EV71多表位抗原亲和纯化及类病毒颗粒胞外自组装

利用肠道病毒71型（EV71）衣壳蛋白优势表位肽段构建融合蛋白抗原能有效抑制病毒感染，有望成为继灭活病毒后更为安全有效的疫苗品种。该融合蛋白能通过原核体系有效表达但形成无序包涵体，采用常规层析介质难以实现目标蛋白与宿主杂质的有效分离，阻碍了对该抗原蛋白进行全面临床前活性及安全性评价。在原有融合蛋白抗原N端插入组氨酸标签，对形成的包涵体变性溶解后直接采用镍金属螯合亲和介质进行分离纯化，获得了纯度大于95%的抗原纯品，目标蛋白收率46.8%。采用透析方式脱除纯化样品中高浓度脲，发现直接透析至无脲的缓冲液中蛋白质大量沉淀，而先稀释至2mol/L脲的缓冲液中然后用G25脱盐柱完全脱除脲则无任何沉淀形成，获得近100%的蛋白质收率。透射电镜分析最终样品发现融合蛋白形成了10nm左右粒径均一的类病毒蛋白颗粒，且在pH 8.0的磷酸盐缓冲液中保持稳定。该研究结果为将EV71融合蛋白抗原发展为安全有效且低成本的手足口疫苗奠定了基础。

Affinity Purification of Enterovirus 71 Fused Multi-Epitope Protein Antigen and Assembling It as Virus-like Particles in Vitro

Human enterovirus 71 (EV71) is one of the main causative pathogens for hand-foot-and-mouth disease (HFMD) which often infects infants and children under 5 years old. Although prophylactic vaccines of inactivated EV71 virus for HFMD have been clinically available, low productivity and safety concerns still prompt scientists to develop other vaccine substitutes. Recently, a kind of vaccine candidate VacA for EV71 has been designed and reported, which contained the main antigen peptides, was fused as a single protein and expressed through recombinant E. coli system. This candidate was demonstrated to have high neutralizing antibody titers as well as efficiently protect against virus infection. However, forming inclusion bodies in E. coli and failure of being separated from the host contaminants through route chromatographs hampered its path to go forward to whole pre-clinic and safety assessments.Another form of VacA as His-VacA was constructed by inserting a His tag at the N-terminal of VacA and explored to be separated through metal affinity chromatography. As still forming inclusion bodies, the fusion protein of His-VacA was directly purified at denatured state through a special mental affinity medium of cOmplete His-tag, which could tolerate low concentration of EDTA and DTT. Purity of about 95% for His-VacA was achieved after such one step chromatographic procedure, with a recovery of 46.8%. Dialysis was then adopted to remove urea in the purified protein, but protein almost completely precipitated when dialyzing against PB buffer without any additives. Nevertheless, in the case that the purified denatured protein was firstly diluted in a buffer containing 2mol/L urea and then exchanged to a buffer without urea through a desalting column of G25, no protein precipitate could be found, resulting in nearly 100% recovery. More inspirationally, His-VacA was found in the form of virus-like particles with a size of 10nm through transmission electron microscopy. Moreover, this protein particle is very stable in the final buffer and then could be used for the further pre-clinic assessments. All these results lay a foundation for developing His-VacA as a new kind of EV71 vaccine with the advantages of safer and cheaper.