摘要
目的克隆牙龈卟啉单胞菌精氨酸牙龈素催化结构域(RgpAcd)基因,并将其置于大肠杆菌中作融合表达。方法利用PCR技术和基因重组技术,克隆牙龈卟啉单胞菌RgpAcd,然后插入中介载体pMD18-T中并测序鉴定。将目的基因片段插入原核表达载体pET-15b来构建表达质粒pET-15b/RgpAcd。重组原核表达质粒经酶切鉴定后转化大肠杆菌BL21感受态细胞,以异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达融合蛋白。结果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;IPTG诱导后的菌体经SDS-聚丙烯酰胺凝胶电泳后有一个相对分子质量为5×104的融合表达蛋白产生。结论本实验成功克隆了牙龈卟啉单胞菌RgpAcd基因,并在大肠杆菌中表达了RgpAcd蛋白,为进一步研制重组活载体疫苗奠定了基础。
Objective To clone the catalytic domain gene sequence of RgpAcd of Porphyromonas gingivalis （P.gingivalis） and to induce its fusion expression in E. coli. 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 correctly fragment was linked with and cloned into a prokaryotie expression vector pET-15b. The recombinant expression plasmid which had been confirmed by enzymes digestion was transformed to E. coli competent ceils BL21 （DE3） and expression of fusion protein was induced by IPTG. Results A 1 476 bp specific fragment was obtained and DNA sequencing showed that the fragment was consistent with those of the published. After induction with IPTG, a fusion protein of 5×10^4 was visualized on SDS-PAGE gel. Conclusion The protein of RgpAcd will be obtained for further study and its protein was correctly expressed in E. coli BL21 ceils.
出处
《华西口腔医学杂志》
CAS
CSCD
北大核心
2006年第5期400-403,共4页
West China Journal of Stomatology
基金
陕西省科技攻关计划资助项目(2004K11-G(44))
陕西省卫生厅科学研究基金资助项目(04D36)
关键词
牙龈卟啉单胞菌
蛋白酶
原核表达
Porphyromonas gingivalis
gingipain： prokaryotie expression
