医学研究与教育
醫學研究與教育
의학연구여교육
MEDICAL RESEARCH AND EDUCATION
2014年
1期
13-16
,共4页
Survivin%pGEX4T-2%IPTG诱导%纯化
Survivin%pGEX4T-2%IPTG誘導%純化
Survivin%pGEX4T-2%IPTG유도%순화
Survivin%pGEX4T-2%IPTG induction%puriifcation
目的: Survivin是凋亡抑制蛋白(IAP)中最强的凋亡抑制因子,纯化得到其功能结构域(BIR)的融合蛋白可为进一步研究Survivin基因与相关基因的相互作用提供依据。方法利用PCR技术扩增Survivin基因中的BIR序列后连接到pGEX4T-2载体上,经大肠杆菌(DH5α)验证后,转化BL21大肠杆菌菌株并用IPTG进行诱导,以谷胱甘肽琼脂糖亲和层析技术纯化融合蛋白。结果实验扩增得到的BIR片段约为276 bp,纯化得到的融合蛋白分子质量大小约为37 ku。结论 PCR结果与预期的片段大小一致,构建重组体后经IPTG在22℃下诱导2 h,菌体中融合蛋白的表达量显著增加,为下一步研究Survivin基因的功能和与其相互作用的蛋白奠定了基础。
目的: Survivin是凋亡抑製蛋白(IAP)中最彊的凋亡抑製因子,純化得到其功能結構域(BIR)的融閤蛋白可為進一步研究Survivin基因與相關基因的相互作用提供依據。方法利用PCR技術擴增Survivin基因中的BIR序列後連接到pGEX4T-2載體上,經大腸桿菌(DH5α)驗證後,轉化BL21大腸桿菌菌株併用IPTG進行誘導,以穀胱甘肽瓊脂糖親和層析技術純化融閤蛋白。結果實驗擴增得到的BIR片段約為276 bp,純化得到的融閤蛋白分子質量大小約為37 ku。結論 PCR結果與預期的片段大小一緻,構建重組體後經IPTG在22℃下誘導2 h,菌體中融閤蛋白的錶達量顯著增加,為下一步研究Survivin基因的功能和與其相互作用的蛋白奠定瞭基礎。
목적: Survivin시조망억제단백(IAP)중최강적조망억제인자,순화득도기공능결구역(BIR)적융합단백가위진일보연구Survivin기인여상관기인적상호작용제공의거。방법이용PCR기술확증Survivin기인중적BIR서렬후련접도pGEX4T-2재체상,경대장간균(DH5α)험증후,전화BL21대장간균균주병용IPTG진행유도,이곡광감태경지당친화층석기술순화융합단백。결과실험확증득도적BIR편단약위276 bp,순화득도적융합단백분자질량대소약위37 ku。결론 PCR결과여예기적편단대소일치,구건중조체후경IPTG재22℃하유도2 h,균체중융합단백적표체량현저증가,위하일보연구Survivin기인적공능화여기상호작용적단백전정료기출。
Objective Survivin gene is the strongest apoptosis-inhibition factor among inhibitor of apoptosis protein (IAP), and purification of fusion protein of functional domains (baculovirus IAP repeat, BIR) may provide the foundation for interaction research between the gene and related other genes. Methods BIR sequence, after ampliifcation with PCR and validation by Escherichia coli DH5αthrough pGEX4T-2 vector, was transformed into E. coli BL21. Fusion protein of BIR was induced by IPTG and puriifed by Glutathione Agarose Affinity Chromatography. Results The amplified fragment BIR was about 276bp, purified fusion protein molecular weight of approximately 37ku. Conclusion PCR results are consistent with the expected fragment size, recombinant plasmids after 2h after IPTG induction, the cell in the fusion protein increase signiifcantly, which provide basis for further research on Survivin gene function and its interacting protein.