河南师范大学学报(自然科学版)
河南師範大學學報(自然科學版)
하남사범대학학보(자연과학판)
JOURNAL OF HENAN NORMAL UNIVERSITY(NATURAL SCIENCE)
2009年
6期
129-132
,共4页
黄维肖%徐妍%徐水凌%曹丽莉%朱逢佳
黃維肖%徐妍%徐水凌%曹麗莉%硃逢佳
황유초%서연%서수릉%조려리%주봉가
幽门螺杆菌%空泡毒素A%原核表达%纯化%鉴定
幽門螺桿菌%空泡毒素A%原覈錶達%純化%鑒定
유문라간균%공포독소A%원핵표체%순화%감정
Helicobacter pylori%vacuolating cytotoxin A%prokaryotic expression%purification%identification
建立从幽门螺杆菌空泡毒素A(VacA)原核表达系统pET32a-vacA-E.coliBL21 DE3中,表达、纯化和鉴定重组空泡毒素A(rVacA)蛋白的方法.采用不同浓度的IPTG(0.1、0.5和1.0 mmol·L~(-1))诱导原核表达系统表达rVacA,10%SDS-PAGE检测表达产量,Ni-NTA亲和层析法纯化rVacA,采用HPLC检测其纯度,Western-blotting进行免疫学鉴定.从已构建的VacA原核表达系统中成功表达和纯化了rVacA蛋白,产量约占细菌总蛋白的28.4%,纯度为82.3%.为后续进一步研究该蛋白的生物活性和相关检测试剂盒奠定基础.
建立從幽門螺桿菌空泡毒素A(VacA)原覈錶達繫統pET32a-vacA-E.coliBL21 DE3中,錶達、純化和鑒定重組空泡毒素A(rVacA)蛋白的方法.採用不同濃度的IPTG(0.1、0.5和1.0 mmol·L~(-1))誘導原覈錶達繫統錶達rVacA,10%SDS-PAGE檢測錶達產量,Ni-NTA親和層析法純化rVacA,採用HPLC檢測其純度,Western-blotting進行免疫學鑒定.從已構建的VacA原覈錶達繫統中成功錶達和純化瞭rVacA蛋白,產量約佔細菌總蛋白的28.4%,純度為82.3%.為後續進一步研究該蛋白的生物活性和相關檢測試劑盒奠定基礎.
건립종유문라간균공포독소A(VacA)원핵표체계통pET32a-vacA-E.coliBL21 DE3중,표체、순화화감정중조공포독소A(rVacA)단백적방법.채용불동농도적IPTG(0.1、0.5화1.0 mmol·L~(-1))유도원핵표체계통표체rVacA,10%SDS-PAGE검측표체산량,Ni-NTA친화층석법순화rVacA,채용HPLC검측기순도,Western-blotting진행면역학감정.종이구건적VacA원핵표체계통중성공표체화순화료rVacA단백,산량약점세균총단백적28.4%,순도위82.3%.위후속진일보연구해단백적생물활성화상관검측시제합전정기출.
The researds is to express,purify and identify the recombinant VacA(rVacA)protein of Helicobacter pylori from prokaryotic expression system pET32α-wacA-E.coliBL2\DE3.The expression of recombination protein is induced by different concentrations of IPTG(0.1,0.5 and 1.0 mmol ? L~(-1))and the expressed product is identified by 10% SDS-PAGE.The expressed recombinant protein is purified by Ni-NTA affinity chromatography.The purity and immunogencity of rVacA are detected by HPLC and Western-blotting,respectively.The expression output of rVacA is approximate 28.4% of the total bacterial protein and the purity is about 82.3%.In this study,the rVacA can be expressed and purified successfully in prokaryotic expression system,which establishes a foundation for the further study of the protein activity and the relational detection kits.