组成型启动子Pldh对乳酸菌表达系统外源基因表达的影响研究

doi:10.11843/j.issn.0366-6964.2025.06.036

Abstract

Abstract:

Using the AmpR (ampicillin resistance) reporter gene expression system, we constructed Lactobacillus expression vector systems with different numbers of Lactobacillus constitutive promoter P_ldh in tandem, and different sequences of promoter P_ldh and AmpR reporter genes, and detected the AmpR reporter gene transcription level, protein expression and evaluated the AMPR enzyme activity, so as to screen out the constitutive multi-promoter that is stable and strongly expressed. This will provide a theoretical basis for the expression of exogenous antigens or functional proteins by Lactobacilli expression vectors. Bioinformatics analysis of P_ldh promoter was performed to construct recombinant Lactobacillus with different numbers of promoter P_ldh in tandem and recombinant Lactobacillus with different order of promoter and exogenous gene AmpR. The strength of exogenous gene expression in different expression systems was comprehensively evaluated by real-time fluorescence quantitative PCR, Western blot, indirect ELISA, and ampicillin live bacterial screening test. Results were as follows: PCR results showed that the recombinant Lactobacillus reuteri successfully amplified P_ldh-P_ldh, P_ldh-P_ldh-P_ldh, P_ldh-P_ldh-P_ldh, P_ldh-AmpR-P_ldh-AmpR, P_ldh-P_ldh-AmpR, and P_ldh-P_ldh-AmpR-AmpR target fragments of 600, 900, 2 300, and 2 300 bp, respectively; and the Western blot results showed that all recombinant bacteria could express the AmpR reporter gene at different amounts. The results showed that all the recombinant bacteria could express the AMPR protein, and the target proteins were all about 28 ku in size. Indirect ELISA, fluorescence quantitative PCR and AMPR enzyme activity assays all showed that the efficiency of the triple promoter to drive the expression of the AmpR reporter gene was significantly higher than that of the double promoter in the tandem Lactobacillus expression vector system with different numbers of promoter P_ldh(P < 0.05), and that the efficiency of triple promoter and dual promoter drove the efficiency of AmpR reporter gene expression were all highly significantly higher than that of single promoter (P < 0.01), and all of them were highly significantly higher than that of the control bacterium pPG-Ampr/HLJ-27 (P < 0.01); in the Lactobacillus expression vector system with different sequences of promoter P_ldh and AmpR reporter genes, the recombinant bacterium pPG-P_ldh-P_ldh-Ampr-Ampr/HLJ-27 drove the expression of the AmpR reporter gene very significantly more efficiently than recombinant bacterium pPG-P_ldh-Ampr-P_ldh-Ampr/HLJ-27 P < 0.01). This study successfully constructed five constitutive multi promoter P_ldh lactic acid bacteria expression systems using AmpR reporter genes. The results showed that in different numbers of promoter P_ldh cascade driven single exogenous protein vector systems, the promoter regulated transcription and thus drove the expression level of exogenous genes, which was positively correlated with the number of promoters; In the dual promoter driven dual exogenous protein vector system, the continuous cascade of two promoters regulates transcription and drives the expression efficiency of dual exogenous genes, which is significantly higher than the expression efficiency of dual exogenous genes driven by two promoters separately; The above data provides necessary basis for optimizing the expression efficiency of exogenous proteins in lactic acid bacteria expression vectors.

Key words: Lactobacillus expression system, constitutive promoter P_ldh, promoter activity, exogenous gene expression efficiency

CLC Number:

LIU Xinzi, ZHAO Haiyuan, JU Ning, CHEN Ying, WANG Zi, MENG Weijing, LI Jiaxuan, JIANG Yanping, CUI Wen, WANG Xiaona. Effect of Constitutive Promoter P_ldh on Exogenous Gene Expression in Lactic Acid Bacteria Expression System[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(6): 2937-2947.

Figures/Tables 10

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引物名称 Primer name	引物序列(5′→3′) Primer sequences	产物大小/bp Product length	扩增片段 Amplified fragments
pPG-F	CTCCGCTTCTTTTATTTTTTTGT	pPG载体通用引物	目的片段
pPG-R	TCTCTCATCCGCCAAAACAG
P1-F	$\underline{{\rm{GTGATA}}}$TCATCCTTTCTTATGTGCAT	310	P_ldh片段1
P1-R	GGACGGAAAAGGCAAAATGTGATATCATCCTTT
P2-F	GGAAAAGGCAAAATGTGATATCATCCTTTCTTATG	317	P_ldh片段2
P2-R	$\underline{{\rm{CTGCAG}}}$TGCTCTGGACGGAAAAGGCAAAA
P3-F	$\underline{{\rm{TCTAGA}}}$GTGATATCATCCTTTCTTATG	1 130	P_ldh-AmpR片段1
P3-R	$\underline{{\rm{GAGCTC}}}$GTGATATCATCCGCCAAAACAG
P4-F	$\underline{{\rm{GAGCTC}}}$ATCCGCCAAAGTGATATCATCCTTTCTTA	1 130	P_ldh-AmpR片段2
P4-R	$\underline{{\rm{GGGCCC}}}$GATCCGCCAAAACAG
A1-F	$\underline{{\rm{CTGCAG}}}$ATTGAGGGCCCCTGCAGATGTACCAATG	860	AmpR片段1
A1-R	ATGTACCAATGCTTCAATGAGTATTC
A2-F	CATTGGTACATGAGTATTCATGTACCAATG	863	AmpR片段2
A2-R	$\underline{{\rm{GGGCCC}}}$CATTGGTACATCTGCAGGG

元件名称 Element name	核心序列 Core sequence	位置 Position	功能 Function
A-box	CCTGCC	279－	保证转录的正确定位 Cis-acting regulatory element
ABRE	GTGCA	41－	脱落酸诱导元件 Cis-acting element involved in the abscisic acid responsiveness
CAAT-box	CAAT	63+，172+，79－，76+，107－	启动子和增强子区域中常见的顺式作用元件 Common cis-acting element in promoter and enhancer regions
CGTCA-motif	CGTCA	257+	参与MeJA反应性的顺式作用调节元件 Cis-acting regulatory element involved in the MeJA-responsiveness
G-Box	CACGTT	41+	顺式作用调节元件参与光响应性 Cis-acting regulatory element involved in light responsiveness
Ⅰ-box	TGATAATGT	56－	轻型响应元件一部分 Part of a light responsive element
MYB	CAACAG	221+	MYB响应元件 MYB response element
TATA-box	TATA	50+，268－	核心启动子元件 Core promoter element
TGACG-motif	TGACG	257－	茉莉酸甲酯反应的顺式作用调节元件 Cis-acting regulatory element involved in the MeJA-responsiveness
W box	TTGACC	234－	WRKY转录因子结合位点 WRKY transcription factors binding site
as-1	TGACG	257－	氧化应激响应元件 Oxidative stress response elements

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Effect of Constitutive Promoter P_ldh on Exogenous Gene Expression in Lactic Acid Bacteria Expression System

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Effect of Constitutive Promoter Pldh on Exogenous Gene Expression in Lactic Acid Bacteria Expression System

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Effect of Constitutive Promoter P_ldh on Exogenous Gene Expression in Lactic Acid Bacteria Expression System