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目的克隆b型流感嗜血杆菌(Haemophlilus influenza type b,Hib)D蛋白(hpd)基因,原核表达并纯化重组hpd蛋白,为下一步开发以D蛋白为基础的结合疫苗奠定基础。方法从Hib CMCC株基因组DNA中PCR扩增hpd基因片段,克隆入载体pET-30a(+),构建重组原核表达质粒pET-30a-hpd,转化入感受态大肠杆菌BL21(DE3),IPTG诱导表达。表达的重组蛋白经6 mol/L尿素变性、DEAE阴离子交换柱纯化、透析复性后,采用Western blot法鉴定其反应原性。结果重组表达质粒pET-30a-hpd经PCR及测序证明构建正确;表达的重组蛋白以包涵体形式存在,表达量约占菌体总蛋白的40%;经一步过柱纯化可得到纯度达95%左右的重组蛋白;纯化的重组蛋白可与Hib免疫小鼠制备的抗血清发生特异性反应。结论已成功克隆了Hib hpd基因,并在大肠杆菌中表达了重组hpd蛋白,为下一步开发以D蛋白为基础的结合疫苗奠定了基础。
Objective To clone the gene of Haemophilus influenza type b (Hib) D protein (hpd) and express in prokaryotic cells and purify the recombinant hpd protein, which will lay the foundation for the further development of the D protein - based conjugate vaccine. Methods The hpd gene fragment was amplified by PCR from genomic DNA of Hib CMCC strain and cloned into vector pET-30a (+). The recombinant plasmid pET-30a-hpd was transformed into competent E. coli BL21 (DE3) . The expressed recombinant protein was denatured by 6 mol / L urea and purified by DEAE anion exchange chromatography. After dialyzed and refolded, the recombinant protein was identified by Western blot. Results The recombinant plasmid pET-30a-hpd was constructed correctly by PCR and sequencing. The expressed recombinant protein existed in the form of inclusion bodies and accounted for about 40% of the total bacterial proteins. After one-step purification, the purity of recombinant protein reached 95% The purified recombinant protein can react specifically with antisera prepared from Hib immunized mice. Conclusion The Hib hpd gene was successfully cloned and the recombinant hpd protein was expressed in E. coli, which laid the foundation for the further development of the D protein-based conjugate vaccine.