Polypeptide and application thereof in preparation of endotoxin detection kit

Abstract

The invention relates to the technical field of polypeptides, in particular to a polypeptide and application thereof in preparation of an endotoxin detection kit. The polypeptide provided by the invention can inhibit endotoxin from activating the tachypleus tridentatus C factor, and the tachypleus tridentatus C factor can be effectively prevented from being pre-activated by endotoxin in a preparation system or environment by adding the polypeptide in the preparation process of the tachypleus tridentatus C factor, so that the sensitivity of the tachypleus tridentatus C factor in endotoxin detection can be improved, the background value of the tachypleus tridentatus C factor can be reduced, and the detection accuracy can be improved. The polypeptide and the preparation method of the limulus factor C provide an effective means for improving the quality of the limulus factor C and the sensitivity and accuracy of endotoxin detection.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and in particular to a polypeptide and an application thereof in the preparation of an endotoxin detection kit. Background Art

[0002] Endotoxin is the main component of the outer wall of Gram-negative bacteria, also known as lipopolysaccharide (LPS). Endotoxin is released only when the bacteria die and dissolve or the cell wall is destroyed by artificial methods. As an exogenous pyrogen, it can trigger the activation of pro-inflammatory cytokines and ultimately lead to cell death. Injection of even trace amounts of LPS at the pg level can cause severe pyrogenic reactions in patients, causing fever, shock, and even death. Endotoxins are ubiquitous and difficult to inactivate, posing a challenge to the pharmaceutical and medical device industries. Endotoxin detection is a necessary procedure in pharmaceutical manufacturing and medical device manufacturing. Therefore, sensitive and reliable endotoxin analysis technology is very necessary.

[0003] As the gold standard for endotoxin detection in the world, the horseshoe crab reagent endotoxin test method has the characteristics of simplicity, rapidity and high sensitivity. Since the mid-1970s, it has been used in the medical field and has been widely adopted by countries around the world. Many countries have set it as a legal bacterial endotoxin test method. After decades of development and improvement, the horseshoe crab reagent endotoxin test method has been applied to pharmaceuticals, medical devices, non-oral preparations, pharmaceutical products, biological preparations, water quality, food testing and scientific research. However, horseshoe crab reagents can only be produced by collecting horseshoe crab hemolymph. With the rapid growth of production units such as biological products, medicines (such as injectable drugs, chemicals, radiopharmaceuticals, antibiotics, vaccines, dialysis fluids, etc.) and medical devices (such as disposable syringes, implantable biomaterials), the demand for quantitative endotoxin detection reagents is increasing, and the dependence of the horseshoe crab reagent endotoxin test method on horseshoe crabs has led to limited reagent supply due to the reduction in the number of horseshoe crabs. The ethical issues surrounding the continued use of animals have also attracted global attention to animal-derived alternatives.

[0004] Horseshoe crab factor C is a serine protease in horseshoe crab blood cells that is sensitive to bacterial endotoxins. In the horseshoe crab blood coagulation system reaction mediated by bacterial endotoxins, horseshoe crab factor C is the first to be activated by endotoxins to initiate the blood coagulation cascade reaction in the entire horseshoe crab blood cells. After the horseshoe crab factor C recombinant protein (i.e., recombinant factor C) expressed by genetic recombination binds to endotoxin, it is converted from an inactive protease to a biologically active protease, which recognizes and catalyzes downstream fluorescent substrates to produce fluorescent signals. The intensity of the fluorescent signal is positively correlated with the endotoxin concentration, thereby quantitatively detecting endotoxins. This endotoxin detection method does not use natural horseshoe crab blood cells, protects horseshoe crab resources, and is a new generation of endotoxin detection technology.

[0005] However, in the preparation process of recombinant factor C, whether it is the recombinant plasmid prepared by Gram-negative bacteria or the culture medium for cell culture, it is impossible to be completely endotoxin-free. The endotoxin in the preparation system of recombinant factor C binds to the recombinantly expressed factor C and activates it, making it unable to be activated by the endotoxin in the sample to be tested, resulting in a decrease in detection sensitivity, and thus it cannot be used for endotoxin detection. Summary of the invention

[0006] The invention provides a polypeptide and application thereof in the preparation of an endotoxin detection kit.

[0007] In order to solve the problem that horseshoe crab factor C is pre-activated by endotoxin during the preparation process, the present invention obtains a polypeptide through design and screening, which can inhibit the activation of horseshoe crab factor C by endotoxin introduced during the preparation process (such as plasmids used for transfection, culture medium for host cell culture, etc.), thereby ensuring the high sensitivity of horseshoe crab factor C in the endotoxin detection process, and providing an effective means for low-cost preparation of high-quality endotoxin detection reagent-horsehoe crab factor C.

[0008] Specifically, the present invention provides the following technical solutions.

[0009] In a first aspect, the present invention provides a polypeptide having an amino acid sequence as shown in SEQ ID NO.1.

[0010] The present invention finds that a polypeptide having an amino acid sequence of GKQAWPMVLG (SEQ ID NO.1) can inhibit the activation of horseshoe crab factor C by endotoxin. Adding the polypeptide during the preparation of recombinant factor C can reduce or avoid the activation of horseshoe crab factor C by endotoxin present in the preparation system, thereby improving the quality of the prepared horseshoe crab factor C and ensuring the sensitivity of endotoxin detection.

[0011] Those skilled in the art will understand that the function of the polypeptide provided by the present invention is determined by the amino acid sequence GKQAWPMVLG, and therefore, polypeptides having the GKQAWPMVLG sequence in principle all have the above-mentioned function of inhibiting endotoxin from activating horseshoe crab factor C. These polypeptides are not limited to polypeptides having an amino acid sequence as shown in SEQ ID NO.1. Under the premise that the function of the GKQAWPMVLG sequence is not affected, polypeptides obtained by adding other amino acid residues to the N-terminus and / or C-terminus of the GKQAWPMVLG sequence also have the above-mentioned function of inhibiting endotoxin from activating horseshoe crab factor C, and are therefore also within the protection scope of the present invention. These situations include, but are not limited to, adding protein tag sequences, signal peptides and the like to the N-terminus and / or C-terminus of the GKQAWPMVLG sequence; or, connecting the GKQAWPMVLG sequence to a linker and the like sequence; or, fusing the GKQAWPMVLG sequence with other polypeptide sequences.

[0012] In some embodiments of the present invention, the amino acid sequence of the polypeptide is shown as SEQ ID NO.1.

[0013] The amino acid sequence shown in SEQ ID NO.1 is: GKQAWPMVLG.

[0014] The present invention also provides a polypeptide obtained by connecting a signal peptide and / or a tag to the N-terminus and / or C-terminus of a polypeptide having a sequence as shown in SEQ ID NO.1.

[0015] The present invention also provides a polypeptide having at least 60% similarity to the amino acid sequence of the polypeptide described above and having the same function. The similarity is preferably at least 70%, 80% or 90%.

[0016] The present invention also provides a polypeptide having the same function obtained by replacing and / or deleting and / or inserting one or more amino acid residues based on the above polypeptide.

[0017] In a second aspect, the present invention provides derivatives, isomers, pharmaceutically acceptable salts, hydrates or solvates of the above-mentioned polypeptides.

[0018] Derivatives of the above-mentioned polypeptides include recombinant polypeptides derived from the polypeptides, carrier conjugates, derivatives obtained by connecting chemical groups, etc.

[0019] Pharmaceutically acceptable salts of the above polypeptides include sodium salts, potassium salts, calcium salts and the like.

[0020] In a third aspect, the present invention provides a biomaterial, wherein the biomaterial is any one of the following (1) to (3): (1) a nucleic acid molecule encoding the polypeptide; (2) a vector containing the nucleic acid molecule described in (1); (3) A host cell containing the nucleic acid molecule described in (1) or the vector described in (2).

[0021] According to the amino acid sequence and codon rules of the polypeptide provided above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the polypeptide. Due to the degeneracy of codons, the nucleotide sequence encoding an amino acid sequence is not unique, and all nucleic acid molecules capable of encoding the polypeptide are within the scope of protection of the present invention.

[0022] The above-mentioned vectors include plasmids, viruses, etc. Among them, plasmids include replicative vectors or non-replicative vectors, and replicative vectors include cloning vectors, expression vectors, etc.

[0023] The host cells mentioned above may be microbial cells, plant cells or animal cells. The microbial cells include but are not limited to prokaryotic cells (such as Escherichia coli, etc.), yeast, etc.; the animal cells include but are not limited to human embryonic kidney (HEK293) cells, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, African green monkey kidney (Vero) cells, canine kidney cells (MDCK) COS cells, mouse NSO thymoma cells and mouse myeloma SP2 / 0 cells, insect cells, etc.

[0024] In a fourth aspect, the present invention provides a method for preparing the above-mentioned polypeptide, which may be a method for preparing the polypeptide by chemical synthesis or biosynthesis.

[0025] The chemical synthesis method may be a solid phase synthesis method or a liquid phase synthesis method. The biosynthesis method includes: introducing a nucleic acid molecule encoding the polypeptide into a host cell to obtain a recombinant host cell, culturing the recombinant host cell, and collecting the polypeptide.

[0026] In a fifth aspect, the present invention provides use of the above-mentioned polypeptide or its derivatives, isomers, pharmaceutically acceptable salts, hydrates or solvates in inhibiting endotoxin-activated horseshoe crab factor C.

[0027] In the above application, the above polypeptide or its derivatives, isomers, pharmaceutically acceptable salts, hydrates or solvates are used to inhibit endotoxin from activating horseshoe crab factor C in vitro.

[0028] In a sixth aspect, the present invention provides use of the above-mentioned polypeptide or its derivatives, isomers, pharmaceutically acceptable salts, hydrates or solvates in the preparation of horseshoe crab factor C or the preparation of endotoxin detection reagents.

[0029] The above application includes: in the process of preparing horseshoe crab factor C, using the polypeptide with an amino acid sequence as shown in SEQ ID NO.1 to treat the host cell used to express horseshoe crab factor C; or, adding the polypeptide to the system for extracting horseshoe crab factor C.

[0030] Preferably, the host cell for expressing horseshoe crab factor C is cultured in a culture medium supplemented with the polypeptide.

[0031] Further preferably, before the horseshoe crab factor C expression plasmid is transfected into the host cells, the host cells are cultured in a culture medium supplemented with the polypeptide.

[0032] The polypeptide or its derivatives, isomers, pharmaceutically acceptable salts, hydrates or solvates described above can be used to prepare horseshoe crab factor C, and the horseshoe crab factor C prepared thereby can be used to prepare an endotoxin detection reagent.

[0033] In a seventh aspect, the present invention provides a method for preparing a recombinant Factor C, the method comprising: adding the above-mentioned polypeptide or its derivative, isomer, pharmaceutically acceptable salt, hydrate or solvate during the culture process of a host cell for expressing the recombinant Factor C.

[0034] By adding the polypeptide to the culture system of host cells expressing recombinant Factor C, it is possible to effectively avoid pre-activation of the recombinant Factor C by endotoxins in the culture system or environment, thereby obtaining a highly sensitive recombinant Factor C that can be used for endotoxin detection.

[0035] In the present invention, the polypeptide can be added to the host cell culture system to be transfected before the recombinant Factor C expression plasmid is transfected into the host cells; and / or, after the recombinant Factor C expression plasmid is transfected into the host cells, the host cells containing the recombinant Factor C expression plasmid are cultured in a culture medium supplemented with the polypeptide.

[0036] Further preferably, the method comprises: before the recombinant Factor C expression plasmid is transfected into the host cell, adding the polypeptide into the culture system of the host cell.

[0037] In some embodiments of the present invention, the host cells are cultured in a culture medium supplemented with the polypeptide 16-30 hours before transfection of the expression plasmid to obtain a cell culture fluid, and then the expression plasmid is mixed with the cell culture fluid and a transfection reagent for transfection, and the host cells are continued to be cultured to express the recombinant Factor C.

[0038] In the above method, the polypeptide is present in both the transfection of the expression plasmid of recombinant factor C and the system of expressing recombinant factor C. The presence of the polypeptide during transfection and protein expression can inhibit the activation of recombinant factor C by endotoxin introduced into the expression plasmid or reagents such as culture medium.

[0039] Preferably, the added amount of the polypeptide is 1-10 μg / mL, preferably 5-10 μg / mL.

[0040] In the present invention, the host cell may be a microbial cell, a plant cell or an animal cell. The microbial cell includes but is not limited to prokaryotic cells and yeast; the animal cell includes but is not limited to human embryonic kidney (HEK293) cells, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, African green monkey kidney (Vero) cells, canine kidney cells (MDCK) COS cells, mouse NSO thymoma cells and mouse myeloma SP2 / 0 cells, insect cells, etc.

[0041] In some embodiments of the present invention, a method for preparing a recombinant Factor C is provided, the method comprising the following steps: (1) Construction of horseshoe crab factor C protein recombinant expression plasmid; (2) 18-26 hours before plasmid transfection, culturing host cells in a culture medium containing the polypeptide to obtain a cell culture fluid; (3) The factor C protein recombinant expression plasmid in step (1) is mixed with the cell culture medium and transfection reagent in step (2) for transfection to recombinantly express horseshoe crab factor C in host cells; and the culture supernatant containing the recombinant factor C is harvested.

[0042] Preferably, the host cell is a HEK293 cell.

[0043] Preferably, after the culture supernatant containing the recombinant Factor C is harvested, the polypeptide is separated and removed.

[0044] In an eighth aspect, the present invention provides a recombinant Factor C prepared by the method described above.

[0045] In the present invention, the amino acid sequence of the recombinant factor C is shown in SEQ ID NO. 2. Compared with the method without adding the polypeptide treatment, the recombinant factor C prepared by the above recombinant factor C preparation method has a lower background value and higher sensitivity when used for endotoxin detection.

[0046] In a ninth aspect, the present invention provides a method for preparing the recombinant Factor C or use of the recombinant Factor C described above in preparing an endotoxin detection reagent.

[0047] In the present invention, the endotoxin detection reagent includes recombinant Factor C and may also include one or more selected from a fluorescent substrate and a buffer.

[0048] In some embodiments of the present invention, the fluorescent substrate is BOC-Val-Pro-Arg-AMC.

[0049] In a tenth aspect, the present invention provides use of the recombinant Factor C prepared by the method described above in detecting endotoxin for purposes other than disease diagnosis and treatment.

[0050] In an eleventh aspect, the present invention provides an endotoxin detection kit, the kit comprising recombinant factor C; the recombinant factor C is prepared using the above-described method for preparing recombinant factor C.

[0051] Preferably, the kit further comprises one or more selected from fluorescent substrates and buffer solutions.

[0052] The above fluorescent substrate can be cleaved by recombinant Factor C to generate free fluorescent groups.

[0053] The above-mentioned buffer may be a buffer commonly used in recombinant Factor C catalytic reactions, and its components may include Tris, NaCl, Triton X-100, and the like.

[0054] The above-mentioned kit preferably uses a substrate fluorescence method to detect endotoxin.

[0055] In a twelfth aspect, the present invention provides use of the kit in detecting endotoxin for purposes other than disease diagnosis and treatment.

[0056] The above-mentioned tests for non-disease diagnosis and treatment purposes may be endotoxin tests for drugs, biological products or medical devices.

[0057] The beneficial effects of the present invention include at least: the polypeptide provided by the present invention can inhibit endotoxin from activating horseshoe crab factor C, and the addition of the polypeptide during the preparation of horseshoe crab factor C can effectively prevent horseshoe crab factor C from being pre-activated by endotoxin in the preparation system or environment, thereby improving the sensitivity of horseshoe crab factor C when used for endotoxin detection, reducing its background value, and improving the accuracy of detection. The polypeptide and horseshoe crab factor C preparation method provided by the present invention provide an effective means for improving the quality of horseshoe crab factor C and the sensitivity and accuracy of endotoxin detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0059] Figure 1 The results of endotoxin standard detection of recombinant Factor C were obtained for the experimental group with added polypeptide and the control group without added polypeptide in Example 4 of the present invention.

[0060] Figure 2 A standard curve for measuring endotoxin by recombinant factor C was prepared for the experimental group to which the polypeptide was added and the control group to which the polypeptide was not added in Example 4 of the present invention. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0062] Example 1 Construction of horseshoe crab factor C recombinant expression vector

[0063] The coding sequence of horseshoe crab factor C was connected to the expression plasmid to construct the horseshoe crab factor C recombinant expression vector pHEK-C1, and the specific method is as follows.

[0064] The polynucleotide encoding the amino acid sequence of horseshoe crab factor C shown in SEQ ID NO.2 was synthesized by Shanghai Bioengineering Co., Ltd., and a restriction endonuclease was added to the 5' end. Bam H Ⅰ restriction site and Kozak sequence; add stop codon TAA and restriction endonuclease at the 3' end XOt Ⅰ restriction endonuclease site. Bam H Ⅰ and XOt I double digestion, followed by agarose gel electrophoresis, gel excision to recover the target fragment. Bam H Ⅰ and XOt Ⅰ Double-enzyme-digested pcNDA3.1(+) was connected to construct the recombinant eukaryotic expression vector pHEK-C1.

[0065] Transformation of pHEK-C1 into E. coli E. coli DH5α, cultured at 37℃ for 16h, single colonies were picked for plasmid extraction. The extracted plasmid was purified by restriction endonuclease Bam H Ⅰ and XOt After double restriction digestion, agarose gel electrophoresis was performed for identification, and the positive clones were sent to Shanghai Bioengineering Co., Ltd. for sequencing. The clones with correct sequencing were selected for plasmid amplification and extraction, and the plasmids extracted were sterile filtered using a low endotoxin plasmid extraction kit (Tiangen Biochemical Technology Beijing Co., Ltd., catalog number DP120-01) and stored at -20°C for future use.

[0066] Example 2 Pre-incubation of polypeptides with HEK293 cells to be transfected

[0067] The polypeptide of the present invention was synthesized by Shanghai Bioengineering Co., Ltd. The amino acid sequence of the polypeptide is shown in SEQ ID NO.1. The polypeptide was prepared into a 10 mg / mL mother solution with DMSO and stored at -20°C for future use.

[0068] 24 h before transfection, HEK293 cells were plated at 1×10 6 / mL for passage, and added the polypeptide with a final concentration of 5 μg / mL to the cell culture system (CD 293TGE, Acrobiosystems, CM-1156-11) (experimental group), while the control group did not add the polypeptide, and the cells were cultured at 37°C.

[0069] Example 3: Transfection of HEK293 cells with horseshoe crab factor C recombinant expression vector and expression of recombinant factor C

[0070] The constructed recombinant plasmid pHEK-C1 was transfected into the HEK293 cells cultured in Example 2 using PEI transfection reagent to perform recombinant expression of horseshoe crab factor C protein. On the day of transfection, the cells in Example 2 were counted and the cell density was adjusted to 2×10 6 / mL, and the activity rate is above 95%.

[0071] The dosage of each component in the transfection complex was calculated based on the cell density: the corresponding relationship between the plasmid dosage and the number of cells was 1×10 6 Each cell corresponds to 1 μg of plasmid, and the corresponding relationship between PEI dose and plasmid dose is that the mass of PEI is 3 times the mass of DNA.

[0072] The transfection complex was prepared according to the above calculated component dosage: 50 μg of the recombinant plasmid at a concentration of 200 μg / mL was added to 0.5 mL of CD 293 TGE medium (liquid A), and 150 μg of the PEI transfection reagent at a concentration of 1 mg / mL was added to 0.5 mL of CD293 TGE medium (liquid B), and after mixing them, liquid B was slowly added to liquid A, and after mixing and standing at room temperature for 10-15 minutes, the transfection mixture was slowly added dropwise to 10 mL of HEK293 cells (the culture medium obtained by cell culture at 37°C in Example 2). After culturing at 37°C, 5% CO2, and 135 rpm for 96 h, the supernatant was collected and dialyzed using PES material to remove free polypeptides, and the dialyzed supernatant was stored at 4°C for later use.

[0073] Example 4 Endotoxin detection experiment

[0074] The cell culture supernatant containing recombinant Factor C (after dialysis) harvested in Example 3 was used as the recombinant Factor C raw material in the endotoxin detection kit to perform an endotoxin detection experiment.

[0075] 1. Treatment of bacterial endotoxin working solution

[0076] 1) Use bacterial endotoxin detection water (purchased from Xiamen Limulus Reagent Biotechnology Co., Ltd., catalog number TRW50) to dissolve the bacterial endotoxin standard (purchased from Xiamen Limulus Reagent Biotechnology Co., Ltd., catalog number CSE10V) to 20EU / mL according to the endotoxin titer displayed on the label (Note: the stock solution concentration after dissolution is 20EU / mL, and it needs to be vortexed on an oscillator at 1000rpm / min for ≥10min).

[0077] 2) Take 4 disposable non-pyrogenic glass test tubes and mark the concentrations of the endotoxin working standard solution on the glass test tubes (0.005, 0.05, 0.5 and 5 EU / mL respectively). Use the endotoxin working standard solution with a concentration of 20 EU / mL to prepare a series of endotoxin standard solutions. The dilution method is shown in Table 1.

[0078] Table 1

[0079] 2. Add sample

[0080] 1) Add 100 μL of the sample to the corresponding wells of a 96-well sterile pyrogen-free white plate in the order of negative control (pyrogen-free water) 0 EU / mL, endotoxin working standard solution 0.005 EU / mL, 0.05 EU / mL, 0.5 EU / mL, and 5 EU / mL, respectively, and set up at least two replicate wells.

[0081] 2) After adding samples, cover the 96-well plate and place it in an incubator at 37°C±1°C for 10 minutes.

[0082] 3. Prepare the substrate and recombinant factor C mixed working solution

[0083] 1) After equilibration at room temperature, the fluorescent substrate dry powder (BOC-Val-Pro-Arg-AMC, commissioned by Shanghai Bioengineering Co., Ltd.) was reconstituted to a final concentration of 0.15 mmol / L with endotoxin test water (purchased from Xiamen Limulus Reagent Biotechnology Co., Ltd., catalog number TRW50), mixed well, and allowed to stand for 10 minutes.

[0084] 2) Preparation of substrate and recombinant Factor C mixed working solution: Mix the recombinant Factor C protein sample (prepared by the method of Example 3) and the fluorescent substrate in a ratio of 1:1, and mix gently and thoroughly.

[0085] 4. Add substrate and recombinant factor C to mix the working solution

[0086] 1) Take out the preheated 96-well plate, use an eight-channel pipette to add 100 μL of the above substrate and recombinant factor C mixed working solution to each sample well, and use a microplate to shake and mix; 2) After mixing, immediately read the fluorescence value at 0h; 3) Place the microplate in an incubator at 37°C ± 1°C and incubate for 1 hour before reading the fluorescence value for 1 hour.

[0087] 5. Data Analysis

[0088] 1) Export the raw fluorescence values ​​(0h time point and 1h time point) read by the microplate reader into a spreadsheet; 2) For all wells, the fluorescence value at 0 h was subtracted from the fluorescence value at 1 h to obtain ΔRFU; 3) Subtract the ΔRFU of the negative control wells from the ΔRFU data of the standard to obtain the net ΔRFU value of the standard; 4) Calculate the logarithm of the endotoxin working standard concentration (EU / mL) and the net ΔRFU; 5) Plot the logarithm of the concentration of the endotoxin working standard solution as the abscissa and the logarithm of the net ΔRFU as the ordinate and perform a linear fit: log(ΔRFU)=A×log(endotoxin concentration EU / mL)+B. The correlation coefficient of the standard curve is R 2 The value of should be ≥0.990.

[0089] As an example, the recombinant factor C prepared by the method of Example 3 of the present invention was used to determine different endotoxin content standard curves using a BMG FLUOstar Omega device.

[0090] The results are as follows Figure 1 As shown, the control group without the addition of the polypeptide of the present invention had a background signal value of 0EU / mL of 48791, while the experimental group with the addition of the polypeptide of the present invention had a background signal value of 0EU / mL reduced to 395; more importantly, the sensitivity of the detection kit was increased from 0.5EU / mL in the control group to 0.005EU / mL in the experimental group. The above results show that the polypeptide of the present invention can significantly reduce the degree of activation of the recombinantly expressed C factor by endotoxin in the system during cell culture, thereby significantly improving the sensitivity of the detection kit.

[0091] The recombinant factor C prepared by the method of Example 3 of the present invention was used as an enzyme raw material in an endotoxin assay kit, and the standard curve fitting method of Example 4 of the present invention was used to fit the signal values ​​of the four concentration points of the standard curve of the experimental group and the control group and the log values ​​of the endotoxin concentration. The results are shown in FIG. Figure 2 As shown, the R 2 (0.9979) was significantly better than the R of the control group without peptide addition. 2 (0.8309), and the slope of the standard curve of the experimental group with added polypeptide (0.9147) was significantly better than the slope of the control group without added polypeptide (0.6917), indicating that the recombinant factor C prepared by the present invention can meet the requirements of the endotoxin detection kit as an enzyme raw material.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polypeptide, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. A derivative, isomer, pharmaceutically acceptable salt, hydrate or solvate of the polypeptide according to claim 1.

3. Biomaterial, characterized in that The biological material is any one of the following (1)-(3): (1) a nucleic acid molecule encoding the polypeptide of claim 1; (2) a vector containing the nucleic acid molecule described in (1); (3) A host cell containing the nucleic acid molecule described in (1) or the vector described in (2).

4. Any of the following uses of the polypeptide according to claim 1 or its derivatives, isomers, pharmaceutically acceptable salts, hydrates or solvates: (1) Application in inhibiting endotoxin-activated horseshoe crab factor C; (2) Application in the preparation of horseshoe crab factor C; (3) Application in the preparation of endotoxin detection reagents.

5. A method for preparing a recombinant factor C, characterized in that: The method comprises: adding the polypeptide or its derivative, isomer, pharmaceutically acceptable salt, hydrate or solvate according to claim 1 during the culture process of host cells for expressing recombinant Factor C.

6. The method according to claim 5, characterized in that The added amount of the polypeptide is 1-10 μg / mL.

7. Use of the method according to claim 5 or 6 or the recombinant Factor C prepared by the method in the preparation of an endotoxin detection reagent.

8. Use of the recombinant Factor C prepared by the method according to claim 5 or 6 in detecting endotoxin for purposes other than disease diagnosis and treatment.

9. An endotoxin detection kit, characterized in that: The kit comprises recombinant factor C; the recombinant factor C is prepared by the method described in claim 5 or 6.

10. Use of the kit according to claim 9 in detecting endotoxin for purposes other than disease diagnosis and treatment.

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