Recombinant factor C tachypleus amebocyte lysate and kit

Abstract

The invention relates to a recombinant C factor tachypleus amebocyte lysate and a kit. A bacterial endotoxin detection reagent comprises the following components: a recombinant C factor, a fluorescent substrate and a balance enhancer, and the balance enhancer is selected from one or more of alkyl pyranoside, polyethylene glycol ether, dextran and dioxane derivatives. According to the recombinant factor C tachypleus amebocyte lysate disclosed by the invention, the specific balanced enhancer is added, so that the reaction activity of the recombinant factor C tachypleus amebocyte lysate and endotoxin can be enhanced, a detection signal is enhanced, the detection time is shortened, meanwhile, the reactivity consistency on endotoxin of different species sources is good, and the recombinant factor C tachypleus amebocyte lysate has excellent universality.

Description

Technical Field

[0001] The present application belongs to the technical field of detection reagents, and in particular, relates to a recombinant factor C horseshoe crab reagent and a kit, which is suitable for quality control in the drug production process and belongs to the technical field of drug production quality detection. Background Art

[0002] Existing bacterial endotoxin detection technologies mainly include gel method, photometry, recombinant factor C method and biosensor detection method. The gel method uses horseshoe crab reagent to react with bacterial endotoxin to generate gel, and the presence of endotoxin is judged by observing the formation of gel. However, the operation is complicated, the sensitivity is low, it is easily interfered by external factors, and it requires a large amount of horseshoe crab blood, which poses ecological protection problems. The photometry method includes turbidity method and colorimetric matrix method. The turbidity method quantitatively detects endotoxin by measuring the turbidity change of the reaction mixture, and the colorimetric matrix method quantitatively detects endotoxin by detecting the number of chromophores released during the reaction. Although the photometry method has high sensitivity, it has more operating steps and is easily interfered by the sample matrix, which may lead to false positive or false negative results.

[0003] Existing bacterial endotoxin detection technologies have certain shortcomings in terms of operational complexity, anti-interference and ecological protection. Future development directions include simplifying the operating process, improving anti-interference and reducing the impact on the ecological environment. The biosensor detection method uses biosensors to detect the binding reaction of endotoxins with specific probes to generate measurable electrical signals or light signals. It has strong anti-interference ability, but the technology is complex, the equipment is expensive, and it lacks verification. It has not yet been widely used in actual production. The recombinant factor C method uses recombinant factor C produced by genetic engineering technology to replace the traditional horseshoe crab reagent for endotoxin detection. The principle is that endotoxin activates the recombinant factor C zymogen, and the activated factor C cuts fluorescent synthetic substrates such as coumarin-labeled polypeptides to produce detectable fluorescent signals. Since the recombinant factor C reagent lacks the factor B and coagulase cascade amplification process in the horseshoe crab reagent, although a more sensitive fluorescence detection method is used to replace the colorimetric method of the traditional horseshoe crab reagent, its sensitivity is still low.

[0004] In traditional endotoxin detection, surfactants have a complex effect on the activity of horseshoe crab reagents, and the mechanism is not very clear. Different surfactants show different enhancing or inhibiting effects on the reaction of horseshoe crab reagents with endotoxins. The practice of adding surfactants to recombinant Factor C reagents to enhance the sensitivity actually utilizes this enhancing effect. However, since bacterial endotoxins are a large class of substances, not a chemical substance with a single structure, the surfactants currently used have different enhancing effects on the recombinant Factor C horseshoe crab reagents for detecting different types of bacterial endotoxins, which will lead to differences in the reactivity of recombinant Factor C to different types of endotoxins. For example, the endotoxin detection method described in Patent ZL02814296.9 only screens surfactants with better activity enhancement effects on endotoxins used as reference substances. In actual sample testing, the accuracy of detecting different types of bacterial endotoxins often deviates greatly. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a recombinant Factor C horseshoe crab reagent and a kit.

[0006] The first aspect of the present invention provides a recombinant Factor C horseshoe crab reagent, comprising the following components: recombinant Factor C, a fluorescent substrate and a balanced enhancer, wherein the balanced enhancer is selected from one or more of alkyl pyranoside, polyethylene glycol ether, dextran, and dioxane derivatives.

[0007] Preferably, the alkyl pyranoside comprises a hydrophilic portion selected from glucose, fucose, mannose, galactose, xylose, maltose or lactose and a hydrophobic portion selected from an alkyl group.

[0008] Furthermore, the alkyl group of the alkyl pyranoside is octyl, heptyl or hexyl, and the alkyl pyranoside is further preferably alkyl glucopyranoside.

[0009] Preferably, the polyethylene glycol ether is polyethylene glycol alkyl ether, and the alkyl chain length of the polyethylene glycol alkyl ether is preferably C5-C15, and more preferably polyethylene glycol monododecyl ether.

[0010] Preferably, the dioxane derivative is a 1,3-dioxane derivative, and more preferably 5-bromo-5-nitro-1,3-dioxane.

[0011] Preferably, the balance enhancer is selected as a combination of two or more, preferably polyethylene glycol monododecyl ether and 5-bromo-5-nitro-1,3-dioxane in a mass ratio of 0.005-0.05:0.02-0.2, or octyl pyranose glucoside and 5-bromo-5-nitro-1,3-dioxane in a mass ratio of 0.1-1:0.02-0.2, or dextran, octyl pyranose glucoside and 5-bromo-5-nitro-1,3-dioxane in a mass ratio of 0.2-2:0.1-1:0.02-0.2.

[0012] Preferably, the content of the equalization enhancer in the recombinant Factor C horseshoe crab reagent is 0.04wt%-4wt%.

[0013] Preferably, the sequence of the recombinant Factor C is selected from SEQ ID NO: 1, or is a polypeptide having ≥ 90% homology with the amino acid sequence shown in SEQ ID NO: 1 and having recombinant Factor C activity.

[0014] Preferably, the recombinant Factor C horseshoe crab reagent further comprises a buffer, and the buffer is preferably 50 to 500 mM Tris-HCl buffer with a pH of 7.4 to 8.0, and more preferably comprises 2 to 20 mM magnesium ions and / or sodium ions.

[0015] Preferably, the fluorescent substrate is selected from DPR-AMC.

[0016] Preferably, the recombinant Factor C is expressed by an insect cell expression system, a mammalian cell expression system or a wheat germ cell-free protein expression system.

[0017] Preferably, the recombinant Factor C is produced by a method in which a host cell comprising a vector encoding the Factor C protein is cultured in the supernatant under conditions such that the vector is expressed in the supernatant.

[0018] The second aspect of the present invention provides a kit comprising the recombinant Factor C horseshoe crab reagent described in the first aspect of the present invention.

[0019] The beneficial effects of the present invention are as follows: the recombinant Factor C horseshoe crab reagent of the present invention can enhance the reactivity of the recombinant horseshoe crab reagent with endotoxin, enhance the detection signal, shorten the detection time, and at the same time has good consistency in reactivity to endotoxins from different species, and has excellent universality. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The technical solution of the present application is further described below in conjunction with the accompanying drawings and embodiments.

[0021] Figure 1 Represents the differences in reactivity of different Factor C horseshoe crab reagents to endotoxins from six different strains.

[0022] Figure 2 Represents the effect of adding different balanced enhancers on the reactivity of the recombinant factor C horseshoe crab reagent. DETAILED DESCRIPTION

[0023] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application may be combined with each other.

[0024] The technical solution of the present application will be described in detail below with reference to the accompanying drawings and in combination with embodiments.

[0025] Example 1 Expression of recombinant factor C, recombinant factor B and recombinant prothrombin

[0026] According to the protein sequence of the recombinant C factor (SEQ ID NO: 1), the corresponding gene fragments were synthesized, and the synthesized gene fragments were connected to pTA2Vector at room temperature to obtain the first recombinant plasmid, and then the first recombinant plasmid was transformed into DH5α competent cells, and the positive clone colonies were selected by blue-white screening, amplified by shaking, identified by PCR and sequenced, and the recombinant plasmid with correct sequencing was double-enzyme digested to obtain the first product.

[0027] The first product was connected with the pFastBac-Dual vector to obtain a second recombinant plasmid, and the second recombinant plasmid was transformed into DH10Bac competent cells, positive monoclonal colonies were screened by blue-white staining, plasmids were extracted by shaking, and the sequences were verified by sequencing after double enzyme digestion;

[0028] The second recombinant plasmid was transformed into DH10Bac competent cells, coated on a plate containing gentamicin, kanamycin sulfate and tetracycline, cultured overnight at 37°C, blue-white screening and picking of positive monoclonal colonies, shaking to extract plasmids, PCR identification and sequencing to obtain bacmid recombinant bacmids.

[0029] The Bacmid recombinant bacmid was transfected into the Sf9 monolayer cells. When the cells showed obvious symptoms of virus infection, the supernatant was collected as the P1 virus. P1 was taken to infect Sf9 cells at an MOI value of 0.1, and the culture supernatant was taken to obtain P2. The Sf9 cells were infected again, and the culture supernatant was taken again. After two times, a high-titer P3 virus was obtained, and the virus titer was measured by the plaque method. Sf9 cells were infected with the P3 virus, and after culturing for 48 to 72 hours, the cell culture supernatant of the recombinant C factor was collected.

[0030] Example 2 Preparation method of recombinant factor C horseshoe crab reagent containing balanced enhancer

[0031] Preparation of a series of buffers: The buffer consists of 200 mM pH 7.5 Tris-HCl, 10 mM magnesium sulfate, 10 mM sodium chloride and different types of enhancers (dextran, octylpyranoglucoside, polyethylene glycol monododecyl ether, 5-bromo-5-nitro-1,3-dioxane).

[0032] Prepare substrate solution: The substrate solution is a DPR-AMC solution with a concentration of 0.2wt‰.

[0033] Preparation of recombinant Factor C horseshoe crab reagent: Mix the cell culture supernatant of recombinant Factor C, substrate solution and buffer solution in a volume ratio of 1:5:4.

[0034] Example 3 Endotoxin Detection Process

[0035] Preheat the instrument and set the incubation temperature to 37°C. Set the template and program. Set the excitation wavelength to 380nm and the emission wavelength to 440nm. Prepare the endotoxin standard solution (5, 0.5, 0.05, 0.005EU / mL) according to a 10-fold gradient dilution. The negative control is water for bacterial endotoxin test. Mix the recombinant factor C horseshoe crab reagent solution. Remove the pyrogen-free microplate and add 100μL of bacterial endotoxin test water or endotoxin standard solution or test sample to each well. After preheating for ≥10 minutes, add 100μL of recombinant factor C horseshoe crab reagent to each well with a pipette or multichannel pipette, vortex mix for 10 seconds, put it in the microplate reader for incubation for 30 to 60 minutes, and read according to the set parameters.

[0036] Example 4 Determination of the difference in the reactivity of recombinant Factor C horseshoe crab reagent to endotoxins from different bacterial species

[0037] Bacterial endotoxins from different bacteria (Serratia marcescens, Salmonella enterica (Minnesota serotype), Escherichia coli 0111:B4, Escherichia coli 055:B5, Salmonella enterica (Minnesota serotype), Pseudomonas aeruginosa 10) were purchased and packaged into frozen endotoxin stock solutions. The frozen different endotoxin stock solutions were diluted to the required concentrations, calibrated using the national endotoxin standard, and the recombinant Factor C horseshoe crab reagent prepared in Example 2 was used to detect the concentration values ​​of different endotoxin solutions. At the same time, endotoxin detection was performed using the recombinant Factor C horseshoe crab reagent without the addition of an equalizing enhancer (no addition) and a commercial natural horseshoe crab reagent. The reaction differences were analyzed by calculating the correlation coefficients of the endotoxin concentrations determined by different reagents (see for details). Figure 1In the figure, O represents a recombinant Factor C horseshoe crab reagent containing 0.5wt% of the balanced enhancer octylpyranoglucoside, B represents a recombinant Factor C horseshoe crab reagent containing 0.02wt% of the balanced enhancer polyethylene glycol monododecyl ether, C represents a recombinant Factor C horseshoe crab reagent containing 0.5wt% CHAPS, C is a comparative example, A, X, and R are commercial natural horseshoe crab reagents of natural dynamic colorimetric method from different manufacturers.

[0038] From the test results, it can be seen that the recombinant Factor C horseshoe crab reagent with the addition of equalizing enhancer O has good consistency with the natural horseshoe crab reagents of manufacturers A and R, and the recombinant Factor C horseshoe crab reagent with the addition of equalizing enhancer B has good consistency with the natural horseshoe crab reagent of manufacturer X. The consistency of the recombinant Factor C horseshoe crab reagent with the addition of equalizing enhancer C and the natural horseshoe crab reagents of manufacturers A, R, and X is not good. This proves that the recombinant Factor C horseshoe crab reagent containing equalizing enhancers O and B of the present invention has good consistency in reactivity to endotoxins from different sources.

[0039] Example 5 Activity enhancement effect determination

[0040] According to the method of Example 2, recombinant Factor C horseshoe crab reagents containing different balanced enhancers were prepared respectively, and the recombinant Factor C horseshoe crab reagent without balanced enhancer was used as a control. The detection was carried out according to the method of Example 3. For details, see Figure 2 . Figure 2 From top to bottom, the recombinant Factor C horseshoe crab reagents prepared respectively include no addition of an equalizing enhancer, 0.04 wt % of 5-bromo-5-nitro-1,3-dioxane, 0.5 wt % of octylpyranoglucoside, 0.5 wt % of octylpyranoglucoside and 0.04 wt % of 5-bromo-5-nitro-1,3-dioxane, and 0.5 wt % of octylpyranoglucoside, 0.04 wt % of 5-bromo-5-nitro-1,3-dioxane, and 1.5 wt % of dextran.

[0041] Depend on Figure 2 It can be seen that, compared with the recombinant Factor C horseshoe crab reagent without the addition of the balanced enhancer, the recombinant Factor C horseshoe crab reagent with the addition of different balanced enhancers can significantly improve the reaction activity to endotoxin and enhance the detection signal. This proves that the balanced enhancer of the present invention can significantly improve the detection activity to endotoxin.

[0042]

[0043] Based on the above ideal embodiments of this application, the relevant staff can make various changes and modifications without departing from the technical concept of this application through the above description. The technical scope of this application is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A recombinant factor C horseshoe crab reagent, characterized in that: The invention comprises the following components: recombinant factor C, a fluorescent substrate and a balanced enhancer, wherein the balanced enhancer is selected from one or more of alkyl pyranoside, polyethylene glycol ether, dextran and dioxane derivatives.

2. The recombinant Factor C horseshoe crab reagent according to claim 1, characterized in that: The alkyl pyranoside comprises a hydrophilic part selected from glucose, fucose, mannose, galactose, xylose, maltose or lactose and a hydrophobic part selected from an alkyl group, the alkyl group of the alkyl pyranoside is preferably an octyl group, a heptyl group or a hexyl group, and the alkyl pyranoside is further preferably an alkyl glucopyranoside.

3. The recombinant Factor C horseshoe crab reagent according to claim 1, characterized in that: The polyethylene glycol ether is a polyethylene glycol alkyl ether, and the alkyl chain length of the polyethylene glycol alkyl ether is preferably C5-C15, and more preferably polyethylene glycol monododecyl ether.

4. The recombinant Factor C horseshoe crab reagent according to claim 1, characterized in that: The dioxane derivative is a 1,3-dioxane derivative, and more preferably 5-bromo-5-nitro-1,3-dioxane.

5. The recombinant Factor C horseshoe crab reagent according to any one of claims 1 to 4, characterized in that: The balance enhancer is polyethylene glycol monododecyl ether and 5-bromo-5-nitro-1,3-dioxane in a mass ratio of 0.005-0.05:0.02-0.2, or octyl pyranoglucoside and 5-bromo-5-nitro-1,3-dioxane in a mass ratio of 0.1-1:0.02-0.2, or dextran, octyl pyranoglucoside and 5-bromo-5-nitro-1,3-dioxane in a mass ratio of 0.2-2:0.1-1:0.02-0.

2.

6. The recombinant Factor C horseshoe crab reagent according to any one of claims 1 to 4, characterized in that: The content of the equilibrium enhancer in the recombinant factor C horseshoe crab reagent is 0.04wt%-4wt%.

7. The recombinant Factor C horseshoe crab reagent according to any one of claims 1 to 4, characterized in that: The sequence of the recombinant Factor C is selected from SEQ ID NO: 1, or is a polypeptide having a homology of ≥ 90% with the amino acid sequence shown in SEQ ID NO: 1 and having recombinant Factor C activity.

8. The recombinant Factor C horseshoe crab reagent according to any one of claims 1 to 4, characterized in that: The recombinant Factor C horseshoe crab reagent further comprises a buffer, and the buffer is preferably 50 to 500 mM Tris-HCl buffer with a pH of 7.4 to 8.0, and more preferably comprises 2 to 20 mM magnesium ions and / or sodium ions.

9. The recombinant Factor C horseshoe crab reagent according to any one of claims 1 to 6, characterized in that: The fluorescent substrate is DPR-AMC.

10. A kit, characterized in that: A recombinant Factor C horseshoe crab reagent comprising any one of claims 1 to 9.

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