Anti-KPC-2 carbapenemase nano antibody Nb-KPC-2 and application thereof

Abstract

The invention belongs to the technical field of carbapenem-resistant enterobacter spp. Bacterium detection, and provides an anti-KPC-2 type carbapenemase nano antibody Nb-KPC-2 and application of the anti-KPC-2 type carbapenemase nano antibody Nb-KPC-2. The anti-KPC-2 type carbapenemase nano antibody Nb-KPC-2 has an amino acid sequence as shown in SEQ ID NO. 1, and the amino acid sequence is as shown in SEQ ID NO. 2. The invention lays a foundation for constructing an immunoassay method of KPC-2 type carbapenemase with low cost and stable performance. By utilizing the characteristics of small volume, good stability and easiness in marking of the nano antibody, the nano antibody can be applied to rapid detection of pathogenic microorganisms, such as preparation of diagnostic test strips, reagents, kits and the like; the method can also be applied to basic research and tool development, such as molecular probes, separation and purification kits, construction of targeted therapy molecular core elements and the like; the early-stage and accurate screening and identification of the drug-resistant gram-negative bacteria for producing the KPC-2 type carbapenemase are realized, and the clinical precise medication and infection control are guided.

Description

Technical Field

[0001] The present invention belongs to the technical field of carbapenem-resistant Enterobacteriales bacteria detection, and in particular relates to an anti-KPC-2 carbapenemase nanoantibody Nb-KPC-2 and applications thereof. Background Art

[0002] With the widespread global use of carbapenem antibiotics, the infection rate of carbapenem-resistant Enterobacteriaces (CRE) has increased annually. Amid this resistance crisis, strains producing Klebsiella pneumoniae carbapenemase (KPC) have demonstrated strong transmission capabilities and the ability to horizontally transfer resistance genes. These strains secrete KPC-type carbapenemases, which specifically hydrolyze the β-lactam ring of carbapenems, resulting in loss of the drugs' key antimicrobial activity. Notably, the KPC-2 subtype dominates the KPC-type carbapenemases family. Its encoding gene can be transmitted between Enterobacteriaces bacteria via plasmids, even crossing species barriers and causing the spread of multidrug resistance.

[0003] The rapid detection and typing of carbapenem-resistant Enterobacteriales bacteria are crucial for the rational use of antibiotics, the determination of treatment plans, and the rapid control of infections. At present, due to the lack of rapid typing detection methods, clinical treatment generally adopts an empirical broad-spectrum medication model, which leads to the abuse of antibiotics, increased treatment failure rates, and accelerated evolution of drug-resistant mutants. Establishing a precise medication strategy based on carbapenemase typing is one of the core measures to slow the spread of drug resistance. The current clinical typing technology lags seriously: the molecular diagnostic method based on nucleic acid amplification is time-consuming and relies on molecular biology laboratories and professional technicians; although immunological testing is simple to operate and has a short detection time, it relies on high-performance antibodies.

[0004] Nanobodies (Nb), single-domain antibodies derived from the variable region of camelid heavy-chain antibodies, have a molecular weight of only 15 kDa, less than one-tenth the size of traditional IgG antibodies. They can penetrate the outer membrane barrier of Gram-negative bacteria and specifically bind to KPC-2 carbapenemases. Due to their lack of light chains and their rigid structure stabilized by only two disulfide bonds, nanobodies are resistant to inactivation at high temperatures, extreme pH values, or in the presence of proteases. Lyophilized nanobodies coated on nitrocellulose membrane strips can be stored at room temperature for over 18 months without the need for cold chain transportation, thus overcoming the storage and transportation limitations of traditional reagents at temperatures between 2 and 8°C. Furthermore, nanobodies can be efficiently expressed in Escherichia coli or Pichia pastoris systems, simplifying purification processes and resulting in production costs that are only 10%-20% of those of traditional monoclonal antibodies. In summary, the identification of nanobodies that specifically recognize KPC-2 carbapenemases will pave the way for the development of low-cost, high-performance KPC-2 immunoassays. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the present invention provides an anti-KPC-2 type carbapenemase nanobody Nb-KPC-2 and its application, which aims to solve the problems mentioned in the background technology.

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

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

[0008] Furthermore, a method for producing an anti-KPC-2 type carbapenemase nanobody Nb-KPC-2 comprises the following steps: Step S1: preparing KPC-2 type carbapenemase protein; Step S2: The KPC-2 carbapenemase protein is screened by phage display and subjected to multiple rounds of panning to obtain the anti-KPC-2 carbapenemase nanobody Nb-KPC-2; Step S3: Expression and purification of anti-KPC-2 type carbapenemase nanobody Nb-KPC-2.

[0009] Furthermore, an anti-KPC-2 carbapenemase nanobody Nb-KPC-2 is used in the preparation of a reagent or kit for 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.

[0010] Furthermore, the clinical sample includes blood, urine, sputum or wound secretions.

[0011] Furthermore, the drug-resistant Gram-negative bacteria include Klebsiella pneumoniae, Escherichia coli or Pseudomonas aeruginosa.

[0012] In a second aspect, the present invention provides a KPC-2 carbapenemase separation and purification kit based on immunoaffinity purification, comprising the anti-KPC-2 carbapenemase nanobody Nb-KPC-2.

[0013] In a third aspect, the present invention provides a highly specific molecular probe, comprising the anti-KPC-2 type carbapenemase nanobody Nb-KPC-2.

[0014] The present invention has the following beneficial effects: providing a nanobody capable of recognizing KPC-2 carbapenemase, having an amino acid sequence as shown in SEQ ID NO.1, laying the foundation for constructing a low-cost and stable immunoassay method for KPC-2 carbapenemase. Taking advantage of the small size, good stability, and easy labeling characteristics of nanobodies, they 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, separation and purification kits, and the construction of core components of targeted therapeutic molecules; achieving early and accurate screening and identification of drug-resistant Gram-negative bacteria that produce KPC-2 carbapenemase, and guiding clinical precision medication and infection control. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] A more complete understanding of exemplary embodiments of the present invention may be obtained by referring to the following drawings: Figure 1 The expression of KPC-2 carbapenemase was identified by SDS-PAGE in Example 1 of the present invention. In the figure, M is a marker and 1 is a sample lane. Figure 2 The purified KPC-2 carbapenemase was identified by SDS-PAGE in Example 1 of the present invention. In the figure, M is a marker and 1 is a sample lane. Figure 3 This is the anti-KPC-2 type carbapenemase nanobody phage identified and screened by Phage-ELISA in Example 1 of the present invention. In the figure: the control is bovine serum albumin; Figure 4 This is the identification result of the purified anti-KPC-2 type carbapenemase nanobody Nb-KPC-2 by SDS-PAGE in Example 2 of the present invention, in which: M is a marker and 1 is a sample lane. DETAILED DESCRIPTION

[0016] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0017] 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 belongs; the terms used herein are only for the purpose of describing specific embodiments rather than limiting the present invention.

[0018] An embodiment of the present invention provides an anti-KPC-2 type carbapenemase nanobody Nb-KPC-2, and the anti-KPC-2 type carbapenemase nanobody Nb-KPC-2 has the amino acid sequence shown in SEQ ID NO.1.

[0019] In some embodiments, the nucleotide sequence encoding the anti-KPC-2 type carbapenemase nanobody Nb-KPC-2 is shown as SEQ ID NO.2.

[0020] In some embodiments, a method for producing an anti-KPC-2 type carbapenemase nanobody Nb-KPC-2 comprises the following steps: Step S1: preparing KPC-2 type carbapenemase protein; Step S2: The KPC-2 carbapenemase protein is screened by phage display and subjected to multiple rounds of panning to obtain the anti-KPC-2 carbapenemase nanobody Nb-KPC-2; Step S3: Expression and purification of anti-KPC-2 type carbapenemase nanobody Nb-KPC-2.

[0021] In some embodiments, the reagent or kit is used to rapidly detect whether drug-resistant Gram-negative bacteria produce KPC-2 type carbapenemase.

[0022] In some embodiments, the clinical sample comprises blood, urine, sputum, or wound secretions.

[0023] In some embodiments, the drug-resistant Gram-negative bacteria include Klebsiella pneumoniae, Escherichia coli, or Pseudomonas aeruginosa.

[0024] In some embodiments, the present invention provides a KPC-2 carbapenemase separation and purification kit based on immunoaffinity purification, comprising an anti-KPC-2 carbapenemase nanobody Nb-KPC-2.

[0025] 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, drug resistance transmission, and interaction of the KPC-2 enzyme with the host.

[0026] Example 1: Screening of anti-KPC-2 carbapenemase nanobodies (1) Preparation of KPC-2 carbapenemase protein: The KPC-2 carbapenemase protein sequence was obtained from the NCBI database. The corresponding DNA gene fragment was synthesized after codon optimization, and the pET-22b-KPC-2 recombinant expression plasmid was constructed by homologous recombination. After successful sequencing, the expression plasmid was transformed into chemically competent cells E. coli Rosetta. A single colony was picked the next day after coating the LB / Amp resistance plate. 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 in Figure 2, there is a clear band at 33 kDa, indicating that the expression of KPC-2 carbapenemase is good. It was further purified using a nickel column and identified using SDS-PAGE. The results are shown in Figure 2. Figure 2 As shown in the figure, there is a high-purity protein band at 33 kDa with a purity greater than 90%, indicating that the purification effect is good.

[0027] (2) Selection of nanoantibodies against KPC-2 carbapenemase: First, the KPC-2 carbapenemase protein was diluted to the required concentration with 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 3 times with PBS; after washing, 300 μL of protein blocking solution was added to each well, and the protein blocking solution was 3% bovine serum albumin solution (BSA) and 3% ovalbumin solution (OVA), which were used alternately at 37°C for 2 h; after incubation, the 96-well plate was washed 3 times with PBS; after washing, 100 μL of diluted phage-displayed natural nanoantibody library (AlpSDab-P) was added to each well, and the library input was 1×10 11pfu / well, incubate at 37°C for 1 hour; after incubation, wash the 96-well plate 10 times with PBST solution, and then wash the 96-well plate 10 times with PBS; after washing, add 94.5 μL of Gly-HCl eluent (pH = 2.2) to each well and incubate on a horizontal shaker at room temperature for 8 minutes; after incubation, aspirate the eluent and add it to 5.5 μL of Tris-HCl neutralization buffer (pH = 9.0) for neutralization to obtain 100 μL of neutralization solution; take 10 μL of neutralization solution for serial dilution and infect E. coli ER2738, spread it on LB / Amp resistance plate to measure the titer of eluted phage; the remaining 90 μL of neutralization solution is infecting E. coli ER2738 for phage amplification, and the obtained phage is used for the next round of panning; In order to obtain nanobodies with high affinity to the antigen, four rounds of panning were carried out. The panning conditions and experimental parameters of 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). The results are as follows Figure 3 As shown, most of the 16 selected clones bind to KPC-2 carbapenemase. After sequencing, it was determined that their amino acid sequences were the same sequence, indicating that the anti-KPC-2 carbapenemase nanoantibody was effectively enriched, and the nanoantibody was named Nb-KPC-2.

[0028] Table 1 Panning conditions and experimental results of anti-KPC-2 carbapenemase nanobodies

[0029] The anti-KPC-2 type carbapenemase nanobody Nb-KPC-2 has the amino acid sequence shown in SEQ ID NO.1.

[0030] Amino acid sequence: SEQ ID NO: 1: QVQLVESGGGLVQPGGSLRLSCAFSGSIFHIYAMGWYRQAQGNQRELVAIITPGGRTNYADPVKGRFAISRDSEKNSAYLEMNSLNPEDTAVYYCYAKRLVSGVGDGNYWGQGTQVTVSS

[0031] The nucleotide sequence encoding the anti-KPC-2 type carbapenemase nanobody Nb-KPC-2 is shown in SEQ ID NO.2.

[0032] Nucleotide sequence: SEQ ID NO.2: CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCATTCTCTGGAAGCATCTTCCATATCTATGCCATGGGCTGGTACCGCCAGGCTCAAGGGAATCAGCGCGAGTTGGTCGCGATTATCACTCCTGGCGGCAGGACAAACTATGCA GACCCCGTGAAGGGCCGATTCGCCATCTCCAGAGACAGCGAAAAAAACTCTGCGTATTTGGAAATGAACAGCCTGAACCCTGAGGACACGGCCGTCTATTACTGTTATGCAAAAAGATTAGTGAGTGGCGTAGGGGACGGTAACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCG

[0033] Example 2: Expression and purification of anti-KPC-2 carbapenemase nanobody Nb-KPC-2 (1) The phage vector pComb3XSS-Nb-KPC-2 was transformed into chemically competent E. coli Rosetta cells by heat shock at 42°C for 90 seconds, spread on LB / Amp resistance plates, and single colonies were picked the next day; (2) Add the above single colony and 5 μL of 100 mg / mL ampicillin to 5 mL of LB liquid culture medium, and culture at 37°C and 180 rpm on a shaking platform for 12 h. After the culture is completed, obtain the bacterial solution; (3) Inoculate the bacterial suspension into 100 mL of autoinduction medium at a 1% (v / v) inoculum volume and add 50 μL of 100 mg / mL ampicillin. Incubate at 37°C, 180 rpm, and shake for 3 h until the logarithmic phase (OD600 reaches 0.5-0.7). Change the culture conditions to 23°C, 130 rpm, and incubate for 12 h to induce protein expression. (4) Centrifuge at 8000 rpm for 10 min to obtain the bacterial cells, and resuspend them in 20 mL of purification equilibration buffer to obtain a bacterial resuspension; (5) Add 20 mg of lysozyme to the bacterial resuspension to a final concentration of 1 mg / mL. Perform enzymatic hydrolysis on a horizontal shaker at 4°C for 30 min. Then, use a cell ultrasonic disruptor to disrupt the bacteria and obtain a broken product. (6) The crushed product was centrifuged at 13000g and 4°C for 30 min to obtain a protein supernatant; the protein supernatant was filtered through a 0.22 μm water filter membrane to remove impurities and then purified to obtain a purified protein supernatant; (7) Add 2 mL of Ni-NTA column material to the purification column. After the Ni-NTA is naturally settled, wash it with at least 5 column volumes of purified water to remove impurities, and then equilibrate the purification column with 15 mL of equilibration buffer; (8) Add the purified protein supernatant to the purification column in batches, allow the purified protein supernatant to bind to Ni-NTA for 10 minutes, then allow it to flow out slowly, and collect the flow-through each time; (9) Use equilibrium buffer (containing imidazole) for washing and purification, eluting gradually from low imidazole concentration to high imidazole concentration. Low imidazole concentration is used to wash the impurities bound to the purification column. Each imidazole concentration should be washed for at least 20 mL. (10) Finally, 250 mM imidazole was used to elute the nanobody protein bound to the nickel column, and 5 mL of the eluate was collected; (11) The collected target protein solution was transferred to a 3 kDa dialysis bag for desalting. The solution was placed in a 4 °C refrigerator in PBS dialysis solution with a pH of 7.4 and a concentration of 10 mmol / L for dialysis. The dialysis solution was replaced every 8 hours for a 1-day dialysis period. (12) After dialysis, the protein solution was concentrated using a 3 kDa ultrafiltration tube and centrifuged multiple times at 4 °C, 3000 g / min, 10 min until the final protein solution retained about 1 mL. The protein concentration was determined using a NanoDrop 2000 microspectrophotometer. After the determination was completed, half the volume of glycerol was added to a final glycerol concentration of 50%. The solution was divided into 500 μL centrifuge tubes and stored in a -80 °C refrigerator.

[0034] The purified anti-KPC-2 carbapenemase nanobody Nb-KPC-2 was identified by SDS-PAGE. Figure 4 As shown, the results showed that a clear band appeared at 17 kDa, which was consistent with the theoretical molecular weight of the nanobody, and the protein was highly pure (single band, no obvious interference from other bands), indicating that the purified anti-KPC-2 type carbapenemase nanobody Nb-KPC-2 was successfully obtained.

[0035] In summary, the present invention prepares KPC-2 carbapenemase protein, and then the KPC-2 carbapenemase protein is subjected to phage display and four rounds of panning to obtain the anti-KPC-2 carbapenemase nanoantibody Nb-KPC-2. The anti-KPC-2 carbapenemase nanoantibody Nb-KPC-2 is then expressed and purified, laying the foundation for the construction of a low-cost and stable immunoassay method for KPC-2 carbapenemase. Utilizing the characteristics of small size, good stability, and easy labeling, nanoantibodies 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, separation and purification kits, and the construction of core components of targeted therapeutic molecules; achieving early and accurate screening and identification of drug-resistant Gram-negative bacteria that produce KPC-2 carbapenemase, guiding clinical precision medication and infection control.

[0036] 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 in the scope of protection of the present invention.

Claims

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

2.

3. The method for producing an anti-KPC-2 type carbapenemase nanobody Nb-KPC-2 according to claim 1 or 2, characterized in that: The following steps are involved: Step S1: preparing KPC-2 type carbapenemase protein; Step S2: The KPC-2 carbapenemase protein is screened by phage display and subjected to multiple rounds of panning to obtain the anti-KPC-2 carbapenemase nanobody Nb-KPC-2; Step S3: Expression and purification of anti-KPC-2 type carbapenemase nanobody Nb-KPC-2.

4. Use of an anti-KPC-2 carbapenemase nanobody Nb-KPC-2 according to claim 1 or 2 in the preparation of a reagent or kit for rapid detection of drug-resistant Gram-negative bacteria in clinical samples, characterized in that: The reagent or kit can quickly detect whether the drug-resistant Gram-negative bacteria produce KPC-2 type carbapenemase.

5. The use according to claim 4, characterized in that: The clinical samples include blood, urine, sputum or wound secretions.

6. The use according to claim 5, characterized in that: The drug-resistant Gram-negative bacteria include Klebsiella pneumoniae, Escherichia coli or Pseudomonas aeruginosa.

7. A KPC-2 carbapenemase separation and purification kit based on immunoaffinity purification, characterized by: It comprises the anti-KPC-2 type carbapenemase nanobody Nb-KPC-2 according to claim 1 or 2.

8. A highly specific molecular probe, characterized in that: It comprises the anti-KPC-2 type carbapenemase nanobody Nb-KPC-2 according to claim 1 or 2.

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