CN112457392B - Soluble ST2 protein antigenic determinant polypeptide and application thereof - Google Patents
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Abstract
The invention provides a soluble ST2 protein antigenic determinant polypeptide and application thereof. The amino acid sequence of the soluble ST2 protein antigenic determinant polypeptide is one of the sequence shown in SEQ ID NO.1 and the sequence shown in SEQ ID NO. 2. The soluble ST2 protein antigen is prepared by coupling the epitope polypeptide and a carrier protein, the soluble ST2 protein antibody is a monoclonal antibody or a polyclonal antibody prepared from the antigen, and the monoclonal antibody and the polyclonal antibody are used for preparing a soluble ST2 protein detection kit. The soluble ST2 protein epitope polypeptide has good antigenicity, an antigen immune animal prepared by the soluble ST2 protein epitope polypeptide can generate a specific antibody, a prepared soluble ST2 protein antigen has good immunogenicity, a prepared ST2 protein antibody can be specifically combined with soluble ST2 in human serum, and a prepared soluble ST2 detection kit can effectively detect the ST2 protein level in the human serum.
Description
Technical Field
The invention relates to the field of polypeptide chemistry, in particular to soluble ST2 protein antigenic determinant polypeptide and application thereof.
Background
Growth Stimulating factor Gene expression 2 protein (Growth Stimulating Express Gene 2, ST 2) is a member of interleukin I receptor family, and is firstly reported to be found in 1989, but the function and mechanism of the protein are not clear at that time, so the protein is considered as an orphan receptor until the research in 2005 finds a specific ligand of ST2, namely interleukin 33 (IL-33). In recent years, the focus of ST2 research has largely surrounded the area of cardiovascular disease, with cardiovascular-related ST2 comprising mainly two configurations: soluble ST2 (sST 2) and cross-model ST2 (ST 2L). ST2L is mainly distributed in myocardial cells, forms a signal path with IL-33, resists myocardial fibrosis and myocardial hypertrophy, slows down ventricular remodeling and plays a role in protecting the myocardium; sST2 exists mainly in blood and competes with ST2L for IL-33 binding, and sST2 binds IL-33, which impairs the myocardial protection effect of ST2L-IL-33 signaling pathway and promotes ventricular remodeling. On the other hand, sST2 is secreted into the peripheral blood when the myocardium is subjected to stretch stimulation, so sST2 can be a detectable index reflecting myocardial fibrosis and is not affected by age, race, body mass index, renal function, and the like.
Some researches show that the sST2 has good application values in the aspects of clinical application such as auxiliary diagnosis of heart failure, prediction of the prognosis condition of heart failure, thinning of clinical stratification of heart failure, prediction of death probability of patients and the like. A1 year follow-up study of 593 patients admitted to the emergency room due to acute dyspnea found that the blood sST2 levels predicted mortality within 1 year for patients, with plasma sST2 concentrations greater than the mean 11-fold higher mortality within 1 year for patients with lower concentrations. In another follow-up study aiming at 137 patients with acute heart failure, 41 patients die within 1 year, the concentration of sST2 at the initial stage of onset is increased compared with that of patients without death, the concentration of sST2 is obviously related to the long-term mortality of the patients with heart failure, and furthermore, the concentration of sST2 is more than 700ng/L, so that the mortality of the patients with acute heart failure within 1 year can be independently predicted. In addition to the baseline level of sST2, the dynamic detection of sST2 enables more effective risk stratification and prognosis assessment for patients with acute/chronic heart failure, since sST2 has a lower rate of biological variation than other markers and is more suitable for detecting disease-directed therapy. A short-term rapid decrease in sST2 suggests a good response to treatment with a better prognosis for patients with acute heart failure, while an increase in sST2 is more likely to reflect an increase in the risk of death. It has been proposed in the American guidelines for heart failure and the Chinese guidelines for the diagnosis and treatment of heart failure that soluble ST2 can reflect myocardial fibrosis, and is a new generation of heart failure management marker with great clinical potential.
Therefore, the determination of the concentration of sST2 is of great clinical significance and value for clinical and prognostic management of patients with cardiovascular disease. The conventional assay for sST2 is typically an immunoassay, i.e. the level of sST2 is detected using an sST2 antibody, and therefore, the discovery and preparation of a suitable ST2 epitope polypeptide has also become the focus of the sST2 assay.
With the development of the medical examination field, a soluble ST2 protein epitope polypeptide with high specificity and antigenicity is clinically needed to be suitable for the preparation and production of high specificity antibodies. Therefore, the soluble ST2 protein antigenic determinant polypeptide and the application thereof are provided, and the soluble ST2 protein antigenic determinant polypeptide has good application prospect and practical significance.
Disclosure of Invention
Aiming at the defects in the background technology, the invention provides the soluble ST2 protein epitope polypeptide and the application thereof, the epitope polypeptide has high antigenicity and strong specificity, and can be used for preparing the corresponding soluble ST2 specific antigen and antibody, the soluble ST2 detection kit and the application thereof.
In order to achieve the purpose, the invention provides the following technical scheme:
in a first aspect, the present invention provides a soluble ST2 protein epitope polypeptide, wherein the amino acid sequences of epitope polypeptides (1) and (2) are the sequence shown in SEQ ID No.1 and the sequence shown in SEQ ID No.2, and specifically:
(1) Arg-Arg-His-Thr-Val-Arg-Leu-Tyr; and
（2）Gln-Tyr-Asp-Cys-Leu-Ala-Leu-Asn-Leu-His-Gly-Tyr。
in a second aspect, the present invention provides a soluble ST2 protein antigen, wherein the soluble ST2 protein antigen is prepared by conjugating the epitope (1) or (2) polypeptide to a carrier protein.
In a third aspect, the invention provides a soluble ST2 protein antibody, wherein the soluble ST2 protein antibody is a monoclonal or polyclonal antibody prepared from the antigen.
In a fourth aspect, the invention provides an application of the soluble ST2 protein antibody in the third aspect in preparing a soluble ST2 protein detection kit.
In a fifth aspect, the present invention provides a soluble ST2 protein detection kit comprising the antibody of the third aspect as a coating antibody, preferably a monoclonal antibody.
Preferably, the soluble ST2 protein detection kit is used for quantitative detection of soluble ST2 protein levels in human serum.
Compared with the prior art, the invention has the following beneficial effects:
1. the soluble ST2 protein antigenic determinant polypeptide provided by the invention has good antigenicity, and an antigen immune animal prepared by using the polypeptide can generate a specific antibody;
2. the soluble ST2 protein antigen provided by the invention has good immunogenicity, and can stimulate immune response of antigen immune animals;
3. the soluble ST2 protein antibody provided by the invention can be specifically combined with soluble ST2 in human serum;
4. the soluble ST2 detection kit provided by the invention can effectively detect the protein level of ST2 in human serum.
Drawings
FIG. 1 is a concentration versus absorbance standard curve for soluble ST2 calibrator in a soluble ST2 protein detection kit.
Detailed Description
In order that the technical contents of the invention can be more clearly understood, the following detailed description is provided for further explanation.
The data used in the present invention have meanings commonly understood by those of ordinary skill in the relevant art. However, for a better understanding of the present invention, some definitions and related terms are explained as follows:
the term "antigenic determinant", as used in the present invention, refers to an antigenically determining chemical group, which is localized on the surface of an antigenic substance and may consist of a contiguous sequence of amino acids or a discontinuous three-dimensional structure of a protein. As used herein, "antigenic determinant" refers specifically to antigenic determinants (1) and (2) of the human soluble ST2 protein.
Soluble ST2 protein antigenic determinant.
The soluble ST2 protein of the present invention is known in the art, and its amino acid sequence is known and can be found in professional databases.
The invention provides a soluble ST2 protein antigenic determinant polypeptide, wherein the amino acid sequences of the antigenic determinant polypeptides (1) and (2) are the sequence shown in SEQ ID NO.1 and the sequence shown in SEQ ID NO.2, and the soluble ST2 protein antigenic determinant polypeptide specifically comprises the following components:
(1) Arg-Arg-His-Thr-Val-Arg-Leu-Tyr; and
（2）Gln-Tyr-Asp-Cys-Leu-Ala-Leu-Asn-Leu-His-Gly-Tyr。
through theoretical research and experimental exploration, the two screened soluble ST2 protein antigenic determinant polypeptides have good antigenicity and can be specifically combined with corresponding antibodies.
The epitope (1) is a polypeptide fragment from the 85 th to the 91 th positions of the N-terminus of the human ST2 protein (NCBI accession number: BAA 20539.1), and is obtained by adding amino acid Y to the C-terminus, and contains 8 amino acids in total.
The epitope (2) is a polypeptide fragment from 73 th to 83 th positions of the N-terminus of human ST2 protein (NCBI accession number: BAA 20539.1), and is obtained by adding amino acid Y to the C-terminus, and contains 12 amino acids in total.
The addition of amino acid Y to the C-terminus is intended to allow the antigenic determinant to be cross-linked to a carrier protein via Bis-diazotized benzidine (BDB) and thereby allow the corresponding antibody to be prepared as an antigen. The antigenic determinant has the characteristics of high hydrophilicity, strong antigenicity and easy synthesis.
The antigenic determinant provided by the invention has the following characteristics:
1. the antigen is strong, and can be specifically combined with a corresponding antibody;
2. after the antigenic determinant is combined with carrier protein, the antigen immune animal can be stimulated to prepare a monoclonal antibody;
3. antibodies prepared using this epitope can specifically bind to human serum soluble ST 2.
The molecular weight of the antigenic determinant (1) provided by the invention is 1226.28, and the molecular weight of the antigenic determinant (2) is 1532.52, and the molecular weight can be determined by mass spectrometry.
Soluble ST2 protein antigen.
The soluble ST2 protein antigen provided by the invention is prepared by coupling the soluble ST2 protein antigenic determinant (1) or (2) polypeptide provided by the invention with a carrier protein.
The carrier protein includes hemocyanin (KLH), Bovine Serum Albumin (BSA), Ovalbumin (OVA), etc., wherein the preferred carrier protein is KLH, which has strong immunogenicity, good immune effect and is not easy to cause cross reaction.
Soluble ST2 protein antibody.
The soluble ST2 protein antibody provided by the invention is a monoclonal antibody prepared by coupling the soluble ST2 protein antigen provided by the invention with a carrier protein, the preparation method is a conventional technology in the field, the monoclonal antibody can be used for preparing a soluble ST2 protein detection kit, and the kit can detect the soluble ST2 protein in a human blood sample based on an immunization method.
And fourthly, a soluble ST2 protein detection kit.
The kit for detecting the soluble ST2 protein provided by the invention preferably adopts an ELISA double-antibody sandwich method to detect the human soluble ST2 protein, and comprises necessary reagents such as a coating antibody, a binding antibody, an enzyme-labeled secondary antibody and the like.
Preferably, the soluble ST2 protein detection kit uses the soluble ST2 protein monoclonal antibody of the present invention as an envelope antibody.
Wherein, when the coating antibody is the soluble ST2 protein monoclonal antibody (1), the binding antibody is soluble ST2 protein polyclonal antibody (2); when the coating antibody is the soluble ST2 protein monoclonal antibody (2), the binding antibody should be soluble ST2 protein polyclonal antibody (1).
In addition, the soluble ST2 protein detection kit may also include any reagents or tools required for the assay, such as pre-coated plates, wash solutions, stop solutions, and the like.
The invention also provides application of the soluble ST2 protein detection kit in quantitative determination of human serum soluble ST2 protein.
To further illustrate the technical means and effects of the present invention, the present invention is further described with reference to the following embodiments, but the scope of the present invention is not limited by the specific embodiments, and it should be understood that the claims are only directed to the described embodiments, and not to the whole embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Example 1 preparation of soluble ST2 protein epitopes (1) and (2).
The preparation method of the soluble ST2 protein antigenic determinants (1) and (2) adopts a chemical solid phase method to synthesize the antigenic determinants, and adopts the principle that the C-terminal of amino acid is fixed on insoluble resin, and then condensation reaction is carried out in sequence and peptide chain is prolonged until the preparation of polypeptide is finished.
The soluble ST2 protein epitopes (1) and (2) of the present invention were prepared as follows.
1. Reagents and raw materials:
HMP resin (P-hydroxymethylphenoxymethyl polyethylene resin, Wang resin);
Fmoc-AA (9-fluorenylmethoxycarbonyl protected amino acid);
NMP (nitrogen methyl pyrrolidone);
DCM (dichloromethane);
MeoH (methanol);
piperidine (Piperidine);
DMAP (dimethylaminopyridine);
HOBT (hydroxybenzotriazole);
DCC (dicyclohexylcarbodiimide);
TFA (trifluoroacetic acid);
EDT (1, 2-ethanedithiol);
thioanisole;
crystallizing phenol;
and (3) acetonitrile.
2. Using an instrument:
a polypeptide automatic synthesizer;
high performance liquid chromatograph.
3. The synthesis method and the process are as follows:
step 1, activating Fmoc-AA.
The structural formula of Fmoc-AA is shown as follows:
can be abbreviated as:
the Fmoc-AA was activated by reaction with DCC and HOBT, the reaction scheme is as follows:
step 2. attaching the activated amino acid to the resin.
Reacting the activated amino acid with HMP resin under DMAP condition to obtain the resin connected with the amino acid, wherein the reaction formula is as follows:
and 3, removing the Fmoc group of the resin connected with the amino acid.
Under the catalytic action of Piperidine, removing the Fmoc group of the resin connected with the amino acid, wherein the reaction formula is as follows:
and 4, repeating the step 1 and the step 2 to obtain another activated amino acid, coupling the obtained activated amino acid with the resin obtained in the step 3, and removing amino groups in the coupled amino component, wherein the reaction formula is as follows:
and 5, repeating the step 3 and the step 4 until the synthesis is finished to obtain the polypeptide resin connected with all the amino acids.
And 6, separating and purifying the polypeptide resin connected with all the amino acids obtained in the step 5 to obtain the soluble ST2 protein antigenic determinants (1) and (2) with the immunocompetence. The steps for the analysis and purification of both epitopes are as follows: step 6-1, using a scavenging agent mixed by TFA combined with EDT, thioanisole and water to react with the polypeptide resin connected with all amino acids obtained in the step 5, and separating the antigenic determinants (1) and (2) of the soluble ST2 protein with the immunological activity from the polypeptide resin, wherein the reaction time is 3 hours;
step 6-2, removing the scavenging agent, and extracting by using ether to obtain crude soluble ST2 protein antigenic determinants (1) and (2) with immunocompetence;
and 6-3, purifying crude soluble ST2 protein antigenic determinants (1) and (2) with immunocompetence to obtain soluble ST2 protein antigenic determinants (1) and (2) with immunocompetence.
Wherein the purification is performed by high performance liquid chromatography, and the conditions are shown in the following table:
example 2. Using the epitopes (1) and (2) obtained in example 1, monoclonal and polyclonal antibodies were prepared, respectively.
The soluble ST2 protein epitopes (1) and (2) obtained in example 1 were linked to a carrier protein, respectively, and soluble ST2 protein antigens (1) and (2) were prepared, and animals were immunized with the antigens (1) and (2), respectively, to prepare specific monoclonal and polyclonal antibodies.
The soluble ST2 protein antibody of the invention is prepared by the following steps: preparation of soluble ST2 protein antigen.
Step 1. 10mg of soluble ST2 protein epitope (1) or (2) obtained in example 1 was dissolved in 1mL of 0.1M PBS buffer (pH 7.4).
Step 2. Take 10mg of carrier protein (KLH) and dissolve it in 20mL of 0.2M borate buffer (pH 8.6).
And 3, mixing the solutions obtained in the step 1 and the step 2, cooling to 0 ℃, taking 110 mu L of BDB coupling agent chromium chloride, reacting for 1.5 hours at room temperature, dialyzing overnight, subpackaging to obtain the soluble ST2 protein antigen (1) or (2), and storing at-20 ℃.
2. Monoclonal antibodies were prepared using immunized animals.
Step 1, animal immunization. The soluble ST2 protein antigen (1) or (2) prepared in 1 is taken as immunogen, Balb/c mice are taken as immunized animals, and Freund's complete adjuvant and Freund's incomplete adjuvant are used for increasing the immunogenicity of the soluble ST2 protein antigen. Fully and uniformly mixing the same amount of antigen and Freund's complete adjuvant, and injecting the immune mouse subcutaneously at multiple points with the dosage of 50 mu g/mouse to complete primary immunity; after four weeks, the same amount of antigen and Freund's incomplete adjuvant are fully mixed, and then the immune mouse is injected subcutaneously with multiple points at a dose of 50 mug/mouse to complete secondary immunity; after four weeks, the mice were immunized by subcutaneous multi-point injection with 50 μ g/mouse of the antigen to complete three immunizations, and the titer was detected by blood sampling seven days later to obtain immunized mice.
And 2, fusing the cells. Opening the abdomen of the immune mouse obtained in the step 1, taking the spleen, washing, removing the surrounding connective tissues, preparing a cell suspension, and culturing by using an incomplete culture medium to prepare a single-cell suspension; the single cell suspension was used to fuse with the prepared myeloma cell SP2/0 under the mediation of 50% PEG to obtain fused cells.
And 3, screening and cloning culture of the hybridoma cells. And (3) placing the fused cells obtained in the step (2) in an incubator at 37 ℃ for 7-9 days, then generating larger cell clones, screening positive holes for cloning culture, obtaining hybridoma cells, and freezing and storing.
And 4, producing the monoclonal antibody. And (2) inoculating adult Balb/c mice with 0.3-0.5mL of pristane or liquid paraffin on an empty stomach, inoculating hybridoma cells 0.5mL after 7-10 days, collecting mice with obviously enlarged abdomens at five days intervals to collect ascites, centrifuging to collect supernatant, and subpackaging for freezing to obtain the monoclonal antibody (1) or (2) prepared from the soluble ST2 protein epitope polypeptide (1) or (2).
And 5, measuring the titer of the antibody. The titer of the monoclonal antibody (1) or (2) prepared by using the soluble ST2 protein antigenic determinant (1) or (2) polypeptide is over 1:32000 measured by an ELISA method.
3. Polyclonal antibodies were prepared using immunized animals.
Step 1, animal immunization. The soluble ST2 protein antigen (1) or (2) prepared in 1 is taken as immunogen, a New Zealand white rabbit is taken as an immunized animal, and Freund's complete adjuvant and Freund's incomplete adjuvant are used for increasing the immunogenicity of the soluble ST2 protein antigen. Fully and uniformly mixing the same amount of antigen and Freund's complete adjuvant, and injecting the mixture into immunized rabbits at multiple subcutaneous points with the dosage of 100 mu g/rabbit to complete primary immunization; after four weeks, the same amount of antigen and Freund's incomplete adjuvant are fully mixed, and then the mixture is injected into the immunized rabbit subcutaneously at multiple points with the dosage of 100 mu g/rabbit to complete the secondary immunization; after four weeks, the antigen is used for carrying out subcutaneous multi-point injection on the rabbits at a dose of 100 mu g/rabbit to complete three times of immunization; after four times of immunization, blood sampling is carried out at intervals of ten days to detect titer, an immunized rabbit is obtained, and after carotid artery bloodletting and serum separation, the polyclonal antibody (1) or (2) prepared by the soluble ST2 protein antigenic determinant polypeptide (1) or (2) is obtained.
And 2, measuring the titer of the antibody. The titer of the polyclonal antibody (1) or (2) prepared by using the soluble ST2 protein antigenic determinant (1) or (2) polypeptide is over 1:16000 measured by an ELISA method.
Example 3. preparation of a soluble ST2 protein detection kit using the monoclonal and polyclonal antibodies obtained in example 2.
In this example, the monoclonal antibody (1) prepared using the soluble ST2 protein epitope (1) polypeptide in example 2 was used as the coating antibody in the soluble ST2 protein detection kit, and the polyclonal antibody (2) prepared using the soluble ST2 protein epitope (2) polypeptide was used as the binding antibody in the soluble ST2 protein detection kit.
1. Reagents and buffers.
Coating buffer solution: carbonate buffer 0.050M, ph 9.6;
sample/wash buffer: 10 XPBS-Tween 20, pH7.2;
enzyme marker dilutions: 10 XPBS-Tween 20 (10 mL), FCS (calf serum) (20 mL), distilled water diluted to 1000mL, enzyme stabilizer (1 g), biological preservative (1 mL);
color-developing agent A: citric acid (35.5 g), carbamide peroxide (10 g), diluted to 1000mL with distilled water, Tween20 (10 mL);
and a color developing agent B: citric acid (120 g), EDTA-2Na (1 g), TMB-2HCl (2 g), distilled water diluted to 1000mL
Stopping liquid: 2M H2SO4。
2. And preparing a pre-coated plate.
Dissolving soluble ST2 protein monoclonal antibody (1) in 0.05M carbonate buffer solution with pH of 9.6 to prepare pre-coating solution, adding 100 mu L per well of an enzyme label plate according to 0.1 mu g/well, standing at 4 ℃ for 18-24 hours, removing the coating solution, washing, sealing by BSA for 16 hours, drying overnight, filling in an aluminum platinum bag, vacuumizing, sealing, and storing at 4 ℃.
3. And (3) composition of the kit.
Pre-coating a plate: 48/96 holes;
soluble ST2 calibrator: 6, 6 multiplied by 1.0mL, the concentration is respectively 0ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, 25ng/mL and 50 ng/mL;
ST2 binding antibody: 1X 10 mL;
enzyme conjugate: 1X 10 mL;
concentrated wash (25 × PBS-Tween 20): 1X 20 mL;
color-developing agent A: 1X 6.0 mL;
and a color developing agent B: 1X 6.0 mL;
stopping liquid: 1X 6.0 Ml.
4. And (3) using the kit.
Adding 100 mu L/hole of a blood sample to be detected and a standard substance into each hole of the pre-coated plate, incubating for 60 minutes at 37 ℃, washing for 5 times by using 1 multiplied by washing buffer solution, and patting dry; add ST2 binding antibody 100 u L/hole in each hole, incubate 30 minutes at 37 ℃, wash 5 times with 1 × washing buffer, pat dry; then, 100. mu.L/well of the enzyme conjugate was added to each well, incubated at 37 ℃ for 30 minutes, washed 5 times with 1 XWash buffer, and patted dry. Adding 50 μ L of color development agent A, B solution into each well, mixing, and incubating at 37 deg.C for 15 min; the reaction was stopped by adding 50. mu.L of stop solution to each well, and absorbance was measured by using an enzyme-linked immunosorbent assay (ELISA) instrument at two wavelengths (450 nm, 620 n).
5. And (5) judging the detection result of the kit.
And drawing a standard curve according to the concentration and the absorbance of the soluble ST2 calibrator in the kit, and calculating the ST2 concentration of the sample to be detected through the standard curve after the sample is detected. The following table shows the concentration of soluble ST2 calibrator and the corresponding average absorbance value (OD), which is the average absorbance value of several batches, used as reference only, and should be based on the actual absorbance value of soluble ST2 calibrator in actual use.
TABLE 1 soluble ST2 calibrator concentration and corresponding average absorbance (OD)
| Concentration ng/ |
0 | 2 | 5 | 10 | 25 | 50 |
| Average Absorbance (OD) | 0.009 | 0.108 | 0.145 | 0.151 | 0.278 | 0.364 |
As shown in FIG. 1, the goodness of fit R of the standard curve is plotted as the concentration of soluble ST2 calibrator in the above table and the corresponding average absorbance2=0.965。
According to the 4. kit using steps and 5. kit test results, the serum soluble ST2 protein detection is carried out on 102 acute heart failure patients and 136 healthy individuals, and the detection results show that the content of the serum soluble ST2 protein of the heart failure patients is obviously higher than that of the healthy group (40.84 +/-9.58 VS. 28.33 +/-6.96) and the difference has statistical significance (P = 0.0084) as shown in the table 2.
TABLE 2 comparison of serum soluble ST2 levels in acute heart failure patient groups and healthy groups
From the above results, it was found that the kit for detecting soluble ST2 protein of the present invention can specifically detect the content of ST2 in serum.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
The above description is only a preferred example of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Tianjin Qiyunnaods biomedicine Co., Ltd
<120> soluble ST2 protein antigenic determinant polypeptide and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Arg Arg His Thr Val Arg Leu Tyr
1 5
<210> 2
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Gln Tyr Asp Cys Leu Ala Leu Asn Leu His Gly Tyr
1 5 10
Claims (7)
1. A soluble ST2 protein antigenic determinant polypeptide, wherein the amino acid sequence of the soluble ST2 protein antigenic determinant polypeptide is one of the sequence shown as SEQ ID NO.1 and the sequence shown as SEQ ID NO.2, and specifically comprises the following components:
（1）Arg-Arg-His-Thr-Val-Arg-Leu-Tyr
（2）Gln-Tyr-Asp-Cys-Leu-Ala-Leu-Asn-Leu-His-Gly-Tyr。
2. a soluble ST2 protein antigen, said soluble ST2 protein antigen prepared by conjugation to a carrier protein using the soluble ST2 protein epitope (1) polypeptide of claim 1.
3. A soluble ST2 protein antigen, said soluble ST2 protein antigen being produced by conjugating a soluble ST2 protein epitope (2) polypeptide of claim 1 to a carrier protein.
4. A soluble ST2 protein antibody, wherein the soluble ST2 protein antibody is a polyclonal antibody prepared from the soluble ST2 protein antigen of claim 2.
5. A soluble ST2 protein antibody, wherein the soluble ST2 protein antibody is a polyclonal antibody prepared from the soluble ST2 protein antigen of claim 3.
6. Use of the soluble ST2 protein antibody according to claim 4 or claim 5 in the preparation of a soluble ST2 protein detection kit.
7. A soluble ST2 protein assay kit, said soluble ST2 protein assay kit comprising the antibody of claim 4 or claim 5 as a coated antibody.
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| CN114350670B (en) * | 2022-03-18 | 2022-06-14 | 北京市心肺血管疾病研究所 | Aptamer capable of specifically recognizing soluble ST2 protein and application thereof |
| CN117903311B (en) * | 2024-03-20 | 2024-10-25 | 湖南卓润生物科技有限公司 | SST2 specific binding protein, and preparation method and application thereof |
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