Preparation method of myeloid cell triggered receptor 2 protein polyclonal antibody and application of polyclonal antibody in inhibition of renal fibrosis

Abstract

The invention discloses a preparation method of a myeloid cell triggered receptor 2 protein polyclonal antibody and application of the polyclonal antibody in inhibition of renal fibrosis, and belongs to the technical field of medical molecular biology. The myeloid cell triggered receptor 2 protein is expressed by macrophages, and verification in the mechanism aspect is carried out on the protein; meanwhile, the polyclonal antibody is synthesized according to the polypeptide of the protein, then the effect of the antibody on treating the renal fibrosis is verified again, and a more targeted and personalized method is provided for diagnosis, treatment and management of the renal fibrosis and related kidney diseases.

Description

Technical Field

[0001] The present invention relates to a method for preparing a protein and its application in inhibiting renal fibrosis, belonging to the technical field of medical molecular biology. Background Art

[0002] Renal fibrosis is an important stage in the progression of chronic kidney diseases. By studying the occurrence mechanism and influencing factors of renal fibrosis, we can deeply understand the occurrence, development, and progression rules of chronic kidney diseases, providing a scientific basis for the prevention, diagnosis, and treatment of related diseases. Studying renal fibrosis can help doctors more accurately diagnose the condition of patients and formulate personalized treatment plans. Understanding the pathophysiological process and molecular mechanism of renal fibrosis helps to discover potential therapeutic targets and develop more effective treatment strategies. Renal fibrosis is also an important factor leading to the gradual deterioration of renal function. Studying renal fibrosis is crucial for evaluating the prognosis and quality of life of patients with chronic kidney diseases. Timely intervention and treatment of renal fibrosis help delay the deterioration of renal function and improve the quality of life of patients.

[0003] Chronic kidney disease (CKD) is a global public health problem. Currently, there is still a lack of effective treatment measures for CKD. Renal fibrosis (RF) is a common pathological change in all kidney diseases progressing to end-stage renal disease. Triggering receptor expressed on myeloid cells-2 (TREM-2) is highly expressed on myeloid cells such as macrophages and dendritic cells, and can regulate the phenotype and function of macrophages. Macrophages produce a large amount of extracellular matrix (ECM), which in turn leads to tissue fibrosis. The homeostasis of ECM is regulated by matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). MMP-9 has the function of degrading tissue collagen and can decompose ECM, while TIMP-1 is a tissue inhibitor of MMP-9 and can inhibit its decomposition. A large number of studies have shown that exosomes participate in the physiological and pathological processes of various diseases through intercellular communication, and macrophage-derived exosomes play a role in the pathological processes of various diseases. Currently, there is still a lack of effective drugs targeting the key proteins that regulate fibrosis by macrophage-derived exosomes. Summary of the Invention

[0004] Use of a reagent for detecting the expression level of myeloid cell triggering receptor 2 in the preparation of a reagent for inhibiting renal fibrosis.

[0005] Use of a reagent for detecting the expression level of myeloid cell triggering receptor 2 in the preparation of a reagent for diagnosing and / or predicting the prognosis of kidney diseases. The reagent is an antibody capable of specifically binding to myeloid cell triggering receptor 2.

[0006] The antibody is conjugated with or labeled with a detection label.

[0007] The antibody is a monoclonal antibody or a polyclonal antibody against myeloid cell triggering receptor 2.

[0008] A polyclonal antibody against myeloid cell triggering receptor 2 protein, which is obtained by immunizing with a polypeptide having a sequence such as SEQ ID NO.3 and / or SEQ ID NO.4 as an antigen.

[0009] Use of a polyclonal antibody against myeloid cell triggering receptor 2 (TREM-2) protein, which is expressed by macrophages, in inhibiting renal fibrosis.

[0010] Furthermore, the mRNA sequence of the key protein TREM-2 is as shown in SEQ ID NO.1, and the amino acid sequence is as shown in SEQ ID NO.2.

[0011] Furthermore, according to antigenicity prediction and analysis of exposed sites, the two polypeptides for preparing the polyclonal antibody are preferably: SEQ ID NO.3 and SEQ ID NO.4.

[0012] A method for preparing a polyclonal antibody against myeloid cell triggering receptor 2 (TREM-2) protein, comprising the following steps:

[0013] S1: Using the polypeptide as an antigen, after subcutaneous immunization of New Zealand white rabbits, blood is collected to detect the titer of the antiserum. When the titer is greater than 1:50000, final blood collection is performed to prepare the antiserum;

[0014] S2: Coupling the polypeptide with an agarose medium to prepare an antigen affinity purification chromatography column. The obtained antiserum is mixed with PBS in equal amounts and slowly loaded. After the antibody binds, it is eluted with a glycine elution buffer to obtain the required purified antibody, which is immediately dialyzed overnight in PBS, and the purity, concentration and titer are measured the next day;

[0015] S3: Detecting the titer of the purified antibody by ELISA, and measuring the concentration of the obtained antibody using a BCA concentration assay kit; observing the purity of the purified antibody by SDS-PAGE electrophoresis and Coomassie brilliant blue staining;

[0016] S4: Indirect ELISA titer detection of the purified antibody.

[0017] Further, in step S1, the New Zealand white rabbits weigh 2 - 2.5 kg, are subcutaneously immunized at 350 - 500 μg / time, immunized once every 2 - 3 weeks, and the titer of the antiserum is determined by the indirect ELISA method.

[0018] Further, in step S2, the temperature for dialysis overnight in PBS is 3 - 5°C.

[0019] Further, the specific method of step S4 is as follows:

[0020] (1) Design a coated plate according to the experimental requirements and make marks on the strips.

[0021] (2) Dilute the protein / polypeptide antigen with PBS coating solution to the required concentration, mix well and add it to the strips, 80 - 120 μL per well, and incubate overnight in a refrigerator at 3 - 5°C;

[0022] (3) After coating, discard the coating solution, wash the plate 3 times, add 200 μL of blocking solution to each well, and incubate in an incubator at 37°C for 1 h. Take out the enzyme - labeled plate, discard the internal liquid, and wash the plate 1 time;

[0023] (4) Purify the antibody and dilute it 3 - fold at 1:500, 100 μL per well, and incubate in an incubator at 37°C for 1 h.

[0024] (5) Take out the enzyme - labeled plate, discard the internal liquid, wash the plate 3 times, add 100 μL of diluted enzyme - labeled secondary antibody to each well, enzyme - labeled secondary antibody: goat anti - rabbit - HRP, 1:50000, and incubate in an incubator at 37°C for 1 h;

[0025] (6) Take out the enzyme - labeled plate, discard the internal liquid, wash the plate 4 times, add 100 μL of TMB chromogenic solution to each well first, and determine the chromogenic time according to the color depth, generally at 37°C for 15 min;

[0026] (7) Add 100 μL of 1M HCl solution to each well to terminate the reaction. Immediately read the value at 450 nm on an enzyme - labeled immunosorbent assay instrument, and define the dilution corresponding to the well with an OD value greater than 2.1 times the OD value of the set negative control as the titer of the sample.

[0027] Preferably, the coating concentration is: 4 - 6 μg / mL, 80 - 120 μL / well, and the coating buffer: phosphate - buffered saline (PBS, pH 7.4).

[0028] Beneficial effects

[0029] The present invention has discovered a key protein that regulates renal fibrosis. This protein is expressed by macrophages, and the mechanism has been verified for this protein. Meanwhile, the present invention synthesized a polyclonal antibody based on the polypeptide of the protein, and then verified the effect of this antibody in treating renal fibrosis, providing a more targeted and personalized method for the diagnosis, treatment, and management of renal fibrosis and related kidney diseases. Description of the Drawings

[0030] Figure 1 It is a fluorescence staining map of the expression of macrophage TREM-2 in the kidneys of patients with (None-mild) none-mild renal fibrosis (fibrosis area < 25%), (Moderate) moderate (fibrosis area 25% - 50%), and (Severe) severe fibrosis (fibrosis area > 50%).

[0031] Figure 2 It is a schematic diagram of the expression level of TREM-2 mRNA in kidney tissues at 1 week (1w) and 2 weeks (2w) after UUO (unilateral ureteral obstruction) found by RT-PCR experiment in A; a schematic diagram of the expression level of TREM-2 protein in kidney tissues at 1 week and 2 weeks after UUO (*P < 0.05 vs control group and sham group) in B.

[0032] Figure 3 It is a Western blot (WB) picture of macrophage TREM-2 in A; a quantitative analysis of the expression level of TREM-2 protein in B (**P < 0.01, ***P < 0.001).

[0033] Figure 4 It is a Western blot of exosome marker proteins CD63, CD81, and TSG101 in A; a particle size analysis of exosomes in B; a transmission electron microscopy picture of exosomes in C.

[0034] Figure 5 It is a Western blot of MMP-9 and TIMP-1 in A; a quantitative graph of the MMP-9 protein level in B; a quantitative graph of the TIMP-1 protein expression level in C; a quantitative graph of the MMP-9 / TIMP-1 ratio in D (**p < 0.01, ***p < 0.001, ****p < 0.0001). Figure 6A: Volcano plot of RNA sequencing of macrophages in the TREM-2- / - + TGF-β1 group and the WT + TGF-β1 group; B: Heat map of RNA sequencing of macrophages in the TREM-2- / - + TGF-β1 group and the WT + TGF-β1 group; C: Schematic diagram of KEGG signaling pathway; D: Western blot of the expression levels of HSPa1b and p-AKT; E: Quantitative analysis chart of AKT (**p < 0.01, ***p < 0.001, ****p < 0.0001); F: Quantitative analysis chart of HSPa1b (**p < 0.01, ***p < 0.001, ****p < 0.0001),

[0035] Figure 7 A: Western blot of HSPa1b, AKT, and p-AKT; B: Expression level of its mRNA after transfection of macrophages with siRNA-HSPa1b; C: Quantitative analysis of p-AKT (**p < 0.01, ***p < 0.001, ****p < 0.0001); D: Quantitative analysis of HSPa1b (**p < 0.01, ***p < 0.001, ****p < 0.0001),

[0036] Figure 8 A: Western blot of exosome marker proteins TSG101, CD63, and CD81; B: Transmission electron micrograph of exosomes; C: Exosome particle size analysis; D: Western blot of MMP-9 and TIMP-1 in exosomes; E: Quantitative analysis of exosomal MMP-9; F: Quantitative analysis of exosomal TIMP-1; G: Quantitative analysis of the MMP-9 / TIMP-1 ratio (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001),

[0037] Figure 9 A: Schematic diagram of the Transwell system and Dil staining; B: Fluorescent staining of TEC phagocytosing Dil-labeled exosomes (200× under confocal microscope, Dil is red fluorescence),

[0038] Figure 10A: Western blot images of macrophage TREM-2, HSPa1b, and p-AKT; B: Western blot images of TEC collagenⅠ and α-SMA; C: Quantitative graph of collagenⅠ; D: Quantitative graph of α-SMA; E: Quantitative graph of TREM-2 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001); F: Quantitative graph of p-AKT (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001); G: Quantitative graph of HSPa1b (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001),

[0039] Figure 11 Fluorescence staining images of renal tubular epithelial cells after co-culture with macrophages for 24 h (photographed by confocal microscope at 400×). CollagenⅠ shows red fluorescence, and DAPI-stained cell nuclei show blue fluorescence,

[0040] Figure 12 Fluorescence staining images of renal tubular epithelial cells after co-culture with macrophages for 24 h (photographed by confocal microscope at 400×). α-SMA shows green fluorescence, and DAPI-stained cell nuclei show blue fluorescence,

[0041] Figure 13 GFP fluorescence images of renal tissues after injection of AAV-shTREM-2 into the renal pelvis,

[0042] Figure 14 HE staining images of the kidney (photographed under the microscope at 200×, scale bar 20 μm),

[0043] Figure 15 A: Pathological images of Masson trichrome staining of the kidney; B: Quantitative graph of the fibrotic area stained by Masson (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001),

[0044] Figure 16 A: Western blot images of TREM-2, HSPa1b, and p-AKT in the kidney; B: Western blot images of CollagenⅠ and α-SMA in the kidney; C: Quantitative graph of collagenⅠ; D: Quantitative graph of α-SMA; E: Quantitative graph of TREM-2 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001); F: Quantitative graph of HSP70 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001); G: Quantitative graph of p-AKT (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001),

[0045] Figure 17 Visualized collagen deposition and cell structure diagram, photographed by confocal microscope at 200× (scale bar 50 μm), collagen Ⅰ red fluorescence, DAPI stained cell nuclei blue,

[0046] Figure 18 Visualized collagen deposition and cell structure diagram, photographed by confocal microscope at 200× (scale bar 50 μm), α-SMA green fluorescence, DAPI stained cell nuclei blue,

[0047] Figure 19 A: HE staining and Masson staining of kidney (photographed under microscope at 200×); B: Quantitative analysis of fibrosis area by Masson staining (****p < 0.0001); C: Coomassie brilliant blue staining after polyclonal antibody protein electrophoresis.

[0048] Figure 20 A: Western blot images of TREM-2, collagen Ⅰ, α-SMA in kidney tissues; B, C, D: Quantitative analysis of TREM-2, collagen Ⅰ, α-SMA (**p < 0.01, ***p < 0.001, ****p < 0.0001).

[0049] Figure 21 , visualized collagen deposition and cell structure diagram. Detailed implementation manners

[0050] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0051] Example 1

[0052] 1. Design and synthesis of proteins

[0053] (1) The expression of TREM-2 in kidney macrophages of patients with kidney fibrosis is increased.

[0054] Kidney tissue sections of patients with kidney fibrosis were obtained by renal puncture biopsy. Immunofluorescence staining showed that compared with patients with no-mild kidney fibrosis (fibrosis area < 25%), the expression of macrophage TREM-2 in the kidneys of patients with moderate (fibrosis area 25%-50%) and severe fibrosis (fibrosis area > 50%) was significantly increased (as Figure 1 shown).

[0055] (2) The expression of TREM-2 is increased in the kidneys of wild-type (WT) mice with unilateral ureteral obstruction (UUO). A mouse UUO model was constructed, and kidney tissue samples were taken 1 week and 2 weeks after modeling. The results of fluorescence quantitative PCR (RT-PCR) and Western blot (WB) experiments showed that the expression levels of TREM-2 mRNA and protein in kidney tissues were significantly increased 1 week and 2 weeks after UUO (as Figure 2 shown).

[0056] (3) TGF-β1 can induce the upregulation of TREM-2 expression in mouse macrophages.

[0057] Mouse peritoneal macrophages were induced with murine TGF-β1 (15 ng / mL, intervened for 24 hours), and the protein expression level of TREM-2 in macrophages was significantly increased. The protein expression level of TREM-2 in macrophages of TREM-2 gene knockout (TREM-2- / -) mice did not increase significantly after TGF-β1 intervention (as Figure 3 shown).

[0058] (4) Changes in the expression levels and ratios of MMP-9 and TIMP-1 in exosomes derived from macrophages of TREM-2- / - mice. Exosomes in the macrophage culture supernatant were collected by ultracentrifugation. The expression levels of exosome marker proteins such as CD63, CD81, and TSG101 were detected by Western blotting, and the concentration and particle size distribution range of exosomes after centrifugation were detected using a nanoparticle tracking analyzer. The results showed that 80% of the TIMP-1 exosomes had a particle size distribution between 30 - 150 nm. Finally, transmission electron microscopy observed vesicle structures close to circular in shape in the exosome dilution (as Figure 4 shown). The expression levels of MMP-9 in exosomes derived from wild-type (WT) macrophages (exo-WT+TGF-β1) and exosomes derived from TREM-2- / - macrophages (exo-TREM-2- / -+TGF-β1) after TGF-β1 intervention were significantly upregulated compared to their respective control groups. Moreover, the expression level of TIMP-1 in the exo-WT+TGF-β1 group was significantly upregulated compared to the WT control group, while the expression level of TIMP-1 in the exo-TREM-2- / -+TGF-β1 group was significantly lower than that in the exo-WT+TGF-β1 group. Further calculating the ratio of MMP-9 / TIMP-1, the ratio in the exo-WT+TGF-β1 group was lower than that in the exo-WT control group. The ratio in the exo-TREM-2- / -+TGF-β1 group was higher than that in the TREM-2- / - control group and was significantly higher than that in the exo-WT+TGF-β1 group (as Figure 5 shown).

[0059] (5)RNA sequencing analysis of macrophages from WT mice and TREM-2- / - mice after TGF-β1 intervention. The RNA sequencing results showed that compared with the WT+TGF-β1 group, 4004 genes were significantly up-regulated, 7512 genes were significantly down-regulated, and 9916 genes had no significant difference in expression in the TREM-2- / -+TGF-β1 group. The top 5 differentially expressed genes were: heat shock protein a1b (HSPa1b), heat shock protein a1a (HSPa1a), H2-Q6 protein, H2-Q7 protein, and Basign protein. KEGG enrichment analysis found that HSPa1b could up-regulate the DNA level of MMPs through the Toll-like receptor signaling pathway, and AKT could up-regulate the DNA level of MMPs through AP1. Then, Western blotting was used to verify the RNA sequencing results. The results showed that the expressions of HSPa1b and p-AKT in the TREM-2- / - macrophage+TGF-β1 group were significantly higher than those in the WT macrophage+TGF-β1 group, which was consistent with the sequencing results (as Figure 6 shown).

[0060] (6) TREM-2- / - macrophages regulate the expression levels of exosomal MMP-9 and TIMP-1 through HSPa1b / AKT. siRNA-HSPa1b and Ly294002 were used to inhibit the expression of HSPa1b and p-AKT in TREM-2- / - macrophages respectively, and WB was used to detect the inhibitory effect. After siRNA-HSPa1b transfection into TREM-2- / - macrophages, the rt-PCR results showed that the mRNA level of HSPa1b was significantly decreased. WB was used to detect the expression levels of HSPa1b and p-Akt in the control group of TREM-2- / - macrophages, the TREM-2- / - macrophage group intervened by TGF-β1 (TREM-2- / - macrophages + TGF-β1), and the TREM-2- / - macrophages + TGFβ1 intervened by siRNA and Ly294002 respectively (TREM-2- / - + TGF-β1 + siRNA-HSPa1b group, TREM-2- / - + TGF-β1 + Ly2940024 group). Compared with the control group of TREM-2- / - macrophages, the expression levels of HSPa1b and p-AKT in the TREM-2- / - macrophage + TGF-β1 group were significantly up-regulated, and the expression level of HSPa1b in the TREM-2- / - + TGFβ1 + siRNA-HSPa1b group and the expression level of p-AKT in the TREM-2- / - + TGF-β1 + Ly2940024 group were significantly decreased. This indicated that siRNA-HSPa1b and Ly294002 effectively inhibited the expression of HSPa1b and p-AKT in TREM-2- / - macrophages (as Figure 7 shown).

[0061] Further detect the expression levels of MMP-9 and TIMP-1 in the above four groups of macrophage-derived exosomes. First, after identifying exosomes using WB, transmission electron microscopy, and nanoparticle tracking analyzer, then use WB to detect the exosomes of the TREM-2- / - macrophage control group (exo-TREM-2- / - macrophage control group), the exosomes of the TREM-2- / - macrophage + TGF-β1 group (exo-TREM-2- / - macrophage + TGF-β1), and the exosomes of the TREM-2- / - macrophage group after intervention with siRNA and Ly294002 (exo-TREM-2- / - macrophage + TGF-β1 + siRNA-HSPa1b, exo-TREM-2- / - macrophage + TGF-β1 + Ly2940024) for the expression levels of MMP-9 and TIMP-1. The expression level of MMP-9 in the exo-TREM-2- / - macrophage + TGF-β1 group was significantly higher than that in the exo-TREM-2- / - macrophage control group. After intervention with siRNA-HSPa1b and Ly294002, the expression level of MMP-9 in TGF-β1-intervened TREM-2- / - macrophages was significantly downregulated. Therefore, the expression of MMP-9 decreased after inhibiting the expression of HSPa1b and AKT in TREM-2- / - macrophages; there was no significant difference in the expression level of TIMP-1 protein in the exo-TREM-2- / - macrophage + TGF-β1 group compared with the exo-TREM-2- / - macrophage control group. However, after intervention with siRNA-HSPa1b and Ly294002, the expression level of TIMP-1 in TREM-2- / - macrophages was significantly upregulated. Therefore, after inhibiting the expression of HSPa1b and AKT in TREM-2- / - macrophages, the expression of TIMP-1 increased. Calculate the MMP-9 / TIMP-1 ratio in the exosomes of the four groups: the ratio in the exo-TREM-2- / - macrophage + TGF-β1 group was higher than that in the exo-TREM-2- / - macrophage control group, while the MMP-9 / TIMP-1 ratios in the exosomes of the TREM-2- / - macrophage + TGF-β1 group after intervention with siRNA-HSPa1b and Ly294002 were significantly decreased compared with the exo-TREM-2- / - macrophage + TGF-β1 group. It shows that the MMP-9 / TIMP-1 ratio of TREM-2- / - + TGF-1 decreased after inhibiting HSPa1b and AKT, and the ECM regulation point shifted towards the synthesis direction (as Figure 8 shown).

[0062] (7) Exosomes derived from TREM-2- / - macrophages inhibit TEC fibrosis

[0063] Macrophages were seeded in the upper chamber of a transwell co-culture system, and the macrophage cell membrane was stained with Dil dye. Then, the transwell insert was transferred into a 6-well plate seeded with renal tubular epithelial cells (TECs). After 24 hours of co-culture, confocal microscopy was used to observe and capture that the TECs in the lower chamber phagocytosed Dil-labeled exosomes derived from macrophages in the upper chamber, emitting red fluorescence signals (as Figure 9 shown).

[0064] After the TECs in the lower chamber were co-cultured with the WT macrophage control group, the TREM-2 - / - macrophage control group, and the co-culture of TREM-2 - / - macrophages and WT macrophages intervened with TGF-β1 for 24 hours, Western blotting was used to detect the expression of collagen Ⅰ and α-SMA in the TECs in the lower chamber. No fibrosis occurred in the TECs co-cultured with the WT macrophage control group and the TREM-2 - / - macrophage control group, and collagen Ⅰ and α-SMA were hardly expressed. However, obvious fibrosis occurred in the TECs co-cultured with the WT macrophages intervened with TGF-β1, and the expression of collagen Ⅰ and α-SMA was significantly upregulated. However, fibrosis in the TECs was significantly inhibited in the co-culture system with the TREM-2 - / - macrophages intervened with TGF-β1, and the expression of collagen Ⅰ and α-SMA was significantly decreased. In addition, the results of immunofluorescence staining of collagen Ⅰ and α-SMA in renal tubular epithelial cells were consistent with the Western blotting results. The fluorescence intensities of collagen Ⅰ and α-SMA were both weakened in the co-culture system of TECs with the TREM-2 - / - macrophages intervened with TGF-β1 (as Figure 10 , Figure 11 , Figure 12 shown).

[0065] (8) GW4869 (an exosome inhibitor) blocked the inhibitory effect of TREM-2 - / - macrophages on TEC fibrosis

[0066] After pre - treating the upper chamber TREM - 2 - / - macrophages with GW4869 (an exosome inhibitor), and then intervening with TGF - β1, co - culture them with the TEC in the lower chamber for 24 h. Then use WB to detect the expression levels of collagen Ⅰ and α - SMA. After the exosome inhibitor intervenes with TREM - 2 - / - macrophages, it cannot effectively inhibit the fibrosis of TEC in the TGF - β1 co - culture environment, and the expressions of collagen Ⅰ and α - SMA are significantly up - regulated. And the immunofluorescence staining of collagen Ⅰ and α - SMA is also similar to the Western blot results. After intervening with TREM - 2 - / - macrophages with GW4869, in the TGF - β1 co - culture environment, the fluorescence intensity of TEC collagen Ⅰ and α - SMA is significantly enhanced. This indicates that after using the exosome inhibitor GW4869, the inhibitory effect of TREM - 2 - / - macrophages on TEC fibrosis is blocked (as Figure 10 、 Figure 11 、 Figure 12 shown).

[0067] (9) siRNA - HSPa1b and Ly294002 reversed the inhibitory effect of TREM - 2 - / - macrophage exosomes on TEC fibrosis

[0068] Pre - treat TREM - 2 - / - macrophages with siRNA - HSPa1b and Ly294002, then intervene with TGF - β1, and then co - culture with TEC for 24 h. Use WB to detect the expressions of TEC collagen Ⅰ and α - SMA. After siRNA - HSPa1b and Ly294002 intervene with TREM - 2 - / - macrophages, they cannot effectively inhibit the fibrosis of TEC in the TGF - β1 co - culture environment, and the expressions of collagen Ⅰ and α - SMA are significantly up - regulated. The immunofluorescence staining of TEC collagen Ⅰ and α - SMA is also consistent with the WB results. After siRNA - HSPa1b and Ly294002 intervene with TREM - 2 - / - macrophages, in the TGF - β1 co - culture environment, the fluorescence intensity of TEC collagen Ⅰ and α - SMA is significantly enhanced (as Figure 10 、 Figure 11 、 Figure 12 shown).

[0069] On the basis of siRNA-HSPa1b and Ly294002 intervention, GW4869 was used to intervene in the upper chamber TREM-2- / - macrophages. In the co-culture environment of TEC with TGF-β1, TREM-2- / - macrophages could not effectively inhibit the fibrosis of TEC, and the expressions of collagen Ⅰ and α-SMA in TEC were significantly up-regulated. The results of immunofluorescence were also consistent with those of WB. After siRNA-HSPa1b, Ly294002, and GW4869 intervened in the upper chamber TREM-2- / - macrophages, in the co-culture system of TEC with TGF-β1, the fluorescence intensities of collagen Ⅰ and α-SMA in TEC were significantly enhanced. As described above, after inhibiting the expressions of HSPa1b and AKT in TREM-2- / - macrophages and blocking the secretion of exosomes, the inhibitory effect of TREM-2- / - macrophages on TEC fibrosis was reversed (as Figure 10 、 Figure 11 、 Figure 12 shown).

[0070] Finally, WB was used to detect the expression levels of proteins in the TREM-2, p-AKT, and HSPa1b pathways of the upper chamber macrophages. TGF-β1 induced an up-regulation of the expression of TREM-2 in WT macrophages, while TREM-2 was hardly expressed in TREM-2- / - macrophages. Compared with WT macrophages, the expressions of HSPa1b and p-AKT in TREM-2- / - macrophages after TGF-β1 intervention were significantly up-regulated. Secondly, after siRNA-HSPa1b and Ly294002 intervened in TREM-2- / - macrophages, the expressions of HSPa1b and p-AKT in TREM-2- / - macrophages were significantly down-regulated. In summary, TREM-2- / - macrophages inhibit the fibrosis of TEC by up-regulating the ratio of MMP-9 / TIMP-1 in their exosomes through the HSPa1b / AKT pathway (as Figure 10 shown).

[0071] (10)AAV-shTREM-2 decreased the expressions of Collagen Ⅰ and α-SMA in the kidneys of UUO mice

[0072] Adeno-associated virus (AAV) vectors (AAV-shTREM-2-eGFP, AAV-shTREM-2) were injected into the renal pelvis. The transfection of AAV was evaluated by observing the fluorescence signal of GFP in the frozen sections of renal tissue with a confocal microscope. Starting from the 3rd day after AAV injection into the renal pelvis, unevenly distributed green strip fluorescence could be observed in the renal tubules, indicating successful local transfection of AAV in the kidneys. By the 5th and 7th days, green fluorescence could be seen evenly distributed in the renal tubules, showing an approximately oval green fluorescence. It shows that the transfection effect of AAV is obvious (asFigure 13 as shown

[0073] The animal models were grouped as follows: normal control group, UUO model group, UUO model group injected with AAV empty vector (AAV-NC) into the renal pelvis (UUO + AAV-NC), and UUO model group injected with AAV-shTREM-2 into the renal pelvis (UUO + AAV-shTREM-2). HE staining results showed that compared with the normal control group, there were a large number of inflammatory cell infiltrations in the renal interstitium of the UUO group and the UUO + AAV-NC group, and atrophy of the morphology of a large area of renal tubular epithelial cells was visible, with the renal tubular lumen narrowed or dilated, showing severe kidney injury and structural damage. However, the infiltration of inflammatory cells in the UUO + AAV-shTREM-2 group was significantly reduced, and kidney injury and structural damage were significantly alleviated. Masson trichrome staining results showed that compared with the normal control group, a large amount of blue-stained collagen was deposited in the kidney tissues and around blood vessels of UUO mice and UUO + AAV-NC mice, while the collagen deposited in the kidneys and around blood vessels of mice in the UUO + AAV-shTREM-2 group was significantly reduced (as Figure 14 , Figure 15 shown

[0074] To further verify that AAV-shTREM-2 inhibits renal fibrosis in UUO mice, WB was used to detect the expression of collagenⅠ and α-SMA in the kidneys of mice. Compared with the normal control group, the expression levels of collagenⅠ and α-SMA in the kidneys of the UUO group and the UUO + AAV-NC group were significantly up-regulated, while the expression levels of collagenⅠ and α-SMA in UUO mice injected with AAV-shTREM-2 into the renal pelvis were significantly down-regulated compared with the UUO group and the UUO + AAV-NC group (as Figure 16 shown

[0075] The fluorescence intensities of collagenⅠ (red fluorescence) and α-SMA (green fluorescence) in the UUO group and the UUO + AAV-NC group were significantly higher than those in the normal control group, while the fluorescence intensities of collagenⅠ and α-SMA in UUO mice injected with AAV-shTREM-2 into the renal pelvis were significantly decreased. The above results indicate that AAV-shTREM-2 can significantly inhibit renal fibrosis in UUO mice (as Figure 17 , Figure 18 shown

[0076] (11) VER-155008 and ly294002 reversed the effect of AAV-shTREM-2 on inhibiting renal fibrosis

[0077] To further verify that TREM-2- / - macrophages inhibit the progression of renal fibrosis in UUO mice through the HSPa1b / AKT pathway, after injecting AAV-shTREM-2 into the renal pelvis of UUO mice, VER-155008 (HSP70 inhibitor, 16 mg / Kg, UUO+AAV-shTREM-2+VER-155008 group) and Ly294002 (10 uM / Kg, UUO+AAV-shTREM-2+Ly294002 group) were intraperitoneally injected. HE staining results showed that after intraperitoneal injection of VER-155008 and Ly294002, there were a large number of inflammatory cell infiltrations in the renal interstitium of UUO mice injected with AAV-shTREM-2 in the renal pelvis, and extensive atrophy of renal tubular epithelial cells occurred, with the renal tubular lumen narrowed or dilated, showing severe renal injury. Masson trichrome staining results showed that compared with the UUO+AAV-shTREM-2 group, the UUO+AAV-shTREM-2+VER-155008 group and the UUO+AAV-shTREM-2+Ly294002 group showed a large amount of blue collagen tissue deposition around the renal tissue and blood vessels (as Figure 14 , Figure 15 shown).

[0078] To further verify that VER-155008 and Ly294002 reversed the inhibitory effect of AAV-shTREM-2 on UUO-induced renal fibrosis, WB was used to detect the expression of collagenⅠ and α-SMA in the two groups of UUO+AAV-shTREM-2+VER-155008 and UUO+AAV-shTREM-2+Ly294002. Compared with UUO mice injected with AAV-shTREM-2 in the renal pelvis, the expression of collagenⅠ and α-SMA in the kidneys of UUO+AAV-shTREM-2 mice after administration of VER-155008 and Ly294002 was significantly up-regulated (as Figure 16 shown).

[0079] The fluorescence intensities of collagenⅠ (red fluorescence) and α-SMA (green fluorescence) in the kidneys of UUO+AAV-shTREM-2 mice after administration of VER-155008 and Ly294002 were significantly enhanced. Therefore, the above experimental results all showed that VER-155008 and Ly294002 reversed the inhibitory effect of AAV-shTREM-2 on the progression of renal fibrosis (as Figure 17 , Figure 18 shown).

[0080] Finally, Western blotting was used to detect the protein expression levels of TREM-2, p-AKT, and HSPa1b in the kidneys of the above-grouped mice. Compared with the control group, the expression level of TREM-2 was significantly upregulated in the kidneys of UUO mice and UUO+AAV-NC mice, and it was hardly expressed in the kidneys of UUO mice injected with AAV-shTREM-2 into the renal pelvis, indicating that AAV-shTREM-2 effectively inhibited the expression of TREM-2. The protein expression levels of HSPa1b and p-AKT in the kidneys of UUO mice injected with AAV-shTREM-2 into the renal pelvis were significantly higher than those of the control group, and slightly higher than those of the UUO group and UUO+AAV-NC group. Finally, the expression levels of HSPa1b and p-AKT in UUO+AAV-shTREM-2 mice intraperitoneally injected with VER-155008 and Ly294002 were significantly decreased. Therefore, VER-155008 and Ly294002 effectively inhibited the expression of HSPa1b and p-AKT in the kidneys of mice( Figure 16 as shown).

[0081] Example 2 Synthesis of Polyclonal Antibody

[0082] (1) Protein Sequence Analysis

[0083] The mRNA sequence of TREM-2 (NCBI Reference Sequence: NM_031254.3) is as follows:

[0084] ATGGGACCTCTCCACCAGTTTCTCCTGCTGCTGATCACAGCCCTGTCCCAAGCCCTCAA

[0085] CACCACGGTGCTGCAGGGCATGGCCGGCCAGTCCTTGAGGGTGTCATGTACTTATGACG

[0086] CCTTGAAGCACTGGGGGAGACGCAAGGCCTGGTGTCGGCAGCTGGGTGAGGAGGGCC

[0087] CATGCCAGCGTGTGGTGAGCACACACGGTGTGTGGCTGCTGGCCTTCCTGAAGAAGCG

[0088] GAATGGGAGCACAGTCATCGCAGATGACACCCTTGCTGGAACCGTCACCATCACTCTGA

[0089] AGAACCTCCAAGCCGGTGACGCGGGCCTCTACCAGTGTCAGAGTCTCCGAGGCCGAGA

[0090] GGCTGAGGTCCTGCAGAAAGTACTGGTGGAGGTGCTGGAGGACCCTCTAGATGACCAA

[0091] GATGCTGGAGATCTCTGGGTCCCCGAGGAGTCATCGAGTTTCGAGGGTGCCCAAGTGGA

[0092] ACACAGCACCTCCAGGAATCAAGAGACCTCCTTCCCACCCACCTCCATTCTTCTCCTCCT

[0093] GGCCTGCGTTCTCCTGAGCAAGTTTCTTGCAGCCAGCATCCTCTGGGCTGTGGCCAGGG

[0094] GCAGGCAGAAGCCGGGAACACCTGTGGTCAGAGGGCTGGACTGTGGCCAAGATGCTGGGCACCAACTTCAGATCCTCACTGGACCCGGAGGTACGTGA。(SEQ ID NO.1)

[0095] The amino acid sequence is:

[0096] MGPLHQFLLLLITALSQALNTTVLQGMAGQSLRVSCTYDALKHWGRRKAWCRQLGEEGP

[0097] CQRVVSTHGVWLLAFLKKRNGSTVIADDTLAGTVTITLKNLQAGDAGLYQCQSLRGREAE

[0098] VLQKVLVEVLEDPLDDQDAGDLWVPEESSSFEGAQVEHSTSRNQETSFPPTSILLLLACVLLSKFLAASILWAVARGRQKPGTPVVRGLDCGQDAGHQLQILTGPGGT。(SEQ ID NO.2) Based on antigenicity prediction and analysis of exposed sites, the following two polypeptides were selected to prepare polyclonal antibodies: HSTSRNQETSFP-C (SEQ ID NO.3)

[0099] LEDPLDDQDAGD-C (SEQ ID NO.4).

[0100] (2) Animal immunization

[0101] Using the polypeptide as an antigen, immunize New Zealand white rabbits (2 - 2.5 kg), immunize subcutaneously at 400 μg per time, and immunize once every 2 - 3 weeks. Collect blood for detection, and determine the titer of the antiserum by the indirect ELISA method. Wait until the titer is greater than 1:50,000 for final blood collection to prepare the antiserum and prepare for purification.

[0102] (3) Antibody purification

[0103] Couple the polypeptide with an agarose medium to prepare an antigen affinity purification chromatography column. Mix the obtained antiserum with PBS in equal amounts and slowly load the sample. After the antibody binds, elute with a glycine elution buffer to obtain the required purified antibody. Immediately perform dialysis overnight at 4°C in PBS, and measure the purity, concentration, and titer the next day.

[0104] (4) Detect the titer of the purified antibody by ELISA, measure the concentration of the obtained antibody using a BCA concentration assay kit; observe the purity of the purified antibody by SDS - PAGE electrophoresis and Coomassie brilliant blue staining.

[0105] (5) Indirect ELISA titer detection of the purified antibody

[0106] Design a coated plate according to the experimental needs and make marks on the strips.

[0107] Dilute the protein / polypeptide antigen with PBS coating solution to the required concentration, mix well and add it to the strips, 100 μL per well, and incubate overnight at 4°C in a refrigerator. Coating concentration: 5 μg / mL, 100 μL / well, coating buffer: phosphate buffer (PBS, pH 7.4). After coating, discard the coating solution, wash the plate 3 times, add 200 μL of blocking solution to each well, and incubate in a 37°C incubator for 1 h. Take out the enzyme - labeled plate, discard the internal solution, and wash the plate 1 time.

[0108] Dilute the purified antibody 3 - fold at 1:500, 100 μL per well, and incubate in a 37°C incubator for 1 h.

[0109] Take out the enzyme - labeled plate, discard the internal solution, wash the plate 3 times, add 100 μL of diluted enzyme - labeled secondary antibody to each well, enzyme - labeled secondary antibody: goat anti - rabbit - HRP, 1:50000. Incubate in a 37°C incubator for 1 h.

[0110] ⑥ Take out the enzyme - labeled plate, discard the internal solution, wash the plate 4 times, add 100 μL of TMB chromogenic solution to each well first, and determine the chromogenic time according to the depth of the color. Generally, at 37°C for 15 min.

[0111] ⑦ Add 100 μL of 1M HCl solution to each well to terminate the reaction. Immediately read the value at 450 nm on an enzyme - labeled instrument, and the OD value is greater than

[0112]

[0113] The dilution corresponding to the well with an OD value 2.1 times that of the set negative control is defined as the titer of the sample.

[0114] Protein gel electrophoresis and Coomassie brilliant blue staining showed that the antibody purity met the requirements of subsequent experiments ( Figure 19 C, the middle lane is the target antibody). The titer of the prepared polyclonal antibody was not less than 3280000 (Table 1, using polypeptide SEQ ID NO.3 as the antigen).

[0115] Table 1. Detection of antibody titer gradient

[0116] 3. Experimental procedures for verifying the therapeutic effect on mouse cells or animal models

[0117] (1) Starting from the 14th day after UUO modeling, polyclonal antibody against TREM-2 was intraperitoneally injected into mice. The results of HE staining of the kidneys showed that compared with the normal control group, a large number of inflammatory cells infiltrated the renal interstitium of mice in the UUO group, and there were varying degrees of atrophy and edema of renal tubular epithelial cells, with the lumen narrowed or dilated, showing severe kidney damage. In the UUO mice intraperitoneally injected with polyclonal antibody against TREM-2 (UUO+TREM-2 pAb), the infiltrating inflammatory cells in the renal interstitium were significantly reduced, and the kidney damage was significantly alleviated.

[0118] The results of Masson trichrome staining showed that compared with the control group, a large amount of blue-stained collagen was deposited in the renal interstitium of UUO mice, with severe fibrosis, while the deposition of blue-stained collagen in the UUO+TREM-2 mab group was significantly reduced ( Figure 19 ).

[0119] (2) Further, WB (western blot) was used to detect the expression levels of collagen Ⅰ and α-SMA in the kidneys of mice. The expression levels of collagen Ⅰ and α-SMA in the UUO group were significantly up-regulated compared with the control group, while the expression levels of collagen Ⅰ and α-SMA in the mice injected with polyclonal antibody against TREM-2 were significantly down-regulated compared with the UUO group. The expression level of TREM-2 in UUO mice was significantly higher than that in the control group, and the expression of TREM-2 in the UUO mice injected with polyclonal antibody against TREM-2 was significantly reduced. Therefore, the polyclonal antibody against TREM-2 effectively inhibited the expression of TREM-2 in the kidneys and the progression of fibrosis ( Figure 20 ).

[0120] (3) Immunofluorescence staining results of renal tissue collagen I and α-SMA showed that the fluorescence intensities of collagen I and α-SMA in the UUO group were significantly higher than those in the control group, while the fluorescence intensities of collagen I and α-SMA in the UUO + TREM-2 pAb group were significantly weaker than those in the UUO group. Therefore, the above results all indicate that the polyclonal antibody against TREM-2 effectively inhibits UUO-induced renal interstitial fibrosis( Figure 21 ).

Claims

1. Use of a polyclonal antibody against myeloid cell triggering receptor 2 protein in the preparation of a reagent for inhibiting renal fibrosis, wherein the mRNA sequence of the myeloid cell triggering receptor 2 is as shown in SEQ ID NO.1, and the amino acid sequence is as shown in SEQ ID NO.

2.

2. A drug for inhibiting renal fibrosis reaction, characterized in that, The drug includes a substance that specifically inhibits the expression of myeloid cell triggering receptor 2.

3. A polyclonal antibody against myeloid cell triggering receptor 2 protein, characterized in that, The antibody is obtained by immunizing with a polypeptide having a sequence such as SEQ ID NO.3 and / or SEQ ID NO.4 as an antigen.

4. A method for preparing a polyclonal antibody against myeloid cell triggering receptor 2 protein, characterized in that, It includes the following steps: S1: Using the polypeptide as an antigen, after subcutaneous immunization of New Zealand white rabbits, blood is collected for detection to determine the titer of the antiserum. Final blood collection is performed to prepare the antiserum when the titer is greater than 1:50000; the polypeptide sequences are as shown in SEQ ID NO.3 and SEQ ID NO.

4. S2: Coupling the polypeptide with an agarose medium to prepare an antigen affinity purification chromatography column. The obtained antiserum is mixed with PBS in equal amounts and slowly loaded. After the antibody binds, it is eluted with a glycine elution buffer to obtain the required purified antibody. The obtained purified antibody is dialyzed overnight in PBS, and the purity, concentration, and titer are measured the next day. S3: Detect the titer of the purified antibody by ELISA, and measure the concentration of the obtained antibody using a BCA concentration assay kit. Observe the purity of the purified antibody by SDS-PAGE electrophoresis and Coomassie brilliant blue staining. S4: Indirect ELISA titer detection of the purified antibody.

5. The preparation method of a myeloid cell triggering receptor 2 protein polyclonal antibody according to claim 4, characterized in that, In step S1, the weight of the New Zealand white rabbit is 2 - 2.5 kg, the subcutaneous immunization dose is 350 - 500 μg / time, immunization is performed once every 2 - 3 weeks, and the titer of the antiserum is determined by the indirect ELISA method.

6. The preparation method of a myeloid cell triggering receptor 2 protein polyclonal antibody according to claim 4, characterized in that, In step S2, the temperature for dialysis overnight in PBS is 3 - 5 °C.

7. A method for preparing a polyclonal antibody against myeloid cell triggering receptor 2 (TREM-2) protein according to claim 4, characterized in that, The specific method of step S4 is as follows: ① Design a coated plate according to experimental needs and mark it on the strip. ② Dilute the protein / polypeptide antigen with a PBS coating solution to the required concentration, mix well and add it to the strip, 80 - 120 μL per well, and incubate overnight in a refrigerator at 3 - 5 °C. ③ After coating, discard the coating solution, wash the plate 3 times, add 200 μL of blocking solution to each well, and incubate in an incubator at 37 °C for 1 h. Take out the enzyme-linked immunosorbent assay (ELISA) plate, discard the internal solution, and wash the plate once. ④ Dilute the purified antibody 3-fold at 1:500, 100 μL per well, and incubate in an incubator at 37 °C for 1 h. ⑤ Take out the ELISA plate, discard the internal solution, wash the plate 3 times, add 100 μL of diluted enzyme-labeled secondary antibody to each well. The enzyme-labeled secondary antibody: goat anti-rabbit - HRP, 1:50000, and incubate in an incubator at 37 °C for 1 h. ⑥ Take out the ELISA plate, discard the internal solution, wash the plate 4 times, add 100 μL of TMB chromogenic solution to each well first, and determine the chromogenic time according to the color depth, generally at 37 °C for 15 min. ⑦ Add 100 μL of 1M HCl solution to each well to terminate the reaction. Immediately read the absorbance at 450 nm on an ELISA reader, and define the dilution corresponding to the well with an OD value greater than 2.1 times the OD value of the set negative control as the titer of the sample.

8. A method for preparing a polyclonal antibody against myeloid cell triggering receptor 2 (TREM-2) protein according to claim 4, characterized in that, The coating concentration is: 4 - 6 μg / mL, 80 - 120 μL / well, and the coating buffer: phosphate buffer (PBS, pH 7.4).

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