A recombinant limulus amebocyte lysate and kit

Abstract

The application relates to a recombinant three-factor limulus reagent and a kit, and the bacterial endotoxin detection reagent comprises the following components: a recombinant C factor, a recombinant B factor, a recombinant coagulogen, a chromogenic substrate and a balance enhancer selected from one or more of alkyl pyranoside, polyethylene glycol ether, PEG and dioxane derivatives. The recombinant three-factor limulus reagent of the application can enhance the reaction activity of the recombinant limulus reagent and endotoxin, enhance the detection signal, shorten the detection time, and has good consistency in the reactivity to endotoxins from different species and excellent universality.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of detection reagents, and particularly relates to a recombinant Limulus three-factor reagent and a kit, which are suitable for quality control in the production process of drugs and belong to the technical field of drug production quality detection. BACKGROUND

[0002] The existing bacterial endotoxin detection technology mainly includes gel method, photometric method, recombinant factor C method and biosensor detection method. The gel method utilizes the reaction of Limulus reagent and bacterial endotoxin to generate a gel, and the presence of endotoxin is determined by observing the gel formation. However, the operation is complex, the sensitivity is low, it is easily disturbed by external factors, and a large amount of Limulus blood is needed, which has ecological protection problems. The photometric method includes turbidity method and chromogenic substrate method. The turbidity method quantitatively detects endotoxin by measuring the turbidity change of the reaction mixture, and the chromogenic substrate method quantitatively detects endotoxin by detecting the number of chromophore released in the reaction process. Although the photometric method has high sensitivity, it has many operation steps and is easily disturbed by sample matrix, which may lead to false positive or false negative results.

[0003] The existing bacterial endotoxin detection technology has certain deficiencies in operation complexity, anti-interference and ecological protection, and the future development direction includes simplifying the operation process, improving the anti-interference and reducing the impact on the ecological environment. The biosensor detection method utilizes the biosensor to detect the binding reaction of endotoxin and specific probes to generate measurable electrical or optical signals, which has strong anti-interference ability, but the technology is complex, the equipment is expensive, and lacks verification, which has not been widely used in actual production. The recombinant factor C method utilizes the recombinant factor C produced by genetic engineering technology to replace the traditional Limulus reagent for endotoxin detection. The principle is that endotoxin activates the recombinant factor C zymogen, and the activated C factor cuts the fluorescence synthesis substrate such as coumarin-labeled polypeptide to generate a detectable fluorescence signal. Since the recombinant C factor reagent lacks the factor B and coagulase cascade amplification process in the Limulus reagent, although a more sensitive fluorescence detection method is used to replace the traditional Limulus reagent chromogenic method, its sensitivity is still low. The recombinant Limulus reagent utilizes the genetic engineering technology to produce the factor C and factor B contained in the natural Limulus reagent to convert the pro-coagulase into coagulase. The coagulase cuts the chromogenic synthesis substrate such as pNA-labeled polypeptide to generate a detectable chromogenic signal. The detection principle, method and equipment are completely the same as those of the natural chromogenic method Limulus reagent, and have higher sensitivity.

[0004] In traditional endotoxin detection, the activity of surfactants on limulus reagent has a complex effect, the mechanism of which is not very clear. Different surfactants show different enhancement or inhibition effects on the reaction of limulus reagent and endotoxin. The method of adding surfactants to recombinant limulus reagent to enhance sensitivity actually utilizes this enhancement effect. However, since bacterial endotoxin is a large class of substances, not a single-structure chemical substance, but the currently used surfactants do not have the same enhancement effect on the detection of different types of bacterial endotoxin by recombinant limulus reagent, which may cause differences in the reactivity of factor C to different types of endotoxin. For example, the method for detecting endotoxin described in patent ZL02814296.9 only selects surfactants that have better activity enhancement effect on the reference endotoxin. In actual sample detection, the accuracy of detection of different types of bacterial endotoxin is often greatly deviated. SUMMARY

[0005] To solve the above problems, the present application provides a recombinant limulus three-factor reagent and a kit.

[0006] The first aspect of the present application provides a recombinant limulus three-factor reagent, comprising the following components: recombinant C factor, recombinant B factor, recombinant coagulogen, chromogenic substrate and balanced enhancer. The balanced enhancer is selected from one or more of alkyl pyranoside, polyethylene glycol ether, PEG, dioxane derivative. The molecular weight of the PEG is preferably 6000-20000.

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

[0008] Further, the alkyl of the alkyl pyranoside is octyl, heptyl or hexyl, and the alkyl pyranoside is further preferably alkyl pyranoglucoside.

[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 is further preferably polyethylene glycol monododecyl ether.

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

[0011] Preferably, the balance enhancer is selected from two or more combinations, preferably polyethylene glycol monododecyl ether, 5-bromo-5-nitro-1,3-dioxane in a mass ratio of 0.005-0.05:0.02-0.2, or octyl pyranoglucoside, 5-bromo-5-nitro-1,3-dioxane in a mass ratio of 0.1-1:0.02-0.2, or PEG, polyethylene glycol monododecyl ether, 5-bromo-5-nitro-1,3-dioxane in a mass ratio of 0.2-2:0.005-0.05:0.02-0.2, or PEG, 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.

[0012] Preferably, the content of the balance enhancer in the recombinant Leptospira three-factor reagent is 0.04wt%-4wt%.

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

[0014] Preferably, the sequence of the recombinant B factor is selected from SEQ ID NO: 2, or is a polypeptide having homology of ≥90% with the amino acid sequence shown in SEQ ID NO: 2 and having the activity of the recombinant B factor.

[0015] Preferably, the sequence of the recombinant coagulogen is selected from SEQ ID NO: 3, or is a polypeptide having homology of ≥90% with the amino acid sequence shown in SEQ ID NO: 3 and having the activity of the recombinant coagulogen.

[0016] Preferably, the recombinant Leptospira three-factor reagent further comprises a buffer, and the pH of the buffer is preferably 7.4-8.0.

[0017] Preferably, the chromogenic substrate is selected from pNA chromogenic substrate peptides.

[0018] Preferably, the recombinant C factor, the recombinant B factor and the recombinant coagulogen are expressed by an insect cell expression system, a mammalian cell expression system or a wheat germ cell-free protein expression system.

[0019] Preferably, the recombinant C factor, the recombinant B factor and the recombinant coagulogen are prepared by a method of culturing host cells containing a vector encoding the C factor protein, the B factor and the coagulogen under conditions in which they are expressed in the supernatant.

[0020] The second aspect of the present application provides a kit comprising the recombinant Leptospira three-factor reagent of the first aspect of the present application.

[0021] The recombinant leucocin factor agent of the present application can enhance the reaction activity of the recombinant leucocin factor agent and endotoxin by adding a specific balanced enhancer, enhance the detection signal, shorten the detection time, and has excellent universality with good consistency of reactivity to endotoxins from different species. BRIEF DESCRIPTION OF DRAWINGS

[0022] The technical solutions of the present application will be further described below in combination with the drawings and examples.

[0023] Figure 1 The figure represents the difference in reactivity of different leucocin factor agents to endotoxins from six different strains.

[0024] Figure 2 The figure represents the influence of adding different balanced enhancers on the reaction activity of the recombinant leucocin factor agent. DETAILED DESCRIPTION

[0025] It should be noted that the examples in the present application and the features in the examples can be combined with each other without conflict.

[0026] The technical solutions of the present application will be further described below in combination with the drawings and examples.

[0027] Example 1 Expression of recombinant C factor, recombinant B factor and recombinant coagulogen

[0028] According to the protein sequences of the recombinant C factor, the recombinant B factor and the recombinant coagulogen (SEQ ID NO: 1-3 in turn), the corresponding gene fragments were synthesized, and the synthesized gene fragments were respectively connected with pTA2Vector under room temperature conditions to obtain first recombinant plasmids. Then each first recombinant plasmid was respectively transformed into DH5α competent cells, and positive clone colonies were selected by blue-white spot screening and shaking culture amplification, PCR identification and sequencing were performed, and the correct sequencing recombinant plasmid was double enzyme cut to obtain the first product.

[0029] The first product was connected with pFastBac-Dual vector to obtain second recombinant plasmids, and each second recombinant plasmid was respectively transformed into DH10Bac competent cells, and positive monoclonal colonies were selected by blue-white spot screening, and plasmids were extracted by shaking culture, and the sequences were verified by double enzyme cutting and sequencing.

[0030] Each second recombinant plasmid was respectively transformed into DH10Bac competent cells, and the plates containing gentamicin, kanamycin sulfate and tetracycline were coated, and cultured at 37℃ overnight, and positive monoclonal colonies were selected by blue-white spot screening, and plasmids were extracted by shaking culture, and PCR identification and sequencing were performed, and bacmid recombinant bacmids were obtained.

[0031] Each Bacmid recombinant bacmid was transfected into Sf9 monolayer cells, and when the cells showed obvious viral infection symptoms, the supernatant was collected as P1 generation virus. The P1 was used to infect Sf9 cells at a MOI value of 0.1, and the culture supernatant was collected to obtain P2. The Sf9 cells were infected again, and the culture supernatant was collected again. After two times, high-titer P3 virus was obtained, and the virus titer was measured by plaque method. The Sf9 cells were infected with P3 generation virus, and the cell culture supernatant of recombinant C factor, recombinant B factor and recombinant coagulogen was collected after 48-72 hours of culture.

[0032] Example 2 Preparation method of recombinant Limulus three-factor reagent containing balance enhancer

[0033] Prepare 1.5M Tris-HCl buffer solution at pH 7.5 and 200mM Tris-HCl buffer solution at pH 7.5.

[0034] Preparation of freeze-dried Limulus reagent: freeze-dry the solution formed by mixing 1 part of 0.9wt% LGR-pNA stock solution, 0.02 parts of 3wt% balance enhancer stock solution, 1 part of 1.5M Tris-HCl buffer solution, and 1 part of cell culture supernatant of recombinant C factor, recombinant B factor and recombinant coagulogen.

[0035] Preparation of recombinant Limulus three-factor reagent: add 200mM pH 7.5 Tris-HCl buffer solution to the freeze-dried reagent to reconstitute it.

[0036] Example 3 Endotoxin detection process

[0037] Preheat the instrument and set the incubation temperature to 37°C. Set the template and program. Set the plate reading wavelength to 405nm, and set the kinetic parameters: read the plate for 60 minutes, read every 30-60 seconds. Prepare the endotoxin standard solution (5, 0.5, 0.05, 0.005 EU / mL) according to the 10-fold gradient dilution. The negative control is bacterial endotoxin test water. After the recombinant Limulus three-factor reagent reconstitution solution is naturally equilibrated to room temperature, gently shake or blow it with a pyrogen removal tip to fully mix the recombinant Limulus three-factor reagent. Take the pyrogen removal microplate and add 100μL of bacterial endotoxin test water or endotoxin standard solution or test sample to each well. Add 100μL of recombinant Limulus three-factor reagent to each well with a pipette or multichannel pipette, vortex for 10 seconds, and place it in the microplate reader according to the set parameters.

[0038] Example 4 Determination of the difference in reaction activity of recombinant Limulus three-factor reagent to endotoxins from different bacterial species

[0039] The 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 the endotoxin mother liquor was stored. The frozen endotoxin mother liquor was diluted to the required concentration, calibrated with the national standard endotoxin, and the concentration values of different endotoxin solutions were detected with the recombinant Limulus Tachypleus amebocyte lysate prepared in Example 2. At the same time, the recombinant Limulus Tachypleus amebocyte lysate without the addition of the balance enhancer (no addition) and the commercial natural Limulus reagent were used for endotoxin detection. The reaction differences were analyzed by calculating the correlation coefficients of the endotoxin concentrations determined by different reagents (see Figure 1 , in which O represents the recombinant Limulus Tachypleus amebocyte lysate containing 0.5wt% of the balance enhancer octyl glucopyranoside, B represents the recombinant Limulus Tachypleus amebocyte lysate containing 0.02wt% of the balance enhancer polyethylene glycol monododecyl ether, C represents the recombinant Limulus Tachypleus amebocyte lysate containing 0.5wt% of CHAPS, C is a comparative example, and A, X and R are commercial natural Limulus reagents from different manufacturers.

[0040] From the detection results, it can be seen that the recombinant Limulus Tachypleus amebocyte lysate with the balance enhancer O has good consistency with the natural Limulus reagents from manufacturers A and R, and the recombinant Limulus Tachypleus amebocyte lysate with the balance enhancer B has good consistency with the natural Limulus reagent from manufacturer X. The concentrations of all endotoxin samples detected by the endotoxin detection reagent with the balance enhancer C are significantly higher by about 10 times, indicating that the balance enhancer C significantly reduces the activity of the corresponding strain endotoxin in the standard. In addition, the consistency of the recombinant Limulus Tachypleus amebocyte lysate with the balance enhancer C with the natural Limulus reagents from manufacturers A, R and X is not good. Therefore, it can be proved that the recombinant Limulus Tachypleus amebocyte lysate with the balance enhancers O and B has good consistency in reactivity to endotoxins from different sources.

[0041] Example 5: Activity enhancement effect determination

[0042] According to the method of Example 2, the recombinant Limulus Tachypleus amebocyte lysate containing different balance enhancers was prepared, and the recombinant Limulus Tachypleus amebocyte lysate without the balance enhancer was used as a control. The detection was performed according to the method of Example 3, and the details are shown in Table 2. Figure 2 . Figure 2From top to bottom, the following represent the following: no addition of a balancing enhancer; addition of 0.04 wt% 5-bromo-5-nitro-1,3-dioxane; addition of 0.5 wt% octylpyranoside; addition of 0.5 wt% octylpyranoside and 0.04 wt% 5-bromo-5-nitro-1,3-dioxane; and addition of 0.5 wt% octylpyranoside, 0.04 wt% 5-bromo-5-nitro-1,3-dioxane, and 2 wt% PEG in a recombinant horseshoe crab three-factor reagent.

[0043] Depend on Figure 2 It is evident that, compared to the recombinant horseshoe crab trifactor reagent without the addition of a balancing enhancer, the recombinant horseshoe crab trifactor reagent with the addition of different balancing enhancers all significantly improved the reactivity against endotoxins, and the time to reach the threshold was shortened. This demonstrates that the balancing enhancer of the present invention can significantly improve the detection activity against endotoxins.

[0044] Table 1 below shows the relevant sequences of the recombinant C factor, recombinant B factor, and recombinant coagulaseogen involved in this invention.

[0045] Table 1

[0046]

[0047]

[0048] Based on the above-described preferred embodiments according to this application, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the technical concept of this application. The technical scope of this application is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A recombinant Limulus factor C reagent, characterized in that, The recombinant Lm three-factor reagent comprises the following components: recombinant factor C, recombinant factor B, recombinant coagulogen, a chromogenic substrate, and a balance enhancer selected from one or more of an alkyl glucopyranoside, a polyethylene glycol alkyl ether, PEG, 5-bromo-5-nitro-1,3-dioxane, wherein the alkyl of the alkyl glucopyranoside is octyl, heptyl or hexyl, and the alkyl chain length of the polyethylene glycol alkyl ether is C5-C15.

2. The recombinant Limulus factor three agent according to claim 1, characterized by The alkyl glucopyranoside is octyl glucopyranoside.

3. The recombinant Limulus factor three agent according to claim 1, characterized by The polyethylene glycol alkyl ether is polyethylene glycol monododecyl ether.

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

2.

5. The recombinant Limulus factor C reagent according to any one of claims 1 to 3, characterized in that, The content of the balance enhancer in the recombinant Lm three-factor reagent is 0.04 wt%-4 wt%.

6. The recombinant Limulus factor C reagent according to any one of claims 1 to 3, characterized by, The sequence of the recombinant factor C is SEQ ID NO: 1, and / or the sequence of the recombinant factor B is SEQ ID NO: 2, and / or the sequence of the recombinant coagulogen is SEQ ID NO:

3.

7. The recombinant Limulus factor C reagent according to any one of claims 1 to 3, characterized by, The recombinant Lm three-factor reagent further comprises a buffer.

8. The recombinant Limulus factor three agent according to claim 7, characterized by The pH of the buffer is 7.4-8.

0.

9. The recombinant Limulus factor C reagent according to any one of claims 1 to 3, characterized by, The chromogenic substrate is a pNA chromogenic substrate peptide.

10. A kit characterized in that: A recombinant Lm three-factor reagent as claimed in any one of claims 1-9.

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