Anti-kpc-2 carbapenemase nanobody nb-kpc-2 and use thereof

Abstract

This invention belongs to the field of detection technology for carbapenem-resistant Enterobacteriaceae, and provides an anti-KPC-2 carbapenemase nanobody Nb-KPC-2 and its applications. The anti-KPC-2 carbapenemase nanobody Nb-KPC-2 has the amino acid sequence shown in SEQ ID NO.1. This invention lays the foundation for constructing a low-cost and stable immunoassay method for KPC-2 carbapenemases. Utilizing the characteristics of small size, good stability, and ease of labeling, nanobodies can be applied to the rapid detection of pathogenic microorganisms, such as the preparation of diagnostic test strips, reagents, and kits; they can also be applied to basic research and tool development, such as as molecular probes, isolation and purification kits, and the construction of core components for targeted therapy molecules; enabling early and accurate screening and identification of drug-resistant Gram-negative bacteria producing KPC-2 carbapenemases, guiding precise clinical medication and infection control.

Description

Technical Field

[0001] This invention belongs to the field of detection technology for carbapenem-resistant Enterobacteriaceae, and particularly relates to an anti-KPC-2 type carbapenemase nanobody Nb-KPC-2 and its application. Background Technology

[0002] With the widespread use of carbapenem antibiotics globally, the infection rate of carbapenem-resistant Enterobacterales (CRE) is increasing year by year. In this resistance crisis, strains producing Klebsiella pneumoniae carbapenemase (KPC) exhibit strong transmissibility and horizontal transfer characteristics of resistance genes. These strains, by secreting KPC-type carbapenemase, can target and hydrolyze the β-lactam ring of carbapenem drugs, leading to the loss of key antibacterial activity. Notably, the KPC-2 subtype is absolutely dominant in the KPC-type carbapenemase family; its encoding gene can be transmitted among Enterobacterales bacteria via plasmids, even crossing species barriers, triggering the spread of multidrug resistance.

[0003] Rapid detection and typing of carbapenem-resistant Enterobacteriaceae are crucial for the rational use of antibiotics, the determination of treatment regimens, and the rapid control of infections. Currently, due to the lack of rapid typing and detection methods, clinical treatment generally relies on empirical broad-spectrum drug use, leading to antibiotic overuse, increased treatment failure rates, and accelerated evolution of drug-resistant mutants. Establishing a precision drug use strategy based on carbapenemase typing is one of the core measures to slow the spread of drug resistance. Current clinical typing technologies are severely lagging: molecular diagnostic methods based on nucleic acid amplification are time-consuming and dependent on molecular biology laboratories and skilled technicians; while immunological tests are simple to perform and quick, they rely on high-performance antibodies.

[0004] Nanobodies (Nb), single-domain antibodies derived from the variable region of camel heavy chain antibodies, have a molecular weight of only 15 kDa, less than one-tenth that of traditional IgG antibodies. They can penetrate the outer membrane barrier of Gram-negative bacteria and target and bind to KPC-2 carbapenemase. Due to their rigid structure, lacking light chains and secured only by two disulfide bonds, nanobodies are not easily inactivated by high temperatures, extreme pH levels, or protease environments. Lyophilized nanobodies coated onto nitrocellulose membrane test strips can be stored at room temperature for more than 18 months without cold chain transportation, overcoming the limitations of traditional reagents stored and transported at 2-8℃. Furthermore, nanobodies can be efficiently expressed using E. coli or Pichia pastoris systems, with simple purification processes and production costs only 10%-20% of traditional monoclonal antibodies. In conclusion, screening for nanobodies that specifically recognize KPC-2 carbapenemase can lay the foundation for constructing low-cost, high-performance KPC-2 immunoassay methods. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an anti-KPC-2 carbapenemase nanobody Nb-KPC-2 and its applications, aiming to solve the problems mentioned in the background art.

[0006] In a first aspect, the present invention provides an anti-KPC-2 carbapenemase nanobody Nb-KPC-2, wherein the anti-KPC-2 carbapenemase nanobody Nb-KPC-2 has the amino acid sequence shown in SEQ ID NO.1.

[0007] Furthermore, the nucleotide sequence encoding the anti-KPC-2 carbapenemase nanobody Nb-KPC-2 is shown in SEQ ID NO.2.

[0008] Furthermore, a method for producing an anti-KPC-2 carbapenemase nanobody Nb-KPC-2 includes the following steps:

[0009] Step S1: Prepare KPC-2 type carbapenemase protein;

[0010] Step S2: KPC-2 carbapenemase protein was obtained through phage display and multiple rounds of screening to obtain anti-KPC-2 carbapenemase nanobody Nb-KPC-2;

[0011] Step S3: Expression and purification of Nb-KPC-2 anti-KPC-2 carbapenemase nanobody.

[0012] Furthermore, the application of an anti-KPC-2 carbapenemase nanobody Nb-KPC-2 in the preparation of a reagent or kit for the rapid detection of drug-resistant Gram-negative bacteria in clinical samples, wherein the reagent or kit rapidly detects whether the drug-resistant Gram-negative bacteria produce KPC-2 carbapenemase.

[0013] Furthermore, the clinical samples include blood, urine, sputum, or wound secretions.

[0014] Furthermore, the drug-resistant Gram-negative bacteria include Klebsiella pneumoniae, Escherichia coli, or Pseudomonas aeruginosa.

[0015] Secondly, the present invention provides a KPC-2 type carbapenemase isolation and purification kit based on immunoaffinity purification, comprising the aforementioned anti-KPC-2 type carbapenemase nanobody Nb-KPC-2.

[0016] Thirdly, the present invention provides a highly specific molecular probe, including the aforementioned anti-KPC-2 carbapenemase nanobody Nb-KPC-2.

[0017] This invention offers the following advantages: It provides a nanobody capable of recognizing KPC-2 carbapenemase, possessing the amino acid sequence shown in SEQ ID NO.1, laying the foundation for constructing a low-cost and stable immunoassay method for KPC-2 carbapenemase. Utilizing the characteristics of small size, good stability, and ease of labeling, nanobodies can be applied to the rapid detection of pathogenic microorganisms, such as in the preparation of diagnostic test strips, reagents, and kits; they can also be applied to basic research and tool development, such as as molecular probes, isolation and purification kits, and the construction of core components for targeted therapeutic molecules; enabling early and accurate screening and identification of drug-resistant Gram-negative bacteria producing KPC-2 carbapenemase, guiding precise clinical medication and infection control. Attached Figure Description

[0018] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures:

[0019] Figure 1 Example 1 of the present invention uses SDS-PAGE to identify the expression of KPC-2 type carbapenemase. In the figure: M is the marker, and 1 is the sample lane.

[0020] Figure 2 This is the KPC-2 type carbapenemase identified and purified by SDS-PAGE in Example 1 of the present invention. In the figure: M is the marker, and 1 is the sample lane.

[0021] Figure 3 The anti-KPC-2 carbapenemase nanoparticle phage identified and screened by Phage-ELISA in Example 1 of this invention is shown in the figure. The control is bovine serum albumin.

[0022] Figure 4The figure shows the identification results of the purified anti-KPC-2 carbapenemase nanobody Nb-KPC-2 by SDS-PAGE in Example 2 of the present invention. In the figure, M is the marker and 1 is the sample lane. Detailed Implementation

[0023] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0025] This invention provides an anti-KPC-2 carbapenemase nanobody Nb-KPC-2, which has the amino acid sequence shown in SEQ ID NO.1.

[0026] In some embodiments, the nucleotide sequence encoding the anti-KPC-2 carbapenemase nanobody Nb-KPC-2 is shown in SEQ ID NO.2.

[0027] In some embodiments, a method for producing an anti-KPC-2 carbapenemase nanobody Nb-KPC-2 includes the following steps:

[0028] Step S1: Prepare KPC-2 type carbapenemase protein;

[0029] Step S2: KPC-2 carbapenemase protein was obtained through phage display and multiple rounds of screening to obtain anti-KPC-2 carbapenemase nanobody Nb-KPC-2;

[0030] Step S3: Expression and purification of Nb-KPC-2 anti-KPC-2 carbapenemase nanobody.

[0031] In some embodiments, the reagent or kit rapidly detects whether drug-resistant Gram-negative bacteria produce KPC-2 type carbapenemase.

[0032] In some embodiments, clinical samples include blood, urine, sputum, or wound secretions.

[0033] In some embodiments, drug-resistant Gram-negative bacteria include Klebsiella pneumoniae, Escherichia coli, or Pseudomonas aeruginosa.

[0034] In some embodiments, the present invention provides a KPC-2 carbapenemase isolation and purification kit based on immunoaffinity purification, comprising an anti-KPC-2 carbapenemase nanobody Nb-KPC-2.

[0035] In some embodiments, the present invention provides a highly specific molecular probe, including an anti-KPC-2 carbapenemase nanobody Nb-KPC-2, for studying the structure, function, catalytic mechanism, resistance transmission, and interaction with the host of the KPC-2 enzyme.

[0036] Example 1: Screening of anti-KPC-2 carbapenemase nanobodies

[0037] (1) Preparation of KPC-2 carbapenemase protein: The KPC-2 carbapenemase protein sequence was obtained from the NCBI database. After codon optimization, the corresponding DNA gene fragment was synthesized, and the pET-22b-KPC-2 recombinant expression plasmid was constructed through homologous recombination. After successful sequencing, the expression plasmid was transformed into chemically competent E. coli Rosetta cells. Single colonies were picked the day after plating on LB / Amp resistance plates. After bacterial culture and IPTG induction, the expression of KPC-2 carbapenemase was identified by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The results are as follows: Figure 1 As shown, a distinct band was observed at 33 kDa, indicating good expression of KPC-2 carbapenemase. Further purification was performed using a nickel column, and identification was conducted using SDS-PAGE. The results are shown below. Figure 2 As shown, there is a high-purity protein band at 33 kDa, with a purity greater than 90%, indicating good purification effect.

[0038] (2) Panning of nanobodies against KPC-2 carbapenemase: First, KPC-2 carbapenemase protein was diluted to the required concentration using carbonate buffer at pH 8.6, as shown in Table 1. 100 μL of KPC-2 carbapenemase protein was added to a 96-well plate and incubated at 37°C for 2 h. After incubation, the 96-well plate was washed three times with PBS. After washing, 300 μL of protein blocking buffer was added to each well. The protein blocking buffer consisted of 3% bovine serum albumin (BSA) and 3% ovalbumin (OVA) solution, used alternately, and incubated at 37°C for 2 h. After incubation, the 96-well plate was washed three times with PBS. After washing, 100 μL of diluted phage display native nanobody library (AlpSDab-P) was added to each well. The library loading was 1 × 10⁻⁶. 11PFU / well, incubate at 37℃ for 1 h; after incubation, wash the 96-well plate 10 times with PBST solution, then wash the 96-well plate 10 times with PBS; after washing, add 94.5 μL of pH 2.2 Gly-HCl elution buffer to each well and incubate on a horizontal shaker at room temperature for 8 min; after incubation, add 5.5 μL of pH 9.0 Tris-HCl neutralization buffer to neutralize, obtaining 100 μL of neutralization solution; take 10 μL of neutralization solution for serial dilution and infect E. coli ER2738, plate on LB / Amp resistance plates to measure the eluted phage titer; the remaining 90 μL of neutralization solution is used to infect E. coli ER2738 for phage amplification, and the obtained phage is used for the next round of panning;

[0039] To obtain nanobodies with high affinity for the antigen, four rounds of panning were conducted. The panning conditions and experimental parameters for each round are shown in Table 1. After the fourth round of panning, the nanobodies were identified by Phage-ELISA (phage enzyme-linked immunosorbent assay), and the results are as follows. Figure 3 As shown, most of the 16 selected clones bind to KPC-2 carbapenemase. Sequencing confirmed that their amino acid sequences were the same, indicating that the anti-KPC-2 carbapenemase nanobody was effectively enriched. The nanobody was named Nb-KPC-2.

[0040] Table 1. Panning conditions and experimental results of anti-KPC-2 carbapenemase nanobodies

[0041]

[0042] The anti-KPC-2 carbapenemase nanobody Nb-KPC-2 has the amino acid sequence shown in SEQ ID NO.1.

[0043] amino acid sequence:

[0044] SEQ ID NO:1:

[0045] QVQLVESGGGLVQPGGSLRLSCAFSGSIFHIYAMGWYRQAQGNQRELVAIITPGGRTNYADPVKGRFAISRDSEKNSAYLEMNSLNPEDTAVYYCYAKRLVSGVGDGNYWGQGTQVTVSS

[0046] The nucleotide sequence encoding the anti-KPC-2 carbapenemase nanobody Nb-KPC-2 is shown in SEQ ID NO.2.

[0047] Nucleotide sequence:

[0048] SEQ ID NO.2:

[0049] CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCATTCTCTGGAAGCATCTTCCATATCTATGCCATGGGCTGGTACCGCCAGGCTCAAGGGAATCAGCGCGAGTTGGTCGCGATTATCACTCCTGGCGGCAGGACAAACTATGCA GACCCCGTGAAGGGCCGATTCGCCATCTCCAGAGACAGCGAAAAAAACTCTGCGTATTTGGAAATGAACAGCCTGAACCCTGAGGACACGGCCGTCTATTACTGTTATGCAAAAAGATTAGTGAGTGGCGTAGGGGACGGTAACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCG

[0050] Example 2: Expression and purification of Nb-KPC-2 anti-KPC-2 carbapenemase nanobody

[0051] (1) The phage vector pComb3XSS-Nb-KPC-2 was transformed into chemically competent cells E. coli Rosetta by heat shock at 42℃ for 90s, plated on LB / Amp resistance plates, and single colonies were picked the next day;

[0052] (2) Add the above single colony and 5 μL of 100 mg / mL ampicillin to 5 mL of LB liquid medium, and culture at 37 °C and 180 rpm for 12 h. After the culture is completed, the bacterial solution is obtained.

[0053] (3) Inoculate the bacterial culture at a rate of 1% (v / v) into 100 mL of self-induction medium and add 50 μL of 100 mg / mL ampicillin; culture at 37 ℃ and 180 rpm for 3 h to the logarithmic phase (OD600 reaches 0.5-0.7), then change the culture conditions to 23 ℃ and 130 rpm for 12 h to induce protein expression;

[0054] (4) Centrifuge at 8000 rpm for 10 min to obtain bacterial cells, and resuspend them in 20 mL of purification equilibration buffer to obtain bacterial resuspension;

[0055] (5) Add 20 mg of lysozyme to the bacterial suspension, with a final concentration of 1 mg / mL. Incubate at 4 °C on a horizontal shaker for 30 min. Then, use a cell sonicator to break down the bacteria and obtain the broken product.

[0056] (6) The broken product was centrifuged at 13000g, 4℃ for 30min to obtain protein supernatant; and the protein supernatant was purified by filtration using a 0.22μm aqueous filter membrane to remove impurities and obtain purified protein supernatant.

[0057] (7) Add 2 mL of Ni-NTA column material to the purification column. After the Ni-NTA settles naturally, wash with at least 5 column volumes of purified water to remove impurities, and then equilibrate the purification column with 15 mL of equilibration buffer.

[0058] (8) Add the purified protein supernatant to the purification column in batches, allowing the purified protein supernatant to bind with Ni-NTA for 10 min, and then let it flow out slowly, collecting each flow-through.

[0059] (9) Wash the purified material with equilibration buffer (containing imidazole), eluting stepwise from low imidazole concentration to high imidazole concentration. The low imidazole concentration is used to wash away any contaminating proteins in the purification column. Each imidazole concentration requires at least 20 mL of rinsing.

[0060] (10) Finally, the nickel column-bound nanobody protein was eluted with 250 mM imidazole and 5 mL of eluent was collected.

[0061] (11) The collected target protein solution was transferred to a 3kDa dialysis bag for desalting. The solution was placed in PBS dialysis solution with pH=7.4 and concentration of 10mmol / L at 4℃ for dialysis. The dialysis solution was changed every 8 hours and the dialysis time was 1 day.

[0062] (12) After dialysis, the protein solution was concentrated using a 3 kDa ultrafiltration tube and centrifuged multiple times at 4 °C, 3000 g / min, for 10 min until the final protein solution was retained at about 1 mL. The protein concentration was determined using a NanoDrop 2000 micro spectrophotometer. After the determination, half the volume of glycerol was added, and the final glycerol concentration was 50%. The solution was dispensed into 500 μL centrifuge tubes and stored in a freezer at -80 °C.

[0063] The purified anti-KPC-2 carbapenemase nanobody Nb-KPC-2 was identified by SDS-PAGE, and the identification results are as follows: Figure 4As shown, the results indicate that a clear band appears at 17 kDa, which is consistent with the theoretical molecular weight of the nanobody, and the protein has high purity (single band with no obvious interference), indicating that the purified anti-KPC-2 carbapenemase nanobody Nb-KPC-2 was successfully obtained.

[0064] In summary, this invention prepared KPC-2 carbapenemase protein, which was then processed using phage display and four rounds of panning to obtain the anti-KPC-2 carbapenemase nanobody Nb-KPC-2. The Nb-KPC-2 nanobody was further expressed and purified, laying the foundation for constructing a low-cost and stable immunoassay method for KPC-2 carbapenemase. Utilizing the characteristics of small size, good stability, and ease of labeling, nanobodies can be applied to the rapid detection of pathogenic microorganisms, such as the preparation of diagnostic test strips, reagents, and kits; they can also be applied to basic research and tool development, such as as molecular probes, isolation and purification kits, and the construction of core components for targeted therapy molecules; enabling early and accurate screening and identification of drug-resistant Gram-negative bacteria producing KPC-2 carbapenemase, guiding precise clinical medication and infection control.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A nanobody Nb-KPC-2 against KPC-2 type carbapenemase, characterized in that: The anti-KPC-2 carbapenemase nanobody Nb-KPC-2 has the amino acid sequence shown in SEQ ID NO.

1.

2. The anti-KPC-2 carbapenemase nanobody Nb-KPC-2 as described in claim 1, characterized in that: The nucleotide sequence encoding the anti-KPC-2 carbapenemase nanobody Nb-KPC-2 is shown in SEQ ID NO.

2.

3. The application of the anti-KPC-2 carbapenemase nanobody Nb-KPC-2 as described in claim 1 or 2 in the preparation of reagents or kits for rapid detection of drug-resistant Gram-negative bacteria in clinical samples, characterized in that: The reagent or kit is used to rapidly detect whether the drug-resistant Gram-negative bacteria produce KPC-2 carbapenemase, including Klebsiella pneumoniae, Escherichia coli, or Pseudomonas aeruginosa.

4. The application as described in claim 3, characterized in that: The clinical samples include blood, urine, sputum, or wound secretions.

5. A kit for isolating and purifying KPC-2 type carbapenemase based on immunoaffinity purification, characterized in that: Including the anti-KPC-2 carbapenemase nanobody Nb-KPC-2 as described in claim 1 or 2.

6. A highly specific molecular probe, characterized in that: Including the anti-KPC-2 carbapenemase nanobody Nb-KPC-2 as described in claim 1 or 2.

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