人C/EBP β结构与功能的生物信息学分析及原核表达

Bioinformatics analysis and prokaryotic expression of human C/EBPβstructure and function

目的 研究人CCAAT/增强子结合蛋白β(CCAAT/enhancer binding proteins, C/EBP β)原核表达载体的构建和蛋白表达,并通过生物信息学分析其结构和生物学功能。方法 收集对数生长期的肝癌细胞SMMC-7721,通过TRIzol法提取SMMC-7721细胞内总RNA,并以RNA为模板采用RT-PCR获得C/EBP β基因的编码序列,通过同源重组的方法将目的基因与原核表达质粒pET-28a(+)相连接,经过大肠杆菌转化、抗性筛选、质粒提取、酶切和DNA测序获得重组质粒pET-28a(+)-C/EBP β。将重组质粒导入大肠杆菌BL21中,通过异丙基-β-D-硫代半乳糖苷(isopropyl β-D-1-thiogal, IPTG)诱导C/EBP β重组蛋白表达,采用镍离子亲和层析法纯化C/EBP β融合蛋白,通过蛋白免疫印迹验证目的蛋白并分析C/EBP β的蛋白纯度。同时对人C/EBP β编码的蛋白质进行理化性质、亲水/疏水性、二级结构以及磷酸化位点等生物信息学分析。结果 通过RT-PCR成功获得人C/EBP β基因,构建的pET-28a(+)-C/EBP β重组质粒经测序鉴定C/EBP β DNA序列完全正确,表明重组pET-28a(+)-C/EBP β质粒构建成功。C/EBP β蛋白是一个性质不稳定的亲水蛋白质,由345个氨基酸组成,分子量为36.105 kD,等电点为8.55。其是一种胞内蛋白质,主要分布在细胞质和细胞核。对其结构分析发现:无规则卷曲是其主要的二级结构,为60.87%。蛋白免疫印迹结果显示通过亲和层析纯化后获得较高纯度的C/EBP β。结论 本实验成功克隆并构建人pET-28a(+)-C/EBP β重组质粒,获得较高纯度的C/EBP β,为进一步C/EBP β蛋白功能的研究和抗体的制备奠定了良好的基础。

Objective To study the construction of a prokaryotic expression vector of human CCAAT/enhancer binding proteinβ(C/EBPβ),investigate the protein expression of C/EBPβand analyze its structure and biological functions by bioinformatic analysis.Methods The total RNA was extracted from human hepatocellular carcinoma cells SMMC-7721 in the logarithmic growth phase using the TRIzol method.The coding sequence of C/EBPβgene was obtained by RT-PCR with RNA as a template.The target gene was linked to the prokaryotic expression plasmid pET-28a(+)through homologous recombination method.After transformation into E.coli,antibiotic selection,plasmid extraction,restriction enzyme digestion,and DNA sequencing,the recombinant plasmid pET-28a(+)-C/EBPβwas obtained.The recombinant plasmid was transformed into E.coli BL21,and the expression of C/EBPβrecombinant protein was induced by isopropylβ-D-1-thiogalactopyranoside(IPTG).The C/EBPβfusion protein was purified using nickel ion affinity chromatography,and the target protein was verified and its purity was analyzed by Western blotting.Additionally,bioinformatics analysis was performed to analyze the physicochemical properties,hydrophilicity/hydrophobicity,secondary structure,and phosphorylation sites of human C/EBPβprotein.Results The human C/EBPβgene was successfully obtained by RT-PCR,and the constructed pET-28a(+)-C/EBPβrecombinant plasmid was verified by sequencing to have a completely correct C/EBPβDNA sequence,indicating successful construction of the recombinant pET-28a(+)-C/EBPβplasmid.C/EBPβwas an unstable hydrophilic protein,and composed of 345 amino acids,with a molecular weight of 36.105 kD and an isoelectric point of 8.55.C/EBPβwas an intracellular protein,mainly distributed in the cytoplasm and nucleus.Structural analysis showed that the primary secondary structure was mainly random coils,accounting for 60.87%.Western blotting results indicated that highly pure C/EBPβwas obtained after purification using affinity chromatography.Conclusion The human pET-28a(+)-C/EBPβrecombinant plasmid is successfully cloned and constructed,and high purity of C/EBPβis obtained,which lay a solid foundation for further research on the function of C/EBPβprotein and the preparation of antibodies.