To Investigate The Expression Of Pig-derived Epidermal Growth Factor In Vitro By C.butyricum Homologous Protein Signal Peptide

Clostridium butyricum(C.butyricum)is one of the probiotics that can be widely used as feed additives for livestock breeding.Supplementation of exogenous pEGF(Porcine epidermal growth factor)benefits the intestinal health of pigs.It will repair the damaged colonic mucosal barrier in the gastrointestinal tract of pigs,promote the proliferation and differentiation of gastrointestinal cells,induce the secretion of digestive enzymes,and inhibit the colonization of pathogens.The genome-wide information of C.butyricum has been revealed.The gene structure and function of the secreted protein signal peptide from C.butyricum can be used to express biological active pEGF secretely,thus enhancing the function of C.butyricum.In this study,C.butyricum KNU/L09 was used as a vector to express and secret FLAG-pEGF protein.The main results were as follows.To replace the abuse of antibiotics in the breeding industry and improve the immunity of pigs to pathogen-borne diseases,this study intended to analysis the genome of C.butyricum and test the ability of C.butyricum to express pEGF in vitro as a probiotic vector.1.The signal peptides analysis showed that there were 12 proteins secreted by Sp Ⅰ signal peptide,10 signal peptide sequences could successfully construct a recombinant C.butyricum with FLAG-pEGF gene.However,recombinant C.butyricum with signal peptide genes from ESBP and NLA cannot grow.2.The qPCR results of the recombinant bacteria genome showed that except for pCBP,pPD3,and pPFT,the number of copies of the recombinant plasmids in C.butyricum increased,and the copy number from plasmid with pGHF signal peptide increased most,which was13.1 times higher than that of recombinant plasmids without signal peptide.Followed by pPD1 signal peptide,increased by 4 times,copy number from plasmid pPD2,pMFP,pSPP,pDBD or pCWP increased by 1.5,1,0.7,0.69,0.35 times.2.Western-Blot and ELISA results showed that the predicted signal peptide from GHF,SPP,PD1 and DBD relatively increased the secretion and expression of pEGF protein in C.butyricum(secretion amount: KNU/pGHF: 51.5 pg/m L;KNU/pSPP: 40.5 pg/m L;KNU/pPD1: 32 pg/m L;KNU/pDBD: 27.5 pg/m L).3.Co-cultivation with Staphylococcus aureus and Salmonella choleraesuis on these four recombinant strains,KNU/pGHF、KNU/pSPP、KNU/pPD1 and KNU/pDBD,showed that the recombinant strains had different degrees of inhibition.KNU/pPD1 had the most obvious inhibitory effect on the growth of co-cultured Staphylococcus aureus.KNU/pSPP had the most obvious inhibitory effect on the growth of co-cultured Salmonella choleraesuis.4.IPEC-J2 cell proliferation had different degrees of promotion effect.KNU/pGHF was68.1%.KNU/pSPP was-33%.KNU/pPD1 was 45.3%.KNU/pDBD was 19%.Among them,the supernatant of KNU/pPD1,KNU/pGHF,and KNU/pDBD had the highest effect on cell survival when the content was 40%;KNU/pSPP had the highest effect on cell survival at100%,reaching 27%.In summary,this study used the homologous signal peptide of Clostridium butyricum to realize the secretion and expression of the heterologous protein FLAG-pEGF,which enhanced the antibacterial and proliferation effects of recombinant strains.This explored a new way to enhance the function of Clostridium butyricum.