A single-chain antibody 8H1 targeting binding to mobile polymyxin resistance protein and application thereof

Abstract

The application discloses a single-chain antibody 8H1 capable of targeting and combining with a movable polymyxin drug resistance protein and an application thereof. An amino acid sequence of the single-chain antibody 8H1 is shown in SEQ ID NO. 2, the movable polymyxin drug resistance protein is MCR-1 protein and / or MCR-2 protein, the single-chain antibody 8H1 can be specifically combined with a functional catalytic region of the movable polymyxin drug resistance protein, thereby inhibiting the function thereof; the single-chain antibody 8H1, a coding gene thereof and an expression vector containing the coding gene can be used for preparing a detection reagent of the movable polymyxin drug resistance protein or a recombinant bacterium with inhibited polymyxin drug resistance, the recombinant bacterium is a gram-negative bacterium and can express the single-chain antibody 8H1. The single-chain antibody 8H1 provided by the application can efficiently target the polymyxin drug resistance protein, block modification of a gram-negative enterobacteriaceae outer membrane lipid A by the polymyxin drug resistance protein and further reverse the drug resistance of the bacterium to polymyxin.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a single-chain antibody 8H1 that targets and binds to a mobile polymyxin resistance protein and its applications. Background Technology

[0002] Polymyxins are a class of polypeptide antibiotics produced by *Paenibacillus polymyxa*, effective against Gram-negative bacteria. The basic structure of polymyxins consists of a polypeptide ring and a polypeptide side chain, with the amino terminus of the side chain acylated by a fatty acid. Due to their positive charge, hydrophobic amino acid composition, and fatty acyl chain, polymyxins are both hydrophilic and lipophilic. Polymyxins kill bacteria primarily through electrostatic interactions; their positive charge interacts with the negative charge on the lipid A of Gram-negative bacteria, disrupting the outer membrane potential balance and increasing membrane permeability. Polymyxins then cross the outer membrane due to this osmotic imbalance, damaging the inner membrane and ultimately killing the bacteria. Polymyxin B and polymyxin E are mainly used to treat Gram-negative bacterial infections, especially those caused by carbapenem-resistant Gram-negative bacilli.

[0003] However, some Gram-negative bacteria develop resistance by modifying Lipid A, altering the amount of negative charge on their outer membrane to reduce electrostatic interactions with polymyxins. The sources of polymyxin resistance in Gram-negative bacteria mainly include two types: intrinsic resistance and acquired resistance. Intrinsic resistance is caused by mutations in certain genes on the bacterial chromosome, leading to chemical modifications of Lipid A on the outer membrane. Compared to intrinsic resistance, acquired resistance poses a greater threat. This resistance can spread rapidly through multiple pathways, leading to the rapid proliferation of resistant strains. This horizontal spread between different bacteria, and the genes that cause bacterial resistance to polymyxins, are called mobile polymyxin resistance genes (mcr).

[0004] Since the discovery of the plasmid-mediated polymyxin resistance gene mcr-1, the mcr family has evolved into 11 variant genes. The mcr gene family encodes a phosphatidylethanolamine-N-methyltransferase (PEA-N-MTase), which can acquire a phosphate ethanolamine group (pEtN) from phosphatidylethanolamine (PE) on the host cell membrane and transfer it to the bacterial Lipid A, thereby altering the charge properties of the outer membrane of Gram-negative bacteria, preventing polymyxin from binding to the cell membrane and thus inducing resistance.

[0005] Several approaches exist to address polymyxin resistance, including CRISPR / Cas system modification, novel antibiotic development, and the application of novel antibiotic adjuvants. Among these, selecting small molecule compounds to inhibit MCR-1 protein function is relatively common. However, small molecule compounds have poor specificity and, besides inhibiting MCR-1 protein function, may also produce unknown side effects. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a single-chain antibody (Single-Chain Variable Fragment, scFv) 8H1 that targets and binds to mobile polymyxin resistance proteins and its applications.

[0007] The technical solution adopted in this invention is:

[0008] I. A single-chain antibody 8H1 that targets and binds to a mobile polymyxin resistance protein

[0009] The amino acid sequence of the single-chain antibody 8H1 is shown in SEQ ID NO.2.

[0010] The mobile polymyxin resistance protein is MCR-1 protein and / or MCR-2 protein.

[0011] The single-chain antibody 8H1 can specifically bind to the functional catalytic region of the mobile polymyxin resistance protein, thereby inhibiting the function of the mobile polymyxin resistance protein.

[0012] II. A biomaterial

[0013] The biomaterial includes at least one of the following biomaterials:

[0014] a) A polynucleotide used to express the single-chain antibody 8H1, the nucleotide sequence of which is shown in SEQ ID NO.1;

[0015] b) An expression vector containing the polynucleotide.

[0016] III. Application of the single-chain antibody 8H1 or the biomaterial described herein

[0017] Both the single-chain antibody 8H1 and the biomaterial can be used to prepare detection reagents for mobile polymyxin resistance proteins or to prepare recombinant bacteria with inhibited polymyxin resistance.

[0018] IV. A recombinant bacterium

[0019] The recombinant bacteria comprise the biological material, and the recombinant bacteria are Gram-negative bacteria.

[0020] More specifically, the recombinant bacteria are either Escherichia or Klebsiella.

[0021] V. A method for inhibiting bacterial polymyxin resistance

[0022] The method includes:

[0023] Step S1) Construct an expression vector; the expression vector contains a polynucleotide capable of expressing the single-chain antibody 8H1 and an expression regulatory element operatively linked to the polynucleotide that is active in bacteria.

[0024] Step S2) The expression vector is introduced into host bacteria using electroporation technology to obtain recombinant bacteria.

[0025] Step S3) The polynucleotide is expressed in recombinant bacteria to form single-chain antibody 8H1. Single-chain antibody 8H1 can specifically bind to the functional catalytic region of the mobile polymyxin resistance protein, thereby inhibiting the function of the mobile polymyxin resistance protein and thus inhibiting the polymyxin resistance of the recombinant bacteria.

[0026] The beneficial effects of this invention are as follows:

[0027] The single-chain antibody 8H1 provided by this invention can efficiently target polymyxin resistance protein (MCR-1 / 2), block the modification of outer membrane lipid A (Lipid A) of Gram-negative Enterobacteriaceae by MCR-1 / 2, and thus reverse the bacterial resistance to polymyxin. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the 8H1 antibody expression and extraction process according to an embodiment of the present invention.

[0029] Figure 2 This is a graph showing the affinity results between the single-chain antibody 8H1 and the MCR-1 protein in Example 1 of the present invention.

[0030] Figure 3 This is a fluorescence co-localization result of single-chain antibody 8H1 and MCR-1 protein in bacteria in Example 1 of the present invention.

[0031] Figure 4 This is a graph showing the resistance results of the engineered strain obtained from Escherichia coli SHuffle strain as the background strain to polymyxin MCR-1 protein in Example 1 of the present invention; where A is the negative control group, B is the positive control group, and C is the experimental group.

[0032] Figure 5This is a graph showing the resistance results of the engineered strain obtained in Example 1 of the present invention, using Klebsiella pneumoniae ATCC 43816 strain as the background strain, to polymyxin MCR-1 protein; where A is the negative control group, B is the positive control group, and C is the experimental group.

[0033] Figure 6 This is a graph showing the resistance results of the engineered strain obtained using Escherichia coli SHuffle strain as the background strain to polymyxin MCR-2 protein in Example 2 of the present invention; where A is the negative control group, B is the positive control group, and C is the experimental group.

[0034] Figure 7 This is a schematic diagram illustrating the binding of the single-chain antibody 8H1 of the present invention to the MCR-1 protein; wherein, A is a surface view and B is a cartoon image. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0036] This invention provides a single-chain antibody 8H1 that targets and binds to a mobile polymyxin resistance protein. The amino acid sequence of the single-chain antibody 8H1 is shown in SEQ ID NO.2.

[0037] The single-chain antibody 8H1 comprises a single light chain and a heavy chain variable region linked by a hinge region. The preferred amino acid sequence of the hinge region is shown in SEQ ID NO.19.

[0038] Among them, the mobile polymyxin resistance proteins are MCR-1 protein and / or MCR-2 protein. The PBD numbers for MCR-1 protein and MCR-2 protein are 5ZJV and 5MX9, respectively.

[0039] Among them, the single-chain antibody 8H1 can specifically bind to the functional catalytic region of mobile polymyxin resistance protein, thereby inhibiting the function of mobile polymyxin resistance protein.

[0040] The present invention also provides a biomaterial, comprising at least one of the following biomaterials:

[0041] a) Polynucleotides used to express the single-chain antibody 8H1;

[0042] b) An expression vector containing a polynucleotide, the expression vector further comprising an expression regulatory element operatively linked to the polynucleotide and active in bacteria, the expression regulatory element being used to express the polynucleotide within the engineered strain.

[0043] The single-chain antibody 8H1 and biomaterials provided by this invention can be used to prepare detection reagents for mobile polymyxin resistance proteins or to prepare recombinant bacteria with inhibited polymyxin resistance.

[0044] Preferably, the recombinant bacteria are Gram-negative bacteria.

[0045] Preferably, the recombinant bacteria are Escherichia coli or Klebsiella spp.

[0046] The present invention also provides a method for inhibiting bacterial polymyxin resistance, comprising:

[0047] Step S1) Construct an expression vector; the expression vector contains a polynucleotide capable of expressing the single-chain antibody 8H1 and an expression regulatory element operatively linked to the polynucleotide that is active in bacteria.

[0048] Step S2) Using electroporation technology, the expression vector is introduced into host bacteria, and recombinant bacteria are obtained after screening.

[0049] Step S3) enables the expression of polynucleotides in recombinant bacteria, thereby inhibiting polymyxin resistance in recombinant bacteria.

[0050] Since the primary structural similarity between MCR-1 and MCR-2 exceeds 80%, the inhibitory effect of 8H1 on the functions of MCR-1 and MCR-2 was verified simultaneously. The results showed that 8H1 was effective in inhibiting both MCR-1 and MCR-2.

[0051] Specific embodiments of the present invention are as follows:

[0052] Example 1

[0053] In this embodiment, firstly, inclusion body protein of the single-chain antibody 8H1 was prepared in large quantities using the *E. coli* BL21 expression system. The inclusion body protein was then refolded using a redox method and purified multiple times using an AKTA separation system to finally obtain the active single-chain antibody protein. Its affinity was subsequently evaluated, and the results are as follows: Figure 2As shown. Molecular cloning technology was used to ligate the mcr-1 gene and its natural promoter (sequence shown in SEQ ID NO. 18), and a Strep tag was inserted at the C-terminus of the mcr-1 gene. The low-copy plasmid pWSK129 was selected as the expression vector for the mcr-1 gene. After ligation, a 7133 bp pWSK129-mcr-1 plasmid was obtained, carrying a kanamycin sulfate resistance gene. The MCR-1 protein is located in the bacterial inner membrane, but its functional catalytic region faces the bacterial periplasmic space. To enable the transport of single-chain antibodies into the bacterial periplasmic space and to bind to and inhibit the function of the MCR-1 protein within the periplasmic space, the scFv gene was fused using the pelB signal peptide and ligated into the pSGKM plasmid. After ligation with a single-chain antibody, a 5449 bp pSGKM-8H1 plasmid was obtained, and the plasmid was modified to have rifampicin resistance. Subsequently, the pWSK129 empty vector plasmid (pWSK129-Empty), pWSK129-mcr-1 plasmid, pSGKM empty vector plasmid (pSGKM-Empty), and pSGKM-8H1 plasmid were introduced into the background strain in various combinations. The resistance values ​​of the resulting strains to polymyxin were determined using the E-test (Epsilometer Test) strip method. The results are shown in [Figure 1]. Figure 4 and Figure 5 .

[0054] In this embodiment, commercially available *Escherichia coli* SHuffle strain and *Klebsiella pneumoniae* ATCC 43816 were selected as background strains (abbreviated as SHuffle and KP43816 in the attached figures, respectively). Furthermore, in this embodiment, the pWSK129 empty vector plasmid and the pSGKM empty vector plasmid were introduced into the background strains as negative controls. Figure 4 A and Figure 5 (A); pWSK129-mcr-1 plasmid and pSGKM empty vector plasmid were introduced into the background strain as positive controls ( Figure 4 B and Figure 5 (B); pWSK129-mcr-1 plasmid and pSGKM-8H1 plasmid were introduced into the background strain as the experimental group ( Figure 4 C and Figure 5 (C).

[0055] like Figure 1 As shown, the specific steps of this embodiment are as follows:

[0056] 1. Expression and purification of single-chain antibodies

[0057] 1.1 Resuscitation of expression strains

[0058] The standard *E. coli* protein expression strain (BL21) was removed from a -80℃ freezer. After transforming the single-chain antibody 8H1 gene into *E. coli*, an antibody expression strain (BL21 carrying pET21a-8H1) was successfully constructed. Single colonies were picked and streaked in three zones on LB agar plates containing the corresponding antibiotic, and then inverted and incubated overnight at 37℃. The next day, single colonies from the LB agar plates were picked and transferred to 500 μL of LB liquid medium containing the corresponding antibiotic. The plates were then placed in a shaker at 37℃ and 220 rpm for overnight amplification and a second activation to obtain the seed culture.

[0059] 1.2. Amplification and culture of expression strains and induction of expression by single-chain antibody

[0060] Further large-volume amplification culture induced the expression of the target protein in *E. coli* BL21, obtaining a large amount of protein samples. The seed culture obtained in the previous step was transferred to 50 mL LB liquid medium containing the corresponding antibiotic for further amplification (culture conditions: 37℃, 220 rpm) until OD. 600 The value was approximately 0.5. The amplified bacterial culture was then added to 1 L of LB liquid medium at a transfer ratio of 1:100, along with 1 mL of the corresponding antibiotic. The medium was then incubated at 37°C and 180 rpm for 4 hours to further amplify the culture until the OD value reached 0.5. 600 The value was approximately 0.5. IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 1 mM, and induction continued for 4 hours at 37°C and 180 rpm. After the induction program was complete, the cells were centrifuged at 4°C and 6000 rpm for 5 minutes. The supernatant was discarded, and the precipitated bacterial cells were collected. The cells were resuspended in 1×PBS and washed, then transferred to 50 mL centrifuge tubes. The tubes were centrifuged at 4200 rpm for 10 minutes and washed. This step was repeated twice to completely remove the culture medium. Finally, the tubes were stored at -20°C.

[0061] 1.3 High-pressure disruption of bacteria

[0062] Large-volume bacterial cell lysis was performed using a high-pressure homogenizer. The bacterial cell membrane was lysed at 1000 MPa, ultimately releasing the target protein into 1×PBS solution (1×PBS, pH 7.4, 1000 mL formulation: NaCl 8 g, KCl 0.2 g, Na₂HPO₄ 1.44 g, KH₂PO₄ 0.24 g). The bacterial cells were resuspended in an appropriate amount of 1×PBS (5 mL of cells per 40 mL of 1×PBS), and thoroughly stirred and vortexed with a glass rod to form a homogenous suspension. The high-pressure homogenizer was set to 1000 MPa and 4°C. After adding the bacterial suspension to the homogenizer, it was homogenized 4-5 times under high pressure, observing the suspension gradually change from an initially viscous state to a non-viscous state. The suspension was then centrifuged at 4°C and 13000 rpm for 30 minutes. After removing the supernatant, the precipitate contained single-chain antibody proteins in inclusion body state and some unlysed bacterial cells.

[0063] 1.4 Inclusion body protein washing

[0064] 1.4.1 Preparation of Inclusion Body Washing Reagent

[0065] Washing buffer: 0.5% Triton-100 (v / v), 50 mM Tris pH 8.0, 300 mM NaCl, 10 mM EDTA, 10 mM DTT (add fresh before use);

[0066] Resuspension buffer: 50 mM Tris pH 8.0, 100 mM NaCl, 10 mM EDTA, 10 mM DTT (add fresh before use);

[0067] Dissolution buffer: 6 M Gua-HCl or 8 M Urea, 10% (v / v) glycerol, 50 mM Tris pH 8.0, 100 mM NaCl, 10 mM EDTA;

[0068] Protein buffer: 20 mM Tris pH 8.0, 300 mM NaCl.

[0069] 1.4.2 Preparation of SDS-PAGE protein gel

[0070] First, select a 1.5 mm thick glass plate, clamp it in the gel casting tank, add water and wait 15 minutes to check the seal. Add 7.5 mL of 12% (v / v) SDS-PAGE separating gel to each glass plate (separating gel 15 mL system formula: ddH2O 4.9 mL, acrylamide / methylenebisacrylamide 30% (v / v) solution 6 mL, 1.5 M Tris-HCl, pH 8.8 3.8 mL, 10% (v / v) SDS 150 μL, 10% (v / v) ammonium persulfate 150 μL, tetramethylethylenediamine 6 μL). Slowly add ddH2O from one side, flatten to remove air bubbles, let stand for 30 minutes to solidify, pour off the ddH2O, and invert for 10 minutes to air dry. Then add 3 mL of stacking gel (the 6 mL stacking gel system formula is: 4.1 mL ddH2O, 1 mL acrylamide / methylenebisacrylamide 30% (v / v) solution, 750 μL 1.5 M Tris-HCl, pH 6.8, 60 μL 10% (v / v) SDS, 60 μL 10% (v / v) ammonium persulfate, and 6 μL tetramethylethylenediamine) and slowly insert it at an angle into a 10-hole 1.5 mm comb. Let it stand for another 20 minutes until the stacking gel solidifies, then remove the comb and set aside.

[0071] 1.4.3 Inclusion body washing

[0072] First, it was observed that inclusion body proteins appeared pure white at the bottom of the precipitate, while bacterial debris and unlysed cells appeared pale yellow on top. The bacterial debris (unlysed cells) covering the inclusion bodies was gently scraped off with a metal spoon, while simultaneously rinsing the surface of the inclusion bodies with ddH2O to remove bacterial debris. Next, the inclusion bodies were resuspended in 30 mL of washing buffer by pipetting, then vortexed thoroughly, and centrifuged at 12000 rpm, 4°C for 20 minutes, discarding the supernatant. This step was repeated three times. The inclusion bodies were then resuspended in resuspension buffer. 80 μL of the resuspended sample was taken and mixed with SDS protein loading buffer (formulation: 60% glycerol, 2% SDS, 10% β-mercaptoethanol, 0.5 M Tris-HCl, and 0.01% bromophenol blue) at a molar ratio of 1:5. The mixture was then heated in a 100°C metal bath for 10 minutes, and the purity of the inclusion body proteins was determined by 12% (v / v) SDS-PAGE gel electrophoresis (140 V, 50 minutes). Weigh a clean centrifuge tube using an analytical balance and record the weight (accurate to milligrams). Pour the sample into the clean centrifuge tube and centrifuge again at 4°C and 12,000 rpm for 20 minutes. Discard the supernatant, wipe the tube wall dry with absorbent paper, and invert it onto absorbent paper. After 10 minutes, weigh and record the weight. Add dissolution buffer at 30 mg / mL, and slowly scrape the inclusion body precipitate with a metal spoon until the precipitate is completely suspended in the solution. Finally, invert the tube at 4°C until the inclusion body protein is completely dissolved (approximately 8 hours). Remove the dissolved sample and centrifuge again at 4°C and 12,000 rpm for 20 minutes. Pour the dissolved inclusion body protein (supernatant) into a new EP tube and store at -20°C for later use.

[0073] 1.5 Inclusion body protein refolding

[0074] 1.5.1 Preparation of inclusion body protein refolding reagent

[0075] Each time you refold, you need to prepare a fresh 2 L volume of refolding buffer and pre-cool it on ice for 30 minutes before use.

[0076] Preparation of refolding buffer: Add 100 mM Tris pH 8.0, 400 mM L-Arg HCl, 2 mM EDTA in sequence, add an appropriate volume of ddH₂O (about 80% of the total volume of the solution to be prepared), stir on a magnetic stirrer until the L-arginine hydrochloride powder is completely dissolved, add a small amount of sodium hydroxide to adjust the pH value to 8.0, and add ddH₂O again to make up the constant volume. Subsequently, clean the Buchner flask for suction filtration with ddH₂O, perform suction filtration once using a 0.45 nm filter membrane, then replace it with a 0.22 nm filter membrane for filtration again. Cover the mouth of the beaker with plastic wrap, and place it on ice for an ice bath for 30 minutes. Place it on a magnetic stirrer, then add oxidized glutathione (final concentration: 5 mM GSSG), reduced glutathione (final concentration: 0.5 mM GSH) and phenylmethylsulfonyl fluoride (final concentration: 0.5 mM PMSF) respectively. Keep stirring for 20 minutes until the glutathione is completely dissolved for later use.

[0077] 1.5.2. Refolding and Concentration of Inclusion Bodies

[0078] First, use a 5 mL syringe to draw 1-2 mL of the dissolved inclusion body protein solution, then draw 3-4 mL of refolding buffer and mix thoroughly. Next, cover the mouth of the beaker with aluminum foil and fix the syringe on the foil, allowing the inclusion body protein solution to slowly drip into the refolding buffer. Add a stir bar to the beaker and adjust the speed to slowly stir the refolding solution, maintaining a temperature of 4°C throughout the refolding process. After 12 hours, begin concentrating the sample and changing the buffer. Take one concentrating ultrafiltration membrane (10 kDa) and soak it in a small amount of refolding buffer for 5 minutes. Unscrew the bottom of the concentration cup, remove the O-ring, add a small amount of refolding buffer to cover the bottom groove, gently pinch the ultrafiltration membrane and slowly flatten it into the base (smooth side up), put the O-ring back and gently press it flat, and finally tighten the base to the cup body. Pour the refolded protein solution into the concentration cup, add a stir bar, and tighten the cap clockwise. Place the magnetic stirrer on the tank, connect the nitrogen blow-off tube, and turn on the stirrer (Note: a. Observe whether the rotor makes a sound, eliminating the possibility of friction between the bottom filter membrane and the cup wall; b. Use stirring speed level 4 to avoid creating a vortex). Open the main valve of the nitrogen cylinder and observe whether there is nitrogen in the cylinder; open the flow rate valve and observe the pressure (it is recommended to control the pressure at 0.1~0.2 MPa; note: the maximum pressure in the concentration cup should not exceed 0.4 MPa; the pressure of the 10 kDa ultrafiltration membrane should not exceed 0.2 MPa). First open the switch at the connecting tube, then open the nitrogen switch in the chromatography cabinet, and observe whether the waste liquid drips slowly. After use, first close the main nitrogen valve, allowing the excess nitrogen in the pipeline to continue concentrating the protein. Wait until the waste liquid stops dripping slowly, then close the flow rate valve, the connecting tube switch, and the nitrogen valve switch in the refrigerator in sequence. Turn off the magnetic stirrer, disconnect the nitrogen connecting tube, and unscrew the cap to pour out the protein. Subsequently, transfer the protein solution to the ultrafiltration concentration tube (10 kDa) and centrifuge for concentration (4℃, 4000 rpm). Meanwhile, add appropriate protein buffer and change the buffer three times. After the protein solution volume is concentrated to 1 mL, aspirate the protein sample and transfer it to a 1.5 mL EP tube. Centrifuge again at 12000 rpm for 10 minutes at 4℃. Remove the precipitate and retain the supernatant. Quickly freeze in liquid nitrogen and store at -80℃ for later use.

[0079] 1.6 Purification of single-chain antibody protein AKTA

[0080] Because single-chain antibodies may aggregate to varying degrees during renaturation, secondary purification using the AKTA pure protein purification system and a Superdex 75 10 / 300 GL column is necessary. Further separation is performed based on the protein aggregation state and molecular weight. First, AKTA buffer is prepared (formulation: final concentration 20 mM Tris pH 8.0, 300 mM NaCl, ddH2O to 1 L), filtered once through a 0.22 μm filter, and placed on ice for 30 minutes. The antibody is then further purified using gel affinity chromatography. The purity of the antibody protein at different peak positions is identified by 12% (v / v) SDS-PAGE. The monomeric single-chain antibody peak is collected, concentrated to 800 μL using a 10 kDa ultrafiltration tube, and purified again using a gel affinity chromatography column (Superdex 75 10 / 300 GL). The concentration is then determined using Nanodrop. Finally, after concentration and measurement, the samples were dispensed into 1.5 mL EP tubes (100 μL per tube), labeled with the sample name and date, flash-frozen in liquid nitrogen, and stored at -80°C.

[0081] 2. Antibody affinity assessment: Microscale Thermophoresis (MST)

[0082] 2.1 Protein expression and purification

[0083] The expression and purification methods for the catalytic functional regions of single-chain antibody 8H1 and MCR-1 proteins are the same as before.

[0084] 2.2 Protein staining labeling

[0085] This embodiment uses a commercially available RED-NHS second-generation protein labeling kit, primarily suitable for preparing red light detectors NT.115 and NT.Automated. In this embodiment, cMCR-1 protein was selected as the target protein, and a single-chain antibody (8H1) was selected as the ligand.

[0086] ① The expressed and purified target protein and three ligand proteins were all subjected to medium replacement using 10 kDa ultrafiltration tubes (MST buffer: final concentration 20 mM HEPES pH8.0, 300 mM NaCl, 0.05% Tween-20). The concentration was measured after the medium replacement was completed for later use.

[0087] ② Add 25 μL of fresh DMSO solution to the RED-NHS second-generation dye and gently pipette several times to dissolve it completely (the solution will be dark blue; store at room temperature before use to prevent the DMSO solution from freezing). The final dye concentration is 600 μM. Take a sterile 1.5 mL EP tube, add 5 μL of freshly prepared RED-NHS second-generation dye, then pipette 5 μL of LMS buffer and mix with it. Gently pipette to mix thoroughly to obtain a dye solution with a final concentration of 300 μM.

[0088] ③ Take another sterilized 1.5 mL EP tube, dilute the target protein, and add 90 μL (approximately 20 μM concentration). Add the entire dye solution (300 μM concentration) from the previous step to the target protein, and gently pipette several times to mix thoroughly, obtaining 100 μL of dye-protein mixture. Incubate at room temperature in the dark for 30 minutes. (Extend incubation time to 60 minutes on ice)

[0089] ④ Equilibrate the B-column: Remove the top cap of the B-column and set it aside, allowing the liquid to drip out slowly. Then remove the bottom cap and set it aside as well. Replace the caps of the 15 mL centrifuge tubes with the adapter provided in the kit. Insert the B-column into the centrifuge tube from the top of the adapter. Fill the B-column with MST buffer to equilibrate it; the buffer will flow through the resin in the column under gravity. Discard the liquid collected in the tube. Repeat the B-column equilibration step three to five times.

[0090] ⑤ After the protein labeling step is completed, transfer 100 μL of the dye-protein labeling reaction solution to the equilibrated B-column. Once the dye-protein solution has completely entered the resin, add 550 μL of MST buffer to the B-column. The buffer will completely pass through the resin under gravity. Then prepare a new sterile 1.5 mL EP tube to collect the labeled protein sample. Place the B-column above the new 1.5 mL EP tube, add 500 μL of MST buffer, and collect the filtered liquid containing the labeled protein. Collection is complete when the liquid stops dripping. Measure the concentration using Nanodrop, convert it to molar concentration, record the concentration, and aliquot into 100 μL tubes. Quick-freeze in liquid nitrogen and store at -80°C for later use. Rinse the B-column three times each with MST buffer and ddH2O, then cap the top and bottom of the tube, return it to the kit, and store at 4°C for future use.

[0091] 2.3 MST Affinity Determination

[0092] ① Open the MO. Control software and select the red fluorescence channel (Nano-RED). Then open the sample chamber cover, remove the sample holder, and remove the fixing magnetic strip.

[0093] ② Fluorescence signal intensity measurement (Pretest): Select the Pretest step, enter the target protein name and concentration, select Monolith NT.115 Standard Treated Capillary (k002) for Capillary, set LED power to Auto by default, and MST power to Medium by default. Then, take an eight-tube PCR tube and add 20 μL of MST buffer to each well. Add 20 μL of labeled cMCR-1 protein to the first well, and mix thoroughly with a pipette. Add 20 μL to the second well and mix again, and so on. Reserve the last well (40 μL). Using two capillaries, aspirate the sample from well 8 and place it in positions 1-2 of the sample holder. After securing the capillaries with magnetic strips, gently place the sample holder into the instrument. Finally, use an NT.115 instrument to scan both samples with red light, ensuring the fluorescence signal is within the range of 200-800. Adjust the target protein concentration based on the fluorescence signal intensity and LED power (requirement: at 20% LED excitation intensity, the fluorescence signal should ideally be within the 200-800 range). Once the requirements are met, proceed to the next step. Record the current dilution gradient, and then prepare for the next binding assay.

[0094] ③ Binding Test: Select the second step, Binding Check, and enter the target protein name and concentration. For Capillary, select Monolith NT.115 Standard Treated Capillary (k002), LED power at 20%, and MST power at the default medium. Simultaneously, enter the ligand protein name and concentration. Take an eight-tube PCR tube, add 20 μL of an appropriate concentration of cMCR-1 protein to the first well, then add 20 μL of MST buffer. Mix thoroughly with a pipette. Add another 20 μL of an appropriate concentration of cMCR-1 protein to the second, third, and fourth wells, and then add 20 μL of the maximum concentration of ligand protein (8H1) to each well, mixing thoroughly with the cMCR-1 protein. Use four capillary tubes for each sample. Place the cMCR-1 protein alone in wells 1-4, and the cMCR-1 & scFv mixed sample in wells 5-8. Then click "Test". (Requirements: When the LED power is at 20% excitation intensity, the fluorescence signal should ideally be within the range of 200~800, with the high intensity difference between each sample not exceeding ±20%; the sample should have good homogeneity and no obvious aggregation; the signal-to-noise ratio should indicate a positive binding result.) If the requirements are met, proceed to the next step of binding analysis.

[0095] ④ Binding Affinity Analysis: Select the third step, Binding Affinity Analysis, and fill in the target protein and ligand protein names and concentrations. Select Monolith NT.115 Standard Treated Capillary (k002) for the Capillary, 20% for the LED power, and Medium for the MST power (default). Prepare two octet PCR tubes, labeled 1-16. Add 20 μL of MST buffer to tubes 2 through 16. Add 20 μL of ligand molecule (8H1) to tubes 1 and 2. Tube 1 contains the highest concentration of ligand in this example (the highest ligand concentration is more than 20 times its interaction Kd value). Take 20 μL from tube 2 and add it to tube 3, mixing thoroughly with a pipette. Then take 20 μL from tube 3 and add it to tube 4, mixing thoroughly. Repeat this process until tube 16. Finally, discard 20 μL of solution from tube 16. Then, add 20 μL of labeled cMCR-1 protein sample of appropriate concentration (the concentration should be the protein concentration corresponding to the optimal signal obtained in the Pretest step) to tubes 1 through 16, and mix thoroughly. Use a capillary tube to sequentially pipette samples from tubes 1 through 16 and place them at their respective numbered positions on the sample rack. Place the sample rack, close the chamber door, and begin detection. Observe whether the fluorescence in each tube is uniform (removing a maximum of four sites). After the test is completed, remove outliers and save the data.

[0096] Affinity results as Figure 2 and Figure 3 As shown, the affinity between 8H1 and MCR-1 proteins is in the nanomolar range, indicating a strong affinity between them.

[0097] 3. Functional validation of single-chain antibodies

[0098] 3.1 Primer Design

[0099] To simulate the native state of mcr-1 in E. coli, the pWSK129 low-copy plasmid was selected as the vector. Snapgene was used to ligate the mcr-1 promoter region and target gene to the pWSK129 plasmid, and primers were designed. A strep tag was inserted at the C-terminus of the mcr-1 gene as needed. The pSGKM plasmid was selected as the functional validation vector for the single-chain antibody. Snapgene was used to ligate scFv 8H1 and the pelB signal peptide to the pSGKM plasmid, and primers were designed. The primer names and sequences used are shown in Table 1.

[0100] Table 1 Primer names and sequences

[0101]

[0102] Primers pWSK129-F and pWSK129-R were used to amplify the pWSK129 plasmid backbone; primers pWSK129-Pmcr-1-F and pWSK129-Pmcr-1-R were used to amplify the mcr-1 promoter; primers pWSK129-mcr-1-F, pWSK129-mcr-1-R1 and pWSK129-mcr-1-R2 were used to amplify the mcr-1 gene and insert a STREP tag at the C-terminus of the gene; primers pSGKM-F and pSGKM-R were used to amplify the pSGKM plasmid backbone; primers pSGKM-pelB-F and pSGKM-pelB-8H1-R were used to amplify the pelB signal peptide; and primers pSGKM-8H1-F and pSGKM-8H1-R were used to amplify the 8H1 gene.

[0103] 3.2 Vector Gene Construction

[0104] 3.2.1. Carrier Preparation

[0105] The pWSK129 plasmid backbone was amplified by PCR, followed by DNA agarose gel electrophoresis at 1% (m / v) (voltage: 120 kV, time: 35 min). After electrophoresis, the target band was excised and recovered using a DNA gel purification kit. The pSGKM plasmid was linearized using the same method, and double digestion with commercially available Bsa I and EcoR I fast digestive enzymes was performed.

[0106] 3.2.2 Target gene fragment amplification and fusion

[0107] Step 1: The mcr-1 gene and its natural promoter region (300 bp) were amplified by PCR using a high-fidelity enzyme (PCR reaction program: pre-denaturation 95℃ for 5 minutes, denaturation 95℃ for 30 seconds, annealing 55℃ for 30 seconds, extension 72℃ for 15 seconds, amplification cycle setting 30). The target gene and purity were identified using a 1% (m / v) DNA agarose gel (voltage: 120 kV, time: 20 minutes). Subsequently, the PCR product was purified using a PCR product purification kit for later use.

[0108] Step 2: The purified mcr-1 gene and its natural promoter region fragment were subjected to fusion PCR again (PCR reaction program: ① Fragment fusion: 95℃ for 20 minutes, then incubated at room temperature for 30 minutes; ② Pre-denaturation: 95℃ for 5 minutes, denaturation: 95℃ for 30 seconds, annealing: 55℃ for 30 seconds, extension: 72℃ for 15 seconds, amplification cycle set to 30). DNA was then subjected to 1% (m / v) agarose gel electrophoresis (voltage: 120kV, time: 35 minutes). After electrophoresis, the target band was excised and recovered using a DNA gel purification kit for later use.

[0109] The pelB signal peptide and scFv(8H1) gene were fused in the same manner.

[0110] 3.2.3 Ligation of the vector with the target gene

[0111] Homologous recombination is used to connect the target gene and the corresponding plasmid vector.

[0112] 3.3 Observation of protein subcellular localization using laser scanning confocal microscopy (LSCM)

[0113] ① First, pick a single colony from an LB agar plate and inoculate it into 2 mL of LB liquid medium containing the corresponding antibiotic. Wrap the test tube with aluminum foil to protect it from light, and then place it in a shaker and incubate overnight at 37°C and 220 rpm to activate the strain.

[0114] ② The next day, 20 μL of bacterial culture was transferred from the test tube to 2 mL of LB liquid medium containing the corresponding antibiotic. The test tube was then wrapped with aluminum foil again and placed in a shaker at 37°C and 220 rpm for 4 hours to amplify the strain to the late logarithmic phase. The OD was then adjusted. 600 Approximately 0.5. Transfer 1 mL of bacterial culture to a 1.5 mL brown EP tube. Centrifuge at 6000 rpm for 5 minutes using a benchtop centrifuge, discard the supernatant, and resuspend the bacterial culture in 1 mL of 1×PBS. Centrifuge again and discard the supernatant. Repeat this step 2-3 times. Finally, resuspend the bacterial culture in 500 μL of 1×PBS for later use.

[0115] ③ Preparation of agarose slides: Weigh 0.5 g of agarose powder and mix thoroughly with 50 mL of 1×PBS (final concentration 1% agarose PBS solution). Autoclave at 121℃ for 15 minutes. After sterilization, observe whether the agarose powder is completely dissolved and allow it to cool slowly at room temperature. Then, aliquot the sterilized agarose solution into 1.5 mL EP tubes (1 mL per tube). Before each use, remove the tube and heat it in a metal bath at 100℃ until completely melted. Fix two coverslips with glue to create a spacer. Place the prepared spacer on either side of the slide, leaving a suitable sample loading area in the middle. Add 200 μL of agarose solution to the sample loading area, and immediately cover it with a single layer of coverslips, gently pressing down the agarose solution. Let it stand until the agarose solution solidifies (about 3 minutes). Dilute the bacterial culture in the EP tube from the previous step to a suitable gradient using 1×PBS solution. Remove the coverslip from the agarose gel tray, drop 10 μL of bacterial suspension onto the tray, cover with the coverslip (remove air bubbles as much as possible and avoid breaking the agarose gel tray), and let stand for 5 minutes to allow the bacterial suspension to dry and the bacteria to fix onto the agarose gel tray. Finally, label the sample with the name and dilution gradient, place it in the sample box, and await observation under a laser confocal microscope.

[0116] ④ Observation of subcellular protein localization under laser confocal microscopy: First, turn on the exciter, mercury lamp, control console, and computer control software of the confocal microscope in sequence according to the operation manual. Switch the objective to 100x oil immersion, then wipe the lens with a small amount of anhydrous ethanol using lens paper (repeat this step 3 times) and wait for the anhydrous ethanol to evaporate completely. Take out the sample slide and add a small amount of cedarwood oil to the coverslip. Then, invert it and fix it on the stage, and adjust the condenser to an appropriate height so that the light can evenly illuminate the sample. Using the coarse focus knob, slowly raise the stage so that the oil droplet contacts the objective. Then, use the fine focus knob to search for bacteria in bright field (or use the corresponding fluorescence channel). After finding the bacteria under the microscope, switch the display to the computer software, set the excitation wavelength of green fluorescence to 488 nm and the emission wavelength to 525 nm. For Mcherry, the excitation wavelength is 580 nm and the emission wavelength is 610 nm. Then click Live, observe the sample in this mode, take pictures, label the results, and save them. Three sample slides were prepared for each sample, and images were observed and collected.

[0117] 3.4 Target gene transformation and functional phenotype strain construction

[0118] ① The target plasmid was transformed into Escherichia coli DH5α. After gene sequencing to ensure the sequence was correct, the returned bacterial culture was stored in a -80℃ freezer.

[0119] ② Preparation of electroporation competent cells: *E. coli SHuffle* and *Klebsiella pneumoniae ATCC43816* competent cells, frozen at -80℃, were placed on ice for 5 minutes to allow slow thawing. A disposable inoculation loop was used to pick a small amount of bacterial culture and streak it onto antibiotic-free LB agar medium in four zones. The strain name and streak date were labeled, and the culture was incubated overnight at 37℃. The next day, single clones were picked and inoculated into 50 mL of antibiotic-free LB agar medium. The medium was then incubated at 220 rpm in a 37℃ shaker until the OD value reached approximately 0.5. The culture was then removed and placed on ice for 30 minutes to pre-cool. Simultaneously, a 10% (v / v) glycerol solution, sterilized by autoclaving, was placed on ice for 30 minutes to pre-cool. The culture was then transferred to 50 mL sterile centrifuge tubes and centrifuged at 4℃ and 4200 rpm for 15 minutes to collect the cells. The supernatant was discarded. Add 15 mL of pre-chilled 10% (v / v) glycerol solution to a centrifuge tube, gently resuspend the bacteria by pipetting, cap the tube, and vortex thoroughly until the bacterial suspension is evenly distributed without any large bacterial fragments. Centrifuge again at 4℃ and 4200 rpm for 15 minutes, discarding the supernatant. Repeat this step twice. Then, add 5 mL of pre-chilled 10% (v / v) glycerol solution to the centrifuge tube again and vortex thoroughly. Aliquot the bacterial suspension into 1.5 mL EP tubes, adding 100 μL to each tube. Label the tubes with the name and preparation date, flash-freeze in liquid nitrogen, and then store at -80℃ for later use. (The electroporation method is also applicable to Klebsiella pneumoniae. However, due to the high viscosity of highly virulent carbapenem-resistant Klebsiella pneumoniae, it has some resistance to centrifugation. Therefore, the centrifugation conditions are changed to 4℃ and 12000 rpm for 15 minutes, with the supernatant gently aspirated by pipetting.)

[0120] ③ Electroporation: 10 μL each of the pWSK129 empty vector and pWSK129-mcr-1 plasmid were added to two separate tubes of SHuffle electroporation competent cells. The cells were transferred to 2 mm electroporation cuvettes, the cuvettes were dried, and electroporated at 2.5 kV for 4 ms. The cells were then placed on ice for 3 minutes, and 500 μL of antibiotic-free LB broth was added. The cells were incubated for 50 minutes at 37°C and 220 rpm on a shaker. After centrifugation at 5000 rpm for 10 minutes, 100 μL of the supernatant was collected. The bacterial culture was resuspended by pipetting and spread onto LB agar plates containing 50 μg / mL kanamycin sulfate. The plates were then incubated overnight at 37°C.

[0121] The following day, single colonies were picked and inoculated into 500 μL of LB liquid medium containing 50 μg / mL kanamycin sulfate, and amplified in a shaker at 37°C and 220 rpm. OD was awaited. 600When the OD value reaches approximately 1.0, transfer to 50 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate. Then, incubate at 37°C and 220 rpm on a shaker for amplification. Wait for OD... 600 At approximately 0.5 μL, stop amplification and pre-cool on ice for 30 minutes to obtain SHuffle-carrying pWSK129 and SHuffle-carrying pWSK129-mcr-1 electrocompetent cells. Store the prepared electrocompetent cells at -80°C for later use, following the same procedures as before.

[0122] Take 10 μL each of the pSGKM empty vector and pSGKM-8H1 plasmid and add them to SHuffle-carrying pWSK129 and SHuffle-carrying pWSK129-mcr-1 electroporation competent cells, respectively. Electroporate the above plasmids into the above E. coli. (The specific electroporation procedure is the same as before, but it is plated on LB solid medium plates containing 50 μg / mL kanamycin sulfate and 50 μg / mL rifampin).

[0123] 3.5 E-test test strip method

[0124] 3.5.1 Pick a single colony from the plate and inoculate it into 2 mL of LB liquid medium containing kanamycin sulfate (50 μg / mL) and rifampin (50 μg / mL). Incubate at 220 rpm and 37°C until OD reaches 0.5. 600 The value is approximately 0.5.

[0125] 3.5.2 Preparation of antibiotic-free LB semi-solid culture media discs. First, prepare LB semi-solid culture medium and autoclave at 121℃ for 20 minutes, then place it in a 65℃ oven for later use. Add the bacterial suspension of the model strain to the LB semi-solid culture medium at a ratio of 1:100, invert and mix well, pour into discs, and let stand for 10 minutes to cool and solidify. Label the strain name in each disc for later use.

[0126] 3.5.3. Tear open the E-test test strip packaging, use metal tweezers to pick up the test strip, and insert it into the center of the solidified LB semi-solid culture medium disc. Invert the disc and incubate overnight at 37°C. The next day, observe the bacterial resistance.

[0127] The results showed that introducing MCR-1 into Escherichia coli and Klebsiella pneumoniae models increased the resistance of Escherichia coli and Klebsiella pneumoniae to polymyxins. Figure 4 B and Figure 5(B). When 8H1 was introduced into E. coli and Klebsiella pneumoniae models carrying MCR-1, the resistance of E. coli and Klebsiella pneumoniae to polymyxin decreased, and they returned to a sensitive state. Figure 4 C and Figure 5 (C).

[0128] Example 2

[0129] In this embodiment, an Escherichia coli (SHuffle) model was prepared according to the same procedure as in Example 1. In this embodiment, the base sequences of the primers used for constructing the pWSK129-mcr-2 plasmid were pWSK129-MCR2-F (as shown in SEQ ID NO. 16) and pWSK129-MCR2-R (as shown in SEQ ID NO. 17).

[0130] The results of this embodiment are as follows: Figure 6 As shown, introducing MCR-2 into the E. coli model increases the resistance of the resulting E. coli to polymyxins. Figure 6 (B). When 8H1 was introduced into an E. coli model carrying MCR-2, the resistance of E. coli to polymyxin decreased, and it returned to a sensitive state. Figure 6 (C).

[0131] Example 3

[0132] The results of this embodiment are as follows: Figure 7 As shown, 8H1 forms a complex with the MCR-1 protein ( Figure 7 (A and B) thereby inhibiting the function of MCR-1 protein.

[0133] The above specific embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

[0134] The amino acid and nucleotide sequences involved in this invention are as follows:

[0135] SEQ ID NO.1:

[0136] Name: Base sequence of single-chain antibody 8H1

[0137] Sequence type: DNA (other DNA)

[0138] Biological origin: synthetic construct

[0139] ATGGATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTTCTACACATCAAGATTACACCCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGACCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTGCTCACGTTCGGTGCTGGGACCAAACTGGGGCTGAAAGGAGGAGGAGGATCAGGAGGAGGAGGATCAGGAGGAGGAGGATCAGGAGGAGGAGGATCAGAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAGGATGTCCTGCAAGGCTTCTGGTTACTCATTCACTGCCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAGTGGATTGGACTTATTAATCCTTACAATGGTGGTTCTACCTACAACCAGAAGTTCAAGGGCAAGGCCACATTAACTGTAGACAAGTCATCCAGCACAGCCTACATGGAGCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAAAGGGGAACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCATAA;

[0140] SEQ ID NO.2:

[0141] Name: Amino acid sequence of single-chain antibody 8H1

[0142] Sequence type: AA

[0143] Organism source: synthetic construct

[0144] MDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIFYTSRLHPGVPSRFSGSGSGTDYSLTISNLDQEDIATYFCQQGNTLLTFGAGTKLGLKGGGGSGGGGSGGGGS GGGGSEVQLQQSGPELVKPGASMRMSCKASGYSFTAYTMNWVKQSHGKNLEWIGLINPYNGGSTYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCARKGNYAMDYWGQGTSVTVSS;

[0145] SEQ ID NO.3:

[0146] Name: Nucleotide sequence of primer pWSK129-F

[0147] Sequence type: DNA (other DNA)

[0148] Biological origin: synthetic construct

[0149] CTGGACAAATTGTTTTCCAAGTCGACCTCGAGGGGGGGGCC;

[0150] SEQ ID NO.4:

[0151] Name: Nucleotide sequence of primer pWSK129-R

[0152] Sequence type: DNA (other DNA)

[0153] Biological origin: synthetic construct

[0154] ATCCACAATTCGAGAAGTGAGAATTCCTGCAGCCCGGGGG;

[0155] SEQ ID NO.5:

[0156] Name: Nucleotide sequence of primer pWSK129-Pmcr-1-F

[0157] Sequence type: DNA (other DNA)

[0158] Biological origin: synthetic construct

[0159] TACCGGGCCCCCCCTCGAGGTCGACTTGGAAAACAATTTGTCCAGGCCAG;

[0160] SEQ ID NO.6:

[0161] Name: Nucleotide sequence of primer pWSK129-Pmcr-1-R

[0162] Sequence type: DNA (other DNA)

[0163] Biological origin: synthetic construct

[0164] AGTATGCTGCATCATGAGAAACTACTCAAA;

[0165] SEQ ID NO.7:

[0166] Name: Nucleotide sequence of primer pWSK129-mcr-1-F

[0167] Sequence type: DNA (other DNA)

[0168] Biological origin: synthetic construct

[0169] TTTGAGTAGTTTCTCATGATGCAGCATACT;

[0170] SEQ ID NO.8:

[0171] Name: Nucleotide sequence of primer pWSK129-mcr-1-R1

[0172] Sequence type: DNA (other DNA)

[0173] Biological origin: synthetic construct

[0174] ATGTGACCAAGGCCTGCGGATGAATGCGGT;

[0175] SEQ ID NO.9:

[0176] Name: Nucleotide sequence of primer pWSK129-mcr-1-R2

[0177] Sequence type: DNA (other DNA)

[0178] Biological origin: synthetic construct

[0179] GTGGATCCCCCGGGCTGCAGGAATTCTCACTTCTCGAATTGTGGATGTGACCAAGGCCT;

[0180] SEQ ID NO.10:

[0181] Name: Nucleotide sequence of primer pSGKM-F

[0182] Sequence type: DNA (other DNA)

[0183] Biological origin: synthetic construct

[0184] GTCGGCAGCAGGTATTTCATGGTCTCGACTAGTATTATAC;

[0185] SEQ ID NO.11:

[0186] Name: Nucleotide sequence of primer pSGKM-R

[0187] Sequence type: DNA (other DNA)

[0188] Biological origin: synthetic construct

[0189] ACCACCACCACCACCACTAAGAATTCCTGCAGCCCGGGGG;

[0190] SEQ ID NO.12:

[0191] Name: Nucleotide sequence of primer pSGKM-pelB-F

[0192] Sequence type: DNA (other DNA)

[0193] Biological origin: synthetic construct

[0194] CCTAGGTATAATACTAGTCGAGACCATGAAATACCTGCTGCCGAC;

[0195] SEQ ID NO.13:

[0196] Name: Nucleotide sequence of primer pSGKM-pelB-8H1-R

[0197] Sequence type: DNA (other DNA)

[0198] Biological origin: synthetic construct

[0199] TGTGTCATCTGGATATCCATGGCCATCGCCGGCTGGGCAG;

[0200] SEQ ID NO.14:

[0201] Name: Nucleotide sequence of primer pSGKM-8H1-F

[0202] Sequence type: DNA (other DNA)

[0203] Biological origin: synthetic construct

[0204] CTGCCCAGCCGGCGATGGCCATGGATATCCAGATGACACA;

[0205] SEQ ID NO.15:

[0206] Name: Nucleotide sequence of primer pSGKM-8H1-R

[0207] Sequence type: DNA (other DNA)

[0208] Biological origin: synthetic construct

[0209] GGGCTGCAGGAATTCTTAGTGGTGGTGGTGGTGGTGTGAGGAGACGGTGAC;

[0210] SEQ ID NO.16:

[0211] Name: Nucleotide sequence of primer pWSK129-MCR2-F

[0212] Sequence type: DNA (other DNA)

[0213] Biological origin: synthetic construct

[0214] TATTTTTTGAGTAGTTTCTCATGACATCACATCACTCTTG;

[0215] SEQ ID NO.17:

[0216] Name: Nucleotide sequence of primer pWSK129-MCR2-R

[0217] Sequence type: DNA (other DNA)

[0218] Biological origin: synthetic construct

[0219] TGTGGATGTGACCAAGGCCTCTGGATAAAATGCCGCGCGGT;

[0220] SEQ ID NO.18:

[0221] Name: Nucleotide sequence of the natural promoter of mcr-1

[0222] Sequence type: DNA (other DNA)

[0223] Biological origin: synthetic construct

[0224] TGGAAAACAATTTGTCCAGGCCAGAGAAGAACATCTGGAATTGCAGCAACAGCGTGACAATTCACAGGACATGCACTAATTAAGAAAAAAGCCCGGTTTTTCGGGCTTTTTAAGATACAAATTATAAATACTCTCAAGTGTATATTCAGTA TGGGATTGCGCAATGATTGCCTAATAAAATTTCTGAAATATTTCTGTATCGCATAATTTTTTATATCAGATAAATTGTACTGGATTTCTTAAAAAATTGCAGTAAAATTGCCGCAATTATCCCACCGTTTATTTTTTGAGTAGTTTCTC;

[0225] SEQ ID NO.19:

[0226] Name: Amino acid sequence of the hinge region

[0227] Sequence type: AA

[0228] Biological origin: synthetic construct

[0229] GGGGSGGGGSGGGGSGGGGS.

Claims

1. A single-chain antibody 8H1 that targets and binds to a mobile polymyxin resistance protein, characterized in that, The amino acid sequence of the single-chain antibody 8H1 is shown in SEQ ID NO.2; the mobile polymyxin resistance protein is MCR-1 protein and / or MCR-2 protein.

2. The single-chain antibody 8H1 according to claim 1, characterized in that, The single-chain antibody 8H1 can specifically bind to the functional catalytic region of the mobile polymyxin resistance protein, thereby inhibiting the function of the mobile polymyxin resistance protein.

3. A biomaterial, characterized in that, Includes at least one of the following biological materials: a) A polynucleotide for expressing the single-chain antibody 8H1 as described in any one of claims 1 to 2, the nucleotide sequence of which is shown in SEQ ID NO. 1; b) An expression vector containing the polynucleotide.

4. The application of the single-chain antibody 8H1 as described in any one of claims 1-2 or the biomaterial as described in claim 3, characterized in that, The reagent is used to prepare a detection reagent for mobile polymyxin resistance protein or to prepare recombinant bacteria with inhibited polymyxin resistance; the recombinant bacteria are Escherichia or Klebsiella; the mobile polymyxin resistance protein is MCR-1 protein and / or MCR-2 protein.

5. A recombinant bacterium, characterized in that, The recombinant bacteria comprises the biological material as described in claim 3, wherein the recombinant bacteria is Escherichia or Klebsiella.

6. A method for inhibiting bacterial polymyxin resistance, characterized in that, include: Step S1) Constructing an expression vector; the expression vector comprises a polynucleotide capable of expressing the single-chain antibody 8H1 as described in any one of claims 1 to 2 and an expression regulatory element operatively linked to the polynucleotide that is active in bacteria; Step S2) The expression vector is introduced into host bacteria using electroporation technology to obtain recombinant bacteria; the recombinant bacteria are Escherichia or Klebsiella. Step S3) The polynucleotide is expressed in recombinant bacteria to form single-chain antibody 8H1. Single-chain antibody 8H1 can specifically bind to the functional catalytic region of the mobile polymyxin resistance protein, thereby inhibiting the function of the mobile polymyxin resistance protein and thus inhibiting the polymyxin resistance of the recombinant bacteria.

Images