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  

Expression and Bioactivity of Porcine Epidermal Growth Factor in Lactococcus lactis

作　　者：周鑫鑫 郭莹莹 肖运才 王金硕[1,2] 周祖涛 刘华珍 石德时[1,2] ZHOU Xinxin;GUO Yingying;XIAO Yuncai;WANG Jinshuo;ZHOU Zutao;LIU Huazhen;SHI Deshi(College of Animal Science and Technology,College of Animal Medicine,Huazhong Agricultural University,Wuhan 430070,China;Key Laboratory of Development of Veterinary Diagnostic Products,Ministry of Agriculture and Rural Affairs,Huazhong Agricultural University,Wuhan 430070,China;Beijing San Nuo Jia Yi Biotechnology Company,Beijing 100176,China)

机构地区：[1]华中农业大学动物科学技术学院、动物医学院,武汉430070 [2]华中农业大学,农业农村部兽用诊断制剂创制重点实验室,武汉430070 [3]北京三诺佳邑生物技术有限责任公司,北京100176

出　　处：《中国畜牧兽医》2024年第7期2823-2836,共14页China Animal Husbandry & Veterinary Medicine

基　　金：湖北省重点研发计划项目(2023DJC145);知识创新专项-基础研究项目(2022020801010224)。

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。【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-1αand IL-1βwere 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 co

关 键 词：乳酸乳球菌 pEGF-NZ重组菌 猪表皮生长因子(pEGF) 

分 类 号：S855.3[农业科学—临床兽医学]