CN109467600B - Preparation method of donkey-derived immunoglobulin G of canine distemper virus - Google Patents
Preparation method of donkey-derived immunoglobulin G of canine distemper virus Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention relates to the technical field of preparation of virus antibodies, in particular to a preparation method of donkey-derived immunoglobulin G of canine distemper virus. The preparation method comprises the following operations: immunizing young healthy donkey with canine distemper inactivated vaccine, detecting antibody titer and neutralizing antibody titer after each immunization, collecting blood by vein when the antibody titer reaches more than 1:10000 and the neutralizing antibody titer reaches more than 1:256, separating serum, and extracting and purifying immunoglobulin G. The immunoglobulin G prepared by the method can be used for clinically treating canine distemper virus diseases, the effective rate can reach 100%, the cure rate can reach more than 85%, and the problem of insufficient sources of antiserum or antibodies of the canine distemper virus is solved.
Description
Technical Field
The invention relates to the technical field of preparation of virus antibodies, in particular to a preparation method of donkey-derived immunoglobulin G of canine distemper virus.
Background
Canine distemper (canine distemper) is an acute, highly contagious disease in animals of the families canines, ferrets and raccoons caused by Canine Distemper Virus (CDV). Clinically characterized by biphasic elevation of body temperature, subsequent bronchitis, catarrhal pneumonia, severe gastroenteritis and neurological symptoms. The host animals of canine distemper virus are wide, and not only dogs of different ages, sexes and breeds are infected under natural conditions, but also animals of ferrets, raccoons, pandas and the like (such as pandas, tigers, lions, pandas, lynx, bears, wolves and the like) are infected; meanwhile, the incidence and mortality of canine distemper are high, the incidence of the puppies with the age of less than 10 months without immunization reaches more than 80%, particularly the incidence and mortality of the puppies with the age of 2-5 months are the highest, and the mortality reaches more than 90%. The canine distemper epidemic not only causes great economic damage to the pet breeding industry, the fur-bearing animal breeding industry and the wild animal protection industry, but also causes certain mental stress to pet owners. Therefore, the composition has important economic value and social benefit for effectively preventing and treating the canine distemper.
At present, the canine distemper is mainly prevented by injecting a vaccine, but the immunized puppy cannot be protected by 100 percent due to the quality of the vaccine, the immune response capability of an individual to the vaccine, the interference of maternal antibodies and the like, so the canine distemper becomes an infectious disease which is common in pet clinic. The main methods for treating canine distemper at present comprise: antiserum (antibody) therapy, traditional Chinese medicine adjuvant therapy, antibiotic adjuvant therapy for preventing secondary bacterial infection, etc.
The traditional Chinese medicine treatment is mainly an auxiliary treatment aiming at clinical symptoms; antibiotic therapy is an adjuvant treatment to prevent secondary bacterial infections. Antiserum or antibody treatment is a specific treatment aiming at canine distemper virus, and plays a major role in treating canine distemper. Antiserum or antibody for treatment at present mainly comes from dogs of the same species of dogs, but due to the shortage of dog sources, most dogs are dispersed in dog raising users, limited antiserum and antibody prepared by dog raising factories cannot meet clinical needs, and meanwhile, the preparation cost is high, so that clinical application of the antiserum and antibody for canine distemper heat treatment is seriously influenced.
Disclosure of Invention
Aiming at the problems that antiserum or antibody for treating canine distemper in the prior art is mainly derived from dogs but is insufficient in canine origin, the invention provides a preparation method of donkey-derived immunoglobulin G for canine distemper virus.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
a preparation method of donkey-derived immunoglobulin G of canine distemper virus comprises the following steps:
step a, inoculating a healthy donkey for young people with canine distemper inactivated vaccine for immunization, collecting blood by veins when the antibody titer in blood reaches more than 1:10000 and the neutralizing antibody titer reaches more than 1:256, and separating serum;
b, mixing the serum with PBS with the same volume, salting out with a saturated ammonium sulfate solution, desalting with a gel chromatography column, purifying with a DEAE cellulose column chromatography, salting out with the saturated ammonium sulfate solution again, desalting with the gel chromatography column, and filtering for sterilization to obtain the compound; wherein the PBS and the saturated ammonium sulfate solution both contain 0.05-0.1M urea.
The preparation method of the donkey-derived canine distemper virus antibody provided by the invention takes donkey as an immune animal, and has the advantages of high yield, low feeding cost, high serum yield and good disease resistance to canine distemper. Through repeated inoculation with the canine distemper inactivated vaccine, donkey can be stimulated to generate immune response, a large amount of specific antibodies aiming at the canine distemper virus are generated in donkey serum, and when the antibody titer and the neutralizing antibody titer reach expected values, immunoglobulin G in the serum can be used for clinically treating the canine distemper virus. When the immunoglobulin G is extracted and purified from serum, 0.05-0.1 mol/L urea is added into PBS and saturated ammonium sulfate solution, which is beneficial to improving the stability of the purified immunoglobulin G and maintaining the titer of an antibody and the titer of a neutralizing antibody. But the dosage of the urea cannot be too high, and the high concentration of the urea can denature and lose the immunoglobulin G, thereby reducing the yield of the immunoglobulin G; if the urea concentration is too low, the urea does not exert a protective effect on the antibody activity in the ammonium sulfate salting-out, and the antibody titer of immunoglobulin G in the crude product is reduced. The immunoglobulin G prepared by the preparation method provided by the invention is used for treating canine distemper, the effective rate can reach 100%, and the cure rate can reach more than 85%, so that the problem of insufficient sources of antiserum or antibody of canine distemper virus is solved.
Preferably, the strain of the inactivated canine distemper vaccine in the step a is a CDV-L virulent strain, and the antibody generated by the strain can be used for resisting the canine distemper virulent strain.
Preferably, the inactivated canine distemper vaccine is a 40% (v/v) formaldehyde inactivated vaccine which does not infect the inoculated donkey, but can promote the generation of antibodies to resist canine distemper virus.
Preferably, the operation of inoculating the canine distemper inactivated vaccine to the young healthy donkey in the step a is to perform inoculation three times by neck intramuscular injection, wherein one week is separated between two adjacent inoculations, and the first inoculation is performed by using 3mL and 5 multiplied by 105TCID50Inactivated vaccine/mL, 6mL, 5X 10 for two subsequent inoculations5TCID50and/mL inactivated seedlings.
Preferably, the age of the young healthy donkey in the step a is 3.5-4.5 years, and the body weight is 250-350 kg.
Preferably, the young healthy donkey in step a is negative for equine infectious anemia virus and melioidosis bacteria, so as to prevent other pathogens from interfering with the production of antibodies in serum.
Preferably, step b is performed by filtration sterilization with a filter having a pore size of 0.2. mu.m.
The embodiment of the invention also provides a canine distemper virus immunoglobulin G solution, and the immunoglobulin G in the solution is prepared by the preparation method. Immunoglobulin G in the solution can specifically bind with corresponding canine distemper virus to eliminate virus in sick canine, and the cure rate of canine distemper can reach 85.7%
Preferably, the solvent of the solution is physiological saline containing 0.05-0.1M urea or PBS containing 0.05-0.1M urea, and the concentration of the immunoglobulin G is 50-60 mg/mL. Urea in solution is able to maintain antibody titers and neutralize antibody titers. Immunoglobulin G can achieve better immune effect within the concentration range, the treatment effect is reduced if the concentration is too low, and the treatment effect cannot be stronger if the concentration is too high, but immunoglobulin G is wasted.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
The embodiment provides a preparation method of donkey-derived canine distemper virus immunoglobulin G, which specifically comprises the following steps:
1. immunization of donkey: selecting local young healthy donkey (negative in equine poverty-passing/melioidosis) of 3.5-4.5 years old and 250-350 kg weight, and immunizing and inoculating canine distemper inactivated vaccine (40% v/v formaldehyde inactivated vaccine of CDV-L virulent strain, 5 multiplied by 10) by neck intramuscular injection for 3 times5TCID50mL), 3mL for the first inoculation and 6mL for the second inoculation. And detecting the antibody titer and the neutralizing antibody titer at 7 th, 14 th, 21 th and 35 th days after immunization, wherein the antibody titer reaches 1:16384 and the neutralizing antibody titer reaches more than 1:256 when 35d is immunized.
2. Collecting blood intravenously, placing the blood into a constant temperature cabinet at room temperature or 37 ℃, centrifuging after serum is separated out, and collecting the serum.
3. And (3) extracting and separating immunoglobulin G:
3.1 salting out
(1) A certain volume of donkey serum is taken, added with PBS (containing 0.01M phosphate and 0.1M urea and having a pH value of 7.0) with the same volume, mixed evenly and dripped with saturated ammonium sulfate solution containing 0.1M urea while stirring, so that the final saturation of the ammonium sulfate solution reaches 25 percent. Standing at 4 deg.C for 30min for salting out. Centrifugation was carried out at 4 ℃ for 15min (3000r/min), the precipitate (fibrinogen) was discarded, and the supernatant (albumin and globulin) was collected.
(2) Measuring the volume of the supernatant, continuously dropwise adding a saturated ammonium sulfate solution containing 0.1M urea into the supernatant to make the final saturation degree of the ammonium sulfate solution reach 50%, mixing, and standing at 4 deg.C for 30 min. Centrifugation was carried out at 4 ℃ for 15min (3000r/min), the supernatant (albumin) was discarded, and the precipitate (globulin) was collected.
(3) The precipitate was diluted with 1.5-fold volume of PBS (containing 0.01M phosphate and 0.1M urea, pH7.0) of the original serum, and saturated ammonium sulfate containing 0.1M urea was added dropwise to a saturation of 35%, and the mixture was left at 4 ℃ for 30 min. Centrifugation was carried out at 4 ℃ for 15min (3000r/min), the supernatant (. alpha. -and. beta. -globulins) was discarded, and the precipitate, i.e.crude immunoglobulin G, was collected. The crude immunoglobulin G precipitate was dissolved in 1/10-fold volume of PBS (containing 0.0175M phosphate and 0.1M urea, pH6.7) in the original serum for further use.
3.2 desalination
(1) Swelling of the gel: sephadex G-25 gel was swollen with 20 volumes of distilled water overnight and then soaked with 2 volumes of PBS (containing 0.0175M phosphate and 0.1M urea, pH6.7) overnight.
(2) Column assembling: and (3) filling the treated gel into a chromatographic column.
(3) Balancing: gels were smoothed with 3 bed volumes of PBS (containing 0.0175M phosphate and 0.1M urea, pH 6.7).
(4) Sample loading and elution: the crude immunoglobulin G was applied to the column bed and eluted with PBS (containing 0.0175M phosphate and 0.1M urea, pH 6.7).
(5) Collecting: and collecting the eluted protein solution through a protein detection system, namely a desalted crude product of the immunoglobulin G, wherein the purity of the immunoglobulin G in the crude product is 93.8%.
3.3 purification
(1) Pretreatment of DEAE cellulose: DEAE cellulose was swollen overnight with 10 times the volume of distilled water, and fine gel particles were removed. Soaking with 2 times of 0.5M NaOH for 30min, and repeatedly pumping and washing with distilled water until pH is neutral; then treated with 0.5M HCl. Then washed with deionized water to about pH6, and finally soaked overnight in PBS (containing 0.0175M phosphate and 0.1M urea, pH 6.7).
(2) Column assembling: the treated DEAE cellulose is loaded into a chromatographic column.
(3) Balancing: the bed was equilibrated with PBS (containing 0.0175M phosphate and 0.1M urea, pH 6.7).
(4) Sample loading and elution: the desalted crude immunoglobulin G product obtained in 3.2 was applied to a column bed and eluted with PBS (containing 0.0175M phosphate and 0.1M urea, pH 6.7).
(5) Collecting samples: and collecting a first protein elution peak, namely the purified canine distemper virus donkey-derived immunoglobulin G solution. The purity of the purified immunoglobulin G was 95.7%.
(6) And (3) concentrating a sample: immunoglobulin G was concentrated using an ammonium sulfate precipitation/desalting process.
(7) And (3) sample sterilization: the bacteria were removed by filtration (0.2 μm pore size microfiltration).
Example 2
This example provides a method for preparing immunoglobulin G of canine distemper virus derived from donkey, which comprises using the sera obtained in steps 1 and 2 of example 1, and extracting and separating immunoglobulin G according to the procedure of step 3 of example 1, wherein the concentration of urea in PBS and saturated ammonium sulfate solution is 0.05M.
Example 3
The embodiment provides a canine distemper virus immunoglobulin G solution, and the preparation method of the solution comprises the following steps: the immunoglobulin G obtained in example 1 was added to a physiological saline containing 0.1M urea to prepare a solution having an immunoglobulin G concentration of 50 mg/mL.
Example 4
The embodiment provides a canine distemper virus immunoglobulin G solution, and the preparation method of the solution comprises the following steps: PBS (containing 0.0175M phosphate and 0.1M urea, pH6.7) was added to the immunoglobulin G obtained in example 1 to prepare a solution having an immunoglobulin G concentration of 60 mg/mL.
Example 5
The embodiment provides a canine distemper virus immunoglobulin G solution, and the preparation method of the solution comprises the following steps: the immunoglobulin G obtained in example 2 was added to a physiological saline containing 0.05M urea to prepare a solution having an immunoglobulin G concentration of 50 mg/mL.
Example 6
The embodiment provides a canine distemper virus immunoglobulin G solution, and the preparation method of the solution comprises the following steps: PBS (containing 0.0175M phosphate and 0.05M urea, pH6.7) was added to the immunoglobulin G obtained in example 2 to prepare a solution having an immunoglobulin G concentration of 60 mg/mL.
Comparative example
This example provides a canine distemper virus immunoglobulin G, which was extracted and isolated from the sera obtained in steps 1 and 2 of example 1 by the procedure of step 3 of example 1, but urea was not used in PBS and saturated ammonium sulfate.
Examples of effects
The immunoglobulin G solutions obtained in examples 3 and 4 and comparative example were subjected to treatment effect analysis using commercially available canine distemper virus immunoglobulin G and antiserum as positive controls.
24 healthy puppies (with canine distemper, parvovirus and infectious bronchitis virus negative) of 2-3 months old are selected, the weight of the puppies is 1.5-2 kg, the puppies are divided into an experimental group and a positive control group (12 puppies in each group), the puppies are separately fed in a disinfected animal room, and the puppies are fed with free food and water for 3 days. The puppies of the control and experimental groups (dose 0.125mg/kg) were injected intramuscularly with an ethanol solution (15mg/mL) of hydrocortisone, and were challenged with virulent strains of canine distemper virus by oral and nasal administration, 3mL of virus solution (1.0X 10) per dog6TCID50) (2 ml orally, 1ml nasally), clinical symptoms were observed day by day after challenge and body temperature was measured, and lymphocyte and leukocyte (hematology) changes were measured over time. And simultaneously identifying the suspected disease puppies by using a canine distemper colloidal gold kit.
The two groups of puppies show the phenomenon of body temperature rise and show the characteristic bidirectional heat type of canine distemper, namely the body temperature changes in a wave shape and usually changes between 39 and 40 ℃. Increase of leukocytes is 20-35 × 109The lymphocyte is obviously reduced after toxicity attack and is 1.5-2 multiplied by 109/L。
Injection treatment is carried out by using the canine distemper virus immunoglobulin G solution prepared in examples 3 and 4 and the serum obtained in the step a of the example 1 respectively (the total protein concentration of the commercial canine distemper antiserum obtained in the canine distemper antiserum and the serum obtained in the step a of the example 1 is 70-80%). The dose of the canine distemper virus immunoglobulin G solution is 30 mg/kg/day in terms of immunoglobulin G, and the dose of the antiserum is 80 mg/kg/day. All injections were given continuously for 3 days. Clinical symptoms are observed day by day, body temperature changes are detected, and blood routine is analyzed. The results show that: body temperature gradually decreased and approached normal (38.2-39.2 ℃); the number of the white blood cells is reduced and is close to the normal value, and the white blood cell number is 8-19 multiplied by 109L; gradual recovery of lymphocyte countThe return is normal and is 3-5 multiplied by 109L; the clinical symptoms gradually disappear; nasal secretion viral titre 100.25～100.35。
The positive control group was treated by injection with commercially available canine distemper virus immunoglobulin G or antiserum at the same dose as above for 3 consecutive days. Clinical symptoms are observed day by day, body temperature changes are detected, and blood routine is analyzed. The results show that: treating a canine distemper dog with the same dose of canine immunoglobulin G or antiserum, wherein the body temperature of the canine distemper dog treated by the canine immunoglobulin G or antiserum is gradually reduced and tends to be normal; the number of the white blood cells is reduced and is close to the normal value, and the white blood cell number is 8-18 multiplied by 109L; the lymphocyte number gradually returns to normal and is 2-5 multiplied by 109L; the clinical symptoms gradually disappear; nasal secretion viral titre 100.20～100.35。
The effective rate and cure rate of each group and the positive control group in the examples are shown in table 1.
TABLE 1 results of effective rate and cure rate for the example group and the positive control group
The results show that the immunoglobulin G prepared by the preparation method of the donkey-derived canine distemper virus immunoglobulin G can achieve the same treatment effect as the commercially available canine immunoglobulin G.
The results of comparing the antibody titer and the neutralizing antibody titer for examples 1, 2 and comparative example are shown in table 2.
TABLE 2 antibody Titers and neutralizing antibody Titers of examples and comparative examples
| Group of | Antibody titer | Neutralizing antibody titer |
| Example 1 | 1:15400 | 1:247 |
| Example 2 | 1:14300 | 1:242 |
| Comparative example | 1:13200 | 1:210 |
As can be seen from the results in table 2, the addition of urea in examples 1 and 2 increased the antibody titer and antibody titer of igg.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (6)
1. A preparation method of donkey-derived immunoglobulin G of canine distemper virus is characterized by comprising the following steps:
step a, inoculating a healthy donkey for young people with canine distemper inactivated vaccine for immunization, collecting blood by veins when the antibody titer in blood reaches more than 1:10000 and the neutralizing antibody titer reaches more than 1:256, and separating serum; the strain of the canine distemper inactivated vaccine is a CDV-L virulent strain; the canine distemper inactivated vaccine is a 40% v/v formaldehyde inactivated vaccine; the operation of inoculating the canine distemper inactivated vaccine to the young healthy donkey isThree injections were administered by intramuscular injection into the neck three times, one week apart between two consecutive injections, 3mL of a 5X 10 vaccine for the first injection5 TCID50Inactivated vaccine/mL, 6mL, 5X 10 for two subsequent inoculations5 TCID50Per mL inactivated seedling;
b, mixing the serum with PBS with the same volume, salting out with a saturated ammonium sulfate solution, desalting with a gel chromatography column, purifying with a DEAE cellulose column chromatography, salting out with the saturated ammonium sulfate solution again, desalting with the gel chromatography column, and filtering for sterilization to obtain the compound; wherein the PBS and the saturated ammonium sulfate solution both contain 0.05-0.1M urea.
2. The method for preparing immunoglobulin G of Canine Distemper Virus (CDV) derived from donkey according to claim 1, wherein the young healthy donkey in step a is 3.5-4.5 years old and 250-350 kg in body weight.
3. The method for preparing immunoglobulin G of Canine Distemper Virus (CDV) derived from donkey according to claim 1, wherein the young healthy donkey in step a is negative for equine infectious anemia virus (MDV) and melioidosis.
4. The method for preparing immunoglobulin G of canine distemper virus derived from donkey according to claim 1, wherein the filtration sterilization is performed with a filter with a pore size of 0.2 μm in step b.
5. A canine distemper virus immunoglobulin G solution, wherein the immunoglobulin G in the solution is prepared by the preparation method of any one of claims 1 to 4.
6. The canine distemper virus immunoglobulin G solution according to claim 5, wherein the solvent of the solution is normal saline containing 0.05-0.1M urea or PBS containing 0.05-0.1M urea, and the concentration of the immunoglobulin G is 50-60 mg/mL.
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