人乳头瘤病毒16型晚期蛋白L1的表达及病毒样颗粒的装配

目的　在昆虫细胞中表达人乳头瘤病毒 16型 (HPV16 )哈尔滨地区分离株的晚期蛋白L1,并分离纯化由L1蛋白在细胞中自主组装成的病毒样颗粒 ,为HPV16预防性疫苗的研制奠定基础。方法　获得含有HPV16L1基因的重组杆状病毒并使目的蛋白L1在昆虫细胞中表达 ;对重组杆状病毒的扩增条件及L1蛋白的表达水平进行优化 ;电镜下观察昆虫细胞中晚期蛋白装配成病毒样颗粒的情况 ,经氯化铯密度梯度纯化病毒样颗粒 ,并经Westernblot进行验证。结果　获得了稳定表达L1蛋白的重组杆状病毒 ;以MOI值为 0 .2感染昆虫细胞可获得较高滴度的重组病毒 ,以MOI值为 10感染昆虫细胞可获得较高水平的L1蛋白表达 ;电镜下可观察到昆虫细胞核内直径为 5 5nm的病毒样颗粒 ;病毒样颗粒可通过氯化铯密度梯度离心获得。结论　获得人乳头瘤病毒哈尔滨地区分离株L1蛋白可在昆虫细胞中自主装配的病毒样颗粒并成功分离纯化。

Expression of L1 major capsid protein from human papillomavirus type 16 and the production of virus-like particles

Objective To express the L1 major capsid protein of regional variant strain of human papillomavirus type 16, and to purify HPV16 L1 virus-like particles. Methods The recombinant plasmids including HPV16 L1 gene was constructed and the L1 protein was expressed by infected Sf9 cells. Optimizating the amplification of recombinant virus and expression level of L1 protein by changing the MOI and the infecting time. VLPs of L1 protein in Sf9 cells was analyzed by electron microscopy. VLPs were purified with CsCl gradient centrifugation. Results The stable strain of recombinant baculovirus expressing HPV16 L1 protein was obtained. These 55nm diameter particles were present in the nuclei of recombinant baculovirus-infected insect cells with density of 1.29- 1.30 /cm 3. Conclusion HPV16 L1 protein could be efficiently expressed in Sf9 cells and VLPs of HPV16 L1 protein could be purified by CsCl gradient centifugation.