Fully human-derived antagonistic antibody taking lactylated STAT6 as target spot and application of fully human-derived antagonistic antibody

Abstract

The invention belongs to the technical field of gene engineering and protein engineering, and particularly relates to a fully human antagonistic antibody taking a lactylated STAT6 protein as a target spot and application of the fully human antagonistic antibody. According to the present invention, the completely humanized antagonistic antibody using the lactylated STAT6 protein as the target spot is screened from the human scFv phage antibody library, the amino acid sequence of the completely humanized antagonistic antibody is represented by SEQ ID NO: 1, and the completely humanized antagonistic antibody has the specificity of the targeted lactylated STAT6 protein, and has the application prospect in preparation of the product for diagnosis, treatment or alleviation of sepsis immunosuppression.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and protein engineering technology, specifically to a fully human antagonistic antibody targeting lactolyzed STAT6 and its applications. Background Technology

[0002] Sepsis is a life-threatening syndrome of organ dysfunction caused by a dysregulation of the systemic inflammatory response triggered by infection, commonly seen in patients with severe trauma or infectious diseases. During sepsis, inflammation and immunosuppression may occur sequentially or simultaneously. In the early stages of the systemic inflammatory response, immune balance can be rapidly restored if the immune system can quickly clear pathogens. If pathogens are not cleared in time, it leads to immune dysregulation. In this situation, patients are prone to secondary infections, leading to long-term immunosuppression, immune failure, and bodily dysfunction, also known as persistent inflammatory-immunosuppressive-catabolic syndrome. When sepsis patients require intensive care, one-third survive no more than 30 days. Due to immunosuppression, nearly half of sepsis survivors are readmitted at least once a year, and sepsis survivors have longer readmission times and a higher risk of death. Specifically, the mortality rate for sepsis survivors in the first year after discharge is 15%, and the mortality rate over the next 5 years is 6-8%. Therefore, maintaining immune homeostasis and mitigating immunosuppression play a crucial role in the pathophysiology of sepsis and determine clinical prognosis.

[0003] Currently, there are no effective means to control immunosuppression in sepsis in clinical practice. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the existing technology and to provide a fully human antagonistic antibody targeting lactated STAT6 and its application.

[0005] In a first aspect, the present invention provides a fully human antagonistic antibody targeting lactated STAT6, wherein the fully human antagonistic antibody is a single-chain antibody and its amino acid sequence is shown in SEQ ID NO: 1.

[0006] In a second aspect, the present invention provides a gene encoding a fully human antagonistic antibody targeting lactolyzed STAT6 as described above.

[0007] Preferably, the nucleotide sequence of the gene is shown in SEQ ID NO: 2.

[0008] A third aspect of the present invention provides an expression vector containing the gene described above.

[0009] In a fourth aspect, the present invention provides a recombinant cell containing the expression vector described above.

[0010] In a fifth aspect, the invention provides the use of the fully human antagonistic antibody targeting lactated STAT6 as described above in the preparation of products for diagnosing sepsis immunosuppression.

[0011] In a sixth aspect, the present invention provides a product for diagnosing sepsis-induced immunosuppression, comprising, as described above, a fully human antagonistic antibody targeting lactated STAT6.

[0012] In a seventh aspect, the invention provides the use of the fully human antagonistic antibody targeting lactated STAT6 as described above in the preparation of a medicament for treating or alleviating immunosuppression in sepsis.

[0013] In an eighth aspect, the present invention provides a medicament for treating or alleviating sepsis-induced immunosuppression, comprising a fully human antagonistic antibody targeting lactated STAT6 as described above.

[0014] The beneficial effects of the present invention are as follows: The present invention screens fully human antagonistic antibodies targeting lactated STAT6 protein from a human scFv phage antibody library. The amino acid sequence of the antibody is shown in SEQ ID NO: 1. The fully human antagonistic antibody has specificity in targeting lactated STAT6 protein and has the application prospect of preparing diagnostic, therapeutic or immunosuppressive products for sepsis. Attached Figure Description

[0015] 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 only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0016] Figure 1 The figure shows the STAT6 lactation level of PBMCs and monocytes in the blood of patients with sepsis and immunosuppression and healthy controls in Example 1 of this invention. Figure 2 The image shows a secondary mass spectrum of amino acid sites that have undergone lactation modification in STAT6; Figure 3 The absorbance results of monoclonal phage ELISA detection of lactated STAT6 antibody are shown in the figure. Figure 4 The number of Treg cells in the blood of sepsis-induced immunosuppressed mice after treatment with lactated STAT6 antagonist antibodies; Figure 5 Survival rate of sepsis-infected immunosuppressed mice treated with lactated STAT6 antagonistic antibodies after secondary infection with Aspergillus fumigatus; Figure 6 Survival rate of sepsis-infected immunosuppressed mice treated with lactated STAT6 antagonistic antibodies after secondary infection with Legionella pneumophila; Figure 7 The bacterial load in the lungs of immunosuppressed mice with sepsis treated with lactated STAT6 antagonistic antibodies after secondary infection with Aspergillus fumigatus or Legionella pneumophila. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0018] This invention provides a fully human antagonistic antibody targeting lactolyzed STAT6. It is a single-chain antibody that has specificity in targeting lactolyzed STAT6, and its amino acid sequence is shown in SEQ ID NO: 1.

[0019] The DNA of the aforementioned single-chain antibody can be obtained using conventional gene recombination techniques. After obtaining the DNA sequence encoding the single-chain antibody via PCR, it is cloned into vectors, which can be plasmids, viruses, or gene fragments commonly used in molecular biology. A protein secretion signal peptide sequence is added to the front end of the DNA sequence encoding the antibody to ensure that the antibody can be secreted from the cell. The vector sequence includes a promoter for gene expression, protein translation initiation and termination signals, and a polyadenylate (PolyA) sequence. The vector contains an antibiotic resistance gene to facilitate replication and expression in host cells such as bacteria and eukaryotic cells. Additionally, the vector includes a eukaryotic cell selectivity gene for the stable selection of host cell lines for transfection.

[0020] The single-chain antibody of the present invention can be further fused with other effector molecules to achieve other additional effects without affecting its targeting, and all of these fall within the scope of the present invention.

[0021] After constructing the plasmid containing the DNA sequence encoding the aforementioned antibody, the recombinant vector can be used to transfect or transform host cells to express the corresponding protein. Various expression systems can be used to express antibodies, including eukaryotic and prokaryotic cells, such as mammalian cells, insect cells, yeast, and bacteria. Because prokaryotic cells readily form inclusion bodies when expressing antibodies, mammalian cells are the preferred system for expressing this protein. Various mammalian cells can be used for large-scale antibody expression, such as CHO cells, 293T cells, NSO cells, and COS cells, all of which are included in the cell types applicable to this invention. The recombinant plasmid containing the gene encoding the aforementioned antibody can be transfected into host cells. Various transfection methods exist, including electroporation, liposome transfection, and calcium phosphate transfection.

[0022] Other expression systems besides mammalian cells, such as insect cells, yeast, and bacteria, can also be used to express the antibodies of this invention, and they are also included in the category of host cells that can be used in this invention. These expression systems have higher protein yields than mammalian cells, but they tend to form inclusion bodies, thus requiring further protein refolding.

[0023] The antibodies of the present invention can also be delivered and expressed using viral vectors, including but not limited to adenovirus vectors, adeno-associated viral vectors, retroviral vectors, herpes simplex virus-based vectors, and lentiviral vectors.

[0024] The fully human antagonistic antibody targeting lactated STAT6 of this invention can detect the lactation level of STAT6 in a subject's blood sample based on its specificity in targeting lactated STAT6. Some embodiments of this invention have found a positive correlation between the lactation level of STAT6 in a subject's blood sample and the severity of sepsis immunosuppression. Therefore, detecting the lactation level of STAT6 in a subject's blood sample can determine the severity of sepsis immunosuppression, and thus can be used to prepare products for detecting sepsis immunosuppression. The detection method can be based on any one or more of ELISA, chemiluminescent immunoassay (CLIA), flow cytometry, colloidal gold immunochromatography (GICA), dot blot, Western blot, latex agglutination, and immunohistochemistry. Therefore, products for detecting sepsis immunosuppression can be kits based on any one or more of ELISA, chemiluminescent immunoassay (CLIA), flow cytometry, colloidal gold immunochromatography (GICA), dot blot, Western blot, latex agglutination, and immunohistochemistry.

[0025] The fully human antagonistic antibody targeting lactolyzed STAT6 of the present invention possesses both specificity for targeting lactolyzed STAT6 and the ability to improve sepsis-induced immunosuppression. In some embodiments of the present invention, using the fully human antagonistic antibody targeting lactolyzed STAT6 alone can improve the severity of immunosuppression in a mouse model of sepsis-induced immunosuppression and improve the survival rate of the mouse model. Therefore, the fully human antagonistic antibody targeting lactolyzed STAT6 of the present invention can be used as a single active ingredient in the preparation of drugs for treating or alleviating sepsis-induced immunosuppression.

[0026] The fully human antagonistic antibody targeting lacto-STAT6 of the present invention can also be combined with other active ingredients that have therapeutic or immunosuppressive effects on sepsis to form a pharmaceutical composition. This composition can be used in conjunction with other treatment methods, including chemotherapy, radiotherapy, and biological therapy.

[0027] The antibodies of the present invention can be prepared into various forms of pharmaceutical formulations according to conventional pharmaceutical techniques, with injections being more preferred and freeze-dried injections being the most preferred.

[0028] Example 1: 1) Collect fresh blood samples from 5 patients with sepsis and 5 healthy individuals undergoing physical examinations; 2) Mix fresh blood and Ficoll in a 5:4 ratio. Add the appropriate volume of Ficoll separation solution to a centrifuge tube. Carefully add the blood to the surface of the separation solution using a Pasteur tube. Then centrifuge at 650g for 20 minutes. 3) After centrifugation, it separates into four layers, from top to bottom: plasma layer, PBMC layer, separation solution layer and red blood cell layer; 4) Carefully pipette the second layer of PBMC into another centrifuge tube containing PBS, mix thoroughly, and centrifuge at 350g for 10 minutes. 5) Discard the supernatant, add PBS to wash again, mix well by pipetting, and centrifuge at 250g for 10 minutes. 6) Discard the supernatant; the precipitate is peripheral blood mononuclear cells (PBMCs). 7) Magnetic separation of monocytes: Incubate a portion of PBMCs with CD14 magnetic beads at 37 degrees Celsius for 20 minutes, then pass the cell fluid through a magnetic column to magnetically adsorb CD14+ monocytes. 8) Remove the magnet and wash the monocytes down with PBS to obtain monocytes. 9) Add RIPA protein lysis buffer containing PMSF to PBMCs and monocytes respectively, and lyse the cells on ice for 30 minutes. 10) Centrifuge at 4 degrees Celsius and 13,000 rpm for 10 minutes, and collect the supernatant protein solution into a new tube; 11) Incubate the protein solution with STAT6 antibody (purchased from CST, catalog number: 5397) at 4 degrees Celsius overnight by rotating. 12) On the second day, add Protein A / G magnetic beads to the protein antibody mixture and incubate at room temperature for 2 hours by rotation. 13) Adsorb magnetic beads onto a magnetic rack, remove the liquid, add PBST to wash, and repeat the adsorption and washing process three times; 14) Adsorb magnetic beads onto a magnetic rack, remove liquid, add 1 x loading buffer to elute magnetic beads, and adsorb magnetic beads onto a magnetic rack again to collect liquid; 15) The collected liquid was heated in a 95-degree metal bath for 10 minutes; 16) Electrophoresis: Add the cooked protein and marker to the sample wells, electrophore at 80 V for 30 min, then switch to 120 V until the bromophenol blue reaches the bottom of the gel, then stop. 17) Transfer: Cut a strip of adhesive to size 110kDa, cover it with a PVDF membrane activated by methanol, and place it into the transfer apparatus in the order of filter paper-adhesive-PVDF membrane-filter paper. Place the transfer apparatus in a box containing crushed ice and transfer at a constant current of 250 mA. 18) Sealing: Place the PVDF membrane in a 5% skim milk sealing solution and seal it on a shaker at room temperature for 1 hour.

[0029] 19) Primary antibody incubation: Wash the blocked PVDF membrane to remove the blocking solution, transfer it to antibody dilution buffer containing lactated antibody (purchased from Jingjie Biotechnology, catalog number: PTM-1401RM), and incubate overnight on a shaker at 4 degrees Celsius. 20) Washing: The next day, transfer the incubated PVDF membrane to TBST and wash it on a shaker at room temperature for 10 minutes each time, for a total of three times; 21) Secondary antibody incubation: Prepare the appropriate secondary antibody (purchased from Anbio, catalog number: HA1001), and transfer the washed PVDF membrane to the antibody containing the secondary antibody; 22) Exposure: Prepare the exposure solution and perform exposure imaging on the exposure unit.

[0030] Test results as follows Figure 1 As shown, in peripheral blood mononuclear cells (PBMCs) and monocytes of immunosuppressed sepsis patients, the lactation of STAT6 (Kla band) in the immunosuppressed sepsis patient group (left) was significantly brighter than that in the normal control group (right), indicating that the level of STAT6 lactation in PBMCs and monocytes was significantly upregulated in immunosuppressed sepsis patients, and higher than that in the healthy control group.

[0031] Identification was performed using mass spectrometry, and the detection results were as follows: Figure 2 As shown, the lactation site of STAT6 is the ninth lysine residue (K9).

[0032] Example 2: Obtaining a fully human antagonistic antibody targeting lactolyzed STAT6: The fully human antagonistic antibody targeting lactolyzed STAT6 in this invention was screened from a human ScFv phage antibody library. The specific process is as follows: 1. Explanation of Experimental Examples Antigen: Human lactolyzed STAT6 protein; Phage library used for screening: human scFv phage antibody library; Example Objective: To screen antagonistic antibodies targeting lactated STAT6 from a human scFv phage antibody library.

[0033] 2. Experimental Procedure: Screening of high-affinity single-chain antibodies against lactated STAT6 2.1 Biological washing (1) Block streptavidin magnetic beads and phage libraries (1×10¹² pfu) separately with 3% MILK-PBS blocking buffer (i.e., PBS containing 3% skim milk powder) at room temperature and 20 rpm for 1 hour. Discard the blocking buffer, add 1 mL of 0.1% PBST, vortex to resuspend for 20 seconds, and magnetically separate for 2 minutes to obtain the blocked streptavidin magnetic beads; (2) Add 10 μg of biotinylated antigen protein (lactated STAT6 protein) to each of the blocked streptavidin magnetic beads, and then bind them on a shaker at 20 rpm for 1 hour; discard the antigen and PBS, wash the magnetic beads 3 times with 0.1% PBST, and then wash them once with PBS. Obtain the antigen-coated magnetic beads; (3) Add the blocked phage library to the antigen-coated magnetic beads, and then allow them to bind on a shaker at 20 rpm for 1 hour at room temperature. Magnetic separation for 2 minutes, then remove the supernatant; (4) Add 1 mL of 0.1% PBST, vortex to resuspend the magnetic beads for 20 seconds, perform magnetic separation for 2 minutes, and remove the supernatant. Repeat this step 15 times; (5) Wash the tube once with 0.05% PBST, add 400 μL of glycine hydrochloric acid solution (Glycine-HCl, pH 2.2), mix thoroughly by inverting, and place on a rotary mixer at room temperature at 20 rpm for 10 minutes; (6) Perform magnetic separation for 2 minutes, aspirate the supernatant and add it to a new 1.5 mL EP tube (with 200 μL Tris-HCl neutralization buffer added, pH 8.0), and store at 4°C for later use; (7) In order to increase the number of high affinity phages, more stringent washing conditions will be implemented in the subsequent washing process, such as reducing the amount of coating antigen, increasing the number of washes and the concentration of Tween 20, or using competitive elution methods. See Table 1 for details.

[0034] Table 1. Selection criteria for each round of selection 2.2 Amplification Elution Products (1) Pick a single TG1 clone from the plate, add it to 20 mL of 2YT medium, and incubate at 37°C and 220 rpm until the logarithmic phase OD600 = 0.6-0.8; (2) Add the eluted antibody library and mix well. Incubate at 37°C for 60 minutes. (3) Add 4 μL of Amp, incubate at 37°C and 180 rpm for 60 minutes; (4) Add M13KO7 (helper phage: TG1>=10:1) to the culture and incubate at 37°C for 30 minutes; (5) Add 30 mL of 2YT and 6 μL of Amp, incubate at 37°C and 180 rpm for 60 min; (6) Centrifuge at 5000 rpm for 10 minutes at room temperature, remove the supernatant, collect the bacterial pellet, resuspend the pellet in 50 mL of 2YTAK, and incubate overnight at 30°C and 220 rpm in a 200 mL Erlenmeyer flask. The pellet is then recovered the next day, and the titer of the amplified product is measured. (7) Transfer the culture to a 100 mL centrifuge tube, centrifuge at 4°C and 12000 rpm for 20 minutes, transfer the supernatant to a new centrifuge tube, centrifuge again at 12000 rpm for 20 minutes, and transfer the supernatant to a new centrifuge tube. (8) Add 1 / 4 volume of PEG / NaCl to the supernatant and incubate on ice for 4 hours; (9) Centrifuge at 4°C and 12000rpm for 20 minutes, discard the supernatant, and centrifuge briefly again to remove the supernatant completely; (10) Resuspend the precipitate in 1 mL PBS, centrifuge at 4°C and 12000 rpm for 10 minutes, and transfer the supernatant to a new centrifuge tube; (11) Determine the titer; (12) Add antibody library diluted 10⁻⁹ / 10⁻¹⁰ to 200 μL of logarithmic TG1, incubate at 37°C for 30 minutes, spread on 2YTAG plates, incubate overnight at 37°C, and count colonies the next day.

[0035] 2.3 Monoclonal Phage ELISA Single colonies were picked from the phage library plates eluted in the last round of screening and inoculated into 96-well deep-well plates containing 300 μL of 2YTAG and cultured at 37°C and 700 rpm until the logarithmic growth phase. (2) Take 100 μL of bacterial culture from each well into a sterile 96-well plate and store at 4°C; (3) Add 100 μL of M13KO7 diluted with 2 YT (titer 2 x 10¹⁰, about 10 times the bacterial concentration), incubate at 37°C for 30 minutes, then incubate at 37°C and 600 rpm for 60 minutes. (4) Centrifuge the 96-well deep plate at 4000 rpm for 10 minutes, discard the supernatant, add 300 μL of 2TYAK to each well to resuspend the bacterial cells, and incubate at 700 rpm and 30°C overnight. (5) Coating target protein: Dilute the biotinylated target molecule with 0.05% PBST solution to 1 μg / ml, add it to the streptavidin microplate, 100 μl / well, mix at 37°C for 1 hour; (6) Washing: Discard the supernatant of the coating, pat dry, and wash 3 times with 0.05% PBST for 1 minute each time; (7) Blocking: Add 300 μL of 4% skim milk powder (PBS) blocking solution per well and let stand at 37°C for 1 hour; (8) Washing: Discard the blocking solution, pat dry, and wash 3 times with 0.05% PBST for 1 minute each time; (9) phag supernatant binding: 50 μL of 2% skim milk powder (PBS) and monoclonal supernatant were mixed at a ratio of 1:1 and added to the corresponding wells of the microplate. The mixture was then shaken slowly at 100 rpm for 1 hour at 37°C. (10) Washing: Discard the supernatant, pat dry, and wash 5 times with 0.1% PBST; (11) Secondary antibody binding: Anti-M13-HRP antibody was diluted 1:5000 in blocking buffer 1% skim milk powder (PBS), 100 μL / well, added to the microplate, and shaken slowly at 37°C and 100 rpm for 1 hour. (12) Washing: Discard the liquid, pat dry, and wash 5 times with 0.1% PBST; (13) Add 100 μL TMB, incubate in the dark for 5-10 minutes, then add 100 μL 2M hydrochloric acid to stop the reaction. Read the OD450 value using a microplate reader. The detection results are as follows: Figure 3 As shown. Clones marked with a yellow background (samples with the highest OD450 value) were selected for sequencing, ultimately yielding an antibody protein sequence with lactation-targeting specificity for STAT6 (sequences shown in SEQ ID NO: 1). The corresponding coding gene sequence is shown in SEQ ID NO: 2. The absorbance of the single-chain antibody shown in SEQ ID NO: 1 is as follows. Figure 1 As shown, it is 4.886.

[0036] Example 3 Expression of lactated STAT6 high-affinity single-chain antibody: (1) Construction of recombinant expression vector: Based on the sequencing results, the antibody gene shown in SEQ ID NO 2 was amplified using primers; the antibody gene fragment was inserted into the expression vector PET28A using homologous recombination; 100 μL of DH5α competent cells were thawed on ice, the tube wall was gently tapped to resuspend the cells, 10 μg of plasmid was added to the competent cells, the tube was tapped a few times, and the cells were incubated on ice for 30 minutes; the cells were heat-shocked in a 42°C water bath for 90 seconds and then quickly placed on ice for 5 minutes; 500 μL of SOC medium was added and incubated at 37°C for 60 minutes; the cells were centrifuged at 5000 rpm for 3 minutes, and a certain amount of bacterial cells were plated on LB / Amp (50 μg / mL) plates as needed to screen for positive clones; PCR was used to identify whether the transformation was successful; the cells were sent to Qingke Biotechnology for sequencing. After confirming that the sequencing results were correct, the plasmid was extracted using a plasmid extraction kit. The plasmid was then transformed into BL-21 (DE3) Escherichia coli. (2) Expression of lactated STAT6 antagonist antibody: BL-21(DE3) was inoculated into 3 mL LB / Amp (50 μg / mL) liquid medium and cultured at 37°C with shaking. The next day, the overnight culture was inoculated into 1 L LB / Amp (50 μg / mL) liquid medium at a ratio of 1 / 100. When the bacterial cells grew to an OD600 of about 0.6, they were cultured at 15°C for 15 minutes. Then, an appropriate amount of 100 mM IPTG was added to a final concentration of 1 mM, and the cells were induced to grow at 15°C for 22 hours. After centrifugation, the bacteria were suspended in 40 mL PBS buffer and then sonicated on ice. The lysate was then centrifuged (4000 rpm, 40 minutes). 8 μL of each extract (total protein, supernatant, and precipitate) was added to 2 μL of 5×SDS Loading Buffer and heated at 95°C for 10 minutes. SDS-PAGE electrophoresis was then performed. The protein was found to be mainly expressed in the supernatant; the protein supernatant was passed through a 0.45 μm sieve for further purification. (3) Purification of lactated STAT6 antagonist antibody: Wash 2 mL of Ni column (G&E) with at least 5 column volumes of binding buffer; add filtered protein supernatant and allow it to flow slowly, retaining the percolation fluid for detecting binding efficiency; wash the column with 5 column volumes of binding buffer, repeating three times; elute with 5 volumes of 200 nM imidazole solution; replace and concentrate the imidazole eluent using an ultrafiltration tube, then calculate the concentration and analyze the purity using SDS-PAGE.

[0037] Example 4: Treatment with lactated STAT6 antagonist antibody reduces the number of Treg cells in the blood of immunosuppressed mice with sepsis: Treg cells are a type of T lymphocyte with immunosuppressive function. Their main role is to maintain the balance and homeostasis of the immune system, prevent excessive immune responses or autoimmune damage, and they are one of the core cells of the immune system's "self-regulation." Detecting the proportion of peripheral blood Treg cells can help determine the immune status of sepsis patients.

[0038] (1) Establishment of a sepsis immunosuppression model: A sepsis animal model was established using cecal ligation and puncture (CLP). Mice were anesthetized by intraperitoneal injection of 100 μL of 1.5% sodium pentobarbital, fixed to a manipulation board, and their abdomens were depilated with hair removal cream. The skin was disinfected, and a 1 cm incision was made in the midline of the abdomen. The cecum was ligated, and a 21G sterile needle was used to puncture the cecal wall at the midpoint between the ligation site and the top of the cecum, creating a cecal perforation. The cecum was gently squeezed with forceps to expel a small amount of intestinal contents to ensure the patency of the perforation channel. The expelled contents were wiped clean with a sterile cotton ball, and then the cecum was slowly pushed back into the abdominal cavity. The layers were sutured, and the skin was disinfected. This modeling method is a classic method for establishing a sepsis animal model and is currently the gold standard animal model for sepsis research. Six hours after CLP modeling, mice were treated with intraperitoneal injection of eltapenem sodium (30 mg / kg), followed by injections every 12 hours for the next three days. Mice that survived this treatment were designated as sepsis-immunosuppressed mice. (2) Five mice with sepsis immunosuppressed by lactated STAT6 antagonist antibody were injected intraperitoneally daily after CLP modeling, designated as the sepsis immunosuppressed group + lactated STAT6 antagonist antibody as a control. Five mice with sepsis immunosuppressed by lactated STAT6 antagonist antibody were injected intraperitoneally daily after CLP modeling, designated as the sepsis immunosuppressed group. On the 7th day after CLP surgery, surviving sepsis immunosuppressed mice were sacrificed, blood samples were obtained, and PBMCs were isolated. The number of Treg cells was detected by flow cytometry. The results are as follows: Figure 4 As shown, treatment with lactated STAT6 antagonist antibodies can reduce the number of Treg cells in the blood of septic immunosuppressed mice.

[0039] Example 5: Treatment with lactated STAT6 antagonist antibody improves survival in sepsis-induced immunosuppressed mice after secondary infection: (1) Constructing a sepsis immunosuppression model: The specific steps are the same as in Example 3; (2) Five septic immunosuppressed mice (n=200 μg each) were intraperitoneally injected daily after CLP modeling, designated as the sepsis immunosuppressed group + lactated STAT6 antagonist antibody control. Five septic immunosuppressed mice (n=200 μg each) were also injected intraperitoneally daily after CLP modeling, designated as the sepsis immunosuppressed group. On day 7 post-CLP surgery, surviving sepsis immunosuppressed mice were intratracheally instilled with either Aspergillus fumigatus spores or Legionella pneumophila suspension. Both Aspergillus fumigatus (fungus) and Legionella pneumophila (bacteria) are opportunistic pathogens that infect the body when immunosuppressed or weakened. Patients with sepsis and cancer, among other immunosuppressed individuals, are susceptible to infection, while healthy individuals can kill them. Therefore, this indicator reflects the level of immunosuppression in the body. Mice mortality was assessed within 7 days after secondary infection. Results are as follows: Figure 5 and Figure 6 As shown, treatment with lactated STAT6 antagonist antibodies can improve the survival rate of sepsis-immunosuppressed mice after secondary infection.

[0040] Example 6: Treatment with lactated STAT6 antagonist antibody reduces pulmonary bacterial load in septic immunosuppressed mice after secondary infection: (1) Constructing a sepsis immunosuppression model: The specific steps are the same as in Example 3; (2) Five septic immunosuppressed mice (n=200 μg each) were intraperitoneally injected daily after CLP modeling, designated as the sepsis immunosuppressed group + lactated STAT6 antagonist antibody control. Five septic immunosuppressed mice (n=5 per group) were also injected intraperitoneally daily after CLP modeling, designated as the sepsis immunosuppressed group. On day 7 post-CLP surgery, surviving sepsis immunosuppressed mice were intratracheally instilled with Aspergillus fumigatus spores or Legionella pneumophila suspension. The mice were sacrificed the day after injection, and lung tissue was collected. The lung and spleen homogenates were diluted using a homogenizer in sterile water for 30 seconds and inoculated onto activated charcoal yeast extract agar to determine the number of colony-forming units in each organ. Results are as follows: Figure 7 As shown, treatment with lactated STAT6 antagonist antibodies can reduce the bacterial load in the lungs of septic immunosuppressed mice during secondary infection.

[0041] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A fully human antagonistic antibody targeting lactolyzed STAT6, characterized in that: This fully human antagonistic antibody is a single-chain antibody, and its amino acid sequence is shown in SEQ ID NO:

1.

2. The gene encoding the fully human antagonistic antibody targeting lactolyzed STAT6 as described in claim 1.

3. The gene according to claim 2, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

4. An expression vector containing the gene as described in claim 2 or 3.

5. A recombinant cell containing the expression vector as described in claim 4.

6. The application of the fully human antagonistic antibody targeting lactated STAT6 as described in claim 1 in the preparation of products for diagnosing sepsis immunosuppression.

7. A product for diagnosing sepsis-induced immunosuppression, characterized in that: This includes the fully human antagonistic antibody targeting lactolyzed STAT6 as described in claim 1.

8. The use of the fully human antagonistic antibody targeting lactated STAT6 as described in claim 1 in the preparation of a medicament for treating or alleviating immunosuppression in sepsis.

9. A medicament for treating or alleviating sepsis-induced immunosuppression, characterized in that: This includes the fully human antagonistic antibody targeting lactolyzed STAT6 as described in claim 1.

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