粪肠球菌Ace抗原表位预测及重组表位疫苗载体构建与表达

Epitope prediction of Enterococcus faecalis Ace protein and construction and expression of recombinant epitope vaccine

利用生物信息学软件预测抗原表位,然后将筛选出的6个抗原表位基因片段分别与铁蛋白H亚基基因片段连接,并克隆到pET-32a表达载体中,构建能融合表达Ace抗原表位和铁蛋白H亚基的重组质粒pET-32a-ace1、pET-32a-ace2、pET-32a-ace3、pET-32a-ace4、pET-32a-ace5、pET-32a-ace6,并经测序验证;优化诱导条件获得原核表达的重组蛋白后,用SDS-PAGE和WesternBlot验证其相对分子质量大小和免疫原性;将重组蛋白进行纯化超滤,然后进行透射电镜成像检查。结果显示,本研究成功构建了7种原核表达载体;在IPTG诱导下获得了5种可溶性表达的融合蛋白,且均有较强的免疫原性;重组融合蛋白Ace1-H、Ace3-H、Ace5-H和Ace6-H在透射电镜下观察到了具有与天然铁蛋白类似的特殊中空结构,且其直径与天然铁蛋白纳米笼类似;而带多表位的重组蛋白Ace-H虽然也能自组装成纳米级颗粒,但没有中空核心,其直径也比天然铁蛋白略大。

Bioinformatics software was used to predict potential Ace antigenic epitopes,and then 6 selected antigenic epitope gene fragments were linked with the ferritin H gene fragment,respectively,which were inserted within prokaryotic expression vector pET-32a to obtain recombinant plasmids.The plasmids were correctly constructed,designated as pET-32a-ace1,pET-32a-ace2,pET-32a-ace3,pET-32a-ace4,pET-32a-ace5 and pET-32a-ace6.The recombinant proteins were expressed through IPTG induction,and the SDS-PAGE and Western Blot methods were applied to verify the molecular weight and immunogenicity of the recombinant proteins.After purification and ultra-filtration,the recombinant proteins were examined by transmission electron microscopy(TEM).The results showed that 7 prokaryotic expression plasmids were successfully constructed;all the fusion proteins were expressed and five of them were soluble,presenting strong immunogenicity.Under TEM imaging,the 5 fusion proteins demonstrated ferritin-like nano structures,among which,proteins Ace1-H,Ace3-H,Ace5-H and Ace6-H had a hollow core with similar diameters to that of natural ferritin,whilst Ace-H nanoparticls looked solid rather than hollow,with a larger diameter than the natural ones.