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WO2023141744A1 - Lactococcus lactis recombiné, microcapsule et utilisation associée - Google Patents

Lactococcus lactis recombiné, microcapsule et utilisation associée Download PDF

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Publication number
WO2023141744A1
WO2023141744A1 PCT/CN2022/073656 CN2022073656W WO2023141744A1 WO 2023141744 A1 WO2023141744 A1 WO 2023141744A1 CN 2022073656 W CN2022073656 W CN 2022073656W WO 2023141744 A1 WO2023141744 A1 WO 2023141744A1
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Prior art keywords
lactococcus lactis
recombinant
recombinant lactococcus
fusion gene
expression vector
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Ceased
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PCT/CN2022/073656
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English (en)
Chinese (zh)
Inventor
金万洙
蒋笑笑
严春龙
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Institute of Zoology of CAS
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Institute of Zoology of CAS
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Priority to PCT/CN2022/073656 priority Critical patent/WO2023141744A1/fr
Publication of WO2023141744A1 publication Critical patent/WO2023141744A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora

Definitions

  • the invention belongs to the technical field of genetic engineering technology, and in particular relates to a recombinant Lactococcus lactis, microcapsules and applications thereof.
  • the object of the present invention is to provide a recombinant Lactococcus lactis, microcapsules and applications thereof, which can secrete hADH1B enzyme and ALDH2 enzyme in the intestinal tract to decompose alcohol after oral administration of the recombinant Lactococcus lactis.
  • the present invention provides a recombinant Lactococcus lactis, including the first recombinant Lactococcus lactis and/or the second recombinant Lactococcus lactis;
  • the first recombinant Lactococcus lactis comprises a first recombinant expression vector;
  • the second recombinant Lactococcus lactis comprises a second recombinant expression vector;
  • the backbone plasmids used for the construction of the first recombinant expression vector and the second recombinant expression vector are respectively PNZ8149;
  • a first fusion gene is inserted into the first recombinant expression vector, and the first fusion gene includes signal peptide SPusp45, probiotic LEISS, enterokinase recognition site DDDDK and human alcohol dehydrogenase in series;
  • a second fusion gene is inserted into the second recombinant expression vector, and the second fusion gene includes signal peptide SPusp45, probiotic LEISS, enterokinase recognition site DDDDK and human acetaldehyde dehydrogenase in series.
  • the nucleotide sequence of the first fusion gene is shown in SEQ ID NO: 1; the nucleotide sequence of the second fusion gene is shown in SEQ ID NO: 2.
  • the first fusion gene and the second fusion gene are respectively inserted between the SphI and XbaI restriction enzyme cutting sites of PNZ8149.
  • the ratio of the effective viable count of the first recombinant Lactococcus lactis and the second recombinant Lactococcus lactis is ( 1 ⁇ 3): (1 ⁇ 3).
  • the present invention also provides a microcapsule containing the recombinant Lactococcus lactis described in the above scheme.
  • the present invention also provides the application of the recombinant Lactococcus lactis described in the above scheme or the microcapsules in the preparation of food, health products or medicines for preventing drunkenness;
  • the recombinant Lactococcus lactis includes the first recombinant Lactococcus lactis, or , the recombinant Lactococcus lactis comprises a first recombinant Lactococcus lactis and a second recombinant Lactococcus lactis.
  • the prevention of drunkenness includes reducing alcohol absorption and/or prolonging alcohol tolerance time.
  • the present invention also provides the application of the recombinant Lactococcus lactis or the microcapsules described in the above scheme in the preparation of food, health products or medicines for hangover.
  • the hangover recovery includes shortening the recovery time after drinking and/or reducing the acute injury of the liver and/or intestinal tract caused by excessive drinking and/or alleviating the vomiting and headache after drinking.
  • the dosage form of the drug includes oral preparations.
  • the present invention provides a recombinant Lactococcus lactis.
  • the present invention uses PNZ8149 (lactic acid bacteria food-grade expression vector) as the base carrier, and a first fusion gene or a second fusion gene is inserted into PNZ8149, and the first fusion gene includes sequentially connected
  • the second fusion gene includes the signal peptide SPusp45 in series, probiotic LEISS, enterokinase recognition site DDDDK and human alcohol dehydrogenase The aldehyde dehydrogenase was fused separately.
  • the target enzyme separates the signal peptide sequence SPusp45 and LEISS sequence from the fusion protein, and releases free alcohol dehydrogenase or acetaldehyde dehydrogenase at the same time, and the released ethanol Dehydrogenase, or aldehyde dehydrogenase, breaks down alcohol.
  • the present invention provides a highly active probiotic expressing human ADH1B (hADH1B and/or acetaldehyde dehydrogenase (ALDH2), which secretes hADH1B enzyme and ALDH2 enzyme to decompose alcohol after oral administration.
  • the results show that oral administration of probiotics expressing hADH1B reduces Alcohol absorption, prolonged alcohol tolerance time. Oral expression of hADH1B and or ALDH2 shortens the recovery time after drinking, and more importantly, the liver and intestinal tract are protected from acute damage caused by excessive drinking. Therefore, the present invention
  • the recombinant Lactococcus lactis has the potential to be prepared as an alcohol partner.
  • the present invention proposes for the first time that ectopically expressed hADH1B and ALDH2 are used to catalyze ethanol decomposition, which provides a practical basis for the subsequent efficient delivery of a variety of enzyme superbiological detoxification agents.
  • Fig. 1 is the schematic diagram of the construction and function of recombinant Lactococcus lactis of the present invention
  • Figure 2 is the result of using anti-hadh antibody to detect recombinant Lactococcus lactis expressing ADH enzyme
  • Figure 3 is the result of using hALDH antibody to detect recombinant Lactococcus lactis expressing ALDH enzyme
  • Fig. 4 is the result that culture plate counting method detects viable bacteria quantity
  • Figure 5 is the situation of prolonging alcohol tolerance time by oral administration of recombinant Lactococcus lactis
  • Figure 6 is the time for drunken mice to become drunk
  • Figure 7 is the statistics of the number of exercises of drunk mice after sobering up
  • Figure 8 shows the reduction of liver-intestinal damage caused by acute alcohol intake by oral administration of recombinant Lactococcus lactis, where A is serum alcohol residue, B is blood triglyceride concentration, C is intestinal mucosal lesions, and D is mouse liver lipid levels.
  • the present invention provides a recombinant Lactococcus lactis, including the first recombinant Lactococcus lactis and/or the second recombinant Lactococcus lactis;
  • the first recombinant Lactococcus lactis comprises a first recombinant expression vector;
  • the second recombinant Lactococcus lactis comprises a second recombinant expression vector;
  • the backbone plasmids used for the construction of the first recombinant expression vector and the second recombinant expression vector are respectively PNZ8149;
  • a first fusion gene is inserted into the first recombinant expression vector, and the first fusion gene includes signal peptide SPusp45, probiotic LEISS, enterokinase recognition site DDDDK and human alcohol dehydrogenase in series;
  • a second fusion gene is inserted into the second recombinant expression vector, and the second fusion gene includes signal peptide SPusp45, probiotic LEISS, enterokinase recognition site DDDDK and human acetaldehyde dehydrogenase in series.
  • nucleotide sequence of the first fusion gene (Noc1-Usp45-LESS-EK-ADH1B) is shown in SEQ ID NO: 1, specifically:
  • nucleotide sequence of the second fusion gene (Noc1-Usp45-LESS-EK-ALDH2) is shown in SEQ ID NO: 2, specifically:
  • the first fusion gene and the second fusion gene are preferably obtained through full-sequence chemical synthesis.
  • the first fusion gene and the second fusion gene are respectively inserted between the SphI and XbaI restriction enzyme cutting sites of PNZ8149.
  • the method of inserting the first fusion gene and the second fusion gene into PNZ8149 is not particularly limited in the present invention, and conventional methods in the art can be used.
  • the original strains of the first recombinant Lactococcus lactis and the second recombinant Lactococcus lactis are preferably Lactococcus lactis NZ3900.
  • the method of transferring the first recombinant expression vector and the second recombinant expression vector into Lactococcus lactis NZ3900 is preferably electroporation; the conditions of electroporation are preferably: 2000V, 25 ⁇ F and 200 ⁇ .
  • the present invention uses PNZ8149 (lactic acid bacteria food-grade expression carrier) as the basic carrier, and the first fusion gene or the second fusion gene is inserted into the PNZ8149, and the first fusion gene includes signal peptide SPusp45, probiotic LEISS, and enterokinase in series.
  • the recognition site DDDDK and human alcohol dehydrogenase the second fusion gene includes sequentially tandem signal peptide SPusp45, probiotic LEISS, enterokinase recognition site DDDDK and human acetaldehyde dehydrogenase respectively fused.
  • the target enzyme in the intestinal tract under the action of endogenous enterokinase in the intestinal tract separates the signal peptide sequence SPusp45 and LEISS sequence from the fusion protein, and releases free alcohol dehydrogenase or acetaldehyde dehydrogenase at the same time.
  • the ratio of the effective number of viable bacteria of the first recombinant Lactococcus lactis and the second recombinant Lactococcus lactis It is (1-3): (1-3), preferably 1:1.
  • the present invention also provides a microcapsule containing the recombinant Lactococcus lactis described in the above scheme.
  • the first recombinant Lactococcus lactis or the second recombinant Lactococcus lactis is respectively embedded; the effective number of viable bacteria of the recombinant Lactococcus lactis in each microcapsule is preferably 1 ⁇ 5*10 9 cfu.
  • the present invention also provides the application of the recombinant Lactococcus lactis described in the above scheme or the microcapsules in the preparation of food, health products or medicines for preventing drunkenness;
  • the recombinant Lactococcus lactis includes the first recombinant Lactococcus lactis, or , the recombinant Lactococcus lactis comprises a first recombinant Lactococcus lactis and a second recombinant Lactococcus lactis.
  • the prevention of drunkenness includes reducing alcohol absorption and/or prolonging alcohol tolerance time.
  • the present invention also provides the application of the recombinant Lactococcus lactis or the microcapsules described in the above scheme in the preparation of food, health products or medicines for hangover.
  • the hangover includes shortening the recovery time after drinking and/or reducing the acute injury of the liver and/or intestinal tract caused by excessive drinking and/or alleviating the vomiting and headache after drinking.
  • the dosage form of the drug preferably includes an oral preparation; the oral preparation preferably includes microcapsules.
  • a plasmid pNZ8149 (Cat#VS-ELV00300-01) carrying a high-efficiency constitutive promoter (GapA promoter, patent CN111518801A) was selected as the vector ( Figure 1).
  • the human ADH1B gene and the human ALDH2 gene were cloned into multiple cloning sites, and the N-terminal was fused with the Usp45-LESS-EK sequence.
  • the present invention uses PNZ8149 (lactic acid bacteria food-grade expression carrier) as the basic carrier, and a fusion gene is inserted into PNZ8149, and the fusion gene includes signal peptide SPusp45, probiotic LEISS, enterokinase recognition site DDDDK and human ethanol deactivation gene in series. hydrogenase or acetaldehyde dehydrogenase, respectively.
  • the full sequence is chemically synthesized SphI-SPusp45-LEISS-DDDDK-ADH1B-XbaI, and SphI-SPusp45-ALDH2-DDDDK-ADH1B-XbaI.
  • the target enzyme in the intestinal tract under the action of endogenous enterokinase in the intestinal tract separates the signal peptide sequence SPusp45 and LEISS sequence from the fusion protein, and releases free alcohol dehydrogenase or acetaldehyde dehydrogenase at the same time.
  • the expression construct was transformed into Lactococcus cremoris NZ3900 (Cat#VS-ELS03900-01) by electroporation, and recombinant bacteria were screened on Elliker agar plates.
  • the electroconversion conditions were 2000V, 25 ⁇ F and 200 ⁇ .
  • forward 5'-catgccatggtcatgaaaaaaagattatcagct-3', shown in SEQ ID NO: 3
  • reverse 5'-gctctagatcaaaacgtcagacggtacg-3', shown in SEQ ID NO: 4
  • primers primers, PCR identification and sequencing of recombinant bacteria .
  • the recombinant Lactococcus lactis was resuspended (1%) in M17 liquid medium, incubated at 30°C for 8h, centrifuged at 4°C (4000g for 10min), and the supernatant was collected. Supernatant proteins were then precipitated by cryogenic ethanol precipitation. The same amount of protein samples were separated by electrophoresis on 10% sds-polyacrylamide gel, the protein was transferred to PVDF membrane, incubated with blocking buffer (5% skimmed milk) at room temperature for 1h, and treated with anti-hadh antibody and hALDH antibody (SantaCruz) Perform blotting. The results of specific antibody detection showed that our recombinant bacteria could express ADH enzyme (Fig. 2) and ALDH (Fig. 3) smoothly.
  • the two recombinant bacteria were cultured for 6-8 hours and collected by centrifugation (4000g, 4°C, 10min). Then microcapsule embedding is carried out on the bacteria: the recombinant bacteria are suspended in 3% (w/v) sodium alginate solution. The suspension was dropped into soybean oil containing 0.2% Tween-80 at a ratio of 1:5, and then stirred with a magnetic stirrer at 600 rpm for 10 minutes. Then slowly add 0.05M CaCl2 solution, stir (600rpm, 10min). Microcapsules were collected by post-centrifugation for 10 min (350 g at 4°C).
  • microcapsules Add the microcapsules to the lyoprotectant (19.5% maltodextrin and 2.5% skimmed milk powder by mass), resuspend and transfer to a petri dish, store at -80°C until completely frozen, and then transfer to A vacuum freeze dryer, the two kinds of bacteria were freeze-dried recombinant Lactococcus lactis microcapsules.
  • the microcapsules of Lactococcus lactis reconstituted by the two kinds of bacteria were dissolved in artificial gastric acid (0.2% sodium chloride, pH value 1.2), and stood still for 2 hours. Afterwards, add capsule breaking fluid (19:81NaH 2 PO 4 0.2 to 0.2Na 2 HPO 4 ) and use a rotator to break at 37°C for 30 minutes at a speed of 200 rpm, and the recombinant Lactococcus lactis is released, and the number of live bacteria is detected by culture plate counting ( Figure 4).
  • the time point when the righting reflex disappeared was defined as the intoxication point, and the time point between the first drinking and intoxication was defined as the alcohol tolerance time.
  • the alcohol tolerance time of the three groups of mice was less than 20 minutes. In order to exclude the influence of individual differences, we defined that the mice that did not lose the righting reflex within 1 hour were considered not to be acutely drunk. Considering the rate of drunkenness and safety, the dose of 6mg/g BW was selected for follow-up experiments (Table 1).
  • mice After drinking alcohol for 1 hour, the mice were placed in the motion detection system, and their motion was detected every 15 seconds. When the rat was drunk, the machine read 0. Mice were considered to have regained locomotor capacity when the number of locomotor movements at four consecutive recording time points was non-zero.
  • Hematoxylin and eosin (H&E) staining paraffin-embedded 4% paraformaldehyde-fixed tissues, stained 5 ⁇ m thick sections, and observed under a 10X or 20X objective lens.
  • the three groups of experimental mice were fed with ADH, ALDH recombinant Lactococcus lactis and control bacteria (transferred pNZ8149 empty Lactococcus lactis, replaced by pNZ), the results showed that the alcohol tolerance time of the recombinant Lactococcus lactis mice expressing hadh1b (ie, the time from drinking to loss of righting reflex) was significantly prolonged, while ALDH probiotics had no significant effect (Table 2, Figure 5).
  • mice were fed with ADH, ALDH recombinant Lactococcus lactis and control bacteria (transferred pNZ8149 empty Lactococcus lactis, replaced by pNZ), the results showed that the drunk rate of recombinant Lactococcus lactis mice expressing hadh1b decreased significantly , while ALDH probiotics had no significant effect (Table 3).

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Abstract

La présente invention concerne un Lactococcus lactis recombiné, une microcapsule et une utilisation associée, qui relèvent du domaine technique du génie génétique. Un premier gène de fusion ou un second gène de fusion est inséré dans le PNZ8149 en utilisant le PNZ8149 en tant que vecteur principal, le premier gène de fusion comprenant le peptide signal SPusp45, le probiotique LEISS, le site de reconnaissance de l'entérokinase DDDDK et l'éthanol déshydrogénase humaine connectés successivement dans la séquence, et le second gène de fusion comprenant le peptide signal SPusp45, le probiotique LEISS, le site de reconnaissance de l'entérokinase DDDDK et l'acétaldéhyde déshydrogénase humaine connectés successivement dans la séquence. Une enzyme cible est utilisée pour séparer la séquence du peptide signal SPusp45 et la séquence LEISS d'une protéine de fusion au moyen d'une digestion enzymatique sous l'action de l'entérokinase endogène dans l'intestin, et en même temps, l'éthanol déshydrogénase ou l'acétaldéhyde déshydrogénase libre est libérée, et l'éthanol déshydrogénase ou l'acétaldéhyde déshydrogénase libérée peut décomposer l'éthanol.
PCT/CN2022/073656 2022-01-25 2022-01-25 Lactococcus lactis recombiné, microcapsule et utilisation associée Ceased WO2023141744A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2019195592A1 (fr) * 2018-04-04 2019-10-10 The University Of Chicago Microbes génétiquement modifiés et leurs compositions
CN111500615A (zh) * 2020-03-20 2020-08-07 中国科学院动物研究所 一种表达ll-37多肽的重组表达载体、重组乳酸乳球菌、抗病毒药物及构建方法和应用
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CN112210519A (zh) * 2019-07-09 2021-01-12 深伦生物科技（深圳）有限公司 一种以食用菌分泌乙醛脱氢酶的基因工程菌

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Publication number Priority date Publication date Assignee Title
WO2019195592A1 (fr) * 2018-04-04 2019-10-10 The University Of Chicago Microbes génétiquement modifiés et leurs compositions
CN111518801A (zh) * 2019-04-11 2020-08-11 中国科学院动物研究所 一种组成型乳酸菌启动子、重组载体及其重组菌和应用
CN112175890A (zh) * 2019-07-02 2021-01-05 深伦生物科技（深圳）有限公司 一种以食用菌分泌乙醇脱氢酶的基因工程菌
CN112210519A (zh) * 2019-07-09 2021-01-12 深伦生物科技（深圳）有限公司 一种以食用菌分泌乙醛脱氢酶的基因工程菌
CN111500615A (zh) * 2020-03-20 2020-08-07 中国科学院动物研究所 一种表达ll-37多肽的重组表达载体、重组乳酸乳球菌、抗病毒药物及构建方法和应用

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Title
LYU YUNBIN, ZHONG LEI, LIU YANAN, LU JING, LAPOINTE GISÈLE, LU FENGXIA, LU ZHAOXIN: "Protective effects of Lactococcus lactis expressing alcohol dehydrogenase and acetaldehyde dehydrogenase on acute alcoholic liver injury in mice : Protective effects of Lactococcus lactis expressing alcohol", JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY, WILEY, HOBOKEN, USA, vol. 93, no. 5, 1 May 2018 (2018-05-01), Hoboken, USA, pages 1502 - 1510, XP093083189, ISSN: 0268-2575, DOI: 10.1002/jctb.5521 *
MA CHAO, ET AL: "Reconstruction of a Food-grade Expression Vector in Lactococcus lactis", ANHUI NONGYE KEXUE (JOURNAL OF ANHUI AGRICULTURAL SCIENCES), ANHUI SHENG NONGYE KEXUEYUAN, CN, vol. 38, no. 19, 31 December 2010 (2010-12-31), CN , pages 10247 - 10250, XP093083201, ISSN: 0517-6611, DOI: 10.13989/j.cnki.0517-6611.2010.19.058 *
ZHANG DANYU, CHEN YANG-YANG; LIU XIAO-YANG; WANG JIA-XING; ZHANG WEI-CAI; BU NING; XIONG XIANG-HUA: "Expression and Purification of Acetaldehyde Dehydrogenase in Lactococcus lactis", LETTERS IN BIOTECHNOLOGY, JUNSHI YIXUE KEXUEYUAN, CN, vol. 31, no. 1, 31 January 2020 (2020-01-31), CN , pages 39 - 43, XP093083193, ISSN: 1009-0002, DOI: 10.3969/j.issn.1009-0002.2020.01.008 *

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