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构建不可分型流感嗜血杆菌(Non-typeable Haemophilus influenzae,NTHi)Hap基因的原核表达载体,并诱导其表达Hap融合蛋白。以NTHi标准株ATCC49247基因组DNA为模板扩增出Hap目的基因片段,双酶切后连接于原核表达载体pET-32a(+),构建Hap重组原核表达质粒pET-32a(+)-Hap,将经PCR、酶切及DNA测序鉴定正确者转化E.coliBL21(DE3),诱导表达带有His标签的Hap融合蛋白。SDS-PAGE电泳分析重组蛋白的相对分子量大小及表达形式,Western blot进一步鉴定表达产物的特异性。通过PCR成功扩增出NTHi Hap基因,构建了具有正确基因序列的Hap原核表达质粒pET-32a(+)-Hap,SDS-PAGE电泳分析成功表达出相对分子量(Mr)为176000的重组融合蛋白,该蛋白主要以包涵体的形式存在;Western blot结果表明该重组融合蛋白可与抗His-tag单克隆抗体发生特异性结合。NTHi Hap蛋白在原核表达系统中的成功表达,将为Hap蛋白免疫活性的深入研究及NTHi新型保护性疫苗的开发奠定实验基础。
The prokaryotic expression vector of non-typeable Haemophilus influenzae (HTH) Hap gene was constructed and induced to express Hap fusion protein. The Hap gene fragment was amplified by using the NTHi standard strain ATCC49247 genomic DNA as a template, and double digested and ligated into the prokaryotic expression vector pET-32a (+) to construct the Hap recombinant prokaryotic expression plasmid pET-32a (+) - Hap. The recombinant plasmid was transformed into E.coli BL21 (DE3) by PCR, enzyme digestion and DNA sequencing. His tag fusion protein Hap was induced. SDS-PAGE electrophoresis analysis of recombinant protein relative molecular weight size and expression, Western blot further identify the specificity of the expression product. The NTHI Hap gene was successfully amplified by PCR and the prokaryotic expression plasmid pET-32a (+) - Hap with the correct gene sequence was constructed. The recombinant fusion protein with a relative molecular weight of 176000 was successfully expressed by SDS-PAGE electrophoresis analysis, The protein mainly exists in the form of inclusion body. Western blot results showed that the recombinant fusion protein could specifically bind with anti-His-tag monoclonal antibody. The successful expression of NTHi Hap protein in prokaryotic expression system will lay the experimental foundation for the further study of Hap protein immunological activity and the development of NTHi novel protective vaccine.