摘要
目的克隆牙龈卟啉单胞菌菌毛蛋白FimA基因,构建原核表达载体,诱导其在大肠杆菌中融合表达,并鉴定、纯化其表达产物。方法克隆牙龈卟啉单胞菌菌毛蛋白FimA基因,构建表达载体pET15b-FimA,转化大肠杆菌BL21(DE3)pLyS感受态细胞;异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达融合蛋白,以抗6×HisTag单克隆抗体为一抗,Western blot鉴定、Co2+柱亲和层析纯化融合蛋白。结果克隆的FimA基因序列及插入表达载体中的FimA序列均与GenBank数据库中的序列呈现100%同源性;IPTG诱导后Western blot鉴定4.1×104处有目的蛋白表达;Co2+柱亲和层析法获得纯化的高浓度FimA蛋白。结论本实验成功构建了牙龈卟啉单胞菌菌毛蛋白FimA基因的原核表达载体pET15b-FimA,并在大肠杆菌中获得成功表达和纯化,为进一步制备牙龈卟啉单胞菌菌毛蛋白单克隆抗体和研制开发预防牙周炎的亚单位蛋白疫苗奠定了实验基础。
Objective To clone the FimA gene of fimbriae from Porphyromonas gingivalis（P.gingivalis） and to construct prokaryotic expression vector which was induced in E.coli BL21（DE3）pLyS in the form of fusion protein expression and to identify, purify the product of its expression. Methods To clone the FimA gene of fimbriae from P.gingivalis and to construct prokaryotic expression vector pET15b-FimA vector which was transformed into the competent cells of BL21（DE3）pLyS. The expression of fusion protein was induced by isopropyl β-D-1-thiogalactopyranoside（IPTG）. With anti-6×His Tag monoclonal antibody as the first antibody, the expressed fusion protein was characterized by Western blot and purified by Co^2＋-NTA affinity chromatography. Results Cloned FimA gene sequences and inserted into expression vector of the FimA sequences were related to the sequence in GenBank database showed 100% homology. IPTG induced and then identified by Western blot showed a fragment of 4.1×10^4 has been expressed. Co^2＋-NTA affinity chromatography column was used to obtain high concentrations of FimA purified protein. Conclusion The recombinant prokaryotic expression vector of pET15b-FimA was constructed and was expressed and purified successfully in E.coli BL21（DE3）pLyS. This study laid the experimental foundation to further prepare for monoclonal antibodies of fimbriae of P.gingivalis and to develop the subunit protein vaccine of prevention of periodontitis.
出处
《华西口腔医学杂志》
CAS
CSCD
北大核心
2010年第3期241-245,共5页
West China Journal of Stomatology
基金
国家自然科学基金资助项目(30500560)
陕西省自然科学基础研究计划资助项目(2006C242)
西安市科技攻关计划资助项目(GG05159)
