不可分型流感嗜血杆菌Hap基因的克隆及原核表达

Cloning Hap Gene from Non-typeable Haemophilus Influenzae And Expression of Hap Protein in Prokaryotic Cell

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。

This study was aimed to construct a prokaryotic expressing vector of Hap gene from Nontypeable Haemophilus influenzae, and express and identify the fusion proteins of Hap-His in E. coll. The gene encoding protein Hap was amplified from Nontypeable Haemophilus influenzae ATCC49247 chromosomal DNA by PCR, then it was cloned into prokaryotie expression plasmid pET-32a （＋）. The recombinant plasmid pET-32a（＋）-Hap was transformed into E. coli BL21 and expression was induced by Isopropy-β-D-thiogalatoside（IPTO）. The Hap-His fusion protein expressed so was analyzed by SDS-PAGE and Western-blot. The results showed that the recombinant expressive plasmid pET-32a （＋）-Hap was constructed successfully, and the recombinant plasmid expressed Hap- His fusion protein with relative molecule mass 176 000 and mainly existed in inclusion body. This fusion protein could combine with anti-His monoclonal antibody specifically through Western blot analysis. Successful expression of Hap-His fusion protein in prokaryotic cell could lay a basis for further study of immunocompetence of Hap protein and development of NTHi vaccine.