CAJ | 학술논문

目的克隆表达EC-SOD3-凋亡素融合蛋白,并检测其生物活性.方法 PCR扩增出apopfin序列,与表达载体EC-SOD3-Pet-28a连接后在大肠杆菌BL21(DE3)中经IPTG诱导,表达产物进行Ni2+-NTA纯化和MIT活性检测.结果表达载体Pet-28a-EC-SOD3-apoptin经酶切鉴定和序列分析,证明质粒构建正确,转化E. Coli BL21(DE3)后,重组蛋白获得表达,Ni2+-NTA纯化后的凋亡素融合蛋白纯度达到85%以上,经噻唑蓝(MTT)法测定具有活性.结论成功构建了表达载体Pet-28a-EC-SOD3-apopfin,并在大肠杆菌中表达了EC-SOD3-凋亡素融合蛋白,纯化的蛋白质具有诱导HeLa细胞凋亡的能力.
목적 극륭표체EC-SOD3-조망소융합단백,병검측기생물활성.방법 PCR확증출apopfin서렬,여표체재체EC-SOD3-Pet-28a련접후재대장간균BL21(DE3)중경IPTG유도,표체산물진행Ni2+-NTA순화화MIT활성검측.결과 표체재체Pet-28a-EC-SOD3-apoptin경매절감정화서렬분석,증명질립구건정학,전화E. Coli BL21(DE3)후,중조단백획득표체,Ni2+-NTA순화후적조망소융합단백순도체도85%이상,경새서람(MTT)법측정구유활성.결론 성공구건료표체재체Pet-28a-EC-SOD3-apopfin,병재대장간균중표체료EC-SOD3-조망소융합단백,순화적단백질구유유도HeLa세포조망적능력.
Objective To clone and express apoptin-EC-SOD3 fusion protein and determine its bioactivity in vitro. Methods Apoptin gene was amplified by PCR and cloned into prokaryotic expression vector EC-SOD3-pET-28a. The constructed recombinant plasmid pET-28a-EC-SOD3-apoptin was transformed to E. Coli BL21 (DE3) for the expression under the induction of IPTG. The expressed protein was purified by Ni2+ -NTA affinity chromatography and its activity was identified by MTr. Results PCR analysis proved that the recombinant plasmid pET-28a-EC-SOD3-apoptin was correctly constructed. The expressed soluble fusion protein was consistent with that expected. Its purity was over 85 % after being purified with Ni2+-NTA agarose and the purified protein showed bioactivity by MTr assay. Conclusion The recombinant plasmid for prokaryotic expression of pET-28a-EC-SOD3-apoptin can be constructed and the apoptin-EC-SOD3 fusion protein can be expressed successfully in E. Coli BL21 (DE3). MTT result indicates that HeLa cells are highly sensitive to the purified apoptin-EC-SOD3.