CN113831401A

CN113831401A - SLE epitope polypeptide and application thereof in SLE diagnosis

Info

Publication number: CN113831401A
Application number: CN202110720644.8A
Authority: CN
Inventors: 裘宇容; 李欣
Original assignee: Southern Hospital Southern Medical University
Current assignee: Southern Hospital Southern Medical University
Priority date: 2021-05-19
Filing date: 2021-06-28
Publication date: 2021-12-24
Anticipated expiration: 2041-06-28
Also published as: CN113831401B

Abstract

The invention discloses an SLE epitope polypeptide and an effect thereof in SLE diagnosis, the SLE epitope polypeptide can provide a new target for SLE diagnosis, is used for better distinguishing SLE patients from healthy people and other autoimmune disease patients, is convenient to provide a new diagnosis strategy and reagent, and makes up for the vacancy of the existing SLE diagnosis technology. The epitope polypeptide provided by the invention has excellent diagnostic efficacy, so that the sensitivity and specificity of diagnosis can be ensured while the novel diagnostic strategy, technology and reagent are provided. The multiple epitope polypeptides of the invention or the combination of the multiple epitope polypeptides and the existing autoantibodies can also improve the sensitivity and specificity of diagnosis so as to obtain more effective diagnosis results. Besides the above benefits, the epitope polypeptide is also beneficial to providing a research basis for the elucidation of the SLE occurrence mechanism, so that the deep research of the occurrence mechanism is carried out based on the epitope polypeptide, and the development of SLE diagnosis and treatment is promoted.

Description

SLE epitope polypeptide and application thereof in SLE diagnosis

Technical Field

The invention relates to the technical field of immunological diagnosis, in particular to an SLE epitope polypeptide and an application thereof in SLE diagnosis.

Background

Systemic Lupus Erythematosus (SLE) is a common and complex autoimmune disease, also called "prototype" of autoimmune disease, with complex and diverse clinical manifestations, and often involving multiple systems throughout the body, including kidney, skin, joints, nervous system, serosa, etc., leading to damage of relevant tissues and organs. In severe cases, multiple organ failure, central nervous system injury, etc. may also occur, leading to death of the patient. Therefore, early diagnosis is crucial to timely alleviating or avoiding the involvement of important organs, improving the prognosis of patients and increasing the survival rate of patients.

However, the diagnostic efficacy of SLE is often limited, since the pathogenesis of SLE is not fully elucidated at present. For example, although conventional diagnosis based on the clinical phenotype of the patient can achieve certain diagnosis results, the diagnosis may be misdiagnosed because SLE is early-stage and only appears as one or two systems affected and is easily confused with other diseases; when multiple organs are affected, the diagnosis is not difficult, but the patient may have progressed to the middle and advanced stage of the disease, and the organ involvement is severe, which is not favorable for improving the prognosis and treatment effect of the patient. Therefore, early diagnosis is often difficult to achieve based on clinical phenotype alone, and more effective diagnostic means are needed.

For this reason, on the basis of the pathology that one of the important features of autoimmune diseases is the appearance of a large number of autoantibodies, the prior art provides means for the diagnosis of SLE by detection of autoantibodies, and this means is also the diagnostic means commonly used at present; detection of autoantibodies, including detection of ANA (antinuclear antibodies), anti-nucleosome antibodies, anti-dsDNA antibodies (anti-double stranded DNA antibodies), anti-Sm antibodies, etc., while detection by autoantibodies is more effective than diagnosis by clinical phenotype alone, it also has many drawbacks. For example, positive results are also observed in some autoimmune diseases other than SLE, and it is difficult to identify SLE from other autoimmune diseases, and the efficacy of detection of each antibody is also deficient in one or more aspects, which makes it increasingly difficult to meet the requirements for SLE diagnosis. For example, anti-dsDNA antibodies and anti-Sm antibodies have high specificity for SLE diagnosis, and can well identify SLE from other autoimmune diseases, but have the defect of low positive rate.

Therefore, there is a need in the art for a target that can be applied to SLE diagnosis to achieve effective diagnosis or can provide reference information for diagnosis, so as to construct a corresponding diagnostic reagent or method based on the target, provide a new diagnostic strategy, to meet the requirements of SLE clinical diagnosis method diversity and accuracy of diagnosis results, and make up for the shortcomings of the existing SLE diagnostic technology.

Disclosure of Invention

The invention aims at least one deficiency of the prior art, and provides an SLE epitope polypeptide and application thereof, the SLE epitope polypeptide can provide a new target for SLE diagnosis, can be used for better distinguishing SLE patients from healthy people and other autoimmune disease patients, has good diagnostic efficiency, and can be combined with the existing clinical diagnostic marker detection application to realize a diagnostic process with better diagnostic efficiency; the epitope polypeptide is also convenient to be applied to the preparation of SLE diagnostic reagents so as to obtain new effective diagnostic reagents and corresponding diagnostic strategies, improve the sensitivity and specificity of diagnosis and fill the vacancy in SLE diagnostic technology in the prior art.

One objective of the present invention is to provide an SLE epitope polypeptide, which comprises one or more of a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide and a fifth polypeptide, wherein the amino acid sequences of the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide and the fifth polypeptide are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No. 5. In one embodiment of the invention, the inventor screens SLE related polypeptides with specificity by a phage display technology, and obtains SLE epitope polypeptides with good diagnostic efficacy and amino acid sequences shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5 by a polypeptide chip and an ELISA method verification. The antigen polypeptide is specifically combined with IgG in serum of an SLE patient and can be used as a diagnostic target of SLE; based on the target, the method is helpful for providing a new SLE diagnosis strategy and preparing a new SLE diagnosis reagent.

Furthermore, the invention also provides the SLE epitope polypeptide, which is derived from the SLE epitope polypeptide by substituting, deleting or adding one or more amino acids and has the epitope activity reacting with IgG antibodies of systemic lupus erythematosus patients. When the derived polypeptide of the epitope polypeptide shown by SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5 has epitope activity specifically reacting with the systemic lupus erythematosus IgG antibody, the derived polypeptide can also be used as a diagnosis target of SLE, so that a corresponding diagnosis strategy and a diagnosis effect can be provided conveniently, and research in SLE diagnosis is promoted.

The first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide and the fifth polypeptide epitope polypeptide can be used for preparing antigens independently or combined with each other or other SLE epitope polypeptides, but carrier proteins are required in the detection process of antibody reaction or connection among the epitope polypeptides, so that the conjugate formed by the systemic lupus erythematosus epitope polypeptide and the carrier proteins is protected. The epitope polypeptide can be applied to the research on the treatment of SLE, and the application of the epitope polypeptide can provide a basis for the research on the treatment of SLE; the coupling with the carrier protein is helpful for the accurate and rapid transportation of the epitope polypeptide and promotes the rapid stress of the organism, so that the carrier protein conjugates corresponding to the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide and/or the fifth polypeptide in the epitope polypeptide are protected.

The nucleotide sequence for coding the systemic lupus erythematosus epitope polypeptide is beneficial to preparing a diagnostic reagent of a target specificity or specificity high-low expression nucleotide sequence at a gene level, and the structure and the characteristics of the epitope polypeptide can be more accurately obtained at the nucleotide sequence level. Furthermore, the application of the method to the detection of nucleotide sequences or the further research of the epitope polypeptides often requires the use of expression vectors or host cells of expression vectors containing the nucleotide sequences, so as to facilitate the multi-aspect research on the epitope polypeptides; and the research is beneficial to the disclosure and the exposition of SLE occurrence mechanism, so the expression vector for expressing the nucleotide sequence and the host cell containing the expression vector are also protected. Similarly, the expression vector based on the nucleotide sequence and the host cell of the expression vector are also helpful for providing research basis for diagnosing and treating SLE, and can even be applied to the preparation of reagents for diagnosing and treating SLE.

The ELISA method can well detect the reaction of the epitope polypeptide and the corresponding antibody, so the systemic lupus erythematosus epitope polypeptide can be used as a competitive/solid phase antigen of an enzyme-linked immunosorbent assay.

The invention also aims to provide application of the systemic lupus erythematosus epitope polypeptide and/or the nucleotide sequence in preparation of a systemic lupus erythematosus diagnostic reagent. By taking the epitope polypeptide or the corresponding nucleotide sequence as a target, the method is beneficial to researching a targeted diagnostic reagent according to the binding site of the epitope polypeptide or the corresponding nucleotide sequence, thereby overcoming the defect of nonspecific detection of the autoantibody in the prior art and improving the diagnostic accuracy; also is convenient for realizing early diagnosis, discovering SLE in time and adopting effective treatment mode, improving the prognosis of patients and improving the treatment effect and survival rate. Further, the diagnostic reagent at least comprises the function of distinguishing healthy people from SLE patients and/or distinguishing patients with other autoimmune diseases from SLE patients, wherein the other autoimmune diseases comprise rheumatoid arthritis, connective tissue diseases, autoimmune liver diseases, xerosis, polymyositis/dermatomyositis, scleroderma, ankylosing spondylitis, ANCA-related vasculitis and autoimmune hemolytic anemia. In one embodiment of the present invention, the epitope polypeptides described above are effective in distinguishing SLE from other autoimmune diseases described above.

The invention also aims to provide the application of the SLE epitope polypeptide and/or the nucleotide sequence in combination with SLE autoantibodies in the preparation of SLE diagnostic reagents. Data analysis shows that the combined detection of the plurality of antigen polypeptides and the autoantibodies has obvious diagnostic value and excellent diagnostic efficacy, so that the combined application of the antigen epitope polypeptides and the SLE autoantibodies is beneficial to the subsequent preparation of SLE diagnostic reagents and SLE diagnosis based on the antigen epitope polypeptide targets and the SLE autoantibodies, so that effective diagnosis with good specificity and sensitivity is realized, and the defects in the prior art when the autoantibodies are used alone are overcome.

Further, SLE autoantibodies include ds-DNA antibodies and/or Sm antibodies. In one embodiment of the invention, the SLE autoantibody is a ds-DNA antibody and/or an Sm antibody, and the above epitope polypeptide in combination with SLE autoantibody diagnostic data has an AUC above 0.874 and up to 0.916.

It is still another object of the present invention to provide a SLE diagnostic kit comprising the SLE epitope polypeptide as described above.

Further, SLE kits also comprise SLE autoantibodies.

Further, the epitope polypeptide is obtained by the following steps:

s1, detecting serum IgG antibodies of SLE group, health group and other disease groups by using phage display technology to screen and obtain the polypeptide only appearing in SLE group and the sequence thereof; s2, sorting according to copy number values, screening M high-copy-number polypeptides ranked at the front, matching the M polypeptides with human-derived and virus-derived protein databases, screening N polypeptides with matching results and trying to be included in a verification polypeptide set, wherein N is less than or equal to M; s3, performing copy number value high-low sequencing on the rest M-N polypeptides, screening P high-copy-number polypeptides ranked at the front and fitting the P high-copy-number polypeptides into a verification polypeptide set, wherein P is less than or equal to M-N; s4, constructing a corresponding polypeptide chip by taking the polypeptides in the verification polypeptide set as the site information of the polypeptide chip, detecting the polypeptide chip by utilizing SLE group, health group and serum samples of other disease groups, and obtaining the systemic lupus erythematosus epitope polypeptide with diagnostic value by AUC screening on the basis of the detection result.

Specifically, the M, N, P is determined based on the accuracy required.

Further, after step S4, the method further includes:

and step S5, carrying out diagnosis value identification and diagnosis efficiency evaluation on the systemic lupus erythematosus epitope polypeptide obtained in the step S4 on the basis of the SLE group, the health group and other disease groups by using an ELISA method, and determining the final systemic lupus erythematosus epitope polypeptide.

Further, the ELISA detection experiment conditions are as follows: the concentration of the polypeptide with the amino acid sequence shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 as antigen coating is respectively 0.1 mu g/mL, 0.05 mu g/mL, 0.02 mu g/mL and 0.02 mu g/mL, the second antibody is Goat anti-Human IgG, the dilution ratio of the second antibody is 1:2000, the dilution multiple of sample serum is 1: 1000 times.

A method for detecting whether serum samples contain antibodies which react with the SLE epitope polypeptide, the SLE epitope polypeptide is taken as an antigen, and the antibodies in the serum are detected by a polypeptide chip or an ELISA method.

Compared with the prior art, the invention has the beneficial effects that: the invention firstly screens SLE related polypeptide by phage display peptide technology, and further obtains the final SLE related polypeptide with potential diagnostic value according to copy number and database comparison after obtaining SLE related polypeptide. And then, obtaining the epitope polypeptide with diagnostic value by polypeptide chip verification and ELISA method verification, wherein the epitope polypeptide comprises a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide and a fifth polypeptide, the amino acid sequences of which are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5. The epitope polypeptide is subjected to multiple screening, the result reliability is high, and the example data show that the epitope polypeptide provided by the invention has better diagnostic efficiency; only a serum sample is needed to realize an effective diagnosis process, and the method is rapid, simple and convenient. Then, the application also aims at the combined application of the epitope polypeptides and SLE autoantibody detection to carry out diagnostic value and efficiency evaluation; the data of the examples show that compared with the detection of the SLE autoantibody alone, the combined detection of the SLE autoantibody and the epitope polypeptide provided by the invention has higher specificity and sensitivity; therefore, based on the combined application of the epitope polypeptide and the SLE autoantibody detection provided by the invention, a diagnostic reagent and a diagnostic strategy with high specificity and sensitivity can be conveniently provided, and a diagnostic result with high accuracy can be obtained; can also make up the vacancy of SLE detection technology and the deficiency of SLE autoantibody diagnosis mode in the prior art. Realizes early diagnosis, enables the SLE patient to be treated in time, and improves prognosis and treatment effect. And the epitope polypeptide is also beneficial to providing a basis for the elucidation of the SLE occurrence mechanism, so that the deep research of the occurrence mechanism is carried out on the basis of the epitope polypeptide, and the development of SLE diagnosis and treatment is promoted.

Drawings

FIG. 1 is a diagram showing the site information of the polypeptide chip in the examples;

FIG. 2 is a schematic view showing the loading of a polypeptide chip in the detection process of the polypeptide chip according to the embodiment;

FIG. 3 is a schematic diagram showing the results of chip detection of SLE-related polypeptides in the examples;

FIG. 4 is ROC graph of the embodiments SLE _ P19, SLE _ P20, SLE _ P27, SLE _ P28, SLE _ P29 and joint diagnosis;

FIG. 5 is a ROC graph of examples SLE _ P19, SLE _ P20, SLE _ P27, SLE _ P28 and SLE _ P29 in different autoimmune diseases;

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The present invention will now be further described with reference to specific examples, which are provided for the purpose of illustration only and are not to be construed as limiting the invention. The test samples and test procedures used in the following examples include the following (generally, according to the conventional conditions or according to the conditions recommended by the reagent company if the specific conditions of the experiment are not specified in the examples; and reagents, consumables and the like used in the following examples are commercially available unless otherwise specified).

Examples

First, preliminary screening

1. SLE-related polypeptide screening

Screening and comparing the binding polypeptides by using a phage display random dodecapeptide library technology through serum IgG antibodies of an SLE group, a healthy control group and other autoimmune disease control groups respectively. Specifically, this example uses the Ph.D. -12 phage display peptide library kit (NEW ENGLAND BIOLABS Co.) for screening.

Wherein, the information of three groups of samples of the SLE group, the healthy control group and the other autoimmune disease control group is shown in the table 1 and the table 2.

TABLE 1 three groups of basic information of specimens

TABLE 2 disease control autoimmune disease composition ratio

Respectively screening positive clones of each group, and then performing sequencing comparison to obtain the polypeptide only appearing in the SLE group and the sequence thereof; specifically, the polypeptide sequence obtained from the SLE group is compared with a healthy control group and a disease control group, and a repetitive sequence is removed, namely the sequence appearing in the control group is removed; and deleting the polypeptide sequences with copy number less than 500 to obtain 1374 polypeptides in total related to SLE. Then, according to the copy number (read count #) value of the polypeptide, sorting from large to small, selecting the first 100 polypeptides with large copy number value as the determined SLE related polypeptides, and performing bioinformatics analysis according to the above.

2. Bioinformatic analysis of SLE-related polypeptides

Because the purpose of the test is to obtain a new target antigen, 100 obtained polypeptides are compared with human-derived and virus-derived protein data (completed by south China medical biology company), wherein human autoantigens or related proteins are expected to be obtained for further research by comparing the human autoantigens and the related proteins with the human-derived protein data; meanwhile, some studies show that SLE onset is related to virus infection, so, and the SLE onset is also compared with virus-derived protein data to obtain related virus proteins. Specifically, 100 polypeptide sequences are compared with human-derived and virus-derived protein data in Ensemble and NCBI databases respectively, and the operation instructions are as follows: "blastp-task blastp-short-query data, fast-db database _ index-outfmt"6qseqid qlen sseqid slide even score parameter length mismatch gain positive qstart qtend sstart send "-evalue 1 e-5".

The comparison results are shown in tables 3 and 4;

TABLE 3 alignment of the preliminary screening polypeptide to the human-derived protein data (wherein qseqid: polypeptide sequence number; pident: probability of correct identification; length: length of correctly matched polypeptide; mismatch: number of mis-matched polypeptide; gene _ ID: number of gene ID; transcript _ ID: number of transcribed RNA; gene _ symbol: number of gene tag; description: description)

TABLE 4 alignment of the preliminary screening polypeptides with the viral-derived protein data (qseqid: polypeptide sequence number; cadent: probability of correct identification; length: length of correctly matched polypeptide; mismatch: number of mismatched polypeptides; dis: protein name; species name)

Bioinformatics analysis results show that 9 SLE-related polypeptides are well matched with human-derived protein data (one polypeptide sequence (seq-12422) can be matched with two proteins at the same time), and 12 SLE-related polypeptides are well matched with virus-derived protein data. Then, the polypeptides (21 in total) capable of matching protein data are firstly included in the polypeptides to be verified, the remaining 79 polypeptides are screened according to the sequence of copy number, then the high-copy-number polypeptides (29 in total) which are ranked at the top are included in the polypeptides to be verified, and the polypeptides are planned to be site information in a subsequently constructed polypeptide chip.

Second, polypeptide chip verification

1. Polypeptide chip construction

Constructing a polypeptide chip according to the screened polypeptide to be verified for subsequent verification, and specifically, regarding 50 screened polypeptides as site information incorporated into the polypeptide chip; the polypeptide chip in this example was manufactured by south China medical Limited, Guangzhou, 16 persons/sheet, wherein the chip substrate sheet was purchased from Arrayit001, USA, and the polypeptide was synthesized by Gill Biochemical (Shanghai) Limited. In a specific embodiment, 7 of the 50 polypeptides selected by the phage display technology (seq-36882, seq-41138, seq-15708, seq-42780, seq-22436, seq-7683, seq-22783) are discarded because they cannot be dissolved, and finally 44 polypeptides, including a random peptide (sequence: DIHRHVVGARTL), are co-spotted.

In addition, in order to ensure the quality of the experimental result, bovine serum albumin with two concentrations is coupled with Biotin, namely Biotin-BSA-250X and Biotin-BSA-500X are used as positive controls; 1xPBS as a blank control, and random peptide as a negative control; each polypeptide was subjected to 2 multiple wells to ensure quality, and the information on the specific polypeptide chip is shown in FIG. 1.

After the polypeptide chip was constructed, the examination was carried out using 644 cases of serum samples (296 cases of SLE group samples, 168 cases of disease control group samples, and 180 cases of healthy control group samples, among them), and the sample information is shown in tables 5, 6, and 7.

TABLE 5 three sets of basic information of specimens

TABLE 6SLE cohort basic clinical information

TABLE 7 disease composition ratio of autoimmune diseases in control group

2. The detection and verification process of the polypeptide chip comprises the following steps:

(1) the main experimental materials:

the instrument equipment comprises: high-speed low-temperature refrigerated centrifuge: hermle, Germany (HERMLE Labortechnik GMbH); SK-D1807-E three-dimensional decoloration shaking table: sailyker, USA; chip LuxScan 10K/B microarray chip scanner: boao biology ltd;

the main reagents are as follows: polypeptide, synthesized by gill biochemical (shanghai) ltd; polypeptide chips from Arrayit, usa (Arrayit 001); confining liquid, bio-engineering (Shanghai) corporation; goat anti-human IgG labeled with biotin, Biotechnology (Shanghai) Ltd; streptavidin labeled with fluorescein, bio-engineering (Shanghai) Co., Ltd; 25x lotion i (PBST), bio-engineering (shanghai) gmbh; 25 XWash solution II (PBS), Biotechnology engineering (Shanghai) Co., Ltd.

(2) Collection of serum samples

Samples are all from residual specimens of southern hospital clinical laboratory of southern medical university, and are centrifuged for 10min at 3500r/min after venous blood specimens are collected; the selected samples have no lipemia and hemolysis, the residual serum of each patient is subpackaged into 2-3 tubes and 400 mu L/tube at 200-.

(3) Polypeptide chip detection

1) Preparing a reagent: assembling the constructed SLE polypeptide chip as shown in FIG. 2; preparing diluted washing liquid: the 25 XWash I was diluted to 1X to 1L and the 25 XWash II was diluted to 1X to 1L with deionized water, respectively, and labeled for future use.

2) The experimental steps are as follows: firstly, after loading chips, adding 90 mu L of 1x confining liquid into each array of the chips, and incubating for 1 hour at room temperature on a shaking table; beating to remove the sealing liquid, adding 90 mu L of samples diluted by 2 times of the sealing liquid into each array, and placing the arrays on a shaking table to shake for 4 hours; and thirdly, beating the sample and cleaning the slide. Repeatedly washing with 1x lotion I for 5 times, 5min each time, repeatedly washing with 1x lotion II for 5 times, 5min each time, and drying; fourthly, diluting the biotin-human IgG by using confining liquid 10⁶And (5) doubling and uniformly mixing. Adding 90 mu L of diluted biotin-human IgG into each hole, placing on a shaking table, and shaking and incubating for 3 hours at room temperature to avoid generating bubbles; cleaning and synchronizing; sixthly, diluting the fluorescence-labeled streptavidin by 2000 times by using a confining liquid, uniformly mixing, adding 90 mu L of diluted streptavidin to each hole, sticking a slide by using an aluminum foil sealing strip, and then incubating for 1 hour at room temperature on a shaking table; seventhly, cleaning and synchronizing; taking the chip matrix out of the frame and scanning by a fluorescence scanner.

3. And (3) analyzing a detection and verification result of the polypeptide chip: after polypeptide chips of the three groups of samples are detected according to the detection process, a chip result is scanned by a crystal core LuxScan 10K/B microarray chip scanner, wherein the wavelength of excitation light is 555 nm; then, chip analysis software GenePix Pro 6.0software is used for extracting data, a positive control Biotin-BSA point signal value is more than 1000, the result of the experiment is qualified, the area under the curve of the random peptide is about 0.5, the positive control is regarded as effective, the detection result of each serum sample is the mean value of two multi-well fluorescence signal values minus a blank control signal value, and specifically, a detection result representative graph of the SLE related polypeptide chip is shown in figure 3.

4. Polypeptide diagnosis efficacy analysis: performing ROC Curve drawing according to the fluorescence signal value, respectively calculating the Area Under the Curve (AUC) of the SLE group and the healthy control group, the SLE group and the disease control group, and the SLE group and the healthy control group combined with the disease control group to evaluate the diagnosis efficiency, selecting the polypeptide with AUC >0.650 and P <0.05 as the subsequent second round verification polypeptide, and selecting 5 polypeptides in the embodiment as follows; SLE _ P19, SLE _ P20, SLE _ P27, SLE _ P28, SLE _ P29, the amino acid sequences of which are SLE _ P19: NSLLNLEKTMVR, respectively; SLE _ P20: DSTCPMVTAPCS, respectively; SLE _ P27: GLYHSNASFRVP, respectively; SLE _ P28: LTNPGLGSSPKA, respectively; SLE _ P29: SCYPAVPQCSTT are provided.

And determining cut-off value of the selected polypeptide according to Youden's Index (YI), and calculating common evaluation index of corresponding diagnostic test according to the cut-off value: sensitivity (SEN), Specificity (SPE), Positive Predictive Value (PPV), Negative Predictive Value (NPV), Positive Likelihood Ratio (PLR), Negative Likelihood Ratio (NLR), accuracy (Ac); performing combined diagnostic value evaluation on the selected polypeptide through logistic regression based on Spss 23.0; the results are shown in tables 8 and 9.

TABLE 8 preliminary screening of polypeptide diagnostic value analysis results

Table 9SLE _ P19, SLE _ P20, SLE _ P27, SLE _ P28, SLE _ P29 and associated diagnostic test evaluation indices for joint diagnosis;

the results of the polypeptide chip screening show that 5 epitope polypeptides (SLE _ P19, SLE _ P20, SLE _ P27, SLE _ P28 and SLE _ P29, the sequences of which are respectively shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No. 5) have better diagnostic efficacy, SLE _ P20 performs optimally compared with a healthy control group, the AUC is 0.864, the AUC of SLE _ P27 is the largest compared with a disease control group and is 0.844, the AUC after the combined diagnosis of 5 polypeptides is 0.956, and the accuracy is 92.80%, as shown in FIG. 4.

Third, ELISA method verification

The above 5 polypeptides were further verified by ELISA using 500 serum samples (200 SLE group samples, 150 disease control group samples, and 150 healthy control group samples). The specimen information is shown in tables 10 to 12.

TABLE 10 three sets of specimen basic information

TABLE 11 SLE cohort basic clinical information

TABLE 12 disease composition ratio of autoimmune diseases in control group

The specific test process comprises the following steps:

1. the main reagents are as follows: antigenic polypeptides, synthesized by gill biochemical (shanghai) ltd; high adsorption ELISA plate, Beijing Sizhengbai Biotech limited; coating liquid, Beijing Sizhengbai Biotech Co., Ltd; bayonet membranes, Beijing Sizhengbai Biotech Co., Ltd; sample diluent, Beijing Sizhengbai Biotech limited; confining liquid, Beijing Sizhengbai Biotech limited; concentrating the washing solution, Beijing Sizhengbai Biotech limited; enzyme conjugate diluent, beijing quanzi bio-technology ltd; color developing solution/single component ultrasensitive TMB, Beijing Sizhengbai Biotech Co., Ltd; stop solution, Beijing Sizhengbai Biotech limited; goat anti-Human IgG (H + L), HRP, Beijing Sizhengbai Biotech Co., Ltd; anti-ds-DNA antibody detection kit (ELISA method), Askeu, Germany; anti-Sm antibody detection kit (ELISA method), Europe and Mongolian medicine, Co., Ltd, Germany.

2. Determination of conditions for polypeptide detection

(1) Reagent preparation

1) Washing liquid: measuring 25mL of 20 Xconcentrated washing solution, and diluting the washing solution to 500mL by using pure water; 2) diluting the antigen polypeptide to 0.02. mu.g/mL, 0.05. mu.g/mL, 0.1. mu.g/mL, 1. mu.g/mL and 10. mu.g/mL respectively by using the coating buffer; 3) three groups of serum samples, three of each group, are respectively diluted to 100 times, 1000 times and 2000 times by using sample diluent; 4) secondary antibody (coat anti-Human IgG) was diluted with enzyme conjugate according to 1:2000, diluting;

(2) experimental procedure

1) Envelope antigen polypeptide: adding the diluted antigen polypeptides with different concentrations into the concave holes of the ELISA plate, adding 100ul of antigen polypeptides into each hole, and standing overnight at 4 ℃; 2) washing: removing the coating solution, washing the concave hole with 300 μ L of washing buffer solution for 3 times, each time for 30 s; 3) adding 200 μ L of sealing liquid into each well, sticking a sealing plate membrane, and incubating in an electrothermal blowing dry oven at 37 deg.C for 1 h; 4) washing; removing the confining liquid, and washing according to the step 2); 5) adding 100 μ L of serum with different dilution ratios into each concave hole, sticking a sealing plate film, and incubating in an electrothermal blowing dry oven at 37 deg.C for 1 h; 6) washing: removing the diluted sample, and washing according to the step 2); 7) adding 100 mu L of color developing agent into each concave hole, keeping out of the sun, and incubating for 30 minutes at room temperature; 8) adding 100 mu L of stop solution into each concave hole; 9) and detecting the OD value of the ELISA plate by using the ELISA reader within 30 minutes, wherein the wavelength is set to 405 nm.

(3) Result analysis method

1) The OD value of the SLE group is larger than that of two groups of control groups as much as possible under the unified experimental condition, and the disease control group is not different from the healthy group; 2) the same sample is compared with different antigen coating concentration differences, and coating concentrations with large OD values are selected as much as possible (when the OD values are reduced along with the increase of the coating concentrations, the antigen saturation is indicated); 3) selecting the condition that the OD value of the SLE group is about 1 as much as possible, wherein the OD values of the disease control group and the healthy group are as small as possible but need to be different from those of blank wells; 4) when the above conditions are satisfied, the sample dilution factors are as uniform as possible.

The finally determined five polypeptide detection experimental conditions are as follows: the dilution ratio of the secondary antibody is 1:2000, and the serum dilution multiple is 1: 1000 times, the concentration of five polypeptides as antigen coating is respectively: SLE _ P19 was 0.1. mu.g/mL, SLE _ P20 was 0.05. mu.g/mL, SLE _ P27 was 0.05. mu.g/mL, SLE _ P28 was 0.02. mu.g/mL, and SLE _ P29 was 0.02. mu.g/mL.

3. Second round verification experiment of polypeptide-successive chip

(1) Reagent preparation

1) Diluting 25mL of 20 Xconcentrated washing solution to 500mL with deionized water; 2) according to the concentration of the antigen polypeptide in the detection experiment condition determined in the previous period, SLE _ P19, SLE _ P20, SLE _ P27, SLE _ P28 and SLE _ P29 are respectively diluted to 0.1 mu g/mL, 0.05 mu g/mL, 0.02 mu g/mL and 0.02 mu g/mL by using coating diluent; 3) sample dilution: samples were diluted with sample dilutions according to 1: diluting at a ratio of 1000; 4) secondary antibody: dilution with enzyme conjugate according to 1: diluting at a ratio of 2000;

(2) the experimental steps are as follows:

1) antigen coating: adding 100 mu L of diluted antigen polypeptide into each concave hole of the enzyme label plate, and incubating overnight at 4 ℃; 2) washing: removing the coating solution, washing the concave hole with 300 μ L of washing buffer solution for 3 times, each time for 30 s; 3) adding 200 μ L of sealing liquid into each well, sticking a sealing plate membrane, and incubating in an electrothermal blowing dry oven at 37 deg.C for 1 h; 4) washing; removing the confining liquid, and washing according to the step 2); 5) adding 100 mu L of serum with different dilution ratios into each concave hole, preparing two compound holes for each sample, sticking a sealing plate film, and putting the samples into an electrothermal blowing dry box at 37 ℃ for incubation for 1 h; 6) washing: removing the diluted sample, and washing according to the step 2); 7) adding 100 mu L of color developing agent into each concave hole, keeping out of the sun, and incubating for 30 minutes at room temperature; 8) adding 100 mu L of stop solution into each concave hole; 9) within 30 minutes, detecting the OD value of the ELISA plate on an ELISA reader, wherein the wavelength is set at 405 nm;

4. statistical method

The data statistics and mapping of the experiment are completed by IBM SPSS statistics 23.0, Graphpad prism 8.0.

1) Sample OD value results: subtracting the blank control OD value average value of the plate from the average value of two repeated measurement OD values of each sample OD value to obtain a final OD value result;

2) statistical description of sample clinical data: the measurement data is described by Mean + -SEM for normal distribution, and M (P) for biased distribution₂₅，P₇₅) The qualitative data is expressed by composition ratio and positive rate. The described subjects include three groups of baseline data (age and sex), and the SLE group basic clinical characteristic data include disease activity composition ratio, disease course, ANA positive rate, ds-DNA antibody positive rate, Sm antibody positive rate, positive rate of different involved system organs, and common rheumatism treatment drugs (including usage glucocorticoid, hydroxychloroquine, cyclophosphamide, methotrexate, tacrolimus, cyclosporine, mycophenolate mofetil and calcitriol). The disease control group comprises the constitution ratio of the non-SLE autoimmune disease, the ds-DNA antibody positive rate and the anti-Sm antibody positive rate. Specifically, the statistical description of the samples in this embodiment is shown in tables 10 to 12 above.

3) Evaluation of diagnostic efficacy: processing and analyzing the result in the step 4 in the verification of the polypeptide chip; specifically, the results of this example are shown in tables 13 and 14. Specifically, the analysis results of the diagnostic efficacy of SLE _ P19, SLE _ P20, SLE _ P27, SLE _ P28 and SLE _ P29 in different autoimmune diseases are shown in table 15, and the ROC curve graph thereof is shown in fig. 5.

4) Further, five polypeptide results were compared with statistical differences in levels between the three groups: performing a normal variance homogeneity test on the data result, if the variances are uniform, performing One-Way ANOVA (One-Way ANOVA), if the variances are not uniform, performing Kruskal-walls rank sum test by adopting a nonparametric test, and drawing a scatter diagram; further, to preliminarily investigate the correlation of the polypeptides with SLE clinical symptoms, correlation analysis of five polypeptides with other clinical data: converting the polypeptide result into qualitative data (negative result and positive result) according to the cut-off value, analyzing the correlation between the activity degree of diseases and the incidence rate of organ involvement between the positive result and the negative result of the polypeptide, and mainly adopting Mann-Whitney U test for analysis and Chi-Squre test;

5) furthermore, this example also explores whether the five polypeptides could improve the diagnostic efficacy of SLE in combination with two clinically used SLE-specific markers, anti-ds-DNA antibody and anti-Sm antibody. Respectively describing ROC curves of the anti-ds-DNA antibody and the anti-Sm antibody by taking a disease control group as a reference, and obtaining corresponding AUC values; then 5 polypeptides are respectively combined with the anti-ds-DNA antibody and/or the anti-Sm antibody for diagnosis, and corresponding AUC values are obtained; the results are shown in Table 16 (where SLE _ P19/P20/P27/P28/P29 represents SLE _ P19, SLE _ P20, SLE _ P27, SLE _ P28, SLE _ P29 union).

The results were considered statistically significant with P < 0.05.

TABLE 13SLE _ P19, SLE _ P20, SLE _ P27, SLE _ P28, SLE _ P29 diagnostic value validation results

TABLE 14 SLE _ P19, SLE _ P20, SLE _ P27, SLE _ P28, SLE _ P29 and associated diagnostic test evaluation index for joint diagnosis

TABLE 15 analysis of the diagnostic potency of SLE _ P19, SLE _ P20, SLE _ P27, SLE _ P28, SLE _ P29 in different autoimmune diseases

TABLE 16 diagnosis results of SLE _ P19, SLE _ P20, SLE _ P27, SLE _ P28, SLE _ P29 in combination with anti-ds-DNA antibody and/or anti-Sm antibody

The results show that: as shown in tables 13-15, the results of ELISA experiments show that the AUC of 5 epitope polypeptides is greater than 0.7, and the AUC after combined detection is 0.943. The result trend of the 5 polypeptide verification results is consistent with that of the polypeptide chip, five polypeptides have diagnostic value, can exist as epitope polypeptides, and can effectively distinguish SLE from other autoimmune diseases. And wherein the AUC of SLE _ P27 is 0.938, the sensitivity is 76.00%, the specificity is 92.70%, the positive likelihood ratio is 10.411, the negative likelihood ratio is 0.259, and the accuracy is 84.40%; SLE _ P27 is superior to other four polypeptides in the diagnostic efficiency in the differential diagnosis of SLE and RA, and AUC is 0.842; SLE _ P29 outperformed the other four polypeptides in distinguishing SLE from other autoimmune diseases. In two rounds of verification, the SLE _27 has the overall performance of the evaluation indexes of the related diagnostic tests superior to other four polypeptides, so the comprehensive performance of the SLE _27 is relatively better.

As shown in table 16 (where SLE _ P19/P20/P27/P28/P29 represents SLE _ P19, SLE _ P20, SLE _ P27, SLE _ P28, SLE _ P29 combinations), the diagnostic efficacy results for SLE with five polypeptides combined with two SLE-specific markers commonly used clinically showed: the AUC value of ds-DNA antibody alone was 0.848, while the AUC of Sm antibody alone was only 0.575; after the 5 screened polypeptides are respectively combined with the two markers for ROC analysis, the AUC after combined diagnosis is respectively 0.912 and 0.848, and the AUC after the 5 polypeptides are simultaneously combined with the two markers for diagnosis is 0.916, which indicates that the combined detection of the 5 polypeptides and the common clinical SLE specific markers has excellent SLE diagnosis efficiency, and the combination of the 5 polypeptides and the common clinical SLE specific markers can obviously improve the diagnosis efficiency on SLE. Therefore, five polypeptides can not only provide effective diagnostic targets by themselves, but also can be combined with the existing autoantibody detection to obtain diagnostic reagents or diagnostic strategies with more diagnostic value.

It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention claims should be included in the protection scope of the present invention claims.

SEQUENCE LISTING

<110> southern hospital of southern medical university

<120> SLE epitope polypeptide and its use in SLE diagnosis

<130>

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 12

<212> PRT

<213> unknown

<400> 1

Asn Ser Leu Leu Asn Leu Glu Lys Thr Met Val Arg

1 5 10

<210> 2

<211> 12

<212> PRT

<213> unknown

<400> 2

Asp Ser Thr Cys Pro Met Val Thr Ala Pro Cys Ser

1 5 10

<210> 3

<211> 12

<212> PRT

<213> unknown

<400> 3

Gly Leu Tyr His Ser Asn Ala Ser Phe Arg Val Pro

1 5 10

<210> 4

<211> 12

<212> PRT

<213> unknown

<400> 4

Leu Thr Asn Pro Gly Leu Gly Ser Ser Pro Lys Ala

1 5 10

<210> 5

<211> 12

<212> PRT

<213> unknown

<400> 5

Ser Cys Tyr Pro Ala Val Pro Gln Cys Ser Thr Thr

1 5 10

Claims

1. The SLE epitope polypeptide is characterized by comprising one or more of a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide and a fifth polypeptide, wherein the amino acid sequences of the first polypeptide, the second polypeptide, the third polypeptide, the fourth polypeptide and the fifth polypeptide are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No. 5.

2. An SLE epitope polypeptide, which is derived from the SLE epitope polypeptide of claim 1 by substitution, deletion or addition of one or more amino acids and has an epitope activity reactive with IgG antibodies of patients with systemic lupus erythematosus.

3. A nucleotide sequence encoding a SLE epitope polypeptide according to claim 1 or 2.

4. Use of the SLE epitope polypeptide of claim 1 or 2 as a competitive/solid phase antigen for enzyme linked immunosorbent assay.

5. Use of a SLE epitope polypeptide of claim 1 or 2 and/or a nucleotide sequence of claim 3 for the preparation of a SLE diagnostic reagent.

6. Use of a SLE epitope polypeptide of claim 1 or 2 and/or a nucleotide sequence of claim 3 in combination with SLE autoantibodies for the preparation of a SLE diagnostic reagent.

7. The use of claim 6, wherein the SLE autoantibodies comprise a ds-DNA antibody and/or an Sm antibody.

8. A SLE diagnostic kit comprising a SLE epitope polypeptide of claim 1 or 2.

9. The SLE diagnostic kit of claim 8, further comprising SLE autoantibodies.

10. A method for detecting whether a serum sample contains an antibody reactive with the SLE epitope polypeptide of claim 1 or 2, wherein the SLE epitope polypeptide of claim 1 or 2 is used as an antigen, and the antibody in the serum is detected by a polypeptide chip or ELISA method.