齐齐哈尔医学院学报
齊齊哈爾醫學院學報
제제합이의학원학보
JOURNAL OF QIQIHAR MEDICAL COLLEGE
2015年
14期
2029-2032
,共4页
杜昭弘%王婉%戴京京%何江%王文洋%杨小康%邢应如%尤其%穆敏%胡东%吴静%张荣波
杜昭弘%王婉%戴京京%何江%王文洋%楊小康%邢應如%尤其%穆敏%鬍東%吳靜%張榮波
두소홍%왕완%대경경%하강%왕문양%양소강%형응여%우기%목민%호동%오정%장영파
Ag85B%TAT-PTD%原核表达%融合蛋白
Ag85B%TAT-PTD%原覈錶達%融閤蛋白
Ag85B%TAT-PTD%원핵표체%융합단백
Ag85B%TAT-PTD%Prokaryotic expression%Fusion protein
目的：构建pET28a-PTD-Ag85B原核表达载体，并对其在E．coli BL21（DE3）中表达条件进行优化。方法将TAT-PTD序列插入到用限制性内切酶Nhe I和BamH I双酶切的pET28a质粒中，获得pET28a-PTD重组质粒；RT-PCR法扩增Ag85B基因并将其克隆至pET28a-PTD质粒中，获得pET28a-PTD-Ag85B重组质粒。将重组质粒pET28a-PTD-Ag85B转化至大肠杆菌，异丙基硫代半乳糖苷（ IPTG）诱导PTD-Ag85B融合蛋白表达，聚丙烯酰胺凝胶电泳（ SDS-PAGE）鉴定表达产物。筛选最适诱导剂浓度、诱导温度和时间，尿素复性包涵体，并用His-tag离子亲和层析柱分离纯化融合蛋白。结果成功构建pET28a-PTD-Ag85B重组质粒，在E．coli BL21中能高效表达PTD-Ag85B融合蛋白。结论成功表达及纯化了PTD-Ag85B蛋白，为获得大量PTD-Ag85B融合蛋白提供了技术储备。
目的：構建pET28a-PTD-Ag85B原覈錶達載體，併對其在E．coli BL21（DE3）中錶達條件進行優化。方法將TAT-PTD序列插入到用限製性內切酶Nhe I和BamH I雙酶切的pET28a質粒中，穫得pET28a-PTD重組質粒；RT-PCR法擴增Ag85B基因併將其剋隆至pET28a-PTD質粒中，穫得pET28a-PTD-Ag85B重組質粒。將重組質粒pET28a-PTD-Ag85B轉化至大腸桿菌，異丙基硫代半乳糖苷（ IPTG）誘導PTD-Ag85B融閤蛋白錶達，聚丙烯酰胺凝膠電泳（ SDS-PAGE）鑒定錶達產物。篩選最適誘導劑濃度、誘導溫度和時間，尿素複性包涵體，併用His-tag離子親和層析柱分離純化融閤蛋白。結果成功構建pET28a-PTD-Ag85B重組質粒，在E．coli BL21中能高效錶達PTD-Ag85B融閤蛋白。結論成功錶達及純化瞭PTD-Ag85B蛋白，為穫得大量PTD-Ag85B融閤蛋白提供瞭技術儲備。
목적：구건pET28a-PTD-Ag85B원핵표체재체，병대기재E．coli BL21（DE3）중표체조건진행우화。방법장TAT-PTD서렬삽입도용한제성내절매Nhe I화BamH I쌍매절적pET28a질립중，획득pET28a-PTD중조질립；RT-PCR법확증Ag85B기인병장기극륭지pET28a-PTD질립중，획득pET28a-PTD-Ag85B중조질립。장중조질립pET28a-PTD-Ag85B전화지대장간균，이병기류대반유당감（ IPTG）유도PTD-Ag85B융합단백표체，취병희선알응효전영（ SDS-PAGE）감정표체산물。사선최괄유도제농도、유도온도화시간，뇨소복성포함체，병용His-tag리자친화층석주분리순화융합단백。결과성공구건pET28a-PTD-Ag85B중조질립，재E．coli BL21중능고효표체PTD-Ag85B융합단백。결론성공표체급순화료PTD-Ag85B단백，위획득대량PTD-Ag85B융합단백제공료기술저비。
Objective To construct pET28a-PTD-Ag85B prokaryotic expression vector, and to explore the optimization of expression conditions in E.coli BL21.Methods Using restriction enzymes Nhe I and BamH I to digest plasmid pET28a and then insert the TAT-PTD sequence into it to obtain pET28a-PTD recombinant plasmid, using RT-PCR for amplification of Ag85B gene, and then cloning to pET28a-PTD plasmid to obtain pET28a-PTD-Ag85B recombinant plasmid.Conversion pET28a-PTD-Ag85B recombinant plasmid to E.coli, isopropyl thiogalactoside ( IPTG ) induces PTD-Ag85B fusion protein expression, using polyacrylamide gel electrophoresis ( SDS-PAGE ) identify the expression product.Screening optimal inducer concentration, the optimum induction temperature and induction time, refolding inclusion by urea, separation and purification the fusion protein with a His-tag ion affinity chromatography.Results The recombinant plasmid pET28a-PTD-Ag85B was successfully constructed, could highly express PTD-Ag85B fusion protein in E.coli BL21 ( DE3 ). Conclusions Successful expression and purification the PTD-Ag85B proteins, in order to obtain a large number of PTD-Ag85B fusion protein provides technical reserves.
