CAJ | 학술논문

目的通过原核表达系统可溶性表达重组D蛋白,为多糖结合疫苗的制备提供蛋白载体.方法将Hib CMCC株基因组DNA中去除信号肽的hpd基因片段插入pET43.1a表达质粒,转化入感受态大肠埃希菌BL21 (DE3),IPTG诱导表达.表达的重组蛋白,经过硫酸铵沉淀和DEAE阴离子交换柱层析纯化后,用Western Blot的方法鉴定其免疫反应原性.结果表达的重组蛋白以可溶性形式存在,表达量约占菌株总蛋白的44％,经过初步纯化后的重组蛋白可以和Hib抗血清发生特异性反应.结论成功构建了D蛋白重组表达质粒,并在原核细胞中以可溶性形式表达出具有良好免疫反应原性的D蛋白.
목적 통과원핵표체계통가용성표체중조D단백,위다당결합역묘적제비제공단백재체.방법 장Hib CMCC주기인조DNA중거제신호태적hpd기인편단삽입pET43.1a표체질립,전화입감수태대장애희균BL21 (DE3),IPTG유도표체.표체적중조단백,경과류산안침정화DEAE음리자교환주층석순화후,용Western Blot적방법감정기면역반응원성.결과 표체적중조단백이가용성형식존재,표체량약점균주총단백적44％,경과초보순화후적중조단백가이화Hib항혈청발생특이성반응.결론 성공구건료D단백중조표체질립,병재원핵세포중이가용성형식표체출구유량호면역반응원성적D단백.
Objective To express the recombinant D protein in prokaryotic expression system solubly and make preparation for producing D-carrier conjugate vaccine next step.Methods The hpd gene fragment removed of signal peptide from genomic DNA of Hib CMCC was inserted into pET43.1a.The recombinant plasmid was transformed to competent E.coli BL21 (DE3) for expression under induction of IPTG.The expressed recombination protein was precipitated with ammonium sulfate,purified by DEAE anion exchange column chromatography and identified for reactogenicity by Western Blot.Results The expressed recombination protein,in a soluble form,constained about 50％ of total somatic protein and showed specific reaction with the HIB antisera after preliminary purification.Conclusion The D protein recombined expression plasmid was constructed successfully and expressed D protein in prokaryotic cells in a solube form.