中华口腔医学杂志
中華口腔醫學雜誌
중화구강의학잡지
Chinese Journal of Stomatology
2010年
8期
482-486
,共5页
李璐%段君兰%王晓茜%杨迷芳%徐艳
李璐%段君蘭%王曉茜%楊迷芳%徐豔
리로%단군란%왕효천%양미방%서염
放线杆菌,伴放射菌%毒素类,生物学%脱氧核糖核酸酶Ⅰ%基因克隆
放線桿菌,伴放射菌%毒素類,生物學%脫氧覈糖覈痠酶Ⅰ%基因剋隆
방선간균,반방사균%독소류,생물학%탈양핵당핵산매Ⅰ%기인극륭
Actinobacillus actinomycetemcomitans%Toxins,biological%Deoxyribonuclease Ⅰ%Gene cloning
目的 体外构建伴放线放线杆菌(Actinobacillus actinomycetemcomitans,Aa)CdtB蛋白的原核表达载体并诱导其表达,通过变性、复性获得有Ⅰ型脱氧核糖核酸酶(deoxyribonuclease Ⅰ,DNase Ⅰ)样活性的重组CdtB蛋白,为进一步研究AaCdtB的功能以及Aa细胞致死性扩张毒素三聚体全毒素在牙周炎发生、发展过程中的分子致病机制奠定基础.方法 以AaATCC29522基因组DNA为模板,采用聚合酶链反应(PCR)法获得cdtB基因,经双酶切、连接的定向克隆技术构建原核表达载体pET-15b-cdtB.转化大肠杆菌感受态细胞BL21(DE3),异丙基-β-D-硫代半乳糖苷诱导CdtB蛋白表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)及蛋白质印迹法检测并鉴定蛋白表达.纯化的重组CdtB体外与超螺旋质粒(pET-32a)DNA孵育,观察其生物学活性.结果 经测序鉴定,构建的原核表达载体pET-15b-cdtB转染的感受态大肠杆菌携带有cdtB基因,该基因片段与GenBank中已收录的AacdtB基因序列一致性高达99%.SDS-PAGE观察到32 000左右的高表达蛋白条带,蛋白质印迹法发现带有6个组氨酸蛋白标签标记的目的 蛋白CdtB.超螺旋质粒DNA与CdtB体外孵育后发生了解螺旋现象.结论 本项研究成功构建了AaCdtB的原核表达载体并诱导CdtB蛋白的体外表达,获得了具有DNaseⅠ样活性的重组CdtB蛋白.
目的 體外構建伴放線放線桿菌(Actinobacillus actinomycetemcomitans,Aa)CdtB蛋白的原覈錶達載體併誘導其錶達,通過變性、複性穫得有Ⅰ型脫氧覈糖覈痠酶(deoxyribonuclease Ⅰ,DNase Ⅰ)樣活性的重組CdtB蛋白,為進一步研究AaCdtB的功能以及Aa細胞緻死性擴張毒素三聚體全毒素在牙週炎髮生、髮展過程中的分子緻病機製奠定基礎.方法 以AaATCC29522基因組DNA為模闆,採用聚閤酶鏈反應(PCR)法穫得cdtB基因,經雙酶切、連接的定嚮剋隆技術構建原覈錶達載體pET-15b-cdtB.轉化大腸桿菌感受態細胞BL21(DE3),異丙基-β-D-硫代半乳糖苷誘導CdtB蛋白錶達,十二烷基硫痠鈉-聚丙烯酰胺凝膠電泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)及蛋白質印跡法檢測併鑒定蛋白錶達.純化的重組CdtB體外與超螺鏇質粒(pET-32a)DNA孵育,觀察其生物學活性.結果 經測序鑒定,構建的原覈錶達載體pET-15b-cdtB轉染的感受態大腸桿菌攜帶有cdtB基因,該基因片段與GenBank中已收錄的AacdtB基因序列一緻性高達99%.SDS-PAGE觀察到32 000左右的高錶達蛋白條帶,蛋白質印跡法髮現帶有6箇組氨痠蛋白標籤標記的目的 蛋白CdtB.超螺鏇質粒DNA與CdtB體外孵育後髮生瞭解螺鏇現象.結論 本項研究成功構建瞭AaCdtB的原覈錶達載體併誘導CdtB蛋白的體外錶達,穫得瞭具有DNaseⅠ樣活性的重組CdtB蛋白.
목적 체외구건반방선방선간균(Actinobacillus actinomycetemcomitans,Aa)CdtB단백적원핵표체재체병유도기표체,통과변성、복성획득유Ⅰ형탈양핵당핵산매(deoxyribonuclease Ⅰ,DNase Ⅰ)양활성적중조CdtB단백,위진일보연구AaCdtB적공능이급Aa세포치사성확장독소삼취체전독소재아주염발생、발전과정중적분자치병궤제전정기출.방법 이AaATCC29522기인조DNA위모판,채용취합매련반응(PCR)법획득cdtB기인,경쌍매절、련접적정향극륭기술구건원핵표체재체pET-15b-cdtB.전화대장간균감수태세포BL21(DE3),이병기-β-D-류대반유당감유도CdtB단백표체,십이완기류산납-취병희선알응효전영(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)급단백질인적법검측병감정단백표체.순화적중조CdtB체외여초라선질립(pET-32a)DNA부육,관찰기생물학활성.결과 경측서감정,구건적원핵표체재체pET-15b-cdtB전염적감수태대장간균휴대유cdtB기인,해기인편단여GenBank중이수록적AacdtB기인서렬일치성고체99%.SDS-PAGE관찰도32 000좌우적고표체단백조대,단백질인적법발현대유6개조안산단백표첨표기적목적 단백CdtB.초라선질립DNA여CdtB체외부육후발생료해라선현상.결론 본항연구성공구건료AaCdtB적원핵표체재체병유도CdtB단백적체외표체,획득료구유DNaseⅠ양활성적중조CdtB단백.
Objective To construct the prokayotic expression vector pET-15b-cdtB containing the cdtB gene from Actinobacillus actinomycetemcomitans (Aa) and to test the bioactivity of this recombinant CdtB in vitro. Methods The toxic cytolethal distending toxin (CDT) subunit encoding gene cdtB was amplified by PCR. Through restriction endonuclease digestion,gene cdtB and vector pET-15b were ligated to form pET-15b-cdtB expression system which was transformed into competent cells Escherichia coli BL21 ( DE3 ). Protein expression was induced by isopropyl-beta-D-thiogalactoside and examined by sodium dodecyl sulfate-pelyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Supercoiled plasmid pET-32a DNA was incubated with purified recombinant CdtB protein in vitro to view any changes in the electrophoretic mobility of the plasmid pET-32a DNA band. Results PCR testing results of pET-15b-cdtB transformed cells demonstrated that all strains contained cdtB gene. The DNA sequence was blast with cdtB gene from GenBank and 99% homology was obtained. Both of SDS-PAGE and Western blotting confirmed that recombinant CdtB was obtained. After incubated with the purified recombinant CdtB in vitro, the supercoiled plasmid pET-32a DNA was observed relaxing by 1% ngarose gel electrophoresis test. Conclusions The recombinant plasmid pET-15b-cdtB was successfully constructed and the recombinant CdtB protein which has the Dnase Ⅰ -like activity was obtained.
