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

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 500bp,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52ku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。

The objective of the study was to construct the transgenic Lactobacillus reuteri with the ability of exocellular secreting endoglucanase and hydrolyzingβ-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 amplifica-tion 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.