Method for preparing hog cholera virus specific polyclonal antibody

Abstract

The invention provides a method for preparing a hog cholera virus specific polyclonal antibody, and belongs to the technical field of veterinary biological products. The preparation method comprises the following steps: performing centrifugation, concentration, dilution, ultra-high-speed centrifugation and density gradient centrifugation on hog cholera virus to obtain non-hog cholera virus with relatively high purity, then combining virus liquid with a rabbit anti-hog cholera virus polyclonal antibody to obtain a complex, performing centrifugation, collecting supernate, adding the supernate into a serum-coated ELISA plate, and performing incubation at 37 DEG C for 1 hour, the detection result shows that the concentration of the specific polyclonal antibody reaches 1.0 mg / ml or above, and the specific polyclonal antibody does not react with other proteins in serum after being diluted by 1000 times or above, so that the specific polyclonal antibody shows relatively high specificity and is beneficial to accurate diagnosis and research.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary biological products, and particularly relates to a method for preparing a swine fever virus-specific polyclonal antibody. Background Art

[0002] Classical swine fever is a highly acute, febrile, contagious disease that occurs in pigs and is caused by the classical swine fever virus. The classical swine fever virus can be divided into classical swine fever virus and African swine fever virus. Classical swine fever is widely prevalent, with a high incidence and mortality rate. Its main transmission routes are the digestive tract and respiratory tract. Moreover, after sows are infected, they can vertically infect fetuses through the placenta. This disease prevails throughout the year and can cause a devastating blow to the pig-raising industry. It is one of the most serious infectious diseases that endanger the pig-raising industry. Currently, vaccination is an important means to control classical swine fever, including inactivated vaccines and attenuated vaccines. However, in production practice, there are still phenomena where pigs cannot resist the invasion of classical swine fever virus after vaccination. This is because the importance of detection has been ignored. Therefore, mastering clinical diagnosis and scientific detection methods is necessary to correctly diagnose and effectively contain the prevalence of classical swine fever.

[0003] When detecting classical swine fever virus, specific antibodies are usually used to capture the classical swine fever virus, and specific antibodies are divided into monoclonal antibodies and polyclonal antibodies. The classical swine fever virus contains multiple immunogenic proteins, and the immunogenic proteins of the virus are prone to mutation during the process of virus prevalence. If the antigen of the classical swine fever virus mutates, the original monoclonal antibody is prone to missed detection. Polyclonal antibodies have multiple binding sites with the virus and relatively tight binding. Even if some antigens of the virus mutate, it does not affect the binding of polyclonal antibodies to the virus. However, in addition to specific antibodies, polyclonal antibodies also contain other non-specific antibodies, which restricts the use of polyclonal antibodies.

[0004] Therefore, there is an urgent need to prepare a classical swine fever virus-specific polyclonal antibody with reduced non-specific reactions. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention designs a method for preparing a classical swine fever virus-specific polyclonal antibody. The concentration of the obtained classical swine fever virus-specific polyclonal antibody reaches 1 mg / ml or more, and it does not react with other proteins in the serum even when diluted 1000 times or more, showing high specificity.

[0006] To achieve the above object, the present invention adopts the following technical solutions to solve the technical problems:

[0007] In a first aspect, the present invention provides a method for preparing a classical swine fever virus-specific polyclonal antibody, and the method includes the following steps:

[0008] S1. First, centrifuge the classical swine fever virus culture solution at 3000 - 10000 revolutions per minute for 10 - 60 minutes, then concentrate and dilute the virus solution, and finally obtain classical swine fever virus through ultra - high - speed centrifugation and density - gradient centrifugation;

[0009] S2. Dilute the rabbit anti - classical swine fever virus polyclonal antibody 10 - 20 times, then add it to the classical swine fever virus and incubate overnight at low temperature. Next, collect the complex of virus and specific antibody through ultra - high - speed centrifugation, add PBS solution with a pH value of 2 - 3, and obtain the supernatant containing specific polyclonal antibody through ultra - high - speed centrifugation;

[0010] S3. Coat the ELISA plate with serum, wash it, add the supernatant, incubate at 37 °C for 1 hour, and then detect the content of specific polyclonal antibody.

[0011] In step S1, centrifugation is used to remove large substances such as cell debris in the classical swine fever virus culture solution, which is beneficial to extracting high - purity virus.

[0012] In step S2, adding PBS solution with a pH value of 2 - 3 is to prevent the virus from binding to the antibody, so that it is released into the solution.

[0013] In step S3, the incubation is repeated 2 - 5 times to remove non - specific antibodies.

[0014] In some embodiments, in step S1, the concentration is carried out through a membrane package with a cut - off pore size of 10 - 300K, and the concentration is repeated 2 - 10 times; the dilution is carried out with 0.1M PBS solution with a pH of 7.0 - 7.4 and a volume 10 - 100 times that of the original.

[0015] In some embodiments, in step S1, the concentration of classical swine fever virus is ≥ 0.2 mg / ml.

[0016] In some embodiments, in step S2, the dilution is carried out with 0.1M PBS solution with a pH of 7.0 - 7.4, and the dilution factor is 2 - 50 times.

[0017] In some embodiments, in step S2, the final concentration of the virus solution is 0.02 - 1 mg / ml.

[0018] In some embodiments, in step S3, the dilution is carried out with 0.1M PBS solution with a pH of 7.0 - 7.4; the serum is one or more of bovine serum, chicken serum, porcine serum, equine serum, and ovine serum, and the final concentration is 1 - 10%.

[0019] In some embodiments, the concentration of the specific polyclonal antibody > 1.0 mg / ml.

[0020] Second aspect, the present invention also provides a classical swine fever virus-specific polyclonal antibody, which is prepared by the method for preparing a classical swine fever virus-specific polyclonal antibody described above.

[0021] In some of these embodiments, after dilution, the classical swine fever virus-specific polyclonal antibody does not react with other proteins in the serum, and the dilution factor of the classical swine fever virus-specific polyclonal antibody is ≥ 1000.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a method for preparing a classical swine fever virus-specific polyclonal antibody. By subjecting classical swine fever virus to centrifugation, concentration, dilution, ultra-high speed centrifugation and density gradient centrifugation, a classical swine fever virus with relatively high purity can be obtained, which can more effectively bind to specific antibodies, reduce non-specific reactions, harvest the classical swine fever virus-specific antibody, and then perform non-specific adsorption with ELISA plates coated with sera from various animal sources, thereby reducing non-specific reactions in the classical swine fever virus-specific antibody, which is beneficial to large-scale production and application.

[0024] The concentration of the specific polyclonal antibody prepared by the present invention reaches more than 1.0 mg / ml, and does not react with other proteins in the serum after being diluted 1000 times or more, showing relatively high specificity, which is beneficial to accurate diagnosis and research. Detailed implementation manners

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to specific embodiments. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0026] In the present invention, regarding numerical ranges, unless otherwise specified, the above numerical ranges are considered continuous and include the minimum value and the maximum value of the range, as well as each value between such minimum value and maximum value. Further, when the range refers to integers, it includes each integer between the minimum value and the maximum value of the range. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0027] In the present invention, there is no particular limitation on the specific dispersion and stirring treatment methods.

[0028] In the present invention, the test methods used are all conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0029] In the present invention, the "African swine fever virus culture solution" is derived from Guangdong Wens Dahuayuan Bio-Tech Co., Ltd., [Approval Number] Veterinary Drug Approval No. 190031084. The antigen of the "polyclonal antibody against African swine fever virus in rabbits" is African swine fever virus E0 protein, Npro protein or p72 protein, which is prepared or customized by the laboratory of Tibet Agriculture and Animal Husbandry College.

[0030] The preparation method of polyclonal antibody against classical swine fever in rabbits includes:

[0031] I. Antigen preparation

[0032] 1. Antigen selection and expression

[0033] (1) Select classical swine fever virus specific antigens (such as E0 protein, Npro protein or p72 protein, etc.), and perform recombinant protein expression through a prokaryotic expression system (Escherichia coli BL21).

[0034] (2) Optimize the gene sequence (codon optimization), construct a recombinant plasmid (pET-28a), and purify the recombinant protein by affinity chromatography (Ni column) after induction expression. The purity should reach more than 85%. The primer sequences are as follows:

[0035] E0-F: 5'-(BamHⅠ)GGCGGATCCTGGAACCTGAGTGACAACGG-3'(SEQ ID NO: 1);

[0036] E0-R: 5'-ACTCCCGCCACCTCCGGCATAGGCACCAAACCAG-3'(SEQ ID NO: 2);

[0037] Npro-F: 5'-CGGGGTACCATGGAGTTGAATCATTTTGAAC-3'

[0038] (SEQ ID NO: 3);

[0039] Npro-R: 5'-CCGGAATTCCTAGCAACTGGTAACCCACAATGG-3'(SEQ ID NO: 4).

[0040] 2. Antigen adjuvant mixing

[0041] During the primary immunization, emulsify the recombinant protein (400 μg / animal) with an equal volume of Freund's complete adjuvant (CFA) until it becomes milky white. For subsequent booster immunizations, use Freund's incomplete adjuvant (IFA).

[0042] II. Animal immunization

[0043] 1. Selection of experimental animals

[0044] Six-week-old healthy New Zealand white rabbits (weighing about 2 kg) were selected and pre-bled (negative control) after 1 week of adaptive feeding.

[0045] 2. Immunization protocol

[0046] (1) Primary immunization: Multiple subcutaneous injections (at the back and the root of the thigh, 4 sites, 250 μL of antigen-adjuvant mixture at each site).

[0047] (2) Booster immunization: Repeated immunization every 20 days, with the dose adjusted to 100 μg per time, for a total of 4 - 5 times.

[0048] (3) Serum collection: Blood was collected from the ear vein 7 - 10 days after the last immunization, and the serum was separated (about 40 - 80 mL / rabbit). After inactivating at 37°C for 30 minutes, it was centrifuged (10000 r / min, 10 min) to obtain the supernatant.

[0049] III. Titer detection (ELISA)

[0050] 1. Coating antigen

[0051] A 96-well plate was coated with 1 μg / mL classical swine fever virus antigen (100 μL / well) and blocked (with PBS containing 5% skim milk powder) overnight at 4°C.

[0052] 2. Serum dilution and detection

[0053] (1) The rabbit serum was diluted stepwise (starting from 1:500, serial dilution) and added to the wells for incubation (37°C, 1 h).

[0054] (2) HRP-labeled secondary antibody (such as mouse anti-rabbit IgG, diluted 1:2000) was added. After TMB color development, it was terminated with 2N (equivalent) H2SO4, and the absorbance at 450 nm was measured. The titer should reach 1:10 5 or higher for final release.

[0055] IV. Antibody purification

[0056] 1. Affinity chromatography

[0057] (1) Classical swine fever virus antigen was conjugated to CNBr-Sepharose 4B medium to prepare an antigen affinity column.

[0058] (2) The antiserum was diluted with PBS and passed through the column. After eluting the impurity proteins, the specific antibody was eluted with a low pH buffer (such as 0.1 M glycine-HCl, pH 2.5), neutralized, dialyzed, and concentrated.

[0059] 2. Purity determination

[0060] Verified by SDS-PAGE, the antibody purity was >90%.

[0061] V. Antibody Preservation

[0062] Add 40 - 50% glycerol to the purified antibody. After aliquoting, store it at -20°C for long-term preservation. For short-term use, it can be stored at 4°C (containing 0.02% NaN3). The cross-reactivity of this antibody with the homologous viral protein of classical swine fever virus is <2%.

[0063] Example 1

[0064] A method for preparing a classical swine fever virus-specific polyclonal antibody is as follows:

[0065] 1. First, centrifuge 500 ml of African swine fever virus culture solution at 8000 rpm for 30 minutes, then concentrate it through a membrane package with a cut-off pore size of 100K, and then dilute the virus solution with 30 volumes of 0.1M PBS solution with a pH of 7.4. Repeat the concentration 4 times.

[0066] 2. Prepare African swine fever virus by ultra-high-speed centrifugation and density gradient centrifugation using traditional methods, with a virus concentration of 0.7 mg / ml.

[0067] 3. Prepare a polyclonal antibody against African swine fever virus in rabbits. Dilute the polyclonal antibody against African swine fever virus in rabbits with 0.1M PBS solution with a pH of 7.4 at a dilution factor of 20 times. Add the virus solution to the diluted polyclonal antibody to make the final concentration of the virus reach 0.02 mg / ml, and place it at 2 - 8°C overnight.

[0068] 4. Collect the complex of virus and specific antibody by ultra-high-speed centrifugation. Add 10 - 50 ml of 0.1M PBS solution with a pH of 2.5 to the virus-antibody complex to dissociate the virus from the antibody and release it into the solution. Then use traditional methods for ultra-high-speed centrifugation and collect the supernatant.

[0069] 5. Dilute bovine serum, chicken serum, pig serum, horse serum, and sheep serum with 0.1M PBS solution with a pH of 7.0 to make the final concentration of the serum 1%. Coat the ELISA plate with the diluted serum and place it at 2 - 8°C overnight.

[0070] 6. Wash the ELISA plate 2 times with 0.1M PBS solution with a pH of 7.0. Add the supernatant containing the specific polyclonal antibody to the serum-coated ELISA plate and incubate at 37°C for 1 hour. Incubate sequentially in the ELISA plates coated with bovine serum, chicken serum, pig serum, horse serum, and sheep serum. Repeat 2 times to detect the content of the specific polyclonal antibody, and the antibody concentration is 1.12 mg / ml.

[0071] 7. Dilute the polyclonal antibody 100, 500, 1000, 5000, and 10000 times, and detect the non-specific reaction of the polyclonal antibody by WB.

[0072] Table 1 Non-specific detection results of the polyclonal antibody in Example 1 and different sera

[0073]

[0074]

[0075] In the table, "+" represents that the polyclonal antibody against classical swine fever virus cross-reacts with other proteins in the serum; "-" represents that the polyclonal antibody against classical swine fever virus does not cross-react with other proteins in the serum.

[0076] As shown in Table 1, the concentration of the specific polyclonal antibody against classical swine fever virus in Example 1 reaches 1.0 mg / ml or more, and the specific polyclonal antibody diluted 1000 times or more does not react with other proteins in the serum, showing high specificity.

[0077] Example 2

[0078] A method for preparing a specific polyclonal antibody against classical swine fever virus is as follows:

[0079] 1. First, centrifuge the African swine fever virus culture solution at 10,000 rpm for 10 minutes, then concentrate it through a membrane package with a cut-off pore size of 10K, and then dilute the virus solution with 50 volumes of 0.1M PBS solution with a pH of 7.4, and repeat the concentration 5 times.

[0080] 2. Use the traditional methods of ultra-high-speed centrifugation and density gradient centrifugation to prepare African swine fever virus, and the virus concentration is 0.5 mg / ml.

[0081] 3. Prepare a polyclonal antibody against African swine fever virus in rabbits, and dilute the polyclonal antibody against African swine fever virus in rabbits with 0.1M PBS solution with a pH of 7.4 at a dilution factor of 10 times. Add the virus solution to the diluted polyclonal antibody to make the final concentration of the virus reach 0.05 mg / ml, and place it at 2 - 8°C overnight.

[0082] 4. Ultra-high-speed centrifuge to collect the complex of the virus and the specific antibody. Add PBS solution with a pH of 3 to the virus-antibody complex to dissociate the virus from the antibody and release it into the solution, and then use the traditional method of ultra-high-speed centrifugation to collect the supernatant.

[0083] 5. Dilute bovine serum, chicken serum, porcine serum, equine serum, and ovine serum with 0.1M PBS solution with a pH of 7.0 to make the final concentration of the serum 1%, coat the ELISA plate with the diluted serum, and place it at 2 - 8°C overnight;

[0084] 6. Wash the ELISA plate 3 times with pH 7.0 0.1M PBS. Add the supernatant containing specific polyclonal antibodies to the ELISA plate coated with serum and incubate at 37 °C for 1 hour. Incubate successively in ELISA plates coated with bovine serum, chicken serum, porcine serum, horse serum, and sheep serum. Repeat 5 times to detect the content of specific polyclonal antibodies, and the antibody concentration is 1.21 mg / ml.

[0085] 7. Dilute the polyclonal antibody 100, 500, 1000, 5000, and 10000 times, and then detect the non-specific reaction of the polyclonal antibody by WB.

[0086] Table 2 Non-specific detection results of the polyclonal antibody in Example 2 with different sera

[0087]

[0088] In the table, "+" represents that the polyclonal antibody against classical swine fever virus cross-reacts with other proteins in the serum; "-" represents that the polyclonal antibody against classical swine fever virus does not cross-react with other proteins in the serum.

[0089] As shown in Table 2, the concentration of the specific polyclonal antibody against classical swine fever virus in Example 2 reaches more than 1.0 mg / ml; when the specific polyclonal antibody is diluted 500 times, it does not cross-react with bovine serum, chicken serum, porcine serum, and horse serum, and when the specific polyclonal antibody is diluted 1000 times and above, it does not react with other proteins in the serum, showing high specificity.

[0090] Example 3

[0091] A method for preparing a polyclonal antibody specific to classical swine fever virus is as follows:

[0092] 1. First, centrifuge the African swine fever virus culture solution at 3000 rpm for 60 minutes, then concentrate it through a membrane package with a cut-off pore size of 150K, and then dilute the virus solution with 30 volumes of 0.1M PBS solution with a pH of 7.4. Repeat the concentration 4 times.

[0093] 2. Use the traditional methods of ultra-high-speed centrifugation and density gradient centrifugation to prepare African swine fever virus, and the virus concentration is 0.6 mg / ml.

[0094] 3. Prepare a polyclonal antibody against African swine fever virus in rabbits. Dilute the polyclonal antibody against African swine fever virus in rabbits with 0.1M PBS solution with a pH of 7.4 at a dilution factor of 15 times. Add the virus solution to the diluted polyclonal antibody to make the final concentration of the virus reach 0.06 mg / ml, and place it at 2 - 8 °C overnight.

[0095] 4. Ultracentrifuge to collect the complex of the virus bound to the specific antibody. Add PBS solution with a pH of 2 to the virus-antibody complex to dissociate the virus from the antibody and release it into the solution. Then use the traditional ultracentrifugation method to collect the supernatant.

[0096] 5. Dilute bovine serum, chicken serum, porcine serum, equine serum, and ovine serum with 0.1M PBS solution at pH 7.0 to a final concentration of 2%. Coat the ELISA plate with the diluted serum and incubate it overnight at 2 - 8°C.

[0097] 6. Wash the ELISA plate 5 times with 0.1M PBS at pH 7.0. Add the supernatant containing the specific polyclonal antibody to the serum-coated ELISA plate and incubate at 37°C for 1 hour. Incubate separately and sequentially in the ELISA plates coated with bovine serum, chicken serum, porcine serum, equine serum, and ovine serum. Repeat 3 times to detect the content of the specific polyclonal antibody, and the antibody concentration is 1.02 mg / mL.

[0098] 7. Dilute the polyclonal antibody 100, 500, 1000, 5000, and 10000 times and detect the non-specific reaction of the polyclonal antibody by WB.

[0099] Table 3 Non-specific detection results of the polyclonal antibody in Example 3 with different sera

[0100]

[0101] In the table, "+" represents that the polyclonal antibody against classical swine fever virus cross-reacts with other proteins in the serum; "-" represents that the polyclonal antibody against classical swine fever virus does not cross-react with other proteins in the serum.

[0102] As shown in Table 3, the concentration of the specific polyclonal antibody against classical swine fever virus in Example 3 reaches 1.0 mg / mL or more; when the specific polyclonal antibody is diluted 500 times, it does not cross-react with chicken serum and equine serum, and when the specific polyclonal antibody is diluted 1000 times or more, it does not react with other proteins in the serum, showing high specificity.

[0103] In addition, it should be understood that although this specification is described according to the embodiments, not each embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing a classical swine fever virus-specific polyclonal antibody, characterized in that, The method comprises the following steps: S1. First, centrifuge the classical swine fever virus culture solution at 3000 - 10000 revolutions per minute for 10 - 60 minutes, then concentrate and dilute the virus solution, and then perform ultra - high - speed centrifugation and density - gradient centrifugation to obtain classical swine fever virus; S2. Dilute the rabbit anti - classical swine fever virus polyclonal antibody by 10 - 20 times, then add it to the classical swine fever virus and incubate overnight at low temperature. Then, perform ultra - high - speed centrifugation to collect the complex of the virus and the specific antibody. Add a PBS solution with a pH value of 2 - 3, and perform ultra - high - speed centrifugation to obtain the supernatant containing the specific polyclonal antibody; S3. Coat the ELISA plate with serum, wash it, add the supernatant, incubate at 37°C for 1 hour, and then detect the content of the specific polyclonal antibody.

2. The method for preparing a classical swine fever virus-specific polyclonal antibody according to claim 1, characterized in that, In step S1, the concentration is carried out by using a membrane package with a cut - off pore size of 10 - 300K and repeated 2 - 10 times; the dilution is carried out with a 0.1M PBS solution with a pH of 7.0 - 7.4 and a volume 10 - 100 times that of the original.

3. The method for preparing a classical swine fever virus-specific polyclonal antibody according to claim 1, wherein In step S1, the concentration of the classical swine fever virus is ≥0.2 mg / ml.

4. The method for preparing a classical swine fever virus-specific polyclonal antibody according to claim 1, wherein In step S2, the dilution is carried out with a 0.1M PBS solution with a pH of 7.0 - 7.4, and the dilution factor is 2 - 50 times.

5. The method for preparing a classical swine fever virus-specific polyclonal antibody according to claim 1, characterized in that, In step S2, the final concentration of the virus solution is 0.02 - 1 mg / ml.

6. The method for preparing a classical swine fever virus-specific polyclonal antibody according to claim 1, wherein In step S3, before coating with serum, it is diluted with a 0.1M PBS solution with a pH of 7.0 - 7.4; the serum is one or more of bovine serum, chicken serum, porcine serum, equine serum, and ovine serum, and the final concentration is 1 - 10%.

7. The method for preparing a classical swine fever virus-specific polyclonal antibody according to claim 1, characterized in that, The concentration of the specific polyclonal antibody > 1.0 mg / ml.

8. A classical swine fever virus-specific polyclonal antibody, characterized in that, It is obtained by the method for preparing a classical swine fever virus - specific polyclonal antibody according to any one of claims 1 - 8.

9. The specific polyclonal antibody against classical swine fever virus according to claim 8, characterized in that, The classical swine fever virus - specific polyclonal antibody does not react with other proteins in the serum after dilution, and the dilution factor of the classical swine fever virus - specific polyclonal antibody is ≥1000.

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