表达和纯化HPV16L1蛋白系统,寻找一种经济、简便、省时的HPV16病毒样颗粒(VLP)疫苗的制备方法。方法　将HPV16L1基因重组入带有 6×His标签的杆状病毒基因组, 并转染昆虫细胞 (Sf 9)进行表达。用镍柱纯化表达蛋白;以小鼠红细胞凝集试验和凝集抑制试验鉴定蛋白生物活性,用透射电镜观察VLP形成。结果　使用新的表达和纯化系统获得HPV16L1高效表达。小鼠红细胞凝集试验和凝集抑制试验证实纯化的HPV16L1蛋白具有HPV16L1的生物学活性;透射电镜观察证实纯化蛋白可自组装成VLP。结论　带有 6×His短肽的HPV16L1蛋白具有与HPV16L1蛋白同样的生物学活性和自组装成VLP的能力,现时的表达纯化系统,提供了一个省时、省力、简便、经济的制备HPV16VLP疫苗的方法。Aim To improve the existing HPV16 VLP preparing system and establish a high-efficient, economic, time-saving system for preparing HPV 16 VLP. Methods Sf-9 cells were infected with recombinant baculovirus containing the target gene coding HPV16L1 protein with 6×His tag. After being cultured for 72 hours at 27℃, the postinfected cells that expressed the HPV16L1 protein were harvested. The ProBondTM purification system was used for protein purification. The expressed protein was revealed and confirmed by SDS-PAGE and Western blot. The bio-function of purifed protein was analyzed by mouse erythrocyte hemagglutination assay and hemagglutination inhibition assay. The formation of VLP was visualized with transmission electron microscope.Results HPV16L1 protein was expressed efficiently by using the novel expression and purification system. The expressed HPV16L1 protein with MW 58KD was revealed by SDS-PAGE, and confirmed by Western blot. After purification, the purity of denatured and native HPV L1 proteins could reach 91.9% and 71.5% respectively. The proteins purified in native condition had the same bio-activity as the natural HPVL1 protein, causing murine erythrocyte agglutination and forming VLP by self-assemble in vitro.Conclusion The purified recombinant HPV16L1 protein with 6×His tag could self-reassemble into virions in vitro, and has identical bio-activity to HPV16L1 protein. So this novel expression and purification system provides a convenient,time-saving,economical way for preparing HPV16 VLP vaccine.

关 键 词：人乳头瘤病毒L1蛋白 蛋白纯化 病毒样颗粒 

分 类 号：R373[医药卫生—病原生物学]