转内切葡聚糖酶基因罗伊乳酸杆菌的构建及其肉鸡饲喂效果验证

doi:10.11843/j.issn.0366-6964.2015.05.013

畜牧兽医学报 ›› 2015, Vol. 46 ›› Issue (5): 774-783.doi: 10.11843/j.issn.0366-6964.2015.05.013

转内切葡聚糖酶基因罗伊乳酸杆菌的构建及其肉鸡饲喂效果验证

王磊^1,2，曹平华^1,3，蔡蓓¹，高印¹，高康¹，杨雨鑫¹，杨明明¹，袁超⁴，陈玉林^1*

（1.西北农林科技大学动物科技学院，杨凌 712100； 2.青海大学青海省畜牧兽医科学院，西宁 810016；3.河南科技大学动物科技学院，洛阳471003； 4.中国农业科学院兰州畜牧与兽药研究所，兰州 730050）

收稿日期:2014-07-16 出版日期:2015-05-23 发布日期:2015-05-23
通讯作者: 陈玉林，教授，博士生导师，主要从事动物遗传资源研究、动物饲料资源与开发利用方面的研究，E-mail：chenyulindk@163.com
作者简介:王磊（1983-），男，甘肃永昌人，博士生，主要从事动物分子营养研究，E-mail：277036818@qq.com
基金资助:
国家自然科学基金资助项目（cyh200803）；国家公益性行业（农业）专项（20090300606）

Construction of Transgenic Lactobacillus reuteri with Endoglucanase and Evaluation of Its Feeding Effectiveness on Broilers

WANG Lei^1,2,CAO Ping-hua^1,3,CAI Bei¹,GAO Yin¹,GAO Kang¹,YANG Yu-xin¹,YANG Ming-ming¹,YUAN Chao⁴,CHEN Yu-lin^1*

(1.College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China;2.Qinghai Academy of Animal Science and Veterinary Medicine, Qinghai University, Xining 810016, China;3.College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471003,China;4.Lanzhou Institute of Husbandry and Pharmaceutical Sciences,Chinese Academy of Agricultural Sciences, Lanzhou 730050, China)

Received:2014-07-16 Online:2015-05-23 Published:2015-05-23
Supported by:

摘要/Abstract

摘要：

本研究旨在构建可胞外分泌内切葡聚糖酶，且具有降解β-葡聚糖能力的转内切葡聚糖酶基因罗伊乳酸杆菌，并对其进行肉鸡饲喂效果验证，为后续转基因乳酸杆菌在动物生产中的应用奠定基础。采用PCR和酶切连接方法构建pLEM4157（cel）载体，转化Lactobacillus.reuteri XC1感受态细胞，进行聚丙烯胺凝胶电泳表达谱分析和内切葡聚糖酶活性测定，同时测定其肉鸡饲喂效果。结果表明，克隆的内切葡聚糖酶基因大小为1 500 bp，经测序比对，与已报道基因序列相似性为99%，氨基酸序列相似性为100%；Native-PAGE 和SDS-PAGE分析结果表明，内切葡聚糖酶基因celW成熟肽在罗伊乳酸杆菌的蛋白分子量均为52 ku左右，与理论计算值相符； L.reuteri pLEM4157（cel）培养上清和胞内上清内切葡聚糖酶活性分别为（0.96±0.08）和（0.37±0.09） U•mL^-1； L.reuteri pLEM4157（cel）对肉仔鸡生长性能无显著影响，但可显著降低21日龄肉仔鸡十二指肠、空肠pH，促进21日龄肉仔鸡肾、法氏囊、胸腺和胰腺的生长发育，增加42 日龄肉仔鸡的胸腺和胰腺指数，提高肉仔鸡21日龄血清白球比、钙、磷含量和42 日龄血清葡萄糖含量。L.reuteri pLEM4157（cel）成功分泌表达了内切葡聚糖酶，具有降解羧甲基纤维素的能力和一定的肉鸡饲喂效果。

Abstract:

The objective of the study was to construct the transgenic Lactobacillus reuteri with the ability of exocellular secreting endoglucanase and hydrolyzing β-glucan and evaluate its feeding effect on broilers，which would establish the foundation for application of the transgenic L.reuteri in animal production.pLEM4157（cel） vector was constructed by PCR and restriction enzyme，then transformed into competent cells of L.reuteri XC1 for SDS-PAGE analysis，determination of endoglucanase activity and evaluation of feeding effect on broilers.The results of PCR amplification and sequence blast showed that endoglucanase gene celW was about 1 500 bp，the similarity of gene and amino acid sequence were 99% and 100%，respectively.Native-PAGE amd SDS-PAGE analysis showed that the molecular mass of celW gene mature peptide was approximately 52 ku，which was consistent to the predicted molecular weight.The endoglucanase activity in the extracellular and intracellular fraction in the transformed L.reuteri culture were（0.96±0.08） and（0.37±0.09） U•mL^-1，respectively.L.reuteri pLEM4157（cel） had not significant effect on growth performance of broilers，but could decrease pH in duodenum and jejunum at 21 d，promote the growth development of kidney，cloacal bursa，thymus and pancreatic at 21 d，increase the thymus and pancreatic index at 42 d，improve the A/G and the content of Ca and P at 21 d，and the content of glucose at 42 d.L.reuteri pLEM4157（cel） successfully secreted and expressed endoglucanase，displayed the ability to hydrolyze carboxymethyl cellulose and feeding effect on broier.

中图分类号:

S831
S815.5

王磊，曹平华，蔡蓓，高印，高康，杨雨鑫，杨明明，袁超，陈玉林. 转内切葡聚糖酶基因罗伊乳酸杆菌的构建及其肉鸡饲喂效果验证[J]. 畜牧兽医学报, 2015, 46(5): 774-783.

WANG Lei,CAO Ping-hua,CAI Bei,GAO Yin,GAO Kang,YANG Yu-xin,YANG Ming-ming,YUAN Chao,CHEN Yu-lin. Construction of Transgenic Lactobacillus reuteri with Endoglucanase and Evaluation of Its Feeding Effectiveness on Broilers[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(5): 774-783.

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参考文献

［1］ORLANDO A，MESSA C，LINSALATA M，et al.Effects of Lactobacillus rhamnosus GG on proliferation and polyamine metabolism in HGC-27 human gastric and DLD-1 colonic cancer cell lines［J］. Immunopharmacol Immunotoxicol，2009，31（1）：108-116.
［2］AMIT-ROMACH E，SKLAN D，UNI Z.Microflora ecology of the chicken intestine using 16S ribosomal DNA primers［J］.Poult Sci，2004，83（7）：1093-1098.
［3］DUMONCEAUX T J，HILL J E，HEMMINGSEN S M，et al.Characterization of intestinal microbiota and response to dietary virginiamycin supplementation in the broiler chicken［J］.Appl Env Microbiol，2006，72（4）：2815-2823.
［4］EHRMANN M A，KURZAK P，BAUER J，et al.Characterization of lactobacilli towards their use as probiotic adjuncts in poultry［J］. J Appl Microbiol，2002，92（5）：966-975.
［5］BOTES M，LOOS B，VAN REENEN C A，et al.Adhesion of the probiotic strains Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 to Caco-2 cells under conditions simulating the intestinal tract，and in the presence of antibiotics and anti-inflammatory medicaments［J］.Arch Microbiol，2008，190（5）：573-584.
［6］BATEUP J M，MCCONNELL M A，JENKINSON H F，et al.Comparison of Lactobacillus strains with respect to bile salt hydrolase activity，colonization of the gastrointestinal tract，and growth rate of the murine host［J］.Appl Env Microbiol，1995，61（3）：1147-1149.
［7］ZUO R，CHANG J，YIN Q，et al.Phytase gene expression in Lactobacillus and analysis of its biochemical characteristics［J］.Microbiol Res，2010，165（4）：329-335.
［8］ZEGERS N D，KLUTER E，VAN DER STAP H，et al.Expression of the protective antigen of Bacillus anthracis by Lactobacillus casei：towards the development of an oral vaccine against anthrax［J］.J Appl Microbiol，1999，87（2）：309-314.
［9］LIU J R，YU B，LIU F H，et al.Expression of rumen microbial fibrolytic enzyme genes in probiotic Lactobacillus reuteri［J］.Appl Env Microbiol，2005，71（11）：6769-6775.
［10］BEDFORD M R，SCHULZE H.Exogenous enzymes for pigs and poultry［J］. Nutr Res Rev，1998，11（1）：91-114.
［11］MATHLOUTHI N，MOHAMED M A，LARBIER M.Effect of enzyme preparation containing xylanase and β-glucanase on performance of laying hens fed wheat/barley-or maize/soybean meal-based diets［J］.Br Poult Sci，2003，44（1）：60-66.
［12］MATHLOUTHI N，JUIN H，LARBIER M.Effect of xylanase and β-glucanase supplementation of wheat-or wheat-and barley-based diets on the performance of male turkeys［J］.Br Poult Sci，2003，44（2）：291-298.
［13］LIU J R，YU B，ZHAO X，CHENG K J.Coexpression of rumen microbial β-glucanase and xylanase genes in Lactobacillus reuteri［J］.Appl Microbiol Biotechnol，2007，77（1）：117-124.
［14］SCHULEIN M.Protein engineering of cellulases［J］.Biochim Biophys Acta-Protein Structure Mol Enzymol，2000，1543（2）：39-52.
［15］CHO J S，CHOI Y J，CHUNG D K.Expression of Clostridium thermocellum endoglucanase gene in Lactobacillus gasseri and Lactobacillus johnsonii and characterization of the genetically modified probiotic Lactobacilli［J］.Current Microbiol，2000，40（4）：257-263.
［16］MAPPLEY L J，TCHORZEWSKA M A，NUNEZ A，et al.Oral treatment of chickens with Lactobacillus reuteri LM1 reduces Brachyspira pilosicoli-induced pathology［J］.J Med Microbiol， 2013，62（pt2）：287-296.
［17］PATTERSON J A，BURKHOLDER K M.Application of probiotics and probiotics in poultry production［J］.Poult Sci，2003，82（4）：627-631.
［18］REID G，FRIENDSHIP R.Alternatives to antibiotic use；probiotics for the gut［J］.Anim Biotechnol，2002，13（1）：97-112.
［19］STEPHENSON D P，MOORE R J，ALLISON G E.Transformation of，and heterologous protein expression in，Lactobacillus agilis and Lactobacillus vaginalis isolates from the chicken gastrointestinal tract［J］.Appl Env Microbiol，2011，77（1）：220-228.
［20］YU B，LIU J R，HSIAO F S，et al.Evaluation of Lactobacillus reuteri Pg4 strain expressing heterologous β-glucanase as a probiotic in poultry diets based on barley［J］.Anim Feed Sci Technol，2008，141（1-2）：82-91.
［21］LIU J R，LAI S F，YU B.Evaluation of an intestinal Lactobacillus reuteri strain expressing rumen fungal xylanase as a probiotic for broiler chickens fed on a wheat-based diet［J］.Br Poult Sci，2007，48（4）：507-514.
［22］JIN L Z，HO Y W，ABDULLAH N，et al.Growth performance，intestinal microbial populations，and serum cholesterol of broilers fed diets containing Lactobacillus cultures［J］.Poult Sci，1998，77（9）：1259-1265.
［23］WATKINS B A，MILLER B F.Competitive gut exclusion of avian pathogens by Lactobacillus acidophilus in gnotobiotic chicks［J］.Poult Sci，1983，62（9）：1772-1779.
［24］程相朝，张春杰.中药免疫增强剂对肉仔鸡免疫器官生长发育及免疫活性细胞影响的研究［J］.中兽医学杂志，2002，（3）：6-8.
CHENG X C，ZHANG C J.The effect of Chinese traditional immunopoteniators on growth and development of immune organ and immunocompetence cells in broilers［J］. Chinese Journal of Traditional Veteriuary Science，2002，（3）：6-8.(in Chinese)
［25］张春杨，牛钟相，常维山，等.益生菌剂对肉用仔鸡的营养、免疫促进作用［J］.中国预防兽医学报，2002，24（1）：51-54.
ZHANG C Y，NIU Z X，CHANG W S，et al.The promoting effects of probiotics to the nutrition and immunity of broilers［J］.Chinese Journal of Preventive Veterinary Medicine，2002，24（1）：51-54.(in Chinese)
［26］唐志刚，王俊峰，温超，等.益生菌对肉鸡生产性能、免疫器官指数和血清指标的影响［J］.江苏农业科学，2010，（4）：208-210.
TANG Z G，WANG J F，WEN C，et al.The effect of probiotics on growth performance，immune organ index and serum indicator in broilers［J］.Jiangsu Agricultural Science，2010，（4）：208-210.(in Chinese)
［27］DE FAZIO A，CHIEW Y E，DONOGHUE C，et al.Effect of sodium butyrate on estrogen receptor and epidermal growth factor receptor gene expression in human breast cancer cell lines［J］.J Biol Chem， 1992，267（25）：18008-18012.
［28］HIROMI K，MASAHIRO S，HAJIME N，et al.Effect of antioxidative lactic acid bacteria on rats fed a diet deficient in vitamin E［J］.J Dairy Sci， 1993，76（9）：2493-2499.
［29］GILL H S，RUTHERFURD K J.Viability and dose-response studies on the effects of the immunoenhancing lactic acid bacterium Lactobacillus rhamnosus in mice［J］.Br J Nutr，2001，86（2）：285-289.
［30］潘康成，何明清，刘克琳.地衣芽抱杆菌对家兔细胞免疫功能的影响［J］.四川农业大学学报，1997，15（3）：368-371.
PAI K C，HE M Q，LIU K L.Study of the effect of Bacillus licheniformis to cell-mediated immunity of rabbits［J］.Journal of Sichuan Agricultural University，1997，15（3）：368-371.(in Chinese)
［31］YASUI H，NAGAOKA N，HAYAKAWA K.Augmentation of anti-influenza virus hemagglutinin antibody production by Peyer’s patch cells with Bifidobacterium breve VIT4064［J］.Clin Diagnost Lab Immunol，1994，1（2）：244-246.
［32］刘晓琳，郭世宁，石达友，等.复方紫锥菊对肉鸡部分血液生化指标的影响［J］.中国兽医杂志，2008，（4）：30-31.
LIU X L，GUO S N，SHI D Y，et al.The effect of compound Echinacea purpurea on partial blood biochemical indicator in broilers［J］.Chinese Journal of Veterinary，2008，（4）：30-31.(in Chinese)

转内切葡聚糖酶基因罗伊乳酸杆菌的构建及其肉鸡饲喂效果验证

Construction of Transgenic Lactobacillus reuteri with Endoglucanase and Evaluation of Its Feeding Effectiveness on Broilers

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