猪表皮生长因子在乳酸乳球菌中的表达及其生物活性研究

doi:10.16431/j.cnki.1671-7236.2024.07.009

摘要/Abstract

摘要： 【目的】构建表达猪表皮生长因子(pEGF)的乳酸乳球菌原核表达系统,并探究其对断奶仔猪腹泻的影响。【方法】以pUC57-SP45-LEISSTCDA-pEGF序列为模板,利用PCR扩增获得缺失LEISSTCDA片段的融合产物,将其与酶切pNZ8149表达载体连接后电转至乳酸乳球菌NZ3900,以LacF为筛选标记获得重组菌pEGF-NZ。用nisin诱导重组菌pEGF-NZ表达重组蛋白pEGF,并通过Tricine-SDS-PAGE检测其表达情况。改良实验室及工业发酵条件以提高pEGF表达量。利用猪回肠上皮细胞(IPEC-J2)增殖试验体外验证表达的pEGF生物活性。通过动物试验验证重组蛋白pEGF对小鼠及断奶仔猪腹泻的影响。【结果】试验成功构建重组菌pEGF-NZ,并分泌表达pEGF蛋白。体外试验结果显示,pEGF蛋白能促进细胞增殖。pEGF-NZ重组菌及其表达的pEGF蛋白能延缓小鼠腹泻发生时间,降低腹泻率,保护小鼠肠绒毛的完整性。与对照组相比,pEGF-NZ重组菌处理后,小鼠结肠中乳酸菌数显著增加(P＜0.05),大肠杆菌数、肠球菌数显著降低(P＜0.05),白细胞介素-1α(IL-1α)、IL-1β含量显著降低(P＜0.05),IL-13含量显著升高(P＜0.05)。饲粮中添加pEGF-NZ重组菌后,断奶仔猪腹泻情况明显缓解,平均日增重有升高趋势,但差异不显著(P＞0.05)。【结论】pEGF-NZ重组菌及其分泌表达的pEGF能够修复肠道屏障,抑制病原菌黏附,调控肠道菌群,其主要通过下调部分促炎因子、上调抑炎因子表达发挥抑炎效果,从而保护断奶仔猪肠道健康,有效缓解腹泻。

关键词: 乳酸乳球菌; pEGF-NZ重组菌; 猪表皮生长因子(pEGF)

Abstract: 【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.

Key words: Lactococcus lactis; pEGF-NZ recombinant bacteria; porcine epidermal growth factor (pEGF)

中图分类号:

S855.3

周鑫鑫, 郭莹莹, 肖运才, 王金硕, 周祖涛, 刘华珍, 石德时. 猪表皮生长因子在乳酸乳球菌中的表达及其生物活性研究[J]. 中国畜牧兽医, 2024, 51(7): 2823-2836.

ZHOU Xinxin, GUO Yingying, XIAO Yuncai, WANG Jinshuo, ZHOU Zutao, LIU Huazhen, SHI Deshi. Expression and Bioactivity of Porcine Epidermal Growth Factor in Lactococcus lactis[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(7): 2823-2836.

参考文献

[1] TANG X P,XIONG K N,FANG R J,et al.Weaning stress and intestinal health of piglets:A review[J].Frontiers in Immunology,2022,13:1042778.
[2] BEAUMONT M,CAUQUIL L,BERTIDE A,et al.Gut microbiota-derived metabolite signature in suckling and weaned piglets[J].Journal of Proteome Research,2021,20(1):982-994.
[3] WERNER S,GROSE R.Regulation of wound healing by growth factors and cytokines[J].Physiological Reviews,2003,83(3):835-870.
[4] ST-PIERRE B,PALENCIA J Y P,SAMUEL R S.Impact of early weaning on development of the swine gut microbiome[J].Microorganisms,2023,11(7):1753.
[5] 张婷,魏立民,刘光亮.五指山猪和长白猪断奶仔猪肠道免疫性能及微生物多样性比较分析[J].中国畜牧兽医,2023,50(8):3113-3122. ZHANG T,WEI L M,LIU G L.Comparative analysis of intestinal immune performance and microbial diversity of weaned piglets from Wuzhishan and Changbai pigs[J].China Animal Husbandry&Veterinary Medicine,2023,50(8):3113-3122.(in Chinese)
[6] SHAO Y R,XIONG X,WANG K X,et al.Early weaning leads to the remodeling of lipid profile in piglet jejunal crypt cells during post-weaning days[J].Animal Nutrition,2022,15(11):102-111.
[7] CERA K R,MAHAN D C,CROSS R F,et al.Effect of age,weaning and postweaning diet on small intestinal growth and jejunal morphology in young swine[J].Journal of Animal Science,1988,66(2):574-584.
[8] PLUSKE J R,WILLIAMS I H,AHERNE F X.Maintenance of villous height and crypt depth in piglets by providing continuous nutrition after weaning[J].Animal Science,1996,62(1):131-144.
[9] JAMES P S,SMITH M W,TIVEY D R,et al.Epidermal growth-factor selectively increases maltase and sucrase activities in neonatal piglet intestine[J].Journal of Physiology-London,1987,393(1):583-594.
[10] WANG L X,ZHU F,YANG H S,et al.Epidermal growth factor improves intestinal morphology by stimulating proliferation and differentiation of enterocytes and mTOR signaling pathway in weaning piglets[J].Science China-Life Sciences,2020,63(2):259-268.
[11] LEE D N,KUO T Y,CHEN M C,et al.Expression of porcine epidermal growth factor in and its biology activity in early-weaned piglets[J].Life Sciences,2006,78(6):649-654.
[12] BEDFORD A,CHEN T,HUYNH E,et al.Epidermal growth factor containing culture supernatant enhances intestine development of early-weaned pigs in vivo:Potential mechanisms involved[J].Journal of Biotechnology,2015,196:9-19.
[13] FRELET-BARRAND A,BOUTIGNY S,MOYET L,et al.Lactococcus lactis,an alternative system for functional expression of peripheral and intrinsic membrane proteins[J].PLoS One,2010,5(1):e8746.
[14] YU D M,XIA Y Y,GE L P,et al.Effects of Lactococcus lactis on the intestinal functions in weaning piglets[J]. Frontiers in Nutrition,2021,8:13256.
[15] MENG Q W,ZHANG Y M,LI J B,et al.Lycopene affects intestinal barrier function and the gut microbiota in weaned piglets via antioxidant signaling regulation[J].Journal of Nutrition,2022,152(11):2396-2408.
[16] TANG M,LAARVELD B,VAN KESSEL A G,et al.Effect of segregated early weaning on postweaning small intestinal development in pigs[J].Journal of Animal Science,1999,77(12):3191-3200.
[17] QIN L S,JI W,WANG J L,et al.Effects of dietary supplementation with yeast glycoprotein on growth performance,intestinal mucosal morphology,immune response and colonic microbiota in weaned piglets[J].Food&Function,2019,10(5):2359-2371.
[18] YANG H S,XIONG X,WANG X C,et al.Effects of weaning on intestinal upper villus epithelial cells of piglets[J]. PLoS One,2016,11(3):e0150216.
[19] WANG J J,CHEN L X,LI P,et al.Gene expression is altered in piglet small intestine by weaning and dietary glutamine supplementation[J].Journal of Nutrition,2008,138(6):1025-1032.
[20] ZHU L H,XU J X,ZHU S W,et al.Gene expression profiling analysis reveals weaning-induced cell cycle arrest and apoptosis in the small intestine of pigs[J].Journal of Animal Science,2014,92(3):996-1006.
[21] HU C H,XIAO K,LUAN Z S,et al.Early weaning increases intestinal permeability,alters expression of cytokine and tight junction proteins,and activates mitogen-activated protein kinases in pigs[J]. Journal of Animal Science,2013,91(3):1094-1101.
[22] BOUDRY G,PERON V,LE HUEROU-LURON I,et al.Weaning induces both transient and long-lasting modifications of absorptive,secretory,and barrier properties of piglet intestine[J].Journal of Nutrition,2004,134(9):2256-2262.
[23] WANG D Y,XU S Y,LIN Y,et al.Recombinant porcine epidermal growth factor-secreting promotes the growth performance of early-weaned piglets[J].BMC Veterinary Research,2014,10(1):1-12.
[24] KAWANO S,IYAGUCHI D,OKADA C,et al.Expression,purification,and refolding of active recombinant human E-selectin lectin and EGF domains in Escherichia coli[J].Protein Journal,2013,32(5):386-391.
[25] WANG S J,GUO C H,ZHOU L,et al.Comparison of the biological activities of Saccharomyces cerevisiae-expressed intracellular EGF,extracellular EGF,and tagged EGF in early-weaned pigs[J].Applied Microbiology and Biotechnology,2015,99(17):7125-7135.
[26] BEDFORD A,LI Z,LI M,et al.Epidermal growth factor-expressing enhances growth performance of early-weaned pigs fed diets devoid of blood plasm[J].Journal of Animal Science,2012,90:4-6.
[27] 邓笑颖,杨宇,姜杨.基于乳糖水解酶的乳酸乳球菌乳糖代谢多样性研究[J].食品与发酵工业,2019,45(21):8-14. DENG X J,YANG Y,JIANG Y.Study on the diversity of lactose metabolism in Lactococcus lactis based on lactose hydrolase[J].Food and Fermentation Industry,2019,45(21):8-14.(in Chinese)
[28] ZENG L,DAS S,BURNE R A.Utilization of lactose and galactose by Streptococcus mutans:Transport,toxicity,and carbon catabolite repression[J].Journal of Bacteriology,2010,192(9):2434-2444.
[29] LAZAR Z,GAMBOA-MELENDEZ H,CRUTZ-LE COQ A M,et al.Awakening the endogenous Leloir pathway for efficient galactose utilization by Yarrowia lipolytica[J].Biotechnology for Biofuels,2015,8:1-16.
[30] OTTO R,SONNENBERG A S M,VELDKAMP H,et al.Generation of an electrochemical proton gradient in Streptococcus-cremoris by lactate efflux[J].Proceedings of the National Academy of Sciences of the United States of America,1980,77(9):5502-5506.
[31] GHARSALLAOUI A,OULAHAL N,JOLY C,et al.Nisin as a food preservative:Part 1:Physicochemical properties,antimicrobial activity,and main uses[J].Critical Reviews in Food Science and Nutrition,2016,56(8):1262-1274.
[32] THONES S,ROTHER S,WIPPOLD T,et al.Hyaluronan/collagen hydrogels containing sulfated hyaluronan improve wound healing by sustained release of heparin-binding EGF-like growth factor[J].Acta Biomaterialia,2019,86:135-147.
[33] BARRIENTOS S,STOJADINOVIC O,GOLINKO M S,et al.Growth factors and cytokines in wound healing[J].Wound Repair and Regeneration,2008,16(5):585-601.
[34] TIBERIO R,BOGGIO P,PERTUSI G,et al.Cytokines,chemokines and growth factors in vitro and wound healing[J].Journal of Investigative Dermatology,2010,130:S61.
[35] CHEUNG Q C,YUAN Z F,PAUL W D,et al.Generation of epidermal growth factor-expressing Lactococcus lactis and its enhancement on intestinal development and growth of early-weaned mice[J].American Journal of Clinical Nutrition,2009,89:871-879.
[36] HU C H,XIAO K,LUAN Z S,et al.Early weaning increases intestinal permeability,alters expression of cytokine and tight junction proteins,and activates mitogen-activated protein kinases in pigs[J].Journal of Animal Science,2013,91(3):1094-1101.
[37] TANG J,XU L,ZENG Y,et al.Effect of gut microbiota on LPS-induced acute lung injury by regulating the TLR4/NF-κB signaling pathway[J].International Immunopharmacology,2021,91:107272.
[38] KANG P,TOMS D,YIN Y L,et al.Epidermal growth factor-expressing enhances intestinal development of early-weaned pigs[J].Journal of Nutrition,2010,140(4):806-811.