CN107304230A - A kind of anti-dog parvovirus refines antibody and preparation method thereof - Google Patents

Abstract

The invention discloses a refined anti-canine parvovirus antibody and a preparation method thereof. The method comprises the following steps: S1. Firstly, the canine parvovirus CPV-S5 and CPV-1401 isolated and identified in Guangdong area are used as base strains to prepare canine parvovirus inactivated vaccine; S2. The vaccine immunizes healthy dogs to prepare canine parvovirus hyperimmune serum; S3. Saturated ammonium sulfate step-by-step salting out to separate the anti-canine parvovirus immunoglobulin in the serum prepared in S2; S4. Purification: Sephadex desalting the anti-canine parvovirus immunoglobulin extracted in step S3; S5 . The anti-canine parvovirus immunoglobulin after the desalting treatment in step S4 is concentrated by ultrafiltration, sterilized by filtration, and subpackaged to obtain. The protein content of the anti-canine parvovirus refined antibody prepared by the invention reaches 134 mg/ml, and the hemagglutination inhibitory titer is not lower than 1:10240; it can be applied to the treatment and emergency prevention of various breeds of dogs when they are exposed to parvovirus infection, and can increase the risk of illness The dog's body immunity and resistance have no adverse reactions, no clinical side effects, good safety, and good prospects for popularization and application.

Description

A kind of anti-canine parvovirus refined antibody and its preparation method

technical field

The invention belongs to the technical field of biopharmaceuticals. More specifically, it relates to a refined anti-canine parvovirus antibody and a preparation method thereof.

Background technique

Canine parvovirus disease, also known as canine parvovirus enteritis, is a highly contagious infectious disease caused by canine parbovirus (CPV), mainly causing acute hemorrhagic enteritis in dogs or acute myocarditis in newborn dogs. The main clinical symptoms are elevated body temperature, severe vomiting, hemorrhagic enteritis and significant decrease in white blood cells. The disease occurs all year round, especially in spring and autumn. Dogs of different types and ages can be infected with the disease. Among them, puppies are the most harmful, with the highest infection rate and mortality rate, which seriously endangers the dog industry in my country. At present, vaccine immunization is used to prevent the disease to a certain extent, but due to reasons such as maternal antibody and strain variation, immune failure often occurs and infection with canine parvovirus occurs. CPV has only one antigenic serotype, CPV-2, but due to antigenic drift, changes in the VP2 gene lead to amino acid substitutions leading to different genotypes, CPV-2a, CPV-2b, and CPV-2c mutants discovered in Italy in 2000 , the emergence of these new subtypes may be one of the reasons for immune failure. Huang Yongliang (2012) identified 19 CPV strains in Shenzhen (9 strains) and Guangzhou (10 strains), of which only 1 strain was identified as CPV-2b, 2 strains were New CPV-2b, and the rest were New CPV-2a subtypes. type. This indicated that New CPV-2a was the dominant strain in Guangzhou and Shenzhen. In terms of treatment, there is still no effective prevention and treatment of canine parvovirus drugs and methods, so the cure rate of canine parvovirus is low. The concentrated preparation of anti-canine parvovirus immunoglobulin is a specific biological product for preventing and treating canine parvovirus disease. It is a passive immune preparation and also has the function of emergency vaccination, which has high promotion and application value.

Immunoglobulin (Ig for short) is a type of globulin with antibody activity synthesized by lymphocytes, especially paddle cells, after the body is stimulated by antigens. Immunoglobulins are ubiquitously present in blood, interstitial fluid, lymph fluid and exocrine fluid of mammals. Immunoglobulin plays an important immune and physiological regulatory role in animals, and is one of the most critical components of the immune system in animals. Its main biological characteristics are specific binding to corresponding antigens, as well as functions such as activating complement and promoting phagocytosis. The use of immunoglobulin can be called passive immunity in the theory of immunology. When immunodeficiency, lack of vaccine and immune failure, immunoglobulin injection can be used instead of active immunization to play a role in disease resistance.

Therefore, the development of a high-concentration, high-activity and high-potency anti-canine parvovirus immunoglobulin preparation specifically targeting the two subtypes of canine parvovirus New CPV-2a and New CPV-2b is an important step for clinical treatment and emergency prevention of canine parvovirus. Effective means of parvovirus disease.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the existing canine parvovirus prevention and treatment drugs, and provide a medicine for the prevention and treatment of canine parvovirus disease - anti-canine parvovirus immunoglobulin preparation, that is, an anti-canine parvovirus immunoglobulin preparation Refined antibodies.

The purpose of the present invention is to provide a refined antibody against canine parvovirus.

Another object of the present invention is to provide a preparation method of the purified anti-canine parvovirus antibody.

The above object of the present invention is achieved through the following technical solutions:

A preparation method for anti-canine parvovirus refined antibody, comprising the following steps:

S1. Prepare canine parvovirus inactivated vaccine;

S2. Prepare canine parvovirus hyperimmune serum from healthy dogs immunized with vaccines;

S3. Separation of anti-canine parvovirus immunoglobulin in the serum prepared in S2 by stepwise salting out with saturated ammonium sulfate;

S4. Purification: Sephadex desalting the anti-canine parvovirus immunoglobulin extracted in step S3;

S5. The anti-canine parvovirus immunoglobulin after the desalination treatment in step S4 is concentrated by ultrafiltration, sterilized by filtration, and subpackaged to obtain.

Wherein, preferably, the method of step S1 is: using the canine parvovirus CPV-S5 (NewCPV-2a) and CPV-1401 (New CPV-2b) isolated and identified in Guangdong area as the basic strains, expanding the culture on F81 cells respectively , Small-scale tangential flow ultrafiltration and concentration, mixed with water-soluble adjuvant MONTANIDE GEL at a ratio of 9:1 to prepare inactivated vaccines.

Preferably, the method of step S2 is: select a 5-week-old CPV HI-negative healthy dog, and at the same time subcutaneously inoculate two inactivated vaccines of canine parvovirus CPV-S5 and CPV-1401 on the back for 25-30 days (preferably 28 days) Immunization was then boosted in the same way; 7 to 14 days after immunization, dog blood with a CPV HI antibody titer of not less than 1:3072 was aseptically collected from the carotid artery, left to stand and centrifuged to obtain serum.

Preferably, the immunization doses of the two inactivated vaccines of canine parvovirus CPV-S5 and CPV-1401 are 1 mL per dog.

Preferably, the steps of step S3 saturated ammonium sulfate salting-out are as follows:

(a) Dilute the serum with phosphate buffered solution (PBS), slowly add pre-cooled saturated ammonium sulfate (AS) solution dropwise under magnetic stirring to a final concentration of 15-25%, stir for 10-30 minutes, and stand at 0-10°C for 0.5 After ~2h, centrifuge at 5000~8000r/min for 20~40min, discard the precipitate;

(b) To the supernatant solution obtained in step (a), slowly add pre-cooled saturated ammonium sulfate solution dropwise under magnetic stirring to a final concentration of 40-60%, and after standing at 0-4°C for 0.5-2h, 5000-8000r/ Centrifuge for 2 to 40 minutes, discard the supernatant, and collect the precipitate;

(c) Add PBS twice the volume of the original serum to the precipitate obtained in step (b) to dissolve, slowly add pre-cooled saturated AS solution dropwise under a magnetic stirrer to a final concentration of 25% to 35%, and stand at 0 to 4°C for 0.5 to 2h, centrifuge at 5000-8000r/min for 20-40 min, discard the supernatant, and collect the precipitate;

(d) Repeat step (c) twice.

The step of step S3 saturated ammonium sulfate stepwise salting-out is most preferably as follows:

(a) Dilute the serum by 2 times with phosphate buffer solution (PBS, pH = 7.0), slowly add pre-cooled saturated ammonium sulfate (AS) solution (pH = 7.0) dropwise under magnetic stirring to a final concentration of 20%, and stir for 20 minutes , after standing at 4°C for 1 h, centrifuge at 6500 r/min for 30 min, and discard the precipitate;

(b) Add pre-cooled saturated ammonium sulfate (AS) solution (pH=7.0) dropwise slowly to the supernatant solution obtained in step (a) under magnetic stirring to a final concentration of 50%. After standing at 4°C for 1 h, 6500 Centrifuge at r/min for 30min, discard the supernatant, and collect the precipitate;

(c) Add two volumes of PBS to the precipitate obtained in step (b) to dissolve, slowly add saturated AS solution dropwise under a magnetic stirrer to a final concentration of 33%, let it stand at 4°C for 1 h, and centrifuge at 6500 r/min for 30 min, discard the supernatant, and collect the precipitate;

(d) Repeat step (c) twice.

Preferably, the purification method described in step S4 is to use Sephadex™ G-25 prepacked gel column to desalt and purify the anti-canine parvovirus immunoglobulin extracted in step S3 by using Sephadex. Specific steps are as follows:

(1) Connect the prepacked column to the protein purification system, and prevent air from entering the column during the process;

(2) Equilibrate the column: Equilibrate the column with 5 times the column volume of PBS to completely remove the ethanol for storing the column, and at the same time make the column completely filled with PBS, and control the flow rate at 5 mL/min;

(3) Sample loading and elution: the maximum sample volume is 1.5 mL, and the flow rate is 1 mL/min. After the sample loading is completed, continue to fill with PBS for elution;

(4) Collection: Use a 1.5 mL ep tube to collect the eluate at the peak with reference to the UV absorption value and resistivity, and store it at -20°C for future use;

(5) Wash the column: wash with 5 times the column volume of PBS, and then wash with 5 times the column volume of 20% ethanol to preserve the column.

Preferably, the ultrafiltration concentration in step S5 is concentrated by using small tangential flow ultrafiltration to obtain a refined antibody with an HI titer of 1:10240.

In order to obtain immunoglobulins with higher protein content, better antibody activity and lower endotoxin content, the present invention optimizes the traditional method of separating and extracting immunoglobulins in serum with saturated ammonium sulfate. The results showed that the immunoglobulin concentration of the purified antibody against canine parvovirus prepared by the optimized saturated ammonium sulfate was 134mg/mL; The titer of the purified anti-canine parvovirus antibody reached 1:10240.

Most preferably, the preparation method of the purified antibody against canine parvovirus (CPV) of the present invention is: the canine parvovirus CPV-S5 (New CPV-2a) and CPV-1401 (New CPV-2b) isolated and identified in Guangdong area The two inactivated vaccines prepared for the basic strains were injected subcutaneously at 1 mL per CPV-negative dog, and boosted immunization 28 days later. The HI antibody reached the highest level 7 days after the second immunization. 10 days after the second immunization, blood was collected from the carotid artery aseptically, and after mixing, hyperimmune serum with an HI titer of not less than 1:3072 was obtained. Use 20% to 50% saturated ammonium sulfate step-by-step salting out to roughly purify hyperimmune serum to remove most of the impurity proteins, optimize the ammonium sulfate concentration and pH of salting out precipitation, and determine the saturated ammonium sulfate concentration of 33%, pH=7.0 for the optimal condition. After a large amount of extraction and purification according to this method, it was desalted by HiTrap™ Desalting 5mL prepacked column, and then purified by a small tangential flow ultrafiltration concentration system to obtain a refined antibody against canine parvovirus, with an HI titer as high as 1:10240.

In summary, the concentration and purity of the purified anti-canine parvovirus antibody prepared by the optimized saturated ammonium sulfate salting-out method are relatively high; the hemagglutination inhibition titer of canine parvovirus is significantly improved.

In addition, the purified anti-canine parvovirus antibody and its application prepared according to the above method are also within the protection scope of the present invention.

The anti-canine parvovirus refined antibody includes anti-canine parvovirus immunoglobulin; wherein, the content of the anti-canine parvovirus immunoglobulin is >130 mg/mL, and the titer of CPV HI antibody is above 1:10240.

The purified anti-canine parvovirus antibody prepared by the invention is in liquid or freeze-dried dosage form with a pH of about 7.0; wherein, the purified anti-canine parvovirus antibody mainly exists as IgG monomer. The refined anti-canine parvovirus antibody of the invention has a long storage period and can be stored for more than 2 years at 2-8°C.

A suitable protective agent, such as maltose, glucose, etc., can also be added to the purified anti-canine parvovirus antibody of the present invention. Those skilled in the art can adjust the dosage of the protective agent according to actual needs.

In addition, the anti-canine parvovirus antibody of the present invention can be applied to the prevention and treatment of canine parvovirus disease, can also be applied to the prevention or treatment of canine parvovirus viral enteritis, and can increase the body immunity and resistance of sick dogs.

Therefore, the anti-canine parvovirus refined antibody prepared by the present invention is used as or in the preparation of a drug for the prevention and/or treatment of canine parvovirus disease, or as or in the preparation of a drug for the prevention and/or treatment of canine parvovirus viral enteritis or The application of reagents, or the application in the preparation or preparation of drugs that can increase the body immunity and resistance of diseased dogs (the diseased dogs refer to dogs suffering from canine parvovirus disease) should be covered by the protection of the present invention. within range.

The method of using the refined anti-canine parvovirus antibody of the present invention: generally adopts subcutaneous or intramuscular injection; it is mainly used for the treatment and emergency prevention of parvovirus infection in dogs of various breeds, and effectively increases the body immunity and disease resistance of sick dogs.

The present invention has the following beneficial effects:

The protein content of the anti-canine parvovirus refined antibody prepared by the invention reaches 134mg/ml, and the hemagglutination inhibitory titer is not lower than 1:10240; it can be applied to the treatment and emergency prevention of various breeds of dogs when they are exposed to parvovirus infection, and can increase the risk of illness Canine immunity and resistance.

Moreover, the results of the safety experiment showed that after the antibody preparation was inoculated into dogs (such as intramuscular or subcutaneous injection of dogs), the dogs’ spirit, body temperature, and appetite were normal, there was no lump at the injection site, no adverse reactions, and no clinical side effects. The safety is good, and the application prospect is very good.

The optimized preparation method of the refined anti-canine parvovirus antibody of the present invention not only maintains the integrity and biological activity of the immunoglobulin molecular structure, but also uses cheap reagents and simple operation in the preparation process, and improves protein purity and immunoglobulin content and biosafety.

Description of drawings

Figure 1 is the SDS-PAGE analysis of proteins extracted with different concentrations of AS; M. Standard protein.

Figure 2 is the SDS-PAGE analysis of purified anti-canine parvovirus antibody and original serum; 1. hyperimmune serum 2. purified anti-canine parvovirus antibody, M. protein marker.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Definitions of terms involved in the present invention:

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "hemagglutination test (HA) and hemagglutination inhibition test (HI)", canine parvovirus has the effect of specifically agglutinating porcine red blood cells at 4°C, which is called hemagglutination test. Hemagglutination can be inhibited by the corresponding antibody called hemagglutination inhibition test, the highest serum dilution that can inhibit 100% erythrocyte agglutination is the HI titer of the serum.

Example 1 Preparation of refined antibody against canine parvovirus

1. Test method

1.1 Preparation of hyperimmune serum against canine parvovirus

Two strains of inactivated vaccines, CPV-S5 (New CPV-2a) and CPV-1401 (New CPV-2b), were prepared according to conventional inactivated vaccine preparation methods. Select a certain number of healthy puppies with negative CPV HI titer, inoculate two inactivated vaccines, CPV-S5 and CPV-1401, subcutaneously on the back, and carry out booster immunization when the HI titer drops below 1:80 (about 4 weeks) .

10 to 14 days after the completion of the second vaccination, the antibody titer was measured by the hemagglutination inhibition test (HI). Dogs with an antibody titer of not less than 1:2048 were selected for aseptic blood collection from the carotid artery, mixed and stored at low temperature.

1.2 Saturated ammonium sulfate (AS) step-by-step salting-out

(1) Thaw the serum prepared above, add an equal volume of phosphate buffer solution (PBS, pH = 7.0), slowly add saturated ammonium sulfate (AS, pH = 7.0) solution dropwise under magnetic stirring to a final concentration of 20%, Stir for 20 min, stand in the refrigerator at 4°C for 1 h, take it out and centrifuge at 6500 r/min for 30 min, and harvest the supernatant.

(2) Slowly add saturated AS solution to the supernatant harvested in step (1) under magnetic stirring to a final concentration of 50%, stir for 20 min, let stand at 4°C for 1 h, and centrifuge at 6500 r/min for 30 min after taking it out. Discard the supernatant and collect the precipitate.

(3) Dissolve the precipitate obtained in step (2) with twice the volume of the original serum in PBS, slowly add saturated AS solution dropwise to different final concentrations (25%-35%) under magnetic stirring, stir for 20 min, and refrigerate at 4°C for static Set aside for 1 h, take it out and centrifuge at 6500 r/min for 30 min, discard the supernatant, and collect the precipitate.

(4) Repeat step (3) twice.

1.3 HiTrap™ Desalting 5 mL prepacked column for desalting

(1) Connect the prepacked column to the protein purification system, and prevent air from entering the column during the process.

(2) Equilibrate the column: Equilibrate the column with 5 times the column volume of PBS to completely remove the ethanol that preserves the column, and at the same time make the column completely filled with PBS, and the flow rate is controlled at 5 mL/min.

(3) Sample loading and elution: The maximum sample volume is 1.5 mL, and the flow rate is 1 mL/min. After the sample loading is completed, continue to fill with PBS for elution.

(4) Collection: Use a 1.5 mL ep tube to collect the eluate at the peak with reference to the UV absorption value and resistivity, and store at -20°C for future use.

(5) Wash the column: wash with 5 times the column volume of PBS, and then wash with 5 times the column volume of 20% ethanol to preserve the column.

1.4 Concentrated immunoglobulin by ultrafiltration

(1) Connect the ultrafiltration membrane (Pellicon XL Cassette, Biomax 50kDa) to the small tangential flow ultrafiltration machine according to the instructions.

(2) Add 500 mL triple distilled water into the sample tank to clean the small tangential flow ultrafiltration system and circulate for 20 minutes.

(3) Drain the distilled water, add 500 mL of 0.1M NaOH solution to sterilize the small tangential flow ultrafiltration system, and circulate for 20 min.

(4) Drain the NaOH solution and repeat step (2).

(5) Drain the distilled water, add 200 mL of PBS to rinse for 10 min, and then drain.

(6) Sample loading: Add the sample to be concentrated (filtered by a filter membrane with a diameter of 0.45 μm), and adjust the pressure valve to prevent membrane damage caused by excessive pressure. The whole process is kept at low temperature to prevent virus inactivation.

(7) When the concentrated volume is reached, the sample is exported, subpackaged in a sterile centrifuge tube, and stored at -80°C for later use.

(8) Repeat steps (3)→(2)→(3). After the membrane is filled with fresh 0.1M NaOH solution, remove it and store it at 4°C.

1.5 Anti-canine parvovirus refined antibody protein concentration and purity detection

The protein concentration was detected by BCA method, and the protein purity was analyzed by SDS-PAGE protein electrophoresis.

2. Experimental results

Figure 1 shows the results of SDS-PAGE analysis of proteins extracted with different concentrations of AS. Figure 2 is the result of SDS-PAGE comparison between the purified anti-canine parvovirus antibody and the original serum.

The protein concentration of the purified anti-canine parvovirus antibody prepared by the present invention is 134 mg/mL, and the hemagglutination inhibition titer (HI) is 12800.

Example 2 Safety Detection of Anti-Canine Parvovirus Antibody Refined Antibody

1. Experimental method

5-month-old Beagle dogs were injected subcutaneously and intramuscularly at doses of 1.0, 2.0, and 4.0 mL per kilogram of body weight, respectively. They were observed continuously for 48 hours and kept for 3 months. dietary status.

2. Experimental results

Beagle dogs injected with different doses of purified anti-canine parvovirus antibodies were observed continuously for 48 hours. There was no abnormal phenomenon such as swelling and allergies at the injection site, and their body temperature and mental and dietary conditions were normal. Continue to feed for 3 months, during which there is no abnormality, indicating that the anti-anti-canine parvovirus refined antibody has no side effects and is suitable for clinical promotion.

Claims (10)

1. a kind of anti-dog parvovirus refines the preparation method of antibody, it is characterised in that comprise the following steps：

S1. canine parvovirus is prepared；

S2. vaccine immunity Healthy Dogs only prepare canine parvovirus hyper-immune serum；

S3. the anti-dog parvovirus immunoglobulin in the serum that prepared by saturated ammonium sulfate solution substep salting-out separation S2；

S4. purify：The anti-dog parvovirus immunoglobulin that sephadex is extracted to step S3 carries out desalination；

S5. by the anti-dog parvovirus immunoglobulin ultrafiltration concentration after step S4 desalting processings, filtration sterilization, packing, produce.

2. preparation method according to claim 1, it is characterised in that step S1 method is：Separated and reflected with In Guangdong Province Strain based on fixed canine parvovirus CPV-S5 and CPV-1401, expands culture, small-sized slipstream on F81 cells and surpasses respectively After filter concentration 9 are pressed with water-soluble adjuvant MONTANIDE GEL：1 ratio is mixed with inactivated vaccine.

3. preparation method according to claim 1, it is characterised in that step S2 method is：Choose 5 week old CPV HI Negative Healthy Dogs, while dorsal sc multiple spot is inoculated with canine parvovirus two kinds of inactivated vaccines of CPV-S5 and CPV-1401,25 According to same method booster immunization after~30d；7~14d after immune, arteria carotis aseptic collection CPV HI antibody titers are not less than 1： 3072 dog blood, stands, is centrifugally separating to obtain serum.

4. preparation method according to claim 1, it is characterised in that the step that step S3 saturated ammonium sulfates solution substep is saltoutd It is rapid as follows：

（a）Serum is diluted with phosphate buffer, precooling saturated ammonium sulfate solution is slowly added dropwise under magnetic agitation to final concentration of 15~25%, 10~30min is stirred, 0~10 DEG C stands after 0.5~2h, and 5000~8000r/min centrifuges 20~40min, abandons heavy Form sediment；

（b）To step（a）In obtained supernatant solution, precooling saturated ammonium sulfate solution is slowly added dropwise under magnetic agitation to final concentration 40~60%, 0~4 DEG C stands after 0.5~2h, and 5000~8000r/min centrifuges 2~40min, abandons supernatant, collects precipitation；

（c）To step（b）The PBS that former serum two volumes are added in gained precipitation dissolves, and is slowly added dropwise under magnetic stirring apparatus pre- Cold saturation AS solution is to final concentration 25%~35%, and 0~4 DEG C stands 0.5~2h, and 5000~8000r/min centrifuges 20~40 min, Supernatant is abandoned, precipitation is collected；

（d）Repeat step（c）Twice.

5. preparation method according to claim 1, it is characterised in that the method purified described in step S4 is use Sephadex G-25 pre-install gel column, the anti-dog parvovirus immune globulin extracted using sephadex to step S3 It is white to carry out desalting and purifying.

6. preparation method according to claim 1, it is characterised in that it is using small-sized tangential to be concentrated by ultrafiltration described in step S5 Stream is concentrated by ultrafiltration.

7. the anti-dog parvovirus prepared according to any methods described of claim 1~6 refines antibody.

8. anti-dog parvovirus refines antibody according to claim 7, it is characterised in that immune including anti-dog parvovirus Globulin；Wherein, content ＞ 130 mg/mL, CPV the HI antibody titers of the anti-dog parvovirus immunoglobulin are not less than 1：10240.

9. anti-dog parvovirus described in claim 7 refine antibody as or prepare the prevention of canine parvovirus disease and/or control Treat medicine in terms of application, as or prepare prevent and/or treatment canine parvovirus viral enteritis medicine or reagent The application of aspect.

10. anti-dog parvovirus described in claim 7 refine antibody as or prepare can increase ill dog immunity of organisms and Application in the medicine of resistance, the ill dog refers to the dog for having infected canine parvovirus.