CAJ | 학술논문

背景：体外合成或通过基因工程表达的凋亡素 Apoptin 是一种可以特异性地诱导肿瘤细胞凋亡，而对人体正常细胞无毒性和转化活性的蛋白，为抑制肿瘤的生长提供可能。 　　目的：构建凋亡素基因的原核表达载体，优化诱导蛋白表达的相关条件，检测纯化所得目的蛋白的活性。 　　方法：将已构建好的凋亡素基因亚克隆至原核表达载体 pET-28b(+)中，将该质粒转化至大肠杆菌E.coli宿主菌中，以异丙基硫代半乳糖苷(IPTG)对其进行诱导表达，聚丙烯酰胺凝胶电泳(SDS-PAGE)分析目的蛋白，并检测所表达的目的蛋白对肿瘤细胞的增殖抑制作用。 　　结果与结论：凋亡素基因被成功的克隆至 pET-28b(+)，在26℃、IPTG 0.5 mmol/L诱导8 h的情况下，Apoptin为包涵体表达，表达产物经 SDS-PAGE 分析，在相对分子质量约15000的位置出现目的蛋白条带，大小与预期结果一致，经变复性和亲和层析纯化后，获得高纯度目的蛋白，进一步检测发现其对肺癌细胞 H460及H1299具有一定的促凋亡作用。实验成功构建了凋亡素原核表达载体PET-28b-Apoptin，获得了具有一定生物活性的目的蛋白，为进一步研究凋亡素的功能和开发其应用价值研究奠定了基础。
배경：체외합성혹통과기인공정표체적조망소 Apoptin 시일충가이특이성지유도종류세포조망，이대인체정상세포무독성화전화활성적단백，위억제종류적생장제공가능。 　　목적：구건조망소기인적원핵표체재체，우화유도단백표체적상관조건，검측순화소득목적단백적활성。 　　방법：장이구건호적조망소기인아극륭지원핵표체재체 pET-28b(+)중，장해질립전화지대장간균E.coli숙주균중，이이병기류대반유당감(IPTG)대기진행유도표체，취병희선알응효전영(SDS-PAGE)분석목적단백，병검측소표체적목적단백대종류세포적증식억제작용。 　　결과여결론：조망소기인피성공적극륭지 pET-28b(+)，재26℃、IPTG 0.5 mmol/L유도8 h적정황하，Apoptin위포함체표체，표체산물경 SDS-PAGE 분석，재상대분자질량약15000적위치출현목적단백조대，대소여예기결과일치，경변복성화친화층석순화후，획득고순도목적단백，진일보검측발현기대폐암세포 H460급H1299구유일정적촉조망작용。실험성공구건료조망소원핵표체재체PET-28b-Apoptin，획득료구유일정생물활성적목적단백，위진일보연구조망소적공능화개발기응용개치연구전정료기출。
BACKGROUND:Apoptin is a protein which is synthesized in vitro or expressed by genetic engineering, without toxic and transformation activity of normal cel s. Apoptin can specifical y induce the apoptosis of tumor cel s and provide the opportunity of inhibiting the growth of cancer. OBJECTIVE:To construct a prokaryotic expression vector for apoptin, optimize the expression conditions, and detect the activity of the purified protein. METHODS:The apoptin gene that had been constructed was cloned into prokaryotic expression vector pET-28b (+), which was transformed into E.coli host bacteria. Apoptin was induced by isopropyl-beta-D-thiogalactoside, and analyzed by polyacrylamide gel electrophoresis. The inhibition activity of apoptin on tumor cel s was detected. RESULTS AND CONCLUSION:Apoptin gene was successful y cloned into pET-28b (+). Apoptin protein was induced to express in form of inclusion body by isopropyl-beta-D-thiogalactoside (0.5 mmol/L) at 26 ℃. And the expression of apoptin with relative molecular mass of about 15 000 was identified by polyacrylamide gel electrophoresis. The target protein was purified by denaturation-renaturation and affinity chromatography, which has pro-apoptotic effect on lung cancer cel s H460 and H1299. The prokaryotic expression vector pET-28b-apoptin is successful y constructed. The apoptin protein with bioactivity is obtained, which al ows further functional study of apoptin.