阅读说明：本技术 一种bnp抗原决定簇多肽及其应用 (BNP antigenic determinant polypeptide and application thereof ) 是由 罗奇斌 申玉林 任毅 廖胜光 于 2021-02-07 设计创作，主要内容包括：本发明提供一种BNP抗原决定簇多肽及其应用。其中所述BNP蛋白抗原决定簇多肽的氨基酸序列为Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Tyr。本发明所述的BNP蛋白抗原是通过使用所述的抗原决定簇多肽与载体蛋白偶联制备的。本发明所述的BNP蛋白抗体是由所述抗原制备成的单克隆抗体。本发明所述的单克隆抗体可用于制备BNP蛋白检测试剂盒。本发明所述的BNP蛋白抗原决定簇多肽具有良好的抗原性,使用其制备的抗原免疫动物能够产生特异性的抗体,使用其制备的BNP蛋白抗原具有良好的免疫原性,使用其制备的BNP蛋白可以特异性地结合人血清中的BNP,使用其制备的BNP蛋白检测试剂盒可有效检测人血清中的BNP蛋白水平。(The invention provides a BNP antigenic determinant polypeptide and application thereof. The amino acid sequence of the BNP protein antigenic determinant polypeptide is Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Tyr. The BNP protein antigen is prepared by coupling the antigenic determinant polypeptide and a carrier protein. The BNP protein antibody is a monoclonal antibody prepared from the antigen. The monoclonal antibody can be used for preparing a BNP protein detection kit. The BNP protein antigenic determinant polypeptide has good antigenicity, an antigen immune animal prepared by the BNP protein antigenic determinant polypeptide can generate a specific antibody, a BNP protein antigen prepared by the BNP protein antigenic determinant polypeptide has good immunogenicity, a BNP protein prepared by the BNP protein antigenic determinant polypeptide can be specifically combined with BNP in human serum, and a BNP protein detection kit prepared by the BNP protein detection kit can effectively detect the BNP protein level in the human serum.)

1. A BNP protein antigenic determinant polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1, specifically comprises the following components: Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Tyr.

2. A BNP protein antigen prepared by coupling a BNP protein epitope of claim 1 to a carrier protein.

3. A BNP protein antibody, said BNP protein antibody being a monoclonal antibody prepared from the BNP protein antigen of claim 2.

4. Use of the BNP protein antibody according to claim 3 for preparing a BNP protein detection kit.

5. A BNP protein detection kit comprising the antibody of claim 4 as a coating antibody.

6. The BNP protein detection kit according to claim 5, further comprising a binding antibody, wherein the binding antibody is an anti-human BNP polyclonal antibody.

7. Use of a BNP protein detection kit according to one of claims 5 and 6 for the quantitative measurement of human serum BNP proteins.

Technical Field

The invention relates to the field of polypeptide chemistry, in particular to BNP antigenic determinant polypeptide and application thereof.

Background

Acute myocardial infarction is myocardial necrosis of acute and persistent ischemic rigid qi of coronary artery, and patients mostly occur on the basis of coronary atherosclerotic stenosis, because of certain causes, coronary artery plaque is ruptured, blood platelets in blood are gathered on the surface of the ruptured plaque to form blood clots (thrombus), and the coronary artery lumen is suddenly blocked, so that myocardial ischemic necrosis is caused. Acute myocardial infarction often causes severe clinical consequences such as arrhythmia, shock or heart failure. The incidence rate of acute myocardial infarction in China is in a remarkable rising trend, at least 50 ten thousand new year happens, and more than 200 ten thousand existing patients.

Acute myocardial infarction has the characteristics of acute morbidity, poor prognosis and high mortality, and the prognosis is an important link for reducing the mortality of diseases. Therefore, various clinical methods are used for disease monitoring and prognosis evaluation, and the level of Brain Natriuretic Peptide (BNP) is one of the markers for monitoring the prognosis of disease reaction.

BNP is a member of the natriuretic peptide family, is derived from the heart, and is a cardiac hormone. BNP was first found in the pig brain in 1988, was later isolated and purified in the human heart and was found to secrete higher amounts than the brain. When myocardial cells are stimulated by pressure or tension, a pro-B-type natriuretic peptide precursor (pre-proBNP) is first formed, which is a molecule consisting of 134 amino acids and stored in the myocardial cells, and when pre-proBNP is secreted out of or after secretion in the myocardial cells, it is decomposed by proteases into a signal peptide consisting of 26 amino acids at the N-terminus and pro-B-type natriuretic peptide (proBNP) consisting of 108 amino acids, which further cleaves an active B-type natriuretic peptide (BNP) consisting of 32 amino acids and an inactive B-type natriuretic peptide (NT-proBNP) consisting of 76 amino acids at the N-terminus under the action of endonucleases. Both are secreted into the blood by cardiomyocytes and can serve as markers of cardiovascular disease. However, clinical use of NT-proBNP is complicated and is easily influenced by renal functions, so that BNP is better than NT-proBNP as a heart failure marker.

With respect to the determination of BNP, some methods are by identifying the cyclic structure of BNP and preparing a corresponding antibody therefrom; other methods are those capable of identifying a polypeptide comprising SEQ ID NO. 2: KVLRRH, SEQ ID NO. 3: SPKMVQGSGC, SEQ ID NO. 4: VQGSGCFGR, SEQ ID NO. 5: MDRISSSSGLG to detect BNP content.

The polypeptide sequence is different from any known BNP surface antigenic determinant, has antigenicity and specificity, can be used for preparing a corresponding antibody, and simultaneously the prepared antibody also has good immunogenicity.

Disclosure of Invention

The invention provides a BNP antigenic determinant polypeptide and application thereof, wherein the antigenic determinant has high antigenicity and strong specificity, can be used for preparing corresponding BNP antigens and antibodies, and simultaneously provides a kit for BNP detection by using the antibodies prepared by the antigenic determinant polypeptide.

In a first aspect, the present invention provides a BNP epitope polypeptide, wherein the amino acid sequence of the epitope polypeptide is shown in SEQ ID No.1, specifically: Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Tyr.

The full length of the amino acid sequence of human BNP is 32 amino acids (Swiss-Prot accession number: P16860.1), and the antigenic determinant polypeptide provided by the invention corresponds to the 20 th to 27 th positions of the full-length amino acid sequence, is obtained by adding amino acid Y to the C terminal, and contains 9 amino acids in total.

The screening principle of the BNP antigenic determinant polypeptide is as follows: 1. the hydrophilicity is strong; 2. the antigenicity is strong; 3. is easy to synthesize. Meanwhile, the BNP antigenic determinant polypeptide provided by the invention has strong immunogenicity, after the antigenic determinant is combined with carrier protein, an antigen immune animal can be stimulated to prepare a monoclonal antibody, and the antibody prepared by the antigenic determinant can be specifically combined with human BNP protein.

The amino acid Y is added to the C-terminus in order to crosslink the antigenic determinant to a carrier protein by Bis-diazotized benzidine (BDB) and prepare an antibody as an antigen. In the invention, Y amino acid is added at the C terminal, the finally prepared antibody is an anti-polypeptide N terminal antibody, and Y amino acid can also be added at the N terminal to prepare an anti-polypeptide C terminal antibody.

The BNP epitope polypeptide fragments described above can be synthesized using solid phase methods, see example 1. The molecular weight of the BNP epitope polypeptide is 1045.3, and the molecular weight can be determined by mass spectrometry.

In a second aspect, the present invention provides a BNP protein antigen prepared by conjugation to a carrier protein using an epitope polypeptide as described in the first aspect.

Useful carrier proteins include hemocyanin (KLH), Bovine Serum Albumin (BSA), Ovalbumin (OVA), and the like. The preparation method adopts the existing mature technology, see example 2.

In a third aspect, the present invention provides a BNP protein antibody that is a monoclonal antibody prepared by using the antigen as described in the second aspect. The preparation method adopts the existing mature technology, see example 2.

In a fourth aspect, the invention provides an application of the BNP protein antibody in the third aspect in preparing a BNP protein detection kit.

In a fifth aspect, the invention provides a BNP protein detection kit.

The BNP protein detection kit provided by the invention preferably adopts an ELISA competition method to detect human BNP protein, and comprises necessary reagents such as a coated antibody, an enzyme-labeled second antibody and the like.

In addition, the BNP protein detection kit may further comprise any reagents or tools required for the assay, such as pre-coated plates, washing solutions, stop solutions, and the like.

The invention also provides application of the BNP protein detection kit in quantitative determination of BNP protein, which is shown in example 3.

Compared with the prior art, the invention has the following beneficial effects:

1. the BNP protein antigenic determinant polypeptide provided by the invention has good antigenicity, and an antigen immune animal prepared by using the BNP protein antigenic determinant polypeptide can generate a specific antibody;

2. the BNP protein antigen provided by the invention has good immunogenicity, and can stimulate antigen immune animals to generate immune response;

3. the BNP protein antibody provided by the invention can be specifically combined with BNP in human serum;

4. the BNP protein detection kit provided by the invention can effectively detect the BNP protein level in human serum.

Drawings

Fig. 1 is a BNP standard substance concentration and absorbance standard curve in the BNP 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 in particular to an antigenic determinant polypeptide of BNP protein.

Example 1 preparation of antigenic determinants of BNP protein.

The preparation method of the BNP protein antigenic determinant adopts a chemical solid phase method to synthesize the antigenic determinant, 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 a peptide chain is extended until the preparation of the antigenic determinant is finished.

The steps for preparing the antigenic determinants of the BNP proteins of the present invention are described in detail below.

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. Synthetic method and process.

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 BNP protein antigenic determinant with the immunocompetence. The procedure for epitope purification was as follows:

step 6-1, reacting TFA, EDT, thioanisole and water mixed scavenger with the polypeptide resin connected with all amino acids obtained in the step 5, and separating the BNP protein antigenic determinant with immunocompetence from the polypeptide resin, wherein the reaction time is 3 hours;

6-2, removing the scavenging agent, and extracting by using ether to obtain a BNP protein antigenic determinant crude product with immunocompetence;

and 6-3, purifying the BNP protein antigenic determinant crude product with the immunocompetence to obtain the BNP protein antigenic determinant with the immunocompetence.

Wherein the purification is performed by high performance liquid chromatography, and the conditions are shown in the following table:

example 2. monoclonal antibodies were prepared using the BNP epitopes obtained in example 1.

The BNP protein epitope obtained in example 1 is linked to a carrier protein, and a SNP protein antigen is prepared, and an animal is immunized with the antigen, and a specific monoclonal antibody is prepared.

The preparation steps of the BNP protein antibody are as follows: preparation of BNP protein antigen.

Step 1. taking 2mg of the BNP protein epitope obtained in example 1, dissolving the BNP protein epitope with 500. mu.L of coupling buffer MES (0.1 mol/L, pH 4.5-5.0).

Step 2, 2mg of carrier protein (BSA) was taken and dissolved in 200. mu.L of deionized water.

And 3, mixing the solutions obtained in the step 1 and the step 2, cooling to 0 ℃, taking 200 mu L (10 g/L) of BDB coupling agent chromium chloride, reacting overnight at room temperature, dialyzing for 3 days at 4 ℃, subpackaging to obtain BNP protein antigen (about 2 g/L), subpackaging and storing at-20 ℃.

2. Monoclonal antibodies were prepared using immunized animals.

Step 1, animal immunization. Taking the prepared BNP protein antigen as immunogen, taking male Balb/c mice as immunized animals, and using Freund's complete adjuvant and Freund's incomplete adjuvant to increase the immunogenicity of the BNP protein antigen. Fully mixing and emulsifying the same amount of antigen and Freund's complete adjuvant, and injecting the mixture into an immunized mouse at multiple subcutaneous points with a dose of 50 mu g/mouse to complete primary immunization; after every three weeks, the same amount of antigen and Freund's incomplete adjuvant are fully mixed and emulsified, and then the immune mouse is injected subcutaneously with multiple points at a dose of 50 mu g/mouse to complete secondary immunity; and completing three times of immunization, and collecting blood after seven days to detect titer to obtain an immunized mouse. Three days before fusion, 50 mu g of antigen without Freund's adjuvant is injected into the abdominal cavity, and additional immunization is completed.

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 fused with the prepared myeloma cell SP2/0 at a ratio of 1:10 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. Inoculating 0.5mL of pristane or liquid paraffin to adult Balb/c mice on an empty stomach, inoculating 0.5mL of hybridoma cells to the abdominal cavity after 7-10 days, collecting mice with obviously expanded abdominal cavity after a week interval, collecting ascites, centrifuging, collecting supernatant, subpackaging and freezing to obtain the monoclonal antibody prepared from the BNP protein antigenic determinant polypeptide.

And 5, measuring the titer of the antibody. The valence of the monoclonal antibody prepared by the BNP protein antigenic determinant polypeptide reaches more than 1: 32000.

Example 3. BNP protein detection kit is prepared by using the monoclonal antibody obtained in example 2.

In this example, a monoclonal antibody prepared using the BNP protein epitope polypeptide in example 2 was used as the coating antibody in the BNP protein detection kit.

1. Reagents and buffers.

Coating buffer solution: carbonate buffer 0.050M, ph 9.6;

washing liquid: 10 XPBS-Tween 20, pH7.2;

diluting liquid: 10 XPBS-Tween 20 (10 mL), FCS (calf serum) (20 mL), distilled water diluted to 1000mL, enzyme stabilizer (1 g), biological preservative (1 mL);

substrate solution: diluting the buffer solution with 10mL of citric acid, 2mL of TMB buffer solution with 10mg/mL of 5% hydrogen peroxide solution with 200 mu L of distilled water to 200 mL;

stopping liquid: 2M H₂SO₄。

2. And preparing a pre-coated plate. After the monoclonal antibody described in example 2 was diluted with a coating solution, 100. mu.L of the diluted antibody was added to each well, and the mixture was allowed to stand overnight at low temperature, washed after the coating solution was removed, blocked with BSA for 16 hours, dried overnight, packed in an aluminum foil bag, vacuum-sealed, and stored at 4 ℃.

3. And (3) composition of the kit.

Pre-coating a plate: 96-well;

BNP freeze-dried standard: 2, the number of the cells is 2;

diluting liquid: 1X 50 mL;

washing liquid: 1X 30 mL;

HRP enzyme conjugate: 1X 20 mL;

substrate solution: 1X 6.0 mL;

stopping liquid: 1X 6.0 mL.

4. And (3) using the kit.

Diluting a sample to be detected by using a diluent according to a ratio of 1: 5; dissolving the BNP freeze-dried standard substance in deionized water to be dissolved, wherein the concentration is 2000 pg/mL; diluting the standard solution with the diluent to gradient of 1000, 500, 250, 125, 62.5 and 31.5 pg/mL; uniformly mixing an equal amount of sample/standard substance to be detected and 60 times diluted HPR enzyme conjugate, respectively adding 20 mu L of the mixture into a pre-coated plate, incubating for 30 minutes at 37 ℃, washing for 5 times by using 1 × washing buffer solution, and patting dry; adding 200 mu L of substrate solution into each hole, and incubating for 60 minutes at 37 ℃ for color development; mu.L of stop solution was added to each well to terminate the reaction, and the absorbance was measured by an enzyme-linked immunosorbent assay (450 nm).

5. And (5) judging the detection result of the kit.

And (4) drawing a standard curve of the concentration and the absorbance of the standard substance according to the standard substance solutions with different concentrations in the sample solution 4, wherein the concentration and the absorbance of the sample are inversely related, and calculating the BNP concentration of the sample to be detected through the standard curve after the sample is detected. The following table shows the BNP calibrator concentration and the corresponding average absorbance value (OD), which is the average absorbance value of several batches, and is used only as a reference, and should be based on the actual absorbance value of the BNP calibrator in actual use.

TABLE 1 BNP standard concentrations and corresponding mean absorbances (OD)

Concentration pg/mL	31.5	62.5	125	250	500	1000	2000
								Average Absorbance (OD)	1.966	1.591	1.306	0.862	0.568	0.408	0.342

As shown in FIG. 1, the standard curve is plotted according to the BNP standard concentration and the corresponding average absorbance in the above table, and the goodness of fit R of the standard curve²=0.985。

According to the 4. kit using steps and 5. kit test results, the serum BNP protein detection is carried out on 94 acute heart failure patients and 136 healthy individuals, and the detection results show that the serum BNP protein content of the heart failure patients is obviously higher than that of the healthy group (2133.4 +/-327.7 VS. 104.5 +/-13.1, pg/mL) and the difference has statistical significance (P = 0.0335) as shown in the table 2.

TABLE 2 comparison of serum BNP levels in the acute heart failure patient group with that in the healthy group

And diluting 3 BNP standard products with different concentrations of 100pg/mL, 200pg/mL and 400pg/mL by using a culture solution, loading 3 holes in each concentration, using 4 steps of the kit and 5 steps of the kit to judge the measured concentration and calculating the variation coefficient between batches, wherein the results are shown in Table 3.

TABLE 3 Intra and inter-batch coefficient of variation results

Wherein, the coefficient of variation is the standard used for measuring repeatability, and the smaller the coefficient of variation, the better the repeatability, the higher the precision, calculate as: coefficient of Variation (CV) ═ standard deviation/mean × 100%.

From the above results, it is clear that the BNP protein detection kit of the present invention can specifically detect the BNP content 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

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