Glycosylation modification site of secreting type RCN3 protein and application of glycosylation modification site

Abstract

The invention relates to the technical field of biological medicines, in particular to a glycosylation modification site of a secreting type RCN3 protein and application of the glycosylation modification site, in particular to application of an Asn140 site of the RCN3 protein as a target in diagnosis and / or prevention and treatment of pulmonary fibrosis and application of the Asn140 site of the secreting type RCN3 protein as a target in prognosis of acute respiratory distress syndrome. According to the invention, the secretory Rcn3 protein can interact with TGF [beta] R1, activate a classical TGF [beta] signal channel, and mediate lung epithelial cell-fibroblast communication to regulate and control activation and functions of fibroblasts; besides, a glycosylation modification site, namely the Asn140 site, which plays an important role in secretion of the RCN3 protein is found, a medicine for preventing and treating pulmonary fibrosis can be developed aiming at the target, and the Asn140 site has great clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to glycosylation modification sites of secretory RCN3 protein and applications thereof. Background Art

[0002] ARDS is acute respiratory distress syndrome, which is mainly caused by increased permeability of the lung endothelium and epithelium. Its pathological process is complex and is divided into an acute inflammatory phase and a chronic repair phase. So far, a large number of studies have focused on the pathogenic mechanism of the acute phase of ARDS. However, with the improvement of treatment methods, more and more patients enter the repair phase, so the occurrence of pulmonary fibrosis in the repair phase has become an important factor in the prognosis of ARDS. Studies have shown that chronic inflammation and pulmonary fibrosis are the main causes of morbidity and mortality in chronic phase ARDS patients.

[0003] Reticulocalbin 3 (RCN3) is an endoplasmic reticulum (ER) lumen protein localized in the secretory pathway and belongs to the Ca 2+ -binding protein of 45kDa(Cab45) / Rcn / ER Ca 2+ -binding protein of 55kDa (ERC45) / Calumenin family. RCN3 is widely expressed in human tissues and plays an important role in the development of various organs and physiological diseases. In recent years, with the study of the biological characteristics of RCN3 and its role in various physiological metabolic processes and cardiovascular and cerebrovascular diseases, as well as tumor diseases, it has been found that intracellular RCN3 plays an important regulatory role and clinical significance, especially in the occurrence and development of lung injury and repair diseases; however, there is currently no functional research on secretory RCN3 protein. As a cytokine secreted by alveolar epithelial cells, secretory protein plays an important regulatory role in the occurrence and development of lung injury and repair diseases.

[0004] Glycosylation is one of the most common post-translational modifications of proteins. Protein glycosylation is mainly divided into four types: N-glycosylation, O-glycosylation, glycosylated phosphatidylinositol-anchored protein linkage and other glycosylation. N-glycosylation is the connection of sugar chains to the amide in the asparagine side chain on newly synthesized protein polypeptides. Protein glycosylation can affect the properties of proteins and has the function of regulating protein functions. After glycosylation modification, the thermal stability, biological activity, enzyme stability, conformational stability and solubility of proteins will change significantly. Protein glycosylation is also closely related to the occurrence and development of many diseases. However, there are currently no reports on whether RCN3 protein is glycosylated, and there are no reports on the use of glycosylation modification sites of RCN3 protein as therapeutic targets related to pulmonary fibrosis.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide glycosylation modification sites of secretory RCN3 protein and their application in pulmonary fibrosis and acute respiratory distress syndrome.

[0007] In a first aspect of the present invention, a glycosylation modification site of a secretory RCN3 protein is provided, wherein the glycosylation modification site of the secretory RCN3 protein is the Asn140 site.

[0008] Specifically, the secreted Rcn3 protein can interact with TGFβR1, activate the classical TGFβ signaling pathway, mediate lung epithelial cell-fibroblast communication and regulate the activation and function of fibroblasts, while the Asn140 site is a glycosylation modification site that plays an important role in the secretion of RCN3 protein.

[0009] The second aspect of the present invention provides the application of the glycosylation modification site of the secretory RCN3 protein, wherein the application is the application of the Asn140 site of the secretory RCN3 protein as a target in the diagnosis and / or prevention and treatment of pulmonary fibrosis.

[0010] The third aspect of the present invention provides the application of the glycosylation modification site of the secretory RCN3 protein, wherein the application is the application of the Asn140 site of the secretory RCN3 protein as a target in the prognosis diagnosis of acute respiratory distress syndrome.

[0011] The fourth aspect of the present invention provides an application of a glycosylation modification site of a secretory RCN3 protein, wherein the application is the application of the Asn140 site of the secretory RCN3 protein in the preparation of a product for diagnosing and / or preventing and treating pulmonary fibrosis.

[0012] The fifth aspect of the present invention provides the application of the glycosylation modification site of the secretory RCN3 protein, wherein the application is the application of the Asn140 site of the secretory RCN3 protein in the preparation of a prognostic diagnostic product for acute respiratory distress syndrome.

[0013] Preferably, the product includes but is not limited to a diagnostic reagent, a diagnostic kit or a drug.

[0014] Preferably, the diagnostic reagent or diagnostic kit is a diagnostic reagent or diagnostic kit targeting the Asn140 site of the secretory RCN3 protein.

[0015] Preferably, the drug is a drug targeting the Asn140 site of the secretory RCN3 protein; more preferably, the drug is a monoclonal antibody and / or polyclonal antibody targeting the Asn140 site of the secretory RCN3 protein.

[0016] In a sixth aspect, the present invention provides a diagnostic reagent and a diagnostic kit, wherein the diagnostic reagent and the diagnostic kit are diagnostic reagents and diagnostic kits that target the Asn140 site of the secretory RCN3 protein.

[0017] In a seventh aspect, the present invention provides an antibody, which is a monoclonal antibody and / or a polyclonal antibody targeting the Asn140 site of the secretory RCN3 protein.

[0018] In an eighth aspect, the present invention provides a pharmaceutical composition for preventing and treating pulmonary fibrosis, wherein the pharmaceutical composition comprises any one of the above-mentioned monoclonal antibodies and polyclonal antibodies.

[0019] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0020] Beneficial effects:

[0021] The present invention discovered that the secretory Rcn3 protein can interact with TGFβR1, activate the classical TGFβ signaling pathway, mediate lung epithelial cell-fibroblast communication and regulate the activation and function of fibroblasts; in addition, the present invention discovered a glycosylation modification site that plays an important role in the secretion of RCN3 protein, namely the Asn140 site, and drugs for the prevention and treatment of pulmonary fibrosis can be developed targeting this target, which has great clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The figures are the results of the secretion of secretory Rcn3 protein in alveolar epithelial and fibroblasts; A is the expression of intracellular and secretory Rcn3 proteins in embryonic fibroblast cell lines isolated from wild-type and Rcn3 gene knockout mice, CL represents cell lysate, i.e. intracellular protein; CM represents cell culture supernatant, i.e. secretory protein; B is the expression of intracellular and secretory Rcn3 proteins in human lung epithelial cells and human lung fibroblasts; C is the results of gene editing of human Rcn3 and immunoblotting of intracellular and secretory Rcn3 proteins; D is the amino acid sequence result of secretory Rcn3 protein identification; E is the co-localization of intracellular Rcn3 protein with the endoplasmic reticulum and the co-localization of intracellular Rcn3 protein with the Golgi marker protein Golgi97.

[0024] Figure 2Results of identifying the Asn140 glycosylation modification site of secretory Rcn3 protein; A shows the expression of intracellular and secretory Rcn3 proteins and endoplasmic reticulum stress marker GRP-78 after cells were treated with protein transport inhibitors; B shows the secretion of Rcn3 protein transfected with HDEL plasmid without endoplasmic reticulum localization signal by immunoblotting, W / O HDEL represents no HDEL signal, **p<0.01 compared with the control group; C shows the Asn140 glycosylation modification site of secretory Rcn3 protein detected by mass spectrometry; D shows the expression of secretory Rcn3 protein after cells were transfected with N140A, N140Q and T142A glycosylation modification site mutant plasmids, WT-PNGF represents PNGase F is the treatment control Rcn3, compared with the control group, **p<0.01; E is the co-localization of Calnexin protein and Flag protein (representing intracellular RCN3 protein) co-stained by immunofluorescence experiment after HELA cells were transfected with wild-type, HDEL-free, and N140Q-Rcn3 plasmids; F is the expression of endoplasmic reticulum stress marker protein GRP78 detected by immunoblotting in transfected control, wild-type, and N140Q-Rcn3 plasmids, and the protein ratio calibrated with GAPDH is shown in the graph (n=4), compared with the control group, *p<0.05.

[0025] Figure 3 The results of Rcn3 secretion in bronchoalveolar lavage fluid after LPS-stimulated acute lung injury in mice; A is the pathological result of inducing acute lung injury in mice and transitioning to the pulmonary fibrosis repair stage in the later stage; B is the expression of Collagen1 and αSMA proteins in mouse lung tissue detected by immunoblotting, and the protein ratio after Tubulin calibration is shown in the chart (n=4). Compared with the control group, *p<0.05, **p<0.01; C is the real-time fluorescence semi-quantitative detection of COL1 in mouse lung tissue Compared with day 0, *p<0.05, **p<0.01 were used for gene expression of COL1α1, COL1α2, αSMA and CTGF. D was detected by real-time fluorescence semi-quantitative detection of gene expression of inflammatory factors IL-1β and TNFα in mouse lung tissue. Compared with day 0, **p<0.01, ***p<0.001 were used for gene expression of COL1α1, COL1α2, αSMA and CTGF. Compared with day 0, *p<0.05, **p<0.01 were used for gene expression of COL1α2, αSMA and CTGF.

[0026] Figure 4 The results of secretory Rcn3 protein levels in bronchoalveolar lavage fluid of patients with pulmonary fibrosis and control patients are shown in Figure 2. Compared with the control group, *p<0.05.

[0027] Figure 5Figure 2 is the result of secretory Rcn3 protein mediating lung epithelial cell-fibroblast communication regulating fibroblast function; A is the expression of fibroblast functional genes detected after recombinant Rcn3 stimulated human lung fibroblasts, and the activation of functional genes after TGFβ stimulated fibroblasts, compared with the control group, *p<0.05, **p<0.01; B is the expression of Collagen1 and αSMA proteins in fibroblasts activated by recombinant Rcn3 detected by immunoblotting experiment, and the protein ratio after Tubulin calibration is shown in the graph (n=4), compared with the control group, *p<0.05; C is the human epithelial cells transfected with control, wild type, N140Q-Rcn 3. After the plasmid was injected into the lung, the real-time fluorescence semi-quantitative experiment was used to detect the expression of COL1α1, COL1α2, αSMA and CTGF genes in the fibroblasts of the IgG group and the Rcn3 antibody group. Compared with the control group, *p<0.05, **p<0.01; D was the CCK8 experiment to detect the effect of recombinant Rcn3 on the proliferation ability of fibroblasts CP-H011; E was the alveolar epithelial-fibroblast co-culture experiment: after LPS stimulated human lung epithelial cells, the real-time fluorescence semi-quantitative experiment showed the expression of COL1α1, COL1α2, αSMA, CTGF and PCNA genes in the fibroblasts of the IgG group and the Rcn3 antibody group. Compared with the control group, *p<0.05, compared with the IgG group, #p<0.05.

[0028] Figure 6 The results of the gene expression profile chip after secretory Rcn3 stimulated fibroblasts; A is the clustering analysis showing the clustering of up-regulated genes and down-regulated genes after recombinant Rcn3 stimulated CP-H011 cells; B is the GO analysis showing the enrichment results; C is the KEGG analysis showing the enrichment signal pathway results.

[0029] Figure 7 The results of the interaction between secretory Rcn3 protein and TGFβR1 to activate fibroblast function; A is the immunoblotting detection of the phosphorylation levels of Smad3, AKT, Erk1 / 2 and JNK proteins after recombinant Rcn3 stimulated CP-H011 cells, and the proportion of phosphorylated proteins is represented by the graph (n=4). Compared with the control group, **p<0.01; B is the immunoprecipitation experiment using HIS antibody to precipitate cytoplasmic proteins and membrane proteins respectively, and the IP products are detected by immunoblotting experiments to detect the binding of membrane proteins with TGFβR1 and TGFβR2; C is the result of the Octet BLI non-labeling detection system to detect the affinity of the interaction between Rcn3 and TGFβR1; D is the result of Western blotting detection that Rcn3 failed to activate Smad3 protein phosphorylation after adding TGFβR1 inhibitor and TGFβR1 / 2 inhibitor.

[0030] Figure 8The results of recombinant RCN3 protein-induced pulmonary fibrosis in mice; A is a schematic diagram of the drug administration; B is the Masson staining of mouse lungs; C is the real-time fluorescence semi-quantitative experiment of COL1α1, COL1α2, and αSMA gene expression in mouse lung tissues, compared with the control group, *p < 0.05; D is a protein blot showing the expression of Collagen Ⅰ and αSMA proteins after recombinant RCN3 stimulation, and the protein ratio after Tubulin calibration is represented by a graph (n = 4), compared with the control group, *p < 0.05.

[0031] Fig. 9 The results of antibody neutralization of RCN3 to alleviate LPS-induced pulmonary fibrosis in mice; A is a schematic diagram of the drug administration; B is the Masson staining of mouse lungs; C is the real-time fluorescence semi-quantitative experiment of COL1α1 and COL1α2 gene expression in lung tissues of mice in the IgG group and RCN3 antibody group. Compared with the control group, **p<0.01, compared with the IgG group, #p<0.05, ##p<0.01; D is the expression of type I collagen in the IgG group and the RCN3 antibody group detected by western blotting. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0033] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0034] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are 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 ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Example 1

[0036] Embryonic fibroblast cell lines were isolated from Rcn3 whole-body knockout mice, and the expression of secretory Rcn3 protein was detected by immunoblotting. Figure 1A in the figure shows that RCN3 is a secretory protein. Secretory Rcn3 protein expression was detected in human lung epithelial cells BEAS-2B and human lung fibroblasts CP-H011 ( Figure 1 B) for human Rcn3 Figure 1 The results of immunoblotting after the C in the figure was edited and the Rcn3 protein was purified by immunoprecipitation with Flag antibody. The detection of double tags and HDEL indicated that Rcn3 was secreted as a full-length sequence except for the signal peptide. After 293T cells overexpressed the Rcn3-Flag plasmid, the Rcn3 protein was purified by immunoprecipitation with Flag antibody for mass spectrometry detection. Figure 1 The underlined sequence in D is the identified amino acid sequence: PSPD AGPHGQGRVHQAAPLSDAPHDDAHGNFQYDHEAFLGREVAKEFDQLTPEESQAR LGRIVDR MDRAGDGDGWVSLAE LRAWIAHTQQRHIRDSVSAAWDTYDTDRDGRVGWEELRNATYGHYAPGEEFHDVEDAETYKKMLARDERRFRVADQ DGDSMATREELTAFLHPEEFPHMRDIVIAETLEDLDRNK DGYVQVEEYIADLYSAEPGEEEPAWVQTER QQFRDFR DLNKDGHLDGSEVGHWVLPPAQDQPLVEANHLLHESDTDKDGRLSKAEILGNWNMFVGSQATNYGEDLTR HHDEL (SEQ ID NO: 1). HELA cells were transfected with Dsred2-ER plasmid, and the co-localization of Rcn3 protein and endoplasmic reticulum was detected by immunofluorescence. The co-localization of Rcn3 protein and Golgi97 protein in wild-type HELA cells was detected by immunofluorescence. The results are shown in Figure 1 As can be seen from the figure E, intracellular RCN3 is localized in both the endoplasmic reticulum and the Golgi apparatus, suggesting a possible secretion pathway.

[0037] Example 2

[0038] In the serum-free state, after the protein transport inhibitor (Brefeldin A) was treated for 24 hours in 293T cells, the expression of secretory Rcn3 protein was significantly reduced by immunoblotting, and the expression of endoplasmic reticulum stress marker GRP-78 in cells was upregulated. Figure 2 As shown in A. Immunoblotting detected the expression of Rcn3 protein in the transfected plasmid without the endoplasmic reticulum localization signal HDEL, that is, the absence of HDEL greatly promoted the secretion of Rcn3 protein, as shown in Figure 2 As shown in B. After PNGase F treatment of secreted proteins, mass spectrometry detected that the Rcn3 protein had an Asn140 glycosylation modification site, as shown in Figure 2 As shown in C. Immunoblotting experiments detected that the expression of secretory Rcn3 protein was significantly reduced after 293T cells were transfected with N140A, N140Q and T142A glycosylation modification site mutation plasmids; at the same time, the molecular weight of Rcn3 protein in the 293T cell group treated with PNGase F was the same as that in the mutant plasmid group, as shown in Figure 2As shown in D. After HELA cells were transfected with wild-type, HDEL-free, and N140Q-Rcn3 plasmids, the co-localization of Calnexin protein and Flag protein was observed by immunofluorescence experiments. The results are shown in Figure 2 E in the figure shows that HDEL signal helps RCN3 protein localize in the cell. Immunoblotting was used to detect the expression of GRP78 protein in transfected control, wild type, and N140Q-Rcn3 plasmids. The results are shown in Figure 2 F in the figure shows that the mutation of the glycosylation site of RCN3 causes a severe endoplasmic reticulum stress response in cells, suggesting that glycosylation modification helps maintain endoplasmic reticulum homeostasis.

[0039] Example 3

[0040] LPS (5 mg / kg) was administered intratracheally to induce acute lung injury in mice, which then transitioned to the pulmonary fibrosis repair stage. The pathological results were as follows: Figure 3 As shown in A. Immunoblotting showed that the expression of Collagen1 and αSMA proteins in mouse lung tissues increased significantly. Figure 3 B. Real-time fluorescence semi-quantitative detection of the expression of COL1α1, COL1α2, αSMA and CTGF genes in mouse lung tissue gradually increased over time. The results are shown in Figure 3 C. Real-time fluorescence semi-quantitative detection of the gene expression of inflammatory factors IL-1β and TNFα in mouse lung tissue increased significantly, especially reaching a peak expression on the second day. The results are shown in Figure 3 D in the figure. The expression of Rcn3 protein in the bronchoalveolar lavage fluid of mice was detected by ELISA. The results are shown in Figure 3 As shown in E, it can be seen from the figure that: there was a significant increase on the 2nd and 4th days, and it returned to normal levels after the 7th day, indicating that: after lung injury occurs, RCN3 is secreted in large quantities in the acute phase and returns to normal after the repair phase.

[0041] Example 4

[0042] The expression of Rcn3 in the bronchoalveolar lavage fluid of patients was detected by ELISA. Figure 4 As shown in the figure, it can be seen that the IPF patients are significantly higher than the control patients (pulmonary sarcoidosis).

[0043] Example 5

[0044] Recombinant Rcn3 (0.1ug / ml, ab276552) stimulated human lung fibroblasts CP-H011. Real-time fluorescence semi-quantitative experiments showed that Rcn3 activated the expression of COL1α1, COL1α2, αSMA and CTGF genes in cells, but PCNA expression did not change. After TGFβ (2ng / ml) stimulated cells, the activation effect of Rcn3 was not significant. The results are shown in Figure 5A in Figure 2. Immunoblotting assay detected that recombinant Rcn3 activated the expression of Collagen1 and αSMA proteins in cells. The results are shown in Figure 5 B. After 293T cells were transfected with control, wild-type, and N140Q-Rcn3 plasmids (N140Q: 418-420 sites were mutated from AAC to CAA), the culture supernatant was collected to stimulate CP-H011 cells. Real-time fluorescence semi-quantitative experiments showed that wild-type Rcn3 could activate the expression of COL1α1, COL1α2, αSMA, and CTGF genes in cells, while N140Q mutant Rcn3 had no such function. Figure 5 C. CCK8 assay detected that recombinant Rcn3 did not affect the proliferation ability of CP-H011 cells after stimulation. The results are shown in Figure 5 D in the figure. LPS (1ug / ml) stimulated human lung epithelial cells BEAS-2B, and the culture supernatant was collected and pretreated with rabbit IgG and Rcn3 antibodies before stimulating CP-H011. Real-time fluorescence semi-quantitative experiments showed that the expression of COL1α1, COL1α2, αSMA, CTGF and PCNA genes in the IgG group was significantly upregulated, while the Rcn3 antibody group had no such effect. The results are shown in Figure 5 E in.

[0045] Example 6

[0046] Cluster analysis showed that the up-regulated and down-regulated genes clustered after recombinant Rcn3 (0.1ug / ml) stimulated CP-H011 cells. Figure 6 A in the figure shows that secreted RCN3 induces 328 genes to be significantly upregulated and 215 genes to be significantly downregulated in fibroblasts. GO analysis shows the enrichment results. Figure 6 In B, it can be seen from the figure that secreted RCN3 stimulates significant changes in collagen and extracellular matrix-related pathways. KEGG analysis shows that the results of enriched signaling pathways are shown in Figure 6 As can be seen from the figure C, secreted RCN3 induces a significant upregulation of the TGFβ signaling pathway.

[0047] Example 7

[0048] Immunoblotting was used to detect the phosphorylation levels of Smad3, AKT, Erk1 / 2 and JNK proteins after recombinant Rcn3 stimulated CP-H011 cells. Figure 7 As shown in A. The cytoplasmic proteins and membrane proteins were precipitated by immunoprecipitation experiments using HIS antibodies. The IP products were detected by immunoblotting experiments to show that the membrane proteins had TGFβR1 binding, but not TGFβR2 binding. Figure 7 As shown in B. The OctetBLI label-free detection system detected that Rcn3 and TGFβR1 had a direct interaction with an affinity of 26.91 nM, see Figure 7C. Western blot detection showed that after adding LY364947 (TGFβR1 inhibitor) and LY2109761 (TGFβR1 / 2 inhibitor), Rcn3 stimulation failed to activate Smad3 protein phosphorylation, see Figure 7 D in the figure shows that inhibiting TGFβ receptor expression can block the activation of Smad3 protein phosphorylation by secretory RCN3.

[0049] Example 8

[0050] like Figure 8 As shown in A, recombinant RCN3 protein (0.1 mg / kg) was intravenously administered on days 0 and 4, and mouse samples were collected on day 14. Masson staining showed that fibrosis occurred in the mouse lungs. Figure 8 B. Real-time fluorescence semi-quantitative experiments found that COL1α1, COL1α2, and αSMA gene expressions were upregulated in mouse lung tissues. Figure 8 C. Western blot showed that after stimulation with recombinant RCN3, the expression of CollagenⅠ and αSMA proteins increased, see Figure 8 D in.

[0051] Example 9

[0052] like Fig. 9 As shown in A, LPS (5 mg / kg) was intravenously administered on day 0, and RCN3 antibody (80 ug / kg) was intravenously administered on day 2 and 4, and mouse samples were collected on day 7. Masson staining showed that lung fibrosis occurred in mice in the IgG group, while fibrosis was alleviated in the RCN3 antibody group. Fig. 9 B. Real-time fluorescence semi-quantitative experiments found that the expression of COL1α1 and COL1α2 genes in the lung tissue of mice in the IgG group was upregulated; the expression of COL1α1 and COL1α2 genes in the RCN3 antibody group was significantly reduced. Fig. 9 C. Western blot showed that the expression of type I collagen increased in the IgG group, but not in the RCN3 antibody group. Fig. 9 D in.

[0053] In summary, the present invention shows through the above experiments that: Rcn3 is a full-length secretory protein located in the endoplasmic reticulum-Golgi secretion pathway, and its Asn140 glycosylation modification site plays an important role in secretion; in the LPS-stimulated acute lung injury mouse model, Rcn3 secretion increased significantly during the repair period of pulmonary fibrosis, and Rcn3 protein was significantly increased in the alveolar lavage fluid of ARDS patients during the repair period; secretory Rcn3 protein mediates lung epithelial cell-fibroblast communication and regulates the function of fibroblasts--LPS stimulates lung epithelial cells and Rcn3 secretion increases significantly, and co-cultured fibroblasts The results showed that Rcn3 can promote the activation and repair of fibroblasts, and this phenomenon can be inhibited by Rcn3 antibody neutralization experiment. The gene expression changes after fibroblasts stimulated by recombinant Rcn3 were detected by gene expression profile chip, and it was found that the expression of genes related to TGFβ signaling pathway was significantly upregulated. Cell model studies showed that secreted Rcn3 protein can interact with TGFβR1, activate the classical TGFβ signaling pathway, and promote the activation and function of fibroblasts. Tracheal administration of Rcn3 recombinant protein can induce pulmonary fibrosis in mice. Specific RCN3 antibodies can neutralize lipopolysaccharide-induced pulmonary fibrosis.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The glycosylation modification site of the secretory RCN3 protein is characterized in that: The glycosylation modification site of the secretory RCN3 protein is the Asn140 site.

2. Application of glycosylation modification sites of secretory RCN3 protein, characterized in that: The application is the use of the Asn140 site of the secretory RCN3 protein as a target in the diagnosis and / or prevention and treatment of pulmonary fibrosis.

3. Application of glycosylation modification sites of secretory RCN3 protein, characterized in that: The application is the use of the Asn140 site of the secretory RCN3 protein as a target in the prognosis diagnosis of acute respiratory distress syndrome.

4. Application of glycosylation modification sites of secretory RCN3 protein, characterized in that: The application is the application of the Asn140 site of the secretory RCN3 protein in the preparation of products for diagnosing and / or preventing and treating pulmonary fibrosis.

5. Application of glycosylation modification sites of secretory RCN3 protein, characterized in that: The application is the use of the Asn140 site of the secretory RCN3 protein in the preparation of an acute respiratory distress syndrome prognostic diagnosis product.

6. The use according to any one of claims 4 to 5, characterized in that: The product is a diagnostic reagent, a diagnostic kit or a drug.

7. The use according to claim 6, characterized in that: The drug is a drug targeting the Asn140 site of the secretory RCN3 protein.

8. The use according to claim 7, characterized in that: The drug is a monoclonal antibody targeting the Asn140 site of the secretory RCN3 protein.

9. The use according to claim 7, characterized in that: The drug is a polyclonal antibody targeting the Asn140 site of the secretory RCN3 protein.

10. An antibody, characterized in that The antibody is a monoclonal antibody and / or a polyclonal antibody targeting the Asn140 site of the secretory RCN3 protein.

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