CN114369152A - Recombinant chicken interleukin-9 protein and preparation and application of antibody thereof - Google Patents

Abstract

The invention relates to the preparation and application of a recombinant chicken interleukin-9 protein and an antibody thereof. The technical problem to be solved by the present invention is to prepare recombinant chicken IL-9 prokaryotic protein and eukaryotic protein with biological functions, and to measure its biological activity. The technical problem to be solved by the present invention is to prepare monoclonal and polyclonal antibodies for detecting chicken IL-9. And use these antibodies to detect the natural IL-9 protein expressed by immune cells in chickens. The present invention successfully solves the problem of difficulty in recombinant expression of chicken IL-9 cytokines, and the recombinant chicken IL-9 protein has the biological activity of activating chicken macrophages and promoting chicken T cell proliferation, suggesting that it has an immune-enhancing function. The chicken IL-9 protein and antibody in the present invention are easy to mass-produce, and are convenient for later application in chicken industry and basic research on poultry immunology.

Description

Preparation and application of a recombinant chicken interleukin-9 protein and its antibody

technical field

The invention relates to the preparation and application of a recombinant chicken interleukin-9 protein and an antibody thereof, in particular to recombinant protein expression, preparation and application of monoclonal and polyclonal antibodies, and belongs to the fields of avian immunology, biological products and diagnostic reagents .

Background technique

Interleukin-9 (IL-9) is a pleiotropic cytokine that belongs to the γ-chain receptor cytokine family together with IL-2, IL-4, IL-7, IL-15 and IL-21 [ 1]. IL-9 was originally found to be a growth factor secreted by Th2-type T cells, but was later found to be mainly secreted by Th9-type T cells [2,3]; in addition, Th17, regulatory T cells, CD8 ⁺ T cells, Natural killer cells, mast cells and innate lymphoid cells can all secrete IL-9 [4]. IL-9 can act on mast cells, B cells, T cells, hematopoietic progenitor cells, macrophages, DC cells, etc. [5]. Although the chicken IL-9 gene has been annotated in the whole chicken genome, it has not been identified at the protein level, and its biological function is still blank; the key reasons are the difficulty of recombinant expression of chicken cytokines and the lack of specific detection An immunological tool for chicken IL-9 protein.

In humans and mice, the IL-9 gene encodes a 144-amino acid glycoprotein with a molecular weight of 14 kD, including an 18-amino acid signal peptide. However, the native IL-9 protein is highly glycosylated with a molecular weight between approximately 32 and 39 kD [3]. In contrast, the chicken IL-9 gene coding region contains 417 bases, encoding 138 amino acids, including a 20 amino acid signal peptide and 3 glycosylation sites. However, due to the lack of corresponding detection tools, the real molecular weight of natural chicken IL-9 is still unclear.

IL-9 has multiple functions in humans or mice. IL-9 induces autoimmune diseases such as inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis, etc., and plays a role in allergic diseases such as food allergy and asthma [6]. In cancer, IL-9 has pro-tumor effects on hematological tumors and solid tumors, but in melanoma, IL-9 and Th9 cells can exert anti-tumor activity [7]. In addition, IL-9 has been shown to modulate immune responses or play a protective role after bacterial, viral and parasitic infections [8]. Avian infectious diseases such as chicken Marek's disease, infectious bursal disease, chicken infectious bronchitis, etc. have plagued the healthy development of my country's chicken industry. Does chicken IL-9 have immune regulation functions and promote the development of avian infectious disease vaccines And the immune protection effect needs to be further studied.

In the present invention, we disclose a method for preparing chicken IL-9 eukaryotic and prokaryotic recombinant protein with biological functions; and prepare chicken IL-9 mouse monoclonal antibody and rabbit polyclonal antibody. Using the prepared monoclonal antibody or polyclonal antibody, the natural chicken IL-9 protein can be detected by Western blot and other techniques. The preparation and acquisition of recombinant chicken IL-9 protein, monoclonal antibody and polyclonal antibody provide an important tool for the development of chicken IL-9 as an immune enhancer, adjuvant for avian infectious diseases, new avian disease vaccines and basic research on avian immunology and means.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the preparation and application of recombinant chicken interleukin-9 protein and its antibody in order to solve the deficiencies of the prior art; the technical problem to be solved by the present invention is to prepare a recombinant chIL-9 prokaryotic with biological functions protein and eukaryotic protein, and determine their biological activity. The technical problem to be solved by the present invention is to prepare monoclonal and polyclonal antibodies for detecting chicken chIL-9. And use these antibodies to detect the natural chIL-9 protein expressed by immune cells in chickens.

The present invention is realized by the following technical solutions: a prokaryotic expression method for preparing recombinant chicken IL-9 protein, the chicken IL-9 gene without signal peptide is constructed into the prokaryotic expression vector pET-32a, the recombinant plasmid (pET-32a- chIL-9) was induced and expressed by transforming E. coli to obtain soluble recombinant chicken IL-9 protein.

A prokaryotic expression method for preparing recombinant chicken IL-9 protein, the nucleic acid sequence of chicken IL-9 gene without signal peptide is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2;

The recombinant chicken IL-9 protein has biological activity, and co-culturing the recombinant chicken IL-9 protein with a chicken macrophage cell line can activate macrophages to produce inflammatory cytokines; Co-culture of spleen cells for 5 days can promote the proliferation of chicken T cells.

The application of the recombinant chicken IL-9 protein can be used as an immune enhancer to improve the immunity of poultry, especially chickens; it can be used as an adjuvant for the development and application of poultry, especially chicken infectious disease vaccines.

A eukaryotic expression method for preparing recombinant chicken IL-9 protein. The complete chicken IL-9 gene is constructed into a eukaryotic expression vector pEGFP-C, and the recombinant plasmid (pEGFP-chIL-9) is transfected into DF-1 cells to obtain Secreted and expressed recombinant chicken IL-9 protein with biological activity;

Described complete chicken IL-9 gene nucleic acid sequence is shown in SEQ ID NO.3, and its amino acid sequence is shown in SEQ ID NO.4;

Co-culture of recombinant chicken IL-9 protein with chicken macrophage cell lines can activate macrophages to produce inflammatory cytokines; co-culture of recombinant chicken IL-9 protein with isolated chicken spleen cells for 5 days can promote the production of chicken T cells. proliferation.

The application of the recombinant chicken IL-9 protein can be used as an immune enhancer to improve the immunity of poultry, especially chickens; it can be used as an adjuvant for the development and application of poultry, especially chicken infectious disease vaccines.

An anti-chicken IL-9 protein monoclonal antibody 4H7, the monoclonal antibody 4H7 can specifically bind to IL-9 protein expressed by chicken immune cells; it can specifically recognize chicken mononuclear cells activated by phorbol ester and ionomycin Cell-expressed IL-9 native protein.

The recombinant chicken IL-9 protein was immunized into BALB/c mice, and the monoclonal antibody cell line obtained by screening the eukaryotic expression chicken IL-9 protein through hybridoma technology, namely hybridoma cell line 4H7;

The hybridoma cell line 4H7 is deposited in the China Center for Type Culture Collection, whose address is: Wuhan University, Wuhan, China; the deposit date is January 19, 2022; the deposit number is CCTCC NO: C202225.

A method for preparing an anti-chicken IL-9 protein rabbit-derived polyclonal antibody. The recombinant chicken IL-9 protein is immunized with New Zealand white rabbits, and the obtained antiserum can recognize eukaryotically expressed chicken IL-9 protein and activated chicken IL-9 protein. IL-9 native protein expressed by mononuclear cells.

The application of anti-chicken IL-9 protein monoclonal antibody 4H7, which was labeled with fluorescein or enzyme, was used to detect the expression of chicken IL-9 by Western-blot, ELISA, indirect immunofluorescence and flow cytometry.

The application of polyclonal antibodies, labeled with fluorescein or enzyme, was used to detect the expression of chicken IL-9 by Western-blot, ELISA, indirect immunofluorescence and flow cytometry.

The method of the invention is advanced and scientific. Through the invention, the preparation and application of a recombinant chicken interleukin-9 protein and its antibody are provided, pET-32a-chIL-9 and pEGFP-C-chIL-9 plasmids are constructed, and The recombinant chIL-9 protein was expressed in ROSETTA bacteria and DF-1 cells, respectively. The effects of recombinant chIL-9 on chicken macrophage activation and T cell proliferation were detected by qPCR and CFSE staining, respectively. The recombinant chIL-9 prokaryotic protein was immunized three times in BALB/c mice, the spleen was fused with SP2/0 cells, and the positive hybridoma cells were screened. Ascites fluid, purified antibodies, and western blot were used to determine the reactivity of different antibodies with the native chIL-9 protein.

Beneficial effects:

The invention successfully solves the problem of difficulty in recombinant expression of chicken IL-9 cytokines, and the recombinant chIL-9 protein has the biological activity of activating chicken macrophages and promoting chicken T cell proliferation, suggesting that it has an immune enhancing function. The invention successfully prepares anti-chIL-9 monoclonal and polyclonal antibodies, and can efficiently and specifically recognize the chIL-9 natural protein. The invention fills in the knowledge and technology gap of biological function and immune detection of chicken IL-9 protein, and provides effective tools and biological preparations for the application of chIL-9 in chicken immunosuppressive diseases. The chIL-9 protein and antibody in the present invention are easy to mass produce, and are convenient for later application in chicken industry and basic research of poultry immunology.

Description of drawings

Figure 1 shows the construction of pET-32a-chIL-9 plasmid, including chIL-9 PCR amplification (a), plasmid map (b) and plasmid restriction identification (c); M: DNA Marker; 1: chIL-9; 2 : pET-32a-chIL-9 plasmid double digestion product.

Figure 2 shows the prokaryotic expression of chIL-9 (a), protein purification (b), renaturation, and tag protein excision (c); Figure a, M: Prestained Protein Ladder; 1: control group lysis supernatant 2: IPTG group lysis supernatant Clear 3. control group lysate pellet 4. IPTG group lysate pellet. Figure b, M: Prestained Protein Ladder; 5: IPTG group cleavage precipitate 6: Purified Trx-chIL-9 protein. Figure c, M: Prestained Protein Ladder; 7: chIL-9 after tev enzyme cleavage.

Figure 3 shows the construction of pEGFP-C-chIL-9 plasmid, including chIL-9 PCR amplification (a), plasmid map (b) and plasmid restriction identification (c); M: DNA Marker 1: chIL-9-CDS; 2: pEGFP-C-chIL-9 plasmid double digestion product.

Figure 4 is the identification of chIL-9 eukaryotic expression, including ICC (a) and western blot (b); a panel, A: pEGFP-C + negative serum B: pEGFP-C + chIL-9 polyantiserum C: pEGFP- C-chIL-9+negative serum D: pEGFP-C-chIL-9+chIL-9 polyantiserum. Figure b, M: Prestained Protein Ladder 1: DF-1 transfected with pEGFP-C 2: DF-1 transfected with pEGFP-C-chIL-9 3: culture supernatant of DF-1 transfected with pEGFP-C 4: The culture supernatant of DF-1 transfected with pEGFP-C-chIL-9.

Figure 5 shows the effect of recombinant chIL-9 eukaryotic protein on the production of inflammatory factors in monocytes and macrophages; Figure a, the effect of chIL-9 on the production of HD11 inflammatory factors; Figure b, the effect of chIL-9 on inflammatory factors in primary monocytes influence of longevity.

Figure 6 shows the effect of recombinant chIL-9 protein on the proliferation of CD3 ⁺ T cells, including prokaryotic and eukaryotic recombinant chIL-9 protein.

Figure 7 shows the recognition of four strains of chIL-9 monoclonal antibodies to eukaryotic expression of chIL-9.

Figure 8 is a graph showing the reaction between chIL-9 mouse monoclonal antibody and rabbit polyclonal antibody and native chIL-9 protein.

Detailed ways

In order to fully understand and apply the present invention, the present invention will be described in detail below with reference to embodiments and accompanying drawings.

Example 1 Preparation of chIL-9 recombinant protein

1. ChIL-9 prokaryotic protein expression

Due to the low adaptability of chicken codons to E. coli hosts, the cloned chIL-9 nucleotide sequence without signal peptide (as shown in SEQ ID NO. a. Use Primer5.0 software to design PCR primers, and introduce restriction sites BamHI and Xho I and tev protease sites, carry out RT-PCR amplification, and clone into prokaryotic expression vector pET-32a (Figure 1), and finally Transform into a ROSETTA expression host.

PCR primer sequences (shown in SEQ ID NO. 5 and 6):

F: 5'-CGGGATCC(BamHI)GAGAACCTGTACTTCCAAGGG(tev)CAGAATTGCCAGGTT-3'

R: 5’-CCGCTCGAG(XhoI)TCACACGCGGGCTTTTAT-3’.

After the recombinant plasmid containing the target gene was transformed into ROSETTA host bacteria, it was induced by a small amount of IPTG, and the bacterial lysis supernatant protein and bacterial cell protein were extracted respectively; SDS-PAGE analysis showed that the chIL-9 recombinant fusion protein was abundantly expressed in the bacterial lysis pellet. Take 300 mL of bacterial liquid after being induced by IPTG in a large amount, ultrasonicate in ice bath for 20-30 min, centrifuge to remove supernatant, resuspend and disperse precipitate in 10 mL of 8M urea, ultrasonicate again for 10 min in ice bath, and collect supernatant after centrifugation. Purification of his-tagged protein under denaturing conditions was carried out according to the instructions of Ni Sepharose column. The purified protein was renatured by dialysis with gradient urea buffer, and finally concentrated and protein concentration was determined. The trx tag was cleaved with tev to obtain a prokaryotic recombinant protein of about 18kD chIL-9 (Figure 2).

2.chIL-9 eukaryotic protein expression

Chicken spleen mononuclear cells were taken and stimulated with PMA/Iono+BFA for 6 h, then RNA was extracted and reverse transcribed into cDNA as a template. Primers were designed according to the full-length coding region sequence of the chIL-9 gene (as shown in SEQ ID NO. 3), T2A peptide sequence was added to the front end of the upstream primer, and EcoRI and BamHI restriction sites were introduced, and RT-PCR amplification was performed. It was cloned into pEGFP-C plasmid (Figure 3), and the ultrapure plasmid was finally extracted.

PCR primer sequences (shown in SEQ ID NO. 7 and 8):

F: 5'-CCGGAATTC(EcoRI)GGAAGCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGACCTATGAATGCCAGCATGCTG-3'

R: 5'-CGGGATCC(BamHI)TTAAACTCTAGATTTATG-3'

The target plasmid was transfected into DF-1 cells, and 25kD recombinant chIL-9 protein was produced in DF-1 cells and in the culture supernatant by cellular immunochemistry (ICC) and Western blot detection (Figure 4).

Example 2 Biological function of chIL-9 recombinant protein

1. Activation of chIL-9 recombinant eukaryotic protein on chicken macrophages

In this experiment, chicken HD11 macrophage cell line and primary macrophages after chicken peripheral blood mononuclear cells adhered were selected to detect the function of chIL-9 recombinant protein. The control group was added with pEGFP-C plasmid transfection supernatant, the chIL-9 group was added with pEGFP-C-chIL-9 plasmid transfection supernatant, and the LPS group was used as a positive control; RNA was extracted after 6 hours of treatment, reverse transcribed into cDNA, and detected by qPCR Changes of key macrophage activators IL-1β, IL-6 and iNOS. Figure 5 shows that recombinant eukaryotic chIL-9 can activate chicken macrophages to produce inflammatory factors.

2. The effect of chIL-9 recombinant protein on the proliferation of chicken T cells

In this experiment, chicken peripheral blood mononuclear cells (PBMC) were selected as target cells to detect the proliferation effect of chIL-9 recombinant protein on T cells. After PBMCs were stained with CFSE, chIL-9 recombinant protein (100ng/mL) and chicken IL-2 (100ng/mL) were added for five days, and SPRD-CD3 and FVD eFluor 780 were stained. The results of flow cytometry showed (Fig. 6) that recombinant chIL-9 could promote the proliferation of chicken T cells.

Example 3 Preparation of chIL-9 mouse monoclonal antibody

The main steps of monoclonal antibody preparation are: mouse immunization, cell fusion, screening of hybridoma cells, cell monocloning, antibody identification, ascites preparation and purification.

1. Screening of positive hybridoma cells

6-week-old female BALB/c mice were selected, and 40 μg of prokaryotically expressed chIL-9 was mixed with Freund's adjuvant and emulsified. The mice were immunized three times by intraperitoneal injection, with an interval of two weeks between each immunization. Seven days later, blood was collected from the tail vein to measure the titer, and then the immunization was boosted. Three days later, the spleen cells of the mice were fused with SP2/0 cells under the action of 50% PEG-1500. , and added to the feeder cells containing HAT medium and placed in a 37°C, 5% CO ₂ cell incubator. After 5 days, the medium was changed halfway, and after 10 days, it was changed to the complete medium containing HT in DMEM.

The positive wells in 5 96-well plates were determined by ELISA, and the positive hybridoma wells were subcloned 2 to 3 times by limiting dilution method, and finally expanded and cultured, and then cryopreserved. The monoclonal antibody hybridoma cell culture supernatant was collected, and the subtype identification was carried out according to the mouse antibody subtype identification kit.

2. Monoclonal antibody ascites preparation and purification method

BALB/c mice about 10 weeks of age were selected, and 0.5 mL of pristane was inoculated intraperitoneally. After 7 to 10 days, hybridoma cells diluted with PBS were intraperitoneally inoculated, with a cell number of 5×10 ⁵ per mouse. After 7 days, the abdomen was distended, and the ascites was collected from the abdominal cavity, once every two days, the ascites was centrifuged to remove the sediment, the supernatant was collected, the titer was measured, and it was stored at -70°C. Ascites was purified using Protein A+G.

Example 4 Preparation and purification of chIL-9 rabbit polyclonal antibody

100 μg of prokaryotic expressed chIL-9 was mixed with complete Freund's adjuvant and emulsified, and injected subcutaneously at multiple points to immunize New Zealand white rabbits; and immunized twice with the emulsion of recombinant prokaryotic chIL-9 and incomplete Freund's adjuvant, each time Two weeks apart; 7 days later, venous blood was collected to determine the titer, and then boosted immunization (200 μg), and 3 days later, the antiserum was obtained by intravenous blood collection, and the rabbit serum was purified with Protein A+G, and finally the chIL-9 rabbit polyclonal antibody was obtained.

Example 5 The reactivity of chIL-9 monoclonal antibody and rabbit polyclonal antibody with eukaryotic expression chIL-9

The pEGFP-C-chIL-9 plasmid was transfected into DF-1 cells, the culture medium was discarded after 48 h, washed twice with PBS, fixed with ice-cold acetone-ethanol mixture (3:2), and blocked with 2% BSA for 1 h at room temperature After incubation, the culture supernatant of different hybridoma cells, IgG1, κ subtype irrelevant mouse monoclonal antibody (control) and rabbit polyclonal antibody (pAb) were incubated overnight at 4°C, washed three times with PBST, and incubated with HRP-conjugated anti- mouse or anti-rabbit antibody for 1 h, washed 3 times with PBST, added AEC chromogenic solution to react for 15 min, and observed the coloration under bright field conditions. The results (Figure 7, table 1) showed that different chIL-9 monoclonal antibodies and rabbit polyclonal antibodies all reacted with eukaryotic chIL-9, and the monoclonal antibody 4H7 and the other three monoclonal antibodies were all of IgG1, κ subtype.

Example 6 Reactivity of chIL-9 monoclonal antibody and rabbit polyclonal antibody with native chIL-9

Chicken PBMCs were taken and stimulated with PMA/Iono+BFA for 6 h, then the protein was extracted, added with 5× loading buffer, and boiled for 10 min to obtain the chIL-9 natural protein sample. Each histone was subjected to SDS-PAGE electrophoresis, transferred to PVDF membrane, blocked with 5% skim milk at room temperature for 1 h, incubated with different monoclonal antibodies and rabbit polyclonal antibodies (1:1000) overnight at 4°C, and washed 3 times with TBST solution. , incubated with HRP goat anti-mouse secondary antibody (1:8000) at 37°C for 1 h, washed 3 times with TBST, and then added luminescent solution for development. Figure 8 shows that different monoclonal antibodies and rabbit polyclonal antibodies reacted with the native chIL-9 protein with a size of approximately 25 kDa.

Table 1 ChIL-9 mouse monoclonal antibody subtype identification and ascites titer determination

references:

1. Noelle, R.J. and E.C. Nowak, Celular sources and immune functions of interleukin-9. Nature Reviews Immunology, 2010.10(10):p.683-687.

2.Veldhoen,M.,et al.,Transforming growth factor-beta'reprograms'thediferentiation of T helper 2 cels and promotes an interleukin 9-producingsubset.Nature Immunology,2008.9(12):p.1341-1346.

3. Stassen, M., E. Schmitt, and T. Bopp, From interleukin-9 to T helper 9cels. Year in Immunology, 2012.1247:p.56-68.

4. Thi, V., et al., Crosstalk between the Producers and Immune Targets of IL-9. Immune Network, 2020.20(6).

5. Goswami, R. and M.H. Kaplan, A Brief History of IL-9. Journal of Immunology, 2011.186(6): p.3283-3288.

6. Deng, Y.X., et al., Th9 cels and IL-9 in autoimmune disorders: Pathogenesis and therapeutic potentials. Human Immunology, 2017.78(2):p.120-128.

7. Wan, J., et al., IL-9 and IL-9-producing cels in tumor immunity. Cell Communication and Signaling, 2020.18(1).

8. Dodd, J.S., et al., IL-9 Regulates Pathology during Primary and MemoryResponses to Respiratory Syncytial Virus Infection. Journal of Immunology, 2009.183(11):p.7006-7013.

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Claims (10)

1. A prokaryotic expression method for preparing recombinant chicken IL-9 protein is characterized in that a chicken IL-9 gene without signal peptide is constructed into a prokaryotic expression vector pET-32a, and a recombinant plasmid (pET-32a-chIL-9) is induced and expressed by transforming escherichia coli to obtain soluble recombinant chicken IL-9 protein.

2. The prokaryotic expression method for preparing recombinant chicken IL-9 protein according to claim 1, characterized in that the nucleic acid sequence of the chicken IL-9 gene without signal peptide is shown in SEQ ID No.1, and the amino acid sequence thereof is shown in SEQ ID No. 2;

the recombinant chicken IL-9 protein has biological activity, and the proliferation of chicken T cells can be promoted by culturing the recombinant chicken IL-9 protein and the separated chicken spleen cells for 5 days.

3. Use of the recombinant chicken IL-9 protein of claim 1 or 2 as an immunopotentiator to increase the immunological competence of avians, in particular chickens; can be used as adjuvant for poultry, especially for development and application of chicken infectious disease vaccine.

4. A eukaryotic expression method for preparing recombinant chicken IL-9 protein is characterized in that a complete chicken IL-9 gene is constructed to a eukaryotic expression vector pEGFP-C, a recombinant plasmid (pEGFP-chIL-9) transfects DF-1 cells to obtain the recombinant chicken IL-9 protein with secretory expression and biological activity;

the complete chicken IL-9 gene sequence is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4;

co-culturing the recombinant chicken IL-9 protein and a chicken macrophage line can activate macrophages to generate inflammatory cytokines; the recombinant chicken IL-9 protein and the separated chicken spleen cells are cultured for 5 days, and the proliferation of the chicken T cells can be promoted.

5. Use of the recombinant chicken IL-9 protein of claim 3 or 4 as an immunopotentiator to increase the immunological competence of avians, in particular chickens; can be used as adjuvant for poultry, especially for development and application of chicken infectious disease vaccine.

6. An anti-chicken IL-9 protein monoclonal antibody 4H7, wherein the monoclonal antibody 4H7 can specifically bind to chicken immune cell-expressed IL-9 protein; can specifically recognize IL-9 natural protein expressed by chicken mononuclear cells activated by phorbol ester and ionomycin.

7. The method for preparing the chicken IL-9 protein monoclonal antibody 4H7 hybridoma cell line of claim 6, which is characterized in that: immunizing BALB/c mice with the recombinant chicken IL-9 protein of claim 1, and screening a monoclonal antibody cell strain, namely a hybridoma cell strain 4H7, from the eukaryotic expression chicken IL-9 protein of claim 4 by using a hybridoma technology;

the hybridoma cell strain 4H7 is preserved in China center for type culture Collection with the address as follows: wuhan, Wuhan university, China; the preservation date is 2022 years, 1 month and 19 days; the preservation number is CCTCC NO: C202225.

8. A method for preparing rabbit polyclonal antibody against chicken IL-9 protein, characterized in that the recombinant chicken IL-9 protein of claim 1 is used to immunize New Zealand white rabbits, and the obtained antiserum can recognize the eukaryotic expressed chicken IL-9 protein and the activated natural IL-9 protein expressed by chicken mononuclear cells.

9. The application of the chicken IL-9 protein resisting monoclonal antibody 4H7, which is characterized in that the chicken IL-9 expression is detected by Western-blot, ELISA, indirect immunofluorescence and flow cytometry after being marked by fluorescein or enzyme.

10. The use of polyclonal antibodies as defined in claim 8, characterized in that said antibodies are labeled with fluorescein or enzyme and used in Western-blot, ELISA, indirect immunofluorescence and flow cytometry for the detection of chicken IL-9 expression.

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