动物营养学报 >

2022 , Vol. 34 >Issue 1: 159 - 176

DOI: https://doi.org/10.3969/j.issn.1006-267x.2022.01.017

研究论文 RESEARCH PAPER

重组猪源罗伊氏乳酸杆菌分泌表达牛乳铁蛋白肽对新生仔猪生长性能的影响以及抗腹泻病毒感染的保护作用研究

  • 汪昭睿 ,
  • 王雪莹 ,
  • 解伟纯 ,
  • 宋丽影 ,
  • 王晓娜 ,
  • 崔文 ,
  • 姜艳平 ,
  • 周晗 ,
  • 王丽 ,
  • 乔薪瑗 ,
  • 徐义刚 ,
  • 李一经 ,
  • 唐丽杰
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  • 1. 东北农业大学动物医学学院, 哈尔滨 150030;
    2. 农业部动物疫病病原生物学重点实验室东北科学观测实验站, 哈尔滨 150030
汪昭睿(1996-),男,湖北孝感人,硕士研究生,从事微生物学与动物营养免疫研究。E-mail:rui10151108@163.com

收稿日期: 2021-05-31

  网络出版日期: 2022-01-18

基金资助

国家自然科学基金项目(31672461)

收起

Effects of Recombinant Porcine Lactobacillus reuteri Secreting Expression of Bovine Lactoferrin Peptides on Growth Performance of Newborn Piglets and Protective Effect against Diarrhea Virus Infection

  • WANG Zhaorui ,
  • WANG Xueying ,
  • XIE Weichun ,
  • SONG Liying ,
  • WANG Xiaona ,
  • CUI Wen ,
  • JIANG Yanping ,
  • ZHOU Han ,
  • WANG Li ,
  • QIAO Xinyuan ,
  • XU Yigang ,
  • LI Yijing ,
  • TANG Lijie
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  • 1. College of Veterinary Medicines, Northeast Agricultural University, Harbin 150030, China;
    2. Northeast Scientific Inspection Observation, Key Laboratory of Animal Pathogen Biology of Ministry of Agriculture, Harbin 150030, China

Received date: 2021-05-31

  Online published: 2022-01-18

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摘要

本试验旨在研究分泌表达牛乳铁蛋白肽（LFCA）的重组猪源罗伊氏乳酸杆菌对新生仔猪生长性能的影响以及对引起仔猪腹泻的猪传染性胃肠炎病毒（TGEV）感染的保护作用。试验1：选取36头1日龄、体重约为1.5 kg的新生杜长大仔猪，随机分为3组，分别为重组菌组、空载菌组和对照组，每组12头仔猪，分别口服表达LFCA的重组猪源罗伊氏乳酸杆菌pPG-LFCA/LR-CO21、含有空载体质粒的重组猪源罗伊氏乳杆菌pPG/LR-CO21和磷酸盐缓冲液（PBS）各4 mL，连续口服3 d，口服后观测时间为14 d。期间仔猪自由采食，记录各组仔猪体重变化和腹泻情况。试验2：选取30头1日龄、体重约为1.5 kg的新生杜长大仔猪，随机分为5组，分别口服1、3、6、9、12 mL半数组织培养感染剂量（TCID50）为10-7.50/mL的TGEV。设置7 d时间为观察期，分析半数致死量的条件。试验3：另选取1日龄、体重约为1.5 kg的新生杜长大仔猪32头，随机分为4组，分别为重组菌组、空载菌组、对照组、TGEV感染组，每组8个重复，每个重复1头仔猪。试验组每天每头按照2×1010 CFU的剂量口服pPG-LFCA/LR-CO21、pPG/LR-CO21及等体积的PBS，连续口服饲喂1周，在8日龄按照半数致死量口服TGEV感染，感染7 d后进行样品采集。记录各组仔猪体重变化和腹泻情况；苏木精-伊红（HE）染色检测小肠绒毛高度及隐窝深度；酶联免疫吸附试验（ELISA）法检测仔猪血清中总免疫球蛋白G（IgG）和肠黏液总分泌型免疫球蛋白A（sIgA）抗体含量；实时荧光定量PCR检测紧密连接蛋白相关基因封闭蛋白-1（Claudin-1）、闭合蛋白（Occludin）和紧密连接蛋白-1（ZO-1）以及炎性因子白细胞介素-6（IL-6）、白细胞介素-8（IL-8）、干扰素-γ（IFN-γ）、肿瘤坏死因子-α（TNF-α）及Toll样受体2（TLR2）的mRNA相对表达量；对仔猪盲肠内容物进行菌群结构分析。结果表明：口服重组猪源罗伊氏乳酸杆菌后，与对照组相比，重组菌组新生仔猪的平均日增重极显著提高（P<0.01），而腹泻率降低且差异极显著（P<0.01）；与TGEV感染组相比，重组菌组仔猪平均日增重增加且腹泻率降低，差异显著（P<0.05）；空肠和回肠绒毛高度及绒毛高度/隐窝深度的比值显著增加（P<0.05）；仔猪血清中总IgG和肠黏液总sIgA抗体含量显著升高（P<0.05）；肠黏膜组织中紧密连接蛋白相关基因Claudin-1、Occludin、ZO-1 mRNA相对表达量极显著升高（P<0.01），血清中细胞因子TNF-α含量极显著降低（P<0.01），血清IFN-γ、IL-6、IL-8、TLR2含量极显著升高（P<0.01），仔猪存活率提高。对仔猪盲肠内容物进行菌群多样性分析结果显示，TGEV感染仔猪后肠道正常菌群占比下降，相比于TGEV感染组，口服重组菌的仔猪肠道菌群致病菌拟杆菌属占比下降，差异显著（P<0.05），菌群多样性更接近对照组，肠道菌群的代谢、遗传信息处理等功能均有所增强。综上所述，本试验成功构建了分泌表达LFCA的重组猪源罗伊氏乳酸杆菌菌株，经口服途径饲喂新生仔猪后，能够促进仔猪生长，降低新生仔猪的腹泻率，经口服途径饲喂感染TGEV的新生仔猪后，可以改善肠道环境，降低病毒对仔猪肠道的损伤，对仔猪抗TGEV感染提供有效保护。

关键词： 牛乳铁蛋白肽; 重组猪源罗伊氏乳酸杆菌; 黏膜免疫; 抗TGEV感染

本文引用格式

汪昭睿 , 王雪莹 , 解伟纯 , 宋丽影 , 王晓娜 , 崔文 , 姜艳平 , 周晗 , 王丽 , 乔薪瑗 , 徐义刚 , 李一经 , 唐丽杰 . 重组猪源罗伊氏乳酸杆菌分泌表达牛乳铁蛋白肽对新生仔猪生长性能的影响以及抗腹泻病毒感染的保护作用研究[J]. 动物营养学报, 2022 , 34(1) : 159 -176 . DOI: 10.3969/j.issn.1006-267x.2022.01.017

Abstract

This experiment was conducted to investigate the effects of recombinant porcine Lactobacillus reuteri secreting bovine lactoferrin peptide (LFCA) on growth performance of newborn piglets and the protective effect on porcine transmissible gastroenteritis virus (TGEV)infection which caused piglet diarrhea. Experiment 1:thirty-six one-day-old newborn piglets with an average body weight of about 1.5 kg were randomly divided into 3 groups, which were pPG-LFCA/LR-CO21 group, pPG/LR-CO21 group and control group, each group with 12 piglets. Piglets in each group were orally administered recombinant porcine Lactobacillus reuteri expressing LFCA pPG-LFCA/LR-CO21, containing empty vector plasmid PPG/LR-CO21 and equal volume phosphate buffer (PBS); oral administration continued for 3 days, and the observation time after oral administration was 14 d. During the period, piglets were fed freely, and the changes of body weight and diarrhea were recorded. Experiment 2:thirty one-day-old newborn piglets with an average body weight of about 1.5 kg were randomly divided into 5 groups and given TGEV with a half tissue culture infection dose (TCID50) of 10-7.50/mL by oral administration of 1, 3, 6, 9 and 12 mL, respectively. The observation period of 7 d was set to analyze the conditions of half lethal dose. Experiment 3:another thirty-two newborn piglets with an average body weight of about 1.5 kg were selected as experimental animals and randomly divided into 4 groups, with 8 piglets in each group. The groups were pPG-LFCA/LR-CO21 group, pPG/LR-CO21 group, control group and TGEV infect group. There were 8 replicates in each group and 1 piglet in each replicate. Each head of the experimental group was orally fed ppG-LFCA/LR-CO21, pPG/LR-CO21 and equal volume of PBS at a dose of 2×1010 CFU per day for 1 consecutive week. At 8 days of age, TGEV was infected by oral administration at half lethal dose, and samples were collected after 7 days of infection. The weight change and diarrhea of each group of piglets were recorded; hematoxylin-eosin staining was used to detect the length of intestinal villi and the depth of crypts; enzyme linked immunosorbent assay (ELISA) was used to determine total serum total immunoglobulin G (IgG) and total secretory immunoglobulin A (sIgA) antibody contents. RT-qPCR was used to detect the mRNA relative expression levels of Claudin-1, Occludin, tight junction protein-1 (ZO-1), inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and Toll-like receptor 2 (TLR2). The flora structure of the contents of the piglet's cecum was analyzed. After oral recombinant porcine Lactobacillus reuteri, compared with the control group, the average daily gain of newborn piglets in the pPG-LFCA/LR-CO21 group was significantly increased (P<0.01), while the diarrhea rate was significantly decreased (P<0.01). Compared with TGEV infection group, the average daily gain of piglets in pPG-LFCA/LR-CO21 group was increased and diarrhea rate was decreased, and the differences were significant (P<0.05). Villus height and the ratio of villus height to crypt depth in jejunum and ileum were significantly increased (P<0.05). The contents of total IgG and intestinal mucosal total sIgA antibody in serum of piglets were significantly increased (P<0.05); the mRNA relative expression levels of tight junction protein-related genes Claudin-1, Occludin and ZO-1 in intestinal mucosal tissue were extremely significantly increased (P<0.01), and the serum TNF-α content was extremely significantly decreased (P<0.01). Serum IFN-γ, IL-6, IL-8 and TLR2 contents were significantly increased (P<0.01), and the survival rate of piglets was improved. The analysis of the bacterial diversity in the contents of the piglets' cecum showed that the proportion of normal intestinal flora of piglets decreased after TGEV infection. Compared with the TGEV infect group, the proportion of pathogenic bacteria Bacteroides in piglet's intestinal flora decreased by oral recombinant bacteria (P<0.05), the diversity of the flora was closer to the control group, and the metabolism of the intestinal flora and the processing of genetic information had been enhanced. In summary, the recombinant porcine Lactobacillus reuteri expressing LFCA is successfully constructed, which can promote the growth of newborn piglets and reduce the diarrhea rate of newborn piglets after oral feeding. Feeding the recombinant bacteria to newborn piglets infected with TGEV by oral route can improve the intestinal environment, reduce the damage of TGEV to piglets' intestine, and provide effective protection against TGEV infection.

Key words： bovine lactoferrin peptides; recombinant porcine Lactobacillus reuteri; mucosal immunization; anti-TGEV infection

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