牙龈卟啉菌蛋白酶rgpAcd基因表达载体构建.docVIP

牙龈卟啉菌蛋白酶rgpAcd基因表达载体的构建 　　作者：许静，李昂，苟建重，许援朝，饶国洲，刘 蒸，谢红帼　西安交通大学医学院附属口腔医院：1.口腔内科;　2.口腔医学研究中心，陕西西安 710004 　　摘要：目的 构建牙龈卟啉菌蛋白酶rgpA催化结构域(rgpAcd)基因的表达载体。方法 利用PCR技术和基因重组技术，克隆牙龈卟啉菌蛋白酶rgpAcd, 插入中介载体pMD18T中并测序鉴定。将目的基因片断插入原核表达载体pET15b，构建表达质粒pET15b/rgpAcd，通过限制性酶切鉴定。结果 PCR产物电泳结果显示，在大约1.5kb处有一特异的条带，与预期的大小一致，核酸序列测定与分析的结果表明，克隆的1476bp基因序列与GenBank数据库中的序列呈现100%同源性，未发生任何突变;构建的表达质粒经酶切后所得片断与预期大小一致，表明牙龈卟啉菌蛋白酶rgpAcd基因的表达载体构建成功。结论 成功克隆了牙龈卟啉菌rgpAcd基因并构建了表达载体，为进一步研制重组活载体疫苗奠定了基础。 　　关键词：牙龈卟啉菌;蛋白酶;表达载体 　　Construction of a recombinant expression vector of rgpAcd gene of porphyromonas gingivalisXu Jing, Li Ang, Gou Jianzhong, Xu Yuanchao, Rao Guozhou, Liu Zheng, Xie Hongguo 　　(1. Department of Oral Medicine, Affiliated Dental Hospital, Medical School of Xian Jiaotong University; 　　2. Research Center for Stomatology, Affiliated Dental Hospital,Medical School of Xian Jiaotong University, Xian 710004, China) 　　ABSTRACT: Objective To construct prokaryotic expression vector of the catalytic domain of rgpAcd of porphyromonas gingivalis (Pg). Methods The desired DNA fragment rgpAcd was obtained by PCR and was separately sequenced and identified by inserting into intervector pMD18T Vector. The correct fragment was linked with and cloned into an prokaryotic expression vector pET15b. Results A 1476bp specific fragment was obtained and DNA sequencing showed that the fragment was consistent with those of the published. After the recombinant expression plasmid was confirmed by enzymes digestion, it showed that the prokaryotic expression vector of rgpAcd gene was constructed successfully. Conclusion The protein of rgpAcd will be obtained for further study. Successful construction of live attenanated vaccine candidate plasmid lays solid foundation for future animal experiments and clinical trials. 　　KEY WORDS: porphyromonas gingivalis; gingipain; expression vector 　　牙周炎是口腔中的常见病和多发病。其引起的牙龈萎缩、牙槽骨丧失一般难以再生，目前治疗上尚无有效的方法。因此，牙周炎的早期预防至关重要。牙周炎是一种细菌感染性疾病，当牙周致病菌侵袭牙周组织时，人体会通过局部细胞免疫和全身免疫影响牙周炎的发