食品级重组乳酸乳球菌强组成型分泌猪肠道益生的pEGF-p40

doi:10.11843/j.issn.0366-6964.2025.05.023

Abstract

Abstract:

Co-administration of porcine epidermal growth factor (pEGF) and trefoil factor 3 has been shown to be effective in promoting intestinal proliferation and repair in pigs. Herein, this study was to construct a food-grade recombinant Lactococcus lactis strong constitutive expression system for the fusion expression of pEGF as well as a porcine gut beneficial protein p40, derived from Lactobacillus rhamnosus. As such, the researchers sought to overcome the current status quo of the low levels of L. lactis recombinant constitutive expression as well as to avoid the use of antibiotics, and to preliminarily develop a more potent porcine gut beneficial agent. Firstly, using the commonly used L. lactis constitutive promoter P32 as a control and the Staphylococcus aureus nuclease reporter system, this study visually screened out the promoter of the elongation factor Tu of L. lactis NZ9000 (P1) outperformed the other promoters of L. lactis NZ9000 constitutively highly expressed proteins and the previously found strong constitutive promoters of L. lactis. Furthermore, by replacing the chloramphenicol resistance gene of pNZ8148 with the nisin resistance gene, a food-grade recombinant L. lactis strong constitutive expression system named NZ9000/pP1NR was successfully constructed, which possessed good nisin resistance and completely lost the chloramphenicol resistance. Using the system, pEGF and the fusion protein pEGF-p40 were successfully expressed. Cell proliferation assays on IPEC-J2 cells showed that the culture supernatant containing pEGF-p40 was more effectively in promoting the proliferation. SGLT-1, GLUT-2, SUC, EGFR and GLP2R were detected in IPEC-J2 cells after stimulation by qRT-PCR, indicating that the culture supernatant containing pEGF-p40 regulated the expression of the relevant genes differently from that of the culture supernatant containing pEGF alone. In this study, a food-grade recombinant L. lactis strong constitutive expression system was constructed. By which, pEGF-p40 with a beneficial effect on porcine intestines, was successfully expressed. This study provides a reference for the safe production of other probiotic substances, and preliminarily explored a potent beneficial preparation for the porcine intestine.

Key words: Lactococcus lactis, constructive secretion, pEGF, p40, intestinal health

CLC Number:

HE Rongxiao, WU Yangbo, ZHANG Shuxia, ZHAO Zengjue, HUANG Juan, PAN Weixiong, REN Zhixin, HUANG Haobin, WU Jiahui, WU Haiyang, SHEN Shiyan, SUN Chongjun, ZHANG Linghua. Intestinal Beneficial pEGF-p40 Secreted by a Food-grade Recombinant Lactococcus lactis Strong Constitutive Expression System[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(5): 2243-2258.

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质粒名称 Plasmids	用途 Applications	来源 Sources
pDM19-T	T载体 T vector	购自宝日医生物技术(北京)有限公司 Purchased from Takara Biomedical Technology (Beijing) Co., Ltd.
pNZ8148	乳酸乳球菌表达载体 Expression vector for L. lactis	本实验室保存 Stored by the writer’s lab
pMG36e	乳酸乳球菌表达载体 Expression vector for L. lactis	本实验室保存 Stored by the writer’s lab
pDM19-T-SP-nuc	nuc基因的T载体 T vector containing nuc gene	本研究构建 Constucted by the current study
pNZ8148-SP-nuc	启动子PnisA控制下的nuc基因报告载体 Vecter with nuc reporter under control of PnisA
pNZ8148Δ(PnisA)-P1-SP-nuc	启动子P1控制下的nuc基因报告载体 Vecter with nuc reporter under control of P1
pNZ8148Δ(PnisA)-P2-SP-nuc	启动子P2控制下的nuc基因报告载体 Vecter with nuc reporter under control of P2
pNZ8148Δ(PnisA)-P3-SP-nuc	启动子P3控制下的nuc基因报告载体 Vecter with nuc reporter under control of P3
pNZ8148Δ(PnisA)-P4-SP-nuc	启动子P4控制下的nuc基因报告载体 Vecter with nuc reporter under control of P4
pNZ8148Δ(PnisA)-P5-SP-nuc	启动子P5控制下的nuc基因报告载体 Vecter with nuc reporter under control of P5
pNZ8148Δ(PnisA)-P8-SP-nuc	启动子P8控制下的nuc基因报告载体 Vecter with nuc reporter under control of P8
pNZ8148Δ(PnisA)-SP-nuc	启动子P32控制下的nuc基因报告载体 Vecter with nuc reporter under control of P32
pNZ8148Δ(PnisA)-P1	启动子P1控制下的表达载体 Expression vector under control of P1
pNZ8148Δ(PnisA)-P1-nsr	插入nsr基因的表达载体 Expression vector with nsr insertion
pP1NR	删除CmR基因的表达载体 Expression vector with CmR deletion
pP1NR-pEGF	pEGF的表达载体 Expression vector for pEGF
pP1NR-pEGF-p40	pEGF-p40的表达载体 Expression vector for pEGF-p40

基因 Gene	引物序列(5′→3′) Primer sequences	参考序列 References
β-actin	F: CACGCCATCCTGCGTCTGGA	XM_021086047.1
β-actin	R: AGCACCGTGTTGGCGTAGAG	XM_021086047.1
EGFR	F: GCCTTAGCCGTCTTATCCAA	NM_214007.1
EGFR	R: TGGGCACAGATGACTTTGGT	NM_214007.1
SGLT-1	F: CCACTTTCCCTATAAAACCTCAC	NM_001164021.1
SGLT-1	R: CTCCATCAAACTTCCATCCTCAG	NM_001164021.1
GLUT2	F: TTGCCTTGGATGAGTTATGTGA	NM_001097417.1
GLUT2	R: GCGTGGTCCTTGACTGAAAA	NM_001097417.1
SUC	F: TGGCCATCCAGTCATGCC	XM_047755123.1
SUC	R: CCACCACTCTGCTGTGGA	XM_047755123.1
GLP2R	F: CCCTGCTGTTTCTGGTTTCC	NM_001246266.1
GLP2R	R: GGCAGGGAACAGAAACGTTT	NM_001246266.1

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Intestinal Beneficial pEGF-p40 Secreted by a Food-grade Recombinant Lactococcus lactis Strong Constitutive Expression System

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