Avian infectious bronchitis virus hemagglutination antigen of type QX, and preparation method and application thereof

Abstract

The application provides a QX type avian infectious bronchitis virus hemagglutination antigen and a preparation method and application thereof, and belongs to the technical field of biotechnology. The preparation method comprises the steps of preparing the QX type avian infectious bronchitis virus hemagglutination antigen through proliferation of the QX type IBV virus in Vero cells, virus inactivation, concentration and purification, phospholipase C treatment and standing at 2-6 DEG C. The application uses Vero cells as a culture medium to prepare the QX type avian infectious bronchitis virus hemagglutination antigen. The hemagglutination antigen prepared by the application has high specificity and can effectively detect the serum antibody level of QX type vaccine immunized birds. Compared with the traditional chicken embryo virus expansion method, the antigen preparation method in the application is simple and low in cost. The application can be widely applied to IBV diagnosis and serum antibody monitoring after QX type vaccine immunization, and provides an effective technical means for IBV prevention and control.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a QX-type avian infectious bronchitis hemagglutination antigen, its preparation method, and its application. Background Technology

[0002] Infectious bronchitis (IB) is an acute, highly contagious respiratory infectious disease caused by the avian infectious bronchitis virus (IBV). IBV exhibits broad tissue tropism, infecting not only the respiratory system of chickens, but some strains can also replicate efficiently in the epithelial cells of the trachea, proventriculus, kidneys, testes, ovaries, and bursa of Fabricius. Outbreaks of this disease can cause significant economic losses to the poultry industry.

[0003] IBV has numerous serotypes, including Mass, QX, 4 / 91, and TW, with the QX strain remaining the predominant circulating strain. The weak cross-protective ability between different IBV serotypes complicates IB diagnosis and control. Currently, serological diagnostic methods for IBV include chicken embryo neutralization tests, cell micro-neutralization tests, agar amplification tests, enzyme-linked immunosorbent assays (ELISA), and hemagglutination inhibition tests (HI). Among these, the agar amplification test has low sensitivity, and the neutralization test is cumbersome and requires specific laboratory conditions. The hemagglutination inhibition test is simple, rapid, and highly specific, making it better suited for IBV diagnosis and antibody detection in grassroots farms.

[0004] Currently, commercially available hemagglutination antigens are prepared using the M41 strain, which exhibits weak cross-reactivity with the prevalent QX strain, leading to a high false-negative rate and affecting detection accuracy. The conventional method for preparing IBV hemagglutination antigens involves inoculating SPF chicken embryos, a cumbersome procedure unsuitable for large-scale production. Furthermore, chicken embryo allantoic fluid contains numerous impurities, resulting in hemagglutination antigens with low purity and high impurities after ultrafiltration purification, impacting their specific hemagglutination titer and leading to relatively high preparation costs. Summary of the Invention

[0005] The purpose of this invention is to provide a hemagglutination antigen for QX type avian infectious bronchitis, its preparation method, and its application. This hemagglutination antigen has high accuracy and specificity in detecting QX type positive serum and can be used for the detection of HI in QX type IBV and the detection of serum antibody levels in avian immunized with QX type vaccine.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing QX type avian infectious bronchitis hemagglutination antigen, comprising the following steps:

[0008] The QX type IBV Vero cell adapted strain was inoculated into Vero cells, and after culture, the viral supernatant was collected to obtain IBV cell supernatant.

[0009] The IBV cell supernatant was inactivated to obtain an inactivated virus solution.

[0010] The inactivated virus solution was placed into a dialysis bag, and then the dialysis bag containing the inactivated virus solution was buried in PEG20000 powder and left to stand overnight at 2-6°C to obtain the dialyzed virus solution. The dialyzed virus solution was washed and dissolved, and then centrifuged at 19000-21000 rpm at 2-6°C for 1.5-2.5 h to obtain the virus precipitate. The virus precipitate was resuspended in buffer to obtain the concentrated virus.

[0011] The concentrated virus was mixed with phospholipase C and allowed to stand at 2-6°C to obtain the QX type avian infectious bronchitis hemagglutination antigen.

[0012] Preferably, the QX type IBV strain includes the rMJ strain, with accession number CGMCC No. 14681.

[0013] Preferably, the copy number of the QX-type IBV Vero cell-adapted strain to the volume ratio of Vero cells is 0.5–1.5 × 10⁻⁶. 6 Copy number / mL.

[0014] Preferably, the inactivation method is the β-propiolactone inactivation method.

[0015] Preferably, the final concentration of phospholipase C is 1-5 U / mL; the reaction is carried out at 37°C and 180-220 rpm for 1.5-2.5 h.

[0016] The present invention also provides a QX type avian infectious bronchitis hemagglutination antigen prepared by the above-described preparation method.

[0017] The present invention also provides an application of the above-mentioned QX type avian infectious bronchitis hemagglutination antigen, wherein the application is any one of the following:

[0018] (1) Application of the hemagglutination antigen in the preparation of HI detection products for QX type IBV;

[0019] (2) Application of the hemagglutination antigen in the preparation of products for detecting antibody levels in avian serum immunized with QX type vaccine.

[0020] Preferably, the product includes reagents or kits.

[0021] Preferably, the number of days for immunizing poultry is 21 days or more.

[0022] The present invention also provides a reagent or kit containing the above-mentioned QX type avian infectious bronchitis hemagglutination antigen.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] This invention provides a hemagglutination antigen for QX-type avian infectious bronchitis virus (IBV), its preparation method, and its application. The invention uses Vero cells as a culture medium to prepare the QX-type IBV hemagglutination antigen. The hemagglutination antigen prepared by this invention has high specificity and can effectively detect serum antibody levels in poultry immunized with QX-type vaccines. Compared to traditional chicken embryo amplification methods, the antigen preparation method in this invention is simple and low-cost. This invention can be widely applied to IBV diagnosis and serum antibody monitoring after QX-type vaccine immunization, providing an effective technical means for IBV prevention and control. Detailed Implementation

[0025] This invention provides a method for preparing QX type avian infectious bronchitis hemagglutination antigen, comprising the following steps:

[0026] The QX type IBV Vero cell adapted strain was inoculated into Vero cells, and after culture, the viral supernatant was collected to obtain IBV cell supernatant.

[0027] The IBV cell supernatant was inactivated to obtain an inactivated virus solution.

[0028] The inactivated virus solution was placed into a dialysis bag, and then the dialysis bag containing the inactivated virus solution was buried in PEG20000 powder and left to stand overnight at 2-6°C to obtain the dialyzed virus solution. The dialyzed virus solution was washed and dissolved, and then centrifuged at 19000-21000 rpm at 2-6°C for 1.5-2.5 h to obtain the virus precipitate. The virus precipitate was resuspended in buffer to obtain the concentrated virus.

[0029] The concentrated virus was mixed with phospholipase C and allowed to stand at 2-6°C to obtain the QX type avian infectious bronchitis hemagglutination antigen.

[0030] In this invention, a QX-type IBV Vero cell-adapted strain is inoculated into Vero cells. After culture, the viral supernatant is collected to obtain IBV cell supernatant. The preferred QX-type IBV strain includes the rMJ strain, with accession number CGMCC No. 14681. As a preferred embodiment, the inoculated Vero cells are cultured in DMEM medium until a density of 75%–85% is reached. The supernatant is discarded, and the Vero cells are washed three times with 5 mL of sterile PBS each time. After washing, the supernatant is discarded, and 15 mL of DMEM medium is added to obtain Vero cells. These Vero cells are used for subsequent inoculation with the QX-type IBV strain. The preferred copy number to Vero cell volume ratio is 0.5–1.5 × 10⁻⁶. 6 Copy number / mL, more preferably 1×10 6 Copy number / mL. The culture medium is preferably high-glucose DMEM medium. The culture time is preferably 20-28 h, more preferably 24 h. This invention utilizes Vero cells as a culture medium to culture QX type IBV, and the prepared hemagglutination antigen has a strong ability to agglutinate chicken erythrocytes.

[0031] In this invention, after obtaining the IBV cell supernatant, the IBV cell supernatant is inactivated to obtain an inactivated virus solution. The preferred inactivation method is β-propiolactone inactivation, and more preferably, the IBV cell supernatant and β-propiolactone are mixed and incubated at 2–6°C for 22–26 hours. The volume ratio of β-propiolactone to the inactivated virus solution is 1:1000. The IBV cell supernatant also includes a solution incubated at 37°C for 2 hours after inactivation for 22–26 hours to remove unhydrolyzed β-propiolactone from the solution.

[0032] In this invention, the inactivated virus solution is placed into a dialysis bag, and then the dialysis bag containing the inactivated virus solution is embedded in PEG20000 powder and incubated overnight at 2-6°C to obtain the dialyzed virus solution. The dialysis bag can be a 300KD dialysis bag. Further, the dialyzed virus solution is washed and dissolved, and centrifuged at 19000-21000 rpm at 2-6°C for 1.5-2.5 h to obtain a virus precipitate. The virus precipitate is resuspended in a buffer solution to obtain concentrated virus. The washing solution for washing and dissolving the dialyzed virus solution is a buffer solution, such as PBS. When resuspending the virus precipitate in a buffer solution, PBS can be used. This invention selects a dialysis bag concentration + ultracentrifugation method to concentrate and purify the inactivated virus solution, improving the concentration efficiency of the virus and obtaining a high concentration of virus.

[0033] In this invention, concentrated virus is mixed with phospholipase C and allowed to stand at 2–6°C to obtain QX-type avian infectious bronchitis hemagglutination antigen. The final concentration of phospholipase C is preferably 1–5 U / mL; the preferred reaction method is 37°C, 180–220 rpm for 1.5–2.5 h. This invention utilizes phospholipase C to enable infectious bronchitis virus to agglutinate red blood cells, and then processes it using a PLC to obtain the hemagglutination antigen. The QX-type avian infectious bronchitis hemagglutination antigen prepared by this invention can effectively detect antibody levels in serum from poultry immunized with QX-type vaccines and can also be used for the detection of HI in QX-type IBV, exhibiting the advantage of high detection specificity.

[0034] The present invention also provides a QX type avian infectious bronchitis hemagglutination antigen prepared by the above-described preparation method.

[0035] The present invention also provides an application of the above-mentioned QX type avian infectious bronchitis hemagglutination antigen, wherein the application is any one of the following:

[0036] (1) Application of the hemagglutination antigen in the preparation of HI detection products for QX type IBV;

[0037] (2) Application of the hemagglutination antigen in the preparation of products for detecting antibody levels in avian serum immunized with QX type vaccine.

[0038] In this invention, the product preferably includes a reagent or kit. The immunization period for poultry is preferably 21 days or more.

[0039] The present invention also provides a reagent or kit containing the above-mentioned QX type avian infectious bronchitis hemagglutination antigen.

[0040] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0041] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0042] In the following examples, the IBYZ strain is a highly virulent QX strain isolated in the laboratory in the early stage, with the accession number CGMCC No. 14682; the rH120-YZ strain is a constructed attenuated strain that replaces the expression of the IBYZ S protein, with the accession number CGMCC No. 8501; the positive serum is a hyperimmune serum prepared after multiple immunizations of SPF chicks with the anti-QX IBV strain; the negative serum is blood directly collected from 7-day-old SPF chicks, incubated at 37°C for 30 min, centrifuged at 8000 rpm for 5 min, and the supernatant was collected and frozen at -20°C.

[0043] Example 1

[0044] A method for preparing QX type avian infectious bronchitis hemagglutination antigen, comprising the following steps:

[0045] (1) Proliferation of IBV rMJ virus

[0046] Cytotoxicity: Vero cells were placed in a 75 cm medium containing high-glucose DMEM (Hyclone). 2 Cells were grown in culture flasks until the cell density reached 80% the following day. The supernatant was discarded, and the cells were washed three times with sterile PBS (5 mL each time). After washing, the supernatant was discarded, and 15 mL of LDM medium was added. The IBVrMJ virus strain was then inoculated at a concentration of 1×10⁻⁶ cells / mL. 6 The viral supernatant was inoculated into Vero cells at a rate of 500 μL / vial, for a total of 15 vicinities, and incubated in a 37°C CO2 incubator. The viral supernatant was aseptically collected 24 h post-infection, centrifuged at 8000 rpm for 3 min, and the supernatant was collected to obtain IBV rMJ cell supernatant (also known as IBV rMJ cytotoxicity).

[0047] The IBV rMJ virus has the accession number CGMCC No. 14681.

[0048] (2) Inactivation of antigen

[0049] Collect 200 mL of IBV rMJ cell supernatant, add 200 μL of β-propiolactone at a ratio of 1:1000, and incubate at 4°C for 24 h. The next day, inactivate the sample by bathing it in a 37°C water bath for 2 h to remove unhydrolyzed β-propiolactone from the solution, thus obtaining inactivated virus solution.

[0050] (3) Virus concentration and purification

[0051] Dialysis bag concentration + ultracentrifugation method: 200 mL of inactivated virus solution was aliquoted into multiple 300 KD dialysis bags, sealed, and then deeply embedded in PEG20000 powder and incubated overnight at 4°C. The contents of the dialysis bags were dissolved using 20 mL of sterile PBS and transferred to 40 mL centrifuge tubes. The mixture was centrifuged at 20000 rpm at 4°C for 2 h, the supernatant was discarded, and the virus pellet was resuspended in 500 μL of PBS and transferred to 1.5 mL centrifuge tubes to obtain concentrated virus.

[0052] (4) PLC processing

[0053] Take 250 μL of the concentrated virus and add 250 μL of phospholipase C (PLC) at 10 U / mL to make the final concentration of PLC 5 U / mL. After mixing well, incubate at 37℃ for 200 rpm for 2 hours, and then let stand at 4℃ for later use to obtain QX type avian infectious bronchitis hemagglutination antigen.

[0054] Comparative Example 1

[0055] A method for preparing QX type avian infectious bronchitis hemagglutination antigen, comprising the following steps:

[0056] (1) Chicken embryo virus: IBV rMJ virus strain was injected at 1×10 6 100 μL / embryo was inoculated into 10-day-old SPF chicken embryos at a rate of 100 μL / embryo, for a total of 100 embryos. After inoculation, the embryos were incubated at 37°C and examined after 24 hours; dead embryos were discarded. 36 hours after inoculation, the chicken embryos were collected and cooled at 4°C for 30 minutes. The allantoic fluid was aseptically collected, centrifuged at 8000 rpm for 5 minutes, and the supernatant was collected to obtain IBVrMJ chicken embryo allantoic fluid (also known as IBV rMJ chicken embryo allantoic fluid toxin).

[0057] The IBV rMJ virus has the accession number CGMCC No. 14681.

[0058] (2) Inactivation of antigen

[0059] Collect 200 mL of IBV rMJ chicken embryo allantoic fluid, add 200 μL of β-propiolactone at a ratio of 1:1000, and incubate at 4 °C for 24 h. The next day, inactivate the sample and incubate it in a 37 °C water bath for 2 h to remove unhydrolyzed β-propiolactone from the solution, thus obtaining inactivated virus solution.

[0060] (3) Virus concentration and purification

[0061] Dialysis bag concentration + ultracentrifugation method: 200 mL of inactivated virus solution was aliquoted into multiple 300 KD dialysis bags, sealed, and then deeply embedded in PEG20000 powder and incubated overnight at 4°C. The contents of the dialysis bags were dissolved using 20 mL of sterile PBS and transferred to 40 mL centrifuge tubes. The mixture was centrifuged at 20000 rpm at 4°C for 2 h, the supernatant was discarded, and the virus pellet was resuspended in 500 μL of PBS and transferred to 1.5 mL centrifuge tubes to obtain concentrated virus.

[0062] (4) PLC processing

[0063] Take 250 μL of the concentrated virus and add 250 μL of phospholipase C (PLC) at 10 U / mL to make the final concentration of PLC 5 U / mL. After mixing well, incubate at 37℃ for 200 rpm for 2 hours, and then let stand at 4℃ for later use to obtain QX type avian infectious bronchitis hemagglutination antigen.

[0064] Comparative Example 2

[0065] The difference between this comparative example and Example 1 is that the virus concentration and purification in this comparative example uses the PEG8000 concentration method. Specifically, step (3) in this comparative example involves weighing 250g of PEG8000 and 43.83g of NaCl, adding them to 1L of sterile ultrapure water, mixing thoroughly, and preparing a 5×PEG8000+NaCl stock solution, which is then stored at 4℃. 50mL of the 5×PEG8000+NaCl stock solution is added to 200mL of inactivated virus solution and placed in a 4℃ refrigerator for overnight rotation. The solution is centrifuged at 8000rpm for 20min, the supernatant is discarded, the virus precipitate is resuspended in 5mL of PBS, and aliquoted into 1.5mL centrifuge tubes to obtain the concentrated virus. The remaining steps are the same as in Example 1.

[0066] Comparative Example 3

[0067] A method for preparing QX type avian infectious bronchitis hemagglutination antigen, comprising the following steps:

[0068] (1) Chicken embryo virus: IBV rMJ virus strain was injected at 1×10 6 100 μL / embryo was inoculated into 10-day-old SPF chicken embryos at a rate of 100 μL / embryo, for a total of 100 embryos. After inoculation, the embryos were incubated at 37°C and examined after 24 hours; dead embryos were discarded. 36 hours after inoculation, the chicken embryos were collected and cooled at 4°C for 30 minutes. The allantoic fluid was aseptically collected, centrifuged at 8000 rpm for 5 minutes, and the supernatant was collected to obtain IBVrMJ chicken embryo allantoic fluid (also known as IBV rMJ chicken embryo allantoic fluid toxin).

[0069] The IBV rMJ virus has the accession number CGMCC No. 14681.

[0070] (2) Inactivation of antigen

[0071] Collect 200 mL of IBV rMJ chicken embryo allantoic fluid, add 200 μL of β-propiolactone at a ratio of 1:1000, and incubate at 4 °C for 24 h. The next day, inactivate the sample and incubate it in a 37 °C water bath for 2 h to remove unhydrolyzed β-propiolactone from the solution, thus obtaining inactivated virus solution.

[0072] (3) Virus concentration and purification

[0073] Weigh 250g of PEG8000 and 43.83g of NaCl, add them to 1L of sterile ultrapure water, mix well, and prepare a 5×PEG8000+NaCl stock solution. Store at 4℃. Add 50mL of the 5×PEG8000+NaCl stock solution to 200mL of inactivated virus solution, and incubate overnight at 4℃. Centrifuge at 8000rpm for 20min, discard the supernatant, resuspend the virus precipitate in 5mL of PBS, and aliquot into 1.5mL centrifuge tubes to obtain concentrated virus.

[0074] (4) PLC processing

[0075] Take 250 μL of the concentrated virus and add 250 μL of phospholipase C (PLC) at 10 U / mL to make the final concentration of PLC 5 U / mL. After mixing well, incubate at 37℃ for 200 rpm for 2 hours, and then let stand at 4℃ for later use to obtain QX type avian infectious bronchitis hemagglutination antigen.

[0076] Comparative Example 4

[0077] The difference between this comparative example and Example 1 is that the virus concentration and purification in this comparative example uses the ultrafiltration method. Specifically, step (3) of this comparative example involves aliquoting 200 mL of inactivated virus solution into six 40 mL centrifuge tubes, centrifuging at 20,000 rpm and 4°C for 2 hours, discarding the supernatant, resuspending the virus precipitate in the six tubes in 500 μL PBS, and transferring it to a 1.5 mL centrifuge tube to obtain the concentrated virus. The remaining steps are the same as in Example 1.

[0078] Comparative Example 5

[0079] The difference between this comparative example and Comparative Example 3 is that the virus concentration and purification in this comparative example uses the ultrafiltration method. Specifically, step (3) in this comparative example involves aliquoting 200 mL of inactivated virus solution into six 40 mL centrifuge tubes, centrifuging at 20,000 rpm and 4°C for 2 hours, discarding the supernatant, resuspending the virus precipitate in the six tubes in 500 μL PBS, and transferring it to a 1.5 mL centrifuge tube to obtain the concentrated virus. The remaining steps are the same as in Comparative Example 3.

[0080] Example 2: Optimization of Different Concentration Methods

[0081] Take 10 μL of the virus stock solution collected before concentration in Example 1 and Comparative Example 1, and the concentrated virus solution in Examples 1 and Comparative Examples 1-5, respectively, and dilute with 990 μL of PBS. Extract viral RNA using the Trizol method and use PrimeScript. TM The RTreagent Kit (Perfect Real Time) was used for reverse transcription, and the viral copy number obtained by different concentration methods was quantified by q-PCR absolute quantification. The differences in concentration efficiency were analyzed, and the results are shown in Table 1.

[0082] Table 1. Determination of viral copy number of antigens obtained by different concentration methods.

[0083]

[0084] Table 1 Results: The PEG8000 concentration method easily generates a large number of complexes during operation, resulting in a large amount of precipitation after centrifugation at 8000 rpm for 20 min. This necessitates a large volume of PBS for resuspending, making it difficult to achieve optimal concentration. Compared to IBV rMJ cell supernatant, chicken embryo virus has a more complex composition. After inactivation and dialysis, a certain degree of protein complex precipitation occurs, affecting the virus concentration efficiency. In comparing the ultrafiltration method and the dialysis bag concentration + ultrafiltration method, ultrafiltration after dialysis bag concentration reduces the loss of different centrifuge tubes, thus achieving a better concentration effect. Therefore, based on the above results, the dialysis bag concentration + ultrafiltration method yields the highest virus concentration and provides the best concentration effect for cytotoxic viruses.

[0085] Example 3: Detection of antigen hemagglutination titer (HA titer)

[0086] Add 25 μL of PBS to each well of a 96-well microplate. Add 25 μL of the QX-type avian infectious bronchitis hemagglutination antigen sample prepared in Example 1 and Comparative Examples 1-5 (after being stored for 7 days) to the first well, mix thoroughly, and then add 25 μL to the second well. Perform serial dilutions until the 11th well, discarding the 25 μL diluent. Prepare a 0.5% fresh chicken erythrocyte suspension and add it to the 96-well microplate at 25 μL / well. Shake the plate on a shaker and incubate at room temperature for 30 min. Observe the results.

[0087] Result Interpretation: Observe whether red blood cells agglutinate when the reaction plate is held vertically at 90 degrees. If the red blood cells are evenly distributed in the wells, it indicates complete agglutination. If the red blood cells are concentrated and sink to the bottom of the wells in a dotted pattern, and flow and hang in a string after being held vertically to the reaction plate, it indicates no agglutination. The maximum dilution of antigen added at which complete agglutination occurs is taken as the HA titer of the antigen.

[0088] Table 2. Determination of HA titer of antigens obtained by different concentration methods.

[0089]

[0090] Table 2 shows that the hemagglutination antigen prepared from the viral supernatant obtained by inoculating Vero cells has a stronger ability to agglutinate chicken erythrocytes compared to chicken embryo virus. Considering both viral copy number and HA titer, the optimal method for preparing rMJ cytotoxic virus as a hemagglutination antigen was selected as dialysis bag concentration followed by ultrafiltration.

[0091] Example 4: Detection of antigen hemagglutination titer at different standing times

[0092] The HA titer of the QX type avian infectious bronchitis hemagglutination antigen (also known as IBV rMJ hemagglutination antigen or HA antigen) prepared in Example 1 was detected at 0, 4, 8, 12, 16 and 20 days.

[0093] Table 3. HA titers of IBV rMJ hemagglutination antigen at different time points.

[0094] HA valence 1:16 1:256 1:512 1:512 1:512 1:512

[0095] The results in Table 3 show that, since the PLC continues to function even when the antigen is left to stand at 4°C, the HA titer increases continuously with the increase of the antigen sample's standing time. It reaches a peak at 8 days of standing, with an HA ratio of 1:512. After this point, the HA titer of the antigen remains unchanged, demonstrating good stability.

[0096] Example 5: Optimization of PLC Concentration

[0097] Take 250 μL of the concentrated virus prepared in step (3) of Example 1, add 250 μL of phospholipase C (PLC) at concentrations of 0.5, 1, 2, 5, and 10 U / mL respectively, incubate at 37°C for 2 h at 200 rpm, and then place at 4°C on day 7 to detect the IBV rMJ hemagglutination antigen HA titer. The results are shown in Table 4.

[0098] Table 4. HA titer of IBV rMJ hemagglutination antigen after treatment with different PLC concentrations.

[0099] HA valence 1:8 1:32 1:128 1:512 1:512

[0100] The results in Table 4 show that the titers of IBV hemagglutination antigen prepared by treating concentrated virus with different concentrations of PLC vary. PLC concentrations of 1–5 U / mL can be used to treat concentrated virus, and the antigen titer increases with increasing PLC concentration, reaching a peak at 2.5 U / mL with an HA titer of 1:512.

[0101] Example 6: Hemagglutination inhibition test of HA antigen

[0102] Preparation of 4-unit antigen: Before the HI test, the HA titer of the HA antigen prepared in Example 1 was determined by hemagglutination test, and 4-unit antigen was prepared using PBS as the working antigen solution in the HI test. The prepared 4-unit antigen was further serially diluted, and the accuracy of its concentration was verified by hemagglutination test.

[0103] Processing of the serum to be tested: Add 75 μL of 25% kaolin suspension to 25 μL of positive serum or negative serum, shake well, let stand at room temperature for 30 min, centrifuge at 3000 rpm for 10 min, and take the supernatant to obtain the serum to be tested diluted 1:4.

[0104] MJ-positive serum: rMJ strain was administered at a concentration of 1×10⁻⁶. 6 Immunize 1-day-old SPF chicks with EID50 per chick, and repeat the immunization at 14 and 30 days of age. Collect blood from SPF chicks at 45 days of age, incubate at 37°C for 30 minutes, centrifuge at 8000 rpm for 5 minutes, and freeze the supernatant at -20°C.

[0105] Negative serum: Blood was collected directly from 7-day-old SPF chicks, incubated at 37°C for 30 minutes, centrifuged at 8000 rpm for 5 minutes, and the supernatant was collected and frozen at -20°C.

[0106] HI Assay: Add 25 μL of PBS to a 96-well microplate. Add 25 μL of positive and negative serum samples to the first column, mix thoroughly, and then add 25 μL to the second column of wells. Perform serial dilutions until the 10th well, discarding 25 μL of the diluent. Add 25 μL / well of the validated 4-unit antigen to the plate until the 11th column. Shake the plate and incubate at 37°C for 30 min. Then add 25 μL / well of 0.5% chicken erythrocyte suspension, shake thoroughly, and incubate at room temperature for 30 min. Observe the results.

[0107] Result Interpretation: The reaction plate was held vertically at 90 degrees, and red blood cell aggregation was observed. If the red blood cells were evenly distributed in the wells, complete aggregation was observed. If the red blood cells were concentrated and settled at the bottom of the wells in a dotted pattern, and flowed and hung in a string after being held vertically to the reaction plate, no aggregation was observed. The maximum dilution of serum added at which no aggregation occurred was taken as the HI titer of that serum. The results are shown in Table 5.

[0108] Table 5. Results of the HI reaction between IBVrMJ hemagglutination antigen and positive and negative sera.

[0109] HI valence 1:512 1:8

[0110] Table 5 shows that the HA antigen prepared in Example 1 can bind well to its corresponding positive serum, and the HI titer of MJ positive serum is 1:512. After the HA antigen reacts with negative serum, there is a certain degree of non-specific reaction, and its non-specific HI titer is 1:8.

[0111] Example 7: Detection of serum HI titer for different serotypes of IBV

[0112] Hyperimmune sera from different serotypes (MJ, IBYZ, H120, M41, and 4 / 91) were treated with a 25% kaolin suspension. 75 μL of kaolin suspension was added to 25 μL of serum, shaken thoroughly, incubated at room temperature for 30 min, and centrifuged at 3000 rpm for 10 min. The supernatant was collected to obtain a 1:4 diluted serum for testing. The HI test was performed using the HA antigen prepared in Example 1, and the results were observed. The HI test method and result interpretation are described in Example 6.

[0113] Table 6. Results of the HI reaction between IBV rMJ hemagglutination antigen and hyperimmune serum prepared from different serotypes.

[0114]

[0115]

[0116] Table 6 shows that both MJ and IBYZ strains belong to the QX type. The HA antigen prepared from MJ can react well with the hyperimmune serum prepared from these two strains, but the reaction with hyperimmune serum prepared from other serotypes is relatively weak, indicating good specificity.

[0117] Example 8: Specific detection of hemagglutination antigen

[0118] A 25% kaolin suspension was used to process avian influenza virus H5 subtype positive serum (AI H5), avian influenza virus H7 subtype positive serum (AI H7), avian influenza virus H9 subtype positive serum (AI H9), and Newcastle disease virus positive serum (ND) (provided by the Infectious Disease Laboratory of the College of Veterinary Medicine, Yangzhou University). 75 μL of kaolin suspension was added to 25 μL of serum, shaken thoroughly, incubated at room temperature for 30 min, centrifuged at 3000 rpm for 10 min, and the supernatant was collected to obtain a 1:4 diluted serum for testing. The HI test was performed using the HA antigen prepared in Example 1, and the results were observed. The HI test method and result interpretation are described in Example 6. The results are shown in Table 7.

[0119] Table 7. HI titers of IBV rMJ hemagglutination antigen reactions with positive sera from other viruses.

[0120] HI valence 1:512 1:8 1:8 1:8 1:8

[0121] The results in Table 7 show that, except for MJ positive serum, this antigen did not react with positive serum of other viruses diluted 1:4, indicating good specificity.

[0122] Example 9: Application of hemagglutination antigen in serum antibody monitoring of immunized chicks

[0123] Select different serotypes of the virus strains rH120-YZ and H120 according to 10 4 Ten 1-day-old SPF chicks were immunized with EID50, with a PBS-immunized group serving as the negative control and five chicks as the negative control group. Blood samples were collected at 14, 21, and 28 days post-immunization, yielding 75 serum samples. IBV MJ hemagglutination antigen was prepared according to Example 1, and a hemagglutination assay was performed to determine the hemagglutination titer of different serum groups. The hemagglutination assay method and result interpretation are described in Example 6, and the results are shown in Table 8.

[0124] Table 8. HI titer of antibodies in serum from immunized chicks.

[0125]

[0126]

[0127]

[0128] Table 8 shows that the IBV hemagglutination antigen prepared in Example 1 was used to detect the HI titer of chick serum from different immunization groups. The results indicated that the serum from the rH120-YZ immunized group reacted with the hemagglutination antigen, and the antibody level in the chicks gradually increased with immunization time. The serum from the rH120 immunized group could bind to the hemagglutination antigen to a certain extent, but the binding efficiency was lower than that of the rH120-YZ immunized group. The negative control group still had a background value of 1:8, which did not increase with immunization time. The IBV hemagglutination antigen prepared by the method in Example 1 is suitable for evaluating antibody levels in chicks immunized with the QX recombinant vaccine 21 days later.

[0129] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing QX type avian infectious bronchitis hemagglutination antigen, comprising the following steps: The QX-type IBV Vero cell-adapted strain was inoculated into Vero cells, and after culture, the viral supernatant was collected to obtain IBV cell supernatant. The IBV cell supernatant was inactivated to obtain an inactivated virus solution. The inactivated virus solution was placed into a dialysis bag, and then the dialysis bag containing the inactivated virus solution was buried in PEG20000 powder and left to stand overnight at 2-6°C to obtain the dialyzed virus solution. The dialyzed virus solution was washed and dissolved, and then centrifuged at 19000-21000 rpm at 2-6°C for 1.5-2.5 h to obtain the virus precipitate. The virus precipitate was resuspended in buffer to obtain the concentrated virus. The concentrated virus was mixed with phospholipase C and allowed to stand at 2-6°C to obtain QX type avian infectious bronchitis hemagglutination antigen. The QX-type IBV Vero cell-adapted strain includes the rMJ strain, with accession number CGMCC No. 14681; The copy number of the QX-type IBV Vero cell-adapted strain was 1.0 × 10⁻⁶ cells / Vero cell volume ratio. 6 Copy number / mL; The inactivation method is the β-propiolactone inactivation method. β-propiolactone is added to the IBV cell supernatant at a ratio of 1:1000 and incubated at 4°C for 24 hours. The inactivated sample is then placed in a 37°C water bath for 2 hours. The final concentration of phospholipase C is 2.5 U / mL; the reaction is carried out at 37°C and 180-220 rpm for 1.5-2.5 h.

2. The QX type avian infectious bronchitis hemagglutination antigen prepared by the preparation method according to claim 1.

3. The application of the QX type avian infectious bronchitis hemagglutination antigen according to claim 2, characterized in that, The application is any one of the following: (1) The application of the hemagglutination antigen in the preparation of HI detection products for QX type IBV; (2) Application of the hemagglutination antigen in the preparation of products for detecting antibody levels in avian serum immunized with QX type vaccine.

4. The application according to claim 3, characterized in that, The products include reagents or kits.

5. The application according to claim 3, characterized in that, The number of days for immunizing poultry is more than 21 days.

6. A reagent or kit containing the QX type avian infectious bronchitis hemagglutination antigen as described in claim 2.

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