基于鞭毛蛋白的H5亚型禽流感疫苗候选株的构建及鉴定

Construction and identification of H5 subtype avian influenza vaccine candidate based on flagellin

目的构建基于鞭毛蛋白的H5亚型禽流感疫苗候选株。方法以H5N1禽流感病毒血凝素(HA)基因和鼠伤寒沙门菌SL7202基因组为模板,通过重叠PCR扩增出HA1-2-fljB基因片段,将其插入原核表达载体pET32a(+),构建重组质粒pET32a-HA1-2-fljB,再将其转化表达菌E.coli BL21(DE3),IPTG诱导目的蛋白表达。SDS-PAGE和Western blot法鉴定分析融合蛋白HA1-2-fljB。通过刺激HEK293-TLR5细胞,检测融合蛋白的TLR5生物学活性。结果正确构建出重组质粒pET32aHA1-2-fljB,SDS-PAGE显示,融合蛋白HA1-2-fljB相对分子质量(Mr)约为100 000,Western blot法表明融合蛋白具有良好的免疫反应性。TLR5生物活性实验结果显示,与HA1-2蛋白刺激组相比,HA1-2-fljB诱导HEK293-TLR5细胞分泌了高水平的IL-8(P<0.01)。结论成功表达出具有TLR5活性的融合蛋白HA1-2-fljB,为研究H5亚型禽流感新型疫苗奠定了基础。

Objective To develop a novel H5 subtype avian influenza vaccine candidate based on flagellin. Methods HAt-2-f/jBwas amplified through splicing hemagglutinin gene fragment HA1-2 of A/Goose/Jiangsu/1/2000 HSN1 and Salmonella typhimufium I] phase flagellin fljB gene by overlap PCR, and then the gene was inserted to pET32a （＋） to construct the recombinant plasmid pET32a-HAl-2-f/jB. The recombinant plasmid was transformed into E. coli BL21 （DE3） and was induced with IPTG. The expression of the fusion protein was identified by SDS-PAGE and Western blotting. The biological activity of the fusion protein was evaluated by stimulating HEK293-TLFL5 cells. Results The recombinant plasmid pET32a-HAI-2-fljB was constructed. The fusion protein, whose relative molecule weight was about 100 000, had a good immunoreactivity. TLP,5 bioassay demonstrated the flagellin part of the fusion protein could be recognized by TLR5 of HEK293-TLR5 cell line to secrete a higher level of IL-8 when compared with HA1-2 protein （ P 〈 0.01 ）. Conclusion The flagellin-based fusion protein HAI-2-fljB with TLR5 bioactivity was expressed correctly, and the results would establish a basis for the further research on avian influenza virus H5 vaccine.