CAJ | 학술논문

目的构建GST/HIF-1α融合蛋白表达载体,并在大肠埃希菌(E.coli)中诱导表达.方法以质粒pcDNA3.1-HIF-1α为模板,用PCR法扩增HIF-1α全长及各个截短片段,通过BamHI和Not I酶切位点将HIF-1α全长及各个截短突变体定向插入pGEx-4T-2中,构建原核表达质粒pGEX-4T-2-HIF-1α及其截短突变体,并转化E.coli DH5α,筛选阳性重组子,限制性内切酶酶切鉴定和DNA序列测定正确后,转入E.coli BL21中,异丙基硫代β-D半乳糖苷诱导表达,SDS-PAGE和Western blot鉴定.结果酶切及测序结果证明,成功构建了原核表达质粒pGEX-4T-2-HIF-1α及其截短突变体,并用Western blot方法证实了GST/HIF-1α全长及各个截短突变体融合蛋白的表达.结论成功构建了HIF-1α原核表达载体及其截短突变体,并证实了融合蛋白的表达,为进一步纯化HIF-1α蛋白和研究HIF-1α的生物学功能奠定了基础.
목적 구건GST/HIF-1α융합단백표체재체,병재대장애희균(E.coli)중유도표체.방법 이질립pcDNA3.1-HIF-1α위모판,용PCR법확증HIF-1α전장급각개절단편단,통과BamHI화Not I매절위점장HIF-1α전장급각개절단돌변체정향삽입pGEx-4T-2중,구건원핵표체질립pGEX-4T-2-HIF-1α급기절단돌변체,병전화E.coli DH5α,사선양성중조자,한제성내절매매절감정화DNA서렬측정정학후,전입E.coli BL21중,이병기류대β-D반유당감유도표체,SDS-PAGE화Western blot감정.결과 매절급측서결과증명,성공구건료원핵표체질립pGEX-4T-2-HIF-1α급기절단돌변체,병용Western blot방법증실료GST/HIF-1α전장급각개절단돌변체융합단백적표체.결론 성공구건료HIF-1α원핵표체재체급기절단돌변체,병증실료융합단백적표체,위진일보순화HIF-1α단백화연구HIF-1α적생물학공능전정료기출.
Objective To construct GST/HIF1α fusion protein expression vector and induce its expression in Escherichia coli (E.coli). Methods The coding sequence of hypoxia inducible factor-la (HIF-la) and its deletion fragments were amplified from the plasmid pcD-NA3.1-HIF-la by PCR and inserted into pGEX-4T-2 by BamHI and Not I. The positive recombinanls were identified by restriction endonu-clease digestion and DNA sequencing. Then they were transformed into E.coli BL21, induced by IPTG and identified by SDS-PAGE and Western blot. Results The prokaryotic expression plasmid pGEX-4T-2-HIF-lα and its deletion mutants were successfully constructed and confirmed by enzyme digestion and sequencing. The desired CST/HIF-1α fusion proteins were expressed and confirmed by Western blot. Conclusion The prokaryotic expression plasmid of HIF-la and its deletion mutants were successfully constructed and the expression of fu-sion proteins was confirmed. This study provides the basis for the further research on purifying HIF-la protein and the biological function of HIF-lα.