摘要
目的:对在大肠杆菌中表达重组变形链球菌表面蛋白抗原PAc的培养条件进行优化,制备高纯度重组蛋白rPAc。方法:载体构建原核表达载体pET20b(+)-AP对不同培养基、诱导浓度,温度和时间等进行筛选确定最佳条件,采用亲和层析分离纯化rPAc蛋白。结果:含原核表达载体pET20b(+)-AP的大肠杆菌在LB培养基中培养至A600=0.6时,终浓度为1mM的IPTG诱导,30℃继续振荡培养4h,可使rPAc的表达量达到最大。分离纯化的蛋白在SDS-PAGE中显示为单一条带。结论:对PAcA-P区的重组质粒pET20b(+)-AP表达的rPAc经纯化后可用于动物免疫,为探索防龋DNA疫苗pCIA-P的新型免疫策略奠定必要的物质基础。
Objective： To express and purify the surface protein antigen（PAc） of streptococcus mutans in E. coli BL21（DE3） plysS and optimize the cultivation reguIation. Methods： The recombinant plasmid pET20b（＋ ）- AP was expressed in E. coli BL21（DE3）plysS and the expression level of rPAc was detected by SDS--PAGE under dif- ferent cultivation conditions, including concentration of IPTG, growth rate of bacterial, and etc. After that, the expressed recombinant protein rPAc was.purified using immobilized metal ion affinity chromatography and identified by SDS--PAGE. Results： It was found that the expression level of rPAc achieved the highest when the BL21（DE3） plysS had been induced for 4h with 1mM IPTG beginning from strain density of A600=0.6 at 30℃. rPAc was puri- fied from the soluble phase by means of His--tag affinity chromatography on a nickel--charged column, with a yield of approximately 300 mg of protein per liter of culture. Conclusion： The recombinant protein has antigenicity and can be acted as candidate anti--caries vaccine. This study could lay a material foundation for the further research of DNA prime--protein boost inoculation.
出处
《口腔医学研究》
CAS
CSCD
北大核心
2010年第1期60-63,共4页
Journal of Oral Science Research
基金
国家自然科学基金资助项目(编号:30600707)
