Prdx1 igg neutralizing antibodies, methods of making and use in the manufacture of a medicament for acute organ injury or sepsis

Abstract

The application belongs to the field of medicine, and specifically discloses a Prdx1 IgG neutralizing antibody, which is a monoclonal neutralizing antibody against Prdx1 protein or a polyclonal neutralizing antibody against Prdx1 protein. The preparation method of the Prdx1 IgG neutralizing antibody and the application of the Prdx1 IgG neutralizing antibody in preparing drugs for acute organ injury or sepsis are also disclosed. The application uses a recombinant Prdx1 protein to immunize mammals, so as to obtain a monoclonal neutralizing antibody against Prdx1 protein and a polyclonal neutralizing antibody against Prdx1 protein. The monoclonal neutralizing antibody against Prdx1 protein and the polyclonal neutralizing antibody against Prdx1 protein can neutralize and block the proinflammatory effect of Prdx1 in serum, and can be used for treating acute organ injury such as acute liver injury, acute kidney injury and acute lung injury, and sepsis.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to Prdx1 IgG neutralizing antibody, its preparation method, and its application in the preparation of drugs for acute organ injury or sepsis. Background Technology

[0002] Acute liver injury (ALI) is liver damage caused by various factors, and its progression is rapid. Studies have shown that exogenous substances, such as viral infections, alcohol or drug abuse, and exposure to toxins, can all lead to ALI. In my country, the leading cause of ALI is viral infection, followed by medications. Since post-necrotic cirrhosis caused by ALI is incurable, it not only severely impacts patients' quality of life but also places a heavy medical burden on the country. Therefore, preventing ALI is crucial for public health. For severe ALI, liver transplantation is one of the most effective treatments, but statistics show that less than 10% of patients receive liver transplants. Currently, treatment for acute liver failure is very limited both domestically and internationally, with a very high mortality rate. Therefore, finding alternative therapies for ALI is an urgent medical need. Extensive research has been conducted globally to find effective drugs for the prevention and treatment of ALI, some of which have been used clinically for a considerable period, but the therapeutic effects of these drugs remain unsatisfactory.

[0003] Acute kidney injury (AKI) is a common clinical syndrome, often accompanied by prolonged hospital stays and poor prognosis in some patients, leading to chronic kidney disease. Globally, approximately 13 million people develop AKI annually, and about 1.7 million die from AKI and its complications. AKI can occur in multiple clinical disciplines, but its incidence exceeds 50% in the ICU. In my country, a large-scale, multicenter epidemiological survey showed that, according to the KDIGO diagnostic criteria and extended criteria, the incidence of AKI was 0.99% and 2.03%, respectively, while the in-hospital mortality rate was as high as 12.4%. In recent years, the prevalence of AKI has been on the rise, and the mortality rate remains high, imposing a huge economic burden on the country and society. For a long time, clinical treatment for AKI has followed the principle of symptomatic support, waiting for the kidney disease to resolve itself. To date, no drugs have been approved for marketing to reduce kidney tissue damage, promote kidney repair, or prevent later chronic kidney fibrosis in AKI.

[0004] Sepsis is a life-threatening organ dysfunction caused by dysregulation of the host response to infection, and it is one of the leading causes of death in patients in the intensive care unit (ICU). The latest cross-sectional epidemiological survey report on ICUs in China shows that the incidence of sepsis in ICUs is 23.9%, and the 90-day mortality rate is 35.5%. Behind this high mortality rate lies the current situation of limited treatment options and poor efficacy. Therefore, developing a new drug to treat sepsis is an urgent medical need.

[0005] Acute lung injury (ALI) is lung damage caused by various intrapulmonary and extrapulmonary etiologies. Its severe form manifests as acute respiratory distress syndrome (ARDS), characterized by persistent hypoxemia and respiratory failure, and is a significant cause of poor prognosis in critically ill patients. Major causes or triggers of ALI include pneumonia, non-pulmonary sepsis, and aspiration of gastric contents. Globally, 3 million people suffer from ALI annually. The prevalence of ALI in ICU patients can reach 10%, while in patients requiring mechanical ventilation, it reaches 23%. The overall mortality rate of ALI exceeds 40%, with higher mortality rates among more severe cases. Treatment for ALI primarily involves controlling the underlying disease and providing symptomatic and supportive care. Mechanical ventilation is the cornerstone of treatment, but ventilator-induced lung injury (VILI) occurs frequently in clinical practice. Currently, there are no effective drugs for treating acute lung injury. Although numerous studies have been conducted on drugs for treating acute lung injury, including β2 agonists, keratinocyte growth factor (KGF), and statins, these drugs have yielded strong evidence in pathophysiology and preclinical studies, but none have demonstrated clear efficacy in clinical application. Therefore, the development of effective treatments remains extremely urgent. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide Prdx1 IgG neutralizing antibody and its application in the preparation of drugs for acute organ injury or sepsis, so as to solve the technical problem of the lack of drugs for the treatment of acute liver injury, acute kidney injury and sepsis in the current medical field.

[0007] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0008] A Prdx1 IgG neutralizing antibody, which is a class of IgG antibodies that can specifically bind to the Prdx1 protein, including monoclonal neutralizing antibodies against the Prdx1 protein or polyclonal neutralizing antibodies against the Prdx1 protein.

[0009] Preferably, the monoclonal neutralizing antibody against Prdx1 protein described above comprises any one or more amino acid sequences from SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, or comprises an amino acid sequence with more than 80% homology obtained by substituting, deleting, and / or adding one or more amino acids and / or terminal modification of any one or more amino acid sequences from SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6.

[0010] Preferably, the amino acid sequence of the CDR1 region of the heavy chain variable region of the monoclonal neutralizing antibody against Prdx1 protein is shown in SEQ ID NO.1, the amino acid sequence of the CDR2 region of the heavy chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the CDR3 region of the heavy chain variable region is shown in SEQ ID NO.3; the amino acid sequence of the CDR1 region of the light chain variable region of the monoclonal neutralizing antibody against Prdx1 protein is shown in SEQ ID NO.4, the amino acid sequence of the CDR2 region of the light chain variable region is shown in SEQ ID NO.5, and the amino acid sequence of the CDR3 region of the light chain variable region is shown in SEQ ID NO.6.

[0011] Preferably, the monoclonal neutralizing antibody against Prdx1 protein comprises amino acids from the heavy chain variable region and / or the light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.

[0012] Preferably, the polyclonal neutralizing antibody against Prdx1 protein is prepared by the following method: using recombinant prdx1 protein as an immunogen, immunizing mammals to obtain mammals expressing Prdx1 IgG neutralizing antibodies, and finally extracting and purifying the polyclonal neutralizing antibody against Prdx1 protein from the mammalian serum.

[0013] Preferably, the Prdx1 IgG neutralizing antibody mentioned above includes Prdx1 neutralizing antibodies derived from rabbits, humans, mice, rats, or human-mouse chimeric sources.

[0014] The nucleic acid molecule encoding the above-mentioned Prdx1 IgG neutralizing antibody includes nucleotides encoding the heavy chain variable region and / or the light chain variable region of the Prdx1 IgG neutralizing antibody, wherein the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.9, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.10.

[0015] Peroxiredoxin1 (Prdx1, Prx1) is an antioxidant protein belonging to the Peroxiredoxins (Prdxs) superfamily. Prdx1 is the most broadly expressed and most abundant member of this family and is considered one of the most important antioxidant enzymes in the body. Previous studies have found that neutralizing antibodies against Prdx1 have potential therapeutic effects on acute liver and kidney injury. This suggests the potential of Prdx1 as a novel drug for treating acute liver and kidney injury, addressing the current lack of effective treatments for these conditions in the pharmaceutical field.

[0016] Based on a general inventive concept, a method for preparing a Prdx1 IgG neutralizing antibody is also provided, wherein the Prdx1 IgG neutralizing antibody is a monoclonal neutralizing antibody against the Prdx1 protein, comprising the following steps:

[0017] A) Using cDNA from isolated mammalian tumor cells as a template, PCR amplification was performed to obtain the target gene fragment Prdx1, which was ligated into a vector plasmid, transformed into E. coli, and positive clones containing the inserted fragment were screened and verified by sequencing to obtain a prokaryotic expression plasmid containing the correct mammalian Prdx1 gene sequence.

[0018] B) The prokaryotic expression plasmid was transformed into the expression host bacteria and cultured. Single clones were selected and induced to express the bacterial culture. The target expressed protein was then purified, which is the mammalian Prdx1 protein.

[0019] C) The purified mammalian-derived Prdx1 protein was used as an antigen to immunize the animals.

[0020] D) After immunization, tissues from immunized animals were taken, plasma cells were extracted and fused with myeloma cells, and after selective culture with hypoxanthine, aminopterin and thymidine, the cells were coated with the human Prdx1 antigen, positive clones were screened by ELISA, and subcloning was performed by limiting dilution to obtain hybridoma cells.

[0021] E) The hybridoma cells were injected into the peritoneal cavity of mice to prepare ascites. The ascites was then separated and purified to obtain a monoclonal neutralizing antibody against Prdx1 protein.

[0022] The above-mentioned preparation method, preferably, specifically includes the following steps:

[0023] A) PCR amplification was performed using cDNA from human tumor cells U87MG as a template. The primers used for PCR amplification were as follows:

[0024] PRDX1-F:CTGCCAAGTGATTGGTGCTTCTG;

[0025] PRDX1-R:AATGGTGCGCTTCGGGTCTGAT;

[0026] The Prdx1 gene fragment with EcoRI / XhoI restriction sites at both ends was obtained, and the target gene fragment Prdx1 was obtained by double digestion with EcoRI / XhoI. The vector plasmid pET22b was double digested with EcoRI / XhoI, and the vector fragment was recovered after agarose gel electrophoresis. It was then ligated with the target gene fragment Prdx1 by T4 ligase, and transformed into E. coli TOP10. After screening for ampicillin resistance and identification by EcoRI / XhoI restriction, positive clones containing the inserted fragment were screened and verified by sequencing to obtain the prokaryotic expression plasmid pET22b-Prdx1 containing the correct mammalian Prdx1 gene sequence.

[0027] B) The prokaryotic expression plasmid was transformed into the expression host bacterium BL21, and the plasmid was spread on a plate containing ampicillin resistance and cultured upside down overnight. Single clones were picked and induced to express the bacterial culture. The target expressed protein was then purified, which is the human Prdx1 protein.

[0028] C) The purified human Prdx1 protein was used as an antigen and fully emulsified with complete Freund's adjuvant for subcutaneous immunization of 6-8 week old mice. Four weeks later, the human Prdx1 protein was emulsified with incomplete Freund's adjuvant and injected intraperitoneally into the mice. After a two-week interval, intraperitoneal antigen booster immunization was performed. One week after the fourth booster immunization, the mice were coated with the human Prdx1 protein, and the antiserum titer of the mice was detected by ELISA. Booster immunization was continued until the antiserum titer of the mice reached >105. Three weeks after the last booster immunization, the mammalian Prdx1 protein was immunized intraspleurally for later use.

[0029] D) Four days after mice were boosted with intrasplenic immunization, spleens were harvested under sterile conditions, lymphocytes were isolated and removed to obtain plasma cells, which were fused with myeloma cell line SP2 / 0. After selective culture with hypoxanthine, aminopterin and thymidine for 3 days, HT medium was added and cultured for another week. Positive clones were screened by ELISA using the human Prdx1 antigen, and subcloning was performed three times using the limiting dilution method. The cells were cultured continuously for 2 months to finally obtain a stable hybridoma cell line.

[0030] E) Take hybridoma cells at 5 × 10⁻⁶ 5Ascites fluid was prepared by intraperitoneal injection of cells / mouse doses into mice. The ascites fluid was diluted with sodium acetate buffer, caprylic acid was added, and the precipitate was discarded by centrifugation. The supernatant was dialyzed overnight with phosphate buffer. The dialysate was collected, saturated ammonium sulfate was added, and the mixture was allowed to stand. The supernatant was discarded by centrifugation. The supernatant was dissolved in PBS and dialyzed. The dialysate was centrifuged to discard the precipitate and the supernatant was collected. The protein G affinity column was brought to room temperature and equilibrated with 5 column volumes of PBS. The last collected supernatant was loaded onto the column and washed with 5 column volumes of PBS. The supernatant was eluted with glycine hydrochloride solution, and the eluent was neutralized with disodium hydrogen phosphate solution. The solution was dialyzed with PBS, and the dialysate was centrifuged. The supernatant was filtered through a 0.22 μm filter membrane and stored to obtain purified monoclonal neutralizing antibody against Prdx1 protein.

[0031] Based on a general inventive concept, a method for preparing a Prdx1 IgG neutralizing antibody is also provided, wherein the Prdx1 IgG neutralizing antibody is a polyclonal neutralizing antibody against Prdx1 protein, comprising the following steps: using recombinant prdx1 protein as an immunogen to immunize mammals to obtain mammals expressing Prdx1 IgG neutralizing antibodies, and finally preparing and purifying the polyclonal neutralizing antibody against Prdx1 protein.

[0032] The above-mentioned preparation method, preferably, specifically includes the following steps:

[0033] (1) Rabbits were selected as immunized animals, and serum was collected before the preparation of neutralizing antibodies;

[0034] (2) On day 1, the immunogen, namely 0.4-0.6 mg of recombinant prdx1 protein, was administered subcutaneously to the immunized animals with complete Freund's adjuvant.

[0035] (3) On days 14, 35 and 56, the immunogen, namely 0.2-0.3 mg of recombinant prdx1 protein, was administered to the immunized animals via subcutaneous injection with incomplete Freund's adjuvant.

[0036] (4) On day 63, rabbit blood was collected after step (3) and serum was extracted. The serum was used to perform ELISA to detect the presence of Prdx1 IgG neutralizing antibodies.

[0037] (5) Select rabbits that express Prdx1 IgG neutralizing antibodies in their serum and administer 0.2-0.3 mg of immunogen and incomplete Freund's adjuvant to immunize the animals again on day 70;

[0038] (6) Collect rabbit serum after treatment in step (5), purify the antibody using an antigen affinity column to obtain the Prdx1IgG neutralizing antibody; and use ELISA to detect its affinity for Prdx1 recombinant protein; and use Western Blot to detect the specificity of Prdx1IgG neutralizing antibody.

[0039] This invention is the first to discover that the Prdx1 protein is an important target for the treatment of acute organ injury and sepsis. The binding molecules that specifically bind to the Prdx1 protein (including monoclonal and polyclonal antibodies against the Prdx1 protein) have significant alleviating or therapeutic effects on acute organ injury and sepsis.

[0040] Based on a general inventive concept, the invention also provides the use of a Prdx1 IgG neutralizing antibody in the preparation of drugs for acute organ injury or sepsis.

[0041] Preferably, the acute organ injury includes acute liver injury, acute kidney injury, and acute lung injury.

[0042] More preferably, the lipopolysaccharide / D-galactosamine-induced acute liver injury clinically corresponds to acute liver injury caused by viral infection; the acetaminophen-induced acute liver injury clinically corresponds to acute liver injury caused by drugs, toxins, or alcohol. The main manifestations of the lipopolysaccharide / D-galactosamine-induced acute liver injury are primarily inflammation followed by oxidative stress, with cellular changes primarily involving apoptosis followed by necrosis; the main manifestations of the acetaminophen-induced acute liver injury are primarily oxidative stress followed by inflammation, with cellular changes primarily involving necrosis followed by apoptosis.

[0043] The acute liver injury mentioned includes, but is not limited to, acute liver injury induced by lipopolysaccharide / D-galactosamine or acetaminophen; the main manifestations of acute liver injury induced by lipopolysaccharide / D-galactosamine are inflammation, oxidative stress, or cell necrosis and apoptosis, which clinically correspond to acute liver injury caused by viral infection; the manifestations of acute liver injury induced by acetaminophen include oxidative stress, aseptic inflammation, or cell necrosis and apoptosis, which clinically correspond to acute liver injury caused by drugs, toxins, or alcohol.

[0044] More preferably, the acute kidney injury includes, but is not limited to, acute kidney injury induced by renal ischemia-reperfusion injury; the main manifestation of acute kidney injury induced by renal ischemia-reperfusion injury is oxidative stress or inflammatory damage.

[0045] More preferably, the acute lung injury includes bleomycin-induced, pneumonia, non-pulmonary sepsis, aspiration of gastric contents, or ventilator-induced acute lung injury.

[0046] Preferably, the Prdx1 IgG neutralizing antibody can be used to treat acute organ injury or sepsis in mice, but since similar or identical diseases in different animals have similar or identical pathogenic mechanisms, this antibody can also be used in other mammals, including humans.

[0047] Preferably, when the Prdx1 IgG neutralizing antibody is used to treat acute liver injury and acute kidney injury, the dosage for rabbits and mice is 300 μg / mouse.

[0048] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0049] 1. This invention utilizes recombinant Prdx1 protein to immunize rabbits, obtaining polyclonal neutralizing antibodies against Prdx1 protein from serum. These antibodies neutralize and block the pro-inflammatory effects of Prdx1 in serum, and can treat acute organ injuries, including acute liver injury, acute kidney injury, acute lung injury, and sepsis. The advantages of polyclonal neutralizing antibodies are their high titer, ease of handling and quality control, and relatively easy and economical preparation. Furthermore, the raw materials for their preparation are readily available; all mammals can be immunized to produce Prdx1 IgG neutralizing antibodies, and all homologous Prdx1 proteins extracted or recombined are immunogens.

[0050] 2. This invention utilizes recombinant Prdx1 protein to immunize mice, thereby obtaining monoclonal neutralizing antibodies against Prdx1 protein through the fusion of plasma cells and hybridoma cells. The advantages of these antibodies are their high specificity and sensitivity, and the ability to continuously obtain stable products after obtaining hybridoma cells, demonstrating their potential as pharmaceuticals. They also neutralize and block the pro-inflammatory effects of Prdx1 in serum, and can treat acute organ injuries such as acute liver injury, acute kidney injury, and acute lung injury, as well as sepsis. Attached Figure Description

[0051] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0052] Figure 1 The images shown are from Example 1, where 1A shows the control and images of Prdx1 polyclonal antibody binding to Prdx1 protein in mouse liver tissue, kidney tissue, and primary macrophages; 1B shows the control and images of Prdx1 monoclonal antibody binding to Prdx1 protein in mouse liver tissue, lung tissue, kidney tissue, and primary macrophages.

[0053] Figure 2The results of ELISA detection of inflammatory factors secreted by mouse primary macrophages in Example 1;

[0054] Figure 3 HE staining images (200x magnification) of the livers of mice in each group in the lipopolysaccharide / D-galactosamine-induced acute liver injury model treated with polyclonal neutralizing antibodies against Prdx1 protein in Example 2.

[0055] Figure 4 HE staining images (200x magnification) of the livers of mice in each group in the acetaminophen-induced acute liver injury model treated with polyclonal neutralizing antibodies against Prdx1 protein in Example 2.

[0056] Figure 5 HE staining images (200x) of the kidneys of mice in each group in the ischemia-reperfusion-induced acute kidney injury model treated with polyclonal neutralizing antibodies against Prdx1 protein in Example 3.

[0057] Figure 6 This is a diagram illustrating the effect of the monoclonal neutralizing antibody against Prdx1 protein in Example 4 on inhibiting Prdx1 protein.

[0058] Figure 7 HE staining images (200x magnification) of the livers of mice in each group in the lipopolysaccharide / D-galactosamine-induced acute liver injury model treated with monoclonal neutralizing antibodies against Prdx1 protein in Example 5.

[0059] Figure 8 HE staining images (200x magnification) of the kidneys of mice in each group in the LPS-induced acute kidney injury model treated with monoclonal neutralizing antibodies against Prdx1 protein in Example 6.

[0060] Figure 9 Survival curves of mice in each group of the LPS-induced sepsis model treated with monoclonal neutralizing antibodies against Prdx1 protein in Example 7;

[0061] Figure 10 HE staining images (200x magnification) of lung tissue from each group of mice in the bleomycin-induced acute lung injury model treated with monoclonal neutralizing antibodies against Prdx1 protein in Example 8. Detailed Implementation

[0062] This invention is the first to discover that Prdx1 protein is an important target for the treatment of acute organ injury and sepsis, and provides a binding molecule that specifically binds to Prdx1 protein—Prdx1 IgG neutralizing antibody (including monoclonal and polyclonal neutralizing antibodies against Prdx1 protein), which exhibits neutralizing or inhibitory activity against Prdx1 protein and has a significant alleviating or therapeutic effect on acute organ injury and sepsis.

[0063] The binding molecule of the present invention that specifically binds to the Prdx1 protein can be a complete immunoglobulin molecule, such as a polyclonal or monoclonal neutralizing antibody; the binding molecule can also be an antigen-binding fragment, including but not limited to Fab, F(ab'), F(ab')2, Fv, dAb, Fd, complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, single-chain phage antibodies, bispecific double-chain antibodies, triple-chain antibodies, quadruple-chain antibodies, and polypeptides or fragments thereof containing at least a fragment sufficient to confer specific antigen binding to the Prdx1 protein. In a preferred embodiment, the binding molecule of the present invention is a mouse monoclonal neutralizing antibody or a rabbit polyclonal neutralizing antibody.

[0064] This invention also provides functional variants of the binding molecule. A variant molecule is considered a functional variant of the binding molecule of this invention if it can compete with the parent binding molecule for specific binding of Prdx1 or a protein fragment thereof. In other words, the functional variant can still bind Prdx1 or a fragment thereof. Functional variants include, but are not limited to, derivatives with substantially similar primary structural sequences but containing, for example, in vitro or in vivo chemical and / or biochemical modifications not found in the parent binding molecule. Such modifications include acetylation, phthalolation, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, cross-linking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, polyethylene glycolation, proteolytic processing, phosphorylation, etc. In other words, modifications in the amino acid and / or nucleotide sequences of the parent binding molecule do not significantly affect or alter the binding properties of the binding molecule encoded by the nucleotide sequence or containing the amino acid sequence; that is, the binding molecule can still recognize and bind its target site.

[0065] The functional variants may have conserved sequence modifications, including nucleotide and amino acid substitutions, additions, and deletions. These modifications can be introduced using standard techniques known in the art, such as directed mutagenesis and randomized PCR-mediated mutagenesis, and may contain both natural and non-natural nucleotides and amino acids.

[0066] Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar structure or chemical properties. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chain amino acids (e.g., aspartic acid, glutamic acid), uncharged polar side chain amino acids (e.g., aspartamine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chain amino acids (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), branched side chain amino acids (e.g., threonine, valine, isoleucine), and aromatic side chain amino acids (e.g., tyrosine, phenylalanine, tryptophan). Those skilled in the art will understand that other amino acid residue families besides those described above can also be used. Additionally, variants may have non-conservative amino acid substitutions, such as substitutions where an amino acid is replaced by another amino acid residue having a different structure or chemical properties. Similar minor variations may also include amino acid deletions or insertions, or both. Using computer programs well-known in the art, one can discover guidelines for determining which amino acid residues can be substituted, inserted, or deleted without eliminating immunological activity.

[0067] Furthermore, functional variants may comprise truncated amino acid sequences at the amino terminus or carboxyl terminus, or both. The functional variants of the present invention may have the same or different, higher or lower binding affinity compared to the parental binding molecule, but may still bind Prdx1 or fragments thereof. For example, the functional variants of the present invention may have increased or decreased binding affinity for Prdx1 or fragments thereof compared to the parental binding molecule. Functional variants within the scope of the present invention have at least about 80% to about 99% amino acid sequence homology to the parental binding molecule described herein.

[0068] In a preferred embodiment of the invention, the binding molecule is a monoclonal neutralizing antibody. The invention includes: a monoclonal neutralizing antibody having the corresponding amino acid sequence of the monoclonal neutralizing antibody, and a monoclonal neutralizing antibody having the variable region chain of the monoclonal neutralizing antibody. The invention also includes any antibody having a light chain and a heavy chain containing the aforementioned complementarity-determining region (CDR), and any antibody whose CDR region has more than 90% (preferably more than 95%) homology to the CDR of the monoclonal neutralizing antibody of the invention.

[0069] The antigen-binding properties of monoclonal neutralizing antibodies can be described by three specific regions located in the variable regions of the heavy and light chains, called complementarity-determining regions (CDRs). These CDRs divide the variable regions into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, spatially close to each other through β-sheets formed by the FRs. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0070] The monoclonal neutralizing antibodies or antibody fragments used in this invention can be fully human, humanized, chimeric, or murine. "Humanized antibody" as used herein refers to an antibody having an amino acid sequence corresponding to antibodies produced by humans, and / or an antibody prepared using techniques known in the art and disclosed in this application for preparing humanized antibodies. Humanized antibodies mainly refer to murine (or other non-human) monoclonal neutralizing antibodies that have been modified and re-expressed using gene cloning and DNA recombination techniques. Most of their amino acid sequences are replaced by human sequences, essentially retaining the affinity and specificity of the parent murine monoclonal neutralizing antibody while reducing its heterology nature, which is beneficial for human application. Humanized antibodies include chimeric antibodies, modified antibodies (also known as CDR grafting antibodies), surface-remodeled antibodies, or fully humanized antibodies. Humanized antibodies can also be produced using various methods known in the art, for example, humanized antibodies can be selected from a phage library expressing human antibodies. Humanized antibodies can also be prepared by introducing human immunoglobulin sites into transgenic animals, such as mice in which endogenous immunoglobulin genes have been partially or completely inactivated. Furthermore, humanized antibodies can also be prepared by immortalizing human B lymphocytes that produce antibodies against specific antigens.

[0071] This invention also provides nucleic acid molecules encoding at least one binding molecule of the invention. Such nucleic acid molecules can be used as intermediates for cloning, for example, in affinity maturation methods as described above. In a preferred embodiment, the nucleic acid molecule is isolated or purified. The sequence of the DNA molecule can be obtained using conventional techniques or hybridoma techniques. Functional variants of these nucleic acid molecules are also part of this invention. Functional variants are nucleic acid sequences that can be directly translated using the standard genetic code to provide the same amino acid sequence as the sequence translated from the parent nucleic acid molecule. Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. Alternatively, the relevant sequence can be synthesized using artificial synthetic methods.

[0072] The binding molecules of the present invention can be used in either unseparated or separated forms. Furthermore, the binding molecules of the present invention can be applied alone or in a mixture comprising at least one binding molecule of the present invention (or a variant or fragment thereof). In other words, the binding molecules can be used in combination, for example, as a pharmaceutical composition comprising two or more binding molecules of the present invention, their variants, or fragments. For example, binding molecules with different but complementary activities can be combined in a treatment regimen to achieve a desired preventive, alleviating, or therapeutic effect; however, binding molecules with the same activity can also be combined in a treatment regimen to achieve a desired preventive, alleviating, or therapeutic effect.

[0073] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0074] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0075] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0076] The recombinant prdx1 protein used in this invention is a commercially available recombinant peroxidoreductase 1 protein, purchased from Abclone (No. 9, Gaokeyuan Third Road, Bio-Park Third Road, East Lake High-tech Development Zone, Wuhan, 430074, China).

[0077] Experimental methods not specifically described in the following examples are generally performed under conventional conditions, such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002, or as recommended by the manufacturer.

[0078] Example 1: Preparation of a polyclonal neutralizing antibody against Prdx1 protein

[0079] A Prdx1 IgG neutralizing antibody, specifically a polyclonal neutralizing antibody against Prdx1 protein, is a type of IgG antibody that specifically binds to the Prdx1 protein. The preparation method is as follows:

[0080] S1. Rabbits were selected as immunization animals, and serum was collected (5 mL / rabbit) before the preparation of neutralizing antibodies.

[0081] S2. On day 1, the immunogen, namely 0.5 mg of recombinant prdx1 protein, was administered via subcutaneous injection at multiple points (4 points) with complete Freund's adjuvant.

[0082] S3. On days 14, 35, and 56, the immunogen, namely 0.25 mg of recombinant prdx1 protein, was administered via subcutaneous injection at multiple points (4 points) with incomplete Freund's adjuvant.

[0083] S4. On day 63, blood was collected from the above rabbits, 1 mL / rabbit, and serum was extracted. The serum was used to perform ELISA to detect the presence of Prdx1 IgG neutralizing antibodies.

[0084] S5. Select rabbits that express Prdx1 IgG neutralizing antibodies in their serum and administer 0.25 mg of immunogen again on day 70 with incomplete Freund's adjuvant.

[0085] S6. Collect the above-mentioned rabbit serum, purify the antibody using an antigen affinity column to obtain the Prdx1 IgG polyclonal neutralizing antibody; and use ELISA to detect its affinity for human Prdx1 recombinant protein (see Table 1 below), confirming that it has good affinity for human Prdx1; use Western blotting to detect the binding affinity and specificity of Prdx1 IgG neutralizing antibody to mouse Prdx1 protein, and compare it with commercially available mouse Prdx1 Western blotting antibody (purchased from Invitrogen), the bands are basically consistent (see Table 1 below). Figure 1 A) This demonstrates that Prdx1 IgG can effectively recognize mouse Prdx1 protein from different tissue sources.

[0086] Table 1 shows the binding activity of Prdx1 IgG for recombinant prdx1 as detected by ELISA.

[0087]

[0088]

[0089] Note: "precipitate" in the table above indicates a very strong binding ability, resulting in a binding product that is visible to the naked eye.

[0090] Experimental methods:

[0091] This embodiment will verify in vitro that the Prdx1 IgG neutralizing antibody can neutralize the human recombinant Prdx1 protein and block its induction of inflammatory factors secreted by mouse primary macrophages. The in vitro anti-inflammatory effect of the polyclonal neutralizing antibody against Prdx1 protein will be demonstrated below:

[0092] 1. Experimental Methods

[0093] (1) Extraction of mouse primary macrophages

[0094] Three 10-week-old male C57 mice were intraperitoneally injected with 3 mL of 3% thioglycolate solution per mouse. The mice were sacrificed 3 days later, and the peritoneal lavage fluid was collected, plated, and washed away non-adherent cells after 2 hours.

[0095] (2) Recombinant Prdx1 stimulates mouse primary macrophages

[0096] The primary macrophages were cultured in empty 1640 medium and divided into four groups: a blank group, a recombinant Prdx1 stimulation group, a recombinant Prdx1 + Prdx1 polyclonal neutralizing antibody group, and a Prdx1 IgG neutralizing antibody group. 200 nM of recombinant Prdx1 was added to each well for stimulation, and 200 nM of anti-Prdx1 polyclonal neutralizing antibody (calculated based on the average molecular weight of IgG being approximately 160 kDa) was added per well. Cell supernatants were collected after 12 or 24 hours, and the levels of inflammatory factors in the supernatant were detected by ELISA.

[0097] 2. Statistical Methods: All measurement data are expressed as mean ± standard deviation (s). One-way ANOVA was used for comparisons among multiple groups. This statistical method is the most commonly used in pharmaceutical experiments for comparing efficacy differences among multiple groups. After confirming differences between groups, the Turky test was used to further verify differences within groups. A two-sided p-value < 0.05 was defined as statistically significant.

[0098] 3. Experimental Results

[0099] 3.1 Polyclonal neutralizing antibodies against Prdx1 protein can block the effect of recombinant prdx1 stimulating mouse primary macrophages to produce inflammatory factors such as IL-1β, TNF-α, and IL-6 (e.g., Figure 2 The difference demonstrated that the polyclonal neutralizing antibody against Prdx1 protein could block the secretion of inflammatory factors by mouse primary macrophages, which was statistically significant.

[0100] Example 2: Application of polyclonal neutralizing antibodies against Prdx1 protein in the preparation of drugs for acute liver injury

[0101] This embodiment applies the Prdx1 IgG neutralizing antibody from Example 1 to the preparation of a drug for acute liver injury. The therapeutic effect of the Prdx1 IgG neutralizing antibody will be demonstrated below:

[0102] 1. Experimental Methods

[0103] (1) To prepare an experimental animal model of lipopolysaccharide / D-galactosamine-induced acute liver injury (ALI) to observe the efficacy of Prdx1 IgG neutralizing antibody in treating ALI characterized by inflammation, oxidative stress / cell necrosis and apoptosis:

[0104] Seventeen SPF-grade C57BL / 6 mice (10-12 weeks old, male, weighing 25-28g) were randomly divided into three groups: control group, model group, and experimental group. Mice in the model group and the Prdx1 IgG neutralizing antibody treatment group were induced into a model by intraperitoneal injection of 100 μg / kg LPS and 500 mg / kg D-GaIN. Two hours after modeling, mice in the neutralizing antibody treatment group were intraperitoneally injected with 300 μg / mouse of Prdx1 IgG neutralizing antibody, while mice in the normal and model groups were injected with the same dose of control IgG. Six hours after modeling, the mice were anesthetized, blood was collected for serum, and the mice were subsequently sacrificed.

[0105] (2) To establish an experimental animal model of acetaminophen (PAP)-induced acute liver injury (ALI) to observe the efficacy of Prdx1 IgG neutralizing antibody in treating ALI induced by drugs, poisoning, etc.

[0106] Eighteen SPF-grade C57BL / 6 mice (8-10 weeks old, male, weighing 23-25g) were randomly divided into three groups: control group, model group, and experimental group. Mice in the model group and the Prdx1 IgG neutralizing antibody treatment group were induced into a model by intraperitoneal injection of 300 mg / kg APAP. Mice in the control group were injected intraperitoneally with 0.3 mL of physiological saline. Two hours after model initiation, mice in the neutralizing antibody treatment group were injected intraperitoneally with 300 μg / mouse of Prdx1 IgG neutralizing antibody, while mice in the normal and model groups were injected with the same dose of control IgG. Eight hours after model initiation, the mice were anesthetized, blood was collected for serum, and the mice were subsequently sacrificed.

[0107] The livers of mice from (1) and (2) were separated, and the left lateral lobe was used to prepare paraffin sections. The remaining liver tissue was placed in a liquid nitrogen container. Liver function of mice in each group was detected by continuous monitoring, and the paraffin sections were stained with hematoxylin and eosin (HE) to observe liver damage.

[0108] The LPS / D-GaIN model liver tissue damage scoring criteria were used in a semi-quantitative analysis of liver pathological damage, referencing the method employed by CARLOS A. et al. The scoring criteria were as follows: Grade 0: little or no evidence of injury; Grade 1: mild damage, cytoplasmic vacuolation, focal nuclear pyknosis; Grade 2: moderate to severe damage, with widespread nuclear pyknosis, eosinophilia, and loss of intercellular boundaries; Grade 3: severe necrosis with hepatic cord loss, hemorrhage, and neutrophil infiltration. The APAP model liver tissue damage scoring criteria were also used in a semi-quantitative analysis of liver pathological damage, referencing the method employed by Zhang-Xu Liu et al. Scoring criteria: 0 points: No damage; 0.5 points: A single necrotic cell seen in the first cell layer adjacent to the central vein, with sublinear degeneration; 1 point: Necrotic cells extending from the central vein into two to three cell layers; 2 points: Necrotic cells extending from the central vein into three to six cell layers, but confined to the periphery; 3 points: Same as 2, but necrosis extends from one central vein to another; 4 points: More severe than 3, with extensive central necrosis throughout the entire section. Five fields of view at 200x magnification were randomly selected from each section for scoring, and the average score was taken as the sample score.

[0109] 2. Statistical Methods: All measurement data are expressed as mean ± standard deviation (s). One-way ANOVA was used for comparisons among multiple groups. This statistical method is the most commonly used in pharmaceutical experiments for comparing efficacy differences among multiple groups. After confirming differences between groups, the Turky test was used to further verify intra-group differences, i.e., whether the differences between the model group and the control group, and the differences between each dosage group and the model group, were statistically significant. A two-sided p-value < 0.05 was defined as statistically significant.

[0110] 3. Experimental Results

[0111] 3.1 HE staining results

[0112] Six hours after LPS / G-GaIN injury, mouse livers showed extensive loss of intercellular boundaries, hepatic cord loss and necrosis, sinusoidal congestion, and nuclear pyknosis. Compared with the model group, the livers of mice treated with Prdx1 IgG neutralizing antibodies showed significantly reduced lesions and a smaller extent of damage (e.g., Figure 3 As shown in Table 2, the pathological score of liver injury was significantly reduced (P<0.05). Twenty-four hours after APAP injury, extensive apoptosis, nuclear pyknosis, and necrosis were observed in the mouse liver. Compared with the model group, the livers of mice treated with Prdx1 IgG neutralizing antibodies showed significantly reduced lesions and a smaller extent of damage (as shown in Table 2). Figure 4 (As shown in Table 3).

[0113] Table 2. Liver injury scores (X±S) in mice of different groups induced by LPS / G-GaIN.

[0114]

[0115] Table 3. Liver injury scores (X±S) in mice of different groups induced by APAP.

[0116]

[0117] 3.2 Results of serum transaminase detection in each group of mice

[0118] Compared with the control group, the serum transaminase levels in the LPS / D-GaIN model group mice were significantly elevated. After treatment with Prdx1 IgG neutralizing antibody, the serum transaminase levels in the mice significantly decreased, and the difference was statistically significant (P<0.001), as detailed in Table 4. Compared with the control group, the serum transaminase levels in the APAP model group mice were significantly elevated. After treatment with Prdx1 IgG neutralizing antibody, the serum transaminase levels in the mice significantly decreased, and the difference was statistically significant (P<0.01), as detailed in Table 5.

[0119] Table 4. Serum transaminase levels in mice with LPS / D-GaIN-induced liver injury (X±S)

[0120]

[0121] Table 5. Serum transaminase levels in mice with APAP-induced liver injury (X±S)

[0122]

[0123]

[0124] In Tables 2, 3, 4, and 5 above, * compared with the control group, P<0.05; ** compared with the control group, P<0.01; *** compared with the control group, P<0.001; # compared with the control group, P<0.05; ## compared with the control group, P<0.01; ### compared with the control group, P<0.001;

[0125] 4. Experimental conclusion: Prdx1 IgG neutralizing antibody can effectively treat LPS / D-GaIN and APAP-induced acute liver injury in mice.

[0126] Example 3: Application of polyclonal neutralizing antibodies against Prdx1 protein in the preparation of drugs for acute kidney injury.

[0127] This embodiment applies the Prdx1 polyclonal neutralizing antibody from Example 1 to the preparation of drugs for acute kidney injury. The following section will demonstrate the therapeutic effect of Prdx1 IgG polyclonal neutralizing antibody on acute kidney injury caused by ischemia-reperfusion (with oxidative stress and inflammation as the main manifestations of injury):

[0128] 1. Experimental Methods

[0129] (1) Establishment of a mouse model of acute kidney injury (AKI) induced by renal ischemia-reperfusion injury (IRI) to observe the efficacy of Prdx1 IgG neutralizing antibody in treating AKI: Eleven SPF-grade C57BL / 6 mice (8-10 weeks old, male, weighing 23-25g) were divided into three groups: control group, IRI+IgG group, and IRI+Prdx1 neutralizing antibody group. Mice in the IRI+IgG group and the IRI+Prdx1 neutralizing antibody group were anesthetized with chloral hydrate, their back hair was shaved, and incisions were made on both sides of the back to expose and separate the bilateral renal pedicles. The renal pedicles were clamped with non-traumatic microvascular clamps for 30 minutes, and then the clamps were released to start reperfusion. The incisions were then sutured. Throughout the operation, the mouse body temperature was maintained at 36.5-37.5℃ using a temperature-controlled heating device. Twenty hours after modeling, mice in the polyclonal neutralizing antibody treatment group were intraperitoneally injected with 300 μg / mouse of Prdx1 IgG neutralizing antibody, while mice in the normal and model groups were injected with the same dose of control IgG. Twenty-four hours after modeling, mice were anesthetized and blood was collected for serum. The hearts were then perfused with physiological saline, and the mice were sacrificed. The kidneys were separated, and half of the tissue from the same side was fixed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) to observe kidney damage. The remaining tissue was placed in a liquid nitrogen container. Mouse serum was sent for renal function testing.

[0130] 2. Statistical Methods: All measurement data are expressed as mean ± standard deviation (s). One-way ANOVA was used for comparisons among multiple groups. This statistical method is the most commonly used in pharmaceutical experiments for comparing efficacy differences among multiple groups. After confirming differences between groups, the Turky test was used to further verify intra-group differences, i.e., whether the differences between the model group and the control group, and the differences between each dosage group and the model group, were statistically significant. A two-sided p-value < 0.05 was defined as statistically significant.

[0131] 3. Experimental Results

[0132] 3.1 HE staining results

[0133] Following IRI injury, mice exhibited significant tubular epithelial cell necrosis, brush border loss, tubular dilation, and cast formation in the renal tubules. Compared to the model group, treatment with Prdx1 polyclonal neutralizing antibody significantly alleviated these lesions and reduced the extent of damage (e.g., Figure 5 As shown in Table 6, the pathological scores for renal tubular injury were significantly reduced (P < 0.001).

[0134] Table 6. Renal tubular injury scores (X±S) in mice of different groups induced by IRI.

[0135]

[0136] 3.2 Results of blood renal function tests in each group of mice

[0137] Compared with the control group, the serum creatinine of mice in the ischemia-reperfusion model group was significantly increased. After treatment with polyclonal neutralizing antibody against Prdx1 protein, the serum creatinine of mice decreased significantly, and the difference was statistically significant (P<0.01), as detailed in Table 7.

[0138] Table 7. Serum renal function levels (X±S) in mice with IRI-induced kidney injury.

[0139]

[0140] In Tables 6 and 7, * P < 0.05 compared with the control group; ** P < 0.01 compared with the control group; *** P < 0.001 compared with the control group; # Compared with the model group, P<0.05; ## Compared with the model group, P < 0.01; ### Compared with the model group, P < 0.001.

[0141] 4. Experimental conclusion: Polyclonal neutralizing antibodies against Prdx1 protein can effectively treat ischemia-reperfusion induced acute kidney injury in mice.

[0142] Example 4: Preparation of a monoclonal neutralizing antibody against Prdx1 protein

[0143] A monoclonal neutralizing antibody against Prdx1 protein, wherein the neutralizing antibody is a type of IgG antibody that specifically binds to Prdx1 protein, and the preparation method is as follows:

[0144] 1. Prokaryotic expression of human Prdx1 antigen

[0145] 1.1 Construction and identification of human Prdx1 vector

[0146] Using cDNA from human tumor cells U87MG (purchased from Raygene) as a template, PCR amplification was performed using the following primers.

[0147] PRDX1-F: CTGCCAAGTGATTGGTGCTTCTG (SEQ ID NO. 11);

[0148] PRDX1-R: AATGGTGCGCTTCGGGTCTGAT (SEQ ID NO. 12).

[0149] The Prdx1 gene fragment with EcoRI / XhoI restriction sites at both ends was obtained and double-digested with EcoRI / XhoI (purchased from NEB) to obtain the target gene fragment Prdx1. The vector plasmid pET22b (purchased from Novogen) was double-digested with EcoRI / XhoI, and the vector fragment was recovered after agarose gel electrophoresis. This fragment was then ligated to the aforementioned target gene fragment Prdx1 using T4 ligase (purchased from NEB), and subsequently transformed into *E. coli* TOP10 (purchased from LIFE). After ampicillin resistance screening, positive clones containing the inserted fragment were identified by EcoRI / XhoI digestion, and the results were verified by sequencing to obtain the prokaryotic expression plasmid pET22b-Prdx1 containing the correct human Prdx1 gene sequence.

[0150] 1.2 Expression and purification of human Prdx1 protein

[0151] The pET22b-Prdx1 plasmid was transformed into the expression host bacterium BL21(DE3) (purchased from Novagen), and the cells were spread on ampicillin-resistant plates and incubated upside down at 37°C overnight. Single colonies were picked for induction expression. After picking single colonies, the cells were cultured with shaking until the OD600 reached 0.6-0.8. IPTG was added to a final concentration of 1 mM, and the cells were induced at 30°C for 4 h before the bacterial culture was collected. The precipitate was collected by centrifugation, and resuspended in 1 / 10 volume of buffer A (50mM NaH2PO4, 300mM NaCl, 10mM imidazole, pH 8.0). PMSF (final concentration 1mM) was added, and the mixture was sonicated on ice (3 seconds, 10-second intervals, 99 times per round, for a total of 4 rounds). The mixture was then centrifuged (4℃, 12000g) for 15 min. The supernatant was collected by centrifugation and purified by Ni-NTAAgarose (purchased from QIAGEN) affinity column chromatography to obtain the target Prdx1 protein. The protein was then dialyzed against PBS, and the purity of the purified protein after dialyzing was analyzed by 12% SDS-PAGE. The content was detected by A280.

[0152] 2. Preparation and identification of monoclonal neutralizing antibodies against human Prdx1 protein

[0153] 2.1 Recombinant protein immunization

[0154] 1 mL of purified human Prdx1 protein (1.0 mg / mL) was used as the antigen and thoroughly emulsified with 1 mL of complete Freund's adjuvant (purchased from Sigma-Aldrich) to subcutaneously immunize 6-8 week old BALB / c mice, with each mouse receiving 100 μg of human Prdx1 antigen. Four weeks later, human Prdx1 antigen was emulsified with incomplete Freund's adjuvant and injected intraperitoneally into mice at a dose of 50 μg per mouse. A booster immunization of 50 μg of antigen was administered intraperitoneally every two weeks. One week after the fourth booster immunization, the mice were coated with the purified human Prdx1 protein obtained above, and the antiserum titer was measured by ELISA. Continuous booster immunizations were performed until the antiserum titer reached >10⁵. Three weeks after the final booster immunization, 20 μg of the above-mentioned human Prdx1 protein was administered intraspleurally for later use.

[0155] 2.2 Establishment of human Prdx1 hybridoma cell line

[0156] Four days after intrasplenic booster immunization in mice, spleens were aseptically harvested, and lymphocytes were isolated by filtering through a 100-mesh filter. These lymphocytes were then fused with the myeloma cell line SP2 / 0 and selectively cultured for 3 days with hypoxanthine, aminopterin, and thymidine (HAT). HT medium was then added, and the cells were cultured for another week. Positive clones were selected by ELISA using the human Prdx1 antigen described above, and subcloning was performed three times using limiting dilution. The cells were then continuously cultured for two months to obtain a stable hybridoma cell line.

[0157] 2.3 Antibody Purification

[0158] Take the above hybridoma cell clones separately, and use 5 × 10 5 Cells / mouse amounts were injected intraperitoneally into the corresponding groups of mice to prepare ascites. 100 mL of ascites was diluted with twice the volume of 0.06 M pH 4.0 sodium acetate buffer, and 4% caprylic acid was slowly added dropwise while stirring. The mixture was stirred for 30 min, then centrifuged at 10000 g for 30 min. The precipitate was discarded, and the supernatant was dialyzed overnight with 0.01 M pH 7.4 phosphate buffer. The dialysate was collected, and an equal volume of saturated ammonium sulfate was slowly added, and the mixture was allowed to stand for 2 hours. The turbidity was centrifuged at 10000 g for 10 min. The supernatant was discarded, and the solution was dissolved in 0.01 M pH 7.4 PBS. The dissolved solution was dialyzed against 0.01 M pH 7.4 PBS, with two buffer changes, the interval between which should not be less than 5 hours. The dialysate was centrifuged at 10000 g for 10 min, the precipitate was discarded, and the supernatant was collected.

[0159] The protein G affinity column (purchased from GE) was brought to room temperature and equilibrated with 5 column volumes of PBS (0.01M PB, 0.15M NaCl, pH 7.4). The supernatant collected earlier was loaded onto the column and washed with 5 column volumes of PBS. Elution was performed with 0.1M glycine hydrochloride solution at pH 2.3, and the eluent was neutralized with 1 / 10 volume of 1M disodium hydrogen phosphate solution at pH 9.0. The solution was dialyzed against 0.01M PBS at pH 7.4, with two buffer changes at intervals greater than 5 hours. The dialysate was centrifuged at 10000g for 10 min, and the supernatant was filtered through a 0.22μm filter and stored to obtain purified monoclonal neutralizing antibodies corresponding to each clone, hereinafter referred to as "Prdx1 monoclonal antibody".

[0160] The obtained Prdx1 monoclonal neutralizing antibody was tested for its affinity for human Prdx1 recombinant protein using ELISA, confirming that it has good affinity for human Prdx1 (see Table 8).

[0161] Table 8 shows the binding activity of Prdx1 monoclonal antibody to recombinant prdx1 as detected by ELISA.

[0162]

[0163] Monoclonal neutralizing antibodies were used as the primary antibody for Western blot analysis, and compared with the commercially available Prdx1 Western Blot primary antibody to produce immunoblot images of different mouse tissues (see [link]). Figure 1 B) The binding bands of the two are basically the same. It can be seen that the human Prdx1 monoclonal antibody obtained in this embodiment can also bind to mouse Prdx1 protein.

[0164] Mouse primary macrophages were stimulated with human Prdx1 protein (150 nM) for 24 h, and the effect of human Prdx1 protein was antagonized by the aforementioned monoclonal neutralizing antibody (8 μg / ml). The levels of IL-6 and TNF-α in the cell supernatant were detected using an IL-6 and TNF-α ELISA kit (purchased from Invitrogen). The results showed that human Prdx1 protein promoted the secretion of IL-6 and TNF-α by mouse primary macrophages; while the Prdx1 monoclonal antibody inhibited this effect of Prdx1 protein (see...). Figure 6 The difference was statistically significant, indicating that the Prdx1 monoclonal antibody can functionally antagonize the effect of the human Prdx1 protein.

[0165] 3. Sequencing of monoclonal neutralizing antibodies against Prdx1 protein

[0166] The TransGold RNA Extraction Kit (purchased from TransGold Inc.) is used to extract RNA from hybridoma cells (see the TransGold RNA Extraction Kit instruction manual for detailed steps).

[0167] Using the reverse transcription product as a template, the target gene was amplified by high-fidelity enzyme PCR. The primers used were:

[0168] Heavy chain primer: GGGAATTCATGRAATGSASCTGGGTYWTYCTCTT (SEQ ID NO.13);

[0169] Light chain primers:

[0170] 1: ACTAGTCGACATGAGTGTGCYCACTCAGGTCCTGGSGTT (SEQ ID NO. 14);

[0171] 2: ACTAGTCGACATGTGGGGAYCGKTTTYAMMCTTTTCAATTG (SEQ ID NO. 15);

[0172] 3: ACTAGTCGACATGGAAGCCCCAGCTCAGCTTCTCTTCC (SEQ ID NO. 16).

[0173] The PCR products obtained from the above amplification were excised by conventional gel electrophoresis; after dissolving in the gel, the target band was purified using an adsorption column and ligated into 0.2 mL PCR tubes according to the conditions in Table 9 (VH: RT, 3 h; VL: 25 °C, 10 min):

[0174] Table 9 Connection Conditions

[0175]

[0176] Add 5 μL of ligation product to approximately 50 μL of TOP10 competent cells, gently tumble to mix, incubate on ice for 30 min, then place the centrifuge tube in a 42°C water bath for 60 s. Immediately after removal, place on ice for 2 min. Add 200 μL of LB (without antibiotics) medium to the centrifuge tube and incubate at 37°C for 1 h. VH: Blue-white screening. 2 hours in advance, take A+ plates and add 16 μL (50 mg / mL) of IPTG and 40 μL (20 mg / mL) of X-Gal. Mix well and spread evenly on the plate. Incubate at 37°C in the dark until use. VL: No blue-white screening, normal ampicillin or kanamycin plate. Aspirate the bacterial culture onto the plate, spread the cells evenly with a bent glass rod, and after the plate surface dries, invert and incubate at 37°C overnight (16 h).

[0177] Ten clones were selected from each sample for sequencing, and all sequencing results were integrated into a FASTA format file. The heavy and light chain amino acid sequences of the monoclonal neutralizing antibody Prdx1 were then obtained by analysis in the IMGT database.

[0178] The amino acid sequences of the heavy chain variable region and the light chain variable region, determined according to the IMGT database, are shown in SEQ ID NO. 7 and SEQ ID NO. 8, respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region, determined according to the IMGT scheme numbering rules, are shown in SEQ ID NO. 1, 2, and 3, respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region, determined according to SEQ ID NO. 4, 5, and 6, respectively. The nucleotide sequence encoding the total heavy chain variable region is shown in SEQ ID NO. 9, and the nucleotide sequence encoding the total light chain variable region is shown in SEQ ID NO. 10.

[0179] SEQ ID NO.1: Monoclonal neutralizing antibody against Prdx1 protein, heavy chain CDR1: GYTFSNSW.

[0180] SEQ ID NO.2: Monoclonal neutralizing antibody against Prdx1 protein, heavy chain CDR2:ILPGSGDT.

[0181] SEQ ID NO.3: Monoclonal neutralizing antibody against Prdx1 protein, heavy chain CDR3: ASPYGDYFFAN.

[0182] SEQ ID NO.4: Monoclonal neutralizing antibody against Prdx1 protein, light chain CDR1: KSLLYKDGKTY.

[0183] SEQ ID NO.5: Monoclonal neutralizing antibody against Prdx1 protein, light chain CDR2:LMS.

[0184] SEQ ID NO.6: Monoclonal neutralizing antibody against Prdx1 protein, light chain CDR3:QQLVEYPYT.

[0185] SEQ ID NO.7: Amino acid sequence of the heavy chain variable region of a monoclonal neutralizing antibody against Prdx1 protein:

[0186] QVQLQQSGPELMKPGASVKISCKATGYTFSNSWIEWVKQRPGHGLEWIGEILPGSGDTDYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCASPYGDYFFANWGLGTLVTVSA.

[0187] SEQ ID NO.8: Amino acid sequence of the light chain variable region of a monoclonal neutralizing antibody against Prdx1 protein:

[0188] DIVITQDELSNPVTSGESVSISCRSSKSLLYKDGKTYLNWFLQRPGQSPQLLIYLMSTRAAGVSDRFSGSGSGTDFTLEISRVKAEDVGVYYCQQLVEYPYTFGGGTKLEIK.

[0189] SEQ ID NO.9: Nucleotide sequence of the heavy chain variable region of a monoclonal neutralizing antibody against Prdx1 protein:

[0190] ACTTAGGCATCCACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCGAATTCACTAGTGATTCCCAAGCTTCCAGGGGCCAATGGATAAACGGGTGGGGCTGTTGTTTTGGCTGCAGAGACAGTGACCAGAGTCCCTAGGCCCCAGTTAGCAAAGAAGTAGTCACCATAGGGGCTTGCACAGTAATAGACGGCAGAGTCCTCAGATGTCAGGCTGCTGAGTTGCATGTAGGCTGTGTTGGAGGATGTATCTGCAGTGATTGTGGCCTTGCCCTTGAACTTCTCATTGTAGTCAGTATCACCACTTCCAGGTAAAATCTCTCCAATCCACTCAAGGCCATGTCCAGGCCTCTGTTTTACCCACTCTATCCAGGAGTTACTGAATGTGTAGCCAGTAGCCTTGCAGGATATCTTCACTGAGGCCCCAGGCTTCATCAGCTCAGGTCCAGACTGCTGCAGCTGAACCTGGGAGTGGACACCTGCAGTTACTGACAGGAGGAAGAGGAAAACCCAGCTCCATTCCATGAATTCCCAATCGAATTCCCGCGGCCGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTAGTGCTTTACGGCACCTCGACCCCAAAAACTTGATTAGGT。

[0191] SEQ ID NO.10: Nucleotide sequence of the light chain variable region of a monoclonal neutralizing antibody against Prdx1 protein:

[0192] AGTGAAGCATCCACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCGAATTCACTAGTGATTACTAGTCGACATGAGTGTGCTCACTCAGGTCCTGGGGTTGCTTATGTTCTGGATCTCTGGAGTCAGTGGGGATATTGTGATAACCCAGGATGAACTCTCCAATCCTGTCACTTCTGGAGAATCAGTTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTATATAAGGATGGGAAGACATACTTGAATTGGTTTCTGCAGAGACCAGGACAATCTCCTCAGCTCCTGATCTATTTGATGTCCACCCGTGCAGCAGGAGTCTCAGACCGGTTTAGTGGCAGTGGGTCAGGAACAGATTTCACCCTGGAAATCAGTAGAGTGAAGGCTGAGGATGTGGGTGTGTATTACTGTCAACAACTTGTGGAGTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTAAGCTTGGGAATCGAATTCCCGCGGCCGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGG。

[0193] Example 5: Monoclonal neutralizing antibodies against Prdx1 protein alleviated elevated serum transaminase levels and liver pathological damage in a mouse model of acute liver injury.

[0194] 1. Laboratory animals

[0195] This experiment used SPF-grade 10-12 week old male C57BL / 6J mice, weighing 26-28g (provided by the Department of Experimental Animals, Central South University). The mice were housed under SPF conditions, 5-6 mice per cage, with free access to water and feed; the animals were randomly assigned to groups.

[0196] The experiment was randomly divided into three groups:

[0197] (1) Normal control group: Normal mice, no modeling, were given control IgG as a control;

[0198] (2) LPS / D-Galn model group: an acute liver injury model was established, and control IgG was given as a control treatment;

[0199] (3) LPS / D-Galn+ anti-Prdx1 protein monoclonal neutralizing antibody treatment group: 350 μg of monoclonal neutralizing antibody was injected intraperitoneally 2 hours after modeling.

[0200] 2. Experimental Methods

[0201] Mice in the model group and the anti-Prdx1 protein monoclonal neutralizing antibody treatment group were induced by intraperitoneal injection of 100 μg / kg LPS and 500 mg / kg D-GaIN. Two hours after modeling, mice in the neutralizing antibody treatment group were intraperitoneally injected with 300 μg / mouse of Prdx1 IgG neutralizing antibody, while mice in the normal group and model group were injected with the same dose of control IgG. Four hours after modeling, mice were anesthetized and blood was collected for serum, after which the mice were sacrificed.

[0202] Mouse livers were isolated, and the left lateral lobe was used for paraffin sectioning. The remaining liver tissue was placed in a liquid nitrogen container. Liver function in each group of mice was assessed using a continuous monitoring method, and the paraffin sections were stained with hematoxylin and eosin (HE) to observe liver damage.

[0203] The LPS / D-GaIN model liver tissue injury scoring criteria were used to perform a semi-quantitative analysis of liver pathological injury, referencing the method employed by CARLOS A. et al. The scoring criteria were as follows: Grade 0: little or no evidence of injury; Grade 1: mild injury, cytoplasmic vacuolation, focal nuclear pyknosis; Grade 2: moderate to severe injury, with widespread nuclear pyknosis, eosinophilia, and loss of intercellular boundaries; Grade 3: severe necrosis with hepatic cord loss, hemorrhage, and neutrophil infiltration.

[0204] Statistical methods: All measurement data are expressed as mean ± standard deviation (s). One-way ANOVA was used for comparisons among multiple groups; this is the most commonly used statistical method in pharmaceutical experiments for comparing efficacy differences among multiple groups. After confirming differences between groups, the Turky test was used to further verify intra-group differences, i.e., whether the differences between the model group and the control group, and the differences between each dose group and the model group, were statistically significant. A two-sided p-value < 0.05 was defined as statistically significant.

[0205] 3. Experimental Results:

[0206] 3.1 HE staining results

[0207] Four hours after LPS / G-GaIN injury, mouse livers showed extensive loss of intercellular boundaries, hepatic cord loss and necrosis, sinusoidal congestion, and nuclear pyknosis. Compared with the model group, the livers of mice treated with Prdx1 IgG neutralizing antibodies showed significantly reduced lesions and a smaller extent of damage (e.g., Figure 7 As shown in Table 10, the pathological scores of liver damage were significantly reduced (P<0.05).

[0208] Table 10 Liver injury scores (X±S) in mice of different groups induced by LPS / G-GaIN.

[0209]

[0210] 3.2 Results of serum transaminase detection in each group of mice

[0211] Compared with the control group, the serum transaminase levels in the LPS / D-GaIN model group mice were significantly elevated. After treatment with Prdx1 monoclonal neutralizing antibody, the serum transaminase levels in the mice were significantly reduced, and the difference was statistically significant (P<0.001), as detailed in Table 11.

[0212] Table 11 Serum transaminase levels (X±S) in mice with LPS / D-GaIN-induced liver injury.

[0213]

[0214]

[0215] In Tables 10 and 11 above, * compared with the normal group, P<0.05; ** compared with the normal group, P<0.01; *** compared with the normal group, P<0.001; # Compared with the model group, P<0.05; ## Compared with the model group, P < 0.01; ### Compared with the model group, P < 0.001.

[0216] 4. Experimental conclusion: Prdx1 monoclonal neutralizing antibody can effectively treat LPS / D-Galn-induced acute liver injury in mice.

[0217] Example 6: Monoclonal neutralizing antibodies against Prdx1 protein improve renal function and renal tubular injury in a mouse model of acute kidney injury.

[0218] 1. Laboratory animals

[0219] This experiment used SPF-grade (8-10 weeks old C57BL / 6J male mice, weighing 23-25g) (provided by the Department of Experimental Animals, Central South University). The animals were housed under SPF conditions, 5-6 mice per cage, with free access to water and feed; the animals were randomly assigned to groups.

[0220] The experiment was randomly divided into three groups:

[0221] (1) Normal control group: Normal mice, no modeling, were given control IgG as a control;

[0222] (2) Model group: An acute kidney injury model was established, and control IgG was given as a control treatment;

[0223] (3) Model + anti-Prdx1 protein monoclonal neutralizing antibody treatment group: monoclonal neutralizing antibody was injected intraperitoneally after modeling;

[0224] 2. Experimental Methods

[0225] (1) To prepare an animal model of AKI induced by lipopolysaccharide (LPS) to observe the efficacy of monoclonal neutralizing antibody against Prdx1 protein in treating AKI characterized by inflammation and renal tubular epithelial cell degeneration:

[0226] Mice in the model group and the Prdx1 monoclonal neutralizing antibody treatment group were induced by intraperitoneal injection at a dose of 15 mg / kg LPS. Ten hours after modeling, mice in the neutralizing antibody treatment group were intraperitoneally injected with 50 μg / mouse of Prdx1 monoclonal neutralizing antibody, while mice in the normal group and the model group were injected with the same dose of control IgG. Twelve hours after modeling, mice were anesthetized and blood was collected for serum. Subsequently, the hearts were perfused with physiological saline and the mice were sacrificed.

[0227] Mouse kidneys were isolated, and half of the tissue from the same side was fixed, paraffin-embedded, and stained with hematoxylin and eosin (HE) to observe kidney damage. The remaining kidney tissue was placed in a liquid nitrogen container. Mouse serum was sent for renal function testing.

[0228] The AKI model scoring criteria for renal tubular injury referenced the method used by Fang et al. to perform semi-quantitative analysis of pathological renal tubular injuries (such as renal tubular epithelial cell necrosis, brush border loss, and cast formation). Scoring criteria: 0 = no renal tubular injury; 1 = renal tubular injury range >0% and <25%; 2 = renal tubular injury range 25%-50%; 3 = renal tubular injury range 51%-75%; 4 = renal tubular injury range 76%-100%. For each slide, 10 fields of view at 200x magnification were randomly selected for scoring, and the average score was taken as the sample score.

[0229] (3) Statistical methods: All measurement data are expressed as mean ± standard deviation (s). One-way ANOVA was used for comparisons among multiple groups. This statistical method is the most commonly used method in pharmaceutical experiments for comparing efficacy differences among multiple groups. After confirming differences between groups, the Turky test was used to further verify intra-group differences, i.e., whether the differences between the model group and the control group, and the differences between each dose group and the model group, were statistically significant. A two-sided P < 0.05 was defined as statistically significant.

[0230] 3. Experimental Results

[0231] 3.1 HE staining results

[0232] Following LPS injury, mouse renal tubules exhibited significant vacuolar degeneration and necrosis of tubular epithelial cells, loss of the brush border, luminal dilation, and extensive cast formation. Treatment with monoclonal neutralizing antibodies against Prdx1 protein significantly reduced the renal lesions in mice (see...). Figure 8 Furthermore, the pathological score of renal tubular injury was significantly reduced (P<0.01), as detailed in Table 12.

[0233] Table 12 Renal tubular injury scores (X±S) in mice of different groups induced by LPS-induced kidney injury.

[0234]

[0235] 3.2 Results of blood renal function tests in each group of mice

[0236] Compared with the control group, the serum creatinine levels of mice in the IRI model group were significantly elevated. After treatment with Prdx1 IgG neutralizing antibody, the serum creatinine levels of mice decreased significantly, and the difference was statistically significant (P<0.01), as detailed in Table 11. Compared with the control group, the serum creatinine levels of mice in the LPS model group were significantly elevated. After treatment with Prdx1 monoclonal neutralizing antibody, the serum creatinine levels of mice decreased significantly, and the difference was statistically significant (P<0.001), as detailed in Table 13.

[0237] Table 13 Serum renal function levels (X±S) in mice with LPS-induced kidney injury.

[0238]

[0239] In Tables 12 and 13, * P < 0.05 compared with the control group; ** P < 0.01 compared with the control group; *** P < 0.001 compared with the control group; # Compared with the model group, P<0.05; ## Compared with the model group, P < 0.01; ### Compared with the model group, P < 0.001.

[0240] 4. Experimental conclusion: Prdx1 monoclonal neutralizing antibody can effectively treat IRI / LPS-induced acute kidney injury in mice.

[0241] Example 7: Monoclonal neutralizing antibodies against Prdx1 protein reduce mortality in sepsis model mice

[0242] 1. Laboratory animals

[0243] This experiment used SPF-grade (8-10 weeks old C57BL / 6J male mice, weighing 23-25g) (provided by the Department of Experimental Animals, Central South University). The animals were housed under SPF conditions, 5-6 mice per cage, with free access to water and feed; the animals were randomly assigned to groups.

[0244] The experiment was randomly divided into two groups:

[0245] (1) Model group: A sepsis model was established, and control IgG was given as a control treatment;

[0246] (3) Model + anti-Prdx1 protein monoclonal neutralizing antibody treatment group: monoclonal neutralizing antibody was injected intraperitoneally after modeling;

[0247] 2. Experimental Methods

[0248] (1) To establish an experimental animal model of LPS sepsis in order to observe the efficacy of Prdx1 monoclonal neutralizing antibody in the treatment of sepsis with systemic inflammation as the main manifestation:

[0249] Mice in the model group and the Prdx1 monoclonal neutralizing antibody treatment group were induced by intraperitoneal injection at a dose of 15 mg / kg LPS. Twelve hours after modeling, mice in the neutralizing antibody treatment group were intraperitoneally injected with 75 μg / mouse of Prdx1 monoclonal neutralizing antibody, while mice in the model group were injected with the same dose of control IgG. The mortality rate of the mice was observed.

[0250] (2) Statistical methods: All measurement data are expressed as mean ± standard deviation (s). The t-test was used for comparisons between two groups. This statistical method is the most commonly used method in drug research for comparing the efficacy differences between two groups. A two-sided p < 0.05 is defined as statistically significant.

[0251] 3. Experimental Results

[0252] Following LPS modeling, mice showed significant mortality. However, treatment with monoclonal neutralizing antibodies against Prdx1 protein significantly reduced the mortality rate in mice (see details). Figure 9 ).

[0253] 4. Experimental conclusion: Prdx1 monoclonal neutralizing antibody can effectively treat LPS-induced sepsis in mice.

[0254] Example 8: Monoclonal neutralizing antibodies against Prdx1 protein reduced mortality in mice with bleomycin-induced acute lung injury.

[0255] 1. Laboratory animals

[0256] This experiment used SPF-grade (8-10 weeks old C57BL / 6J male mice, weighing 23-25g) (provided by the Department of Experimental Animals, Central South University). The animals were housed under SPF conditions, 5-6 mice per cage, with free access to water and feed; the animals were randomly assigned to groups.

[0257] The experiment was randomly divided into 3 groups:

[0258] (1) Control group: Normal mice underwent only surgical treatment, were not exposed to bleomycin, and were treated only with control IgG antibody;

[0259] (2) Model group: Bleomycin was used to establish the model; and control IgG antibody was used for treatment.

[0260] (3) Model + anti-Prdx1 protein monoclonal neutralizing antibody treatment group: After modeling, the patient was treated with intraperitoneal injection of anti-Prdx1 protein monoclonal neutralizing antibody.

[0261] 2. Experimental Methods

[0262] (1) To establish an experimental animal model of bleomycin (BLM)-induced acute lung injury in order to observe the efficacy of monoclonal neutralizing antibody against Prdx1 protein in treating acute lung injury characterized by alveolar epithelial cell necrosis, alveolar structural disorder, and inflammatory cell infiltration:

[0263] Mice were anesthetized with pentobarbital. The neck skin was disinfected, and the trachea was exposed by incision of the neck skin. Mice in the model group and treatment group were slowly injected with BLM (5 mg / kg) via insulin injection, while mice in the control group were injected with an equal volume of physiological saline. The skin was then sutured in layers and disinfected. The mice were placed headfirst on the experimental table and manually fixed, then rotated horizontally several times. The mice were placed on a warming blanket and returned to their cages after waking. Three days after modeling, mice in the neutralizing antibody treatment group received an intraperitoneal injection of a monoclonal neutralizing antibody against Prdx1 protein (150 μg / mouse). Seven days after modeling, mice were anesthetized, and lung tissue was collected. The left lung was fixed whole with tissue fixative, and paraffin sections were prepared for HE staining to observe lung tissue damage. The right lung tissue was preserved in a liquid nitrogen tank.

[0264] The lung injury scoring criteria for the acute lung injury model were based on the method used by Barriga M et al., which performed a semi-quantitative analysis of pathological damage to lung tissue (alveolar inflammatory infiltration, alveolar wall thickening, and alveolar hemorrhage). Scoring criteria: 0 points: Normal alveolar structure, no inflammatory cell infiltration. 1 point: Mild alveolar structural disorder, localized small amount of inflammatory cell infiltration, alveolar hemorrhage, damage area <25% of the visual field. 2 points: Moderate alveolar structural destruction. Significant inflammatory cell infiltration, alveolar hemorrhage, damage area 25-50% of the visual field. 3 points: Significant alveolar destruction, abundant inflammatory cell infiltration, alveolar hemorrhage, damage area 51-75% of the visual field. 4 points: Severe alveolar structural destruction, extensive disappearance of alveolar structure, abundant inflammatory cell infiltration, alveolar hemorrhage, alveolar consolidation, damage area 76-100% of the visual field. Ten fields of view (200x magnification) were randomly selected from each slide for scoring, and the average score was taken as the sample score.

[0265] 2. Statistical Methods: All measurement data are expressed as mean ± standard deviation (X±S). One-way ANOVA was used for comparisons among multiple groups. This statistical method is the most commonly used in pharmaceutical experiments for comparing efficacy differences among multiple groups. After confirming differences between groups, the Turky test was used to further verify intra-group differences, i.e., whether the differences between the model group and the control group, and the differences between each dosage group and the model group, were statistically significant. A two-sided p-value < 0.05 was defined as statistically significant.

[0266] 3. Experimental Results

[0267] 3.1 HE staining results

[0268] Following BLM injury, mouse lung tissue exhibited significant alveolar inflammatory cell infiltration and alveolar structural disorder. Compared to the model group, treatment with a monoclonal neutralizing antibody against Prdx1 protein (150 μg / mouse) significantly alleviated these lesions. Figure 10 The lung tissue pathological damage score was significantly reduced (P<0.01), as detailed in Table 14.

[0269] Table 14. Lung pathological damage scores (X±S) in mice of different groups during BLM lung injury.

[0270]

[0271]

[0272] In Table 13, compared with the control group, ***, P<0.001; ## Compared with the model group, P<0.01.

[0273] 4. Experimental conclusion: Administration of 150 μg of monoclonal neutralizing antibody against Prdx1 protein can effectively alleviate bleomycin-induced acute lung injury in mice.

Claims

1. A Prdx1 IgG neutralizing antibody, characterized in that, The amino acid sequence of the CDR1 region of the heavy chain variable region of the antibody is shown in SEQ ID NO. 1, the amino acid sequence of the CDR2 region of the heavy chain variable region is shown in SEQ ID NO. 2, and the amino acid sequence of the CDR3 region of the heavy chain variable region is shown in SEQ ID NO. 3; the amino acid sequence of the CDR1 region of the light chain variable region of the antibody is shown in SEQ ID NO. 4, and the amino acid sequence of the CDR2 region of the light chain variable region is shown in SEQ ID NO.

5. The amino acid sequence of the CDR3 region of its light chain variable region is shown in SEQ ID NO.

6.

2. The Prdx1 IgG neutralizing antibody according to claim 1, characterized in that, The antibody comprises amino acids in a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 7 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8.

3. The Prdx1 IgG neutralizing antibody according to any one of claims 1-2, characterized in that, The antibodies include Prdx1 neutralizing antibodies derived from rabbits, humans, mice, rats, or human-mouse chimeric sources.

4. A nucleic acid molecule encoding the Prdx1 IgG neutralizing antibody according to any one of claims 1-3, characterized in that, It includes nucleotides encoding the heavy chain variable region and the light chain variable region of the antibody, wherein the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO. 9, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.

10.

5. The use of a Prdx1 IgG neutralizing antibody as described in any one of claims 1-3 in the preparation of drugs for acute organ injury or sepsis, characterized in that, The acute organ injury refers to acute liver injury, acute kidney injury, or acute lung injury.

6. The application according to claim 5, characterized in that, The acute liver injury includes lipopolysaccharide / D-galactosamine-induced or acetaminophen-induced acute liver injury.

7. The application according to claim 6, characterized in that, The lipopolysaccharide / D-galactosamine-induced acute liver injury clinically corresponds to acute liver injury caused by viral infection; the acetaminophen-induced acute liver injury clinically corresponds to acute liver injury caused by drugs, toxins, or alcohol.

8. The application according to claim 5, characterized in that, The acute kidney injury includes acute kidney injury induced by renal ischemia-reperfusion injury; the manifestations of acute kidney injury induced by renal ischemia-reperfusion injury include oxidative stress or inflammatory damage.

9. The application according to claim 5, characterized in that, The acute lung injury includes bleomycin-induced, pneumonia, non-pulmonary sepsis, aspiration of gastric contents, or ventilator-induced acute lung injury.

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