Recombinant limulus reagent, its preparation method and application

Abstract

The present application relates to the technical field of bioengineering, and particularly relates to a recombinant limulus reagent, a preparation method and application thereof. The present application obtains a high-activity recombinant limulus three-factor (coagulogen, B and C factors) by using an insect baculovirus expression system and a protein stabilizer, and the recombinant limulus three-factor is used for detecting bacterial endotoxin. The method can exclude G-factor bypass interference, thereby effectively avoiding the possibility of false positive of dextran, and simultaneously solving the problem of lack of natural limulus reagent resources; the recombinant limulus reagent can adopt an end-point colorimetric method, simplifying a detection mode, and completing detection in a conventional enzyme label instrument; a cascade reaction is used, thereby reducing sample usage and improving detection sensitivity; after optimization, the detection time is shortened to 20 min, and the detection efficiency is greatly improved; and the recombinant limulus reagent can be used as a new type of rapid endotoxin detection reagent.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering technology, and particularly relates to a recombinant limulus reagent, a preparation method and application thereof. BACKGROUND

[0002] Endotoxin is a general term for toxic substances present in the cell wall of gram-negative bacteria (such as Salmonella typhi, Shigella dysenteriae, etc.), which is a component of the cell wall of various gram-negative bacteria, and is a toxin released after cell lysis, also known as "pyrogen", unit EU / ml. The toxicity of various bacterial endotoxins is roughly the same, which can cause fever, coagulation, shock in human body, and even death in severe cases. In the quality control of drugs, it must be strictly monitored and detected. In recent years, with the vigorous development of the global biopharmaceutical industry, more and more drugs will involve the fermentation production process of gram-negative bacteria such as Escherichia coli in the production process, so more attention is paid to bacterial endotoxin detection. According to a rough estimate, the United States Pharmacopoeia-National Formulary (USP-NF) has included nearly 4000 quality standards of raw materials, preparations and excipients, of which more than 700 standards involve bacterial endotoxin detection.

[0003] Limulus is an arthropod that has existed on earth for 480 million years. The unique body structure of limulus enables it to have strong adaptability and overcome the survival crisis brought by extreme environment, becoming a "living fossil" in the marine world. The blue blood flowing in the body of limulus has been proved to have unique medical value. The amoeba-like cells in its blood are extremely sensitive to bacteria, and the detection reagent made of limulus blood extract is the most sensitive and efficient bacterial endotoxin detection means in the world.

[0004] The main component of limulus reagent is limulus hemocyte lysate treated with chloroform to remove anti-lipopolysaccharide factor, and appropriate amounts of divalent calcium and magnesium ions are added, containing factor C (Factor C, FC), factor B (Factor B, FB), factor G (Factor G, FG), proclotting enzyme, clotting enzyme, etc. The basic principle of its enzyme cascade reaction has been widely studied, including two agglutination reaction pathways: one is the C factor pathway mediated by endotoxin: C factor is activated by combining with endotoxin, and then activates B factor, and the activated B factor converts proclotting enzyme into clotting enzyme, and the clotting enzyme converts clotting enzyme into clotting protein, and the clotting protein is cross-linked and dehydrated to form a gel; the other is the G factor pathway mediated by 1,3-β-D-glucan, which can also cause similar agglutination reaction, as shown in Figure 1

[0005] ​In summary, it is becoming increasingly important to develop rapid and reliable endotoxin detection methods that do not rely on natural horseshoe crabs as detection raw materials.

[0006] Bacterial endotoxin testing methods include gel-based assays and photometric assays, the latter of which includes turbidimetric and chromogenic substrate assays. Therefore, Limulus amebocyte lysate (LA) reagents can be categorized as: gel-based, kinetic turbidimetric, endpoint turbidimetric, kinetic chromogenic, and endpoint chromogenic. Gel-based LAL reagents use the principle of agglutination with endotoxins to qualitatively or semi-quantitatively detect endotoxins.

[0007] To ensure the consistency and sustainability of the test, replace the use of animal-derived reagents and effectively reduce the cost of testing; in 2010, the United States Pharmacopeial Convention (USP) published a Stimuli Article entitled "Methods for the detection of Gram-negative bacterial endotoxins using recombinant Factor C" in Pharmacopeial Forum PF36 (1), which detailed the validation of the method using the fluorescence method of recombinant Factor C for the detection of bacterial endotoxins in pharmaceuticals; in 2020, another article was published comparing the recombinant Factor C method with the traditional Limulus amebocyte lysate method, and in the same year, detailed the detection of bacterial endotoxins using fluorescence spectrophotometry and spectrophotometry based on recombinant reagents, providing guidance for the industry to establish endotoxin detection methods based on recombinant reagents. There are not many methods for the detection of endotoxins using recombinant factors in China, and they are still mainly concentrated on the recombinant Factor C method; in 2016, the Chinese Academy of Sciences detailed the method of using recombinant Limulus amebocyte lysate tri-factor to detect endotoxins using fluorescence method in the patent "Recombinant Limulus amebocyte lysate tri-factor reagent and its method for detecting endotoxins". Comprehensive findings show that the reactivity of factor C alone is far lower than that of the combined use of multiple factors, or the dynamic detection method has a long reaction time and low detection efficiency. Therefore, it is of great significance to provide a new and rapid botulinum toxin detection reagent and method. Summary of the Invention

[0008] In view of this, the present invention provides a recombinant limulus amebocyte lysate reagent and its preparation method and application. The recombinant limulus amebocyte lysate reagent prepared by the present invention has high activity and stability, and the detection results are accurate and reliable.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] A method for preparing a recombinant limulus amebocyte lysate reagent comprises the following steps:

[0011] Step 1): The gene fragments encoding horseshoe crab factor C, factor B, and procoagulant are respectively connected to the baculovirus expression vector and transformed, and recombinant plasmids 1 to 3 are obtained after extraction;

[0012] Step 2): Infect insect cells with the recombinant plasmids 1 to 3, culture them, and collect the culture supernatants to obtain recombinant horseshoe crab factor C, recombinant horseshoe crab factor B, and recombinant horseshoe crab coagulase factor, respectively;

[0013] Step 3): recombinant horseshoe crab factor C, recombinant horseshoe crab factor B and recombinant horseshoe crab coagulase factor are mixed with pyrogen-free Tris buffer, horseshoe crab tripeptide substrate and endotoxin-free water to obtain a recombinant horseshoe crab reagent;

[0014] After the infection in step 2), the method further comprises the step of adding a protein stabilizer;

[0015] The protein stabilizer consists of a protease inhibitor and an amino acid stabilizer; the protease inhibitor includes a serine protease inhibitor, and the amino acid stabilizer includes a neutral amino acid and / or a basic amino acid.

[0016] In this study, we used the insect-baculovirus expression system to express recombinant horseshoe crab factors (procoagulase, factors B and C), and established a novel and rapid endotoxin detection method using the cascade reaction of recombinant proteins and an end-point colorimetric method. The detection principle is shown in Figure 1 The results showed that the recombinant Limulus amebocyte lysate reagent of the present invention can maintain high activity and stability at room temperature, and the detection results are accurate and reliable, laying the foundation for the development of low-cost, fast, and new endotoxin detection reagents.

[0017] In some embodiments, in step 2), the amino acid stabilizer is added 0 to 24 hours after the infection.

[0018] In some embodiments, in step 2), the final concentration of the protease inhibitor by mass volume percentage is ≤0.25% (the converted concentration is ≤2.5g / L), specifically 0.1%, 0.15%, 0.2%, 0.25%. The final concentration of the amino acid stabilizer is 0.5g / L-5g / L, preferably 2-3g / L, specifically 0.5g / L, 0.8g / L, 1g / L, 2g / L, 3g / L, 4g / L, 5g / L. Wherein, the concentration of the protein stabilizer is the concentration of the active ingredient (such as protease inhibitory ingredient, amino acid substance) that plays a stabilizer role in the protein stabilizer system. The protease inhibitor is preferably phenylmethylsulfonyl fluoride (PMSF), and the basic amino acid is preferably arginine. In some specific embodiments, the protein stabilizer includes phenylmethylsulfonyl fluoride (PMSF) and arginine.

[0019] In some embodiments, in step 2), the insect cells are Sf9 cells, Sf21 cells, or other types of insect cells. The baculovirus expression vector includes a pFastbac1 vector, a pFBDM vector, or other insect expression vectors commonly used in the art. In a specific embodiment of the present invention, the insect cells are Sf9 monolayer cells, and the baculovirus expression vector is a pFastbac1 vector.

[0020] In some embodiments, in step 2), the culture supernatant is the culture supernatant obtained by culturing insect cells infected with P3 virus for 48 hours.

[0021] In some embodiments, in step 3), the mass ratio of the recombinant horseshoe crab factor C, the recombinant horseshoe crab factor B and the recombinant horseshoe crab procoagulant is (40-70):(15-25):(15-35), specifically 55:20:25.

[0022] In the present invention, the amino acid sequence of the horseshoe crab factor C is shown in SEQ ID NO: 1, and the nucleic acid sequence encoding the horseshoe crab factor C is shown in SEQ ID NO: 2;

[0023] The amino acid sequence of the horseshoe crab factor B is shown in SEQ ID NO: 3, and the nucleic acid sequence encoding the horseshoe crab factor B is shown in SEQ ID NO: 4;

[0024] The amino acid sequence of the procoagulant is shown in SEQ ID NO: 5, and the nucleic acid sequence encoding the procoagulant is shown in SEQ ID NO: 6.

[0025] The present invention also provides a recombinant limulus amebocyte lysate reagent prepared by the above preparation method.

[0026] The present invention also provides the use of the recombinant Limulus amebocyte lysate reagent in the preparation of a product for detecting endotoxin.

[0027] The present invention provides a recombinant limulus amebocyte lysate reagent and its preparation method and application. The present invention has the following beneficial effects:

[0028] 1) The recombinant limulus amebocyte lysate prepared by the present invention can replace the traditional limulus amebocyte lysate, solving the problem of the rapid decline in the number of marine limulus amebocyte lysate while avoiding the false positive results of endotoxin detection caused by the interference of factor G in the traditional limulus amebocyte lysate. It can be used as a new and rapid endotoxin detection reagent.

[0029] 2) Compared with traditional limulus amebocyte lysate reagents, the recombinant limulus amebocyte lysate reagents of the present invention have a single component and can be produced using bioengineering technology and strict quality control, thereby avoiding batch instability caused by raw materials. Endotoxin detection reagents containing these recombinant limulus amebocyte lysate factors can be produced quickly and at low cost;

[0030] 3) The present invention not only effectively improves the activity and stability of the recombinant Limulus amebocyte lysate reagent by adding a protein stabilizer during the expression of the recombinant three factors, but also helps to reduce background reactivity and improve the accuracy of endotoxin detection.

[0031] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram showing the principle of endotoxin detection by recombinant horseshoe crab three-factor according to the present invention;

[0033] Figure 2 14 shows the standard curve of the recombinant limulus amebocyte lysate of the present invention;

[0034] Figure 3 Shows the effect of different concentrations of arginine on the reaction activity;

[0035] Figure 4 The effects of different concentrations of arginine on cell viability are shown. DETAILED DESCRIPTION

[0036] The present invention provides a recombinant limulus amebocyte lysate reagent, its preparation method, and its application. Those skilled in the art can refer to the contents of this document and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0037] In the present invention, the amino acid sequence of the horseshoe crab factor C is shown in SEQ ID NO: 1, and the nucleic acid sequence encoding the horseshoe crab factor C (GenBank: D90271.1) is shown in SEQ ID NO: 2.

[0038] SEQ ID NO: 2:

[0039]

[0040] SEQ ID NO: 1:

[0041] MVLASFLVSGLVLGILAQQMRPVQSRGVDLGLCDETRFECCKCGDPGYVFNVPMKQCTYFYRWRPYCKPCDDLEAKDICPKYKRCQECKAGLDSCVTCPPNKYGTWCSGECQCKNGGICDQRTGACTCRDRYEGAHCEILKGPLLPSDSQVQEVRNPPDNPQTIDYSCSPGFKLKGVARISCLPNGQWS SFPPKCIRECAKVSSPEHGKVNAPSGNMIEGATLRFSCDSPYYLIGQETLTCQGNGQWSGQIPQCCLKLVFCPDLDPVNHAEHQVKIGVEQKYGQFPQGTEVTYTCSGNYFLMGFNTLKCNPDGSWSGSQPSCVKVADREVDCDSKAVDFLDDVGEPVRIHCPAGCSLTAGTVWGTAIYHELLSSCRAAI HAGKLPNSGGAVHVVNNGPYSDFLGSDLNGIKSEELKSLARSFRFDYVSSSTAGRSGCPDGWFEVEENCVYVTSKQRAWERAQGVCTNMAARLAVLDKDLIPSLTETLRGKGLTTTWIGLHRLDAEKPFVWELMDRSNVVLNDNLTFWASGEPGNETNCVYLDIRDQLQPVWKTKSFQPSSFACMMDLSDRNKAKCDDPGPLENGHATLHGQSIDGFYAGSSIRYSCEVLHYLSGTETVTCTTNGTWSAPKPRCIKVITCQNPPVPSYGSVEIKPPSRTNSISRVGSPFLRLPRLPLPLARAAKPPPKPRSSQPSTVDLASKVKLPEGHYRVGSRAIYTCESRYYELLGSQGRRCDSNGNWSGRPASCIPVCGRSDSPRSPFIWNGNSTEIGQWPWQAGISRWLADHNMWFLQCGGSLLNEKWIVTAAHCVTYSATAEIIDPSQFKIYLGKYYRDDSRDDDYVQVREALEIHVNPNYDPGNLNFDIALIQLKTPVTLTTRVQPICLPT DITTREHLKEGTLAVVTGWGLNENNTYSEMIQQAVLPVVAASTCEEGYKEADLPLTVTENMFCAGYKKGRYDACSGDSGGPLVFADDSRTERRWVLEGIVSWGSPSGCGKANQYGGFTKVNVFLSWIRQFI.

[0042] The amino acid sequence of the horseshoe crab factor B is shown in SEQ ID NO: 3, and the nucleic acid sequence encoding the horseshoe crab factor B (GenBank: D14701.1) is shown in SEQ ID NO: 4.

[0043] SEQ ID NO: 4

[0044]

[0045] SEQ ID NO: 3:

[0046] MTWICVITLFALASATLGNKVSRVGVLFPKTRNDNECTARGGLKGSCKSLIDCPSVLATLKDSFPVVCSWNGRFQPIVCCPDAIAPPPVTTTAVTVISTKEPKLPRLHISGCGKRKVKIDITTVGRSGSPILPPISTPQNSTGGRGIIAGGVEAKIGAWPWMAAVFVKNFGIGRFHCAGSIISNKYILSAAHAFLIGGRK LTPTRLAVRVGGHYIKRGQEYPVKDVIIHPHYVEKENYNDIAIIELKEELNFTDLVNPICLPDPETVTDPLKDRIVTAAGWGDLDFSGPRSQVLREVSIP VVPVDKCDQAYEKLNTPSLKNGITNNFLCAGLEEGGKDACQGDSGGPLMLVNNTRWIVVGVVSFGHKCAEEGYPGVYSRVASYLDWIAKVTNSLDHAVTN.

[0047] The amino acid sequence of the procoagulant (GenBank: M58366.1) is shown in SEQ ID NO: 5, and the nucleic acid sequence encoding the procoagulant is shown in SEQ ID NO: 6.

[0048] SEQ ID NO: 6:

[0049] ATGTTGGTGAATAACGTGTTTTCACTACTGTGTTTCCCACTCTTGATGTCTGTGGTTAGATGCAGTACTCTCAGCAGACAGCGTAGACAGTTTGTTTTCCCTGACGAGGAAGAACTTTGCTCAAACCGATTTACTGAAGAAGGAACATGCAAAAATGTCTTGGATTGTAGAATACTTTTACAAAAAAATGATTATAATTTACTCAAAGAATCAATATGCGGCTTTGAAGGCATAACACCCAAAGTTTGTTGTCCGAAATCAAGCCATGTAATTTCAAGTACACAGGCACCTCCAGAAACCACTACGACTGAACGCCCACCAAAACAGATACCACCCAATCTTCCTGAAGTGTGTGGAATTCACAATACTACAACTACCAGGATTATTGGAGGTCGGGAAGCACCTATTGGAGCCTGGCCGTGGATGACTGCTGTCTACATAAAACAAGGAGGAATCAGAAGTGTTCAGTGTGGTGGCGCACTTGTCACTAACAGGCACGTGATTACAGCTTCGCACTGTGTTGTAAACAGTGCAGGAACAGATGTGATGCCAGCTGATGTATTCTCGGTTCGTCTGGGTGAACACAATTTATACAGTACCGATGACGATTCGAATCCAATAGATTTTGCAGTTACGTCGGTGAAACATCACGAACACTTTGTACTCGCGACGTATTTGAATGACATCGCAATTCTAACGTTAAATGACACAGTTACGTTTACAGACAGAATTCGACCCATTTGTCTACCTTATCGTAAGTTGAGATACGATGATCTAGCAATGAGAAAACCGTTTATCACTGGATGGGGAACAACAGCATTTAACGGCCCATCTAGTGCAGTGTTGAGAGAAGTACAGTTACCAATATGGGAACACGAGGCCTGTAGACAGGCCTACGAGAAGGATTTAAATATTACAAACGTGTATATGTGTGCTGGCTTTGCAGATGGCGGGAAGGATGCTTGCCAGGGTGATTCTGGAGGTCCAATGATGTTGCCTGTTAAAACCGGAGAGTTTTATCTCATTGGAATTGTGTCTTTCGGAAAGAAATGCGCATTGCCTGGATTTCCTGGGGTTTACACAAAAGTGACAGAGTTTTTAGATTGGATTGCAGAACATATGGTGTAG。

[0050] SEQ ID NO:5:

[0051] MLVNNVFSLLCFPLLMSVVRCSTLSRQRRQFVFPDEEELCSNRFTEEGTCKNVLDCRILLQKNDYNLLKESICGFEGITPKVCCPKSSHVISSTQAPPETTTTERPPKQIPPNLPEVCGIHNTTTTRIIGGREAPIGAWPWMTAVYIKQGGIRSVQCGGALVTNRHVITASHCVVNSAGTDVMPADVFSVRLGEHNLYSTDDDSNPIDFAVTSVKHHEHFVLATYLNDIAILTLNDTVTFTDRIRPICLPYRKLRYDDLAMRKPFITGWGTTAFNGPSSAVLREVQLPIWEHEACRQAYEKDLNITNVYMCAGFADGGKDACQGDSGGPMMLPVKTGEFYLIGIVSFGKKCALPGFPGVYTKVTEFLDWIAEHMV。

[0052] 本发明采用的试材皆为普通市售品,皆可于市场购得。

[0053] 下面结合实施例,进一步阐述本发明:

[0054] Example 1 Preparation of recombinant limulus amebocyte lysate

[0055] Step 1) Referring to NCBI, the nucleic acid sequences of horseshoe crab factor C (GenBank: D90271.1), factor B (GenBank: D14701.1), and procoagulant (GenBank: M58366.1) were synthesized, and primers were designed using Primer 5.0. The primers were amplified to obtain PCR amplified horseshoe crab factor C, factor B, and procoagulant gene fragments, respectively;

[0056] Step 2) The amplified horseshoe crab factor C, factor B, and coagulase factor target gene fragments were ligated with the pfastbac1 vector at room temperature (15-25°C) for 30 minutes to obtain first recombinant plasmids. Each first recombinant plasmid was then transformed into DH5α competent cells, coated on plates containing gentamicin, carnamycin sulfate, and tetracycline, and cultured at 37°C overnight. Positive monoclonal colonies were screened for blue-white spots and picked, and cultured in liquid medium at 37°C overnight. The plasmids were extracted, identified by PCR, and sequenced to obtain bacmid recombinant bacmids.

[0057] Step 3) Each Bacmid recombinant bacmid was transfected into Sf9 cell monolayers (six-well plates). When the cells showed obvious symptoms of viral infection, the culture supernatant was collected and centrifuged at 1450 rpm for 5 min. The resulting supernatant was P1. P1 was used to infect Sf9 cells at an MOI of 0.1, and the culture supernatant was obtained to obtain P2. The Sf9 cells were then infected, and the culture supernatant was obtained again. After two rounds of infection, a high-titer P3 virus was obtained, and the virus titer was measured by plaque assay. Sf9 cells were infected with the P3 virus, and the culture supernatant was collected 48 hours later to obtain horseshoe crab recombinant factor C, factor B, and procoagulant, respectively.

[0058] Step 4) A stabilizer solution was added during the expression process, wherein arginine was added within 24 hours of expression to a final concentration of 2 g / L, and PMSF was added after 24 hours of expression to a final concentration of 0.125%.

[0059] Step 5) Using the recombinant horseshoe crab factor C, recombinant horseshoe crab factor B and recombinant horseshoe crab procoagulant obtained in step 4), a recombinant horseshoe crab reagent is prepared. The reaction system is configured as follows:

[0060] 15 μl of recombinant limulus amebocyte lysate (Factor C:Factor B:Procoagulant ratio of 55:20:25);

[0061] Pyrogen-free Tris buffer: 30ul;

[0062] Limulus tripeptide substrate: 5ul;

[0063] 1% Tween-20: 10ul;

[0064] Endotoxin-free water: make up to 100ul.

[0065] Endotoxin was detected using the recombinant three-factor limulus amebocyte lysate reagent obtained above:

[0066] Step 1: Prepare at least 6 endotoxin standard solutions of different concentrations and plate them in a 96-well plate in ascending order of concentration, with 3 replicates for each concentration. Add 0.5 EU / ml and 4 EU / ml standards for recovery testing.

[0067] Step 2: Prepare the reagent for endotoxin detection, mix well, and add it to the 96-well plate paved with endotoxin; place the 96-well plate in a microplate incubator, set the reaction temperature of the microplate incubator to 37°C, and the reaction time to 20 minutes;

[0068] Step 3: After the reaction is completed, add 50ul of color development solution 1, 50ul of color development solution 2, and 50ul of color development solution 3 to each reaction well in sequence;

[0069] Step 4: Set the microplate reader to an excitation wavelength of 545 nm and an emission wavelength of 620 nm; detect and record the absorbance value; use the absorbance value (OD) as the vertical axis after log conversion to base 10, and the endotoxin standard solution concentration (EU / ml) as the horizontal axis to create a four-parameter standard curve. The linear determination results are shown in Tables 1 and Figure 2 The endotoxin spike recovery results are shown in Table 2. The results show that both linearity and endotoxin spike recovery meet the requirements.

[0070] Table 1

[0071] Endotoxin concentration 1st time 2nd time 3rd time 0EU / ml 0.0365 0.036 0.035 0.005EU / ml 0.0405 0.038 0.0395 0.05EU / ml 0.073 0.0715 0.067 0.1EU / ml 0.1175 0.1165 0.1115 0.5EU / ml 0.424 0.424 0.377 1EU / ml 0.783 0.7855 0.7355 2.5EU / ml 1.7575 1.761 1.7085 5EU / ml 2.8575 2.9025 2.9085

[0072] Table 2 Results of spike recovery determination

[0073]

[0074] Example 2

[0075] Referring to Example 1, the timing of adding the protein stabilizer in step 4) was adjusted. The implementation scheme is shown in Table 3. The recombinant three-factor limulus amebocyte lysate was prepared according to the method of Example 1, and endotoxin was detected. The results are shown in Table 4.

[0076] Table 3

[0077]

[0078]

[0079] Table 4

[0080]

[0081] The results showed that adding arginine within 24 hours did not affect the activity, and adding PMSF after 24 hours did not affect the activity. The optimal choice was to add arginine at 0 hours and PMSF at 48 hours.

[0082] Test Example 1

[0083] During the expression process in step 4), different concentrations of protease inhibitor PMSF were added, and the other conditions were the same as in Example 1. Endotoxin was detected according to the method in Example 1, and the results were as follows: the addition of PMSF can effectively reduce the background, and the optimal final concentration is 0.125%.

[0084] Table 5

[0085] Endotoxin concentration PMSF 0% PMSF 0.0625% PMSF 0.125% PMSF 0.25% 0EU / ml 0.233 0.021 0.012 0.10 1.0EU / ml 0.976 0.866 0.814 0.787 2.0EU / ml 1.973 2.097 2.153 1.762

[0086] Test Example 2

[0087] Refer to Example 1, wherein, in step 4), only different concentrations of arginine were added during the expression process, and PMSF was not added. The other conditions were the same as in Example 1. Endotoxin was detected by referring to the method in Example 1, and the results are shown in FIG. Figures 3-4 .

[0088] The results showed that the cell viability gradually increased with the addition of arginine concentration, but the reaction activity gradually decreased, and the optimal final concentration was 2-3 g / L.

[0089] Test Example 3

[0090] Control group 1: referring to Example 1, wherein no stabilizer component was added during the expression process in step 4), and the remaining conditions were the same as in Example 1.

[0091] After the preparation of the limulus amebocyte lysate in control group 1 and Example 1, an accelerated test was conducted at room temperature for 8 hours. Endotoxin was detected according to the method of Example 1. The results are as follows:

[0092] It can maintain good room temperature stability (Table 6). When no stabilizer is added, the background value increases significantly after 8 hours at room temperature, and the high value decreases by more than 20% (Table 7).

[0093] Table 6 Measurement results of Example 1

[0094] Verification items: -20℃ control Room temperature 8h Room temperature drop in 8 hours 0EU / ml 0.087 0.092 5.7％ 2EU / ml 2.777 2.815 1.4％

[0095] Table 7 Measurement results of control group 1

[0096] Verification items: -20℃ control Room temperature 8h Room temperature drop in 8 hours 0EU / ml 0.087 0.238 173.5％ 2EU / ml 2.777 2.180 -22.6％

[0097] Test Example 4: Verification of the Effect of Other Stabilizers

[0098] Refer to Example 1, wherein, in step 4), the expression process is not added with the protein stabilizer according to the present application, but other forms of stabilizers (non-amino acids) are added, and the rest is the same as Example 1.

[0099] Set a control: do not add any form of stabilizer, and the other conditions are the same as Example 1. Refer to the method of Example 1 to detect endotoxin, and the results are shown in Table 8.

[0100] The results show that other stabilizers Halt, EDTA, glycerol, trehalose, glycine, sorbitol cannot achieve the control effect.

[0101] Table 8

[0102]

[0103] The above only describes the preferred embodiments of the present application, and the protection scope of the present application is not limited to the above examples. Any technical solutions that fall within the concept of the present application are within the protection scope of the present application. It should be noted that, for ordinary skilled persons in the art, improvements and refinements without departing from the principles of the present application are also considered to be within the protection scope of the present application.

Claims

1. A method for preparing a recombinant limulus amebocyte lysate reagent, characterized in that: The steps include: Step 1): The gene fragments encoding horseshoe crab factor C, factor B, and procoagulant are respectively connected to the baculovirus expression vector and transformed, and recombinant plasmids 1 to 3 are obtained after extraction; Step 2): Insect cells are infected with the recombinant plasmids 1 to 3 respectively, cultured, and the culture supernatant is collected to obtain recombinant horseshoe crab factor C, recombinant horseshoe crab factor B, and recombinant horseshoe crab coagulase factor, respectively; Step 3): recombinant horseshoe crab factor C, recombinant horseshoe crab factor B and recombinant horseshoe crab coagulase factor are mixed with pyrogen-free Tris buffer, horseshoe crab tripeptide substrate and endotoxin-free water to obtain recombinant horseshoe crab reagent; Step 2) further comprises the step of adding a protein stabilizer after the infection; The protein stabilizer is composed of a protease inhibitor and an amino acid stabilizer; the protease inhibitor is phenylmethylsulfonyl fluoride, and the amino acid stabilizer is arginine; The amino acid stabilizer is added 0 to 24 hours after the infection; The protease inhibitor is added 24 to 48 hours after the infection; The final concentration of the protease inhibitor by weight volume percentage is 0.0625% to 0.125%, and the final concentration of the amino acid stabilizer is 0.5g / L to 5g / L; The amino acid sequence of the horseshoe crab factor C is shown in SEQ ID NO: 1; The amino acid sequence of the horseshoe crab factor B is shown in SEQ ID NO: 3; The amino acid sequence of the procoagulant is shown in SEQ ID NO:

5.

2. The preparation method according to claim 1, characterized in that In step 2), The insect cells are Sf9 cells, sf21 cells or other types of insect cells; The baculovirus expression vector includes a pfastbac vector or a pFBDM vector or other insect expression vectors.

3. The preparation method according to claim 1, characterized in that In step 2), the culture supernatant is the culture supernatant obtained by culturing virus-infected insect cells for 48-72 hours.

4. The preparation method according to claim 1, characterized in that In step 3), the mass ratio of the recombinant horseshoe crab factor C, the recombinant horseshoe crab factor B and the recombinant horseshoe crab procoagulant is (40-70): (15-25): (15-35).

5. The preparation method according to any one of claims 1 to 4, characterized in that The nucleic acid sequence encoding the horseshoe crab factor C is shown in SEQ ID NO: 2; The nucleic acid sequence encoding the horseshoe crab factor B is shown in SEQ ID NO: 4; The nucleic acid sequence encoding the procoagulant is shown in SEQ ID NO:

6.

6. The recombinant limulus amebocyte lysate reagent prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the recombinant Limulus amebocyte lysate prepared by the preparation method according to any one of claims 1 to 5 or the recombinant Limulus amebocyte lysate according to claim 6 in the preparation of a product for detecting endotoxin.

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