多表位流感纳米粒构建表达及验证

Construction, expression and verification of multi-epitope influenza nanoparticles

研制具有广谱作用的新型纳米重组亚单位疫苗,为新型流感纳米疫苗的研发奠定基础。将跨膜蛋白M2的胞外域(M2e)和H1、H3、B亚型流感血凝素的第2亚单位的保守区域通过4GS蛋白Linker连接至铁蛋白,将M2e-HA2-Ferrtin基因(MHF基因)克隆至原核表达载体pET32a,将重组质粒转化到感受态细胞BL21(DE3),经IPTG(异丙基-β-D-硫代半乳糖苷)体外诱导表达,通过镍柱纯化,利用不同浓度的咪唑洗脱液洗脱蛋白,通过SDS-PAGE和Western blot鉴定蛋白产物。经透射电子显微镜和纳米粒度仪观察其构象及大小。结果表明成功构建表达纯化重组流感蛋白,经PCR扩增出MHF为1 071 bp大小片段,经SDS-PAGE和Western blot检测蛋白产物,重组流感蛋白以可溶性形式存在,大小为61 kDa且具有生物学活性,电子透射显微镜观察其构型,可见pET32a-M2e-HA2-Ferrtin融合蛋白形成类病毒样颗粒,大小均一。经纳米粒度仪分析,平均粒径Xav=61.58 nm。本研究成功表达纯化并验证多表位流感纳米蛋白,为新型流感纳米疫苗的研制奠定了基础。

To develop a new type of nano-recombinant subunit vaccine with a broad-spectrum effect and lay the foundation for the development of a novel influenza nano-vaccine. The extracellular domain(M2 e) of transmembrane protein M2 and the conserved region of the second subunit of hemagglutinin of H1,H3,B subtype influenza were connected to ferritin through 4 GS protein linker, the M2 e-HA2-Ferrtin gene(MHF gene) was cloned into the prokaryotic expression vector pET32 a, then the recombinant plasmid was transformed into competent cells BL21(DE3). Protein expression was induced in vitro by IPTG(isopropyl-β-D-thiogalactoside) and protein was purified by nickel column, different concentrations of imidazole eluent were used to elute protein. Protein products were identified by SDS-PAGE and Western blot. Their conformation and size were observed by transmission electron microscope and nano-particle size analyzer. The results showed that M2 e-HA2-Ferrtin recombinant influenza protein was successfully constructed and purified, which was soluble, 61 kDa, biologically active and formed uniformly sized virus-like particles with an average particle size of 61.58 nm. In this research, multi-epitope influenza nanoprotein is successful expressed and purified, which will lay a foundation for the development of new influenza nanovaccine.