摘要
目的研发基于鞭毛蛋白的新型甲型H1N1流感疫苗。方法分别以鼠伤寒沙门菌SL7202基因组和含有甲型H1N1流感病毒HA基因的重组质粒pET-HA为模板,通过PCR扩增Ⅱ相鞭毛蛋白fljB和HA1-2基因,再通过overlapPCR将这两基因片段拼接成HA1-2-fljB,将其插入原核表达质粒pET30a(+),构建重组质粒pET-HA1-2-fljB,并转化表达宿主菌E.coli BL21(DE3)中,以IPTG诱导表达。SDS-PAGE和Western blotting鉴定融合蛋白HA1-2-fljB的表达。结果正确构建出重组质粒pET-HA1-2-fljB,SDS-PAGE和Western blotting结果显示,融合蛋白分子量约84kD,并具有良好的免疫反应性,能强烈诱导HEK293-mTLR5细胞分泌IL-8。结论成功表达具有TLR5生物学活性的融合蛋白HA1-2-fljB,为研究甲型H1N1流感疫苗奠定了基础。
The aim of this study was to develop a novel kind of flagellin-based influenza A virus subtype H1N1 vaccine.The genes coding for fljB and HA1-2 were respectively amplified by polymerase chain reaction（PCR） and the 2 genes were merged into HA1-2-fljB by overlap PCR,and then the gene was inserted to vector pET30a（＋） to construct the recombinant plasmid.The recombinant plasmid was transformed into expressive vector E.coli BL21（DE3） and was induced with IPTG.The expression of fusion protein was analyzed by SDS-PAGE and Western blotting.The results indicated that the recombinant plasmid pET-HA1-2-fljB was constructed.The fusion protein,whose molecule weight was about 84 kD,had a good immunoreactivity and strongly induced HEK293-mTLR5 cells to secrete IL-8.In conclusion,a flagellin-based fusion protein HA1-2-fljB with TLR5 bioactivity was expressed correctly,and these results would establish the basis for the further research in influenza virus subtype H1N1 vaccine.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2012年第1期19-23,共5页
Chinese Journal of Zoonoses
基金
国家自然科学自然基金项目(30871860
31172299)
江苏省自然科学基金项目(BK2010039)
江苏省青蓝工程项目联合资助(苏教师200830号)
江苏省2011年度普通高校研究生科研创新计划项目(CXLX11_0999)
关键词
鞭毛蛋白
血凝素
原核表达
疫苗
flagellin
hemagglutinin
prokaryotic expression
vaccine
