Expression and Bioactivity of Porcine Epidermal Growth Factor in Lactococcus lactis
[Objective]The objective of this experiment was to construct the prokaryotic expression system of Lactococcus lactis expressing porcine epidermal growth factor(pEGF)and to explore its effect on diarrhea of weaned piglets.[Method]Using pUC57-SP45-LEISSTCDA-pEGF sequence as the template,the fusion product of the missing LEISSTCDA fragment was obtained by PCR amplification,which was connected with the expression vector pNZ8149 and transferred to Lactococcus lactis NZ3900 by electric transmission,and the recombinant pEGF-NZ was obtained by using LacF as the screening marker.Recombinant strain PEGF-NZ was induced by nisin to express recombinant protein pEGF,and its expression was detected by Tricine-SDS-PAGE.The pEGF expression was increased by improving the laboratory and industrial fermentation conditions.The bioactivity of pEGF expressed in porcine ileal epithelial cells(IPEC-J2)was verified in vitro by proliferation assay.The effects of recombinant protein pEGF on diarrhea in mice and weaned piglets were verified by animal experiments.[Result]The recombinant strain PEGF-NZ was successfully constructed and secreted to express pEGF protein.In vitro experiment results showed that pEGF protein could promote cell proliferation.pEGF-N recombinant bacteria and its expression of pEGF could delay the occurrence time of diarrhea in mice,reduce the diarrhea rate,and protect the integrity of intestinal villi in mice.Compared with control group,after treatment with recombinant pEGF-NZ,the number of lactic acid bacteria of mice was significantly increased(P＜0.05),the number of Escherichia coli and Enterococcus were significantly decreased(P＜0.05),and the contents of IL-1α and IL-1β were significantly decreased(P＜0.05).The content of IL-13 were significantly increased(P＜0.05).After supplemented with pEGF-NZ,the diarrhea of weaned piglets was significantly relieved,and the average daily gain tended to increase,but the difference was not significant(P＞0.05).[Conclusion]Recombinant PEGF-NZ and its expression pEGF could repair intestinal barrier,inhibit the adhesion of pathogenic bacteria,regulate intestinal flora,and exert anti-inflammatory effects mainly by down-regulating the expression of some pro-inflammatory factors and up-regulating anti-inflammatory factors,so as to protect intestinal health of weaned piglets and effectively relieve diarrhea.
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