Polypeptide composition and application thereof

Abstract

The invention relates to a polypeptide composition and application thereof, and relates to the technical field of brucellosis detection. The polypeptide composition comprises at least one of an OPM25 polypeptide library and an OPM31 polypeptide library; the OPM25 polypeptide library comprises at least one polypeptide, and the amino acid sequence of any polypeptide is the same as the amino acid sequence of a section of polypeptide in the OPM25 protein; the OPM31 polypeptide library comprises at least one polypeptide, and the amino acid sequence of any polypeptide is the same as the amino acid sequence of a section of polypeptide in the OPM31 protein. The application is the application of the polypeptide composition in preparation of a product for detecting brucellosis.

Description

Technical Field

[0001] The present disclosure relates to the technical field of ELISPOT detection, and in particular to a polypeptide composition and application thereof. Background Art

[0002] Brucellosis is a zoonotic infectious disease caused by Brucella, which is mainly transmitted through contact with infected animals or their products. The disease is prevalent in many countries and regions in the world, posing a serious threat to human health and animal husbandry production. Due to the long incubation period of the disease, complex clinical manifestations, and difficulty in early diagnosis, the development of a new, highly sensitive and specific detection method is of great significance for the prevention and treatment of the disease.

[0003] At present, the detection of Brucella mainly relies on bacterial culture, serological tests and molecular biological methods. Bacterial culture is the gold standard, but it is time-consuming and requires special equipment and conditions. Serological tests such as serum agglutination test (SAT) and enzyme-linked immunosorbent assay (ELISA) are simple to operate, but their sensitivity and specificity are not high, and false positive and false negative results are prone to occur. Molecular biological methods such as polymerase chain reaction (PCR) have high sensitivity, but require special instruments and equipment and are easily affected by contamination.

[0004] There are some problems and limitations in the existing detection methods. First, the sensitivity and specificity of these methods are not high, and misdiagnosis and missed diagnosis are prone to occur. Secondly, the operation of these methods is complicated, and special equipment and technicians are required, which is not conducive to the promotion and application in primary medical institutions. In addition, the detection time of these methods is long, which cannot meet the needs of clinical rapid diagnosis. Therefore, it is urgent to develop a new, simple, fast, sensitive and specific detection method to improve the detection level and detection efficiency of brucellosis.

[0005] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute the prior art known to ordinary technicians in the field. Summary of the invention

[0006] The purpose of the present disclosure is to overcome the deficiencies of the above-mentioned prior art and to provide a polypeptide composition and application thereof, using the OPM25 polypeptide library and / or the OPM31 polypeptide library as polypeptide antigens to accurately and quickly detect brucellosis.

[0007] According to one aspect of the present disclosure, there is provided a polypeptide composition, the polypeptide composition comprising at least one polypeptide library of the OPM25 polypeptide library and the OPM31 polypeptide library;

[0008] The OPM25 polypeptide library includes at least one polypeptide, and the amino acid sequence of any polypeptide is the same as the amino acid sequence of a polypeptide in the OPM25 protein; the OPM31 polypeptide library includes at least one polypeptide, and the amino acid sequence of any polypeptide is the same as the amino acid sequence of a polypeptide in the OPM31 protein.

[0009] In an exemplary embodiment of the present disclosure, the amino acid sequence of any one of the polypeptides in the OPM25 polypeptide library is selected from the amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 40;

[0010] The amino acid sequence of any one polypeptide in the OPM31 polypeptide library is selected from the amino acid sequences SEQ ID NO: 41 to SEQ ID NO: 86.

[0011] According to another aspect of the present disclosure, there is provided a use of the above polypeptide composition in preparing a product for detecting brucellosis.

[0012] In an exemplary embodiment of the present disclosure, the product for detecting brucellosis includes a kit, a detection reagent or a biological detection chip.

[0013] In an exemplary embodiment of the present disclosure, the kit includes a specific stimulation antigen, and the specific stimulation antigen includes the polypeptide composition.

[0014] In an exemplary embodiment of the present disclosure, the kit is an ELISPOT kit or an ELISA kit; both the ELISPOT kit and the ELISA kit are used to detect the level of the first type of specific cytokine, for distinguishing brucellosis from non-brucellosis;

[0015] The first type of specific cytokines includes one or more of TNF-β, IL-5, IP-10, IFN-γ, IL-6 and MCP-1.

[0016] In an exemplary embodiment of the present disclosure, the kit is an ELISPOT kit or an ELISA kit; both the ELISPOT kit and the ELISA kit are used to detect the level of the second type of specific cytokines, and are used to distinguish acute brucellosis from chronic brucellosis;

[0017] The second type of specific cytokines includes one or more of IP-10, IFN-γ and MCP-4.

[0018] In an exemplary embodiment of the present disclosure, the ELISPOT kit further comprises an experimental plate, a capture antibody and a detection antibody;

[0019] The detection method of the ELISPOT kit comprises the following steps:

[0020] S1. Obtain a cell sample to be tested, a negative control solution, and a positive control solution;

[0021] S2, coating with a capture antibody that specifically binds to interferon-γ, adding a negative control solution to the negative control group, adding a positive control solution to the positive control group, and adding the polypeptide composition to the test group;

[0022] S3, adding equal amounts of the cell samples to be tested to the negative control group, positive control group and detection group described in step S2; culturing at 37±1.0°C After 1 hour, anti-γ-interferon detection antibody was added;

[0023] S4, developing color for the negative control group, the positive control group and the test group and measuring the number of spots, and judging the result of the test group according to the results of the negative control group and the positive control group;

[0024] If the average number of spots in the positive control group is greater than 20, and the average number of spots in the negative control group is less than 10, then the average number of spots in the test group minus the average number of spots in the negative control group is greater than or equal to 7, and the test result is positive; if the average number of spots in the test group minus the average number of spots in the negative control group is less than or equal to 6, then the test result is negative;

[0025] If the average number of spots in the positive control group is greater than 20, and the average number of spots in the negative control group is greater than 10, the cell sample to be tested needs to be measured repeatedly;

[0026] If the average number of spots in the positive control group is less than 20, and the average number of spots in the negative control group is less than 10, then the average number of spots in the test group minus the average number of spots in the negative control group is greater than or equal to 7, and the test result is positive; if the average number of spots in the test group minus the average number of spots in the negative control group is less than or equal to 6, the cell sample to be tested needs to be measured repeatedly;

[0027] If the average number of spots in the positive control group is less than 20, and the average number of spots in the negative control group is greater than 10, the cell sample to be tested needs to be measured repeatedly.

[0028] In an exemplary embodiment of the present disclosure, in step S4, when the test result is positive, if the average number of spots in the test group minus the average number of spots in the negative control group is greater than or equal to 7 and less than or equal to 60, the cell sample to be tested corresponds to chronic brucellosis;

[0029] If the average number of spots in the test group minus the average number of spots in the negative control group is greater than or equal to 61, the sample cells to be tested correspond to acute brucellosis.

[0030] In an exemplary embodiment of the present disclosure, in step S2, the concentration of each polypeptide in the polypeptide composition is 10-15 ug / ml;

[0031] In step S1, the cell sample to be tested includes peripheral blood mononuclear cells, and the concentration of the peripheral blood mononuclear cells is 2.5×10 6 / mL~3.0×10 6 / mL.

[0032] According to another aspect of the present disclosure, there is provided a use of the above polypeptide composition in the preparation of a product for identifying brucellosis in the asymptomatic infection period.

[0033] Beneficial effects of the present disclosure: The present disclosure uses a polypeptide composition composed of an OPM25 polypeptide library and / or an OPM31 polypeptide library as a specific stimulation antigen for the detection of brucellosis. The product for detecting brucellosis prepared using the above polypeptide composition can not only be used to distinguish non-brucellosis patients from brucellosis patients, but also can be used to distinguish acute brucellosis from chronic brucellosis, and can be used for early screening of asymptomatic brucellosis infection, which makes up for the deficiencies in the prior art and expands the application scope of brucellosis detection.

[0034] The ELLISPOT detection method and ELISA detection method provided in this application have high sensitivity and specificity, can improve the accuracy of brucellosis detection, and are conducive to earlier discovery and control of the epidemic, and facilitate early prevention and control of the disease. At the same time, the ELISPOT detection method and ELISA detection method are simple to operate, do not require special equipment and technicians, are applicable to a wide range of scenarios, and can be used for basic medical services.

[0035] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings herein are incorporated into the specification and constitute a part of the specification, illustrate embodiments consistent with the present disclosure, and together with the specification are used to explain the principles of the present disclosure. Obviously, the accompanying drawings described below are only some embodiments of the present disclosure, and for ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without creative work.

[0037] Figure 1 This is a heat map of the changes in multiple cytokines during the treatment of brucellosis patients in one embodiment of the present disclosure.

[0038] Figure 2This is a schematic diagram of the changes in cytokines IFN-γ, IP-10, and MCP-4 during the treatment of brucellosis patients in one embodiment of the present disclosure.

[0039] Figure 3 This is a schematic diagram of ROC curve results of an ELISpot assay method for distinguishing non-brucellosis patients from brucellosis patients in one embodiment of the present disclosure.

[0040] Figure 4 This is a schematic diagram of the consistency analysis results of the test tube agglutination test and the ELISpot test in one embodiment of the present disclosure.

[0041] Figure 5 This is a schematic diagram of the ROC curve results of the enzyme-linked immunosorbent spot test method for distinguishing acute brucellosis from chronic brucellosis in one embodiment of the present disclosure. DETAILED DESCRIPTION

[0042] Example embodiments will now be described more fully with reference to the accompanying drawings. However, example embodiments can be implemented in many forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be comprehensive and complete and will fully convey the concept of the example embodiments to those skilled in the art.

[0043] The terms "first class specific cytokine" and "second class specific cytokine" are not biologically classified. In the embodiments of the present disclosure, in order to distinguish between different application scenarios (differentiation of brucellosis and non-brucellosis, or differentiation of acute brucellosis and chronic brucellosis), the polypeptide composition provided by the present disclosure is used as a specific stimulation antigen to stimulate peripheral blood mononuclear cells and can be used as a cytokine for detection indicators. For example, if brucellosis and non-brucellosis are distinguished, an ELISPOT kit or ELISA kit for detecting the first class specific cytokine (at least one of TNF-β, IL-5, IP-10, IFN-γ, IL-6 and MCP-1) can be used; if acute brucellosis and chronic brucellosis are distinguished, an ELISPOT kit or ELISA kit for detecting the second class specific cytokine (at least one of IP-10, IFN-γ and MCP-4) can be used.

[0044] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present invention is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0045] The disclosed embodiments provide a polypeptide composition, which includes at least one of the OPM25 polypeptide library and the OPM31 polypeptide library; and the amino acid sequence of any one of the polypeptides is the same as the amino acid sequence of a polypeptide in the OPM25 protein; the OPM31 polypeptide library includes at least one polypeptide, and the amino acid sequence of any one of the polypeptides is the same as the amino acid sequence of a polypeptide in the OPM31 protein. For example, to evaluate the structure of the OPM25 protein, N2 amino acids can be selected as a polypeptide at intervals of N1 amino acids. Wherein N1 and N2 can be selected based on the protein structure, the immunogenicity of the polypeptide, etc. For example, N1 can be 3, 5, 7 or 10, etc., and N2 can be 10, 15 or 20, etc.

[0046] In one embodiment of the present disclosure, the amino acid sequence of any polypeptide in the OPM25 polypeptide library is selected from the amino acid sequence of SEQ ID NO: 1 to the amino acid sequence of SEQ ID NO: 40; the amino acid sequence of any polypeptide in the OPM31 polypeptide library is selected from the amino acid sequence of SEQ ID NO: 41 to the amino acid sequence of SEQ ID NO: 86. That is, the OPM25 polypeptide library includes at least part of the polypeptides shown in the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 40, and the OPM31 polypeptide library includes at least part of the polypeptides shown in the amino acid sequences of SEQ ID NO: 41 to SEQ ID NO: 86. The polypeptide composition can be the OPM25 polypeptide library, the OPM31 polypeptide library, or a mixture of the OPM25 polypeptide library and the OPM31 polypeptide library.

[0047] The polypeptide composition provided by the present disclosure can be used to prepare products for detecting brucellosis. The products include test kits, detection reagents or biodetection chips. For example, the products can be ELISPOT test kits, ELISA test kits, biochips with biological probes, etc.

[0048] In one embodiment of the present disclosure, the kit includes specific stimulation antigens, and the specific stimulation antigens include at least one of the OPM25 polypeptide library and the OPM31 polypeptide library.

[0049] In one embodiment of the present disclosure, the kit is an ELISPOT kit or an ELISA kit. Both the ELISPOT kit and the ELISA kit are used to detect the level of the first type of specific cytokines for distinguishing brucellosis and non-brucellosis; the first type of specific cytokines include one or more of TNF-β, IL-5, IP-10, IFN-γ, IL-6 and MCP-1. That is to say, in the application scenario of distinguishing brucellosis and non-brucellosis, the ELISPOT kit and the ELISA kit contain a polypeptide composition (specific stimulating antigen), and a capture antibody and a detection antibody that can specifically bind to the first type of specific cytokines. One type of cytokine in the first type of specific cytokines can be measured for identification, and multiple cytokines can also be measured for joint judgment to improve the accuracy of identification.

[0050] In one embodiment of the present disclosure, the kit is an ELISPOT kit or an ELISA kit. The ELISPOT kit and the ELISA kit are both used to detect the level of the second class of specific cytokines, which are used to differentiate acute brucellosis and chronic brucellosis; the second class of specific cytokines include one or more of IP-10, IFN-γ and MCP-4. That is to say, in the application scenario of differentiating acute brucellosis and chronic brucellosis, the ELISPOT kit and the ELISA kit include a polypeptide composition (specific stimulating antigen), and a capture antibody and a detection antibody that can be specifically bound to the second class of specific cytokines. A cytokine in the second class of specific cytokines can be measured for differentiation, and a plurality of cytokines can also be measured for joint judgment to improve the accuracy of differentiation.

[0051] In one embodiment of the present disclosure, the ELISPOT kit for brucellosis also includes an experimental plate, a capture antibody and a detection antibody. Further, the ELISPOT kit also includes a negative control solution and a positive control solution. Exemplarily, the negative control solution can be a culture solution of a cell sample to be tested; the positive control solution can be a plant lectin solution. Exemplarily, the experimental plate is a microplate arranged in an array. For example, it can be a 96-well plate, a 24-well plate or a 384-well plate, etc. The microplate can be an integrated structure or a detachable microplate.

[0052] In one embodiment of the present disclosure, the detection method using the above ELISPOT kit comprises the following steps:

[0053] S1. Obtain a cell sample to be tested, and obtain a negative control solution and a positive control solution.

[0054] In one example, the cell sample to be tested is peripheral blood mononuclear cells, wherein the concentration of the peripheral blood mononuclear cells is 2.5×10 6 / mL~3.0×10 6 / mL. Within this concentration range, the cells to be tested can be evenly distributed in the culture medium, avoiding excessively high cell concentrations that lead to cell stacking and affect the judgment of the test results, and also avoiding too low a dilution concentration of the cells to be tested that reduces the sensitivity and accuracy of the test.

[0055] In one example, the negative control solution may be a culture medium of a cell sample to be tested. The positive control solution may be a lectin solution, for example, the lectin solution may be concanavalin A (ConA). The negative control solution and the positive control solution may be included in the kit, or may be prepared as needed.

[0056] S2. Coating with a capture antibody that specifically binds to interferon-γ, adding a negative control solution to the negative control group, adding a positive control solution to the positive control group, and adding the polypeptide composition to the detection group. For example, when testing a cell sample to be tested, first coat the capture antibody on a microplate, add a negative control solution to at least one negative control well on the microplate to form a negative control group, add a positive control solution to at least one positive control well on the microplate to form a positive control group, and add a polypeptide composition to at least one detection well on the microplate to form a detection group.

[0057] In one example, the concentration of each polypeptide in the polypeptide composition is 10-15 ug / ml.

[0058] S3, adding equal amounts of the cell samples to be tested to the negative control group, positive control group and detection group described in step S2; culturing at 37±1.0°C After 1 hour, a detection antibody is added. The detection antibody may be an enzyme-labeled detection antibody, such as an alkaline phosphatase-labeled detection antibody.

[0059] S4. Add a color developer to the negative control group, the positive control group and the test group to develop color and measure the number of spots, and judge the result of the test group based on the results of the negative control group and the positive control group.

[0060] If the average number of spots in the positive control group is greater than 20, and the average number of spots in the negative control group is less than 10, then the average number of spots in the test group minus the average number of spots in the negative control group is greater than or equal to 7, and the test result of the cell sample to be tested is positive; the average number of spots in the test group minus the average number of spots in the negative control group is less than or equal to 6, and the test result of the cell sample to be tested is negative; if the average number of spots in the positive control group is greater than 20, and the average number of spots in the negative control group is greater than 10, the cell sample to be tested needs to be measured repeatedly; if the average number of spots in the positive control group is less than 20, and the average number of spots in the negative control group is less than 10, then the average number of spots in the test group minus the average number of spots in the negative control group is greater than or equal to 7, and the test result of the cell sample to be tested is positive; the average number of spots in the test group minus the average number of spots in the negative control group is less than or equal to 6, and the cell sample to be tested needs to be measured repeatedly; if the average number of spots in the positive control group is less than 20, and the average number of spots in the negative control group is greater than 10, the cell sample to be tested needs to be measured repeatedly.

[0061] In one embodiment of the present disclosure, when the test result is positive, if the average number of spots in the test group minus the average number of spots in the negative control group is greater than or equal to 7 and less than or equal to 60, the cell sample to be tested corresponds to chronic brucellosis. If the average number of spots in the test group minus the average number of spots in the negative control group is greater than or equal to 61, the cell sample to be tested corresponds to acute brucellosis.

[0062] The disclosed embodiment also provides the use of the polypeptide composition in the preparation of a product for identifying brucellosis in the asymptomatic infection period. In this way, early diagnosis of brucellosis can be achieved, which is particularly suitable for early screening in animal husbandry to avoid economic losses caused by the pandemic of brucellosis and the resulting risk of illness for animal husbandry workers.

[0063] The following is further described in conjunction with specific embodiments.

[0064] Example 1: Preparation of polypeptide composition

[0065] OMP25 and OMP31 proteins in the Brucella Outer Membrane Protein (OMP) family were selected as target antigens, and the species specificity of OMP25 and OMP31 proteins was verified using NCBI BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The protein structure was evaluated using DNASTAR software, and 15 amino acids were selected as a peptide every 5 amino acids. The immunogenicity of each peptide was scored by MHC I using the Immune Epitope Database (IEDB) to evaluate the immunogenicity of the peptide.

[0066] The polypeptide is prepared by a polypeptide solid phase synthesis method, and a plurality of polypeptides form a polypeptide composition, which can be used as a specific stimulating antigen to participate in an in vitro immune response; the purity of the polypeptide is more than 95%. That is to say, the polypeptides in the polypeptide composition provided in the embodiment of the present disclosure are synthesized by chemical methods, without the need for biological preparation, with low production costs, simple synthesis processes and high safety. The synthesized polypeptides are dissolved in a preservation solution so that the concentration of each polypeptide in the preservation solution is 1ng / ml and stored in a -80°C refrigerator. The preservation solution is an RMPI 1640 solution containing 10% dimethyl sulfoxide (DMSO), and the RMPI 1640 solution is a type of cell culture solution. Among them, at least part of the polypeptides of the OMP25 protein form an OMP25 polypeptide library, and at least part of the polypeptides of the OMP31 protein form an OMP31 polypeptide library. The polypeptide composition can be an OMP25 polypeptide library and / or an OMP31 polypeptide library. For example, the OMP25 polypeptide library includes polypeptides shown in the amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 40, and the OPM31 polypeptide library includes polypeptides shown in the amino acid sequences SEQ ID NO: 41 to SEQ ID NO: 86. The polypeptide structure and sequence are detected by high performance liquid chromatography (HPLC) and an analytical method for measuring the mass-to-charge ratio of ions (MS) to determine whether the structure and purity of the prepared polypeptide meet the requirements. As shown in Table 1, the HPLC and MS measurement results corresponding to each polypeptide are shown.

[0067] Table 1. HPLC and MS detection results of various peptides

[0068]

[0069]

[0070]

[0071] Embodiment 2 Nineteen patients with acute brucellosis were followed up and sampled. The basic information of the patients is shown in Table 2 (number 1-19), of which 16 / 19 (84.2%) samples were male, with a median age of 51.5 (quartile: 39.25-53.25) years. Fourteen (28.6%) samples had brucellotic osteoarthritis, 14 (28.6%) samples had urogenital tract inflammation, and 2 (4.1%) samples had neurobrucellosis.

[0072] Table 2. Basic characteristics of 19 patients with brucellosis

[0073]

[0074]

[0075] Numbers 1-19 in Table 2 are blood samples collected from 19 patients with brucellosis on day 0 (D0, i.e., the first day of follow-up, the first blood sample collection), and the plasma collected from each sample is the D0 sample. Numbers 20-33 in Table 2 are blood samples collected from 14 patients who are still continuing treatment among the above 19 patients with brucellosis on day 14, and the plasma collected from each sample is the D14 sample. Numbers 34-38 in Table 2 are blood samples collected from 5 patients who are still continuing treatment among the above 19 patients with brucellosis on day 42, and the plasma collected from each sample is the D42 sample. Numbers 39-48 in Table 2 are blood samples collected from 11 patients who are still continuing treatment among the above 19 patients with brucellosis on day 90, and the plasma collected from each sample is the D90 sample. The plasma samples collected at different time periods were tested for multiple cytokines using the V-PLEX Human Cytokine 36-Plex Kit (Meso Scale Discovery, MSD).

[0076] Table 3. Measurement results of various cytokines in D0 samples

[0077]

[0078]

[0079] The result that 19 D0 samples carry out multi-cytokine measurement is as shown in Table 3, and compared with normal value, the cytokines that are higher than the normal value number for more than 30% include TNF-β, IL-5, IP-10, IFN-γ, IL-6 and MCP-1. In other words, compared with healthy people, the cytokines TNF-β, IL-5, IP-10, IFN-γ, IL-6 and MCP-1 of brucellosis patients all have obvious elevation. In this way, it is suggested that the peptide composition provided by the disclosed embodiment can be adopted to carry out immunostimulation again to the serum sample of body, detect one or more of the above-mentioned cytokines (TNF-β, IL-5, IP-10, IFN-γ, IL-6 and MCP-1), with specific identification brucellosis.

[0080] After the test results of multiple cytokines of D0 samples, D14 samples, D42 samples and D90 samples were standardized, the heat map of the changes in cytokine levels during the treatment of brucellosis patients was as follows: Figure 1 Among them, the levels of IFN-γ, IP-10, and MCP-4 in patients with brucellosis during treatment are shown in Figure 2As shown in the figure, the two dotted lines represent the upper and lower limits of the normal range. Compared with patients with acute brucellosis, the IFN-γ level of brucellosis patients after treatment was significantly different (P<0.05) between D0 (44.33±78.52pg / ml) and D90 (1.85±1.79pg / ml), the level of interferon-γ-induced protein 10 (IP-10) was significantly different (P<0.05) between D0 (2659.6±3496.04pg / ml) and D90 (496.05±282.53pg / ml), and the level of monocyte chemoattractant protein 4 (MCP-4) was significantly different (P<0.001) between D0 (84.8±68.02pg / ml) and D90 (200.88±82.56pg / ml). The result shows, from acute stage brucellosis (D0 sample, acute attack phase) to chronic stage brucellosis (D90 sample, chronic treatment phase), the level of IFN-γ and IP-10 is obviously reduced to normal range, and the relative baseline of MCP-4 level further increases within the normal range. In other words, IFN-γ, IP-10 and MCP-4 are in the course of treatment, and the cytokine in the body is all recovered to normal level. So, prompting, can use one or more of IFN-γ, IP-10 and MCP-4 as the index of detecting acute stage and chronic stage brucellosis.

[0081] Embodiment 3

[0082] The polypeptide composition prepared in Example 1 was used as a specific stimulating antigen, and brucellosis was identified by detecting the specific level of gamma-interferon.

[0083] 1. Sample collection and grouping

[0084] This embodiment has collected 97 experimenters from Huashan Hospital Affiliated to Fudan University and Shenyang Sixth People's Hospital, and is divided into experimental group and control group, and experimental group comprises 41 Brucellosis patients, and control group comprises 56 non-brucellosis patients.Collect clinical information and peripheral blood sample of each experimenter.All experimenters have all signed informed consent, and obtained the approval of the Ethics Committee of Huashan Hospital Affiliated to Fudan University and Shenyang Sixth People's Hospital.

[0085] The information statistics of the subjects are shown in Table 4. In terms of clinical characteristics, there were no statistical differences between the experimental group and the control group in terms of gender, age, clinical symptoms and signs (fever, headache), and comorbidities (including diabetes, rheumatic diseases, chronic liver disease, and cardiovascular disease) (all P>0.05). Compared with the control group, the proportion of subjects in the experimental group experiencing fatigue and myalgia was higher (P<0.05). Compared with the experimental group, the proportion of subjects in the control group experiencing hypertension was higher (P<0.05).

[0086] Among the 41 patients with brucellosis in the experimental group, 8 patients (19.5%) had untreated brucellosis in the acute phase, 16 patients (39.0%) had treated brucellosis in the acute phase, and 17 patients (41.5%) had brucellosis in the chronic phase. Among them, 29 patients (70.7%) had arthritis, 6 patients (14.6%) had nervous system inflammation, 2 patients (4.9%) had urinary tract inflammation, and 2 patients (4.9%) had endocarditis.

[0087] The 56 non-brucellosis patients in the control group included 37 patients with other infections (66.1%), 13 non-infected patients (23.2%), and 6 healthy controls (10.7%).

[0088] Table 4 Basic information of subjects in the experimental and control groups

[0089] feature Experimental Group Control group P-value Total [Example] 41.0 56.0 Male [Example (%)] 26(63.4) 34(60.7) 0.787 <![CDATA[Age [years, M(Q1, Q3)]]]> 53.0(43.0-58.0) 60.5(38.8-68.8) 0.095 Disease characteristics [cases (%)] Untreated acute brucellosis 8(19.5) 0(0.0) - Acute brucellosis has been treated 16(39.0) 0(0.0) - Chronic brucellosis 17(41.5) 0(0.0) -

[0090] 2. Prepare the cell samples to be tested

[0091] Step S1: 10 mL of fasting peripheral venous blood of the subject is drawn into an EDTA vacuum anticoagulation tube and allowed to stand at room temperature for 8 hours. The EDTA vacuum anticoagulation tube containing the peripheral venous blood of the subject is centrifuged in a horizontal centrifuge for 10 minutes at a speed of 2500-3000 rpm, with both the speed increasing and decreasing being 9.

[0092] Step S2, after centrifugation, the upper serum is drawn for later use. The buffy coat cells with white blood cells are gently drawn using a Buffy tube, and the red blood cells in the lower layer are not drawn as much as possible. The drawn white blood cells are transferred to a first sterile centrifuge tube containing RPMI 1640 culture medium, and the capacity of the first sterile centrifuge tube can be 15 mL. The total volume is supplemented to 8 mL with RPMI 1640 culture medium, and the cells are fully blown to resuspend to obtain a first cell suspension.

[0093] Step S3, draw 4mL of lymphocyte separation solution into a second sterile centrifuge tube with a capacity of 15mL, tilt the second sterile centrifuge tube, use a bus tube to draw the first cell suspension in the first sterile centrifuge tube, close to the surface of the lymphocyte separation solution in the second sterile centrifuge tube, and slowly drip without destroying the stratification. Centrifuge at room temperature in a horizontal centrifuge, with a speed of 2500rpm, a speed increase and a speed decrease set to 1, and a centrifugation time of 25 minutes. After centrifugation using a bus tube, the middle white film layer of the second sterile centrifuge tube is transferred to a third sterile centrifuge tube, and 4mL of RPMI 1640 culture medium is added to the third sterile centrifuge tube in advance. Add RPMI 1640 culture medium to the third sterile centrifuge tube to make up the total volume to 12mL, and mix thoroughly by blowing to obtain a second cell suspension.

[0094] Step S4: Place the third sterile centrifuge tube in a horizontal centrifuge and centrifuge at room temperature at a speed of 1500 rpm for 10 minutes. Discard the supernatant and retain the cells deposited at the bottom of the third sterile centrifuge tube.

[0095] Step S5, add 1 mL of 37°C preheated AIM-V culture medium to the third sterile centrifuge tube, resuspend the cells, and obtain a third cell suspension. Take 10uL of the third cell suspension and 10uL of 0.4% trypan blue and mix them well, then draw 10uL of the mixed solution and add it to the cell counting plate, and count on the cell counter as soon as possible to record the number of live cells and cell activity.

[0096] Step S6: According to the viable cell count concentration, add AIM-V medium to adjust the peripheral blood mononuclear cell (PBMC) concentration to 2.5×10 6 / mL.

[0097] Thus, according to the operations of step S1 to step S6, the cell samples to be tested of each subject in the experimental group and the control group are obtained respectively, that is, peripheral blood mononuclear cell (PBMC) solutions.

[0098] In one example, if there are more red blood cells in the cells deposited at the bottom of the third sterile centrifuge tube obtained in step S4, 5 ml of red blood cell lysis solution can be added to the third sterile centrifuge tube. After sufficient mixing, incubate at room temperature for 5-10 minutes and then centrifuge at room temperature, with a centrifugal speed of 2000 rpm for 5 minutes. After centrifugation, the supernatant is discarded and the cells deposited at the bottom of the third sterile centrifuge tube are retained. In this way, the red blood cells in the cells at the bottom of the third sterile centrifuge tube can be removed. PMI 1640 medium is added to the third sterile centrifuge tube to resuspend the deposited cells, and PMI 1640 medium is added to make up to 12 mL, and the cells are washed with PMI 1640 medium. After centrifugation at 1500 rpm for 10 minutes, the supernatant is discarded to obtain the cells deposited at the bottom of the third sterile centrifuge tube. Then continue with subsequent steps S5 and S6. In this example, in order to remove excess red blood cells, if there are not many red blood cells in the cells deposited in the third sterile centrifuge tube in step S4, there is no need to lyse the red blood cells.

[0099] 3. Sample testing of the experimental group and the control group

[0100] (1) Enzyme-linked immunospot assay (ELISPOT)

[0101] ELISPOT test principle: Brucella-specific effector T lymphocytes are present in the peripheral blood of people infected with Brucella. When stimulated again by Brucella-specific stimulating antigens, these specific effector T lymphocytes will quickly produce an immune response and secrete the cytokine gamma-interferon (IFN-γ). The ELISPOT test is based on this specific immune response and detects the number of cells that produce gamma-interferon through specific capture antibodies and enzyme-labeled antibodies.

[0102] In the ELISPOT experiment, each subject's sample requires at least three wells, namely, a positive control well, a negative control well, and a detection well. PBMC solution of the same subject's sample is added to at least three wells, and plant lectin PHA is added to the positive control well, cell culture medium is added to the negative control well, and the prepared polypeptide composition is added to the detection well as a specific stimulation antigen. When Brucella-specific effector T lymphocytes are present in the peripheral blood mononuclear cells (lymphocytes) in the subject's sample, the Brucella-specific polypeptide composition will serve as a specific stimulation antigen to stimulate these specific effector T lymphocytes to secrete the cytokine γ-interferon. The secreted cytokine gamma interferon can be captured by the pre-coated first antibody (anti-human gamma interferon antibody). At the same time, the cytokine gamma interferon is also combined with the added second antibody (enzyme-labeled mouse anti-human gamma interferon antibody). After the color development substrate is added, the color development substrate is enzymatically hydrolyzed into insoluble pigment precipitation spots. Each of the pigment precipitation spots represents a gamma interferon secreting cell (that is, the specific effector T lymphocyte of Brucella). By counting the pigment precipitation spots, it can be determined whether there are specific effector T lymphocytes of Brucella in the sample and the number of specific effector T lymphocytes.

[0103] Preparation of detection reagents:

[0104] a. Prepare RPMI 1640 medium and AIM-V medium; prepare 0.4% (w / v) trypan blue solution.

[0105] b. Specific stimulating antigen working solution: In the ELISPOT experiment, the polypeptide composition is used as a specific stimulating antigen to participate in the in vitro immune response. When the polypeptide composition is used, AIM-V cell culture fluid is added to the preservation solution to dilute the concentration of each polypeptide to 10 ng / ml to obtain a specific stimulating antigen working solution. The specific stimulating antigen working solution can be divided and placed in a -20°C refrigerator.

[0106] c. Purchase a cellular immune response detection kit (ELISpot) (Cat. No. 2303013A) produced by Fosun Diagnostics Technology Co., Ltd., and use a 96-well microplate coated with a first antibody (capture antibody), a positive control solution of plant lectin PHA, an alkaline phosphatase-labeled second antibody (detection antibody), and a colorimetric substrate solution.

[0107] Testing steps:

[0108] Step S10, according to the requirement of 3 wells for each sample, take out the microwell strips required for the test from the aluminum foil package. The 3 wells include a negative control well, a positive control well and a detection well, constituting a microwell group. In the same microwell group, 50uL of AIM-V culture medium is added to the negative control well, 50uL of plant lectin PHA is added to the positive control well, and 50uL of specific stimulation antigen working solution is added to the detection well, wherein the concentration of each polypeptide in the specific stimulation antigen working solution is 10ng / ml.

[0109] Step S20: Add 100uL of peripheral blood mononuclear cell (PBMC) solution of the same subject to the negative control well, positive control well and detection well of each microwell group, wherein the cell concentration of the peripheral blood mononuclear cell (PBMC) solution is 2.5×10 6 / mL, cover the cover.

[0110] Step S30: Place the microplate obtained in step S20 in a 5% CO2 incubator and incubate at 37±1.0°C. Hour.

[0111] Step S40: Take the microplate out of the incubator, discard the liquid in each well, and turn the microplate upside down on absorbent paper to pat dry. Add 200 μL of PBS buffer to each well and let it stand. Repeat the washing process with PBS buffer solution for 4 times.

[0112] Step S50, add 50uL of the freshly prepared alkaline phosphatase labeled second antibody working solution to each well, and cover the cover plate. The freshly prepared alkaline phosphatase labeled second antibody (detection antibody) working solution refers to the working solution obtained by diluting the concentrated alkaline phosphatase labeled second antibody by 1:200.

[0113] Step S60: incubate the microplate obtained in step S50 at 2-8°C for 60 minutes, discard the liquid in each well, pat dry, and wash four times with PBS buffer solution in the same manner as in step S40.

[0114] Step S70, add 50uL of the color substrate solution to each well, react in the dark at room temperature for 7 minutes, discard the liquid in each measurement well, wash each well with distilled water or deionized water to terminate the reaction. Use a magnifying glass or ELISPOT automatic plate reader to count the spots and obtain the measurement results of the positive control well, negative control well and detection well corresponding to each sample.

[0115] (2)SAT

[0116] The serum of the subjects in the experimental group and the control group was subjected to a tube agglutination test (SAT) to measure the serum titer of each subject.

[0117] The comparative results of the ELISPOT test and the tube agglutination test are shown in Table 5.

[0118] Table 5 ELISPOT test and tube agglutination test results

[0119]

[0120]

[0121] Among them, SAT is a tube agglutination test, ELISPOT refers to enzyme-linked immunospot test, and SFCs refers to spot-forming cells. a indicates that patients with confirmed brucellosis have a negative SAT after standard antimicrobial treatment, but still have symptoms related to brucellosis. b indicates that patients with confirmed brucellosis have a SAT titer of 1:50 after standard antimicrobial treatment, of which 2 patients have a course of less than 1 year and 6 patients have a course of more than 1 year. c indicates that among 41 patients with brucellosis, 23 received blood culture; among 56 patients with non-brucellosis, 50 received blood culture.

[0122] 4. Data analysis and result judgment

[0123] like Figure 3 As shown in Figure 2, the diagnostic performance of the ELISPOT method was evaluated by receiver operating characteristic (ROC) analysis. The results showed that when the optimal cutoff value was 6.0, the area under the curve was 0.986 (95% CI: 0.966, 1.000, P < 0.05), the sensitivity was 95.1%, and the specificity was 98.2%. Figure 4As shown in the figure, the consistency analysis of the results of tube agglutination test (SAT test) and ELISPOT test of 41 patients with brucellosis was performed, where * represents P<0.05; ** represents P<0.01; ns represents no significant statistical difference between the two groups. With the increase of the titer of SAT test, the SFCs of patients with brucellosis increased significantly (P<0.05). Among them, the SFCs value experimental results ([M(Q1, Q3)]) corresponding to the control group (n=56), the experimental group SAT titer 1:50 (n=8), the experimental group SAT titer 1:100 (n=18), and the experimental group SAT titer 1:200 (n=14) were 0.0 (0.0, 2.0), 15.0 (9.0, 30.0), 62.0 (29.0, 75.0), and 86.0 (49.5, 106.5), respectively.

[0124] Among the 41 brucellosis patients in the experimental group, there were 8 patients with untreated acute brucellosis and 17 patients with chronic brucellosis. ROC curve analysis was performed on the ELISPOT results of patients with untreated acute brucellosis (acute brucellosis patients) and patients with chronic brucellosis. Figure 5 As shown, when the optimal cutoff value was 60.5, the corresponding area under the ROC curve was 0.768 (95% CI: 0.533, 1.000, P < 0.05), the sensitivity of distinguishing acute brucellosis from chronic brucellosis was 76.5%, and the specificity was 75.0%.

[0125] Result judgment:

[0126] Normally, the negative control wells have no or very few spots, while the positive control wells to which lectin PHA is added have more than 20 spots or the spots are spread all over the positive control wells.

[0127] Determine the result based on the reaction of the microwell group corresponding to the same sample:

[0128] 1. When the number of spots in the positive control well exceeds 20 or the spots are spread throughout the positive control well, and the number of spots in the negative control well is greater than 10, the test result is "uncertain" and needs to be rechecked.

[0129] 2. When the number of spots in the positive control well exceeds 20 or the spots are distributed throughout the positive control well, and the number of spots in the negative control well is <10, if (the number of spots in the test well) - (the number of spots in the negative control well) ≥ 7, the test result is "positive"; if (the number of spots in the test well) - (the number of spots in the negative control well) ≤ 6, the test result is "negative".

[0130] If the number of spots in the positive control well exceeds 20 or the spots are all over the positive control well, it means that the positive control lectin PHA can determine the viability of the peripheral blood mononuclear cells (PBMC) added to the microwell group corresponding to each sample and whether the reagents in each detection step are normal. There are also very few people whose T cells are unresponsive to the lectin PHA.

[0131] 3. When the number of spots in the positive control well is less than 20 or there are no spots in the positive control well, and the number of spots in the negative control well is greater than 10, the test result is invalid and the sample needs to be retested.

[0132] 4. When the number of spots in the positive control well is less than 20 or there is no spot in the positive control well, and the number of spots in the negative control well is <10, if (the number of spots in the test well) - (the number of spots in the negative control well) ≥ 7, the test result is "positive"; if (the number of spots in the test well) - (the number of spots in the negative control well) ≤ 6, the test result is invalid and the sample needs to be retested.

[0133] It is understood that a "positive" result indicates that specific effector T lymphocytes against Brucella are present in the corresponding sample. A "negative" result indicates that specific effector T lymphocytes against Brucella may not be present in the sample.

[0134] Furthermore, when the test result is "positive", if 7≤(the number of spots in the test well)-(the number of spots in the negative control well)≤60, the test result is chronic brucellosis; if (the number of spots in the test well)-(the number of spots in the negative control well)≥61, the test result is acute brucellosis.

[0135] Embodiment 4

[0136] The basic information and clinical history of 2482 subjects in brucellosis-endemic areas were collected, and the statistical information is shown in Table 6. All subjects signed informed consent forms, and the study was approved by the Ethics Committee of Huashan Hospital Affiliated to Fudan University and Shenyang Sixth People's Hospital.

[0137] Table 6. Basic information of residents without history of Brucella infection in endemic areas

[0138]

[0139]

[0140] Blood samples were collected from each research subject, and serum and peripheral blood mononuclear cell (PBMC) solution were separated using the method provided in Example 1. The serum was used for SAT detection, and 197 research subjects were screened out based on the results of the SAT detection. The 197 research subjects were then subjected to ELISPOT detection using the polypeptide composition provided in the embodiments of the present disclosure as a specific stimulation antigen. The results of the SAT and ELISPOT tests are shown in Table 7. Using SAT as the gold standard, the sensitivity of ELISPOT for screening asymptomatic infections in residents with no history of brucellosis infection was 83.2%, and the specificity was 94.9%. The sensitivity = true positive / (true positive + false negative), specificity = true negative / (true negative + false positive), where a true positive is when both the SAT test and the ELISPOT test are positive (SAT + ELISPOT + ), true negative means that both SAT and ELISPOT tests are negative (SAT - ELISPOT - ), false negative is SAT + ELISPOT - , false positive is SAT - ELISPOT + .

[0141] Table 7 Evaluation of the diagnostic efficacy of ELISPOT for asymptomatic infection patients

[0142]

[0143] 2484 subjects were followed up for six months. No immune-negative subjects developed brucellosis. Among the 102 immune-positive subjects (the sum of the number of people with SAT titers of 1:50, 1:100, 1:200 and above in Table 6), 67 were visited, of which 7 (10.4%) were diagnosed with brucellosis. This indicates that there is an asymptomatic infection period for brucellosis infection.

[0144] The enzyme-linked immunospot method provided by the present disclosure adopts a polypeptide composition composed of an OPM25 polypeptide library and / or an OPM31 polypeptide library as a specific stimulating antigen to detect the infection of brucellosis. First, the method has high sensitivity and specificity, can improve the accuracy of brucellosis detection, is conducive to earlier discovery and control of the epidemic, is convenient for early prevention and control of the disease, and protects the health of the public. The ELISPOT and ELISA detection methods provided by the present disclosure are simple to operate, do not require special equipment and technicians, are rich in applicable scenarios, and can be used for basic medical services. Secondly, the detection method is short in time during the detection of brucellosis, and while improving the detection efficiency, the early prevention and control efficiency of the disease is further improved. Furthermore, the detection method can not only be used for the diagnosis of brucellosis, but also can be used for the identification of acute brucellosis and chronic brucellosis, and for the early screening of asymptomatic brucellosis infection, which makes up for the deficiencies in the prior art and expands the scope of application of the detection. It is understandable that this brucellosis detection method can be further used for detection in animal husbandry, so as to facilitate the timely detection and treatment of infected animals, prevent the spread of the epidemic in farms, and ensure the healthy development of animal husbandry.

[0145] Those skilled in the art will readily appreciate other embodiments of the present disclosure after considering the specification and practicing the invention disclosed herein. This application is intended to cover any modification, use or adaptation of the present disclosure, which follows the general principles of the present disclosure and includes common knowledge or customary techniques in the art that are not disclosed in the present disclosure. The specification and examples are intended to be exemplary only, and the true scope and spirit of the present disclosure are indicated by the appended claims.

Claims

1. A polypeptide composition, characterized in that: comprising at least one of an OPM25 polypeptide library and an OPM31 polypeptide library; The OPM25 polypeptide library includes at least one polypeptide, and the amino acid sequence of any polypeptide is the same as the amino acid sequence of a polypeptide in the OPM25 protein; the OPM31 polypeptide library includes at least one polypeptide, and the amino acid sequence of any polypeptide is the same as the amino acid sequence of a polypeptide in the OPM31 protein.

2. The polypeptide composition according to claim 1, characterized in that: The amino acid sequence of any one of the polypeptides in the OPM25 polypeptide library is selected from the amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 40; The amino acid sequence of any polypeptide in the OPM31 polypeptide library is selected from the amino acid sequences SEQ ID NO: 41 to SEQ ID NO:

86.

3. Use of the polypeptide composition according to claim 1 or 2 in the preparation of a product for detecting brucellosis.

4. The use according to claim 3, characterized in that: The product for detecting brucellosis includes a test kit, a detection reagent or a biological detection chip.

5. The use according to claim 4, characterized in that: The kit comprises a specific stimulation antigen, and the specific stimulation antigen comprises the polypeptide composition.

6. The use according to claim 5, characterized in that: The kit is an ELISPOT kit or an ELISA kit; The ELISPOT kit and the ELISA kit are both used to detect the level of the first type of specific cytokines, and are used to distinguish between brucellosis and non-brucellosis; The first type of specific cytokines includes one or more of TNF-β, IL-5, IP-10, IFN-γ, IL-6 and MCP-1.

7. The use according to claim 5, characterized in that: The kit is an ELISPOT kit or an ELISA kit; The ELISPOT kit and the ELISA kit are both used to detect the level of the second type of specific cytokines, and are used to distinguish acute brucellosis from chronic brucellosis; The second type of specific cytokines includes one or more of IP-10, IFN-γ and MCP-4.

8. The use according to claim 6, characterized in that: The ELISPOT kit also includes an experimental plate, a capture antibody and a detection antibody; The detection method of the ELISPOT kit comprises the following steps: S1. Obtain a cell sample to be tested, a negative control solution, and a positive control solution; S2, coating with a capture antibody that specifically binds to interferon-γ, adding a negative control solution to the negative control group, adding a positive control solution to the positive control group, and adding the polypeptide composition to the test group; S3, adding equal amounts of the cell samples to be tested to the negative control group, positive control group and detection group described in step S2; culturing at 37±1.0°C After 1 hour, anti-γ-interferon detection antibody was added; S4, developing color for the negative control group, the positive control group and the test group and measuring the number of spots, and judging the result of the test group according to the results of the negative control group and the positive control group; If the average number of spots in the positive control group is greater than 20, and the average number of spots in the negative control group is less than 10, then the average number of spots in the test group minus the average number of spots in the negative control group is greater than or equal to 7, and the test result is positive; if the average number of spots in the test group minus the average number of spots in the negative control group is less than or equal to 6, then the test result is negative; If the average number of spots in the positive control group is greater than 20, and the average number of spots in the negative control group is greater than 10, the cell sample to be tested needs to be measured repeatedly; If the average number of spots in the positive control group is less than 20, and the average number of spots in the negative control group is less than 10, then the average number of spots in the test group minus the average number of spots in the negative control group is greater than or equal to 7, and the test result is positive; if the average number of spots in the test group minus the average number of spots in the negative control group is less than or equal to 6, the cell sample to be tested needs to be measured repeatedly; If the average number of spots in the positive control group is less than 20, and the average number of spots in the negative control group is greater than 10, the cell sample to be tested needs to be measured repeatedly.

9. The use according to claim 8, characterized in that: In step S4, when the test result is positive, if the average number of spots in the test group minus the average number of spots in the negative control group is greater than or equal to 7 and less than or equal to 60, the cell sample to be tested corresponds to chronic brucellosis; If the average number of spots in the test group minus the average number of spots in the negative control group is greater than or equal to 61, the sample cells to be tested correspond to acute brucellosis.

10. The use according to any one of claims 8 to 10, characterized in that: In step S2, the concentration of each polypeptide in the polypeptide composition is 10-15 ug / ml; In step S1, the cell sample to be tested includes peripheral blood mononuclear cells, and the concentration of the peripheral blood mononuclear cells is 2.5×10 6 / mL~3.0×10 6 / mL.

11. Use of the polypeptide composition according to claim 1 or 2 in the preparation of a product for identifying brucellosis in the asymptomatic infection period.

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