Soluble Expression Of Human-derived Nanog And Its Fermentation Optimization In Bacillus Subtilis And Escherichia Coli

Nanog is a key transcription factor for maintaining the self-renewal and pluripotency of embryonic stem cells,especially in tumor stem cells.Nanog expression is closely related to the development of a tumor.Nowadays,with the investigation of tumor stem cells,the demand of Nanog in the related medical investigation and medicine innovation is increasing.Currently,the production of Nanog in vitro is mainly carried out by heterologous expression.The public reports mainly focus on the prokaryotic expression studies of mouse-derived and pig-derived Nanog.However,there are few reports on the prokaryotic expression of human-derived Nanog.Moreover,the yield is very low.The efficient preparation of human-derived Nanog can effectively solve many adverse factors caused by animal-derived Nanog in clinical applications and meet the requirements of scientific research and production.Therefore,this study applied molecular cloning technology to construct recombinant Bacillus subtilis and recombinant E.coli expression strains.The main results including:?1?The human-derived nanog gene was connected with the plasmid pMA5,achieved soluble expression in Bacillus subtilis WB800.The number of recombinant plasmids dropped to 67%after 96 h.The concentration of Nanog was obtained at 189 mg·L^-1 after optimizing the components of culture medium and cultural conditions.?2?The human-derived nanog gene was ligated to the E.coli expression plasmid pET-32a,and a recombinant strain was obtained.The recombinant strain was induced by IPTG for expression.Then,the product was purified by nickel ion column affinity chromatography and the isolated product was subjected to thrombin digestion to obtain the target protein.Western blot was used to identify the specificity of antibody binding to the protein.The stability determination of the recombinant E.coli pET32a-nanog suggested that the number of colonies containing the recombinant plasmid was 94%after 96 h.These results indicated that the recombinant expression plasmid pET32a-nanog has a good stability in the E.coli and the strain is suitable for large-scale fermentation.Under shaking culture conditions,the best-induced temperature was 37?,IPTG concentration was 0.2 mmol·L^-1,and the best carbon source was glycerin.Through single factor and orthogonal test,the compound nitrogen source 45 g·L^-1,MgSO₄ 0.8 g·L^-1,sorbitol 0.4 mol·L^-1 were the best fermentation condition,and the yield of Nanog was achieved at 850 mg·L^-1.?3?The fermentation of recombinant E.coli was investigated in a 15 L fermentor.In the fermentation process,a three-stage temperature control method was proposed.The highest Nanog yield was up to 1386 mg·L^-11 after 12 hours with the dissolved oxygen set at 30%,37??30?for 30 min during the induction period?.This study realized the efficient production of Nanog and laid the foundation for the subsequent preparation of Nanog polyclonal antibodies and basic medical research.