牙龈卟啉菌蛋白酶基因rgpA与rgpB蛋白酶区的克隆和表达  

Molecular cloning and expression of rgpA and rgpB proteinase region genes of Porphyromonas gingivalis

作　　者：石卓瑾[1] 严杰[2] 陈莉丽[1] 

机构地区：[1]浙江大学医学院附属第二医院口腔科,杭州310009 [2]浙江大学医学院病原微生物学教研室,杭州310058

出　　处：《口腔医学》2008年第7期340-342,346,共4页Stomatology

基　　金：国家自然科学基金资助项目(30471888)

摘　　要：目的克隆牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)rgpA、rgpB蛋白酶区,构建rgpA、rgpB蛋白酶区原核表达系统。方法采用聚合酶链式反应(PCR)从PgATCC33277菌株中扩增rgpA、rgpB蛋白酶区,T-A克隆后测定核苷酸序列,构建pET42a的rgpA/rgpB蛋白酶区表达载体,在E.coli BL21DE3宿主菌中用不同浓度的IPTG诱导其表达。结果所克隆的PgATCC33277菌株rgpA、rgpB基因蛋白酶区的核苷酸序列与报道的相应核苷酸序列同源性分别为98.9%、99.0%,氨基酸序列同源性分别高达99.2%、99.1%。pET42a-rgpA/rgpB-E.coli BL21DE3系统的蛋白表达量为细菌总蛋白的60%左右。结论本研究成功地构建PgrgpA、rgpB蛋白酶区高效表达系统,为测定RgpA、RgpB免疫原性及作用提供前提。Objective To clone the rgpA and rgpB proteinase gene regions of Porphyromonas gingivalis （Pg） and to construct their expression system via pET42a vector. Methods The rgpA and rgpB proteinase region genes were amplified by PCR and T- A cloned from Pg ATCC 33277 strain. The nuclcotides of the target DNA fragments were firstly sequenced and then inserted into the pET42a to construct expression vectors. The expressions of rgpA and rgpB proteinase region proteins were induced by IFIG with different dosages in BI21 DE3 E. coli. Results The homology of nucleotides sequence of the cloned rgpA and rgpB proteinase region gene fragments from ATCC 33277 strains were 98.9% and 99.0%, compared with the reported sequences. The homology of the amino acid sequences of the cloned rgpA and rgpB proteinase region gene fragments were 99.2% and 99.1%, compared with the reported sequence. The expression output of RgpA and RgpB proteinase region protein in pET 42a - rgpA/rgpB - BI21 DE3 system was approximately 60 % of the total bacterial proteins. Conclusion An expression system of Pg rgpA and rgpB proteinase region genes with high efficiency was successfully established. This will be the premise for the study on their immunogenicity and biological functions.

关 键 词：牙龈卟啉单胞菌 rgpA rgpB 牙龈素 

分 类 号：R394.2[医药卫生—医学遗传学]