Use of biological protein preparation acetyl esterase KpACE for preparing a medicament for treating hemorrhagic shock

Abstract

The application provides an application of a biological protein preparation acetyl esterase KpACE in preparation of a medicine for treating hemorrhagic shock. The application first uses a mutant protein obtained by site-directed mutation of acetyl esterase KpACE derived from Klebsiella pneumoniae for the treatment of hemorrhagic shock, and the mutant protein can be mixed in a resuscitation fluid for synchronous reinfusion. The resuscitation treatment combined with the mutant protein can significantly prolong the survival time of a mouse with hemorrhagic shock, and reduce the liver and kidney injury after resuscitation, thereby providing an effective new treatment strategy for hemorrhagic shock and having a good clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of the biological protein preparation acetylesterase KpACE in the preparation of drugs for treating hemorrhagic shock. Background Technology

[0002] Hemorrhagic shock is a critical illness caused by acute massive blood loss leading to a sharp decrease in effective circulating blood volume, resulting in insufficient tissue perfusion, cellular hypoxia, and metabolic disorders. It is one of the leading causes of death from trauma. Currently, the standard treatment for hemorrhagic shock mainly includes fluid resuscitation and blood transfusion to restore effective blood volume and tissue perfusion as quickly as possible. However, existing treatments have many limitations: resuscitation with crystalloid and colloid solutions may worsen tissue edema and ischemia-reperfusion injury; blood transfusion is constrained by the shortage of blood products and the risks of transfusion-related infections and immune reactions. More importantly, even if the hypovolemia is corrected in time through the above methods, a considerable proportion of patients still experience continued disease progression, secondary multi-organ damage, and poor prognosis.

[0003] Hemorrhagic shock is a rapidly progressing and clinically challenging condition, posing a significant problem in critical care medicine. Studies have shown that immune dysfunction following hemorrhagic shock is a crucial mechanism leading to septic shock, multiple organ dysfunction, and even death. Therefore, exploring effective interventions to prevent secondary damage following hemorrhagic shock is of great clinical significance in reducing mortality. However, current clinical treatments for secondary damage following severe hemorrhagic shock are very limited, necessitating the development of new and effective interventions. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide the application of a mutant protein of acetylesterase KpACE in the preparation of a drug for treating hemorrhagic shock, wherein the drug is used in combination with resuscitation therapy. The present invention uses acetylesterase KpACE derived from Klebsiella pneumoniae as a base, and obtains the mutant protein KpACE through site-directed mutagenesis. H180A Studies (showing that its enzyme activity is approximately 10 times higher than the wild type) have revealed that synchronous reinfusion of this mutant protein mixed in resuscitation fluid significantly prolongs the survival time of mice with hemorrhagic shock, demonstrating a significant therapeutic effect. Furthermore, biochemical analysis results indicate that this mutant protein can alleviate liver and kidney damage in mice after resuscitation treatment for hemorrhagic shock. To achieve the above-mentioned objectives, this invention provides the following technical solution: A first aspect of the present invention provides the use of the recombinant protein preparation acetylesterase KpACE in the preparation of a drug for treating hemorrhagic shock, wherein the recombinant protein preparation acetylesterase KpACE has a histidine mutated at position 180 to alanine.

[0005] In this invention, KpACE stands for Klebsiella pneumoniae Acetyl Esterase, and its protein sequence corresponds to the acetyl esterase gene of Klebsiella pneumoniae strain ATCC43816 in NCBI, with the standard number WP_038432364.1.

[0006] In an optional implementation, the drug for treating hemorrhagic shock is used in combination with resuscitation therapy.

[0007] In this invention, the resuscitation treatment refers to the resuscitation treatment for hemorrhagic shock, the core of which is rapid hemostasis and fluid resuscitation, with the aim of restoring effective circulating blood volume and ensuring blood perfusion of vital organs. Traditional resuscitation fluids (such as crystalloid or artificial colloid solutions) have significant limitations in the treatment of hemorrhagic shock: large-volume infusions can easily lead to dilutional coagulopathy, destroying existing fragile thrombi and exacerbating bleeding; at the same time, they cannot correct the metabolic acidosis and hypothermia caused by shock, making it difficult to break the vicious cycle of the "death triad." In addition, existing fluids often lack anti-inflammatory and endothelial protective effects, and reperfusion can easily induce secondary injury. The drug for treating hemorrhagic shock provided by this invention can be used in combination with resuscitation fluids to achieve a therapeutic effect on hemorrhagic shock.

[0008] In an optional embodiment, the recombinant protein formulation acetylesterase KpACE has a linker tag at its N-terminus and / or C-terminus.

[0009] In an optional embodiment, the linker tag refers to a tag peptide or protein that is fused and expressed with the target protein using in vitro DNA recombination technology, including one or more of His-tag, Strep-tag, GST-tag, SUMO-tag, MBP-tag, Trx-tag, Flag-tag, MBP-tag, and myc-tag, or a tag containing at least one antigenic epitope or fluorescent labeling site. The linker tag does not affect the native conformation and biological activity of the target protein after elution.

[0010] In a specific embodiment of the present invention, the amino acid sequence of the recombinant protein preparation acetylesterase KpACE is shown in SEQ ID NO:1.

[0011] Furthermore, the drug is used to improve organ damage caused by hemorrhagic shock.

[0012] In an optional embodiment, the organ injury is an acute organ injury, including kidney, lung, liver, heart, brain, and gastrointestinal tract injury or multiple organ dysfunction.

[0013] Furthermore, the organ damage is liver damage and / or kidney damage.

[0014] In a specific embodiment of the present invention, the drug improves liver damage by reducing alanine aminotransferase levels.

[0015] In a specific embodiment of the present invention, the drug improves kidney damage by reducing blood urea nitrogen, creatinine, or their ratio.

[0016] Furthermore, the drug is used to prolong the survival time of individuals with hemorrhagic shock.

[0017] A second aspect of the present invention provides the use of biomaterials in the preparation of drugs for treating hemorrhagic shock, said biomaterials comprising any one of the following: 1) The nucleic acid molecule encoding the recombinant protein preparation acetylesterase KpACE described in the first aspect; 2) An expression cassette containing the nucleic acid molecule described in 1); 3) A recombinant vector containing the nucleic acid molecule described in 1); 4) A recombinant host cell containing the nucleic acid molecule described in 1), the expression cassette described in 2), and / or the expression vector described in 3).

[0018] In this invention, nucleic acid molecules refer to polymeric forms of nucleotides of any length, including DNA, RNA, and DNA / RNA hybrids, and also include DNA or RNA analogs. Therefore, the nucleic acids of this invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc.

[0019] In this invention, an expression cassette refers to a nucleic acid construct containing at least one target gene or coding sequence and regulatory elements necessary for its transcription and / or translation. These regulatory elements include, but are not limited to, promoters, enhancers, terminators, ribosome binding sites, and / or poly(A) signal sequences. The expression cassette can function as an independent functional unit, driving the expression of the target gene in a suitable host cell.

[0020] In this invention, a recombinant vector refers to an artificial construct capable of delivering and preferably expressing one or more target genes or sequences in a host cell. The vector can be a known vector or a self-constructed vector. In a specific embodiment of this invention, the recombinant vector is the pET-28a vector.

[0021] In this invention, recombinant host cells refer to any cell type suitable for transformation, transfection, transduction, etc., using expression cassettes or recombinant vectors containing nucleic acid molecules provided by this invention. The recombinant cells include prokaryotic cells and eukaryotic cells. In a specific embodiment of this invention, the recombinant host cell is an *Escherichia coli* cell.

[0022] Furthermore, the nucleotide sequence of the nucleic acid molecule in 1) is shown in SEQ ID NO:3.

[0023] A third aspect of the present invention provides a pharmaceutical composition for treating hemorrhagic shock, the pharmaceutical composition comprising a therapeutically effective amount of the recombinant protein preparation acetylesterase KpACE described in the first aspect and / or the biological material described in the second aspect, and a pharmaceutically acceptable carrier.

[0024] In this invention, a pharmaceutically acceptable carrier refers to a non-toxic, non-immunogenic excipient or carrier that is physically and chemically compatible with the active ingredient and is used in conjunction with the recombinant protein to form a pharmaceutical composition. The carrier itself does not possess therapeutic activity but ensures the safety, stability, and efficacy of the formulation during storage and use.

[0025] In this invention, a therapeutically effective amount refers to an amount sufficient to produce a foreseeable therapeutic benefit or biological effect when the recombinant protein is administered to a subject in need, the effect including but not limited to at least one of reducing alanine aminotransferase levels, reducing blood urea nitrogen, creatinine or their ratio, and prolonging the survival time of an individual with hemorrhagic shock.

[0026] Furthermore, the effective therapeutic dose is 600-1000 μg.

[0027] Furthermore, the effective therapeutic dose is 800 μg.

[0028] A fourth aspect of the present invention provides a resuscitation fluid composition for treating hemorrhagic shock, the resuscitation fluid composition comprising the recombinant protein preparation acetylesterase KpACE described in the first aspect and a resuscitation fluid.

[0029] In this invention, the resuscitation fluid refers to the basic carrier used clinically for fluid resuscitation of hemorrhagic shock, including but not limited to balanced salt solutions such as lactated Ringer's solution, acetate Ringer's solution, and compound sodium chloride solution, crystalloid solutions such as physiological saline and hypertonic saline solution, as well as artificial colloid solutions such as hydroxyethyl starch and gelatin preparations, and also includes blood products such as thawed fresh frozen plasma or suspended red blood cells.

[0030] In an optional embodiment, the resuscitation fluid is a crystalloid and / or a colloidal solution.

[0031] In a specific embodiment of the present invention, the resuscitation solution is a mixture of compound sodium chloride solution and blood at a volume ratio of 1:1.

[0032] Advantages and beneficial effects of this invention: This invention is the first to use a mutant protein obtained by site-directed mutagenesis of Klebsiella pneumoniae acetylesterase KpACE for the treatment of hemorrhagic shock, serving as an effective supplement to existing fluid resuscitation and blood transfusion therapies. The administration method of this invention involves simultaneously reinfusing the protein mixed in the resuscitation fluid, requiring no additional procedures, and is fully compatible with existing resuscitation protocols, making it convenient for clinical use. The recombinant protein provided by this invention can be produced on a large scale through genetic engineering, with stable and controllable quality, and has good prospects for clinical translation and industrialization value. Attached Figure Description

[0033] Figure 1 The graph shows the purification results of the KpACE mutant recombinant protein, where A is the chromatographic purity analysis graph and B is the electrophoretic purity detection graph.

[0034] Figure 2 This is a representative diagram showing the general condition of mice in each group of hemorrhagic shock models.

[0035] Figure 3 Survival curves for each group of mice.

[0036] Figure 4 A statistical graph showing the plasma alanine aminotransferase levels in mice of each group.

[0037] Figure 5 Statistical graphs of renal function indicators (serum creatinine, blood urea nitrogen, and blood urea nitrogen / creatinine ratio) for mice in each group.

[0038] Figures 2 to 5 In the study, the Shock group was the control group that received blood combined with crystalloid resuscitation after hemorrhagic shock; the Shock+BSA group was the negative control group that added BSA protein to the resuscitation fluid; and the Shock+KpACE group was the experimental group that added KpACE mutant protein (abbreviated as KpACE in the figure) to the resuscitation fluid. Figure 4 and Figure 5 In this study, the Sham group served as a control group of normal mice that did not receive any treatment. Detailed Implementation

[0039] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0040] Example 1 Recombinant protein KpACE H180A Therapeutic effect on mice with hemorrhagic shock 1. Experimental Materials Recombinant KpACE protein expressed and purified in vitro H180A , with a 6His-tag linked to the N-terminus of the recombinant protein, and its amino acid sequence is: (SEQ ID NO: 1). The "KpACE" described in the following examples and accompanying drawings refers to the KpACE mutant KpACE of the present invention H180A protein.

[0041] Isoflurane (batch number: 2023041501, manufacturer: Shandong Ante Animal Husbandry Technology Co., Ltd.).

[0042] Pentobarbital sodium (batch number: P11011, manufacturer: Merck KGaA, Germany).

[0043] Iodophor disinfectant (batch number: 210603, manufacturer: Shandong Dexinkang Medical Technology Co., Ltd.).

[0044] Heparin sodium (batch number: 9041-08-1, manufacturer: Beijing Solarbio Science & Technology Co., Ltd.).

[0045] Compound sodium chloride (batch number: 24090411V, manufacturer: Anhui Shuanghe Pharmaceutical Co., Ltd.).

[0046] 0.9% Sodium Chloride Injection (batch number: 2010102010, manufacturer: Shijiazhuang No. 4 Pharmaceutical Co., Ltd.).

[0047] BSA (bovine serum albumin, batch number: 2024 / 01, manufacturer: Beijing Boaoteda Technology Co., Ltd.).

[0048] SPF-grade healthy male mice (C57BL / 6J, 8-10 weeks old, provided by Beijing Speford Biotechnology Co., Ltd. [production license: SCXK (Jing) 2019-0010]).

[0049] 2. Experimental Methods (1) A hemorrhagic shock model was constructed using C57BL / 6J mice to investigate the protective effect of KpACE H180A on the survival of mice with hemorrhagic shock. The mice were randomly divided into three experimental groups: ① Shock group (given blood combined with crystalloid resuscitation after hemorrhagic shock), ② Shock+BSA group (BSA protein added to the resuscitation solution, negative control), ③ Shock+KpACE H180A group (KpACE added to the resuscitation solution H180A , abbreviated as KpACE in the figures).

[0050] 1) Preparation of the hemorrhagic shock model: SPF-grade healthy male C57BL / 6J mice (8-10 weeks old, weighing 22±2 g) were selected. After anesthesia with isoflurane, 1.2% sodium pentobarbital solution was injected intraperitoneally at a dose of 7 μL / g. After anesthesia, the mice were fixed, their skin was prepared and disinfected, and the femoral arteries on both sides were dissected under a microscope. The distal end of the femoral artery was ligated, and then a glass needle filled with 1% sodium heparin solution was inserted into the femoral artery in a centripetal direction and fixed. One cannula was connected to a signal acquisition system to monitor heart rate and blood pressure; the other cannula was connected to a syringe fixed to a micro-infusion machine for blood aspiration and reinfusion for resuscitation. After the mouse's blood pressure stabilized for 10 min, blood was slowly drawn at a uniform rate until the blood pressure dropped to 40±2 mmHg.

[0051] 2) KpACE H180A Treatment regimen: Resuscitation began after maintaining hypotension for 90 minutes. Collected blood was mixed with compound sodium chloride solution at a 1:1 volume ratio to prepare the resuscitation fluid, and KpACE was added accordingly. H180A BSA, and KpACE, were infused at a constant rate via the femoral artery over 30 minutes. H180A The dosage was 800 µg / animal (the stock solution concentration was 10.027 mg / ml, diluted to 4 mg / ml, and the administration volume was 200 µL / animal); the dosage of BSA was 20 µg / animal (the stock solution concentration was 5 mg / mL, diluted to 100 µg / mL, and the administration volume was 200 µL / animal). The dosage selection was based on the reference [DOI: 10.1093 / femspd / ftae008].

[0052] 2. Observation index detection method: The mice were continuously observed for 96 hours after resuscitation and drug administration, and the survival status of each group of mice was recorded and survival curves were plotted.

[0053] 3. Recombinant protein KpACE H180A Purification and expression method: For purification and expression methods, please refer to patent 202311038814.X, "An Acetylesterase KpACE Derived from Klebsiella pneumoniae and Its Application".

[0054] 1) Obtaining the mutant coding gene: Using site-directed mutagenesis, a specific codon in the gene (SEQ ID NO:2) encoding recombinant acetylesterase rKpACE was replaced to obtain the rKpACE mutant. H180A The gene (SEQ ID NO:3).

[0055] 2) Construction of recombinant plasmid: The substituted sequence was ligated into the pET-28a vector to obtain the recombinant plasmid pET-28a-rKpACE. H180A This plasmid can express the recombinant acetylesterase rKpACE mutant protein rKpACE. H180A .

[0056] 3) Transformation of host bacteria: The recombinant plasmid pET-28a-rKpACE was transformed into the host bacteria. H180A Transformed into Escherichia coli BL21(DE3) competent cells, plated on LB agar plates containing 50 mg / L kanamycin, and positive single clones were selected.

[0057] 4) Activation and expansion culture of strains: Positive Escherichia coli BL21(DE3) containing recombinant plasmids were inoculated into liquid LB medium containing 50 mg / L kanamycin for activation, and then transferred to shake flasks after activation.

[0058] 5) Induction of expression: Cultured at 37℃ and 200 rpm until OD 600nm Once the concentration reaches 0.6-0.8, add IPTG inducer to a final concentration of 0.5 mM and incubate at 28℃ and 200 rpm for 4-6 h.

[0059] 6) Cell Collection and Disruption: After culture, collect the cells by centrifugation at 8000 rpm for 10 min. Wash three times with phosphate buffer, then resuspend the cells in buffer (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0). Disrupt the cells by sonication in an ice-water bath (conditions: 300 W power, 3 s sonication followed by a 7 s pause as one cycle, total time 60 min). After sonication, centrifuge at 10000 rpm for 30 min at low temperature and collect the supernatant.

[0060] 7) Affinity chromatography purification: The collected supernatant was purified by Ni column affinity chromatography, eluting sequentially with the following buffers: Washing buffer: 20 mM Tris-HCl, 300 mM NaCl, 50 mM imidazole, pH 8.0 — used to elute impurities.

[0061] Elution buffer: 20 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, pH 8.0 — used for eluting the target protein (i.e., rKpACE). H180A ).

[0062] 8) Protein purification: The eluted protein solution was purified by gel filtration using a Superdex® 200 Increase 10 / 300 GL size exclusion column (Sigma-Aldrich, GE28990944) with phosphate buffer pH 7.4. The protein was collected at UV280 / UV260 peak.

[0063] 9) Purity identification: The purified rKpACE H180AThe purity of the solution was observed by SDS-polyacrylamide electrophoresis, and the purity was 99%.

[0064] III. Experimental Results Figure 1 The purified recombinant protein KpACE is shown. H180A A protein formulation with a purity of ≥99% was obtained through purification, with SDS-PAGE ≥99% and SEC-HPLC ≥99%, meeting the relevant requirements of the Pharmacopoeia of the People's Republic of China for recombinant protein products and the quality control standards for therapeutic biological products. It can be used as a therapeutic-grade protein formulation for subsequent drug development and clinical application.

[0065] The results are as follows Figure 2 , Figure 3 As shown, there was no significant difference in appearance between the BSA infusion group (Shock + BSA) and the Shock mice. At 48 hours of hemorrhagic shock, both the Shock group and the Shock mice receiving BSA infusion exhibited complete eye closure with copious discharge; subsequently, the vast majority of these mice died. KpACE infusion... H180A Some mice with hemorrhagic shock survived for 96 hours, and the surviving mice had their eyes fully open, no secretions, and a significantly prolonged survival time. A significant increase in the survival time of mice with hemorrhagic shock was observed after 42 hours (P=0.0060, the difference was statistically significant).

[0066] Compared to the Shock group, BSA infusion did not prolong the survival time of mice with hemorrhagic shock, while KpACE... H180A Infusion significantly prolonged the survival time of mice with hemorrhagic shock, indicating that KpACE... H180A Combined resuscitation therapy has a significant therapeutic effect on mice with hemorrhagic shock.

[0067] Example 2 Recombinant protein KpACE H180A Therapeutic effect on liver and kidney injury in mice with hemorrhagic shock I. Experimental Materials 1) Fully automated biochemical analyzer (model: CelercareV5, manufacturer: Tianlv Micro-Nano Technology Co., Ltd.).

[0068] 2) Blood gas test discs (13+4) (Batch No.: 260552, Manufacturer: Tianlv Micro-Nano Technology Co., Ltd.).

[0069] 3) Miniature lithium heparin tube (batch number: 20251125, manufacturer: Hebei Keqiang Trading Co., Ltd.).

[0070] Other materials are the same as in Example 1.

[0071] II. Experimental Methods 1. Experimental Animals and Grouping: C57BL / 6J mice were randomly divided into four groups: ① Sham group (normal mice, no treatment), ② Shock group, ③ Shock+BSA group, and ④ Shock+KpACE group. The definitions of each group, the preparation of the hemorrhagic shock model, and the drug administration regimen were the same as in Example 1.

[0072] 2. Sample Collection and Indicator Detection: 24 hours after resuscitation and drug administration, arterial blood was collected via the abdominal aorta and placed in a lithium heparin anticoagulant tube. After centrifugation, the supernatant plasma was collected and added to a blood gas analysis disc (13+4). The disc was then inserted into a fully automated biochemical analyzer for analysis. Liver injury indicators: alanine aminotransferase; renal function indicators: blood urea nitrogen, creatinine, and the blood urea nitrogen / creatinine ratio.

[0073] III. Experimental Results like Figure 4 As shown, compared with the Sham group, the plasma alanine aminotransferase level in the Shock group was significantly increased (P=0.0005), indicating acute liver injury after resuscitation from hemorrhagic shock; there was no significant difference between the Shock+BSA group and the Shock group; compared with the Shock group, the alanine aminotransferase level in the Shock+KpACE group was significantly decreased (P=0.0053), indicating that KpACE... H180A It can alleviate acute liver injury in mice after recovery from hemorrhagic shock.

[0074] like Figure 5 As shown, compared with the Sham group, the Shock group mice had significantly higher plasma urea nitrogen (P < 0.0001), creatinine (P = 0.004), and urea nitrogen / creatinine ratio (P = 0.0026), indicating impaired renal function after resuscitation; there was no significant difference between the Shock+BSA group and the Shock group; compared with the Shock group, the Shock+KpACE group mice had decreased plasma urea nitrogen (P < 0.0001) and a decreased urea nitrogen / creatinine ratio (P = 0.0007), indicating that KpACE... H180A It can reduce kidney damage in mice after recovery from hemorrhagic shock.

[0075] The above results suggest that KpACE H180A Its therapeutic effect on hemorrhagic shock may be related to its ability to reduce secondary liver and kidney damage after resuscitation.

[0076] Table 1. Sequence List

[0077] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. The application of the recombinant protein preparation acetylesterase KpACE in the preparation of drugs for treating hemorrhagic shock, characterized in that, The recombinant protein preparation acetylesterase KpACE has a histidine at position 180 mutated to alanine. Preferably, the drug for treating hemorrhagic shock is used in combination with resuscitation therapy.

2. The application according to claim 1, characterized in that, The recombinant protein preparation acetylesterase KpACE has a linker tag at its N-terminus and / or C-terminus.

3. The application according to claim 1, characterized in that, The amino acid sequence of the recombinant protein preparation acetylesterase KpACE is shown in SEQ ID NO:

1.

4. The application according to claim 1, characterized in that, The drug is used to improve organ damage caused by hemorrhagic shock.

5. The application according to claim 4, characterized in that, The organ damage refers to liver damage and / or kidney damage.

6. The application according to claim 1, characterized in that, The drug is used to prolong the survival time of individuals with hemorrhagic shock.

7. The application of biomaterials in the preparation of drugs for treating hemorrhagic shock, characterized in that, The biomaterial includes any one of the following: 1) A nucleic acid molecule encoding the recombinant protein preparation acetylesterase KpACE as described in any one of claims 1-3; 2) An expression cassette containing the nucleic acid molecule described in 1); 3) A recombinant vector containing the nucleic acid molecule described in 1); 4) A recombinant host cell containing the nucleic acid molecule described in 1), the expression cassette described in 2), and / or the expression vector described in 3).

8. The application according to claim 7, characterized in that, The nucleotide sequence of the nucleic acid molecule in 1) is shown in SEQ ID NO:

3.

9. A pharmaceutical composition for treating hemorrhagic shock, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of the recombinant protein preparation acetylesterase KpACE as described in any one of claims 1-3 and / or the biological material as described in claim 7, as well as a pharmaceutically acceptable carrier; Preferably, the effective therapeutic dose is 600-1000 μg; Preferably, the effective therapeutic dose is 800 μg.

10. A resuscitation fluid composition for treating hemorrhagic shock, characterized in that, The resuscitation fluid composition comprises the recombinant protein preparation acetylesterase KpACE as described in any one of claims 1-3 and the resuscitation fluid; Preferably, the resuscitation solution is a crystalloid and / or a colloidal solution.

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