由乙肝病毒核心蛋白(HBc-VLP)递载的流感通用疫苗Flu@uV设计构建、表征及初步活性研究  

Design,Construction,Characterization and Preliminary Activity Research of Universal Influenza Virus Vaccine( Flu@ uV) Delivered by Hepatitis B Virus Core Protein( HBc-VLP)

作　　者：张旭凡[1] 胡发彪 马兴元[1] 王天文[1] 王平[1] 王晓丽[1] 

机构地区：[1]华东理工大学生物工程学院生物反应器工程国家重点实验室,上海200237

出　　处：《中国生物工程杂志》2016年第2期7-15,共9页China Biotechnology

摘　　要：目的:构建以HBc为载体的甲型流感病毒HA和M2e流感通用疫苗(Flu@u V),利用大肠杆菌BL21(DE3)表达系统,进行初步的蛋白表达及纯化。在此基础上,构建DNA流感通用疫苗。方法:利用全基因合成的序列为模板,成功构建HA-M2e-HBc、M2e-HBc、HBc、3M2e-HBc和3HA-3M2e-HBc基因的重组质粒,并在大肠杆菌中表达,经SDS-PAGE、Western blot和电镜检测其表达。将纯化的蛋白与弗氏佐剂共同免疫小鼠,取小鼠外周血进行流式细胞分析。通过荧光分析和Western blot初步验证DNA流感通用疫苗在人源胚胎肾细胞(HEK293T)中的表达情况。结果:成功表达纯化了HA-M2e-HBc、M2e-HBc、HBc和3M2e-HBc四种蛋白,经电镜观察到30nm左右的蛋白纳米颗粒样结构。小鼠外周血流式细胞分析显示HBc和3M2e-HBc可以增加小鼠的免疫力,而HA-M2e-HBc和M2e-HBc对小鼠免疫力的提高没有影响。通过荧光检测和Western blot检测说明DNA流感通用疫苗在真核细胞中成功表达。结论:成功构建HBc与甲型流感病毒HA和M2e的病毒样颗粒,为流感通用疫苗的研制奠定了重要基础。Objective： To construct a universal influenza virus vaccines （Flu@ uV）, a universal influenza virus vaccine containing HA and M2e of influenza A virus was disigned and constructed. Hepatitis B virus core protein（HBc-VLP） was used as the delivery vector and adjuvant in the Flu@ uV. The universal influenza antigen proteins were expressed in E. coli BL21 （DE3） and purified for preliminary activity assay. Method： Using the synthesized gene sequence as a template,recombinant plasmids expressing HA-M2e-HBc,M2e-HBc,HBc ,3M2e- HBc and 3HA-3M2e-HBe were successful constructed. Their expression in E. coli was tested through SDS-PAGE, Western blot and electron microscopy. Mices were co-inoculated by purified protein with Freund＇ s adjuvant. Flow cytometry was used to analyze peripheral blood of immunized mice. Expression of the corresponding DNA vaccine in human embryonic kidney cells（HEK293T） was preliminary validated by fluorescence analysis and Western blot. Result： Four kinds of HA-M2e-HBc, M2e-HBc, HBc and 3M2e-HBc proteins were successfully expressed and purified. Protein nanoparticles about 30 nm could be observed through electron microscope. Flow cytometry analysis of the peripheral blood showed that HBc and 3M2e-HBe increased the immunity of mice, and HA-M2e-HBc and M2e-HBe had no effect on that. DNA universal influenza virus vaccines were successfully expressed in eukaryotic cells through fluorescence detection and Western blot. Conclusions： A virus-like particles containing HBc, HA, and M2e of influenza A virus was successfully constructed, it laying a base for the development of universal influenza virus vaccine.

关 键 词：流感通用疫苗 HBc-VLP 设计构建与表征 免疫活性 

分 类 号：Q819[生物学—生物工程]