Salmonella typhimurium endotoxin specific binding polypeptide and application thereof

Abstract

The invention belongs to the technical field of biology, and provides a salmonella typhimurium endotoxin specific binding polypeptide and application thereof. The amino acid sequence of the polypeptide is selected from any one of SEQ ID No.1 to SEQ ID No.5 in a sequence list. Experiments prove that the polypeptide can be specifically bound with the salmonella typhimurium endotoxin, and compared with an existing anti-endotoxin clinical drug polymyxin B, the polypeptide has obviously higher specific binding capacity with the salmonella typhimurium endotoxin and also has obviously better salmonella typhimurium endotoxin detoxification capacity. On the basis, the invention also provides application of the polypeptide in removal, separation or analysis of salmonella typhimurium endotoxin and preparation of salmonella typhimurium endotoxin detoxification drugs. The polypeptide can be applied to the biomedical fields of biological separation, biological detection, disease diagnosis and treatment and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to Salmonella typhimurium endotoxin specific binding polypeptides and applications thereof. The present application obtains a plurality of polypeptides specifically targeting Salmonella typhimurium endotoxin through phage display screening technology, and the polypeptides have high affinity and affinity specificity with Salmonella typhimurium endotoxin, and can be widely applied to various fields of biomedicine, such as biological separation, biological detection, disease diagnosis and treatment, and the like. BACKGROUND

[0002] Endotoxin is a kind of toxin produced by gram-negative bacteria (such as Escherichia coli, Salmonella, Pseudomonas aeruginosa, etc.), which widely exists in nature. The chemical nature of endotoxin is lipopolysaccharide, which is the main component of the outer membrane of bacterial cell wall. When bacteria die or lyse, endotoxin is released into the surrounding environment, inducing host innate immune response, causing expression and systemic release of various cytokines such as tumor necrosis factor, interleukin and other inflammatory mediators. In severe infection, a large amount of endotoxin invades the blood from the intestinal tract or the infected site, and causes a series of critical conditions such as septic shock, disseminated intravascular coagulation, acute respiratory distress syndrome, systemic inflammatory response syndrome, multiple organ failure, and finally leads to patient death. Therefore, the precise separation and analysis of endotoxin has important significance in the fields of biological medicine, clinical diagnosis and treatment, etc.

[0003] However, the precise separation and analysis of endotoxin faces many difficulties and challenges. In the aspect of separation, the traditional method mainly relies on the negative charge or hydrophobicity of endotoxin to separate by anion exchange chromatography or hydrophobic interaction chromatography, but these methods based on physical and chemical properties show significant limitations in complex biological samples (such as plasma, recombinant protein solution). The physicochemical properties of endotoxin overlap with those of other components in the sample, resulting in insufficient separation selectivity, difficulty in effectively enriching pathogenic dose endotoxin at ng / mL level, and non-specific loss of valuable biomolecules in the sample. In addition, endotoxin is prone to form aggregates with a molecular weight of more than 100 kDa in solution, and its heterogeneity further increases the difficulty of separation. In the aspect of analysis, the existing endotoxin analysis means highly depends on limulus reagent, and the main raw material is limulus blood products. Due to the ecological protection and sustainable development of limulus resources, the detection system relying on limulus reagent faces ethical and supply chain risks, therefore, developing sustainable endotoxin detection technology has become an urgent need of the biological medicine industry.

[0004] The bacterial heterogeneity of endotoxin and its complex supramolecular aggregate structure make it extremely difficult to be precisely separated and analyzed. The key to breaking through these technical bottlenecks lies in obtaining endotoxin recognition molecules with high specificity and high affinity. Phage display technology constructs a library with a library capacity of more than 1012 by using the genetic recombination technology of Escherichia coli and the packaging capacity of bacteriophage, and can obtain a large number of polypeptides with high specificity and high affinity to target molecules through screening. Therefore, the present application uses phage display technology to obtain a plurality of polypeptides specifically targeting Salmonella typhimurium endotoxin, and the polypeptides have high affinity and affinity specificity with Salmonella typhimurium endotoxin, and can be widely applied to various fields of biomedicine, such as biological separation, biological detection, disease diagnosis and treatment, and the like. 11The random peptide library and the biological panning strategy of "adsorption-elution-amplification" can realize efficient screening of specific recognition molecules for specific biological targets. Although there are polypeptides designed for E. coli endotoxin, due to the bacterial heterogeneity of endotoxin, polypeptides designed for other bacterial endotoxins cannot specifically bind to Salmonella typhimurium endotoxin. Salmonella typhimurium is a common bacterium that causes infection in clinical practice, but there is currently no report of endotoxin recognition polypeptides. In order to meet the needs of precise separation, detection analysis or detoxification of Salmonella typhimurium endotoxin in practical applications, it is necessary to design molecules that can specifically bind to Salmonella typhimurium endotoxin. SUMMARY

[0005] In view of the fact that there is no report of recognition molecules that can specifically bind to Salmonella typhimurium endotoxin in the prior art, there is a technical gap in the field of precise separation and analysis of Salmonella typhimurium endotoxin, and there is an urgent need for molecules that can effectively detoxify Salmonella typhimurium endotoxin. One of the purposes of the present application is to provide a series of Salmonella typhimurium endotoxin specific binding polypeptides. The second purpose of the present application is to provide the application of the polypeptides in the removal or separation or analysis of Salmonella typhimurium endotoxin. The third purpose of the present application is to provide the application of the polypeptides in the preparation of Salmonella typhimurium endotoxin detoxification drugs. By using the polypeptides as specific binding ligands for Salmonella typhimurium endotoxin, the specific removal or detection of Salmonella typhimurium endotoxin in blood or other biological products can be achieved, and the detoxification of Salmonella typhimurium endotoxin poisoning can be achieved, thereby achieving the purpose of precise separation, analysis and efficient detoxification of Salmonella typhimurium endotoxin.

[0006] To achieve the above-mentioned purposes of the application, the technical solutions adopted by the present application are as follows:

[0007] In a first aspect of the present application, a Salmonella typhimurium endotoxin specific binding polypeptide is provided, wherein the amino acid sequence of the polypeptide is selected from any one of SEQ ID No. 1 to SEQ ID No. 5 in the sequence listing, and the polypeptide can specifically bind to Salmonella typhimurium endotoxin. More specifically, the amino acid sequence of the polypeptide is as follows:

[0008] SEQ ID No. 1: Gly-Ser-Gly-Asp-Thr-Met-Pro-Ser-Lys-Ser-Leu-Val (PEP-1 for short),

[0009] SEQ ID No. 2: Thr-Pro-Leu-Thr-Ala-Phe-Leu-Ala-Gly-Pro-Arg-Pro (PEP-2 for short),

[0010] SEQ ID No. 3: Ala-Ser-Leu-Arg-Asp-Ile-Gln-Thr-Gly-Ser-Leu-Lys (abbreviation: PEP-3),

[0011] SEQ ID No. 4: Thr-Ser-Leu-Pro-Arg-Tyr-Tyr-Ala-Met-Asp-Gly-Ser (abbreviation: PEP-4),

[0012] SEQ ID No. 5: Gly-Thr-Asn-Trp-Ser-Ile-His-Glu-Asn-Asn-Met-Gly (abbreviation: PEP-5).

[0013] The present application proves by experiments that the polypeptide provided by the present application can specifically bind to Salmonella typhimurium endotoxin, and compared with the current anti-endotoxin clinical drug polymyxin B, the polypeptide has obviously higher affinity and binding force with Salmonella typhimurium endotoxin, and the polypeptide has obviously better Salmonella typhimurium endotoxin detoxification capacity.

[0014] Based on the above experimental results, the second aspect of the present application provides application of the above Salmonella typhimurium endotoxin specific binding polypeptide in Salmonella typhimurium endotoxin removal or separation or analysis.

[0015] Based on the above experimental results, the third aspect of the present application provides application of the above Salmonella typhimurium endotoxin specific binding polypeptide in preparation of Salmonella typhimurium endotoxin detoxification drugs.

[0016] The technical solution provided by the present application can produce the following beneficial technical effects:

[0017] The present application provides a series of Salmonella typhimurium endotoxin specific binding polypeptides, which can specifically bind to Salmonella typhimurium endotoxin, have high affinity and strong specificity for Salmonella typhimurium endotoxin, and have good detoxification effect. For example, experiments have shown that the polypeptides have significantly higher affinity and binding capacity for Salmonella typhimurium endotoxin than the current clinical drug polymyxin B, and have significantly better Salmonella typhimurium endotoxin detoxification capacity. Based on this, the polypeptides can be used as specific binding ligands for Salmonella typhimurium endotoxin to specifically remove or detect Salmonella typhimurium endotoxin in blood or other biological products, and can also achieve detoxification of Salmonella typhimurium endotoxin poisoning, thereby achieving the purpose of precise separation, analysis and efficient detoxification of Salmonella typhimurium endotoxin, and can be widely used in biomedical fields such as biological separation, biological detection, clinical diagnosis and treatment, and help to fill the technical gap in the field of biological medicine preparations for precise separation, analysis and detoxification of Salmonella typhimurium endotoxin. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a screening process schematic diagram of Salmonella typhimurium endotoxin specific binding polypeptides in the embodiments of the present application.

[0019] Figure 2 is the enzyme-linked immunoassay detection result of the affinity capacity of the polypeptide and Salmonella typhimurium endotoxin.

[0020] Figure 3 is the binding dissociation curve of PEP-1 and Salmonella typhimurium endotoxin.

[0021] Figure 4 is the binding dissociation curve of PEP-2 and Salmonella typhimurium endotoxin.

[0022] Figure 5 is the binding dissociation curve of PEP-3 and Salmonella typhimurium endotoxin.

[0023] Figure 6 is the binding dissociation curve of PEP-4 and Salmonella typhimurium endotoxin.

[0024] Figure 7 is the binding dissociation curve of PEP-5 and Salmonella typhimurium endotoxin.

[0025] Figure 8 is the binding dissociation curve of polymyxin B and Salmonella typhimurium endotoxin.

[0026] Figure 9 is the Salmonella typhimurium endotoxin detoxification capacity test result of PEP-2 and polymyxin B. DETAILED DESCRIPTION

[0027] The Salmonella enterica endotoxin-specific binding polypeptide and its application provided by the present application are further illustrated by the following examples. It should be noted that the following examples are only used to further illustrate the present application and should not be construed as limiting the scope of the present application. Based on the above description of the present application, those skilled in the art can make some non-essential improvements and adjustments to the specific implementation of the present application, which still falls within the scope of the present application.

[0028] Example 1

[0029] In this example, the Salmonella enterica endotoxin-specific binding polypeptide is screened by the following steps:

[0030] (1) Bacterial endotoxin extraction

[0031] First, single colonies of Salmonella enterica (S. typhimurium, ATCC 14028, American Type Culture Collection) were cultured in 500 mL Luria-Bertani (LB) liquid medium at 37 °C overnight to OD 600 = ~1.0), and then the bacteria were collected by centrifugation (5000 rpm, 10 min), washed with ultrapure water for 3 times, and resuspended in ultrapure water to 10 8 CFU / mL. After three freeze-thaw cycles (freezing temperature: -80 °C, time 20 min; melting temperature: room temperature, time 30 min), the bacterial endotoxin was extracted by the hot phenol water method: equal volume of phenol was added to the bacterial suspension obtained by the above operation, stirred at 66 °C for 30 min, and then placed at 4 °C overnight, and the upper aqueous phase (containing endotoxin) was collected. Equal volume of ultrapure water was added to the remaining phenol phase, and the above extraction process was repeated (stirred at 66 °C for 30 min, and then placed at 4 °C overnight, and the upper aqueous phase was collected). After combining the water phases obtained by the two extractions, the solution in the dialysis bag was dialyzed in ultrapure water for 7 days (dialysis bag molecular weight cut-off: 14000 Da), and then the solution in the dialysis bag was freeze-dried to obtain 316 mg of white fluffy Salmonella enterica endotoxin powder.

[0032] (2) Screening of endotoxin-specific binding polypeptide by phage random peptide library

[0033] Pre-screening: (i) Bovine serum albumin (BSA) coated 96-well plate preparation: 100 μL of 5 wt% BSA solution was added to each well of a 96-well plate, which was then incubated at 4 ℃ with shaking at 100 rpm overnight, and then all the remaining BSA solution in the wells was poured out, and the plate was washed 3 times with TBST solution (Tris buffer saline solution containing 0.1 wt% Tween-20). (ii) 10 μL of phage solution in the phage display random dodecapeptide library kit (Ph.D.-12, New England Biolabs, USA) was dissolved in 90 μL of Tris buffer saline solution (TBS) and added to the above BSA-coated 96-well plate, which was then incubated at 37 ℃ with shaking at 100 rpm for 30 min, and then the supernatant was collected after standing for 30 min, and phage amplification was performed according to the following steps.

[0034] Phage amplification: (i) 10 μL of the above supernatant containing the phage to be amplified (phage solution) was added to 2 mL of logarithmic growth phase Escherichia coli ER2738 (New England Biolabs, USA) LB bacterial solution (OD 600 = ~1.0). (ii) The bacterial solution containing the phage was added to 20 mL of LB liquid medium containing 0.02 wt% tetracycline (LB-Tet), and the medium was incubated at 37 ℃ with shaking at 250 rpm for 5 h. (iii) The above medium was centrifuged at 4 ℃ at 10000 rpm for 10 min, and 80% of the total volume of the supernatant was transferred to a new centrifuge tube, and 1 / 6 of the volume of PEG / NaCl solution (PEG / NaCl solution preparation: 20 g PEG8000, 14.6 g NaCl dissolved in 100 mL ultrapure water) was added to the centrifuge tube, and the mixture was mixed uniformly and precipitated at 4 ℃ overnight. (iv) The precipitated solution was centrifuged at 4 ℃ at 10000 rpm for 15 min, and all the supernatant was discarded. (v) The precipitate was resuspended with 1 mL of TBS, and the suspension was centrifuged at 4 ℃ at 10000 rpm for 5 min. (vi) The supernatant obtained by centrifugation was transferred to a new centrifuge tube, and 1 / 6 of the volume of PEG / NaCl solution was added to the centrifuge tube, and the solution was precipitated on ice for 1 h. (vii) The precipitated solution was centrifuged at 4 ℃ at 10000 rpm for 10 min, and the supernatant was discarded, and the precipitate was resuspended with 200 μL of TBS and centrifuged at 4 ℃ at 10000 rpm for 1 min. (viii) The supernatant obtained by centrifugation was transferred to a new centrifuge tube, and an equal volume of sterilized glycerol was added, and the mixture was stored at -20 ℃ for later use.

[0035] Endotoxin-specific binding polypeptide affinity screening: (i) Endotoxin-coated 96-well plate preparation: 100 μL of Salmonella typhimurium endotoxin solution (1 mg / mL, obtained by dissolving in water after step (1) above) was added to a single well of a 96-well plate, which was shaken at 4 °C and 100 rpm overnight, the remaining liquid was poured out, and 200 μL of a 5 wt% BSA solution was added for blocking (4 °C, 100 rpm, incubation for 1 h); then the plate was washed with TBST solution 3 times. (ii) 10 μL of phage solution (obtained after amplification in the previous step, titer 10 12 pfu / mL) was dissolved in 90 μL of TBS and added to a single well of the above endotoxin-loaded 96-well plate, which was shaken at 37 °C and 200 rpm for 30 min, and then the supernatant was discarded after standing. The phage bound to the plate was eluted according to the following steps.

[0036] Phage elution: 200 μL of glycine-hydrochloric acid buffer (0.2 M, pH = 2.2) was added to a single well of the plate for elution, which was shaken at 37 °C and 200 rpm for 10 min, and then the supernatant was aspirated, followed by the addition of 50 μL of Tris-hydrochloric acid buffer (pH = 9.1) for neutralization, to obtain the phage eluate, and finally an equal volume of sterilized glycerol was added, which was stored at -20 °C for later use.

[0037] The obtained phage eluate was used as a phage solution, and the above phage amplification-endotoxin-specific binding polypeptide affinity screening-phage elution steps were repeated for 3 rounds (a total of 4 rounds) of affinity screening.

[0038] (3) Phage monoclonal extraction

[0039] (i) Gradient dilute the eluate of phage after the 4th round of affinity screening with LB liquid medium, take 10 µL of phage liquid diluted by 10 times, 100 times and 1000 times of the original volume respectively, and add them into 190 µL of E. coli ER2738 liquid of logarithmic growth phase, mix evenly, and incubate in a constant temperature incubator at 37 ℃ for 10 min. (ii) Take the above mixture and add it into 3 mL of LB agar medium containing 0.1 wt% MgCl2 (Beijing Solabio Technology Co., Ltd.) which is melted and cooled to 45-50 ℃, shake thoroughly, and then pour onto a LB solid medium plate containing 0.005 wt% isopropyl-β-D-thiogalactopyranoside (IPTG) and 0.004 wt% 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) (LB / X-gal / IPTG), and culture at 37 ℃ overnight to obtain single clone phage colonies. (iii) Pick a single clone phage colony from the plate and add it into 1 mL of E. coli ER2738 liquid of logarithmic growth phase, and culture at 37 ℃, 250 rpm for 4.5 h. (iv) Centrifuge the culture in a centrifuge tube for 1 min, transfer the supernatant into a fresh tube, centrifuge again, and then take 80% of the total volume of the supernatant and transfer it into a new centrifuge tube, which is the amplified phage stock solution. (v) Use the M13 phage single-stranded DNA rapid extraction kit (DP2301, Beijing Biotek Biotechnology Co., Ltd.) to extract the phage plasmid according to the steps in the product manual, and determine the polypeptide sequence displayed on the surface of the corresponding phage through DNA sequencing and analysis.

[0040] (4) Sequencing and analysis

[0041] The sequencing of the DNA sample was commissioned by Bioengineering (Shanghai) Co., Ltd., and the sequencing primer was -96gIII (-96gIII sequence: 5´-HOCCCTCATAGTTAGCGTAACG-3'). The sequencing results are shown in Table 1, and the #1-#7 sequences in Table 1 are shown in SEQ ID No. 1-SEQ ID No. 7 of the sequence listing.

[0042] Table 1

[0043] No. Polypeptide sequence #1 Gly-Ser-Gly-Asp-Thr-Met-Pro-Ser-Lys-Ser-Leu-Val #2 Thr-Pro-Leu-Thr-Ala-Phe-Leu-Ala-Gly-Pro-Arg-Pro #3 Ala-Ser-Leu-Arg-Asp-Ile-Gln-Thr-Gly-Ser-Leu-Lys #4 Thr-Ser-Leu-Pro-Arg-Tyr-Tyr-Ala-Met-Asp-Gly-Ser #5 Gly-Thr-Asn-Trp-Ser-Ile-His-Glu-Asn-Asn-Met-Gly #6 Gly-Ser-Gly-Asp-Thr-Met-Pro-Ser-Lys-Ser-Leu-Val #7 Gly-Asn-Asn-Trp-Ser-Ile-His-Glu-Asn-Thr-Ala-Gly

[0044] Example 2

[0045] In this example, the affinity of the polypeptide to Salmonella enterica endotoxin was detected by enzyme-linked immunoassay (ELISA).

[0046] The titers of the single clone colonies of the screened phage (obtained in step (3)(ii) in Example 1, a total of 7 kinds) were all adjusted to 1.2×1011 pfu / mL, 100 μL of the phage suspension was added to the endotoxin-coated 96-well plate (the process was the same as step (i) of the endotoxin-specific polypeptide screening in step (2) of Example 1 above), and incubated at 25 °C, 150 rpm for 2 h; after washing with TBST solution for 6 times, 200 μL of horseradish peroxidase (HRP)-labeled anti-M13 phage antibody (GE HealthCare Technologies Inc., USA; diluted at a ratio of 1:5000 in TBST solution) was added, and incubated at 25 °C, 150 rpm for 1 h, followed by washing with TBST solution for 6 times, and finally, ELISA was determined by ELISA color developing kit (C510031, Shanghai Generay Biotech Co., Ltd.), and BSA was used as a negative control. According to the results obtained by ELISA method (the greater the absorbance, the stronger the affinity of the phage to Salmonella typhimurium endotoxin), the absorbance (absorbance value at 405 nm) = 0.2 was used as the threshold value, and the phage monoclonal with strong affinity to Salmonella typhimurium endotoxin was screened out, and the corresponding amino acid sequence was: Figure 1

[0047] Information of SEQ ID No. 1

[0048] (a) Sequence characteristics

[0049] * Length: 12 amino acids

[0050] * Type: amino acid

[0051] * Chain type: single chain

[0052] (b) Molecular type: protein

[0053] Sequence description:

[0054] SEQ ID No. 1: Gly-Ser-Gly-Asp-Thr-Met-Pro-Ser-Lys-Ser-Leu-Val (abbreviation: PEP-1);

[0055] Information of SEQ ID No. 2

[0056] (a) Sequence characteristics

[0057] * Length: 12 amino acids

[0058] * Type: amino acid

[0059] * Chain type: single chain

[0060] (b) Molecular type: protein

[0061] Sequence description:​

[0062] SEQ ID No. 2: Thr-Pro-Leu-Thr-Ala-Phe-Leu-Ala-Gly-Pro-Arg-Pro (abbreviation: PEP-2);

[0063] Information of SEQ ID No. 3

[0064] (a) Sequence characteristics

[0065] * Length: 12 amino acids

[0066] * Type: Amino acid

[0067] * Chain type: Single chain

[0068] (b) Molecular type: Protein

[0069] Sequence description:

[0070] SEQ ID No. 3: Ala-Ser-Leu-Arg-Asp-Ile-Gln-Thr-Gly-Ser-Leu-Lys (abbreviation: PEP-3);

[0071] Information of SEQ ID No. 4

[0072] (a) Sequence characteristics

[0073] * Length: 12 amino acids

[0074] * Type: Amino acid

[0075] * Chain type: Single chain

[0076] (b) Molecular type: Protein

[0077] Sequence description:

[0078] SEQ ID No. 4: Thr-Ser-Leu-Pro-Arg-Tyr-Tyr-Ala-Met-Asp-Gly-Ser (abbreviation: PEP-4);

[0079] Information of SEQ ID No. 5

[0080] (a) Sequence characteristics

[0081] * Length: 12 amino acids

[0082] * Type: Amino acid

[0083] * Chain type: Single chain

[0084] (b) Molecular type: Protein

[0085] SEQUENCE DESCRIPTION:

[0086] SEQ ID No. 5: Gly-Thr-Asn-Trp-Ser-Ile-His-Glu-Asn-Asn-Met-Gly (abbreviated as: PEP-5)

[0087] Example 3

[0088] In this example, the affinity of different polypeptides to Salmonella typhi endotoxin was evaluated based on the bio-layer interferometry technology.

[0089] The affinity between Salmonella typhi endotoxin and five polypeptides (PEP-1, PEP-2, PEP-3, PEP-4, PEP-5) screened and polymyxin B (PMB, Beijing Solabio Technology Co., Ltd.) was measured by bio-layer interferometry technology (BLI) on Octet K2 molecular interaction analysis system (FortéBio). According to the instruction, the polypeptides (PEP-1, PEP-2, PEP-3, PEP-4, PEP-5 or PMB) to be tested were immobilized on the surface of Octet® Amine Reactive Second Generation (AR2G) probes to form polypeptide-modified probes. Then, according to the instruction of FortéBio Octet K2 system, the polypeptide-modified probes were immersed in TBS solution with different concentrations of Salmonella typhi endotoxin (for PEP-1, PEP-2, PEP-3, PEP-4, PEP-5, the concentrations of endotoxin were 8320, 4160, 2080, 1040, 520, 260, 130 nM, respectively; for PMB, the concentrations of endotoxin were 80, 60, 40, 30, 20, 10, 5 μM, respectively), and then dissociated in TBS. The adsorption-dissociation kinetic data were recorded using the software provided by FortéBio, and the association rate constant (K a ), dissociation rate constant (K d ) and equilibrium dissociation constant (K D ) were calculated by curve fitting. The test results of equilibrium dissociation constant (K D ) are shown in Table 1. Figures 3-8 Figures 3-8 ​It can be seen that the equilibrium dissociation constant of the five polypeptides (PEP-1, PEP-2, PEP-3, PEP-4, and PEP-5) with Salmonella typhimurium endotoxin is significantly smaller than that of PMB with Salmonella typhimurium endotoxin, which is at least three orders of magnitude different from that of PMB with Salmonella typhimurium endotoxin. The smaller the equilibrium dissociation constant, the stronger the affinity of the corresponding polypeptide with Salmonella typhimurium endotoxin. The experimental results show that the above species polypeptides have strong binding capacity with Salmonella typhimurium endotoxin.

[0090] Example 4

[0091] In this embodiment, the detoxification ability of polypeptides on Salmonella typhimurium endotoxin was evaluated based on limulus reagent.

[0092] The polypeptides (PEP-1, PEP-2, PEP-3, PEP-4, PEP-5, or PMB) were dissolved in ultrapure water to obtain a polypeptide solution with a polypeptide concentration of 100 μg / mL. 300 μL of polypeptide (PEP-1, PEP-2, PEP-3, PEP-4, PEP-5, or PMB) solution was mixed with 300 μL of Salmonella typhimurium solution (5 ng / mL), and incubated at 30 ℃ for 30 min. Then, the Salmonella typhimurium endotoxin level in the solution was detected by dynamic turbidity method limulus reagent (KT-125, Zhanjiang Andus Biological Co., Ltd.) according to the product instruction manual. The Salmonella typhimurium endotoxin level in the solution was detected by endotoxin detector. The same amount of ultrapure water was added to the Salmonella typhimurium solution as a blank control group (Ctrl). The detection results are shown in Figure 9 The experimental results show that PEP-1, PEP-2, PEP-3, PEP-4, and PEP-5 can significantly reduce the level of Salmonella typhimurium endotoxin in the Salmonella typhimurium solution, and have better Salmonella typhimurium endotoxin detoxification ability than the current anti-endotoxin clinical drug PMB.

Claims

1. A Salmonella typhimurium endotoxin-specific binding polypeptide, characterized in that, The amino acid sequence of the polypeptide is selected from any sequence in SEQ ID No. 1 to SEQ ID No. 5 of the sequence listing.

2. The application of the Salmonella Typhimurium endotoxin-specific binding polypeptide of claim 1 in the removal, separation or analysis of Salmonella Typhimurium endotoxin.

3. The use of the Salmonella Typhimurium endotoxin-specific binding polypeptide of claim 1 in the preparation of an antidote for Salmonella Typhimurium endotoxin.

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