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目的克隆人八聚体结合蛋白4(Oct4)c DNA,研究其在原核及真核系统中的表达。方法提取人宫颈癌He La细胞中的总RNA,经反转录PCR获得c DNA,PCR扩增Oct4 c DNA并将其分别克隆入表达载体p ET-22b(+)原核质粒和pc DNATM3.1(+)真核质粒,构建含该序列的重组原核表达载体p ET-22b(+)-Oct4和真核表达载体pc DNA3.1(+)-his-Oct4。然后分别进行双酶切和测序鉴定。前者在E.coli BL21(DE3)中诱导表达目的蛋白,后者以脂质体介导法转染至HEK293T细胞,SDS-PAGE和Western blot法检测细菌和细胞中OCT4蛋白的表达水平。结果 PCR扩增得到约1100 bp目的 DNA片段;重组表达质粒p ET-22b(+)-Oct4和pc DNA3.1(+)-his-Oct4经双酶切鉴定和测序分析证明载体构建成功;p ET-22b(+)-Oct4经异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达后SDS-PAGE分析表明,目的蛋白在宿主E.coli BL21(DE3)中高效表达OCT4融合蛋白,相对分子质量(Mr)约38 000,与其理论Mr基本相符;pc DNA3.1(+)-his-Oct4转染HEK293T细胞经Western blot法检测,证实导入的Oct4成功表达。结论在大肠杆菌和HEK293T细胞成功表达OCT4蛋白。
Objective To clone human octamer binding protein 4 (Oct4) c DNA and study its expression in prokaryotic and eukaryotic systems. Methods Total RNA was extracted from human cervical cancer HeLa cells and c DNA was obtained by reverse transcription PCR. Oct4 c DNA was amplified by PCR and cloned into expression vector pET-22b (+) prokaryotic plasmid and pcDNA3.1 (+) Eukaryotic plasmid was constructed. The recombinant prokaryotic expression vector p ET-22b (+) - Oct4 and the eukaryotic expression vector pcDNA3.1 (+) - his-Oct4 were constructed. Then double digestion and sequencing were identified. The former was expressed in E.coli BL21 (DE3) and the latter was transfected into HEK293T cells by lipofectamine. The expression of OCT4 protein in bacteria and cells was detected by SDS-PAGE and Western blot. Results The DNA fragment of about 1100 bp was amplified by PCR. The recombinant plasmid p ET-22b (+) - Oct4 and pcDNA3.1 (+) - his-Oct4 were identified by double enzyme digestion and sequencing analysis. After SDS-PAGE analysis of ET-22b (+) - Oct4 induced by isopropyl-β-D-thiogalactopyranoside (IPTG), the target protein was highly expressed in E. coli BL21 (DE3) OCT4 fusion protein, relative molecular mass (Mr) about 38 000, and its theoretical Mr basically consistent; pcDNA3.1 (+) - his-Oct4 transfected HEK293T cells detected by Western blot, confirmed the successful expression of Oct4. Conclusion OCT4 protein was successfully expressed in E. coli and HEK293T cells.