中华口腔医学杂志
中華口腔醫學雜誌
중화구강의학잡지
Chinese Journal of Stomatology
2012年
z1期
63-67
,共5页
何钟勤%钟丞%高心%薛莹%孙晓宇%刘晓红
何鐘勤%鐘丞%高心%薛瑩%孫曉宇%劉曉紅
하종근%종승%고심%설형%손효우%류효홍
韦荣球菌属%乳酸脱氢酶类%重组表达%蛋白纯化
韋榮毬菌屬%乳痠脫氫酶類%重組錶達%蛋白純化
위영구균속%유산탈경매류%중조표체%단백순화
Veillonella%Lactate dehydrogenases%Preliminary expression%Protein activity
目的 进一步测定殊异韦荣菌中依赖性乳酸脱氢酶(lactate dehydrogenase,LDH)的活性,以期为研究该酶的具体功能及作用机制奠定基础,并为龋病预防和治疗提供新的思路.方法 重组质粒转入大肠杆菌BL21(DE3)和Rosetta(DE3)中,采用不同时间及6种不同浓度的异丙基硫代-β-D-半乳糖苷(isopropyhhio-β-D-galactoside,IPTG)诱导乳酸脱氢酶蛋白表达,选择最佳条件诱导LDH蛋白表达.并经His-标记蛋白纯化柱(HisTRAP FF柱)提纯,用LDH活性试剂盒测定蛋白活性.结果 pET-28a-LDH转入BL21(DE3)和Rosetta(DE3)中十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示最适表达条件分别为IPTG终浓度0.4 mmol/L、30℃诱导8h,IPTG终浓度0.4 mmol/L、30 ℃诱导6h.经BandScan 5.0软件分析,pET-28a-LDH质粒转化BL21(DE3)表达相对含量为9.0％,高于Rosetta(DE3)的6.1％；LDH活性试剂盒测定的蛋白活性值为13.79.结论 本项研究获得了LDH蛋白的最适诱导条件,并测出殊异韦荣菌中依赖性LDH的活性,为进一步研究LDH的功能及在龋病防治中的作用奠定了基础.
目的 進一步測定殊異韋榮菌中依賴性乳痠脫氫酶(lactate dehydrogenase,LDH)的活性,以期為研究該酶的具體功能及作用機製奠定基礎,併為齲病預防和治療提供新的思路.方法 重組質粒轉入大腸桿菌BL21(DE3)和Rosetta(DE3)中,採用不同時間及6種不同濃度的異丙基硫代-β-D-半乳糖苷(isopropyhhio-β-D-galactoside,IPTG)誘導乳痠脫氫酶蛋白錶達,選擇最佳條件誘導LDH蛋白錶達.併經His-標記蛋白純化柱(HisTRAP FF柱)提純,用LDH活性試劑盒測定蛋白活性.結果 pET-28a-LDH轉入BL21(DE3)和Rosetta(DE3)中十二烷基硫痠鈉-聚丙烯酰胺凝膠電泳顯示最適錶達條件分彆為IPTG終濃度0.4 mmol/L、30℃誘導8h,IPTG終濃度0.4 mmol/L、30 ℃誘導6h.經BandScan 5.0軟件分析,pET-28a-LDH質粒轉化BL21(DE3)錶達相對含量為9.0％,高于Rosetta(DE3)的6.1％；LDH活性試劑盒測定的蛋白活性值為13.79.結論 本項研究穫得瞭LDH蛋白的最適誘導條件,併測齣殊異韋榮菌中依賴性LDH的活性,為進一步研究LDH的功能及在齲病防治中的作用奠定瞭基礎.
목적 진일보측정수이위영균중의뢰성유산탈경매(lactate dehydrogenase,LDH)적활성,이기위연구해매적구체공능급작용궤제전정기출,병위우병예방화치료제공신적사로.방법 중조질립전입대장간균BL21(DE3)화Rosetta(DE3)중,채용불동시간급6충불동농도적이병기류대-β-D-반유당감(isopropyhhio-β-D-galactoside,IPTG)유도유산탈경매단백표체,선택최가조건유도LDH단백표체.병경His-표기단백순화주(HisTRAP FF주)제순,용LDH활성시제합측정단백활성.결과 pET-28a-LDH전입BL21(DE3)화Rosetta(DE3)중십이완기류산납-취병희선알응효전영현시최괄표체조건분별위IPTG종농도0.4 mmol/L、30℃유도8h,IPTG종농도0.4 mmol/L、30 ℃유도6h.경BandScan 5.0연건분석,pET-28a-LDH질립전화BL21(DE3)표체상대함량위9.0％,고우Rosetta(DE3)적6.1％；LDH활성시제합측정적단백활성치위13.79.결론 본항연구획득료LDH단백적최괄유도조건,병측출수이위영균중의뢰성LDH적활성,위진일보연구LDH적공능급재우병방치중적작용전정료기출.
Objective To determine dependence lactate dehydrogenase activity in Veillonella dispar.Methods Transformed the recombinant plasmid pET-28a-LDH in BL21 (DE3) and Rosetta(DE3).Recombinant lactate dehydrogenase(LDH) protein was induced at different conditions.Induction condition was optimized to obtain proper yield of recombinant protein.After purification by HisTRAP FF column,the protein activity was determined with LDH reactive kit.Results Sodium dodecylsulfate-polyacrylamide gel electrophoresis showed that the best protein expression conditions were isopropylthio-β-D-galactoside (IPTG)end for 0.4 mmol/L concentration in 30 degrees to induce 8 hours,or IPTG end for 0.4 mmol/L concentration in 30 degrees to induce 6 hours.Through the analysis of BandScan 5.0,the expression in BL21 (DE3) was 9.0％,higher than 6.1％ in Rosetta(DE3).After purification by HisTRAP FF column,the protein active value of 13.79.Conclusions Recombinant LDH protein could be induced by IPTG with an optimal condition.
