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WO2022227187A1 - Biocapteur, son procédé de préparation et son utilisation - Google Patents

Biocapteur, son procédé de préparation et son utilisation Download PDF

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Publication number
WO2022227187A1
WO2022227187A1 PCT/CN2021/096921 CN2021096921W WO2022227187A1 WO 2022227187 A1 WO2022227187 A1 WO 2022227187A1 CN 2021096921 W CN2021096921 W CN 2021096921W WO 2022227187 A1 WO2022227187 A1 WO 2022227187A1
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WIPO (PCT)
Prior art keywords
heme
biosensor
gene
nucleoprotein
subunit
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Ceased
Application number
PCT/CN2021/096921
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English (en)
Chinese (zh)
Inventor
周明
付卫雷
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangzhou Tebsun Bio Tech Development Co Ltd
Huazhong Agricultural University
Original Assignee
Guangzhou Tebsun Bio Tech Development Co Ltd
Huazhong Agricultural University
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Application filed by Guangzhou Tebsun Bio Tech Development Co Ltd, Huazhong Agricultural University filed Critical Guangzhou Tebsun Bio Tech Development Co Ltd
Priority to CN202180001959.5A priority Critical patent/CN113454205A/zh
Publication of WO2022227187A1 publication Critical patent/WO2022227187A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present application belongs to the technical field of genetic engineering, and relates to a biosensor and its preparation method and application, in particular to a heme-sensitive biosensor and its preparation method and application.
  • Microbes in the human body affect human health and can also be used to detect or treat certain diseases.
  • artificially modified microorganisms have been widely used in the diagnosis and treatment of diseases.
  • Synthetic biology is an efficient method of artificially modifying microorganisms. It mainly uses basic DNA and RNA elements to construct gene circuits that can respond to certain small molecules, and can output quantifiable signals, such as fluorescent signals, to confer
  • the new function of microbial engineering bacteria is to construct related gastrointestinal microorganisms into molecular sensors (ie related engineered bacteria), and then through oral administration of these molecular sensors, the related microbial engineered bacteria can colonize the host. Molecular sensors are able to respond to this disease and convert it into an output signal that enables disease detection.
  • Heme and globin combine to form hemoglobin, and hemoglobin is an important component in red blood cells. Therefore, heme can be used as a marker for blood testing. In Escherichia coli, heme synthesis starts from 5-aminolevulinic acid (5-ALA).
  • 5-aminolevulinic acid 5-ALA
  • the precursor of 5-ALA undergoes a three-step enzymatic reaction of HemB, HemC, and HemD in turn, and passes through an intermediate: porphyrinogen (porphobilinogen, PBG) and uroporphyrinogen precursor (pre-uroporphyrinogen), generate the first cyclotetrapyrrole uroporphyrinogen III (Uroporphyrinogen III, UROGEN), and then sequentially in HemE, HemF (or HemN), HemG and Under the continuous catalytic reaction of HemH enzyme, uroporphyrinogen III passes through intermediates: coproporphyrinogen III (COPROGEN), protoporphyrinogen IX (protoporphyrinogen IX, PROTOGEN) and protoporphyrin IX (protoporphyrin IX, PROTO) , converted to heme, wherein coproporphyrinogen III can be catalyzed by either an oxygen-dependent oxid
  • the present application provides a biosensor, a preparation method and application thereof, in the presence of heme, the biosensor can emit far-red light FR or near-infrared light NIR after being excited by a light source of a specific wavelength, and has high specificity and sensitivity, thus enabling the detection of heme as well as blood.
  • the present application provides a biosensor comprising engineered cells simultaneously expressing heme oxidase-1, phycobilisome nucleoprotein subunit ApcF2 and heme transmembrane transporter.
  • the heme transmembrane transporter can transfer heme into cells, and the heme oxidase-1 (Ho1) oxidizes heme to biliverdin (BV).
  • Ho1 heme oxidase-1
  • the phycobilisome nucleoprotein subunit ApcF2 can autocatalyze covalently binding biliverdin based on an enzymatic reaction. After binding, it can emit far-red light FR or near-infrared light NIR after being excited by a light source of a specific wavelength, thereby realizing detection. heme.
  • the engineered cells are deficient in the heme synthase gene.
  • the heme synthase genes include hemB, hemC, hemD, hemE, hemF, hemG, hemH and hemN.
  • the heme synthase gene in the engineered cells is knocked out to weaken the heme synthesis of the engineered cells themselves, thereby reducing the background interference of heme in the engineered cells.
  • the phycobilisome nucleoprotein subunit ApcF2 is a group of fluorescent proteins derived from the algal species PCC7203 that can emit far-red light (about 670 nm) and near-infrared light (about 710 nm), including wild-type protein ApcF2 (such as SEQ ID NO: 1) and its mutated series proteins BDFP1.1, BDFP1.2, BDFP1.3, BDFP1.4, BDFP1.5, BDFP1.6, BDFP1.7, BDFP1.8 and BDFP1.9, amino acid sequence As shown in SEQ ID NOs: 2-10, respectively.
  • BDFP1.1, BDFP1.2, BDFP1.3, BDFP1.4, BDFP1.5, BDFP1.6, BDFP1.7, BDFP1.8 or BDFP1.9 All proteins can achieve similar effects in the biosensor of the present application.
  • the heme transmembrane transporter comprises the transmembrane transporter ChuA.
  • the engineered cells comprise engineered E. coli.
  • the engineered cells contain a gene encoding heme oxidase-1, a gene encoding a phycobilisome nucleoprotein subunit ApcF2, and a gene encoding a heme transmembrane transporter.
  • the heme oxidase-1 comprises the amino acid sequence shown in SEQ ID NO: 11.
  • the encoding gene of the heme transmembrane transporter comprises the nucleic acid sequence shown in SEQ ID NO: 12.
  • the biosensor comprises an engineered Escherichia coli containing the gene encoding heme oxidase-1, the gene encoding the phycobilisome nucleoprotein subunit ApcF2BDFP1.6 and the transmembrane transporter chuA
  • the coding gene of the heme synthase gene hemF is deleted, and the detection principle of the biosensor is shown in Figure 1.
  • the transmembrane transporter chuA can transfer heme (Heme) into the cell, and heme oxidase-1 ( Ho1) oxidizes heme to biliverdin (BV), and the phycobilisome nucleoprotein subunit ApcF2 (BDFP1.6) can autocatalyze covalent binding to biliverdin based on an enzymatic reaction. Excited, it can emit far-red light FR or near-infrared light NIR, thus realizing the detection of heme. In addition, knocking out hemF ( ⁇ hemF) can weaken the self-synthesized heme of the engineered cells, thereby reducing the background interference of heme in the engineered cells. .
  • the present application provides a preparation method of the biosensor according to the first aspect, the preparation method comprising:
  • gene knockout is performed using the ⁇ -red recombination system in this application.
  • the host cell comprises E. coli.
  • the method of introduction comprises any one of electrotransduction, viral vector system, non-viral vector system or direct gene injection.
  • the gene is introduced into the host cell by a plasmid vector in the present application.
  • the plasmid vector comprises pACYCDuet.
  • the present application provides a composition comprising the biosensor of the first aspect.
  • the composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, electronic circuit or diluent.
  • the present application provides the application of the biosensor of the first aspect or the composition of the third aspect in detecting heme and/or blood.
  • the biosensor of the present application can simultaneously express heme oxidase-1, phycobilisome nucleoprotein subunit ApcF2 and heme transmembrane transporter, and has low expression of heme, which can effectively respond to extracellular heme and generate fluorescence , and its own heme background fluorescence is low, which can effectively perform heme and/or blood detection;
  • the biosensor of the present application is simple in preparation, low in cost, and can effectively detect hemorrhage in the body, and has a broad development prospect.
  • FIG. 1 is a schematic diagram of the biosensor of the present application.
  • Fig. 2 is the fluorescence intensity diagram of the biosensor and blood reaction of the application, the error bar indicates that the standard deviation SD comes from the statistics of 3 independent samples;
  • Figure 3 is a graph of the fluorescence intensity of the biosensor and heme reaction of the application, and the error bars indicate that the standard deviation SD comes from 3 independent sample data statistics;
  • FIG. 4 is a graph of the fluorescence intensity of the biosensor and buffer reaction of the application, and the error bar indicates that the standard deviation SD comes from 3 independent sample data statistics;
  • Figure 5 is a graph of the fluorescence intensity of the reaction between the biosensor of the application and biliverdin, and the error bars indicate that the standard deviation SD comes from 3 independent sample data statistics;
  • Fig. 6 is a picture of No. 1 culture solution in Test Example 1;
  • Fig. 7 is the picture of No. 2 culture solution in Test Example 1;
  • Fig. 8 is a picture of No. 3 culture solution in Test Example 1;
  • Fig. 9 is the picture of No. 4 culture solution in Test Example 1;
  • Fig. 10 is the picture of No. 5 culture solution in Test Example 1;
  • Figure 11 is a graph of fluorescence intensity of different engineered cells
  • Figure 12 is a dose-response curve diagram of the fluorescence intensity of the biosensor and blood
  • Figure 13 is a dose-response curve diagram of the fluorescence intensity of the biosensor and heme
  • Figure 14 is a flow chart of the zebrafish in vivo test
  • Figure 15 is an imaging diagram of zebrafish
  • Figure 16 is a graph of zebrafish fluorescence intensity.
  • the recombinant plasmid was constructed in this example, and the gene experiment operation was carried out according to "Molecular Cloning", and the primer sequences are shown in Table 1.
  • the gene sequence encoding the transmembrane transporter chuA (SEQ ID NO: 12) was synthesized by Wuhan Tianyi Huiyuan Biotechnology Co., Ltd., and inserted into the plasmid pACYCDuet (Novagen Company) through restriction enzyme sites NcoI and XhoI, The recombinant plasmid pACYCDuet-chuA was obtained, which contained chloramphenicol resistance.
  • Heme oxidase-1 was amplified from plasmids pACYCDuet-ho1 (containing the heme oxidase-1 gene ho1, the ho1 gene sequence was derived from all1897 (GeneBank), algal species PCC7120) and pET28a-bdfp1.6 (using primers) P3, P4) and the gene sequence of phycobilisome nucleoprotein subunit ApcF2 (using primers P5, P6), wherein, the amino acid sequence of heme oxidase-1 is SEQ ID NO: 11, phycobilisome nucleoprotein subunit ApcF2 The amino acid sequence is SEQ ID NO: 7, and then, the heme oxidase-1 gene ho1 is inserted into the first open reading frame (ORFs) of the vector plasmid pCDFDuet using restriction sites NcoI and PstI; phycobili The somatic nucleoprotein subunit ApcF2 gene bdfp1.6 was
  • Escherichia coli heme synthase gene hemF was knocked out.
  • the hemF gene sequence is as shown in SEQ ID NO:13
  • the hemF upstream region sequence is as shown in SEQ ID NO:14
  • the hemF downstream region sequence is shown in SEQ ID NO: 15
  • the kanamycin gene sequence is shown in SEQ ID NO: 16
  • the FRT site sequence is shown in SEQ ID NO: 17.
  • the plasmid pKD46 was transformed into E. coli BL21 with ampicillin resistance gene, ⁇ -red recombinase gene and arabinose promoter, and the transformed plasmid was cultured in LB medium containing ampicillin (50g/mL). When the OD 600 reaches 0.3, add 2% arabinose to induce E.
  • coli to express ⁇ -red recombinase, and then add hemF and its flanking region genes, kanamycin gene and FRT site
  • the gene fragment was transformed into the above-mentioned Escherichia coli containing ⁇ -red recombinase, and the LB plate containing kanamycin (30g/mL) was used to screen the successful knockout strain, and further verified by PCR experiment. After overnight culture, plasmid pKD46 was removed to obtain hemF knockout E. coli.
  • This embodiment provides a biosensor, the biosensor is an engineered Escherichia coli, and the engineered Escherichia coli contains the gene encoding heme oxidase-1 and the encoding gene of the phycobilisome nucleoprotein subunit ApcF2BDFP1.6 and the gene encoding the transmembrane transporter chuA, and deletion of the heme synthase gene hemF.
  • the recombinant plasmids pACYCDuet-chuA and pCDFDuet-ho1-bdfp1.6 prepared in Example 1 were introduced into the hemF knockout Escherichia coli prepared in Example 2 to obtain engineered Escherichia coli, and using kanamycin (30g/mL) , chloramphenicol (40g/mL) and streptomycin (50g/mL) in the TB medium to culture the engineered Escherichia coli, when the OD600 of Escherichia coli reaches 0.7, add 1mM IPTG to induce protein expression, at 16°C After induction for 15 h, ChuA, BDFP1.6 and HO1 were co-expressed. Using a 50 mL centrifuge tube, the cells were centrifuged at 16°C and 5000 ⁇ g for 5 min, and washed twice with distilled water to collect the cells.
  • This test example detects the affinity of the biosensor prepared in Example 3 to heme or blood.
  • Figure 2- Figure 5 Figure 2 and Figure 3 are the test results of adding blood and heme, respectively. Reaching a higher level, indicating that the biosensor prepared in this application can effectively respond to heme or blood and generate high-intensity fluorescence.
  • Figure 4 and Figure 5 are the experimental results of adding buffer and biliverdin, respectively. The fluorescence intensity is low and no The obvious change indicates that the biosensor prepared in this application has low background interference, so it can perform effective detection.
  • the biosensor prepared in Example 3 was titrated with different concentrations of heme or blood, specifically including: using kanamycin-containing
  • the biosensors were cultured in TB medium of chloramphenicol (30 g/mL), chloramphenicol (40 g/mL) and streptomycin (50 g/mL), respectively, and when the OD 600 of E.
  • the AB strain zebrafish (purchased from the Institute of Hydrobiology, Chinese Academy of Sciences) were selected for the experiment, and were raised at 28°C with a light cycle of 14 hours and a dark cycle of 10 hours.
  • the zebrafish were anesthetized with ethyl aminobenzoate methanesulfonic acid (MS-222, 100 mg/L), and then 4 ⁇ 10 6 CFU of the biosensor (engineered E.
  • Example 3 prepared in Example 3 was injected into the abdomen of the zebrafish with a microsyringe , 4 days later, 20 of them were randomly selected, and aspirin (20 mg/mL) was injected into the abdomen in the same way, and the remaining 20 were injected with 10 ⁇ L of sterile water as a control.
  • Aspirin (aspirin) is an inflammatory drug. Can cause gastrointestinal bleeding (considering the insolubility of aspirin in water, first grind aspirin into powder, and then prepare a suspension).
  • the zebrafish was imaged using a device QPIX420 (Molecular Devices Company) with an excitation wavelength of 628 nm and an emission wavelength of 692 nm. OriginLab), during the experiment, when calculating the average fluorescence intensity of engineered Escherichia coli, each experiment was repeated 3 times to ensure that the P value was ⁇ 0.05 to reach the significance level.
  • the biosensor of the present application can efficiently react with heme, and has high specificity.
  • its own background fluorescence is low, so it can effectively detect heme, and can effectively detect internal bleeding.
  • the present application illustrates the detailed method of the present application through the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed method, which does not mean that the present application must rely on the above-mentioned detailed method for implementation.
  • Those skilled in the art should understand that any improvement to the application, the equivalent replacement of each raw material of the product of the application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the application.

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Abstract

La présente invention concerne un biocapteur, ainsi que son procédé de préparation et son utilisation. Le biocapteur comprend une cellule modifiée exprimant simultanément une hème oxygénase-1, une sous-unité de nucléoprotéine de phycobilisome ApcF2 et un transporteur transmembranaire d'hème. Le biocapteur de la présente application peut exprimer simultanément l'hème oxygénase-1, la sous-unité nucléoprotéique ApcF2 du phycobilisome et le transporteur transmembranaire de l'hème, est faible quant à l'expression de l'hème, peut répondre efficacement à l'hème extracellulaire et générer une fluorescence, est faible quant à sa propre fluorescence de fond de l'hème, et peut détecter efficacement l'hème et/ou le sang.
PCT/CN2021/096921 2021-04-30 2021-05-28 Biocapteur, son procédé de préparation et son utilisation Ceased WO2022227187A1 (fr)

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CN202180001959.5A CN113454205A (zh) 2021-04-30 2021-05-28 一种生物传感器及其制备方法和应用

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170058282A1 (en) * 2015-07-09 2017-03-02 Massachusetts Institute Of Technology Genetically engineered sensors for in vivo detection of bleeding
CN110684789A (zh) * 2019-10-24 2020-01-14 南京林业大学 融合基因、重组载体及其制备方法、镉离子全细胞生物传感器及其制备方法与应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170058282A1 (en) * 2015-07-09 2017-03-02 Massachusetts Institute Of Technology Genetically engineered sensors for in vivo detection of bleeding
CN110684789A (zh) * 2019-10-24 2020-01-14 南京林业大学 融合基因、重组载体及其制备方法、镉离子全细胞生物传感器及其制备方法与应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DING WENLONG: "The Molecular Evolution of Near-infrared Fluorescence Proteins Based on the Study of Phycobiliproteins and Lyases", CHINESE DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, no. 1, 1 December 2017 (2017-12-01), pages 1 - 183, XP055982392 *

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