A binding protein b binding to klebsiella pneumoniae and its use

Abstract

The present application relates to a kind of binding protein B combined with klebsiella pneumoniae and its application, belong to molecular biology and protein engineering technical field.The binding protein B provided by the present application can specifically bind the membrane outer part of the unique protein P24017 of klebsiella pneumoniae OMPA_KLEPN, and its good recognition and stable binding capacity for klebsiella pneumoniae are verified by double-molecule fluorescence complementation, flow cytometry and other experiments, can be used for the identification and screening of klebsiella pneumoniae, provides key tool for the targeted drug development for the bacteria.

Description

Technical Field

[0001] This invention relates to a binding protein B that binds to Klebsiella pneumoniae and its applications, belonging to the fields of molecular biology and protein engineering technology. Background Technology

[0002] De novo protein design refers to the design of protein sequences that do not exist in nature through computational simulation or experimental methods, giving them a predetermined three-dimensional structure and function (such as catalysis, binding, assembly, etc.).

[0003] Klebsiella pneumoniae ( Klebsiella pneumoniae ) belongs to the genus Klebsiella of the family Enterobacteriaceae. Klebsiella Gram-negative bacilli, with oval or rod-shaped cells, lacking flagella, and possessing a relatively thick capsule. They are widely distributed in nature (such as in water and soil) and in the intestines and respiratory tracts of humans and animals, forming part of the normal flora. As opportunistic pathogens, they can cause infections when the host's immunity is weakened, leading to hospital-acquired pneumonia, urinary tract infections, sepsis, meningitis, liver abscesses, wound infections, etc.

[0004] Protein P24017·OMPA_KLEPN is an extracellular protein unique to Klebsiella pneumoniae. By designing proteins that bind to P24017·OMPA_KLEPN de novo, binding protein B that can recognize and stably bind to Klebsiella pneumoniae can be screened. This can be used for the identification and screening of Klebsiella pneumoniae, as well as the development of targeted drugs against Klebsiella pneumoniae. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a binding protein B that binds to Klebsiella pneumoniae and its applications.

[0006] The technical solution of the present invention is as follows:

[0007] A binding protein B that binds to Klebsiella pneumoniae, the amino acid sequence of said binding protein B being shown in SEQ ID NO. 3.

[0008] A coding gene that encodes the aforementioned binding protein B.

[0009] According to a preferred embodiment of the present invention, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.4.

[0010] An expression vector comprising the above-described encoding gene.

[0011] According to a preferred embodiment of the present invention, the expression vector used is a prokaryotic or eukaryotic expression vector.

[0012] Further preferred, the expression vector used is pETDuet-1.

[0013] A recombinant bacterium, wherein the recombinant bacterium contains the above-described coding gene or the above-described expression vector.

[0014] According to a preferred embodiment of the present invention, the host bacterium used is Escherichia coli BL21(DE3).

[0015] The application of the above-mentioned binding protein B in the identification and screening of Klebsiella pneumoniae.

[0016] The application of the aforementioned binding protein B in the development of targeted drugs against Klebsiella pneumoniae.

[0017] Beneficial effects:

[0018] This invention provides a binding protein B that can specifically bind to the extracellular portion of the Klebsiella pneumoniae-specific protein P24017·OMPA_KLEPN. Experiments such as bimolecular fluorescence complementation and flow cytometry have verified that it has good recognition and stable binding ability for Klebsiella pneumoniae, and can be used for the identification and screening of Klebsiella pneumoniae, providing a key tool for the development of targeted drugs against this bacterium. Attached Figure Description

[0019] Figure 1 This is the AlphaFold predicted structure diagram of protein P24017·OMPA_KLEPN;

[0020] Figure 2 The AlphaFold predicted structure diagram of the extracellular portion A212-341 of protein P24017·OMPA_KLEPN.

[0021] Figure 3 The diagram shows the binding of the protein backbone to protein P24017·OMPA_KLEPN in the extracellular region A212-341 as predicted by RFdiffusion.

[0022] Figure 4 Map of recombinant plasmid pETDuet-1-BIFC-FC;

[0023] Figure 5 Line graph showing the change in protein fluorescence intensity over time for the protein B expressing bacteria and control bacteria;

[0024] Figure 6 This is an SDS-PAGE electrophoresis analysis of the target protein after induction by the protein B-expressing bacteria.

[0025] Figure 7 Line graph showing the change in fluorescence intensity of Klebsiella pneumoniae after binding to fluorescent binding protein B with the number of washes;

[0026] Figure 8 This is an SDS-PAGE electrophoresis analysis of fluorescent binding protein B;

[0027] Figure 9 This is a flow cytometry result for the control group.

[0028] Figure 10 This is a flow cytometry image of the fluorescent binding protein B binding sequence.

[0029] Figure 11 This is a line graph showing the change in protein fluorescence intensity over time for the protein I expressing bacteria and the control bacteria.

[0030] Figure 12 This is an SDS-PAGE electrophoresis analysis of the target protein after induction by the protein I-expressing bacteria.

[0031] Figure 13 This is an SDS-PAGE electrophoresis analysis of fluorescent binding protein I;

[0032] Figure 14 This is a flow cytometry result for the control group.

[0033] Figure 15 This is a flow cytometry image of the fluorescent binding protein I binding sequence. Detailed Implementation

[0034] The technical solution of the present invention will be further described below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solution of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0035] Unless otherwise specified in the examples, the procedures were performed under standard conditions; reagents or instruments used without a specified manufacturer were all commercially available products.

[0036] Among them, the Klebsiella pneumoniae used in the examples ( Klebsiella pneumoniae The bacterial culture was purchased from the China Industrial Microbial Culture Collection Center (CICC), with the number NCTC 13442. It was prepared to a bacterial concentration of 1000 cfu / mL for use in experiments.

[0037] The composition of LB liquid culture medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, ultrapure water as solvent, pH value is natural pH, and it is autoclaved at 121℃ for 20 min.

[0038] LB liquid medium containing ampicillin sodium: Add 100 μg / mL (final concentration) of ampicillin sodium to LB liquid medium before use.

[0039] Example 1

[0040] De novo design of protein P24017·OMPA_KLEPN binding protein

[0041] AlphaFold is an artificial intelligence (AI) system developed by DeepMind, primarily used to predict the three-dimensional structure of proteins. The amino acid sequence of protein P24017·OMPA_KLEPN (as shown in SEQ ID NO.1, and its encoding nucleotide sequence as shown in SEQ ID NO.2) was imported into AlphaFold, and the predicted structure was obtained through calculation and execution. Figure 1 As shown.

[0042] RFdiffusion is a deep learning model specifically developed for de novo protein design and molecular docking. Based on predicted structural diagrams ( Figure 1 Select the portion of protein P24017·OMPA_KLEPN that is free outside the membrane, such as... Figure 2 As shown, this protein sequence is A212-341. This sequence was introduced into RFdiffusion to obtain binding maps of multiple binding protein backbones with selected proteins free on the extracellular membrane. Based on the predicted binding sites, appropriate binding protein backbones were selected, such as... Figure 3 As shown.

[0043] ProteinMPNN was used to fill the amino acid sequence, resulting in multiple binding protein amino acid sequences. The obtained binding proteins were then docked with protein P24017·OMPA_KLEPN via an HDOCK server. Based on the binding strength and binding site, an amino acid sequence with high binding strength was selected and named binding protein B. Its amino acid sequence is shown in SEQ ID NO.3, and its encoding nucleotide sequence is shown in SEQ ID NO.4.

[0044] Example 2

[0045] The binding affinity of protein B to protein P24017·OMPA_KLEPN was verified using bimolecular fluorescence complementation (BiFC). BiFC is a powerful tool for studying protein-protein interactions in living cells. Its core principle involves splitting a fluorescent protein into two non-fluorescent fragments, fusing each fragment with a target protein. When the target protein interacts, the two fragments spatially approach each other and reassemble into a complete fluorescent protein, emitting fluorescence. This allows for the direct detection of protein-protein interactions.

[0046] Two gene fragments, gene fragment 1 and gene fragment 2, were synthesized, ligated into the same dual promoter vector pETDuet-1, and transformed into Escherichia coli BL21(DE3) for expression.

[0047] Gene fragment 1: protein P24017·OMPA_KLEPN (amino acid sequence as shown in SEQ ID NO.1, encoding nucleotide sequence as shown in SEQ ID NO.2) + flexible linker peptide (amino acid sequence as shown in SEQ ID NO.5, encoding nucleotide sequence as shown in SEQ ID NO.6) + EYFP-A (amino acid sequence as shown in SEQ ID NO.7, encoding nucleotide sequence as shown in SEQ ID NO.8), was ligated into vector pETDuet-1 by double digestion with NcoI and EcoRI. An intermediate vector containing gene fragment 1 was obtained.

[0048] Gene fragment 2: binding protein B (amino acid sequence as shown in SEQ ID NO.3, encoding nucleotide sequence as shown in SEQ ID NO.4) + flexible linker peptide (amino acid sequence as shown in SEQ ID NO.5, encoding nucleotide sequence as shown in SEQ ID NO.6) + EYFP-B (amino acid sequence as shown in SEQ ID NO.9, encoding nucleotide sequence as shown in SEQ ID NO.10), was ligated into an intermediate vector containing gene fragment 1 by double digestion with NdeI+XhoI.

[0049] The two synthesized gene fragments, fragment 1 and fragment 2, were ligated as two CDS into the vector pETDuet-1 to obtain the recombinant plasmid pETDuet-1-BIFC-FC. A simplified diagram of the plasmid is shown below. Figure 4 As shown. The bacteria were transformed into Escherichia coli BL21(DE3) to obtain the binding protein B expressing strain, which was then preserved in glycerol tubes.

[0050] Gene fragment 1 and EYFP-B were ligated into the vector pETDuet-1 in the same manner and transformed into Escherichia coli BL21(DE3) as a control strain, and preserved in glycerol tubes.

[0051] Example 3

[0052] Validation of the binding affinity between protein B and protein P24017·OMPA_KLEPN

[0053] Activation: 100 μL of the protein B-expressing bacteria obtained in Example 2 was inoculated into 50 mL of LB liquid medium containing ampicillin sodium in the experimental group; the control group was inoculated with an equal amount of control bacteria. Both cultures were incubated at 37°C and 200 rpm for 12 h to obtain activated bacteria.

[0054] Transfer: Inoculate 1 mL of activated bacteria into 50 mL of LB liquid medium containing ampicillin sodium, and incubate at 37°C and 200 rpm for about 1.5 h.

[0055] Induction: until the bacterial culture reaches OD 600 When the concentration of 1 mol / L IPTG is 0.8, add 10 μL of 1 mol / L IPTG. Incubate at 26 °C and 200 rpm on a shaker.

[0056] Sampling: Sample every 2 hours within 0-16 hours after adding IPTG.

[0057] Sample processing: After sampling, the fluorescence intensity after removing interference from LB liquid culture medium was measured. 1 mL of sample was transferred to a 1.5 mL centrifuge tube, centrifuged at 12000 rpm for 2 min, the supernatant was removed, and the sample was resuspended in 1 mL PBS buffer (pH 7.0).

[0058] Fluorescence intensity measurement: Add 200 μL of sample to each well of a black 96-well plate; incident light 480 nm, reflected light 520 nm for the microplate reader.

[0059] Fluorescence intensity measurement results are as follows Figure 5 As shown, the results indicate that the fluorescence intensity of the protein B-expressing bacteria was significantly higher than that of the control bacteria, and the fluorescence intensity of the protein B-expressing bacteria gradually increased with the increase of induction time, indicating that the protein B-expressing bacteria have a strong binding ability to protein P24017·OMPA_KLEPN, and can generate a fluorescence signal through the complementarity of the EYFP fragment after binding.

[0060] Example 4

[0061] Electrophoresis verification

[0062] Activation: 100 μL of protein B-expressing bacteria was inoculated into 50 mL of LB liquid medium containing ampicillin sodium and cultured at 37°C and 200 rpm for 12 h to obtain activated bacteria.

[0063] Transfer: Inoculate 1 mL of activated bacteria into 50 mL of LB liquid medium containing ampicillin sodium, and incubate at 37°C and 200 rpm for about 1.5 h.

[0064] Induction: The bacterial culture reaches OD 600 When the concentration of 1 mol / L IPTG is 0.8, add 10 μL of 1 mol / L IPTG. Incubate at 26℃ and 200 rpm for 10 h on a shaker.

[0065] Remove culture medium: Centrifuge 40 mL of culture at 6000 rpm for 10 min, remove supernatant, add 10 mL of PBS buffer (pH 7.0), resuspend, centrifuge again, add another 10 mL of PBS buffer (pH 7.0), and resuspend.

[0066] Disruption of bacterial cells: set power ratio to 80% (950w, 80%), sonication cycle: 4s on / 6s off, total duration 16min; after sonication, centrifuge at 5000rpm for 10min to obtain supernatant.

[0067] Preparation of gel electrophoresis samples: Take 80 μL of supernatant and 20 μL of protein loading buffer into a 1.5 mL centrifuge tube and boil in boiling water for 10 min.

[0068] Gel chromatography: 15 μL sample, 10 μL marker; 140 V voltage, 30 min running time. After the gel, stain with Coomassie Brilliant Blue for 2 h, then destain with destaining agent.

[0069] The results of the photo observation are as follows Figure 6 As shown, gene fragments 1 and 2 correspond to 50kD and 24kD respectively, and the protein bands are clear, proving that the protein expressed by the protein-binding bacteria is correct.

[0070] Example 5

[0071] Verification of the binding affinity of protein B to Klebsiella pneumoniae

[0072] The gene fragment: binding protein B (amino acid sequence as shown in SEQ ID NO.3, encoding nucleotide sequence as shown in SEQ ID NO.4) + flexible linker peptide (amino acid sequence as shown in SEQ ID NO.5, encoding nucleotide sequence as shown in SEQ ID NO.6) + EYFP (amino acid sequence as shown in SEQ ID NO.11, encoding nucleotide sequence as shown in SEQ ID NO.12) + His6 tag was ligated into the vector pETDuet-1 by double digestion with NcoI and EcoRI.

[0073] The recombinant vector was transformed into Escherichia coli BL21(DE3) to obtain fluorescent binding protein B expressing bacteria, which were then preserved in glycerol tubes.

[0074] The gene fragment: EYFP (amino acid sequence as shown in SEQ ID NO.11, nucleotide sequence as shown in SEQ ID NO.12) + His6 tag was ligated into the vector pETDuet-1 by double digestion with NcoI + EcoRI, and then transformed into Escherichia coli BL21(DE3) as a control strain and preserved in glycerol tubes.

[0075] The culture conditions for the activation, transfer, and induction steps are the same as in Example 4.

[0076] Remove culture medium: Centrifuge 40 mL of culture at 6000 rpm for 10 min, remove supernatant, add 10 mL of PBS buffer (pH 7.0), resuspend, centrifuge again, add another 10 mL of PBS buffer (pH 7.0), and resuspend.

[0077] Disruption of bacterial cells: set power ratio to 80% (950w, 80%), sonication cycle: 4s on / 6s off, total duration 16min; after sonication, centrifuge at 5000rpm for 10min to obtain supernatant.

[0078] Incubation: Take 5 mL of the supernatant of the fluorescent binding protein B expressing bacteria obtained in the above steps and mix it with 5 mL of Klebsiella pneumoniae solution. Incubate at 4°C for 2 h as the fluorescent binding protein B binding group. Under the same conditions, mix the supernatant of the Klebsiella pneumoniae and control bacteria and incubate as the control group.

[0079] Washing after incubation: Centrifuge at 12000 rpm for 2 min to remove supernatant. Due to the large mass of Klebsiella pneumoniae, all of them were in the precipitate after centrifugation. The precipitate was resuspended in 5 mL PBS buffer (pH 7.0).

[0080] The fluorescence intensity of the resuspended solution was measured using an ELISA reader with incident light at 480 nm and reflected light at 520 nm.

[0081] The results are as follows Figure 7 As shown, the fluorescence intensity in the resuspension of the fluorescent binding protein B group remained at a high level after one wash, proving that the de novo-designed binding protein B has good binding to Klebsiella pneumoniae. As the number of washes increased, the fluorescence intensity gradually decreased, but it was always higher than that of the control group, indicating that the two have a strong binding ability.

[0082] Further electrophoresis experiments were conducted to verify protein expression levels. The electrophoresis procedures and experimental conditions were the same as in Example 4, and the results are as follows: Figure 8 As shown, the protein is expressed correctly.

[0083] The incubated samples from the control group and the fluorescent protein B binding group were diluted 10-fold and then analyzed by flow cytometry. The results are as follows: Figure 9 , Figure 10 As shown, compared with the control group, the binding group showed a significant high-intensity fluorescence peak, proving that the binding protein B can recognize and stably bind to Klebsiella pneumoniae, verifying its good targeting binding ability.

[0084] Comparative Example 1

[0085] De novo design of protein P24017·OMPA_KLEPN binding protein

[0086] According to the design method of Example 1, multiple binding protein amino acid sequences were obtained. The obtained binding proteins were docked with protein P24017·OMPA_KLEPN through an HDOCK server. An amino acid sequence with high binding strength was selected and named binding protein I. Its amino acid sequence is shown in SEQ ID NO.13, and its encoding nucleotide sequence is shown in SEQ ID NO.14.

[0087] The binding affinity of binding protein I to protein P24017·OMPA_KLEPN was verified using bimolecular fluorescence complementation (BFF). The verification method was the same as in Examples 2 and 3. Bacteria expressing binding protein I were constructed according to the method in Example 2. The fluorescence intensity measurements of the BFF expression bacteria and the control bacteria are shown below. Figure 11 As shown, the results indicate that the fluorescence intensity of the protein I-expressing bacteria was significantly different from that of the control bacteria, suggesting that protein I has the ability to bind to protein P24017·OMPA_KLEPN.

[0088] Electrophoresis verification was performed according to the method in Example 4, and the SDS-PAGE electrophoresis analysis of the target protein after induction by protein I expression bacteria is shown in the figure below. Figure 12 As shown, gene fragments 1 and 2 correspond to 55kD and 33kD respectively, and the protein bands are clear, proving that the protein expressed by the protein I-binding bacteria is correct.

[0089] Control bacteria and fluorescent binding protein I expressing bacteria were constructed according to the method in Example 5. The product of the fluorescent binding protein I expressing bacteria induced by the method in Example 4 was verified by electrophoresis, yielding the following results: Figure 13 The electrophoresis image shown confirms correct protein expression. Control bacteria and bacteria conjugated with fluorescent protein I were incubated with Klebsiella pneumoniae culture, and the incubated samples were diluted 10-fold before flow cytometry analysis. The results are as follows. Figure 14 , Figure 15 As shown, compared with the control group, no significant high-intensity fluorescence peak was observed in the fluorescent binding protein I binding group, proving that binding protein I cannot recognize and stably bind to Klebsiella pneumoniae.

[0090] This comparative example shows that binding protein I has the ability to bind to protein P24017·OMPA_KLEPN. However, in real bacterial surface environments, due to the spatial conformation of protein P24017·OMPA_KLEPN, the degree of surface exposure, and other molecular interferences on the bacterial surface, binding protein I may not be able to recognize and stably bind to Klebsiella pneumoniae.

Claims

1. A binding protein B that binds to Klebsiella pneumoniae, characterized in that, The amino acid sequence of the binding protein B is shown in SEQ ID NO.

3.

2. A gene encoding a gene, characterized in that, The encoding gene encodes the binding protein B as described in claim 1.

3. The encoding gene as described in claim 2, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

4.

4. An expression carrier, characterized in that, The expression vector contains the coding gene as described in claim 2 or claim 3.

5. The expression vector as described in claim 4, characterized in that, The expression vectors used are prokaryotic or eukaryotic expression vectors.

6. The expression vector as described in claim 5, characterized in that, The expression vector used was pETDuet-1.

7. A recombinant bacterium, characterized in that, The recombinant bacteria comprises the coding gene of claim 2 or claim 3 or the expression vector of claim 4.

8. The recombinant bacteria as described in claim 7, characterized in that, The host bacterium used was Escherichia coli BL21(DE3).

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