A high expression-based CRM197 protein specific neutralization detection method

Abstract

The application discloses a high-expression-based CRM197 protein specific neutralization detection method and belongs to the technical field of biological detection. The method comprises the following steps: S1, strain resuspension: Corynebacterium diphtheriae is resuspended to obtain a bacterial suspension; S2, bacterial liquid culture: the bacterial suspension is inoculated into an iron trichloride YC liquid culture medium to be cultured to obtain a bacterial liquid; and S3, specific neutralization: the bacterial liquid is inoculated on Elek's medium to be subjected to virulence determination. The application establishes a novel CRM197 protein specific neutralization detection method, greatly improves the technical requirements of detection effect and time limit, and provides a novel detection method for biological science.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, more particularly, to a high-expression-based CRM197 protein specificity neutralization detection method. BACKGROUND

[0002] CRM197 protein is obtained by culturing Corynebacterium diphtheriae in a suitable culture medium, producing protein through fermentation, and then centrifuging, filtering and purifying. Quality control of the bacterial strain is required during the culture process, including culture characteristics, staining microscopy, biochemical response, and specificity neutralization control. The current specificity neutralization test uses a method of recovering the bacterial strain and inoculating it in Lysenko's medium for 3-7 days, scraping the bacterial body and transferring it to Elek's agar medium and culturing with filter paper containing diphtheria antitoxin for 5-7 days to form a specificity neutralization phenomenon. The process requires sterile wet box culture to prevent contamination of other bacteria on the culture medium and cause the specificity neutralization phenomenon to fail. However, this method has a long detection window period, poor repeatability, and the problem of unclear specificity neutralization phenomenon. Therefore, a detection method for CRM197 specificity neutralization is needed to improve the detection period, specificity neutralization effect, and repeatability of the detection method. SUMMARY

[0003] The present application aims to overcome the shortcomings of the prior art and provide a high-expression-based CRM197 specificity neutralization detection method. Iron ions are added to YC medium to accelerate the secretion of specific proteins by Corynebacterium diphtheriae, and the high-expression-specific proteins are used as antibodies to form an immune complex precipitate line with diphtheria antitoxin at a certain ratio to achieve specificity neutralization. This method is simple, short in cycle, high in sensitivity, good in repeatability, clear in phenomenon, economical and practical, and provides an effective detection means for CRM197 protein specificity neutralization.

[0004] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0005] A high-expression-based CRM197 protein specificity neutralization detection method, comprising the following steps:

[0006] S1: Resolving the bacterial strain: Corynebacterium diphtheriae strain is resolved to obtain a bacterial suspension;

[0007] S2: Culturing the bacterial liquid: inoculating the bacterial suspension into a YC liquid culture medium containing ferric chloride to culture and obtain a bacterial liquid;

[0008] S3: Specificity neutralization: inoculating the bacterial liquid into Elek's medium for virulence determination.

[0009] The application is further provided with the step S1, physiological saline or culture medium is used to reconstitute the Corynebacterium diphtheriae strain.

[0010] The application is further provided with the step S2, the YC liquid medium containing ferric chloride is prepared by mixing 0.2wt% ferric chloride solution and YC liquid medium with the volume ratio of 1000:1;

[0011] The YC liquid medium is prepared by mixing YC base liquid, solution A, solution B and solution C with the volume ratio of 967:2:1:30; wherein,

[0012] Each 100mL solution A contains magnesium sulfate heptahydrate 22.5g, beta-alanine 0.115g, nicotinic acid 0.115g, pimelic acid 0.0075g, copper sulfate pentahydrate 0.5g, zinc sulfate heptahydrate 0.229g, manganese sulfate monohydrate 0.057g, 3mL of 11.6mol / L hydrochloric acid, and the rest is water;

[0013] Each 100mL solution B contains L-cystine 20g, 20mL of 11.6mol / L hydrochloric acid, and the rest is water;

[0014] Each 100mL solution C contains maltose monohydrate 50g, calcium chloride dihydrate 0.5g, and the rest is water;

[0015] The preparation method of YC base liquid is as follows: after the base liquid is prepared, the pH value is adjusted to 7.2-7.6 by sodium hydroxide solution, and the supernatant is clear and filtered, and then the YC base liquid is obtained; each 1000mL base liquid contains yeast extract powder 20g, acid hydrolysis casein 10g, L-tryptophan 0.05g, calcium chloride dihydrate 1.324g, potassium dihydrogen phosphate 5g, and the rest is water.

[0016] The application is further provided with the step S2, the inoculation amount of the strain is 1%.

[0017] The application is further provided with the step S2, the specific culture method is as follows: the YC liquid medium containing ferric chloride inoculated with the bacterial suspension is placed in a constant temperature shaker, and cultured at 35℃ and 250rpm for 8-10 hours, and then cooled to 30℃ and continuously cultured for 8-10 hours.

[0018] The application is further provided with the step S3, which is specifically as follows:

[0019] a. Elek's medium is heated and boiled to dissolve in water, and after high-pressure sterilization, newborn calf serum is added, mixed uniformly, and then poured into a flat plate; before the medium solidifies, a filter paper strip soaked with diphtheria antitoxin is pasted in the center of the plate, and then the medium is dried after solidification;

[0020] b. Using a swab to take the bacterial solution obtained in step S2, draw a line along the vertical direction of the filter paper strip soaked with diphtheria antitoxin, and then perform wet box culture to observe whether an immune complex precipitate line is formed on the Elek agar medium.

[0021] The application is further provided that in step a, the dosage ratio of Elek medium, water and newborn calf serum is 2.185g:40mL:10mL.

[0022] The application is further provided that in step a, the concentration of diphtheria antitoxin on the filter paper strip is 1000Lf / mL.

[0023] The application is further provided that in step b, after the bacterial solution is inoculated, the wet box culture is performed at 35-37℃ for 3-7 days.

[0024] In summary, the application has the following beneficial effects:

[0025] After the Corynebacterium diphtheriae is recovered, iron ions are used to quickly promote the secretion of specific proteins by Corynebacterium diphtheriae, so as to form a precipitate line of an immune complex between the specific protein antigen and the specific antibody in a proper proportion. The method uses the biological characteristics of Corynebacterium diphtheriae, and by adding a proper amount of ferric chloride solution, the highest efficient secretion expression of specific proteins is achieved, and the specificity of the test is improved. The method is simple, short in cycle, high in sensitivity, good in specificity, economical and practical, and provides an effective detection means for the research of CRM197 protein specificity neutralization test. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 A diagram of specific neutralization phenomenon. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative efforts fall within the protection scope of the application.

[0028] A detection method based on high-expression CRM197 specificity neutralization, comprising the following steps:

[0029] 1. Solution preparation

[0030] 1.1 Preparation of solution A, solution B, solution C and YC base solution

[0031] Solution A, solution B and solution C are prepared according to the formula shown in Table 1 using purified water as a solvent.

[0032] The base solution was prepared according to the formula shown in Table 2 using purified water as the solvent, and the pH value of the base solution was adjusted to 7.4 (the pH value can be adjusted to 7.2-7.6 according to the actual situation) using 1 mol / L sodium hydroxide solution. The supernatant was clarified and filtered through a 0.45 μm filter membrane to obtain the YC base solution.

[0033] Table 1

[0034]

[0035] Table 2

[0036]

[0037] 1.2 Preparation of YC liquid medium:

[0038] The YC base solution, solution A, solution B and solution C (the volume ratio of the YC base solution to solution A, solution B and solution C was 967:2:1:30) were mixed and sterilized to obtain the YC liquid medium.

[0039] 1.3 Preparation of solution D (0.2 wt% ferric chloride solution):

[0040] 0.35 g of ferric chloride was precisely weighed, and water for injection was added to make up to 50 mL to obtain a 0.7 wt% ferric chloride solution. 15 mL of the 0.7 wt% ferric chloride solution was taken, water for injection was added to make up to 50 mL, and sterilization and filtration were performed to obtain the solution D.

[0041] 1.4 Preparation of YC liquid medium containing ferric chloride:

[0042] 50 μL of solution D (0.2 wt% ferric chloride solution) was added to 50 mL of YC liquid medium, and the mixture was mixed to obtain the YC liquid medium containing ferric chloride.

[0043] 1.5 Preparation of Elek's medium plate and filter paper strip:

[0044] 2.185 g of Elek's medium was heated and dissolved in 40 mL of purified water, and was divided and sterilized at 121°C for 15 hours. When the medium cooled to 55°C, 10 mL of sterile newborn calf serum was immediately mixed and poured into a plate. Before the medium solidified, a filter paper strip soaked with 1000 Lf / mL diphtheria antitoxin was placed in the center of the plate with sterile forceps, and the medium was dried after solidification.

[0045] 2 Bacterial liquid culture

[0046] Under aseptic conditions in a biosafety cabinet, reconstitute the Corynebacterium diphtheriae seed batch with 1 mL of 0.9 wt% sterile sodium chloride solution (or use culture medium for reconstitution). Vortex for 5 seconds and repeatedly pipette to dissolve it into a bacterial suspension. Inoculate an appropriate amount of the bacterial suspension into YC liquid medium containing ferric chloride, with an inoculum volume of 1%. Incubate in a constant temperature shaker at 35°C and 250 rpm for 8 hours (the incubation time can be adjusted between 8 and 10 hours depending on the actual situation). Cool down to 30°C and continue incubating for 8 hours (the incubation time can be adjusted between 8 and 10 hours depending on the actual situation) to obtain the bacterial suspension.

[0047] 3. Specific neutralization test

[0048] Dip an inoculation loop into the bacterial solution and streak it vertically along a filter paper strip soaked in diphtheria antitoxin. Let it sit for 10 hours, then place it in a biochemical incubator at 35-37°C and incubate for 3 days (the incubation time can be adjusted between 3 and 7 days depending on the actual situation).

[0049] 4. Result Determination

[0050] according to Figure 1 As shown, a distinct white precipitate line is visible on Elek's agar medium.

[0051] In another embodiment of the above-mentioned method for the specific neutralization detection of CRM197 protein based on high expression, a comparative example was set up (i.e., bacterial culture was performed according to the above method but using YC liquid medium without the addition of ferric chloride). It was found that the detection cycle of this embodiment was shortened by 50% compared with the comparative example.

[0052] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A high expression-based CRM197 protein-specific neutralization detection method, characterized by, The method comprises the following steps: S1: Resolving the strain: resolving the strain of Corynebacterium diphtheriae to obtain a bacterial suspension; S2: Culturing the bacterial liquid: inoculating the bacterial suspension into a YC liquid medium containing ferric chloride to culture, and obtaining a bacterial liquid; The specific method of culturing in step S2 is as follows: placing the YC liquid medium containing ferric chloride inoculated with the bacterial suspension into a constant-temperature shaker, culturing at 35℃ and 250 rpm for 8-10 hours, then reducing the temperature to 30℃, and continuing to culture for 8-10 hours; The YC liquid medium containing ferric chloride in step S2 is prepared by mixing 0.2wt% ferric chloride solution and YC liquid medium at a volume ratio of 1000:1; The YC liquid medium is prepared by mixing YC base liquid, solution A, solution B and solution C at a volume ratio of 967:2:1:30; wherein, Each 100mL of solution A contains magnesium sulfate heptahydrate 22.5g, beta-alanine 0.115g, nicotinic acid 0.115g, pimelic acid 0.0075g, copper sulfate pentahydrate 0.5g, zinc sulfate heptahydrate 0.229g, manganese sulfate monohydrate 0.057g, 3mL of 11.6mol / L hydrochloric acid, and the rest is water; Each 100mL of solution B contains L-cystine 20g, 20mL of 11.6mol / L hydrochloric acid, and the rest is water; Each 100mL of solution C contains maltose monohydrate 50g, calcium chloride dihydrate 0.5g, and the rest is water; The preparation method of the YC base liquid is as follows: after the base liquid is prepared, the pH value is adjusted to 7.2-7.6 by using sodium hydroxide solution, and the supernatant is filtered after being clarified, thereby obtaining the YC base liquid; each 1000mL of the base liquid contains yeast extract powder 20g, acid hydrolyzed casein 10g, L-tryptophan 0.05g, calcium chloride dihydrate 1.324g, potassium dihydrogen phosphate 5g, and the rest is water; S3: Specific neutralization: inoculating the bacterial liquid on Elek's medium, and culturing the bacterial liquid at 35-37℃ for 3 days in a wet box to perform virulence determination.

2. The neutralization assay based on high expression of CRM197 protein specificity according to claim 1, characterized in that, In step S1, the strain of Corynebacterium diphtheriae is resolved by using physiological saline or culture medium.

3. The neutralization assay based on high expression of CRM197 protein specificity according to claim 1, characterized in that, In step S2, the inoculation amount of the strain is 1%.

4. The neutralization assay based on high expression of CRM197 protein specificity according to claim 1, characterized in that, Step S3 is specifically as follows: a. Dissolving Elek's medium in water by heating and boiling, sterilizing under high pressure, and then adding newborn calf serum, and pouring the mixture into a flat plate; before the medium solidifies, a filter paper strip soaked with diphtheria antitoxin is pasted in the center of the plate, and then the medium is dried after solidification; b. Using an inoculation ring to dip the bacterial liquid obtained in step S2, inoculating along the vertical direction of the filter paper strip soaked with diphtheria antitoxin, and then performing wet box culture to observe whether an immune complex precipitate line is formed on Elek's agar medium.

5. The neutralization assay based on high expression of CRM197 protein specificity according to claim 4, characterized in that, In step a, the dosage ratio of Elek's medium, water and newborn calf serum is 2.185g:40mL:10mL.

6. The neutralization assay based on high expression of CRM197 protein specificity according to claim 4, characterized in that, In step a, the concentration of diphtheria antitoxin on the filter paper strip is 1000Lf / mL.

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