Coupling Design,optimized Expression And Anti-tumor Activity Of Onconase

Onconase is a ribonuclease extracted from the oocytes of the North American Rana Pipiens.It has RNA degrading activity and is approved by the FDA for the treatment of mesenchymal tumors.The clinical application shows that Onconase has low immunogenicity,little damage to normal tissues and organs,and has good application prospects in other tumor treatments.However,the number of North American Rana Pipiens used to extract Onconase has been declining year by year.The source of Onconase is limited,and my country has not introduced this species,which limits the basic research and clinical application of Onconase in our country to a certain extent.Therefore,the use of genetic engineering technology to obtain Onconase is of great significance.Other study has shown that Onconase can form dimers through the exchange of N-terminal peptide chains during the drying process,and its anti-tumor effect is better than that of monomer,which is expected to further promote the research and application of Onconase.Among genetic engineering expression systems,the Pichia pastoris expression system has gradually been widely used.There are many types of promoters available in this expression system,among which p GAP occupies an important advantage.Pichia pastoris uses p GAP to express foreign proteins without changing the carbon source,and can avoid the use of methanol.Therefore,the fermentation cycle is short,the production cost is low,and the safety is high,which is conducive to large-scale production.The most important thing is that Pichia pastoris is a eukaryotic organism,which can process and modify proteins without producing endotoxin.The FDA recognizes it as a safe expression system and has broad application prospects in the field of medicine.At present,there are few reports on the expression of Onconase dimers in Pichia pastoris.The purpose of this study is to design the onconase gene for coupling.It is expected to form disulfide bonds to form dimers,to study its expression in Pichia pastoris,and to explore whether it has anti-tumor activity.The specific research content is as follows:1.The construction of recombinant Pichia pastoris expression systemThe Onconase gene was designed for even polymerization,optimized according to the Pichia pastoris codon preference,XhoⅠand Eco RⅠrecognition sites were introduced at both ends of the gene,and the gene fragments were integrated into the p GAPZαMUT vector designed by our teaching and research office to construct The recombinant plasmid p GAPZαMUT-D-ONC,double enzyme digestion results showed that the recombinant plasmid was successfully constructed.The linearized plasmid was electrotransformed into Pichia pastoris GS115,and engineering bacteria were screened by Zeocin resistance,gene sequencing and SDS-PAGE.The supernatant obtained from shake flask culture was purified by Ni-Sepharose 6FF affinity chromatography column.The results showed that Pichia pastoris successfully expressed the target protein.The target protein concentration determined by the BCA method was 102.25 mg/L.DTT was used to reduce the D-ONC protein.The result showed that the dimer was successfully reduced to monomer,indicating that the target protein expressed by Pichia pastoris was composed of Onconase single chain.2.The culture conditions of the engineered strain were optimizedThe influence of pH,temperature,rotation speed,carbon source and time on protein expression was studied by single factor experiment and uniform design experiment.The single factor experiment was used to optimize.The results showed that the best pH was 6.0,the best temperature was 29°C,the best rotation speed was200 rpm,the best carbon source was glucose,and the best time was 72 h.On this basis,a four-factor six-level uniform design experiment was designed,with protein concentration as an indicator,pH,temperature,rotation speed and carbon source concentration as dependent variables,and the regression model was established by stepwise regression analysis.The results showed that the concentration of the carbon source had the most significant effect on the expression of D-ONC,and there was an interaction between the various culture conditions.The optimal culture conditions showed that:the pH was 5.6,the temperature was 28℃,the rotation speed was 220rpm,the carbon source concentration was 3.5%,and the time was 72 h.The protein concentration under this condition combination was 119.03 mg/L.Compared with conditions,the protein concentration was increased by 16.41%.3.Pilot trail and purification of D-ONCBased on the results of optimization of expression conditions,the engineered strain was subjected to fermentation at the level of a 50 L fermentor.Feeding was carried out by means of dissolved oxygen feedback,and fermentation was stopped after 72 h.Filtration of fermentation brothed after centrifugation.The suction filtrate was concentrated by using an ultrafiltration membrane with a cut-off flow of 10 k Da.The obtained retentate was separated and purified by Ni-Sepharose 6FF affinity column to purify the D-ONC.The results showed that the protein concentration of D-ONC was the highest at 72 h,and the pure target protein D-ONC could be obtained after purification.The target protein concentration was 496.36 mg/L.4.The activity determination of D-ONCMTT method was used to to analyze whether D-ONC has biological activity on MCF-7 and Hep G2 cells.After different concentrations of D-ONC acted on MCF-7and Hep G2 cells for 48 h,the cell morphology was observed and the absorbance was measured.The results showed that the cell inhibition rate and tumor cell morphology were different between different concentration groups.The IC₅₀of D-ONC to MCF-7and Hep G2 cells were 2.8 and 3.3μmol/L,respectively.In summary,this study successfully constructed the recombinant plasmid p GAPZαMUT-D-ONC,and correctly expressed the Onconase conjugate in Pichia pastoris;after optimizing the expression conditions of Pichia pastoris,the expression of D-ONC was increased;the fermentation product was separated and purified,and the obtained D-ONC was used for in vitro activity research,which preliminarily confirmed that D-ONC has a certain anti-tumor activity,laying a theoretical foundation for further research on D-ONC.