《猪用抗生素的替代》PPT课件.ppt

Alternatives of antibiotics for pigs猪用抗生素的替代,Defa Li 李德发,Ministry of Agriculture Feed Industry Centre 农业部饲料工业中心,2006年11月30日于北京,猪营养,营养需要与代谢,分子生物学,Who operates MAFIC Research,植物提取物,免疫学,微生物学,分析化学,营养与免疫,基因工程菌,细胞生物学,分子营养与肉品质,团队研究,Antibiotics抗生素,Residue残留,Resistance耐药性,Harmful to animal and human health影响动物和人类的健康,Reduce the use of antibiotics减少抗生素的使用,EU欧盟,China中国,Current situation and future development 现状与展望,Alternatives of antibiotics 抗生素替代,Antimicrobial peptides 抗菌肽,Herbal extracts 天然提取物,Enzyme preparations 酶制剂,Probiotics 益生素,Probiotics益生素,Lactobacillus乳酸杆菌,Mechanism机理研究,Application应用研究,绿色荧光蛋白(GFP)标记,蛋白组学,粘附位点,无抗发酵饲料,蛋白差异,复合发酵培养,悉生动物模型,信号传导,作用机制,Targeting distribution 目的细菌在肠道中的定位分布情况，其与肠道菌系之 间的关系Specific passway and function 具体作用途径与功能的研究,Experimental model of gnotobiotic animals 悉生动物实验模型,悉生动物（已知菌动物）,空白组,益生菌,致病菌,益生菌+致病菌,Development of antibiotics-free fermented feed无抗发酵饲料的开发,Fermentative respiratory membrane发酵呼吸膜,Single way valve 单向阀门,CO2 venting,preventing air to enter 排出袋内微生物产生的CO2，阻止大气进入,A,E.coli K88 大肠杆菌K88B,E.coli K99 大肠杆菌K99C,S.typhimurium 鼠伤寒沙门氏菌 D,Staph.Aureus 金黄色葡萄球菌,Int.J.Gener.Mol.Microbiol.,X.H.Guo,Defa Li,2006,Inhibitory activity of Bacillus spp.139 against pathogenic bacteria,but not Lactobacillus外源芽孢杆菌可抑制有害菌，对乳酸菌无影响,Multi-strains combination,creating anaerobic condition automatically 多菌种组合，自身创造厌氧环境,Yeast酵母菌,Bacillus spp.芽胞杆菌,好氧,兼性,兼性,Lactobacillus reproduction in anaerobic condition 乳酸杆菌厌氧发酵,Inoculating,packing,then fermentation先接种、包装，后发酵,发酵,轻工业副产物：玉米浆、果渣,Effect of fermentation time on energy and gossypol能量与游离棉酚随发酵时间的变化,Decrease the content of gossypol 降低游离棉酚的含量,Effects of antibiotics-free fermented feed on growth performance in pigs无抗发酵饲料对猪生产性能的影响,P 0.05,RFID systemRFID 追溯系统,基因芯片,Gene chip：The on line examination technique of antibiotics 基因芯片：快速检测分割肉中的抗生素含量（20-100 ppb）,Proteomics 蛋白质组学,Proteomics analysis of interaction between Lactobacillus and intestine乳酸杆菌和肠道细胞相互作用的蛋白质组学研究,J.Nutr.,J.J.Wang,Defa Li,2006,蛋白质组学,双向电泳,样品制备,数据库比对鉴定蛋白,相互作用机制,生物功能,差异蛋白,质谱分析,图像分析,胶内酶切,蛋白,Lactobacillus乳酸杆菌,Intestinal epithelial cells肠道上皮细胞,等点聚焦,SDS-PAGE,Experimental design试验设计,Intestinal cells,Adhered or not,Lactobacillus fermentum I5007 cells,Culture medium,37C 37C温箱,4 h,Rabbit jejunum兔子空肠,Two-dimensional gel electrophoresis 双向电泳,37C 1 h,Mass spectrometry 质谱,Differential proteins差异蛋白,Lactobacillus in rabbit jejunum culture兔子空肠培养条件下乳酸杆菌表达差异的蛋白,Electrophoresis,F.Yang,Defa Li,2006,Fructose-6-phosphate Phosphoketolase(F6PPK)6-磷酸果糖磷酸酮酶,Lactate dehydrogenase乳酸脱氢酶,Dihydrolipomide dehydrogenase二氢硫辛酸脱氢酶（丙酮酸脱氢酶系）,糖酵解,三羧酸循环,磷酸戊糖途径,减少氧自由基产生,NADPH生成增加,有利于乳酸杆菌存活,Intestinal cells after Lactobacillus adhesion肠细胞被乳酸杆菌粘附条件下表达差异的蛋白,Electrophoresis,F.Yang,Defa Li,2006,Voltage-dependent anion channel 1 电压依从性阴离子通道1,Glutathione transferase 谷胱甘肽转移酶,Heat shock protein gp96 热应激蛋白gp96,调控膜通透性,改善机体免疫,减少氧化应激,改善肠道完整性及其健康,Synbiotics 合生素,Functional mechanism of synbiotics 合生素的作用机理,Synbiotics 合生素,Bacillus subtilis枯草芽孢杆菌,Astragalus,aloe polysaccharide,etc.黄芪多糖，芦荟多糖等,Immunity免疫力,Antibacterial substance 抑菌物质,Organic acid有机酸,Health动物健康,Bacteriophage产生抗菌素挥发性脂肪酸：乙酸、丙酸、丁酸Tolerance of the intestinal environment 耐受肠道内环境,Bacillus subtilis枯草芽孢杆菌,ZnO氧化锌,Hypothesis假设,Intestine function肠道功能,Enteric nervous system 肠神经系统,Feed intake采食量,Diarrhea腹泻,生产性能Growth performance,Gastrointestinal hormones胃肠激素,Mast cell mediators肥大细胞介质,Intestinal IGF-1 system小肠IGF-1系统,Hormone on eating采食相关激素,ZnO,Effects of dietary supplement of 3000 mg/kg ZnO on the expression of fundic gland Ghrelin gene in piglets日粮中添加3000 mg/kg氧化锌对断奶仔猪胃底腺Ghrelin基因表达的影响,P=0.008,Increase the expression of the Ghrelin gene 显著提高断奶仔猪胃底腺Ghrelin基因的表达,提高采食量,Improve the intestinal function 药理剂量氧化锌促进仔猪肠道健康,The small intestinal morphology of weaned pigs receiving a diet with or without supplementation of 3000 mg/kg Zn 日粮中添加3000 mg/kg氧化锌ZnO对仔猪小肠形态的影响,J.Nutr.,X.L.Li,Defa Li,2006,Increase the expression of IGF-1 gene 提高小肠 IGF-1 基因的表达 通过IGF-1的旁分泌作用促进仔猪肠道健康和生长,Effects of dietary supplement of 3000 mg/kg ZnO on the expression of small-intestinal mucosa IGF-1 gene in piglets日粮中添加3000 mg/kg氧化锌对仔猪小肠粘膜IGF-1基因表达的影响,J.Nutr.,X.L.Li,Defa Li,2006,Histamine concentrations in the small intestine of weanling piglets fed diets containing normal and high-levels of ZnO高锌日粮对断奶仔猪小肠组胺浓度的影响,Decrease the release of the histamine in the small intestine 高锌显著降低断奶仔猪小肠组胺的释放稳定小肠通透性,The numbers of mast cells(cell/cm2)in the small intestine of weanling piglets fed diets containing normal or high-levels of zinc oxide高锌日粮对断奶仔猪小肠肥大细胞数量的影响,Decrease the numbers of the mast cells in the small intestine 高锌显著降低断奶仔猪小肠各层肥大细胞数改善小肠功能,mRNA and protein levels for the stem cell factor(SCF)gene in the mid jejunum of weanling piglets fed diets containing normal and high-levels of ZnO高锌对空肠中段干细胞因子表达（基因与蛋白）的影响,Decrease the expression of SCF 高锌显著降低空肠干细胞因子的表达 调控肥大细胞数量及介质释放,P=0.016,Protein,Normal ZnO,High ZnO,P=0.024,mRNA,Normal ZnO,High ZnO,The effects of dietary zinc on the changes in Isc(IscZn)and in G(GZn)日粮锌对短路电流和电导率改变的影响,Comp.Biochem.Physiol.,Z.K.Feng,2006,Zn improves tight junction permeability高锌降低断奶仔猪小肠粘膜跨上皮电位差维持肠道紧密连接的完整性,Enzyme preparations酶制剂,Complex enzyme-mutation and selection复合酶诱变选育,Aspergillus sulphureus-MAFIC001 可高产复合酶的硫色曲霉及其诱变筛育方法和固态发酵制备饲用复合酶的应用,High yield of complex enzyme:xylanase,-glucanase,pectinase,etc.高产复合酶：木聚糖酶、-葡聚糖酶、果胶酶等Industrial production 工业化生产，商业利润超过1000万元,Aspergillus niger-MAFIC005 饲用非淀粉多糖酶高产菌种的选育及其固态发酵生产技术,High yield of complex enzyme:xylanase,-glucanase,pectinase,etc.高产复合酶：木聚糖酶、葡聚糖酶、果胶酶等,Existing questions and the methods to increase the yield of-mannanase存在问题及提高-甘露聚糖酶产量的方法,Low yield and enzymatic activity,difficult for massive application of-mannanase 产量低，酶活力不高，使-甘露聚糖酶不能大量推广使用,Screening high yielding A.sulphureus MAFIC001-mannanase strains by a mutant treatment 诱变选育高产硫色曲霉MAFIC001-甘露聚糖酶菌种,Modifying-mannanase gene sequence according to codon bias 根据密码子偏好性，优化-甘露聚糖酶基因序列,-mannanase-Aspergillus sulplhureus MAFIC001-甘露聚糖酶硫色曲霉MAFIC001,RT-PCR and rapid amplification of cDNA ends(RACE)techniqueRT-PCR和cDNA末端快速扩增技术,Primer 1 and Primer 2 used to amplify the partial sequence of-mannanase 设计引物Primer 1和Primer 2扩增-甘露聚糖酶部分序列Obtain the N-terminal and C-terminal of-mannanase gene sequence using RACE technique 使用RACE技术获得-甘露聚糖酶的N-端和C-端序列,J Biotechnol.,X.L.Chen,Defa Li,2006,The first time clone the full-length gene sequence of A.sulphureus MAFIC001-mannanase(GenBank accession no.DQ328335)首次克隆了硫色曲霉MAFIC001-甘露聚糖酶全基因序列,Modifying the mature-mannanase gene sequence by codon bias 密码子优化改造-甘露聚糖酶成熟编码基因序列Constructing P.pastoris expression vector pPICZA,high density fermentation of recombinant-mannanase in 10 L fermenter 构建毕赤酵母表达载体pPICZA，实现重组-甘露聚糖 酶在10 L发酵罐中高密度发酵,Pichia pastoris expression vector毕赤酵母表达载体,A,Fermentation device（10 L）发酵装置（10升）,SDS-PAGE of expressed-mannanase in fermentor with different induction times 发酵罐中-甘露聚糖酶在不同时间的表达量,Highest enzymatic activity(最高酶活力)：1100 U/mLPurity(纯度)：96,Highest protein expression level(最高蛋白浓度)：3 g/L,Accumulation of-mannanaseactivity with different induction times for fermentation发酵中-甘露聚糖酶活性随诱导时间的积累,1100 U/mL,JAM,X.L.Chen,Defa Li,2006,-mannanase characterization:an extremely broad pH(2.2-8.0)range,optimum pH is 2.4 酶学特性：pH（）范围宽，最适pH 2.4,JAM,X.L.Chen,Defa Li,2006,-mannanase protein expression level-甘露聚糖酶蛋白表达水平,-mannanase activity-甘露聚糖酶活力,Further research进一步的研究,Pilot scale,mass-industrial production 中试，实现工业化生产,Determine the efficacy by animal feeding trials 动物试验验证其效果,National feed engineering technology research center Nankou fermentation base国家饲料工程技术研究中心南口发酵实验基地,Enzyme Microb.Tech.,P.Deng,Defa Li,2006,Xylanase木聚糖酶,Aspergillus niger MAFIC005 Xylanase gene 克隆黑曲霉MAFIC005木聚糖酶xynB基因，GenBank登录号为 实现了在大肠杆菌和毕赤酵母中的表达 酶学性质表明，与动物胃肠道环境相适应 Aspergillus sulphureus MAFIC001 Xylanase gene 克隆硫色曲霉MAFIC001木聚糖酶xynA和xynB基因，GenBank 登录号分别为和 实现了在大肠杆菌和毕赤酵母中的表达 酶学性质表明，与动物胃肠道环境相适应,+,Modified xynA改造xynA,pGAPZA载体,Constitutive expression in Pichia pastoris毕赤酵母中的组成型表达,Aspergillus sulphureus硫色曲霉,Thermostability of the modified recombinant xylanase改造的重组木聚糖酶热稳定性研究,Enzymatic activity:120 U/mL 最高酶活力达120 U/mL,Protein concentration:3.78 mg/mL 蛋白浓度为3.78 mg/mL,Good stability:80C for 90 min 热稳定性好，80C保持90 min,Herbal extracts(Astragalus polysaccharide,APS)天然提取物（黄芪多糖）,Hypothesis 假设,Inflammation reaction of LPS LPS炎性反应,Disorder of GH/IGF-I axisGH/IGF-I轴异常,Immune suppress(cyclophosphamide)免疫抑制（环磷酰胺，CY）,Immune enhancement免疫增强,Immune type免疫类型,APS黄芪多糖,Inflammation factor secretion炎性因子分泌,-？,+,-,-？,-？,+,+,？,-,Cell culture细胞培养,？,(Linear P 0.05),(Linear P 0.05),Effects of APS on ratio of CD4+/CD8+and Lymphocyte proliferation in pigletsAPS对仔猪CD4+/CD8+比值和淋巴细胞增殖的影响,Anim.Sci.,S.L.Yuan,X.S.Piao,Defa Li,2006,Improve cell immune function,the optimal APS level was 500 mg/kgAPS提高断奶仔猪的细胞免疫功能，增强其对入侵微生物和疾病 的抵抗力，促进仔猪健康和生长 APS在断奶仔猪日粮中的最适添加量为500 mg/kg,CD4+/CD8+,淋巴细胞增殖,Effects of dietary supplement of APS on serum cytokines in weaned pigs received cyclophosphamide(CY)APS（注射环磷酰胺,CY）对断奶仔猪血清细胞因子的影响(pg/mL),Anim.Sci.,S.L.Yuan,X.S.Piao,Defa Li,2006,Reverse CY-induced cell immune suppress and humour immune suppress环磷酰胺免疫抑制模型研究发现，APS可促进猪的正常细胞免 疫，也可逆转CY造成的免疫抑制本研究建立了一种评价APS对仔猪营养和免疫作用的有用模型,Effects of APS on serum index in pigletsAPS对仔猪血清指标的影响,Recover the normal function of GH/IGF-I axis炎性细胞因子过度分泌以及GH/IGF-I轴的异常，导致断奶仔猪 发生免疫炎症和生长速度下降APS通过降低炎性细胞因子、GH和皮质醇的含量，增加抗炎性 因子的合成，恢复GH/IGF-I轴的正常而发挥其免疫抗炎作用,Anim.Sci.,S.L.Yuan,X.S.Piao,Defa Li,2006,b,Increase NO and iNOS activity of the peripheral blood lymphocytes in pigletsAPS显著增加仔猪外周血淋巴细胞NO和iNOS的活性，并呈剂量依赖性,Effects of APS on NO and iNOS activity in piglets APS对仔猪NO和iNOS的影响,J.Anim.Sci.,X.F.Mao,Defa Li,2005,(P 0.05),*,*,*,*,*,*,*,(P 0.05),Effects of APS on different cytokines APS对不同细胞因子的影响,Increase the IL-2 and IFN-secretionT细胞可能是APS直接作用的靶细胞之一APS可在体外促进细胞因子IL-2和IFN-的分泌，这 可能是APS的免疫调节作用之一,Anim.Sci.,S.L.Yuan,X.S.Piao,Defa Li,2006,J.Anim.Sci.,X.F.Mao,Defa Li,2005,Mechanism of immune function of APSAPS的免疫作用机理,APS,iNOS,NO,细胞因子表达,淋巴细胞增殖,+,改善动物免疫,IL-2 etc.,IFN-,+,+,Receptor,Gene knockout 基因敲除,Signal transduction pathway 信号传导通路,TLRsToll 样受体,Antimicrobial peptides(AMPs)抗菌肽,AMPs抗菌肽,Abundant and diverse groups of molecules that are encoded by definite genes 特定编码基因产生的一类小分子多肽Important components of the innate immune system 先天免疫系统的重要组成成分,1:control with no insert;2-6:positive recombinants induced 72 h1：对照；2-6：重组菌诱导72 h的正对照组（抗菌肽）,Process biochemistry,J.Zhang,2006,Antibacterial assay抑菌分析,Current situation and existing problems现状及存在问题,Research of higher structure and antibacterial mechanism of AMPs 研究抗菌肽高级结构和抗菌机理Short half-life,easy to lose activity 半衰期短，易失活Toxicity toward prokaryotic cell 抗菌肽对原核细胞的毒性Low yield,difficult to purify 天然抗菌肽纯化难度大，产量低,Modify the molecule of AMPs 优化抗菌肽分子Select definite strain,and fusion expression 原核系统中选育特定抗性菌株，真核系统中采用融合 表达以提高其表达率Special induction mode,reduce product cost 以特殊的诱导模式，降低生产成本,Key points for product development产品研发关键点,Molecule designing:IFN-gene+ABPS1 独特的分子设计:-干扰素基因与抗菌肽ABPS1序列串连Two expression systems 采用两套表达系统，原核及真核表达，择优进行后续试验Conditions for high expression efficiency 摸索提高表达效率的条件,Development work 开展工作,enzyme preparations,herbal extracts,probiotics,antimicrobial peptides酶制剂，天然提取物，益生素，抗菌肽,Recombinant DNA technique重组DNA技术,Microbiology微生物学,Antibiotics 抗生素,Extraction 提取,Conclusions 结论,Animal products绿色畜产品,再次感谢各位对饲料工业中心十年来的关心与支持！,