表达牛乳铁蛋白肽的罗伊氏乳杆菌分泌型信号肽的筛选及鉴定

doi:10.11843/j.issn.0366-6964.2025.03.041

摘要/Abstract

摘要：

本研究旨在构建一株高效表达牛乳铁蛋白肽的重组罗伊氏乳杆菌，通过筛选不同信号肽的方式提高牛乳铁蛋白肽的分泌表达水平，提高重组菌株的应用效率。对实验室分离的一株罗伊氏乳杆菌进行全基因组测序，筛选分泌蛋白的信号肽基因，构建表达牛乳铁蛋白肽(LFCA)的重组菌株，通过Western blot、ELISA、激光共聚焦等技术分析和比较信号肽分泌LFCA的效果，对重组菌分泌的LFCA对金黄色葡萄球菌增殖抑制作用及其半数最低抑菌浓度进行检测。结果显示，A2、A3、A4和A8信号肽能够表达LFCA，其中A3和A8能够使LFCA分泌到培养上清中，且A3信号肽使LFCA的分泌表达水平提高了近2倍，对宿主菌株的生长性能无显著影响，重组菌株分泌的LFCA对金黄色葡萄球菌的半数最低抑菌浓度为97.50 μg·mL^-1。本研究成功筛选出能够高效分泌牛乳铁蛋白肽的信号肽，重组菌株的抑菌效率得到显著提高。

关键词: 牛乳铁蛋白肽, 罗伊氏乳杆菌, 重组蛋白分泌表达信号肽, 抑菌活性

Abstract:

The aim of this study was to construct a recombinant Lactobacillus reuteri expressing bovine lactoferrin peptides, and to enhance the secretion and expression of lactoferrin peptides by screening different signal peptides, thereby improving the application efficiency of the recombinant strains. A strain of Lactobacillus reuteri isolated in the laboratory was subjected to whole genome sequencing, the signal peptide gene for secreted proteins was screened, and recombinant strains expressing bovine lactoferrin peptides(LFCA)were constructed. The effect of LFCA secreted by the signal peptide was analyzed and compared by Western blot, ELISA, and laser confocal techniques, and the inhibition of proliferative activity of Staphylococcus aureus by LFCA secreted by the recombinant bacteria as well as the half-minimum inhibitory concentration were identified. The results showed that A2, A3, A4 and A8 signal peptides were able to express LFCA, of which A3 and A8 were able to successfully make them secreted into the culture supernatant, and A3 signal peptide increased the secretion and expression level of bovine lactoferrin peptides nearly 2-fold, which did not have significant effect on the growth performance of the strains, and the half-minimum inhibitory concentration of LFCA secreted by the strains was 97.50 μg·mL^-1 against Staphylococcus aureus. This study successfully screened the signal peptide which could secrete lactoferrin peptide efficiently, and the bacteriostatic and antiviral efficiency of the recombinant strain was significantly improved.

Key words: bovine lactoferrin, Lactobacillus reuteri, the recombinant protein secreted signal peptide, antibacterial activity

中图分类号:

S852.69

赵文悦, 杨景, 邵怡岚, 李佳璇, 姜艳平, 崔文, 王晓娜, 唐丽杰. 表达牛乳铁蛋白肽的罗伊氏乳杆菌分泌型信号肽的筛选及鉴定[J]. 畜牧兽医学报, 2025, 56(3): 1431-1440.

ZHAO Wenyue, YANG Jing, SHAO Yilan, LI Jiaxuan, JIANG Yanping, CUI Wen, WANG Xiaona, TANG Lijie. Screening and Identification of Secretory Signal Peptide of Lactobacillus reuteri Expressing Lactoferrin Peptide[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1431-1440.

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信号肽名称 Name of signal peptide	信号肽序列 Signal peptide sequence
A1	ATGTTGGATCGACACGTTTGGCAAAGGCTTGCTACCGTGGTACTGGCAGCCTTGGTAAT TTTCAGCATCAATACTTTCTCACCAGTA
A2	ATGAAAAAATATATTATCAGTATTAGTAGTATTGTTGCTTTGTTGGTTATAATTCTT GGGTTATCCTTTGTTCCGTGGAAATCGATGGGG
A3	ATGGTGAACGAGATAAATTTCGTTTTTCGACTGCTTAAGCGGATATTTATAATAGGAC TTTTAGTAGGTGGAGGATGGCTTTATTTCAATGATGCACAAGTTCAAGCAACGGCG
A4	ATGCGATTTCGATTCAATAAAAAGACTCAGTATTTACTTTTATCTTTAGTAGCAGTTCT CGGCATTGGTAGTTTTTCC
A5	ATGAAAAAAGAAACAAAAATCATTTGTGGAAGTGTACTTGCATTTTTGATTATTTTAT TTGGGATTGTGGCG
A6	ATGAGTAAAAAATTACTTGCTGGGATATTATTAGGTGGAGTCGCAACATATGCT
A7	ATGGGCAAGGACACTAAAGGCTTCTTTCGCCGGAAATGGTTTTTGCGAATTATTTCGTT AATCCTAGCACTCTTTCTATTCATGTATGTAAATGGG
A8	ATGAAAAAATTTAAGTTGATTAGCGGTTTATTAGTTATGTTCTTAGCATTCTTTATTT TAACGGGATATACA
A9	ATGAAATTTTGGAAGAAAGCACCATTAACAATTGCAGCCTTAACAGTCAGTACCTCTG CAGGAATCACAAGTGTTTCTGCT
A10	ATGCAATCAAGTTTAAAGAAATCTCTTTACTTGGGCCTTGCCGCATTGAGCTTTGCTG GTGTTGCTGCCGTTTCAACGACTGCTTCAGCT

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