Preparation method of H5 and H7 subtype recombinant avian influenza virus hemagglutination inhibition test antigen

Abstract

The invention provides a preparation method of an H5 and H7 subtype recombinant avian influenza virus hemagglutination inhibition test antigen, which overcomes the defects that in the existing avian influenza virus antibody hemagglutination inhibition test, a standard substance cannot accurately detect the virulence of a strain to be detected and cannot truly reflect the immune effect of an avian influenza vaccine, and the H5 and H7 subtype recombinant avian influenza virus hemagglutination inhibition test antigen can be obtained by adjusting inactivation and purification processes. The pH value is adjusted, so that impure protein is separated out more easily, components influencing a detection result are reduced, the binding capacity of hemagglutinin is changed, the activity of the hemagglutinin is more easily bound with a serum antibody, the impure protein, inorganic salt and urate in an antigen are removed, and errors in the detection process are reduced. The method provided by the invention is high in specificity, the detection result is more accurate and reliable, and the effectiveness of the vaccine can be better evaluated.

Description

Technical Field

[0001] The invention belongs to the field of veterinary biological products, and in particular relates to a method for preparing a hemagglutination inhibition test antigen of H5 and H7 subtype recombinant avian influenza viruses. Background Art

[0002] The avian influenza virus hemagglutination inhibition test is a test method currently commonly used by the WHO for global influenza testing. It can be used to identify the HA subtype of avian influenza virus isolates, and can also be used to detect whether there are infection or immune antibodies consistent with the antigenic subtype in poultry serum. This experimental method requires antigen standardization in each test in order to accurately detect the virulence of the strain to be tested, thereby truly reflecting the immune effect of the avian influenza vaccine.

[0003] Since the hemagglutinin (HA) and neuraminidase (NA) of different avian influenza viruses (AIV) have different antigenicity, 16 specific HAs and 9 specific NAs have been found in poultry, named H1~H16, N1~N9 respectively. Different HAs and different NAs can form more than 100 subtypes of avian influenza viruses. According to the pathogenicity of avian influenza viruses, AIV can be divided into high pathogenic avian influenza viruses (High Pathogenic Avian Influenza Virus, HPAVI) and low pathogenic avian influenza viruses (Low Pathogenic Avian Influenza Virus, LPAIV). At present, the H5 or H7 subtype avian influenza viruses prevalent in China are all HPAIV.

[0004] In the current HA test of H5 and H7 subtype avian influenza virus antigens, the standard antigen has low detection specificity for existing strains, resulting in the inability to truly reflect the level of serum antibodies. Therefore, a stable and reliable hemagglutination inhibition test method is urgently needed. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing H5 and H7 subtype recombinant avian influenza virus hemagglutination inhibition test antigens.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] A method for preparing H5 and H7 subtype recombinant avian influenza virus hemagglutination inhibition test antigens, the preparation method comprising the following steps:

[0008] (1) Inoculating the virus strain, incubating and culturing, and collecting the allantoic fluid for later use;

[0009] (2) Concentrate the allantoic fluid to half of its original volume and measure the viral titer;

[0010] (3) Adjust the pH value of the allantoic fluid to 6.0-6.4;

[0011] (4) Add 0.1-0.2% formaldehyde solution and place in a 37°C constant temperature water bath shaker to inactivate for 24 hours;

[0012] (5) purification by nickel ion column affinity chromatography;

[0013] (6) Add lyophilizing protectant and store at low temperature.

[0014] Furthermore, the H5 subtype avian influenza virus is the H5N2 subtype recombinant avian influenza virus rHN5801 strain or the H5N2 subtype recombinant avian influenza virus rGD59 strain.

[0015] Furthermore, the H7 subtype avian influenza virus is the H7N9 subtype recombinant avian influenza virus rHN7903 strain.

[0016] Furthermore, the virus titer in step (2) is not less than 1:512.

[0017] Furthermore, the nickel ion column affinity chromatography purification method in step (5) is: drain to the chromatography column with a rubber rod and let it stand; then equilibrate the medium with 5 column volumes (CV) of equilibration buffer; slowly allow the lysis supernatant filtered with a 0.45 μm filter membrane to flow through the medium; elute and wash the medium with 5 CV of equilibration buffer; elute the target protein with elution buffer and collect it.

[0018] Furthermore, the freeze-drying protective agent of step (6) is prepared by weighing 20-40 g of sucrose, adding it into 100 ml of PBS solution to fully dissolve it, sterilizing it under high pressure at 100° C. for 30 minutes, and storing it at 2-8° C. for later use.

[0019] The method for preparing the H5 and H7 subtype recombinant avian influenza virus hemagglutination inhibition test antigens is used in the virulence detection of avian influenza vaccine strains.

[0020] By adopting the above technical solution, the present invention has the following beneficial effects:

[0021] The present invention overcomes the defects that the standard products in the existing avian influenza virus antibody hemagglutination inhibition test cannot accurately detect the virulence of the strain to be tested and cannot truly reflect the immune effect of the avian influenza vaccine. The strain used in the present invention has strong specificity, making the test results more accurate and reliable, and better able to evaluate the effectiveness of the vaccine.

[0022] The present invention optimizes the existing inactivation process, and after adjusting the pH value, it is easier to precipitate the impurity protein, thereby reducing the components that affect the detection results; by adjusting the pH value, the hemagglutinin binding ability is changed, so that the hemagglutinin activity is easier to bind to serum antibodies.

[0023] The purification process adopted by the present invention can remove foreign proteins, inorganic salts and urate in the antigen, thereby reducing errors in the detection process.

[0024] The freeze-dried protective agent has a single component, but can achieve a stable and long-term preservation effect. DETAILED DESCRIPTION

[0025] The following specific descriptions are exemplary and are intended to provide further explanation of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements fall within the protection scope of the present invention.

[0026] Example 1: Preparation method of recombinant avian influenza virus hemagglutination inhibition test antigen

[0027] 1. Preparation and testing of antigens for hemagglutination inhibition test

[0028] 1.1 Preparation of virus solution for antigen

[0029] 1.1.1 Vaccination

[0030] Take H5N2 subtype recombinant avian influenza virus rHN5801 strain, H5N2 subtype recombinant avian influenza virus rGD59 strain and H7N9 subtype recombinant avian influenza virus rHN7903 strain for production respectively, dilute them 10000 times with sterile saline, inoculate 0.2 ml into the allantoic cavity of each embryo, seal the pinhole, and continue incubating at 36-37℃.

[0031] 1.1.2 Incubation and observation

[0032] Within 48 hours after inoculation, the eggs are candled once a day, and dead embryos within 48 hours are discarded. Thereafter, the eggs are candled once every 4 to 6 hours, and dead chicken embryos are removed at any time until 72 hours, when all chicken embryos are removed, dead or not, with the air chambers upright, and cooled at 2 to 8 degrees Celsius for 4 to 24 hours.

[0033] 1.1.3 Harvest

[0034] Take out the cooled chicken embryos, disinfect the eggshells at the air chamber, and then peel off the eggshells at the air chamber with aseptic surgery, remove the eggshell membrane, cut the chorioallantoic membrane and amnion (be careful to prevent the yolk from rupturing), and absorb the embryonic fluid. Each chicken embryo should be carefully inspected before absorbing the embryonic fluid. Those with fetal corruption, turbid embryonic fluid, and any suspected contamination should be discarded. Dead embryos and live embryos are harvested separately, and each number of chicken embryos are divided into a group, and the embryonic fluid is absorbed and placed in the same sterilized container, and stored below -15℃ for later use.

[0035] 1.2 Concentration of virus solution

[0036] The harvested rHN5801, rGD59 and rHN7903 virus fluids were concentrated to 1 / 2 of the original volume using ultrafiltration concentration equipment, and samples were taken to determine the virus titer. The HA titer of the virus fluid should not be less than 1:512.

[0037] 1.3 Inactivation

[0038] The pH value of the chicken embryo allantoic fluid that has passed the above inspection is adjusted to 6.0-6.4 using NaOH solution, and a formaldehyde solution with a final concentration of 0.1% is added in a metered manner, and shaken as it is added to fully mix. Then the allantoic fluid is poured into another sterilized container. Inactivate it in a 37°C constant temperature water bath shaker for 24 hours, and store it at 2-8°C.

[0039] 1.4 Purification

[0040] The inactivated antigen was purified by nickel ion column affinity chromatography:

[0041] 1.4.1 Filling

[0042] After the filler replaced with degassed pure water in advance is stirred evenly, it is drained into the chromatography column with a tamping rod and allowed to stand.

[0043] 1.4.2 Balance

[0044] The medium was equilibrated with 5 column volumes (CV) of equilibration buffer and the flow rate was controlled at 100 cm / h.

[0045] 1.4.3 Sample loading

[0046] The lysate supernatant filtered with a 0.45 μm filter membrane slowly flowed through the medium, and the flow rate was controlled at 50 cm / h.

[0047] 1.4.4 Rinse

[0048] The medium was eluted with 5 CV of equilibration buffer at a flow rate of 100 cm / h.

[0049] 1.4.5 Washing

[0050] The medium was washed with 5 CV of wash buffer at a flow rate of 100 cm / h.

[0051] 1.4.6 Elution

[0052] The target protein was eluted with elution buffer at a flow rate of 100 cm / h. The target protein was collected when the UV detection peak began to rise and the collection was stopped when the UV detection value was lower than 100.

[0053] 1.5 Semi-finished product inspection

[0054] 1.5.1 Sterility test

[0055] Take inactivated chicken embryo allantoic fluid of rHN5801, rGD59 and rHN7903 strains and test them according to the methods in the appendix of Part III of the current "Chinese Pharmacopoeia of Veterinary Medicine".

[0056] 1.5.2 Red blood cell agglutination titer

[0057] The completely inactivated rHN5801, rGD59 and rHN7903 virus fluids were subjected to HA titer determination according to the appendix of the current "Chinese Veterinary Pharmacopoeia".

[0058] 1.5.3 Virus content determination

[0059] The inactivated rHN5801, rGD59 and rHN7903 virus solutions were diluted 10-fold in series with sterile saline. -6 , 10 -7 , 10 -8 , 10 -9 4 dilutions, 5 10-11 day old SPF chicken embryos were inoculated into each allantoic cavity, 0.1 ml per embryo, and incubated at 37°C for 72 hours. Chicken embryos that died before 24 hours were discarded, and the chicken embryo fluid was harvested one by one, and the red blood cell agglutination titer was determined respectively; those with red blood cell agglutination titer not less than 1:16 were judged as infected, and the EID was calculated according to the Reed-Muench method. 50 .

[0060] 1.5.4 Inactivation test

[0061] Take the inactivated rHN5801, rGD59 and rHN7903 virus fluids and inoculate 10 9-11 day old SPF chicken embryos into the allantoic cavity, 0.2 ml per embryo, and continue incubation at 37°C. Remove the dead chicken embryos within 24 hours and observe for 3 days. The non-specific death of chicken embryos should not exceed 2. Harvest the embryo fluids of all chicken embryos and measure the hemagglutination titers. If the HA titers are all negative, blind transmission should be carried out according to the same method as above, and the embryo fluids of the passaged chicken embryos should be taken for HA test.

[0062] 1.6 Preparation of lyophilization protectant

[0063] Accurately weigh 20-40 g of sucrose, add it to 100 ml of PBS solution to fully dissolve, sterilize it under high pressure at 100°C for 30 minutes, and store it at 2-8°C for later use.

[0064] 1.7 Preparation and packaging of antigens The qualified concentrated antigens were evenly mixed with the lyophilized protective agent in a ratio of 1:1 (v / v), quantitatively packaged, and freeze-dried in a freeze dryer. Three batches of hemagglutination inhibition antigens of avian influenza virus rHN5801 strain, rGD59 strain and rHN7903 strain were produced successively, with batch numbers Ag2205001, Ag2205002, Ag2205003, Ag2205004, Ag2205005, Ag2205006, Ag2203001, Ag2203002 and Ag2203003, respectively.

[0065] 1.8 Inspection of finished products

[0066] 1.8.1 Properties Nine batches of hemagglutination inhibition test antigens were taken and their color and state were observed. After adding PBS (0.1 mol / L, pH 7.0-7.2), their dissolution was observed.

[0067] 1.8.2 Sterility test Nine batches of hemagglutination inhibition test antigens were taken and tested according to the method in the appendix of Part III of the current Chinese Pharmacopoeia of Veterinary Medicine.

[0068] 1.8.3 Titer determination Nine batches of hemagglutination inhibition test antigens were taken and HA titer was determined according to the appendix of the current Chinese Pharmacopoeia of Veterinary Medicine.

[0069] 1.8.4 Specificity test Nine batches of hemagglutination inhibition test antigens were taken and HI tests were performed on them according to the appendices of the three volumes of the current "Chinese Pharmacopoeia of Veterinary Medicine" with positive sera of avian influenza virus H5 subtype, H7 subtype, H9 subtype, Newcastle disease virus hemagglutination inhibition test positive sera and egg drop syndrome virus hemagglutination inhibition test positive sera.

[0070] 1.8.5 Determination of residual moisture Take 9 batches of hemagglutination inhibition test antigen and conduct the determination according to the appendix of the current Chinese Pharmacopoeia of Veterinary Medicine.

[0071] 1.8.6 Vacuum Degree Determination Take 9 batches of hemagglutination inhibition test antigens and conduct the determination according to the appendix of the current Chinese Pharmacopoeia of Veterinary Medicine.

[0072] 2 Results

[0073] 2.1 Preparation and testing of virus solution

[0074] Under laboratory conditions, three batches of virus liquid of H7N9 subtype recombinant avian influenza virus rHN7903 strain and H5N2 subtype recombinant avian influenza virus rHN5801 strain and rGD59 strain were prepared continuously, with batch numbers S202201, S202202, S202203, S202204, S202205, S202206S202207, S202208 and S202209, respectively. After inspection of semi-finished products, all nine batches of virus liquid met the requirements and could be used for the preparation of hemagglutination inhibition test antigens. The results are shown in Tables 1, 2 and 3 for details.

[0075] Table 1 Inspection results of 3 batches of rHN7903 semi-finished products in the laboratory

[0076]

[0077] Table 2 Inspection results of 3 batches of rHN5801 semi-finished products in the laboratory

[0078]

[0079] Table 3 Inspection results of 3 batches of rGD59 semi-finished products in the laboratory

[0080]

[0081] 2.2 Preparation and testing of antigens for hemagglutination inhibition test

[0082] Under laboratory conditions, three batches of avian influenza virus H5 subtype (rHN5801 strain, rGD59 strain) and avian influenza virus H7 subtype (rHN7903 strain, rHN7903 strain) hemagglutination inhibition test antigens were prepared using an experimental freeze dryer according to the optimized freeze drying curve parameters. The batch numbers were Ag2205001, Ag2205002, Ag2205003, Ag2205004, Ag2205005, Ag2205006, Ag2203001, Ag2203002 and Ag2203003. The total preparation volume was 1726 bottles (1 ml / bottle), and the results are shown in Table 4.

[0083] The properties, sterility test, titer test, specificity test, residual moisture test and vacuum test were carried out on 9 batches of hemagglutination inhibition test antigens of avian influenza virus rHN5801 strain, rGD59 strain and rHN7903 strain prepared in the laboratory. The results showed that the properties of the 9 batches of hemagglutination inhibition test antigens met the requirements of the general rules for veterinary biological products. The sterility test was carried out according to the three appendices of the current "Chinese Veterinary Pharmacopoeia". All 9 batches of hemagglutination inhibition test antigens were qualified. After measurement, the agglutination titers of the 9 batches of hemagglutination inhibition test antigens to 1% chicken red blood cells were all 1:512. The results are shown in Table 5.

[0084] The results of the specificity test showed that all 9 batches of hemagglutination inhibition test antigens were specific, and the results are shown in Tables 6, 7, and 8. The residual moisture and vacuum degree of 9 batches of hemagglutination inhibition test antigens were measured in accordance with the appendix of the current "Chinese Veterinary Pharmacopoeia". The results showed that the residual moisture and vacuum degree of the 9 batches of antigens met the requirements of the General Rules for Veterinary Biological Products, and the results are shown in Tables 9 and 10.

[0085] Table 4 Batch number and batch size of laboratory-prepared hemagglutination inhibition test antigens

[0086]

[0087] Table 5 Properties, sterility test and titer test results of 9 batches of hemagglutination inhibition test antigens

[0088]

[0089]

[0090] Table 6 Specificity test results of three batches of hemagglutination inhibition test antigens

[0091]

[0092] Table 7 Specificity test results of three batches of hemagglutination inhibition test antigens

[0093]

[0094] Table 8 Specificity test results of three batches of hemagglutination inhibition test antigens

[0095]

[0096]

[0097] Table 9 Results of residual water content determination of three batches of hemagglutination inhibition test antigens

[0098]

[0099] Table 10 Vacuum degree determination results of three batches of hemagglutination inhibition test antigens

[0100]

[0101]

[0102] 3. Comparison of the results of hemagglutination inhibition test of H5 and H7 subtype recombinant avian influenza viruses before and after antigen treatment 3.1 Comparison of the results of serum detection after adjusting the pH value of the three antigens

[0103] The test results show that after adjusting the pH of the three antigens to 6.0-6.4, the antibody titer of the serum was 1.2-2.2㏒2 higher than the unadjusted test results when tested according to the 2020 edition of the Chinese Veterinary Pharmacopoeia Part III. This indicates that adjusting the pH of the antigen in the hemagglutination inhibition test to an appropriate value is more conducive to the binding of the antigen and the antibody. The results are shown in Table 11.

[0104] Table 11 Comparison of pH adjustment

[0105]

[0106] 3.2 Comparison of serum test results after purification of three antigens

[0107] The test results show that after the pH value of the three antigens was adjusted to 6.0-6.4 and purified by affinity chromatography, the antibody titer of the serum was tested according to the third part of the 2020 edition of the Chinese Veterinary Pharmacopoeia. The test results are 1.5-2.3㏒2 higher than the test results of the untreated antigen. This shows that after the pH of the antigen in the hemagglutination inhibition test is adjusted to an appropriate value and purified, on the one hand, the influence of substances such as antigenic impurities is reduced, and on the other hand, after adjusting the pH value, the binding ability of hemagglutinin can be more activated, which is conducive to the binding of antigens and antibodies, thereby truly reflecting the effect of the vaccine. The results are shown in Table 12.

[0108] Table 12 Comparison of antigens before and after treatment

[0109]

[0110]

[0111] Example 2. Application effect of H5 and H7 subtype recombinant avian influenza virus hemagglutination inhibition test antigen

[0112] Ten 21-day-old SPF chickens were subcutaneously inoculated with a trivalent inactivated vaccine of recombinant avian influenza virus (H5+H7) (H5N2 rHN5801 strain + rGD59 strain, H7N9 rHN7903 strain) through the neck, 0.3 ml / chicken, and another five SPF chickens of the same age were not immunized as blank controls. Blood was collected and serum was separated 21 days after immunization, and the HI antibody titer was determined using the hemagglutination inhibition test antigen of avian influenza virus H5 subtype (rGD59 strain) (rHN5801 strain), the antigen of recombinant avian influenza virus rHN7903 strain of H7N9 subtype, and the standard antigen H5 Re-13 strain, H5 Re-14 strain and H7 Re-4 strain. The test results showed that the HI antibody titer of rGD59 strain, H5 Re-13 strain, rHN5801 strain, H5 Re-14 strain, rHN7903 strain and H7 Re-4 strain differed by 2 to 3㏒2 when the same serum was tested for antigen. The results are shown in Table 13.

[0113] Table 13 Comparison results between the patented detection antigen and the commercial antigen (unit: ㏒2)

[0114]

[0115] Since the existing highly pathogenic avian influenza trivalent vaccine is a government-mandated immunization, there are two vaccines, one is a recombinant avian influenza virus (H5+H7) trivalent inactivated vaccine (H5N2 rHN5801 strain + rGD59 strain, H7N9 rHN7903 strain), and the other is a recombinant avian influenza virus (H5+H7) trivalent inactivated vaccine (H5N6 H5-Re13 strain + H5N8 H5-Re14 strain + H7N9 H7-Re4 strain). The existing standard antigens H5 Re-13 strain, H5 Re-14 strain and H7 Re-4 strain cannot truly evaluate the recombinant avian influenza virus (H5+H7) trivalent inactivated vaccine (H5N2 rHN5801 strain + rGD59 strain, H7N9 rHN7903 strain). rHN7903 strain), so it is necessary to use the hemagglutination inhibition test antigen of avian influenza virus H5 subtype (rGD59 strain) (rHN5801 strain) and the antigen of H7N9 subtype recombinant avian influenza virus rHN7903 strain for evaluation.

Claims

1. A method for preparing H5 and H7 subtype recombinant avian influenza virus hemagglutination inhibition test antigen, characterized in that: The preparation method comprises the following steps: (1) Inoculating the virus strain, incubating and culturing, and collecting the allantoic fluid for later use; (2) Concentrate the allantoic fluid to half of its original volume and measure the viral titer; (3) Adjust the pH value of the allantoic fluid to 6.0-6.4; (4) Add 0.1-0.2% formaldehyde solution and place in a 37°C constant temperature water bath shaker to inactivate for 24 hours; (5) purification by nickel ion column affinity chromatography; (6) Add lyophilizing protectant and store at low temperature.

2. The method for preparing a H5 and H7 subtype recombinant avian influenza virus hemagglutination inhibition test antigen according to claim 1, characterized in that: The H5 subtype avian influenza virus is the H5N2 subtype recombinant avian influenza virus rHN5801 strain and the H5N2 subtype recombinant avian influenza virus rGD59 strain.

3. The method for preparing a hemagglutination inhibition test antigen of H5 and H7 subtype recombinant avian influenza virus according to claim 1, characterized in that: The H7 subtype avian influenza virus is the H7N9 subtype recombinant avian influenza virus rHN7903 strain.

4. The method for preparing a hemagglutination inhibition test antigen of H5 and H7 subtype recombinant avian influenza virus according to claim 1, characterized in that: The virus titer in step (2) is not less than 1:

512.

5. The method for preparing a H5 and H7 subtype recombinant avian influenza virus hemagglutination inhibition test antigen according to claim 1, characterized in that: The nickel ion column affinity chromatography purification method in step (5) is as follows: drain to the chromatography column with a rubber rod and let it stand; then equilibrate the medium with 5 column volumes (CV) of equilibration buffer; slowly allow the lysate supernatant filtered with a 0.45 μm filter membrane to flow through the medium; elute and wash the medium with 5 CV of equilibration buffer; elute the target protein with elution buffer and collect it.

6. The method for preparing a hemagglutination inhibition test antigen of H5 and H7 subtype recombinant avian influenza virus according to claim 1, characterized in that: The preparation method of the lyophilization protective agent in step (6) is as follows: weigh 20-40 g of sucrose, add it into 100 ml of PBS solution to fully dissolve it, sterilize it under high pressure at 100° C. for 30 minutes, and store it at 2-8° C. for later use.

7. Use of the method for preparing the H5 and H7 subtype recombinant avian influenza virus hemagglutination inhibition test antigen as claimed in claim 1 in the virulence detection of avian influenza vaccine strains.

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