登革病毒3型病毒样颗粒在酵母体内的表达与鉴定  

Expression and identification of dengue virus type 3 like particles in pichica pastoris

作　　者：付春云[1,2] 方丹云[1,2] 刘岩[1,2] 喻治准[1,2] 江汉宁[1,2] 江丽芳[1,2] 周俊梅[1,2] 

机构地区：[1]中山大学中山医学院微生物学教研室,广东广州510080 [2]中山大学热带病防治研究教育部重点实验室,广东广州510080

出　　处：《热带医学杂志》2011年第6期613-616,656,共5页Journal of Tropical Medicine

基　　金：国家自然科学基金(U0632002)

摘　　要：目的探讨登革病毒3型病毒样颗粒(DENV3-VLPs)的免疫原性。方法用PCR法扩增登革病毒3型prME基因,插入载体pGAPZaA构建重组质粒pGAPZaA-prME-D3,将其转化毕赤酵母X33构建转化子pGAPZaA-prME-D3/X33。对转化子表达上清和细胞裂解液进行SDS-PAGE和Western blotting分析。用蔗糖密度梯度离心纯化表达的DENV3-VLPs并进行鉴定和电镜观察。结果成功构建重组载体pGAPZaA-prME-D3,获得酵母转化子pGAPZaA-prME-D3/X33,并应用毕赤酵母表达了DENV3-VLPs,表达蛋白约50000Mr,电镜观察VLPs直径为20～50nm。结论应用酵母表达系统成功表达了DENV-3VLPs,经鉴定表明其具有免疫反应性,为后续免疫原性研究及四价登革VLPs疫苗的构建奠定了基础。Objective To construct DENV3-VLPs recombinant vector,purify and identify DENV3-VLPs expressed in P. pastoris expression system. Methods DEVN-3 prM and E genes were amplified by PCR and inserted into pGAPZaA to construct the recombinant plasmid pGAPZaA-prME-D3.The pGAPZaA-prME-D3/X33 was constructed by transferring the plasmid to P.pastoris X33.The expression superuatant and cell lysate from pGAPZaA-prME-D3/X33 was analyzed by SDS- PAGE and Western blotting.The sucrose density gradient ultracentrifugation was used to purify the DENV3-VLPs.The characteristic of VLPs were identified and observed by electron microscopy. Results The recombinant vector pGAPZaA- prME-D3 was successfully constructed.The pGAPZaA-prME-D3/X33 was obtained and it can express the DENV3-VLPs which is about 50 000 Mr and 20 to 50nm diameter observed by electron microscopy. Conclusions DENV3-VLPs can be successfully expressed using yeast expression system.The experiment indicated that it has perfect immunoreactivity,which important for the follow-up studies of immunogenicity and the development of four serotypes of DENV VLPs subunit vaccine candidates.

关 键 词：登革病毒 病毒样颗粒 蔗糖密度梯度离心 毕赤酵母 

分 类 号：R373.33[医药卫生—病原生物学]