CN106117341B - Antigen epitope peptide of IL-9 and its application - Google Patents

Abstract

The invention discloses the epitope peptide of IL-9 a kind of and its applications.IL-9 epitope peptide provided by the present invention, amino acid sequence are following any: (1) sequence 1；(2) 9-13 of sequence 1；(3) sequence 2；(4) 1-5 of sequence 2；(5) sequence 3；(6) 5-10 of sequence 3；(7) 6-11 of sequence 3；(8) 4-10 of sequence 3；(9) position 6-13 of sequence 3.IL-9 epitope peptide provided by the present invention and the core epitope of determination can be used for preparing the antibody of the anti-IL-9 of efficient and sensible.The present invention is of great significance for the diagnosis and prevention and treatment of IL-9 abnormal expression related disease.

Description

Antigen epitope peptide of IL-9 and its application

technical field

The invention belongs to the field of biotechnology, and relates to an IL-9 antigenic epitope peptide and its application.

Background technique

IL-9 is a cytokine secreted in the supernatant of Th cells for the first time in 1988. It is mainly secreted by new effector Th9 cells. In addition, Th2, Th17 and other cells can also secrete it. Mature IL-9 is a glycoprotein with a molecular weight of 14,000 consisting of 144 amino acids and a signal peptide of 18 amino acids. IL-9 is involved in the occurrence and development of various diseases. Due to the difference in the microenvironment of different diseases, IL-9 plays a role in promoting or inhibiting in different diseases. In the study of autoimmune diseases, it was found that IL-9 was involved in the process of disease inflammation; the study found that IL-9 mRNA in the pathological tissue of patients with autoimmune colitis was higher than that of healthy controls, and exogenous injection of Th9 in mouse models After the cells, the colonic inflammation of the mice was aggravated, and after the IL-9 gene was knocked out or neutralized by the IL-9 antibody, the endoscopic manifestations and histopathological changes of the mouse colitis were significantly alleviated. In addition, elevated levels of IL-9 mRNA and protein were found in the pathological tissues of patients with bronchial asthma, suggesting that IL-9 is involved in the occurrence and development of asthma; studies have confirmed that IL-9 transgenic mice can show respiratory hyperresponsiveness under antigen stimulation, Accompanied by increased infiltration of inflammatory cells, increased IgE levels and other characteristics of acute asthma attacks. However, after culling the mice or introducing IL-9 antibody, the airway inflammation and airway hyperresponsiveness decreased significantly compared with before. After Helicobacter pylori infected human body, it was found that IL-9 and IL-9mRNA in the gastric mucosa pathological tissue were significantly higher than that of the healthy control group. The amount of colonization of the gastric mucosa of the mice and the degree of inflammation of the gastric mucosa of the mice were higher than those of the normal infection group. It was confirmed for the first time that IL-9 plays a protective role in the host immune response induced by Helicobacter pylori. In addition, it was found that IL-9 in the serum of patients with viral hepatitis B was significantly higher than that of healthy controls, and when the viral load was less than 10 ⁵ , the level of IL-9 increased with the increase of viral load, but when the viral load was greater than At 10 ⁵ , the serum level of IL-9 decreased instead. Serum levels of IL-9 were also found to be significantly higher in active tuberculosis patients than in healthy controls, suggesting that IL-9 is involved in the body's immune response to tuberculosis.

In summary, IL-9 is a multi-effect cytokine that participates in the immune response process of various diseases in the body, and IL-9 antibody may be used for the diagnosis and treatment of various diseases.

Contents of the invention

One object of the present invention is to provide an antigenic epitope peptide of IL-9.

The amino acid sequence of the IL-9 epitope peptide provided by the present invention can be any of the following:

(1) Sequence 1 in the sequence listing;

(2) No. 9-13 (core epitope) of sequence 1 in the sequence listing;

(3) Sequence 2 in the sequence listing;

(4) No. 1-5 (core epitope) of sequence 2 in the sequence listing;

(5) Sequence 3 in the sequence listing;

(6) No. 5-10 (core epitope) of sequence 3 in the sequence listing;

(7) No. 6-11 (core epitope) of sequence 3 in the sequence listing;

(8) No. 4-10 (core epitope) of sequence 3 in the sequence listing;

(9) Positions 6-13 of Sequence 3 in the Sequence Listing (core epitope).

Correspondingly, the present invention also provides an IL-9 antigen, which is specifically formed by coupling the IL-9 antigen epitope peptide with a carrier protein.

In the present invention, the carrier protein can specifically be keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA). Of course, other carrier proteins that meet the experimental requirements can be used.

More specifically, in an embodiment of the present invention, when used as an immunogen for immunizing animals to prepare antibodies, the carrier protein is specifically keyhole limpet hemocyanin (KLH); when used as a coating agent for screening monoclonal cells In the case of strains, the carrier protein is specifically bovine serum albumin (BSA).

The application of the IL-9 antigen epitope peptide or the IL-9 antigen as an immunogen in preparing an anti-IL-9 antibody also belongs to the protection scope of the present invention. Wherein, the application can be either a non-disease diagnosis and treatment application or a disease diagnosis and treatment application.

Of course, the anti-IL-9 antibody prepared by using the IL-9 antigen epitope peptide or the IL-9 antigen as an immunogen also belongs to the protection scope of the present invention.

The application of the antibody in detecting IL-9 or preparing products for detecting IL-9 also belongs to the protection scope of the present invention. Wherein, the application can be either a non-disease diagnosis and treatment application or a disease diagnosis and treatment application.

The application of the IL-9 antigen epitope peptide or the IL-9 antigen or the antibody in the following (a) or (b) also belongs to the protection scope of the present invention.

(a) preparing products for diagnosing diseases related to abnormal expression of IL-9;

(b) preparing products for treating and/or preventing diseases related to abnormal expression of IL-9.

The present invention also protects the product whose active ingredient is the IL-9 antigen epitope peptide or the IL-9 antigen or the antibody; the product has at least one of the following functions: (A) diagnosis of abnormal expression of IL-9 related diseases; (B) treating and/or preventing diseases related to abnormal expression of IL-9.

Wherein, the abnormal expression of IL-9 refers to the significant difference in the expression level of IL-9 in patients compared with healthy controls without disease. In an example of the present invention, the disease related to abnormal expression of IL-9 is specifically active tuberculosis.

In addition, the nucleic acid molecule encoding the IL-9 antigen epitope peptide and the expression cassette, recombinant vector or recombinant cell line containing the nucleic acid molecule also belong to the protection scope of the present invention.

In the present invention, the IL-9 is human IL-9.

Experiments have proved that the IL-9 antigen epitope peptide and the determined core epitope provided by the present invention can be used to prepare highly efficient and sensitive anti-IL-9 antibodies. The invention has great significance for the diagnosis and prevention of diseases related to the abnormal expression of IL-9.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, the preparation of IL-9 antigen

1. Determination of the epitope peptide sequence in IL-9 antigen

1. Search the sequence information of IL-9 protein in NCBI as follows:

IL-9蛋白的序列如下：m l l a m v l t s a l l l c s v a g q g c p t l ag i l d i n f l i n k m q e d p a s k c h c s a n v t s c l c l g i p s d n ct r p c f s e r l s q m t n t t m q t r y p l i f s r v k k s v e v l k n n kc p y f s c e q p c n q t t a g n a l t f l k s l l e i f q k e k m r g m r gk i.

The overall protein sequence of IL-9 is relatively specific, and there is no similar protein sequence.

2. According to the analysis of the Uniprot database information, the possible glycosylation sites of IL-9 were obtained, and the signal peptide and glycosylation sites were avoided, and the following three regions were selected as antigenic sites:

IL9-pep1: DINFLINKMQEDPASKC(sequence 1);

IL9-pep2: CIFQKEKMRGMRGKI (sequence 2);

IL9-pep3: SRVKKSVEVLKNNKC (SEQ ID NO: 3).

Among them, the first amino acid "C" in IL9-pep2 does not come from the sequence of IL-9 protein, but is additionally added for coupling to the carrier.

2. Preparation of IL-9 Antigen

Use a peptide synthesizer to artificially synthesize three polypeptide sequences (IL9-pep1, IL9-pep2 and IL9-pep3) shown in Sequence 1-3 in the sequence listing, and combine the three polypeptides with keyhole limpet hemocyanin (KLH) and bovine serum albumin respectively Protein (BSA) was coupled to obtain "epitope peptide-carrier protein conjugate". Among them, the one coupled with KLH is used for immunizing mice to prepare anti-IL-9 antibodies; the one coupled with BSA is used as an ELISA coating agent for screening antisera.

After testing, the purity of the two "epitope peptide-carrier protein conjugates" obtained by coupling is greater than 90%, 10 mg.

Example 2, Preparation of Monoclonal Antibody against IL-9 Antigen in Example 1 and Epitope Identification

1. Preparation and Characterization of Monoclonal Antibody against IL-9 Antigen in Example 1

1. Animal immunity

Four SPF-grade BALB/c female mice were immunized with the three KLH-coupled polypeptides (IL9-pep1, IL9-pep2 and IL9-pep3) prepared in Example 1, respectively. Specific immunization strategies are as follows:

(1) Initial immunization: the dose of KLH-conjugated polypeptide was 60 μg (total mass after coupling), the adjuvant was Freund’s complete adjuvant, the dose of adjuvant was 200 μl, and the abdomen was subcutaneously injected for immunization.

(2) Booster immunization was carried out 14 days after the initial immunization. The dose of KLH-coupled polypeptide was 30 μg (total mass after coupling), and the adjuvant was Freund’s incomplete adjuvant; the dose of adjuvant was 200 μl, which was injected subcutaneously in the abdomen immunity. A total of 3 booster immunizations were carried out, and the time interval between two adjacent booster immunizations was 14 days.

After the third boost, the three polypeptides (IL9-pep1, IL9-pep2 and IL9-pep3) prepared in Example 1 coupled with BSA were used as coating agents respectively, and the antibody titer in the mouse serum was detected by indirect ELISA, and selected The dilution corresponding to the minimum OD reading whose titer is greater than the maximum OD/2 is the experimentally determined titer.

The specific method for detecting the antibody titer in mouse serum by ELISA is as follows:

1) Dilute the coating source with coating solution (sodium carbonate-sodium bicarbonate buffer, pH9.6), the final concentration is 2 μg/ml, 100 μl/well, 4 ° C, overnight; dissolved in PBS) and washed twice.

2) Block with blocking solution (2% milk dissolved in PBS), 200 μl/well, incubate at 37° C. for 2 hours; then wash once with washing solution.

3) Dilute the mouse serum to be tested 2 times from 200 times (the dilution is PBS), the blank control (blank) is PBS, and the negative control (negative) is 1000 times dilution of the unimmunized mouse serum (diluted The solution is PBS); both were 100 μl/well, incubated at 37°C for 1 h; then washed 3 times with washing solution.

4) Add secondary antibody (goat anti-mouse IgG/HRP, BPI, product number: AbP71003-D-HRP) diluted 20,000 times in PBS, 100 μl/well, incubate at 37°C for 1 hour; take it out and wash it 3 times with washing solution.

5) For color development, the color development solution is 100 μl/well, and the color development time is 5-15 minutes. Among them, the chromogenic solution is "1% A solution + 10% B solution"; A solution: 1% TMB in DMSO; B solution: 0.1% H ₂ O ₂ dissolved in citric acid buffer.

6) Add 50 μl of stop solution (2M sulfuric acid) to each well to stop.

7) Measure the absorbance value with dual wavelength (450, 630). The titer is the dilution factor corresponding to 1/2 the maximum OD value.

2. Cell Fusion

1) Gently blow off the well-conditioned sp2/0 cells (ATCC Number CRL-1581) from the wall of the culture flask, and suck it into a 50ml centrifuge tube.

2) The mice were plucked from the eyeballs to collect blood, then killed by pulling the neck, and soaked in 75% alcohol for 5 minutes.

3) Pour a small amount of serum-free IMDM into the plate, put the cell sieve and the inner core of the syringe into the plate. Remove the mouse spleen using scissors and forceps and place it on a cell mesh. Gently crush the spleen with the inner core of the syringe, suck the crushed cells into the centrifuge tube containing sp2/0 cells, and centrifuge at 1500 rad/min for 5 min.

4) Remove the mouse thymus with scissors and tweezers, and crush it. Put the crushed thymocytes into a 15ml centrifuge tube, add 1ml of HAT (Sigma; product number: H0262-10VL), and put it in the incubator for later use.

5) Pour off the supernatant of the centrifuged cells, blow the cells carefully and gently with serum-free IMDM, and centrifuge (1500 rad/min, 5 min).

6) Discard the supernatant of the centrifuged cells as much as possible. Tap the bottom of the centrifuge tube to fully suspend the cells, put the centrifuge tube into warm water at 37°C, slowly add 1ml of PEG1500 (Roche; product number: 78364) within 1 minute, and let stand in warm water for 1min after the addition is complete. Then 2 ml of serum-free IMDM was slowly added over 2 min, followed by 8 ml of serum-free IMDM over 2 min. Centrifuge at 1000rad/min for 5min.

7) Pour off the supernatant, add 10ml of newborn bovine serum, carefully blow the cells evenly, and pour into the thymocytes prepared above. Then add 25ml of sterilized semi-solid medium and mix thoroughly. Then pour evenly into 30 cell culture dishes. Put the cell culture dish into the wet box, and then put it into the incubator for culture. Incubate for 7-10 days.

3. ELISA screening positive hybridoma cells

The above-mentioned monoclonal growth on the semi-solid medium was picked and cultured in a 96-well culture plate with 100 μl of T cells (the medium was IMDM complete medium), and the cell culture supernatant was taken 4 days later for ELISA screening of the monoclonal. Positive hybridoma cells are obtained, and the obtained positive hybridoma cell lines are screened for the second time to obtain hybridoma cell lines that are positive in both screenings. The screened positive clones were successively transferred to 6-well plates and cell culture flasks for expansion and frozen storage.

The steps of the above-mentioned ELISA method for screening positive cells are as follows:

1) The three polypeptides (IL9-pep1, IL9-pep2 and IL9-pep3) coupled with BSA prepared in Example 1 were diluted with coating solution (sodium carbonate-sodium bicarbonate buffer, pH9.6), and the final concentration was 2 μg/ml, 100 μl/well, 4°C, overnight; then wash 3 times with washing solution (0.05% Tween dissolved in PBS).

2) Block with blocking solution (2% milk dissolved in PBS), 200 μl/well, incubate at 37°C for 2 hours; then wash with washing solution for 3 times.

3) Add primary antibody (cell culture supernatant), negative control (SP2/0 cell culture supernatant), blank control (PBS), positive control (mouse positive serum with the highest titer obtained as above, carry out 1000 times with PBS Dilution), both at 100 μl/well, incubated at 37°C for 1 hour; then washed 3 times with washing solution.

4) Add secondary antibody (goat anti-mouse IgG/HRP, BPI, product number: AbP71003-D-HRP) diluted 20,000 times in PBS, 100 μl/well, incubator at 37° C. for 1 hour; remove and wash 3 times with washing solution.

5) For color development, the color development solution is 100 μl/well, and the color development time is about 5 minutes. Among them, the chromogenic solution is "1% A solution + 10% B solution"; A solution: 1% TMB in DMSO; B solution: 0.1% H ₂ O ₂ dissolved in citric acid buffer.

6) Add 50 μl of stop solution (2M sulfuric acid) to each well to stop.

7) Measure the absorbance value at dual wavelengths (450, 630), and if there is a discoloration reaction when observed with the naked eye, it is positive.

The results showed that after the first screening, 5 positive hybridoma cell lines targeting IL-9-pep1, 18 positive hybridoma cell lines targeting IL-9-pep2, and 30 positive hybridoma cell lines targeting IL-9pep3 were obtained cell line. The positive hybridoma cell lines obtained above were screened twice to obtain 2 positive hybridoma cell lines for IL-9pep1, 15 positive hybridoma cell lines for IL-9pep2, and 21 positive hybridoma cell lines for IL-9pep3. cell line.

4. Identification of monoclonal cell subtypes

1) Dilute the coating antibody with 100mM PBS (pH7.4) (this antibody is an antibody that can bind to mouse IgG, IgA, IgM, Southern Biotech product) to 0.5μg/ml, add 0.1ml to each well, 4 ℃, overnight .

2) Wash 3 times with PBS-T, add 200 μl of blocking solution (2% BSA and 3% sucrose in PBS) to each well, and incubate at 37° C. for 2 hours.

3) Washing with PBS-T for 3 times; adding 100 μl of the hybridoma supernatant obtained in step 3 after two screenings to each well, and incubating at 37° C. for 1 hour.

4) Wash 3 times with PBS-T; use blocking solution 1:1000 (κ, λ) (for κ or λ type secondary antibodies) or 1:2000 (for IgM or IgA or IgG1 or IgG3 or IgG2a or (Is IgG2b secondary antibody) diluted HRP-labeled antibody (SouthernBiotech product) 0.1ml per well, were added to appropriate wells, incubated at 37 ℃ for 1h.

5) Wash 3 times with PBS-T; add 50 μl of chromogenic solution to each well, measure absorbance at dual wavelengths (450, 630) within 10-20 min, record and save the data. Among them, the chromogenic solution is "0.2ml A solution + 10μl 30% H ₂ O ₂ dissolved in 10ml B solution; A solution: 15mg/ml ABTS dissolved in H ₂ O; B solution: citric acid buffer, pH 4.0.

The results showed that: the absorbance value (OD) of the supernatant of each hybridoma cell line and the subtype of the secreted monoclonal antibody are shown in Table 1-Table 3. The OD value for the immunogen in the table is the affinity between the antibody and the antigen produced, and generally an OD value greater than 0.15 is defined as positive.

Table 1 Monoclonal cell lines targeting IL9-pep1 and their corresponding monoclonal antibody subtypes

Numbering OD against the envelope subclass 2 1.506 G2b

Table 2 Monoclonal cell lines against IL-9-pep2 and their corresponding monoclonal antibody subtypes

Numbering OD against the envelope subclass 1 0.775 G2a 2 0.936 G2b 3 0.537 G1 4 0.626 G1 5 0.649 G1 10 0.289 G3 11 0.537 G1 12 0.456 G1 14 0.365 G1 16 0.612 G1 17 0.422 G1

Table 3 Monoclonal cell lines against IL-9-pep3 and their corresponding monoclonal antibody subtypes

Numbering OD value against immunogen Subtype 2 0.634 G2a 4 0.589 G2a 6 0.449 G2a 7 0.335 G1 15 0.299 G1 16 0.376 G2a 17 0.514 G1 18 0.582 G3 twenty one 0.307 G3 twenty two 0.358 G1 twenty four 0.638 G1 29 0.461 G1

2. Identification of core epitope of IL-9 antigen

1. Polypeptide synthesis

Use a peptide synthesizer to synthesize three peptides (IL9-pep1, IL9-pep2, and IL9-pep3) on the fiber membrane, and replace the original amino acids with alanine one at a time starting from the first amino acid. The details of all the peptides synthesized are as follows:

IL-9-01-01: A D I N F L I N K M Q E D P A S K C A

IL-9-01-02: A A I N F L I N K M Q E D P A S K C A

IL-9-01-03: A D A N F L I N K M Q E D P A S K C A

IL-9-01-04: A D I A F L I N K M Q E D P A S K C A

IL-9-01-05: A D I N A L I N K M Q E D P A S K C A

IL-9-01-06: A D I N F A I N K M Q E D P A S K C A

IL-9-01-07: A D I N F L A N K M Q E D P A S K C A

IL-9-01-08: A D I N F L I A K M Q E D P A S K C A

IL-9-01-09: A D I N F L I N A M Q E D P A S K C A

IL-9-01-10: A D I N F L I N K A Q E D P A S K C A

IL-9-01-11: A D I N F L I N K M A E D P A S K C A

IL-9-01-12: A D I N F L I N K M Q A D P A S K C A

IL-9-01-13: A D I N F L I N K M Q E A P A S K C A

IL-9-01-14: A D I N F L I N K M Q E D A A S K C A

IL-9-01-16: A D I N F L I N K M Q E D P A A K C A

IL-9-01-17: A D I N F L I N K M Q E D P A S A C A

IL-9-01-18: A D I N F L I N K M Q E D P A S K A A

IL-9-02-01: A A I F Q K E K M R G M R G K I A A

IL-9-02-02: A A A F Q K E K M R G M R G K I A A

IL-9-02-03: A A I A Q K E K M R G M R G K I A A

IL-9-02-04: A A I F A K E K M R G M R G K I A A

IL-9-02-05: A A I F Q A E K M R G M R G K I A A

IL-9-02-06: A A I F Q K A K M R G M R G K I A A

IL-9-02-07: A A I F Q K E A M R G M R G K I A A

IL-9-02-08: A A I F Q K E K A R G M R G K I A A

IL-9-02-09: A A I F Q K E K M A G M R G K I A A

IL-9-02-10: A A I F Q K E K M R A M R G K I A A

IL-9-02-11: A A I F Q K E K M R G A R G K I A A

IL-9-02-12: A A I F Q K E K M R G M A G K I A A

IL-9-02-13: A A I F Q K E K M R G M R A K I A A

IL-9-02-14: A A I F Q K E K M R G M R G A I A A

IL-9-02-15: A A I F Q K E K M R G M R G K A A A

IL-9-03-01: A S R V K K S V E V L K N N K C A

IL-9-03-02: A A R V K K S V E V L K N N K C A

IL-9-03-03: A S A V K K S V E V L K N N K C A

IL-9-03-04: A S R A K K S V E V L K N N K C A

IL-9-03-05: A S R V A K S V E V L K N N K C A

IL-9-03-06: A S R V K A S V E V L K N N K C A

IL-9-03-07: A S R V K K A V E V L K N N K C A

IL-9-03-08: A S R V K K S A E V L K N N K C A

IL-9-03-09: A S R V K K S V A V L K N N K C A

IL-9-03-10: A S R V K K S V E A L K N N K C A

IL-9-03-11: A S R V K K S V E V A K N N K C A

IL-9-03-12: A S R V K K S V E V L A N N K C A

IL-9-03-13: A S R V K K S V E V L K A N K C A

IL-9-03-14: A S R V K K S V E V L K N A K C A

IL-9-03-15: A S R V K K S V E V L K N N A C A

IL-9-03-16: A S R V K K S V E V L K N N K A A

2. Identification of core epitopes of IL-9 antigen and screening of corresponding positive monoclonal antibodies

(1) Hydration: the fiber membrane with the polypeptide synthesized in step 1 was first washed with 40ml of 100% dehydrated ethanol for 15 minutes, then washed with 40ml of 75% dehydrated alcohol for 15 minutes, and then washed with 40ml of 50% dehydrated alcohol Wash with water and ethanol for 15 minutes, and finally wash with 1×PBS for 30 minutes.

(2) Blocking: block with blocking solution for 3-4 hours at room temperature.

(3) Add the culture supernatant prepared in step 1 corresponding to each hybridoma cell line in Table 1-Table 3: the monoclonal antibody sample was diluted with blocking solution 1:100 (volume ratio), and incubated overnight at 4°C.

(4) Membrane washing: wash 3 times with 1×PBS-T, 10 minutes each time.

(5) Adding secondary antibody: Dilute the secondary antibody (horseradish peroxidase-labeled goat anti-mouse antibody, product of IBP Company) at a ratio of 1:10000 (volume ratio), and incubate at room temperature for 2 hours.

(6) Membrane washing: wash 3 times with 1×PBS-T, 10 minutes each time.

(7) Exposure: Fully mix A and B solutions in the ECL chromogenic solution at a ratio of 1:1 (volume ratio).

(8) Put the polypeptide array membrane into the box, add the mixed solution on the membrane, and react for 2 minutes.

(9) Absorb the excess mixed solution on the membrane with filter paper, put the polypeptide membrane on the plastic membrane, and develop the color in a chemiluminescence imaging system.

(10) Developing: ECL developing and exposing the polypeptide film, scanning the film, and analyzing with TotalLabControlCenter v2009 software. If the amino acid at the core epitope is replaced, the antigen-antibody reaction falls off or becomes weaker, and the luminescence becomes weaker or no luminescence when developing the color. The results obtained are analyzed by software, and the gray value is lower than that of the wild type (complete polypeptide without substituted amino acids) 50% of them were identified as the core epitopes.

3. Results

The mAbs identified as positive were as follows:

IL9-pep1: DINFLINKMQEDPASKC; a total of 1 monoclonal antibody (the monoclonal antibody secreted by the hybridoma cell line numbered 2 in Table 1), the 9th-13th amino acid is the core epitope.

IL9-pep2: CIFQKEKMRGMRGKI; a total of 1 monoclonal antibody (the monoclonal antibody secreted by the hybridoma cell line numbered 1 in Table 2), the 1-5th amino acid is the core epitope.

IL9-pep3: SRVKKSVEVLKNNKC; a total of 8 mAbs, the core epitopes of mAbs No. 6, 15, and 17 are at amino acids 5-10; the core epitopes of mAbs 2, 7, and 18 are at positions 6-11 Amino acids; No. 4 mAb core epitope 4-10 amino acids; No. 21 mAb core epitope 6-13 amino acids. Each number corresponds to the number of the hybridoma cell lines in Table 3.

The monoclonal antibodies secreted by the remaining hybridoma cell lines not mentioned in Table 1-Table 3 were negative in the identification test in step 2.

The specific application examples of each positive monoclonal antibody screened in embodiment 3 and embodiment 2

The monoclonal antibody secreted by each hybridoma cell line screened in Example 2 (ie, the culture supernatant of the hybridoma cell line) was subjected to the corresponding serological ELISA detection test to verify the detection effect. details as follows:

Serum of patients to be tested: 10 patients clinically diagnosed with active tuberculosis were anticoagulated with heparin sodium, and the patients voluntarily agreed to participate in the relevant research of this experiment. Serum IL-9 is known to be upregulated in patients with active tuberculosis relative to healthy controls.

1. Coating: Collect 4ml of heparin sodium anticoagulant blood from patients with active tuberculosis, centrifuge at 12000g for 5min, absorb 100μl of the upper serum and coat it on an ELISA plate, overnight at 4°C. At the same time, healthy normal human serum was set as a negative control.

2. Add 100 μl of monoclonal antibody to be tested in each well, incubate at 37°C for 1 hour, and wash the plate.

3. Add 100 μl of horseradish peroxidase-labeled goat anti-mouse IgG (product of BPI Company, product number: AbP71003-D-HRP), and incubate at 37° C. for 30 minutes.

4. Add substrate TMB 100μl, react for 10-30min

5. Add 2M sulfuric acid to stop the reaction, and read the plate at 450nm with a microplate reader.

6) Reading: Measure the OD value of each well to be tested with a single wavelength of 450nm, and take the ratio (P/N) of the OD value of the negative control well greater than 2.1 as the critical point for judging positive. ELISA result judgment method: If P/N>2.1, it is judged as positive.

The experiment was repeated three times, and the results were averaged.

The results show that the monoclonal antibodies secreted by the hybridoma cell lines screened in Example 2 (ie, the culture supernatant of the hybridoma cell lines) were used for the corresponding serological ELISA detection test, and the detection results were accurate and reliable. Among them, Table 4 shows the serological ELISA detection results using the monoclonal antibody secreted by the IL-9-pep-1 monoclonal cell line numbered 2 in Table 1.

Table 4 IL-9-PEP monoclonal antibody to 10 cases of active tuberculosis patients serum ELISA detection results

Claims (6)

1.IL-9 epitope peptide, amino acid sequence are following any:

(1) sequence 1 in sequence table；

(3) sequence 2 in sequence table；

(5) sequence 3 in sequence table.

2.IL-9 epitope peptide-carrier protein couplet object, for by IL-9 epitope peptide described in claim 1 and carrier egg It is white to be coupled.

3. IL-9 epitope peptide-carrier protein couplet object according to claim 2, it is characterised in that: the carrier egg White is keyhole limpet hemocyanin or bovine serum albumin(BSA).

4. IL-9 epitope peptide-carrier protein couplet object described in claim 2 or 3 is preparing anti-IL-9's as immunogene Application in antibody.

5. encoding the nucleic acid molecules of IL-9 epitope peptide described in claim 1.

6. expression cassette, recombinant vector or recombinant cell lines containing nucleic acid molecules described in claim 5.