Detection method of carbapenem-resistant enterobacter (CRE) antibody

Abstract

The invention provides an antigen protein. The antigen protein is any one or a combination of more of proteins as shown in SEQ ID NO: 1-5. The antigen protein provided by the invention can be used for detecting the existence of carbapenem-resistant enterobacter (CRE) in a subject. The invention also provides a detection reagent or a kit and a method for detecting or diagnosing carbapenem-resistant enterobacter (CRE) by using the antigen protein, the detection reagent or the kit. The method disclosed by the invention has the advantages of simple material taking, low price, high detection speed, high detection rate and the like, so that the method is very suitable for on-site detection or bedside detection of CRE.

Description

Technical Field

[0001] The present invention relates to the field of detection. Specifically, the present invention relates to a method for detecting antibodies against carbapenem-resistant Enterobacteriaceae (CRE). Background Art

[0002] Carbapenem-resistant Enterobacteriaceae (CRE) refers to Enterobacteriaceae bacteria that are resistant to carbapenem antibiotics. The Centers for Disease Control and Prevention (CDC) in the United States defines carbapenem-resistant Enterobacteriaceae (CRE) as Enterobacteriaceae bacteria that are insensitive to doripenem, ertapenem, meropenem, or imipenem, or it is confirmed that Enterobacteriaceae bacteria produce carbapenemase. Among them, Enterobacteriaceae includes 62 genera, and 10 genera are commonly involved in human infections, which are important clinical infectious pathogens. Carbapenem antibiotics have the characteristics of a broad antibacterial spectrum, strong antibacterial activity, stability against β-lactamase, and low toxicity. With their widespread application, drug resistance has increased rapidly. Among them, CRE has attracted much attention due to its high drug resistance, fast transmission speed, and high lethality, and has been listed by the CDC as an urgent global drug resistance threat.

[0003] Among various infectious diseases that occur in hospitals, the most common pathogenic bacteria are mainly Enterobacteriaceae bacteria, including Klebsiella pneumoniae and Escherichia coli, which can cause diseases such as wound infections, respiratory system or urinary system diseases in patients. The most effective drug among antibacterial drugs is carbapenem drugs, which belong to a class of β-lactam antibiotics and have the broadest antibacterial spectrum. However, with the widespread use of antibacterial drugs globally and the emergence of carbapenemase, the effectiveness of this antibacterial drug is getting smaller and smaller. Carbapenem-resistant Enterobacteriaceae (CRE) has thus emerged. In recent years, with the widespread application of broad-spectrum antibacterial drugs, the proportion of multi-drug resistant Enterobacteriaceae bacteria has increased, making it more difficult to select drug treatments. Bloodstream infections, urinary tract infections, and respiratory tract infections are the main sources of CRE infections. Among them, respiratory tract infections account for about 40% of the susceptible population, and studies have shown that the common infected departments are mainly the intensive care unit and the respiratory intensive care unit. Among them, the intensive care unit accounts for about 32%, and the respiratory intensive care unit accounts for about 28%. Therefore, the susceptible population of CRE infection is critically ill patients, the respiratory tract is the common infection site, and the use of antibacterial drugs and endotracheal intubation before infection can both lead to CRE infection.

[0004] The existing detection methods for CRE-resistant bacteria infections mainly include two categories: phenotypic identification and genotypic identification, both of which identify resistant bacteria or a certain protein in resistant bacteria. Phenotypic identification methods include the CarbaNP test, the CIM disk synergy test, the modified Hodge test method, etc. These methods have a low cost, but the detection time is long, requiring 2 - 7 days or even longer for bacterial culture; the positive detection rate is low; and they can only detect whether carbapenemase is produced, unable to determine the specific genotype. Genotypic identification methods include enzyme immunoassay chromatography technology and molecular detection technology. These methods have relatively better sensitivity, but are costly and also require time-consuming bacterial culture. If the bacterial culture is unsuccessful, a diagnosis cannot be made.

[0005] Therefore, there is a greater clinical need for a method that can detect CRE-resistant bacteria more quickly and accurately. Summary of the Invention

[0006] The object of the present invention is to develop a low-cost and rapid detection kit for CRE detection and a method for rapidly and simply detecting CRE using the said kit.

[0007] In a first aspect, the present invention provides an antigen protein, which is any one or a combination of more than one of the proteins shown in SEQ ID NO: 1 - 5.

[0008] In a specific embodiment, the antigen protein is the protein shown in SEQ ID NO: 1.

[0009] In a specific embodiment, the antigen protein is used to detect the presence of carbapenem-resistant Enterobacteriaceae (CRE) in a subject.

[0010] In a preferred embodiment, the subject is a mammal; preferably, the subject is a primate; more preferably, the subject is a human.

[0011] In a second aspect, the present invention provides a detection reagent, which contains the antigen protein described in the first aspect.

[0012] In a preferred embodiment, the detection reagent is used for the detection of carbapenem-resistant Enterobacteriaceae (CRE).

[0013] In a third aspect, the present invention provides a kit for detecting or diagnosing carbapenem-resistant Enterobacteriaceae (CRE), which is equipped with:

[0014] 1) The antigen protein described in the first aspect or the detection reagent described in the second aspect;

[0015] 2) An instruction manual for teaching the use of the said antigen protein or detection reagent to detect the presence of carbapenem-resistant Enterobacteriaceae in a sample.

[0016] In a preferred embodiment, "detecting the presence of carbapenem-resistant Enterobacteriaceae in a sample using the antigen protein or detection reagent" means detecting the presence of antibodies against carbapenem-resistant Enterobacteriaceae in the sample using the antigen protein or detection reagent.

[0017] In a preferred embodiment, the kit further contains a well plate for coating the antigen protein.

[0018] In a preferred embodiment, the sample is the serum of a subject.

[0019] In a specific embodiment, the kit further contains a coating solution for coating the antigen protein and a diluent for diluting the sample.

[0020] In a specific embodiment, the coating solution and the diluent for diluting the sample are 1% BSA solution.

[0021] In a fourth aspect, the present invention provides the use of the antigen protein described in the first aspect in the preparation of a detection reagent or kit for detecting or diagnosing carbapenem-resistant Enterobacteriaceae (CRE).

[0022] In a fifth aspect, the present invention provides a method for detecting or diagnosing carbapenem-resistant Enterobacteriaceae (CRE), the method comprising the step of detecting the presence of carbapenem-resistant Enterobacteriaceae in a sample using the antigen protein described in the first aspect or the detection reagent described in the second aspect or the kit described in the third aspect.

[0023] In a preferred embodiment, the method comprises the following steps:

[0024] 1) Coating the antigen protein on a well plate;

[0025] 2) Contacting the sample of the subject with the well plate coated with the antigen protein;

[0026] 3) Detecting the presence of antibodies that bind to the antigen protein.

[0027] In a preferred embodiment, the sample is the serum of a subject.

[0028] In a preferred embodiment, the method is an ELISA method.

[0029] In a specific embodiment, the method is for non-diagnostic purposes.

[0030] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Shows the protein expression of NJSS-1 and KPC-2 compared by SDS-PAGE; where "1" is the whole bacteria before induction; "2" is the whole bacteria after induction; "M" is the protein Marker;

[0032] Figure 2 Shows the protein purification of NJSS-1 and KPC-2 compared by SDS-PAGE; where "1" is the sample before purification; "2" is the flow-through protein after passing through the column; "3" is the purified protein 1 eluted; "4" is the purified protein 2 eluted; "5" is the purified protein 3 eluted;

[0033] Figure 3 Compares the affinity of the full-sequence proteins of NJSS-1 and KPC-2 with the KPC-2 monoclonal antibody (mAb);

[0034] Figure 4 Shows the test results of the binding ability of NJSS-1 and mAb7 (KPC-2 monoclonal antibody);

[0035] Figure 5 Shows the optimization of the blocking conditions;

[0036] Figure 6 Shows the test results of NJSS-1 antibody in the serum samples of CRE-positive patients;

[0037] Figure 7 Shows the effect of classifying and diagnosing two types of testers (patients and normal people) by the CRE antibody detection method through ROC curve analysis. DETAILED DESCRIPTION OF THE INVENTION

[0038] The inventors have conducted extensive and in-depth research and developed a series of CRE structural proteins with brand-new sequences. Based on these CRE structural proteins, the presence of CRE can be determined by directly detecting the samples of the subjects, such as CRE antibodies in the serum. Further, the inventors have developed a low-cost and rapid detection kit for CRE detection, which can be directly applied to the detection of clinical patients, without bacterial culture, can quickly judge the resistant bacteria infection, guide the correct clinical medication, reduce the mortality rate, and finally form a clinically available CRE pre-diagnosis system. This product is an in vitro diagnostic reagent, mainly for the qualitative detection of various major genotype carbapenemase antibodies in human serum, so as to detect and monitor carbapenem resistance, and can be used for the screening of CRE patients. The present invention has been completed on this basis.

[0039] TERM DEFINITION

[0040] The terms used in this text have the meanings commonly understood by those skilled in the art. For a clear understanding of the present invention, some terms are defined as follows.

[0041] Antigen protein

[0042] The terms "antigen protein", "antigen" or "antigen protein of the present invention" used in this text have the same meaning. These terms all refer to the CRE structural protein of a brand-new sequence developed by the present inventors and capable of being used for the detection of carbapenem-resistant Enterobacteriaceae (CRE).

[0043] In a specific embodiment, the antigen protein of the present invention is any one or a combination of more than one of the proteins shown in SEQ ID NO: 1-5. In a preferred embodiment, the antigen protein of the present invention is the protein shown in SEQ ID NO: 1.

[0044] The antigen protein of the present invention can bind well with the antibodies against carbapenem-resistant Enterobacteriaceae in a sample (such as a serum sample) of a subject (including but not limited to mammals; for example, humans), and thus can be used to detect carbapenem-resistant Enterobacteriaceae in the subject; that is, for the preparation of a detection reagent or kit for detecting or diagnosing carbapenem-resistant Enterobacteriaceae (CRE).

[0045] Detection reagent, detection kit and detection method

[0046] In view of the fact that the antigen protein of the present invention can bind well with the carbapenem-resistant Enterobacteriaceae antibodies in a sample, the present invention further provides a detection reagent for the detection of carbapenem-resistant Enterobacteriaceae (CRE), and the detection reagent contains the antigen protein of the present invention.

[0047] Based on the antigen protein or detection reagent of the present invention, the present invention also provides a kit for detecting or diagnosing carbapenem-resistant Enterobacteriaceae (CRE), and the kit contains the antigen protein or detection reagent of the present invention. In a further embodiment, the kit further includes an instruction manual to teach those skilled in the art how to use the antigen protein or detection reagent to detect carbapenem-resistant Enterobacteriaceae in a sample.

[0048] Based on the teachings of the present invention and the common general knowledge in the art, those skilled in the art should also understand that the kit of the present invention further includes other devices or reagents required for carrying out the detection. For example, the kit of the present invention may further include a well plate for coating the antigen protein, or a coating solution for coating the antigen protein and a dilution solution for diluting the sample. In a preferred embodiment, the coating solution and the dilution solution for diluting the sample are 1% BSA solution.

[0049] Using the antigen protein, detection reagent or detection kit of the present invention, a person skilled in the art can detect or diagnose the presence of carbapenem-resistant Enterobacteriaceae (CRE) in a subject sample. In a specific embodiment, a person skilled in the art can coat the antigen protein on a well plate; subsequently, contact or incubate the subject's sample with the well plate coated with the antigen protein for a certain period of time; finally, detect whether an antibody binds to the antigen protein. A person skilled in the art knows how to implement the detection of the carbapenem-resistant Enterobacteriaceae (CRE), including but not limited to using the ELISA method.

[0050] The method of the present invention can detect carbapenem-resistant Enterobacteriaceae (CRE) in a subject sample, and thus can be used for the diagnosis of CRE. However, a person skilled in the art should understand that the method of the present invention can also be applied to other purposes, such as for scientific research purposes or for drug development purposes, etc. Therefore, the method of the present invention is not only used for diagnostic purposes.

[0051] The main advantages of the present invention:

[0052] 1. Simple sampling. Only a small amount of the patient's peripheral blood needs to be drawn for detection in the present invention;

[0053] 2. Low cost. Compared with existing enzyme immunoassay chromatography or molecular detection methods, etc., the price is lower;

[0054] 3. Rapid detection. The experimental results can be obtained in only 2 hours without bacterial culture, so as to enable on-site detection or bedside detection of CRE;

[0055] 4. High detection rate. For many infected patients, especially those with blood infections, it is difficult to culture bacteria, resulting in a low detection rate and difficult detection problems. This method detects the antibody of the corresponding resistant enzyme in the serum, so it can be directly detected without relying on bacterial culture.

[0056] The following specific examples further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following examples are usually carried out under conventional conditions, such as the conditions described in Sambrook and Russell, etc., (Molecular Cloning: A Laboratory Manual (Third Edition) (2001) CSHL Press), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial channels without special instructions.

[0057] Materials and Methods

[0058] Materials:

[0059] 1. Strains and plasmids

[0060] The E. coli BL21(DE3) strain was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0061] The expression plasmids NJSS-1---NJSS-5 were commissioned to be produced by Nanjing Genscript Biotech Co., Ltd.

[0062] 2. Main experimental reagents

[0063]

[0064]

[0065] 3. Experimental instruments and equipment

[0066] Instrument Name Manufacturer Small desktop constant temperature oscillator American Jingqi Company Benchtop centrifuge Thermo Fisher Scientific Vertical Pressure Steam Sterilizer Shanghai Boxun Company Ultra-low temperature refrigerator Thermo Fisher Scientific Electronic balance Shanghai Sunny Hengping Scientific Instruments Co., Ltd. Adjustable high speed homogenizer Changzhou Weijia Instrument Co., Ltd. Gel imaging system Shanghai Tianneng Technology Co., Ltd. Small ice machine Shanghai Yuming Instrument Co., Ltd. Clean bench Sujing Antai Instrument Co., Ltd. High-speed refrigerated centrifuge Thermo Fisher Scientific Ultrasonic crusher American Jingqi Company Protein Chromatography GE Healthcare Shaker American Jingqi Company Electrophoresis Apparatus Bio-Rad Corporation pH meter Mettler Toledo Water Purifier Shanghai Likang Biological Company ELISA reader Thermo Medical refrigerator Haier Company

[0067] Methods

[0068] 1. Plasmid extraction

[0069] Take 1 - 4 ml of the bacterial solution that has been cultured overnight in LB medium, centrifuge at 12000 rpm for 1 min, and discard the supernatant; add 250 μl of Buffer S1 to resuspend the bacterial pellet; add 250 μl of Buffer S2, gently and fully invert 4 - 6 times to fully lyse the bacteria until a clear solution is formed; add 350 μl of Buffer S3, gently and fully invert up and down 6 - 8 times, centrifuge at 12000 rpm for 10 min; then aspirate the supernatant after centrifugation and transfer it to a preparation tube (provided by the kit), centrifuge at 12000 rpm for 1 min, and discard the filtrate; place the preparation tube back into the centrifuge tube, add 500 μl of Buffer W1, centrifuge at 12000 rpm for 1 min, and discard the filtrate; place the preparation tube back into the centrifuge tube, add 700 μl of Buffer W2, centrifuge at 12000 rpm for 1 min, discard the filtrate, and repeat once; place the preparation tube back into a 2 ml centrifuge tube, centrifuge at 12000 rpm for 1 min; transfer the preparation tube into a new 1.5 ml centrifuge tube, add 60 - 80 μl of deionized water to the center of the membrane in the preparation tube, let it stand at room temperature for 1 min; centrifuge at 12000 rpm for 1 min, and store the obtained plasmid at -20°C.

[0070] 2. Preparation and transformation of competent cells

[0071] Under sterile conditions, pick E. coli from the strain and streak it on a plate, label it and culture it overnight in a 37°C incubator; select smooth, bright, and appropriately sized single colonies, inoculate them into 3 ml of LB, culture at 37°C and 200 rpm overnight; dilute it 1:100 into fresh 100 ml of LB medium and culture at 37°C for 2 - 3 hours until OD 600Reach 0.4 - 0.5, 4000 rpm, 4°C, centrifuge for 5 min, discard the supernatant, and collect the bacterial cells; resuspend the bacterial cells in 10 ml of pre-cooled 100 mM sterile CaCl 2 , place on ice for 20 min, centrifuge at 4000 rpm, 4°C for 5 min, discard the supernatant, and collect the bacterial cells; resuspend the bacterial cells in 4 ml of 100 mM sterile CaCl (containing 15% glycerol), aliquot and store at -80°C in the refrigerator.

[0072] Take out a tube of competent cells from the -80°C refrigerator and slowly thaw on ice (all operations need to be completed on ice); take 50 ul of competent cells, add 2 ul of plasmid, place on ice for 30 min, then place in a 42°C water bath for 90 s, and place on ice for 3 min; add 250 ul of antibiotic-free LB medium, culture at 37°C, 250 rpm for 1 h; centrifuge at 3000 rpm for 2 min on the centrifuge, discard 200 ul of the supernatant, and resuspend the remaining part by pipetting; take two plates with the corresponding resistance, evenly spread 1 / 10 of the bacterial solution on one half and 9 / 10 of the bacterial solution on the other half, and culture overnight at 37°C.

[0073] 3. Protein induction expression and purification

[0074] Pick the protein expression strain stored at -80°C and evenly spread it on an LB solid plate containing the corresponding antibiotic, and culture overnight at 37°C; according to the principle of "small bright circle", pick the transformants from the LB plate to the LB liquid medium containing resistance the next day, and shake at 37°C, 250 rpm until the OD 600 reaches 0.8 - 1.0, add IPTG, culture at 37°C, 250 rpm for 4 h, take samples, centrifuge at 12000 rpm for 5 min, discard the supernatant, and prepare samples for SDS-PAGE.

[0075] After fermentation, the bacteria were harvested by centrifugation at 4500 rpm, 4 °C for 30 min; the wet bacteria were added with normal saline at 20 ml / g, homogenized, and magnetically stirred for 15 min until there were no obvious particles, then centrifuged at 4500 rpm, 4 °C for 15 min and washed three times; the wet bacteria were added with ultrasonic buffer at 20 ml / g, homogenized, and magnetically stirred for 15 min until there were no obvious particles, and lysed on ice bath; after the treatment, the supernatant was collected at 4 °C, 18000 rpm for 10 min, and the precipitate was added with 1*PBS at pH 7.4, 10% glycerol, and 10 mM imidazole at 20 ml / g, mixed and stirred, treated on ice for 30 min, centrifuged at 4 °C, 18000 rpm for 10 min, the supernatant was collected, the two supernatants were mixed, centrifuged at 18000 rpm, 4 °C for 30 min, and passed through a membrane to prepare for purification. A 30 ml NI column was selected, loaded at 2.5 ml / min, eluted at 3 ml / min, and linearly eluted with 0-100% B; the different elution groups were dialyzed separately, loaded into a dialysis bag (3500D), placed in 2 L of dialysis solution, dialyzed overnight at 4 °C with magnetic stirring, transferred to a new 2 L of dialysis solution the next day, dialyzed for 4 h with magnetic stirring, and the samples were collected.

[0076] 4. BCA Protein Concentration Determination

[0077] For the specific operation steps, refer to the operation manual of the BCA kit from Solarbio Life Sciences. The general process includes the preparation of BCA working solution; the preparation of standard BSA; the dilution of test samples and the detection with an enzyme-linked immunosorbent assay (ELISA) reader.

[0078] 5. SDS-PAGE Protein Identification

[0079] Prepare the separating gel and stacking gel, prepare a 10% separating gel and a 4.8% stacking gel, slowly drip the gel solution along the inner surface of the long glass plate of the gel chamber with a dropper, being careful not to generate bubbles. Add the gel solution to a position 2 cm from the upper edge of the short glass plate, about 5 ml. Then inject a small amount of water, about 0.5 - 1 ml, with a fine dropper, and let it polymerize at room temperature for 30 - 40 min.

[0080] Take 1 ml of the bacteria before and after IPTG induction, centrifuge, discard the supernatant, treat the precipitate with loading buffer, boil for 10 min, and keep it at 4 °C for later use; first measure the protein concentration of the samples before purification, the effluent, and different elution solutions, then prepare the samples with loading buffer, boil for 10 min, load the samples into the sample wells of the electrophoresis gel, connect the electrophoresis apparatus, control the current at 15 - 20 mA before the sample enters the gel, for about 15 - 30 min, after the bromophenol blue indicator in the sample reaches the separating gel, raise the current to 30 - 45 mA, and keep the current stable during the electrophoresis process. After the electrophoresis is completed, take out the separating gel, put it into 0.25% Coomassie Brilliant Blue for staining, after the staining is completed, remove the staining solution, rinse with distilled water, then add the decolorizing solution, and perform diffusion decolorization until the protein bands are clear.

[0081] 6. Indirect ELISA

[0082] First, coat with antigen at 100 μl / well and incubate overnight at 4°C. The next day, add 250 μl / well of washing solution, let it stand for 1 min, wash once, add 5% BSA blocking solution at 100 μl / well, and block at 37°C for 1 h. Then pat dry the plate, add the diluted sample at 100 μl / well, and incubate at 37°C for 1 h. Wash five times with 200 μl / well of washing solution, add anti-human IgG-HRP (1:10000) at 100 μl / well, and incubate at room temperature for 30 min. Finally, wash five times with 200 μl / well of washing solution, add TMB at 100 μl / well, develop color for 5 min, add 50 μl of stop solution, and read the OD 450 Read the plate.

[0083] Example

[0084] Example 1. Design of antigen protein

[0085] After analyzing the gene sequences of different CRE-resistant strains, the inventors further analyzed the expression sequence composition and structure of KPC, IMP, OXA48like, NDM, VIM, etc. Through sequence optimization, 5 structural proteins were developed on the basis of the original protein sequence, which can be used for antibody detection of resistant enzymes of different genotypes of CRE (KPC, NDM, VIM, IMP, OXA-48 genotypes respectively), and were named NJSS-1, NJSS-2, NJSS-3, NJSS-4, NJSS-5 (corresponding to KPC, IMP, OXA48like, NDM, VIM proteins respectively). The detailed amino acid sequences are as follows:

[0086] NJSS-1 (280aa)

[0087] MPLAGFSATALTNLVAEPFAKLEQDFGGSIGVYAMDTGSGATVSYRAEERFPLCSSFKGFLAAAVLARSQQQAGLLDTPIRYGKNALVPWSPISEKYLTTGMTVAELSAAAVQYSDNAAANLLLKELGGPAGLTAFMRSIGDTTFRLDRWELELNSAIPGDARDTSSPRAVTESLQKLTLGSALAAPQRQQFVDWLKGNTTGNHRIRAAVPADWAVGDKTGTCGVYGTANDYAVVWPTGRAPIVLAVYTRAPNKDDKHSEAVIAAAARLALEGLGVNGQG (SEQ ID NO:1)

[0088] NJSS-2(237aa)

[0089] MSWNAHFTEHKSQGVVVLWNENKQQGFTNNLKRANQAFLPASTFKIPNSLIALDLGVVKDEHQVFKWDGQTRDIATWNRDHNLITAMKYSVVPVYQEFARQIGEARMSKMLHAFDYGNEDISGNVDSFWLDGGIRISATEQISFLRKLYHNKLHVSERSQRIVKQAMLTEANGDYIIRAKTGYSTRIEPKIGWWVGWVELDDNVWFFAMNMDMPTSDGLGLRQAITKEVLKQEKIIP(SEQ ID NO:2)

[0090] NJSS-3(244aa)

[0091] MGSRPKTKLEKGSMSKLSVFFIFLFCSIATAAESLPDLKIEKLDEGVYVHTSFEEVNGWGVVPKHGLVVLVNAEAYLIDTPFTAKDTEKLVTWFVERGYKIKGSISSHFHSDSTGGIEWLNSRSIPTYASELTNELLKKDGKVQATNSFSGVNYWLVKNKIEVFYPGPGHTPDNVVVWLPERKILFGGCFIKPYGLGNLGDANIEAWPKSAKLLKSKYGKAKLVVPSHSEVGDASLLKLTLEQA(SEQ ID NO:3)

[0092] NJSS-4(234aa)

[0093] MFSVDSSGEYPTVSEIPVGEVRLYQIADGVWSHISTQSFDGAVYPSNGLIVRDGDELLLIDTAWGAKNTAALLAEIEKQIGLPVTRAVSTHFHDDRVGGVDVLRAAGVATYASPSTRRLAEVEGNEIPTHSLEGLSSSGDAVRFGPVELFYPGAAHSTDNLVVYVPSASVLYGGCAIYELSRTSAGNVADADLAEWPTSIERIQQHYPEAQFVIPGHGLPGGLDLLKHTTNVVK(SEQ ID NO:4)

[0094] NJSS-5(249aa)

[0095] MLSGCMPGEIRPTIGQQMETGDQRFGDLVFRQLAPNVWQHTSYLDMPGFGAVASNGLIVRDGGRVLVVDTAWTDDQTAQILNWIKQEINLPVALAVVTHAHQDKMGGMDALHAAGIATYANALSNQLAPQEGMVAAQHSLTFAANGWVEPATAPNFGPLKVFYPGPGHTSDNITVGIDGTDIAFGGCLIKDSKAKSLGNLGDADTEHYAASARAFGAAFPKASMIVMSHSAPDSRAAITHTARMADKLR(SEQ ID NO:5)

[0096] Compared with the original protein sequences, these optimized proteins have the advantages of high expression level, easy purification, and high purity.

[0097] Taking NJSS-1 as an example, the expression level of the target protein is as high as 40% of the total bacterial protein, while the expression level of the original protein sequence of KPC-2 is about 10% of the total bacterial protein( Figure 1 ). With the same amount of bacteria, the purified protein yield of NJSS-1 is 5-8 times that of KPC-2, and the purity is increased to 95%( Figure 2 ).

[0098] Example 2. Affinity between antigen protein and carbapenem-resistant Enterobacteriaceae (CRE) antibody

[0099] The inventors coated NJSS-1 or KPC-2 protein respectively, and used the enzyme-linked immunosorbent assay method to compare the binding affinity of both with the KPC-2 monoclonal antibody. The results showed that the binding affinity between NJSS-1 and the monoclonal antibody was significantly better than that of the unoptimized KPC-2 protein( Figure 3 , P<0.001). The lowest detection limit of the KPC-2 monoclonal antibody detected by this enzyme-linked immunosorbent assay method was 0.1 ng / ml( Figure 4 ).

[0100] Example 3. Optimization of coating solution and dilution solution

[0101] The sample to be detected in the detection method developed by the present invention is human serum. When detecting with serum samples, due to the influence of various factors in the serum, high background interference will occur. Taking NJSS-1 coated antigen as an example, the present invention carried out a series of optimizations on the coating solution and the dilution solution of the sample to be detected used in the detection method, and finally selected a solution with 1% BSA as the main active ingredient as the coating solution and sample dilution solution of the final product, thereby effectively reducing the background interference problem in the sample( Figure 5 ).

[0102] Example 4. Detection of Carbapenem-Resistant Enterobacteriaceae (CRE) Antibodies Using Antigen Proteins

[0103] Using NJSS-1 as the coating antigen, serum samples from 20 patients infected with CRE were detected. It was found that the concentration of the corresponding antibodies in the serum of CRE-infected patients was significantly higher than that in the serum of the healthy control group without infection (16 cases) (t-test P<0.0001, Figure 6 ). Analyzing the CRE antibody detection method of the present invention using the Receiver operating characteristic curve (ROC curve), the area under the curve (AUC) was 0.861. When the specificity was 100%, the sensitivity was 65% ( Figure 7 ).

[0104] Example 5. Affinity of Antigen Proteins with Carbapenem-Resistant Enterobacteriaceae (CRE) Antibodies

[0105] Using the same method as in Example 2, the inventors detected the binding affinities of the antigen proteins NJSS-2, NJSS-3, NJSS-4, and NJSS-5 of the present invention with the corresponding monoclonal antibodies. The results showed that the binding affinities of the antigen proteins of the present invention with IMP, OXA48like, NDM, and VIM antibodies were significantly higher than those of the corresponding unoptimized proteins.

[0106] Example 6. Detection of Carbapenem-Resistant Enterobacteriaceae (CRE) Antibodies Using Antigen Proteins

[0107] Using the same method as in Example 4, the inventors used NJSS-2, NJSS-3, NJSS-4, and NJSS-5 as the coating antigens respectively to detect serum samples from CRE-infected patients. Finally, it was found that the concentration of the corresponding antibodies in the serum of CRE-infected patients was significantly higher than that in the serum of the healthy control group without infection. It can be seen from the ROC curve that the method of detecting CRE antibodies using these proteins has excellent specificity and sensitivity.

[0108] Example 7. Detection of Carbapenem-Resistant Enterobacteriaceae (CRE) Antibodies Using Antigen Proteins

[0109] Using the same method as in Example 4, the inventors used different combinations of NJSS-1, NJSS-2, NJSS-3, NJSS-4, and NJSS-5 as the coating antigens to detect serum samples from CRE-infected patients. Finally, it was found that the concentration of the corresponding antibodies in the serum of CRE-infected patients was significantly higher than that in the serum of the healthy control group without infection. It can be seen from the ROC curve that using the combination of the antigen proteins of the present invention can further improve the specificity and sensitivity of the method for detecting CRE antibodies.

[0110] All documents mentioned in this invention are cited herein by reference as if each individual document was cited by reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. An antigen protein, which is any one or a combination of more than one of the proteins shown in SEQ ID NO: 1-5.

2. The antigen protein according to claim 1, characterized in that the antigen protein is the protein shown in SEQ ID NO:

1.

3. The antigen protein according to claim 1, characterized in that the antigen protein is used to detect the presence of carbapenem-resistant Enterobacteriaceae (CRE) in a subject.

4. A detection reagent, which contains the antigen protein according to any one of claims 1-3.

5. A kit for detecting or diagnosing carbapenem-resistant Enterobacteriaceae (CRE), which is equipped with: 1) The antigen protein according to any one of claims 1-3 or the detection reagent according to claim 4; 2) An instruction manual for teaching the use of the antigen protein or the detection reagent to detect the presence of carbapenem-resistant Enterobacteriaceae in a sample.

6. The kit according to claim 5, characterized in that the kit is further equipped with a coating solution for coating the antigen protein and a diluent for diluting the sample.

7. The kit according to claim 6, characterized in that the coating solution and the diluent for diluting the sample are 1% BSA solution.

8. Use of the antigen protein according to any one of claims 1-3 in the preparation of a detection reagent or a kit for detecting or diagnosing carbapenem-resistant Enterobacteriaceae (CRE).

9. A method for detecting or diagnosing carbapenem-resistant Enterobacteriaceae (CRE), which includes the step of using the antigen protein according to any one of claims 1-3 or the detection reagent according to claim 4 or the kit according to any one of claims 5-7 to detect the presence of carbapenem-resistant Enterobacteriaceae in a sample.

10. The method according to claim 9, characterized in that the method is for non-diagnostic purposes.

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