牙龈卟啉菌蛋白酶rgp Acd基因表达载体的构建

Construction of a recombinant expression vector of rgpAcd gene of porphyromonas gingivalis

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pMD18-T中并测序鉴定。将目的基因片断插入原核表达载体pET-15b,构建表达质粒pET-15b/rgpAcd,通过限制性酶切鉴定。结果PCR产物电泳结果显示,在大约1.5 kb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1 476 bp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。

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 inter-vector pMD18-T Vector. The correct fragment was linked with and cloned into an prokaryotic expression vector pET-15b. Results A 1 476 bp 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.