Construction Of Recombinant Bacillus Subtilis Expressing Goat IFN-α And Optimization Of Its Fermentation Conditions

Bacillus subtilis,as a probiotic,has the characteristics of being inexpensive,easy to obtain,and easy to ferment and cultivate,and is currently an ideal expression host for the production of various exogenous proteins,which has been widely studied and applied in agriculture,industry and biomedicine.With the in-depth research,it was found that the Bacillus subtilis expression system still has deficiencies,mainly including short maintenance time of the receptor state,low transformation efficiency,poor stability of recombinant plasmids,degradation of extracellular proteases to exogenous proteins,difficulty in screening the most suitable signal peptide for extracellular secretion of exogenous proteins and high complexity of promoters,so for specific target genes solving the above problems is crucial for protein expression.Therefore,for specific target genes solving the above problems is essential for efficient protein expression.Viral diseases in goat pose a major threat to the goat farming industry in China,and there is no effective drug for the prevention and treatment of these diseases and a lack of effective means.Goat interferon alpha（GOIFN-α）is an effective treatment for viral diseases in goat,and it also has excellent immunomodulatory activity.GOIFN-αexpressed in the Bacillus subtilis expression system can be administered directly via oral administration,which is more convenient and faster and reduces personnel labor on farms.However,there is no report of GOIFN-αexpression in Bacillus subtilis,so there is a need to screen a recombinant GOIFN-α-producing Bacillus subtilis strain and also optimize the fermentation culture conditions to provide a material basis for a low-cost and high-efficiency solution for the prevention and treatment of viral diseases in goat farming.In this study,the selected Bacillus subtilis expression system was first validated by the reporter gene GFP,and the original P₄₃ promoter was found not to be effective in initiating gene expression,so the number of bases between the ribosomal binding site and the start codon and the number of bases contained in the promoter were adjusted.After modification of the original P₄₃ promoter,the new promoter was ligated to the shuttle plasmid p HY300PLK and transformed into the host bacterium B.subtilis WB800N.The observation of the bacteriophage color and the luminescence of GFP by fluorescence microscopy confirmed that the newly constructed P₄₃-F2 was more effective in initiating the expression of the Bacillus subtilis expression system.After that,GFP was replaced with the target gene GOIFN-αand different signal peptides were introduced upstream of this target gene to screen for matching promoters.The signal peptides included SP_{Dac B},SP_{ywb N},SP_{amy E},and the promoters included P₄₃-F2,P_grac,P_glv,P_xylr,P_{srf A},P_apr and P_npr,and the constructed recombinant plasmids were p HY-P₄₃-F2-SP_{Dac B}-GOIFN-α,p HY-P_grac-SP_{ywb N}-GOIFN-α,p HY-P_xylr-SP_{amy E}-GOIFN-α,p HY-P_glv-SP_{amy E}-GOIFN-α,p HY-P_{srf A}-SP_{amy E}-GOIFN-α,p HY-P_apr-SP_{amy E}-GOIFN-αand p HY-P_npr-SP_{amy E}-GOIFN-α.The above recombinant plasmids were transformed,screened,and cultured under the appropriate expression conditions,verified by SDS-PAGE and Western Blot.The final screening showed that the recombinant bacteria p HY-P_xylr-SP_{amy E}-GOIFN-α/B.subtilis WB800N could express the target protein GOIFN-α,and then the shake flask fermentation conditions（1 L）were optimized for this strain.And then the shake flask fermentation culture conditions were optimized,mainly from different gradients of vaccination dose,time,temperature,OD value and inducer concentration,and the antiviral activity of GOIFN-αwas measured.The main results of this study were as follows:（1）The modified P₄₃-F1 and P₄₃-F2 promoters successfully initiate GFP reporter gene expression.（2）The optimal Bacillus subtilis receptor state preparation and transformation methods screened provided for the subsequent expression of exogenous proteins.（3）By screening different types of signal peptides,it was demonstrated that SP_{amy E} signal peptide could be used for the extracellular secretory expression of GOIFN-αfrom three signal peptides,SP_{Dac B},SP_{ywb N},and SP_{amy E}.（4）Based on the SP_{amy E} signal peptide,the P_xylr promoter was screened from six promoters,P_grac,P_glv,P_xylr,P_{srf A},P_apr and P_npr,and the P_xylr promoter was screened for the highest extracellular secretory expression of GOIFN-α.（5）In the optimization of shake flask fermentation conditions of recombinant bacterium p HY-P_xylr-SP_{amy E}-GOIFN-α/B.subtilis WB800N,the highest extracellular secretory expression of GOIFN-αwas achieved at 2%inoculum,33℃,220 r/min,OD_595nm of 0.65,and final concentration of D-xylose inducer of 0.8%.（6）The results of antiviral activity showed that the antiviral activity of GOIFN-αwas for4.6×10⁴ IU/m L.In conclusion,The expression of GOIFN-αin the expression system of Bacillus subtilis and the effective validation of antiviral activity have not been reported yet,providing a production strain for the prevention and treatment of viral diseases in goat,and the optimization of GOIFN-αfermentation production conditions provides a reference for the production of Bacillus subtilis biologics with an efficient preparation protocol and a cost effective production process.