Construction And Expression Of Recombinant Fusion Protein Of Human Apolipoprotein O

ObjectivesTo construct human apolipoprotein O (apoO) expression vector and obtain recombinant fusion protein thioredoxin (trx)-apoO by Escherichia coli prokaryotic expression system.MethodsThe ApoO gene fragment from the human liver cDNA library was amplified by PCR. The resulting product was inserted into pET-32a vector, cloned into plasmid E. coli DH10B and sequenced after double digestion. The confirmed cDNA then transformed into E. coli BL 21 (DE3) where it was induced to express protein by IPTG.The fusion protein was purified by Ni-NTA resin. BCA protein assay was applied to detect the concentration of recombinant apoOResults1. The apoO gene was amplified according to the human liver cDNA library by PCR. The PCR products were put into 1.5% agarose gel for electrophoresis and observed with UV transilluminator. DNA fragment about 519 bp which was in correspondence with the expected relative molecular mass was oberserved.2. The pET-apoO plasmid was established successfully.After double digestion with Bam HI and Xhoâ… , the recombinant plasmid was showed 2 specific bands on 1% agarose gel electrophoresis.One was close to the location of plasmid before digestion; the other had a uniform size as the target gene. The inserted fragment was confirmed correct by DNA sequencing.3. When compared with the control group, the IPTG-induced E. coli BL21 (DE3) that contained the recombinant plasmid pET-ApoO was showed a new band (relative molecular mass was about 32 kD) on the SDS-PAGE which was nearly in accordance with the molecular mass of the target protein.4. After purified by immunoaffinity chromatography, the recombinant fusion protein was showed a relative molecular weight of 34 kD, which was in line with the molecular mass of the target protein.5. The concentration of the recombinant protein was calculated according to the the BCA protein assay curve, and the result was 0.5 mg/ml.ConclusionsWe have cloned human apoO gene and successfully expressed its recombinant fusion protein trx-apoO.The human apoO recombinant protein can be useful in clinical and basic medical researches.On one hand, it can be served as an antigen for developing apoO antibody which could be applied to measure plasma concentration of apoO for clinical use. On the other hand, It can also be used in animal or cellular models to further explore the role of apoO in lipid metabolism in vivo.