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

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (6): 2937-2947.doi: 10.11843/j.issn.0366-6964.2025.06.036

组成型启动子P_ldh对乳酸菌表达系统外源基因表达的影响研究

刘芯孜¹(), 赵海渊¹(), 鞠宁³(), 陈莹¹, 王梓⁴, 孟伟静⁵, 李佳璇¹^,², 姜艳平¹^,², 崔文¹^,², 王晓娜¹^,²^,*()

1. 东北农业大学动物医学学院, 哈尔滨 150030
2. 动物疾病防控技术与制剂创制省重点实验室, 哈尔滨 150030
3. 逊克县农业农村局, 逊克 161499
4. 通辽市农牧科学研究所, 通辽 028000
5. 通辽市农牧业发展中心, 通辽 028000

收稿日期:2024-08-12 出版日期:2025-06-23 发布日期:2025-06-25
通讯作者: 王晓娜 E-mail:m18636724373@163.com;zhywxn1925@163.com;j2645295669@163.com;xiaonawang0319@163.com
作者简介:刘芯孜(2000-)，山西临汾人，硕士，主要从事微生物与免疫学研究，E-mail：m18636724373@163.com
赵海渊(1989-)，黑龙江哈尔滨人，农学博士，主要从事微生物与免疫学研究，E-mail：zhywxn1925@163.com
鞠宁(1997-)，黑龙江肇东人，硕士，主要从事微生物与免疫学研究，E-mail：j2645295669@163.com
第一联系人：
刘芯孜、赵海渊和鞠宁是同等贡献作者
基金资助:
黑龙江省自然科学基金联合引导项目(LH2021C043)；国家自然科学基金青年(32102707)

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

LIU Xinzi¹(), ZHAO Haiyuan¹(), JU Ning³(), CHEN Ying¹, WANG Zi⁴, MENG Weijing⁵, LI Jiaxuan¹^,², JIANG Yanping¹^,², CUI Wen¹^,², WANG Xiaona¹^,²^,*()

1. College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China
2. Provincial Key Laboratory of Animal Disease Prevention and Control Technology and Preparation Creation, Harbin 150030, China
3. Xunke County Agriculture and Rural Bureau, Xunke 161499, China
4. Tongliao Institute of Agriculture and Animal Husbandry, Tongliao 028000, China
5. Tongliao Agricultural and Animal Husbandry Development Center, Tongliao 028000, China

Received:2024-08-12 Online:2025-06-23 Published:2025-06-25
Contact: WANG Xiaona E-mail:m18636724373@163.com;zhywxn1925@163.com;j2645295669@163.com;xiaonawang0319@163.com

摘要/Abstract

摘要：

旨在利用AmpR(氨苄青霉素抗性)报告基因表达系统，构建不同数量乳酸菌组成型启动子P_ldh串联、启动子P_ldh与AmpR报告基因不同顺序的乳酸菌表达载体系统，分别检测AmpR报告基因转录水平、蛋白表达量并评价AMPR酶活性，从而筛选出稳定且强表达的组成型多启动子表达系统，为乳酸菌活菌载体表达外源抗原或功能性蛋白提供理论依据。对P_ldh启动子进行生物信息学分析，构建不同数量启动子P_ldh串联的重组乳酸菌以及启动子与外源基因AmpR不同顺序重组乳酸菌。通过实时荧光定量PCR、Western blot、间接ELISA检测以及氨苄青霉素活菌筛选试验综合评估不同表达系统中外源基因表达的强弱。PCR结果显示重组乳酸菌成功扩增出600、900、2 300和2 300 bp的P_ldh-P_ldh、P_ldh-P_ldh-P_ldh、P_ldh-AmpR-P_ldh-AmpR和P_ldh-P_ldh-AmpR-AmpR目的片段；Western blot结果显示重组菌均可表达AMPR蛋白，目的蛋白大小均约为28 ku。间接ELISA、荧光定量PCR和AMPR酶活性检测结果均显示，在不同数量启动子P_ldh串联乳酸菌表达载体系统中，三启动子驱动AmpR报告基因表达的效率显著高于双启动子(P < 0.05)，三启动子、双启动子驱动AmpR报告基因表达的效率均极显著高于单启动子(P < 0.01)，且均极显著高于对照菌pPG-Ampr/HLJ-27(P < 0.01)；在启动子P_ldh与AmpR报告基因不同顺序的乳酸菌表达载体系统中，重组菌pPG-P_ldh-P_ldh-Ampr-Ampr/HLJ-27驱动AmpR报告基因表达的效率极显著高于重组菌pPG-P_ldh-Ampr-P_ldh-Ampr/HLJ-27(P < 0.01)。本研究利用AmpR报告基因成功构建了5种组成型多启动子P_ldh乳酸菌表达系统，结果显示在不同数量启动子P_ldh串联驱动单外源蛋白载体系统中，启动子调控转录进而驱动外源基因表达量与启动子数量呈正相关；在双启动子驱动双外源蛋白载体系统中，连续串联两个启动子调控转录进而驱动双外源基因表达效率显著高于两个启动子分别驱动双外源基因表达效率；以上数据为乳酸菌表达载体对外源蛋白的表达效率优化提供了必要的依据。

关键词: 乳酸菌表达系统, 组成型启动子P_ldh, 启动子活性, 外源基因表达效率

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

中图分类号:

刘芯孜, 赵海渊, 鞠宁, 陈莹, 王梓, 孟伟静, 李佳璇, 姜艳平, 崔文, 王晓娜. 组成型启动子P_ldh对乳酸菌表达系统外源基因表达的影响研究[J]. 畜牧兽医学报, 2025, 56(6): 2937-2947.

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.

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参考文献 30

1	孙佳丽,董浩,刘郁夫,等.肠道菌群与动物传染病关系研究进展[J].动物医学进展,2020,41(7):107-110.
	SUNJ L,DongH,LIUY F,et al.Research progress on the relationship between gut microbiota and animal infectious diseases[J].Advances in Veterinary Medicine,2020,41(7):107-110.
2	LEMME-DUMITJ M,CAZORLAS I,PERDIGONG D V,et al.Probiotic bacteria and their cell walls induce Th1-type immunity against Salmonella Typhimurium challenge[J].Front Immunol,2021,12,660854. doi: 10.3389/fimmu.2021.660854
3	MORADIM,KOUSHEHS A,ALMASIH,et al.Postbiotics produced by lactic acid bacteria: The next frontier in food safety[J].Compr Rev Food Sci Food Saf,2020,19(6):3390-3415. doi: 10.1111/1541-4337.12613
4	REUTERM A,HANNIFFYS,WELLSJ M.Expression and delivery of heterologous antigens using Lactic acid bacteria[J].Methods Mol Med,2003,8(7):101-14.
5	SYBESMAW,STARRENNBURGM,KLEERBEZEMM,et al.Increased production of folate by metabolic engineering of Lactococcus lactis[J].Appl Environ Microbiol,2003,69(6):3069-3076. doi: 10.1128/AEM.69.6.3069-3076.2003
6	GRACEL,DOUGLAS,YONGJ G,et al.Integrative food grade expression system for lactic acid bacteria[J].Methods in Molecular Biology,2011,16(1):31-37.
7	KLEESBEZEMABM,HOLSP,HUGENHOLTZJ.Lactic acid bacteria as a cell factory: rerouting of carbon metabolism in Lactococcus lactis by metabolic engineering[J].Enzyme Microb Technol,2000,26(9-10):840-848. doi: 10.1016/S0141-0229(00)00180-0
8	HUSSAMM H,GHADAM S,HAMEEDM J.Antibiofilm activity of fructophilic lactic acid bacteria filtrate against Klebsiella pneumoniae virulence gene expression[J].Egy J Hospit Med,2022,89(2):6438-6444. doi: 10.21608/ejhm.2022.270278
9	XIAOY,WANGX N,LIY,et al.Evaluation of the immunogenicity in mice orally immunized with recombinant Lactobacillus casei expressing porcine epidemic diarrhea virus S1 protein[J].Viruses,2022,14(5):890-890. doi: 10.3390/v14050890
10	JINX,HEY F,ZHOUY H,et al.Lactic acid bacteria that activate immune gene expression in Caenorhabditis elegans can antagonise Campylobacter jejuni infection in nematodes, chickens and mice[J].BMC Microbiol,2021,21(1):169. doi: 10.1186/s12866-021-02226-x
11	DOUILLARDF P,O'CONNELL-MOTHERWAYM,CAMBILLAUC,et al.Expanding the molecular toolbox for Lactococcus lactis: construction of an inducible thioredoxin gene fusion expression system[J].Microb Cell Fact,2011,10(1):66. doi: 10.1186/1475-2859-10-66
12	OSCARP. K,PASCALLER,MICHIELK,et al.Quorum sensing-controlled gene expression in lactic acid bacteria[J].J Biotechnol,1998,64(1):15-21. doi: 10.1016/S0168-1656(98)00100-X
13	DUBENDORFFJ,STUDIERF.Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor[J].J Mol Biol,1991,219(1):45-59. doi: 10.1016/0022-2836(91)90856-2
14	JAMESF,CHRISTOPHERW,C.B.COLLINS,et al.Stringent regulation of stably integrated chloramphenicol acetyl transferase genes by E. coli lac repressor in monkey cells[J].Cell,1988,52(5):713-722. doi: 10.1016/0092-8674(88)90409-6
15	陈英,王培娟,张文君,等.mCherry红色荧光标记乳酸菌的融合表达系统构建及应用[J].生物工程学报,2019,35(3):492-504.
	CHENY,WANGP J,ZHANGW J,et al.Construction and application of mCherry red fluorescent protein fusion expression system in lactic acid bacteria[J].Chinese journal of biotechnology,2019,35(3):492-504.
16	袁世豪,张海琳,鞠宁,等.不同组成型启动子对乳酸菌表达系统外源基因表达影响的比较[J].中国畜牧兽医,2023,50(11):4370-4380.
	YUANS H,ZHANGH L,JUN,et al.Comparison of the effects of different composition promoters on exogenous gene expression in lactic acid bacteria expression system[J].Chinese Animal Husbandry and Veterinary Medicine,2023,50(11):4370-4380.
17	JANAS,DEBJ K.Strategies for efficient production of heterologous proteins in Escherichia coli[J].App Microbiol Biotechnol,2005,67,289-298.
18	LIM J,WANGJ S,CHENGY,et al.A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli[J].Microb Cell Fact,2012,11(1):19.
19	ATAÖ,PRIELHOFERR,GASSERB,et al.Transcriptional engineering of the glyceraldehyde-3-phosphate dehydrogenase promoter for improved heterologous protein production in Pichia pastoris[J].Biotechnol Bioeng,2017,114(10):2319-2327.
20	YEOI S,CHOB K,KIMJ H.Conversion of L-arabinose to L-ribose by genetically engineered Candida tropicalis[J].Bioprocess Biosyst Eng,2021,44(6):1147-1154.
21	YEOI S,SHIMW Y,KIMJ H.Construction of genetically engineered Candida tropicalis for conversion of L-arabinose to L-ribulose[J].J Biotechnol,2018,274,9-14.
22	SILVIAC,REBECCAC,KeX,et al.Distinct but spatially overlapping intestinal niches for vancomycin-resistant Enterococcus faecium and carbapenem-resistant Klebsiella pneumoniae[J].Plos Pathog,2015,11(9):1005132.
23	徐惠芬. 基于抗生素基因为报告基因的核酸酶效力检测[D]. 苏州: 苏州大学, 2016.
	XU H F. Detection of nuclease efficacy based on antibiotic genes as reporter genes[D]. Suzhou: Soochow University, 2016. (in Chinese)
24	SPANGLERJ R,CARUANAJ C,PJILLIPSD A,et al.Broad range shuttle vector construction and promoter evaluation for the use of Lactobacillus plantarum WCFS1 as a microbial engineering platform[J].Synth Biol,2019,4(1):ysz012.
25	MAQSOOD I. 组成型表达鸡传染性法氏囊病毒vp2基因重组乳酸菌的构建及免疫原性分析[D]. 哈尔滨: 东北农业大学, 2016.
	MAQSOOD I. Construction and immunogenicity analysis of recombinant lactic acid bacteria expressing the vp2 gene of chicken infectious bursal disease virus with constituent expression[D]. Harbin: Northeast Agricultural University, 2016. (in Chinese)
26	张芙蕊,韩若冰,郭梦雅,等.梅花鹿茸生长过程中顶端不同组织COL1A1基因启动子DNA甲基化模式及差异分析[J].中国农业大学学报,2022,27(10):153-163.
	ZhANGF R,HANR B,GUOM G,et al.Methylation patterns and differential analysis of COL1A1 gene promoter DNA in different tissues during the growth process of velvet antler in plum blossom[J].Journal of China Agricultural University,2022,27(10):153-163.
27	ZHAOP,CHUNLUMA,LIBINXU,et al.Exploiting tandem repetitive promoters for high-level production of 3-hydroxypropionic acid[J].Appl Microbiol Biotechnol,2019,103(10):4017-4031.
28	GUANC G,CUIW J,CHENGJ T,et al.Development of an efficient autoinducible expression system by promoter engineering in Bacillus subtilis[J].Microb Cell Fact,2016,15(1):66.
29	GUANC G,CUIW J,CHENGJ T,et al.Construction of a highly active secretory expression system via an engineered dual promoter and a highly efficient signal peptide in Bacillus subtili[J].New Biotechnology,2016,33(3):372-379.
30	GOODARTS A,GUZOWSKIJ F,RICEM K,et al.Effect of genomic location on expression of β-galactosidase mRNA controlled by the herpes simplex virus type 1 UL38 promoter[J].J Virol,1992,66(5):2973-2981.

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

[1]	陈哲, 陈蓉, 闫乐艳, 陈佳彬, 于建宁. 鹅MyoG基因启动子活性及转录调控元件分析[J]. 畜牧兽医学报, 2021, 52(7): 1869-1879.
[2]	温丽娟, 刘发伟, 林长光, 陈子韬, 张哲, 张豪, 李加琪, 袁晓龙. 猪Nhlh2基因启动子活性及其表达调控的初步研究[J]. 畜牧兽医学报, 2018, 49(4): 667-674.
[3]	王麒, 王晗, 张秀玲, 刘春杨, 张乐超, 李兰会, 李祥龙. 鸡内源白血病病毒ev21与慢羽非连锁分析和LTR区启动子活性分析[J]. 畜牧兽医学报, 2017, 48(5): 930-937.
[4]	刘志永，左其生，肖天荣，韦光辉，陈庭峰，朱睿，张振韬，王怡临，蒋舒颖，张亚妮，李碧春. Am80和TSA对鸡Stra8基因启动子活性的调控作用[J]. 畜牧兽医学报, 2015, 46(6): 896-902.

组成型启动子P_ldh对乳酸菌表达系统外源基因表达的影响研究

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

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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

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

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组成型启动子P_ldh对乳酸菌表达系统外源基因表达的影响研究

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