变形链球菌耐氟菌株耐酸相关基因ropA的克隆及蛋白表达  

Cloning and Protein Expression of Acid-Resistance Gene ropA in Fluoride-Resistance Strain of Streptococcus mutans

作　　者：超博 张红[1] 李文月[1] 邵思奇 张志民[1] 

机构地区：[1]吉林大学口腔医学院牙体牙髓病科,吉林长春130041

出　　处：《微生物学杂志》2017年第4期23-27,共5页Journal of Microbiology

基　　金：大学生创新创业训练计划项目(2014B78315)

摘　　要：克隆并表达变形链球菌耐氟菌株耐酸相关基因ropA。以变形链球菌UA159的耐氟菌株全基因组为模版,PCR扩增ropA基因并与p MD-18T克隆载体连接构建重组克隆质粒,测序鉴定。Bam HⅠ、HindⅢ双酶切重组克隆质粒,回收目的基因片段并与p Pro EX HTa表达载体通过粘性末端连接构建重组表达质粒,转化入大肠埃希菌感受态细胞DH5α,IPTG诱导,SDS-PAGE检测Rop A表达量。测序结果为变形链球菌UA159的耐氟菌株ropA基因碱基序列与亲代菌株UA159完全一致。SDS-PAGE结果显示IPTG成功诱导Rop A蛋白表达,并且随诱导时间的延长蛋白表达增多。变形链球菌UA159耐氟菌株的耐酸相关基因ropA未发生突变,说明变形链球菌耐氟菌株耐酸性增强不是由ropA碱基序列的改变导致。本研究成功诱导Rop A蛋白表达,为后续研究Rop A蛋白功能奠定了基础。Acid-resistance related gene ropA of fluoride-resistance strain of Streptococcus mutaus was cloned and ex- pressed taking template of complete genome of fluoride-resistance strain UA159 of S. mutans, PCR amplified ropA gene and linked with pMD-18T cloned vector to construct recombinant cloned plasmid, sequenced and characterized. The recombinant plasmid was digested with double enzyme BamH I and Hind III, the recovered targeted gene frag- ments were linked with pProEX Hta expressed vector through viscose terminus to construct the expression plasmid and transformed into E. coli＇s receptive state cells DH5c~, induced with IPTG, and detected the expression amount of ropA with SDS-PAGE. The results showed that the sequencing results of ropA gene base sequence of fluoride-resistant strain of S. mutans was completely accord with the parent strain UA159. The results of SDS-PAGE showed that IPTG suc- cessfully induced to express RopA protein, and as the induced time protracted the expressed amount of protein in- creased. Therefore, it was concluded that acid-resistant related gene ropA of fluoride-resistant strain of S. mt^tans did not mutated, suggested that the strengthening of acid-resistance of fluoride-resistant strain of S. mutans was resulted in the changes of ropA base sequence. The successful induction of RopA protein expression in this experiment had laid a foundation to study on the function of RopA protein that follow up.

关 键 词：变形链球菌 耐氟菌株 耐酸相关基因ropA 克隆 蛋白表达 

分 类 号：Q939.93[生物学—微生物学]