牙龈卟啉单胞菌PrtC蛋白体外表达、纯化及功能初探  

Prokaryotic expression,purification and function of prtC gene in Porphyromonas gingivalis

作　　者：高晓雪 汪颖[2] 夏荣[1] Gao Xiaoxue;Wang Ying;Xia Rong(Dept of Stomatology,The Secnd Afiliated Hospital of Anhui Medical University,Hefei 230601;Laboratory of Biochemistry&Strucural Biology,Uniersity of Science and Technology of China,Hefei 230026)

机构地区：[1]安徽医科大学第二附属医院口腔科,合肥230601 [2]中国科学技术大学结构与生物化学实验室,合肥230026

出　　处：《安徽医科大学学报》2020年第10期1515-1519,共5页Acta Universitatis Medicinalis Anhui

基　　金：安徽省自然科学基金(编号:1508085MH156)。

摘　　要：目的牙龈卟啉单胞菌PrtC蛋白体外表达纯化及功能初探。方法全基因合成牙龈卟啉单胞菌(P.gingivalis)ATCC33277中prtC基因,构建重组表达载体p29-prtC,生物信息学分析和低温诱导表达、SDS-PAGE、Western blot鉴定,酶解法检测胶原酶的功能。结果成功构建了PrtC蛋白原核表达载体,在大肠杆菌表达体系内获得可溶蛋白表达,通过亲和层析和分子筛层析纯化出高纯度且较稳定的PrtC蛋白,并且实验证明它具有降解胶原蛋白的能力。结论体外生化实验说明PrtC蛋白具有胶原蛋白酶活性,并且为后续解析PrtC蛋白的结构奠定了基础。Objective To explore the expression and purification of PrtC protein from Porphyromonas gingivalis and its function in vitro.Methods The whole gene was used to synthesize prtC gene in ATCC33277 of Porphyromonas gingivalis, and the recombinant expression vector p29-prtC was constructed,Bioinformatics analysis, low-temperature induced expression,SDS-PAGE and Western blot identification were performed, and the function of collagenase was detected by enzymatic hydrolysis.Results The prokaryotic expression vector of collagenase protein PrtC was successfully constructed, and the soluble protein expression was obtained in the expression system of E.coli. The protein PrtC with high purity and stability was purified by affinity chromatography and molecular sieve chromatography, and it was proved to have the ability to degrade collagen.Conclusion PrtC protein expressed by prokaryotic recombinant protein had collagenase activity, which laid a foundation for subsequent analysis of PrtC structure.

关 键 词：牙龈卟啉胞菌 PrtC蛋白 胶原酶 蛋白表达 

分 类 号：R781[医药卫生—口腔医学]