基于Spyligase技术的禽流感血凝素H5HA10多聚体抗原的高效制备

High-efficient Preparation of Haemagglutinin H5HA10 Polymer Antigen of Avian Influenza Based on Spyligase Technology

利用Spyligase技术在H5HA10序列的C端和N端分别加上SpyTag和KTag序列,并分别与His6-ArsC、His6-PAS200、His6-XTEN、His6-PEAT、SiTag3-SUMO、SiTag3-MBP、SiTag3-PAS200、SiTag3-XTEN和SiTag3-ArsC促溶标签相连,成功构建了含9种不同促溶性标签的H5HA10重组表达载体,同时构建了1种含有Cherry标签的Spyligase重组表达载体。将这10种重组载体分别转化至大肠杆菌BL21(DE3),经IPTG诱导表达和SDS-PAGE分析,筛选出可以显著促进H5HA10重组蛋白表达的融合标签ArsC。通过优化诱导表达条件,在大量诱导表达、纯化后成功获得了高纯度的H5HA10和Spyligase重组蛋白,最终在PCT缓冲体系中, Spyligase促使H5HA10重组蛋白的蛋白聚合,形成了环化单体、三聚体、六聚体以及九聚体。

In this study, SpyTag and KTag were respectively added to the C-terminus and the N-terminus of the hemagglutinin H5 HA10 sequence of H5 N1 avian influenza HA protein by using Spyligase technology, and then they were separately linked with the dissolution-promoting tags His6-ArsC, His6-PAS200, His6-XTEN, His6-PEAT, SiTag3-SUMO, SiTag3-MBP, SiTag3-PAS200, SiTag3-XTEN and SiTag3-ArsC, finally nine H5 HA10 recombinant expression vectors containing different dissolution-promoting tags were successfully established. In addition, one Spyligase recombinant expression vector containing Cherry tag was also constructed. These 10 recombinant expression vectors were respectively transformed into Escherichia coli strain BL21(DE3), and the fusion tag ArsC which could significantly promote the expression of H5 HA10 recombinant protein was screened out through IPTG inducement expression and SDS-PAGE analysis. Through the optimization of conditions for induced expression, after large quantity of induced expression and purification, the highly-purified H5 HA10 and Spyligase recombinant proteins were successfully synthesized. In PCT buffer system, Spyligase prompted the protein polymerization of H5 HA10 recombinant protein, and formed cyclized monomers, trimers, hexamers and nonamers.