Bioinformatics analysis and prokaryotic expression of human C/EBP β structure and function
Objective To study the construction of a prokaryotic expression vector of human CCAAT/enhancer binding protein β(C/EBP β),investigate the protein expression of C/EBP β and 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/EBP β gene 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/EBP β was obtained.The recombinant plasmid was transformed into E.coli BL21,and the expression of C/EBP β recombinant protein was induced by isopropyl β-D-1-thiogalactopyranoside(IPTG).The C/EBP β fusion protein was purified using nickel ion affinity chroma-tography,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/EBP β protein.Results The human C/EBP β gene was successfully obtained by RT-PCR,and the constructed pET-28a(+)-C/EBP β recombinant plasmid was verified by sequencing to have a completely correct C/EBP β DNA sequence,indicating successful construction of the recombinant pET-28a(+)-C/EBP β plasmid.C/EBP β was 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/EBP β was 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/EBP β was obtained after purification using affinity chromatography.Conclusion The human pET-28a(+)-C/EBP β recombinant plasmid is successfully cloned and constructed,and high purity of C/EBP β is obtained,which lay a solid foundation for further research on the function of C/EBP β protein and the preparation of antibodies.
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