人乳头瘤病毒16型嵌合病毒样颗粒体外形成实验方法的建立  

Development of a method for formation of chimeric virus-like particles of human papillomavirus type 16 in vitro

作　　者：张飞齐 杨露 杨晓明 ZHANG Fei-qi;YANG Lu;YANG Xiao-ming(Wuhan Institute of Bio Products Co.,Ltd.,Wuhan 430200,Hubei Province,China)

机构地区：[1]武汉生物制品研究所,湖北武汉430200 [2]中国生物技术股份有限责任公司,北京100029

出　　处：《中国生物制品学杂志》2018年第9期999-1003,1010,共6页Chinese Journal of Biologicals

摘　　要：目的建立人乳头瘤病毒(human papillomavirus,HPV)16型嵌合病毒样颗粒(virus-like particle,VLP)体外形成实验的方法。方法以HPV16型L1为模板,将两个L2保守序列(aa56~75,aa108~120)分别插入L1两个可插入位点(DE袢状区,h4状区),构建出4种嵌合蛋白基因,利用Bac-to-Bac杆状病毒-昆虫表达系统表达嵌合的4种VLP蛋白,经蔗糖密度梯度离心纯化,纯化的目的蛋白进行VLP体外解聚重聚实验,Western blot分析体外解聚重聚前后的蛋白。结果表达及纯化的4种HPV16 L1-L2嵌合蛋白相对分子质量均约60 000,大小与预期相符;经VLP体外解聚重聚实验,使不能很好自组装的4种HPV16 L1-L2嵌合蛋白组装形成VLP,并具有免疫原性。结论建立的方法有助于HPV嵌合VLP的形成,能够明显提高其组装效率,为以L2蛋白为目标的第二代HPV疫苗的嵌合VLP体外组装提供了可行的实验方法。Objective To develop a method for formation of chimeric virus-like particles（VLPs）of human papillomavirus（HPV）type 16. Methods Four chimeric protein genes, Chi-a, Chi-b, Chi-c and Chi-d, were constructed by inserting two HPV16 L2 fragments（aa56 ~ 75 and aa108 ~ 120）, into the two sites（D-E loop and h4 helix） of L1 of HPV16 respectively, based on which four kinds of VLPs were expressed in Bac-to-Bac system and purified by sucrose density gradient centrifugation. The purified VLPs were subjected to disassembly and reassembly test in vitro, and analyzed by Western blot. Results All the relative molecular masses of four chimeric proteins were about 60 000, which were consistent with those expected. The four chimeric proteins which could not be self-assembled well formed VLPs by disassembly and reassembly test in vitro, and showed immunogenicity. Conclusion The developed method was helpful to the formation of VLPs of HPV, by which the assemble efficacy was improved significantly. It contributes to a method of assembly in vitro of chimeric VLPs of the second generation of HPV vaccine targeting L2 protein.

关 键 词：人乳头瘤病毒16型 基因重组 嵌合病毒样颗粒 体外重组实验 

分 类 号：R373.9[医药卫生—病原生物学]