Method for determination of foot-and-mouth disease virus-like particles by sucrose density gradient ultracentrifugation

Abstract

The present invention discloses a method for determining foot-and-mouth disease virus-like particles by sucrose density gradient ultracentrifugation, and relates to the technical field of quantitative analysis of foot-and-mouth disease virus-like particles antigen. The present invention specifically comprises the following steps: preparing a 20%-45% sucrose gradient with an automatic gradient mixer, placing 500µl foot-and-mouth disease virus-like particles on the top of the sucrose gradient; separating virus-like particles by centrifugation at 38000rpm for 3.5h at 10°C; connecting an ultracentrifuge tube to a high performance liquid chromatograph, using the pump head of the chromatograph as the driving force, and detecting a flow rate of 0.28 ml / min; continuously detecting the optical signal of the sucrose gradient solution by using an ultraviolet and laser detector, and analyzing the peak area of ​​the sample by a computer software system; calculating the content of virus-like particles according to the regression equation of the ultraviolet absorption peak area and protein concentration of a thyroglobulin standard. The present invention can be used for determining the assembly efficiency of foot-and-mouth disease virus-like particles and monitoring the antigen content of a variety of foot-and-mouth disease virus-like particle vaccines, and indirectly evaluating the efficacy of the vaccine.

Description

Technical Field

[0001] The invention belongs to the technical field of quantitative analysis of foot-and-mouth disease virus-like particles antigen, and particularly relates to a method for measuring foot-and-mouth disease virus-like particles by using sucrose density gradient ultracentrifugation. Background Art

[0002] Foot-and-mouth disease is an acute, febrile, highly contagious disease with a wide range of natural hosts. It can infect livestock such as cattle, pigs, sheep and other wild animals, and poses a serious threat to the world's livestock industry and international trade in animal products. Inactivated vaccines are the main vaccines currently used and have played an important role in prevention and control. However, the use of live viruses in the production process of inactivated vaccines has the risk of spreading the virus, and it cannot distinguish between infected and immune animals (although the 3B-deficient foot-and-mouth disease whole virus marker vaccine can accurately identify infected and immune animals, its production still requires the use of live viruses), and has shown a trend that does not meet the current requirements for foot-and-mouth disease prevention and control in my country.

[0003] Virus-like particles are composed of one or more structural proteins of the virus. Their mechanisms of entering immune cells and inducing immune responses are similar to those of the whole virus. They are non-infectious due to the absence of nucleic acids. In September 2021, the foot-and-mouth disease VLPs vaccine composed of foot-and-mouth disease structural proteins VP0, VP1 and VP3, which was expressed and self-assembled by the Escherichia coli system, obtained the National Class I New Veterinary Drug Certificate. The advantage of virus-like particle vaccines is that they do not require the use of live viruses in production, have low production costs, high production efficiency, can distinguish between infected and immune animals, and have higher immune efficacy than inactivated vaccines. They are considered by the industry to be the most powerful candidate to replace existing inactivated vaccines.

[0004] The protective effect of the vaccine mainly depends on the homology between the vaccine strain and the epidemic strain and the antigen content in the vaccine. The content of virus-like particles is a key factor in determining the quality and immune efficacy of the foot-and-mouth disease virus-like particle vaccine. At present, there is no relevant report on the quantitative method of foot-and-mouth disease virus-like particle content. Due to the similarity between the structure of virus-like particles and the whole virus, the quantitative technology of foot-and-mouth disease virus can be used for reference, but due to the heterogeneity of artificially assembled foot-and-mouth disease virus-like particles, the quantitative technology of foot-and-mouth disease virus cannot be fully applied. The quantitative methods of foot-and-mouth disease virus particles currently promoted in my country mainly include "determination of foot-and-mouth disease antigen 146S by size exclusion high-performance liquid chromatography column" and "quantification of foot-and-mouth disease 146S by sucrose density gradient centrifugation". Since foot-and-mouth disease virus-like particles have no nucleic acid and their molecular weight is smaller than that of the whole virus, their floating density in sucrose is also smaller than that of foot-and-mouth disease virus, and they are called 75S. It is theoretically inferred that the peak position of foot-and-mouth disease virus-like particles in size exclusion high-performance liquid chromatography column technology and sucrose density gradient centrifugation is later than that of the whole virus. When the content of FMD virus-like particles was determined using a size exclusion HPLC column, it was found that there was no peak at the location where the FMD virus appeared. Due to the heterogeneity of the artificially assembled FMD virus-like particles, it was difficult to distinguish between virus-like particles and monomers and aggregates. In addition, the high pressure of the HPLC system may damage the structure of the virus-like particles, which limits the accuracy of its detection. Sucrose density gradient centrifugation is a classic method for separating viruses. Its action conditions are relatively mild, but the peak position of virus-like particles is different from that of the whole virus. Therefore, the sucrose gradient and conditions for separating virus-like particles are different from those of the whole virus. The traditional sucrose density gradient centrifugation method is all manually operated, with poor repeatability.

[0005] Therefore, it is necessary to improve the prior art to solve the above-mentioned technical problems. Summary of the invention

[0006] The purpose of the present invention is to provide a method for determining foot-and-mouth disease virus-like particles by sucrose density gradient ultracentrifugation. The action conditions of sucrose density gradient ultracentrifugation are mild, the damage to virus-like particles is small, and the optimized sucrose gradient and centrifugation conditions make the resolution of sucrose density gradient higher, and aggregates, virus-like particles, monomers and impurities can be completely distinguished; the special use of high-performance liquid chromatography reduces the damage of pressure to virus-like particles and the influence of human subjective judgment on the results, improves the sensitivity and accuracy of the detection, and ensures the repeatability of the results. This method can be used to determine the assembly efficiency of foot-and-mouth disease virus-like particles and monitor the antigen content of various foot-and-mouth disease virus-like particle vaccines, and indirectly evaluate the efficacy of vaccines.

[0007] In order to solve the above technical problems, the present invention is achieved through the following technical solutions:

[0008] The present invention provides a method for determining foot-and-mouth disease virus-like particles by using sucrose density gradient ultracentrifugation, and the determination method specifically comprises the following steps:

[0009] S1: Prepare 20% and 45% sucrose solutions respectively with TNE buffer of pH 8.0; take a 4.5 ml ultracentrifuge tube and mark the ultracentrifuge tube with the scale of the gradient mixer; first add 20% sucrose solution to the ultracentrifuge tube to the mark, and then use a long needle to add 45% sucrose solution from the bottom to the top of the ultracentrifuge tube; place the ultracentrifuge tube containing the sucrose gradient solution in the gradient mixer, select the 20%-45% sucrose gradient preparation program, and work continuously for 90 seconds to obtain a 20%-45% continuous gradient sucrose solution;

[0010] S2: Take the foot-and-mouth disease virus-like particle assembly mixture, centrifuge it at 8000rpm for 3min in a high-speed centrifuge, and take the supernatant for use; first draw 400µl of waste liquid from the top layer of the prepared sucrose gradient solution, then add 500µl of supernatant, and centrifuge it at 35000rpm for 3.5h in an ultracentrifuge at 10℃ to obtain a centrifuged sample;

[0011] S3: Connect the pump head of the HPLC to the external ultracentrifuge tube using a 0.5 mm diameter tube with a 15 cm long needle at the end, turn on the pumping system and UV detection system of the HPLC, use 45% sucrose solution as the mobile phase, and balance the baseline;

[0012] S4: Insert the long needle into the bottom of the ultracentrifuge tube containing the centrifuged sample; turn on the pump system, set the flow rate to 0.28 ml / min, set the detection wavelength to 280 nm and 259 nm, and the chromatograph computer system automatically draws the concentration curve;

[0013] S5: Prepare 6 standard solution samples with concentration gradients of 12.5-200µg / ml with buffer solution for thyroglobulin standard protein; take 0.5ml of standard solution sample, replace the foot-and-mouth disease virus-like particle assembly mixture and operate according to steps S1-S3, then insert the long needle into the bottom of the high-speed centrifuge tube containing the standard solution sample; turn on the pump system, run at a flow rate of 0.28ml / min, detect at an ultraviolet wavelength of 280nm, integrate the absorption peak of the protein to obtain the peak area, and establish a linear regression equation between the peak area and the concentration;

[0014] S6: According to the linear regression equation of protein concentration and peak area established in S5, the integrated area of ​​the absorption peak of the foot-and-mouth disease virus-like particle assembly mixture is substituted into the regression equation to calculate the content of virus-like particles in the sample to be tested.

[0015] Furthermore, in step S1, before the gradient mixer is used, the gradient mixer should be adjusted to balance using a level.

[0016] Furthermore, in step S3, deionized water is used as the mobile phase to discharge bubbles in the system.

[0017] Furthermore, the determination method is used for the detection of the foot-and-mouth disease type A virus-like particle assembly mixture and the foot-and-mouth disease type O virus-like particle assembly mixture.

[0018] Furthermore, the determination method is also used for the detection of emulsified foot-and-mouth disease virus-like particle vaccines.

[0019] The present invention has the following beneficial effects:

[0020] The invention has good repeatability and small intra-batch error. The configuration of the sucrose density gradient is prepared by a fully automatic gradient instrument. The peak analysis of the virus-like particles is fully automatically analyzed by a high-performance liquid chromatography system. Compared with the manual operation error of the traditional density gradient centrifugation, the detection result is more accurate and has wide applicability. The invention can be used to detect foot-and-mouth disease virus-like particles of different serotypes, solves the problem that there is no quantitative detection method for the current foot-and-mouth disease virus-like particle antigen, and overcomes the problems of poor repeatability and complicated analysis process of the traditional sucrose density gradient ultracentrifugation method. The invention has important significance for the quality control of the foot-and-mouth disease virus-like particle vaccine in my country and the rationality of the production process. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0022] Figure 1 Chromatographic analysis profiles of foot-and-mouth disease virus, foot-and-mouth disease virus-like particle assembly mixture and unassembled foot-and-mouth disease protein;

[0023] Figure 2 This is the ELISA result diagram of the sample after sucrose density gradient ultracentrifugation;

[0024] Figure 3 The SDS-PAGE analysis results of the target collection peak of sucrose density gradient ultracentrifugation and the particle size results of dynamic light scattering detection;

[0025] Figure 4 Transmission electron microscopy analysis results of foot-and-mouth disease virus, foot-and-mouth disease O virus-like particles and foot-and-mouth disease A virus-like particles collected from the sucrose density gradient ultracentrifugation target peak;

[0026] Figure 5 This is a sucrose density gradient ultracentrifugation chromatogram of the thyroglobulin standard protein in Example 1;

[0027] Figure 6 is a linear relationship diagram between the absorption peak area and concentration of thyroglobulin at different dilution concentrations in Example 1;

[0028] Figure 7 This is a graph showing the results of chromatographic analysis of foot-and-mouth disease virus, foot-and-mouth disease type O virus-like particles and foot-and-mouth disease type A virus-like particles after sucrose density gradient ultracentrifugation in Example 2. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0030] The present invention provides a method for determining foot-and-mouth disease virus-like particles by using sucrose density gradient ultracentrifugation, and the determination method specifically comprises the following steps:

[0031] (1) Preparation of sucrose gradient:

[0032] 1. Prepare 20% and 45% sucrose solutions respectively using TNE buffer at pH 8.0;

[0033] 2. Take an ultracentrifuge tube with a specification of 4.5 ml and mark it with the scale provided by the gradient mixer;

[0034] 3. First, add 20% sucrose solution to the ultracentrifuge tube to the mark, and then use a long needle to add 45% sucrose solution from the bottom to the top of the ultracentrifuge tube. Do not generate bubbles during the whole process.

[0035] 4. Turn on the gradient mixer and use a spirit level to adjust the gradient mixer to balance. Place the ultracentrifuge tube containing the sucrose gradient solution in the gradient mixer. Select the 20%-45% sucrose gradient preparation program and work continuously for 90 seconds to obtain a 20%-45% continuous gradient sucrose solution.

[0036] (2) Sucrose density gradient ultracentrifugation:

[0037] 1. Take the foot-and-mouth disease virus-like particle assembly mixture, centrifuge it at 8000 rpm for 3 minutes in a high-speed centrifuge, and take the supernatant for later use;

[0038] 2. First, discard 400µl from the top layer of the prepared sucrose gradient solution, then add 500µl of supernatant, and centrifuge at 35000rpm for 3.5h at 10℃ to obtain the centrifuged sample.

[0039] (3) Preparation of HPLC:

[0040] 1. Connect the HPLC pump head to the external ultracentrifuge tube using a 0.5 mm diameter tube with a 15 cm long needle at the end.

[0041] 2. Turn on the pumping system and UV detection system of the HPLC, and use deionized water as the mobile phase to remove the bubbles in the system;

[0042] 3. Use 45% sucrose solution as the mobile phase to balance the baseline.

[0043] (4) Determination of virus-like particle samples:

[0044] 1. Insert the long needle in (3) into the bottom of the ultracentrifuge tube in (2);

[0045] 2. Turn on the pump system, set the flow rate to 0.28 ml / min, set the detection wavelength to 280 nm and 259 nm, and the chromatograph's computer system automatically draws the concentration curve and integrates the target peak to obtain the peak area.

[0046] (5) Determination of the mixture of foot-and-mouth disease virus and unassembled foot-and-mouth disease protein:

[0047] A centrifuged sample of the mixed solution of foot-and-mouth disease virus and unassembled foot-and-mouth disease protein is prepared according to the method of step (2), and the centrifuged sample is measured according to step (4).

[0048] The curve drawn from the data measured in step (4) is as follows Figure 1 As shown, the chromatographic peak of foot-and-mouth disease virus is at 5-9 min (Figure A), the peak positions of foot-and-mouth disease virus-like particle assembly mixture (type O and type A) are both at 5-12 min (Figure B), and the unassembled foot-and-mouth disease protein mixture has no chromatographic peak (Figure C).

[0049] (6) Open the fraction collector while the chromatograph is detecting, collect samples from the beginning of the detection, and store them in the collection tube at 2-8°C for future use.

[0050] (7) The mixed solution samples of the foot-and-mouth disease type O virus-like particles and the mixed solution samples of the foot-and-mouth disease type A virus-like particles collected in (6) were tested by the foot-and-mouth disease type O inactivated antigen double antibody sandwich ELISA method and the foot-and-mouth disease type A inactivated antigen double antibody sandwich ELISA method, respectively, to determine that the strongest reaction occurred at the position where the target peak appeared, and the OD value reaction curve ( Figure 2 ) corresponds to the chromatogram.

[0051] (8) The target chromatographic peak collection tubes of the determined foot-and-mouth disease virus and foot-and-mouth disease virus-like particle assembly mixture are mixed and detected by SDS-PAGE electrophoresis and dynamic light scattering (DLS), respectively.

[0052] like Figure 3 It can be seen that clear FMD VP0, VP1 and VP3 bands can be seen in the target tubes of the FMD O type and A type virus-like particle assembly mixture (Figure A), and the size of the particles is about 30nm (Figure B).

[0053] (9) Observe the target mixed tube in (8) under transmission electron microscopy, and you can see complete FMD A and O virus-like particles, such as Figure 4 shown.

[0054] Based on the above steps, the present invention makes a corresponding preferred list of the equipment and materials required for the test, but the above list does not exclude the use of other equipment and materials with the same function, as follows;

[0055] (1) Instruments and equipment

[0056] The high-speed centrifuge was purchased from Shuke Company, model TGL-16; the ultraspeed refrigerated centrifuge was purchased from Beckman Company, model Optima XE-90; the high performance liquid chromatograph was purchased from Shimadzu Company, model LC-40; the fully automatic density gradient preparation instrument was purchased from Biocomp Company; the nanoparticle size analyzer was purchased from Malvern Company, model Nano-ZS; and the electrophoresis instrument was purchased from Bio-Rad Company.

[0057] (2) Reagents

[0058] Chromatographic grade methanol and isopropanol were purchased from Sigma; Tris was purchased from VWR Life Science; NaCl was purchased from Sinopharm Chemical Reagent Co., Ltd.; EDTA was purchased from Sinopharm Chemical Reagent Co., Ltd.; and sucrose was purchased from Beijing Solebow Technology Co., Ltd.

[0059] (3) Foot-and-mouth disease virus-like particle assembly mixture

[0060] The foot-and-mouth disease type O virus-like particle assembly mixture and the foot-and-mouth disease type A virus-like particle assembly mixture are both prepared and preserved by the R&D center of Huapai Bioengineering Group. When testing the samples, the foot-and-mouth disease virus-like particle assembly mixture does not require any treatment and can be directly loaded for measurement.

[0061] (4) Foot-and-mouth disease virus

[0062] The foot-and-mouth disease virus in the experiment is an inactivated foot-and-mouth disease type O antigen. The foot-and-mouth disease type O inactivated vaccine was purchased from a commercial channel. 100 ml of the foot-and-mouth disease type O inactivated vaccine was measured with a measuring cylinder and added into a 100 ml to 250 ml container. 10 ml of n-pentanol was aspirated with a pipette and added into 100 ml of the vaccine, covered and thoroughly shaken to mix, and then placed at 2 to 8 ° C for 30 minutes. After the oil phase and the water phase are separated, the lower aqueous phase is completely aspirated and placed into a clean container, which is covered and sealed, and stored at 2 to 8 ° C for later use.

[0063] Embodiment 1

[0064] In order to verify that the method has high accuracy, the present invention is also verified by using thyroglobulin standard protein;

[0065] Thyroglobulin standard protein was purchased from GE. Six standard solution samples with concentration gradients were prepared with buffer (Tris-HCl solution at pH 7.5) in the concentration range of 12.5-200 μg / ml and stored at 2-8°C for later use.

[0066] According to the methods of (1) preparation of sucrose gradient, (2) sucrose density gradient ultracentrifugation, (3) preparation of high performance liquid chromatography and (4) determination of virus-like particle samples, the virus-like particle samples are replaced with thyroglobulin standard protein, and the concentration of the standard protein is chromatographed;

[0067] like Figure 5 As shown, the thyroglobulin standard protein exhibits an absorption peak. The concentrations of the diluted standard solution samples in the experiment are 25µg / ml, 50µg / ml, 100µg / ml, 150µg / ml, 175µg / ml and 200µg / ml respectively.

[0068] The absorption peaks of standard proteins of different concentrations were integrated to obtain the peak area, and a linear response curve was obtained with the peak area as the abscissa and the concentration as the ordinate, and it can be concluded that C=3E-05X+3.497;

[0069] Where C is the protein concentration in µg / ml, X is the absorption peak area in mAU.min, see Figure 6 The R of the obtained standard curve 2 The value is 0.9935, indicating that this method has high accuracy.

[0070] Embodiment 2

[0071] Establishment of quantitative method for assembly mixture of foot-and-mouth disease virus-like particles and repeatability testing;

[0072] 1. Integration of absorption peaks of the foot-and-mouth disease virus-like particle assembly mixture: centrifuge the foot-and-mouth disease virus, foot-and-mouth disease type O, and type A virus-like particle assembly mixture according to the determination method, and perform target peak analysis according to the analysis method of the determination method, and integrate the target peak at the same time;

[0073] 2. Calculation of virus-like particle concentration: Substitute the absorption peak area into the formula C=3E-05X+3.497 obtained in Example 1 to calculate the antigen concentration, where C is the protein concentration in µg / ml, and X is the absorption peak area in mV.min. Repeat the measurement three times.

[0074] The results of HPLC analysis are shown in Figure 6 , Figure A is foot-and-mouth disease virus, Figure B is foot-and-mouth disease type O virus-like particle assembly mixed solution, Figure C is foot-and-mouth disease type A virus-like particle assembly mixed solution content, the measurement results are shown in Table 1;

[0075] Table 1

[0076]

[0077] The inactivated antigen is foot-and-mouth disease virus;

[0078] O-VLPs is an assembly mixture of O-type foot-and-mouth disease virus-like particles;

[0079] A-VLPs is a mixed solution of type A foot-and-mouth disease virus-like particles.

[0080] Sucrose density gradient centrifugation combined with liquid chromatography quantitative method was used to repeatedly detect foot-and-mouth disease virus-like particles. From the statistical results of antigen content in Table 1, it can be seen that the peak positions of foot-and-mouth disease virus-like particles are consistent, proving that the sucrose density gradient centrifugation combined with liquid chromatography quantitative method for detecting foot-and-mouth disease virus-like particles content is repeatable.

[0081] Embodiment 3

[0082] The determination method can also be applied to emulsified foot-and-mouth disease virus-like particle vaccines, and the vaccine can be detected without demulsification treatment.

[0083] The above are only preferred embodiments of the present invention and do not limit the present invention. Any modification to the technical solutions recorded in the aforementioned embodiments, any equivalent replacement of some of the technical features therein, and any modification, equivalent replacement, and improvement made are all within the protection scope of the present invention.

Claims

1. A method for determining foot-and-mouth disease virus-like particles using sucrose density gradient ultracentrifugation, characterized in that: The determination method specifically comprises the following steps: S1: Prepare 20% and 45% sucrose solutions using TNE buffer at pH 8.0; Take an ultracentrifuge tube with a specification of 4.5 ml, and mark the ultracentrifuge tube using the scale provided by the gradient mixer; first add 20% sucrose solution into the ultracentrifuge tube to the mark, and then use a long needle to add 45% sucrose solution into the ultracentrifuge tube from the bottom to the top; place the ultracentrifuge tube containing the sucrose gradient solution into the gradient mixer, select the 20%-45% sucrose gradient preparation program, and work continuously for 90 seconds to obtain a 20%-45% continuous gradient sucrose solution; S2: Take the foot-and-mouth disease virus-like particle assembly mixture, centrifuge it at 8000rpm for 3min in a high-speed centrifuge, and take the supernatant for use; first, discard 400µl from the top layer of the prepared sucrose gradient solution, then add 500µl of the supernatant, and centrifuge it at 35000rpm for 3.5h at 10℃ in an ultracentrifuge to obtain a centrifuged sample; S3: Connect the pump head of the HPLC to the external ultracentrifuge tube using a 0.5 mm diameter tube with a 15 cm long needle at the end, turn on the pumping system and UV detection system of the HPLC, use 45% sucrose solution as the mobile phase, and balance the baseline; S4: Insert the long needle into the bottom of the ultracentrifuge tube containing the centrifuged sample; turn on the pump system, set the flow rate to 0.28 ml / min, set the detection wavelength to 280 nm and 259 nm, and the chromatograph computer system automatically draws the concentration curve to obtain the integrated area of ​​the target peak; S5: Prepare 6 standard solution samples with a concentration gradient of 12.5-200 µg / ml using a buffer solution for the thyroglobulin standard protein; take 0.5 ml of the standard solution sample, replace it with the foot-and-mouth disease virus-like particle assembly mixture, and perform the operation according to steps S1-S3, and then insert the long needle into the bottom of the ultracentrifuge tube containing the standard solution sample; Turn on the pump system and run it at a flow rate of 0.28 ml / min. Detect at an ultraviolet wavelength of 280 nm, integrate the absorption peak of the protein to obtain the peak area, and establish a linear regression equation between the peak area and the concentration; S6: According to the linear regression equation of protein concentration and peak area established in S5, the integrated area of ​​the absorption peak of the foot-and-mouth disease virus-like particle assembly mixture is substituted into the regression equation to calculate the content of virus-like particles in the sample to be tested.

2. The method for determining foot-and-mouth disease virus-like particles using sucrose density gradient ultracentrifugation according to claim 1, characterized in that: In step S1, before using the gradient mixer, the gradient mixer should be adjusted to balance using a level.

3. The method for determining foot-and-mouth disease virus-like particles using sucrose density gradient ultracentrifugation as claimed in claim 2, characterized in that: In step S3, deionized water is used as a mobile phase to discharge bubbles in the system.

4. The method for determining foot-and-mouth disease virus-like particles by sucrose density gradient ultracentrifugation according to claim 1 or 3, characterized in that: The determination method is used for detecting the foot-and-mouth disease type A virus-like particle assembly mixture and the foot-and-mouth disease type O virus-like particle assembly mixture.

5. The method for determining foot-and-mouth disease virus-like particles using sucrose density gradient ultracentrifugation according to claim 4, characterized in that: The assay was also used to test emulsified foot-and-mouth disease virus-like particle vaccines.

Images