Application of ANXA2 protein in improving immunoreaction of sheep to mycoplasma pneumoniae

Abstract

The invention discloses application of ANXA2 protein in improving immunoreaction of sheep to mycoplasma pneumoniae, and finds that the ANXA2 protein is sheep mycoplasma pneumoniae receptor protein and can be combined with the sheep mycoplasma pneumoniae, meanwhile, the protein is also TLR2 receptor inhibition protein, and after expression of an ANXA2 gene is interfered and pulmonary alveolar epithelial cells are infected by the sheep mycoplasma pneumoniae, the protein can be combined with the sheep mycoplasma pneumoniae and can be combined with the TLR2 receptor inhibition protein. The autophagy response of the cells is enhanced, and the cell proliferation function is recovered; and after the ANXA2 gene is interfered, the phagocytosis and clearance of the mycoplasma by the macrophages are also enhanced. It is shown that the immune response of cells to mycoplasma can be improved by knocking out the ANXA2 gene, damage of mycoplasma pneumoniae to the cells or sheep is reduced, and the time required for variety improvement can be remarkably shortened when the ANXA2 gene is used for later disease-resistant variety breeding.

Description

Technical Field

[0001] The invention belongs to the biological field, and particularly relates to application of ANXA2 protein in improving the immune response of sheep to Mycoplasma pneumoniae. Background Art

[0002] Mycoplasma ovipneumoniae (MO) is the most common pathogen causing respiratory diseases in sheep. Pathological sections of the lungs of sick sheep mainly show necrosis and shedding of alveolar epithelial cells, obvious widening of alveolar septa, vasodilation, congestion and edema, and infiltration of a large number of lymphocytes and monocytes. If the pathogens in the body cannot be cleared in time in the early stage of the disease, chronic infection is very likely to occur. However, the receptor protein and its function of Mycoplasma pneumoniae are not fully understood.

[0003] The LPPT protein of MO is highly homologous to the adhesin P110 of Mycoplasma hyopneumoniae (Mhp), and belongs to the P110 / LppT adhesin N-terminal domain family, which is a major adhesion protein. ANXA2 protein is a membrane-linked protein that participates in important cell life activities such as pathogen recognition, cell membrane formation, cell membrane transport, endocytosis and exocytosis, cell proliferation, cell signal transduction, differentiation and apoptosis, and angiogenesis. However, whether it can recognize the LPPT protein of MO has not been studied.

[0004] Sheep alveolar epithelial cells are the first line of defense against pathogens and are also key cells that constitute the integrity of alveoli. Once epithelial cells are damaged, the cells necrotize and fall off, which will destroy the alveolar function of performing gas exchange. The Toll-like receptors on its surface have the function of recognizing pathogens and transmitting signals. When Toll-like receptors recognize pathogen signals, they activate the inflammatory cascade in the cell and even the cell autophagy function to resist the invasion of pathogens. Cell autophagy can remove harmful substances in the body and reduce the impact of mycoplasma on cells, but the impact of mycoplasma on cell autophagy is still unclear. Summary of the invention

[0005] The purpose of the present invention is to provide application of ANXA2 protein in improving the immune response of sheep to Mycoplasma pneumoniae.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] In the first aspect, the present invention claims the use of the ANXA2 gene in the following (a1) or (a2) or (a3) ​​or (a4):

[0008] (a1) Improve the immune response of sheep to Mycoplasma pneumoniae;

[0009] (a2) reducing the damage and / or proliferation inhibition of sheep alveolar epithelial cells caused by Mycoplasma pneumoniae;

[0010] (a3) Cultivate new disease-resistant sheep breeds with improved resistance to Mycoplasma pneumoniae.

[0011] (a4) preparing a preparation for preventing and treating Mycoplasma ovipneumoniae infection.

[0012] In the second aspect, the present invention claims the use of a biological material for silencing, interfering with or inhibiting the expression of the ANXA2 gene or a biological material for reducing the activity or content of a protein encoded by the ANXA2 gene in the following (a1) or (a2) or (a3) ​​or (a4):

[0013] (a1) Improve the immune response of sheep to Mycoplasma pneumoniae;

[0014] (a2) reducing the damage and / or proliferation inhibition of sheep alveolar epithelial cells caused by Mycoplasma pneumoniae;

[0015] (a3) Cultivate new disease-resistant sheep breeds with improved resistance to Mycoplasma pneumoniae.

[0016] (a4) preparing a preparation for preventing and treating Mycoplasma ovipneumoniae infection.

[0017] Furthermore, the above application is to reduce the inhibition of cell proliferation by Mycoplasma ovipneumoniae and enhance the immune response of sheep to Mycoplasma pneumoniae by silencing, interfering with or inhibiting the expression of the ANXA2 gene or reducing the level or activity of the protein encoded by the ANXA2 gene.

[0018] Furthermore, the biological material for silencing, interfering with or inhibiting the expression of the ANXA2 gene or the biological material for reducing the activity or content of the protein encoded by the ANXA2 gene is at least one of the following (b1) to (b3):

[0019] (b1) an interference or silencing fragment of the ANXA2 gene;

[0020] (b2) an interference expression vector or a silencing vector comprising the interference or silencing fragment described in (b1);

[0021] (b3) A recombinant microorganism containing the interfering expression vector or silencing vector as described in (b2).

[0022] Furthermore, the ANXA2 gene interference or silencing fragment in (b1) is siRNA, and the siRNA includes siRNA1 and siRNA2.

[0023] The upstream sequence of the siRNA1 is GGGAUGCUCUGAACAUUGATT, and the downstream sequence is UCAAUGUUCAGAGCAUCCCTT;

[0024] The upstream sequence of the siRNA2 is CCCUGUACUACUACAUUCATT, and the downstream sequence is UGAAUGUAGUAGUACAGGGTT.

[0025] Furthermore, the ANXA2 gene in the above application is a DNA molecule described in any one of the following (c1)-(c4):

[0026] (c1) a DNA molecule whose coding region includes the nucleotide sequence shown in SEQ ID NO.1 in the sequence list;

[0027] (c2) the nucleotide sequence is a DNA molecule as shown in SEQ ID NO.1 in the sequence list;

[0028] (c3) a DNA molecule that hybridizes with the DNA sequence defined in (c1) or (c2) under stringent conditions and encodes the Mycoplasma ovipneumoniae receptor ANXA2 protein;

[0029] (c4) A DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA sequence defined in (c1) or (c2) and encodes the Mycoplasma ovipneumoniae receptor ANXA2 protein.

[0030] Furthermore, the protein encoded by the ANXA2 gene in the above application is as follows (d1) or (d2):

[0031] (d1) a protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing;

[0032] (d2) A protein derived from SEQ ID NO. 2 in the sequence listing, wherein one or more amino acid residues are substituted and / or deleted and / or added and which has the same function as the protein described in (d1).

[0033] In a third aspect, the present invention claims a method for improving the immune response of sheep to Mycoplasma pneumoniae, which reduces the inhibition of cell proliferation by Mycoplasma by silencing, interfering with or inhibiting the expression of the aforementioned ANXA2 gene or reducing the level or activity of the protein encoded by the aforementioned ANXA2 gene, thereby improving the immune response of sheep to Mycoplasma pneumoniae.

[0034] In a fourth aspect, the present invention claims a preparation for enhancing the immune response of sheep to Mycoplasma pneumoniae, wherein the preparation comprises a biological material that silences, interferes with or inhibits the expression of the ANXA2 gene or a biological material that reduces the activity or content of the protein encoded by the ANXA2 gene.

[0035] Furthermore, the biological material for silencing, interfering with or inhibiting the expression of the ANXA2 gene or the biological material for reducing the activity or content of the protein encoded by the ANXA2 gene is preferably an interfering or silencing fragment of the ANXA2 gene. Further, the interfering or silencing fragment of the ANXA2 gene is preferably siRNA, and the siRNA includes siRNA1 and siRNA2.

[0036] The upstream sequence of the siRNA1 is GGGAUGCUCUGAACAUUGATT, and the downstream sequence is UCAAUGUUCAGAGCAUCCCTT;

[0037] The upstream sequence of the siRNA2 is CCCUGUACUACUACAUUCATT, and the downstream sequence is UGAAUGUAGUAGUACAGGGTT.

[0038] Beneficial effects of the present invention: The present invention has found that the ANXA2 protein is a receptor protein of Mycoplasma ovipneumoniae, which can bind to the LPPT protein of Mycoplasma ovipneumoniae. At the same time, the protein is also a TLR2 receptor inhibitory protein. After interfering with the expression of the ANXA2 gene and infecting alveolar epithelial cells with Mycoplasma ovipneumoniae, the autophagy reaction of the cells is enhanced and the cell proliferation function is restored; after interfering with the ANXA2 gene, the phagocytosis and clearance of mycoplasma by macrophages is also enhanced. It shows that knocking out the ANXA2 gene can improve the immune response of cells to mycoplasma, reduce the damage of Mycoplasma pneumoniae to cells or sheep, and can be used for the later stage of disease-resistant variety breeding, which can significantly shorten the time required for variety improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a schematic diagram of the binding between the LPPT protein of Mycoplasma ovipneumoniae and the receptor ANXA2 protein provided in an embodiment of the present invention; wherein (A) is a three-dimensional structure diagram of the LPPT protein; (B) is a three-dimensional structure diagram of the ANXA2 protein; and (C) is a 3D interaction pattern between the ANXA2 protein and the LppT protein.

[0040] Figure 2Figure 1. Result of CO-IP binding of the Mycoplasma ovipneumoniae LPPT protein to the receptor ANXA2 protein. Lysates from 293T cells (+) transfected with pCMV-N-Flag-ANXA2 and pCMV-HA-C-LPPT plasmids were immunoprecipitated using anti-HA antibody and analyzed by western blot using antibodies against FLAG and HA. Lysates from 293T cells (-) not transfected with pCMV-N-Flag-ANXA2 and pCMV-HA-C-LPPT plasmids were used as a control group. Whole cell lysate (WCL) of 293T was used as input.

[0041] Figure 3 Interference with ANXA2 gene expression in sheep lung epithelial cells resulted in a reduction in ANXA2 protein content.

[0042] Figure 4 After interfering with the expression of ANXA2 gene in sheep lung epithelial cells, mycoplasma infection was added, and the results of cell autophagy-related pathways and proteins TLR2, Myd88, mTOR, p-mTOR, LC3Ⅱ / LC3Ⅰ; (A) Western blot analysis of four groups; (B) Numerical values ​​of target protein / reference protein, different superscript letters represent P<0.05, and the same letters represent P>0.05.

[0043] Figure 5 The results of interfering with the expression of ANXA2 gene in sheep lung epithelial cells and then infecting them with mycoplasma showed that the level of cellular autophagy increased. The amount of green fluorescence in the NC+MO group was higher than that in other groups, indicating that the autophagic flow of the cells was blocked, while the amount of green fluorescence was reduced in the siANXA2+MO group.

[0044] Figure 6 Figure 3. The results of mycoplasma infection after interfering with the expression of ANXA2 gene in sheep lung epithelial cells, and the restoration of cell proliferation ability; (A) Representative EdU staining of four groups; (B) Statistical analysis of the proportion of EdU positive cells, different superscript letters represent P<0.05, and the same letters represent P>0.05.

[0045] Figure 7 After ANXA2 gene interference in sheep macrophages, DiO-stained mycoplasma was added, and the results of macrophage phagocytosis and clearance of mycoplasma were enhanced; (A) represents the representative and statistical graphs of the proportion of DiO-positive cells in the two groups of cells; (B) is the statistical graph of the average fluorescence intensity of the two groups of cells. * indicates P < 0.05, ** indicates P < 0.01.

[0046] Figure 8After ANXA2 gene interference in sheep macrophages, DiO-stained mycoplasma was added, and the fluorescence content of mycoplasma in macrophages decreased, but the fluorescence intensity of lysosomal protein LAMP2A increased. * indicates P < 0.05. DETAILED DESCRIPTION

[0047] The present invention is further described below with reference to the examples. The experimental methods in the following examples without specifying specific conditions are generally carried out according to the well-known means in the art.

[0048] Example 1

[0049] Based on the ANXA2 gene (NCBI accession number: NM_001093788.1), ANXA2 protein (protein accession number: NP_001087257.1) and LPPT protein sequence (protein accession number: WDV48811.1) disclosed in the prior art, AlphaFold2 was used to perform de novo modeling to obtain the complete three-dimensional structures of the two proteins. The complete three-dimensional structures are shown in Figure 1 As shown in A and B in Figure 1. In order to study the intermolecular binding region and interaction mode between ANXA2 protein and LPPT protein, this study used the professional protein-protein and protein-DNA / RNA docking HDCOK program for docking, and selected the structure with the best docking score as the standard result for subsequent interaction analysis. The docking score is calculated based on the ITScorePP or ITScorePR iterative scoring function. The more negative the docking score, the greater the binding possibility of the binding model and the stronger the interaction. The confidence score formula is Confidence_score = 1.0 / [1.0+e0.02*(Docking_Score+150)], that is, when the confidence score is higher than 0.7, the two molecules are very likely to bind.

[0050] The results showed that the optimal docking result of ANXA2 protein and LPPT protein molecule was -233.73kcal / mol and Confidence Score was 0.842, indicating that the complex model obtained by docking had a high degree of credibility. Subsequently, the interaction pattern of the spatial structure binding site and binding region between ANXA2 protein and P110 protein molecules was further analyzed. Figure 1 As shown in C. Figure 3The diagram of the interaction pattern between ANXA2 protein and P110 protein shows that 11 groups of hydrogen bond interactions and 2 groups of salt bridge interactions are formed between ANXA2 protein and P110 protein. Specifically, the hydrogen bonds formed by amino acids between the two proteins are from ANXA2 protein His5, Ser12, Arg196, Gln226, Arg230, Glu231, Lys233, Tyr235, Asp239, Glu242 and Gln264; the amino acid residues from P110 protein are Arg444, Glu453, Pro488, Thr490, Lys627, Tyr628, Asn629, Glu654 and Arg657. In addition, there are a large number of hydrophobic interactions between the two proteins to promote further stable binding between the two proteins and form a complex.

[0051] In order to verify that ANXA2 protein can bind to LPPT protein, the ANXA2 gene sequence was depleted of the stop codon and BamHI and HindIII restriction sites were inserted at both ends. The whole gene was synthesized by Nanjing Qingke Biotechnology Co., Ltd. and connected to the pCMV-N-Flag vector to obtain the recombinant plasmid CMV-N-Flag-ANXA2. In addition, the LPPT nucleotide sequence was codon-optimized and EcoRI and XhoI restriction sites were inserted at both ends. The whole gene was synthesized by Nanjing Qingke Biotechnology Co., Ltd. and connected to the pCMV-HA-C vector to construct the pCMV-HA-C-LPPT plasmid. The nucleotide sequence of the LPPT gene after codon optimization is shown in SEQ ID NO.3, and the amino acid sequence of the LPPT protein encoded by it is shown in SEQ ID NO.4. The above gene sequences were synthesized by Nanjing Qingke Biotechnology Co., Ltd.

[0052] 293T cells were seeded into 6-well culture plates and transfected with pCMV-N-Flag-ANXA2 and pCMV-HA-C-LPPT plasmids. Transfected cells were harvested 24 hours after transfection and lysed in NP-40Lysis buffer containing 1mM protease inhibitors. The lysate was centrifuged at 12000×g for 10 minutes, and the supernatant containing 500μg of total protein was immunoprecipitated with 20μL of magnetic beads with anti-HA antibody at 4°C. Western blot analysis was performed using antibodies against FLAG and HA. The results showed that ANXA2 protein was able to bind to LPPT protein, indicating that ANXA2 protein is a receptor for Mycoplasma ovipneumoniae ( Figure 2 ).

[0053] Example 2

[0054] The Mycoplasma ovipneumoniae Y98 standard strain (a conventional strain known in the art, Mesomycoplasma ovipneumoniae ATCC 29419) was provided by Liu Maojun, a researcher at the Veterinary Research Institute of Jiangsu Academy of Agricultural Sciences.

[0055] The sheep alveolar epithelial cell line was provided by Associate Professor Han Hongbing from the College of Animal Science and Technology, China Agricultural University.

[0056] Two siRNAs targeting the sheep ANXA2 gene were designed and synthesized by Genetix. The siRNA sequences are shown in Table 1.

[0057] Table 1 Specific sequences of siRNA

[0058]

[0059] Sheep alveolar epithelial cells were inoculated in 6-well plates, and when the cells grew to 80% confluence, 12 μL of siRNA1 and siRNA2 mixture was transfected. After 24 hours, the cells were collected to extract protein, and Western blot analysis was performed using anti-ANXA2 antibodies. The results showed that siRNA could reduce the protein expression of ANXA2 ( Figure 3 ).

[0060] To verify the phenotypic response of cells to mycoplasma infection after ANXA2 gene interference, cells were divided into four groups: NC, NC+MO, siANXA2+MO and siANXA2. Y98 mycoplasma (MOI=10) was added 6 hours after transfection, and cells were harvested and lysed 24 hours later to extract cell proteins. Western blot analysis was performed using anti-TLR2, Myd88, mTOR, p-mTOR, and LC3 antibodies ( Figure 4 ), the results showed that mycoplasma infection could reduce the levels of TLR2, Myd88 and LC3Ⅱ proteins, and increase the expression of p-mTOR (NC+MO group), but interfering with ANXA2 could restore the levels of TLR2, Myd88 and LC3Ⅱ proteins, and reduce the expression of p-mTOR (siANXA2+MO group), and only interfering with the ANXA2 gene did not affect the expression of the above proteins (siANXA2 group), indicating that interfering with the ANXA2 gene can restore the level of cellular autophagy through the TLR2-Myd88-mTOR pathway.

[0061] To verify the smoothness of the cell autophagy flow, mCherry-eGFP-LC3 autophagy double-labeled adenovirus purchased from Hanheng Bio was added to each group of cells at MOI=1, and the changes in autophagy flow were observed using a laser confocal microscope after 24 hours ( Figure 5), the results showed that compared with the NC group, the number of yellow spots in the NC+MO group was greater than that in the NC group, indicating that cellular autophagy was blocked, while the number of yellow spots in the siANXA2+MO group decreased and the number of red spots increased, indicating that the cellular autophagic flow was restored.

[0062] To detect the effect of cell autophagy recovery on cell proliferation, we used the Beyotime EdU detection kit to detect the proliferation function of the four groups of cells. After 24 hours of transfection and mycoplasma infection, the cells were stained and the percentage of red fluorescent cells was observed and recorded using a fluorescence microscope. The results showed that compared with the NC group, the percentage of proliferating cells in the NC+MO group decreased, while the percentage of proliferating cells increased after ANXA2 gene interference, with no significant difference compared with the NC group, indicating that ANXA2 gene interference can reduce the damage of mycoplasma to cell proliferation function by restoring cell autophagy ( Figure 6 ).

[0063] To detect the changes in the phagocytic and clearance abilities of sheep macrophages after ANXA2 gene interference, we isolated and induced sheep bone marrow mononuclear cell-derived macrophages as follows: femoral and tibial bone marrow was flushed into a 50 ml centrifuge tube with sterile PBS, centrifuged at 300 g for 5 min, erythrocytes were lysed with erythrocyte lysis buffer, centrifuged at 300 g for 5 min, and then 1–2 × 10 6 The cells / ml were resuspended in 1640 culture medium (20% FBS, 1% double antibody, 50 ng M-CSF), and the medium was replaced by half on the third day and fully on the fifth day. After 7-10 days of culture, oval cells appeared, which were macrophages.

[0064] Subsequently, DiO-stained mycoplasma was added to macrophages at an MOI of 10. After 24 hours, flow cytometry was used to detect the percentage of DiO-positive cells and the average fluorescence intensity of positive cells. The results showed that compared with the siNC+MO group, the percentage of DiO-positive cells in macrophages in the siANXA2+MO group increased, indicating that the phagocytic ability of macrophages was enhanced, but the average fluorescence intensity was low, indicating that the intracellular mycoplasma content was low and the cell's ability to clear mycoplasma was enhanced ( Figure 7 ).

[0065] To verify that the autophagic ability of sheep macrophages was enhanced after ANXA2 gene interference, DiO-stained mycoplasma was added to macrophages at MOI=10. After 24 hours, the intracellular mycoplasma content and the fluorescence intensity of the lysosomal marker protein LAMP2A were observed using a laser confocal microscope. The results showed that compared with the siNC+MO group, the fluorescence intensity of mycoplasma in macrophages in the siANXA2+MO group was reduced, but the fluorescence intensity of LAMP2A was increased, indicating that the autophagic ability of macrophages was enhanced after ANXA2 gene interference ( Figure 8 ).

[0066] In summary, the embodiment of the present invention uses the LPPT protein of Mycoplasma ovipneumoniae as a ligand to predict and verify that the sheep ANXA2 protein is an LPPT receptor protein. By interfering with the expression of the ANXA2 gene, it was found that the TLR2-Myd88-mTOR pathway of the cell was activated, thereby activating cell autophagy, reducing the proliferation inhibition of mycoplasma on cells, and enhancing the phagocytosis and clearance of mycoplasma by macrophages.

[0067]

[0068]

[0069]

[0070]

Claims

1. Use of the ANXA2 gene in the following (a1) or (a2) or (a3) ​​or (a4): (a1) Improve the immune response of sheep to Mycoplasma pneumoniae; (a2) reducing the damage and / or proliferation inhibition of sheep alveolar epithelial cells caused by Mycoplasma pneumoniae; (a3) Cultivate new disease-resistant sheep breeds with improved resistance to Mycoplasma pneumoniae. (a4) preparing a preparation for preventing and treating Mycoplasma ovipneumoniae infection.

2. Use of a biological material for silencing, interfering with or inhibiting the expression of the ANXA2 gene or a biological material for reducing the activity or content of the protein encoded by the ANXA2 gene in the following (a1) or (a2) or (a3) ​​or (a4): (a1) Improve the immune response of sheep to Mycoplasma pneumoniae; (a2) reducing the damage and / or proliferation inhibition of sheep alveolar epithelial cells caused by Mycoplasma pneumoniae; (a3) Cultivate new disease-resistant sheep breeds with improved resistance to Mycoplasma pneumoniae. (a4) preparing a preparation for preventing and treating Mycoplasma ovipneumoniae infection.

3. The use according to claim 1 or 2, characterized in that: By silencing, interfering with or inhibiting the expression of the ANXA2 gene or reducing the level or activity of the protein encoded by the ANXA2 gene, the inhibition of cell proliferation by Mycoplasma ovipneumoniae is reduced, and the immune response of sheep to Mycoplasma pneumoniae is improved.

4. The use according to claim 2, characterized in that The biological material for silencing, interfering with or inhibiting the expression of the ANXA2 gene or the biological material for reducing the activity or content of the protein encoded by the ANXA2 gene is at least one of the following (b1) to (b3): (b1) an interference or silencing fragment of the ANXA2 gene; (b2) an interference expression vector or a silencing vector comprising the interference or silencing fragment described in (b1); (b3) A recombinant microorganism containing the interfering expression vector or silencing vector as described in (b2).

5. The use according to claim 4, characterized in that: The ANXA2 gene interference or silencing fragment in (b1) is siRNA, and the siRNA includes siRNA1 and siRNA2. The upstream sequence of the siRNA1 is GGGAUGCUCUGAACAUUGATT, and the downstream sequence is UCAAUGUUCAGAGCAUCCCTT; The upstream sequence of the siRNA2 is CCCUGUACUACUACAUUCATT, and the downstream sequence is UGAAUGUAGUAGUACAGGGTT.

6. The use according to claim 1 or 2 or 3 or 4, characterized in that: The ANXA2 gene is a DNA molecule described in any one of the following (c1)-(c4): (c1) a DNA molecule whose coding region includes the nucleotide sequence shown in SEQ ID NO.1 in the sequence list; (c2) the nucleotide sequence is a DNA molecule as shown in SEQ ID NO.1 in the sequence list; (c3) a DNA molecule that hybridizes with the DNA sequence defined in (c1) or (c2) under stringent conditions and encodes the Mycoplasma ovipneumoniae receptor ANXA2 protein; (c4) A DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA sequence defined in (c1) or (c2) and encodes the Mycoplasma ovipneumoniae receptor ANXA2 protein.

7. The use according to claim 2, 3 or 4, characterized in that , the protein encoded by the ANXA2 gene is as follows (d1) or (d2): (d1) a protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing; (d2) A protein derived from SEQ ID NO. 2 in the sequence listing, wherein one or more amino acid residues are substituted and / or deleted and / or added and which has the same function as the protein described in (d1).

8. A method for improving the immune response of sheep to Mycoplasma pneumoniae, characterized in that: By silencing, interfering with or inhibiting the expression of the ANXA2 gene described in claim 1 or reducing the level or activity of the protein encoded by the ANXA2 gene described in claim 2, the inhibition of cell proliferation by mycoplasma is reduced, and the immune response of sheep to Mycoplasma pneumonia is improved.

9. A preparation for improving the immune response of sheep to Mycoplasma pneumoniae, characterized in that The preparation contains biological materials that silence, interfere with or inhibit the expression of the ANXA2 gene or biological materials that reduce the activity or content of the protein encoded by the ANXA2 gene.

10. The preparation for improving the immune response of sheep to Mycoplasma pneumoniae according to claim 9, characterized in that The biological material for silencing, interfering with or inhibiting the expression of the ANXA2 gene or the biological material for reducing the activity or content of the protein encoded by the ANXA2 gene is preferably an interfering or silencing fragment of the ANXA2 gene, and the interfering or silencing fragment of the ANXA2 gene is siRNA, and the siRNA includes siRNA1 and siRNA2. The upstream sequence of the siRNA1 is GGGAUGCUCUGAACAUUGATT, and the downstream sequence is UCAAUGUUCAGAGCAUCCCTT; The upstream sequence of the siRNA2 is CCCUGUACUACUACAUUCATT, and the downstream sequence is UGAAUGUAGUAGUACAGGGTT.

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