AU2020103111A4

AU2020103111A4 - Preparation Method of Feline Feline panleukopenia virus Therapeutic Antibody

Info

Publication number: AU2020103111A4
Application number: AU2020103111A
Authority: AU
Inventors: Anbin Bai; Fenglian Chen; Jun Lin; Jinfeng Liu; Ling Ma; Shaomin Qin; Shuying Qin; Jianmin Wu; Lei Yang
Original assignee: Guangxi Veterinary Research Institute
Current assignee: Guangxi Veterinary Research Institute
Priority date: 2020-10-29
Filing date: 2020-10-29
Publication date: 2021-01-07
Anticipated expiration: 2028-10-29

Abstract

The invention discloses a feline feline panleukopenia virus therapeutic antibody, which is prepared by taking an FPV inactivated vaccine and an FPV subunit vaccine as immunogens to immunize horses. Based on this, a corresponding preparation method is established and comprises the following steps of: creatively inoculating healthy horses with two different types of FPV immunogens according to a certain process, taking the FPV inactivated vaccine as a basic immunogen, taking the FPV subunit vaccine as a booster immunogen, collecting hyperimmune blood after generating a high-titer antibody, carrying out purification by using IgG, cutting an Fc' fragment, and preparing high-activity immunoglobulin F (ab') 2. The method can be used for preparing a large amount of horse-derived anti-FPV hyperimmune serum, and extracting and purifying the high-titer F (ab') 2 antibody. As shown in experiments, the feline panleukopenia virus therapeutic antibody prepared by the method disclosed by the invention not only has high titer, but also has exact effects in preventing and treating feline panleukopenia virus. It can be applied to emergent prevention and treatment of feline panleukopenia virus and has important practical significance and wide application prospect.

Description

Preparation Method of Feline Feline panleukopenia virus Therapeutic Antibody

TECHNICAL FIELD

The invention belongs to the technical field of feline panleukopenia virus therapy, and particularly relates to a preparation method of a feline panleukopenia virus therapeutic antibody.

BACKGROUND

Feline panleukopenia virus, also known as feline panleukopenia, feline infectious enteritis, etc., is a lethal infectious disease caused by feline parvovirus (FPV) characterized by high fever, vomiting, rapid leukopenia and enteritis in felines. FPV can directly or indirectly infect a plurality of animals such as feline, procyonidae, mustelidae and the like under natural conditions, and is the most widely infecting and most pathogenic virus among the meat animal parvoviruses at present, wherein the feline and mink with small body sizes are most susceptible. FPV is frequently subject to endemia, the mortality rate of the feline infected with the FPV is generally 60%-70%, and the highest rate can reach more than 90%, brining extremely serious harm.

FPV is a parvovirus replication dependent on host cells. VP1 and VP2 are structural proteins that together form the major viral capsid proteins of FPV virus particles. However, among the two structural proteins, VP1 accounts for only 10%, while VP2 accounts for 90%, so VP2 is the main component of FPV capsid protein. As shown in the research, the VP2 protein not only has the coagulation activity, but also is a main protective antigen for FPV to induce organism to generate neutralizing antibodies, and the VP2 protein can be effectively expressed in mammalian cells and baculovirus expression systems to form virus-like particles by self-assembly, and FPV subunit vaccines prepared from the FPV virus-like particles have good immunogenicity.

Feline panleukopenia virus is the key point of feline disease prevention. At present, periodical immune injection is used at home and abroad to prevent feline panleukopenia virus. However, there is no effective and reliable drug therapy for the affected animals except for the injection of monoclonal antibodies and hyperimmune serum. Although the anti-virus hyperimmune serum is a relatively safe immune preparation, the preparation cost of the feline anti-FPV hyperimmune serum is high due to the limited blood content of a feline, and the direct use of the homologous hyperimmune serum can cause adverse reactions to animals due to low purity, and also can cause the diffusion and transmission of certain infectious diseases due to other pathogens in the serum; therefore, the development of the high-purity heterologous anti-FPV antibody is the best choice for treating the feline panleukopenia virus. At present, there are few domestic studies focusing on the treatment of heterologous feline panleukopenia virus therapeutic antibodies.

SUMMARY

The technical problem to be solved by the invention is to provide a preparation method of a feline panleukopenia virus therapeutic antibody so as to relieve the status quo that the feline panleukopenia virus therapeutic antibody is insufficient and the prevention and control effect is not good.

In order to solve the technical problems, the invention adopts the following technical scheme:

A feline panleukopenia virus therapeutic antibody is prepared by immunizing horses with a FPV inactivated vaccine and a FPV subunit vaccine as immunogens.

The feline panleukopenia virus therapeutic antibody is prepared by sequentially immunizing horses with the FPV inactivated vaccine as a basic immunogen and the FPV subunit vaccine as a booster immunogen.

The FPV inactivated vaccine is prepared based on a FPV-GXO1 strain (FPV clinical isolate GXO1), and the FPV subunit vaccine is prepared on the basis of FPV virus-like particles formed by a FPV-VP2 gene in a baculovirus/insect cell expression system by connecting a signal peptide HBM with an N-terminal end and subjected to insect cell codon preference optimization.

A preparation method of the feline panleukopenia virus therapeutic antibody comprises the steps of:

(1) Respectively utilizing the FPV inactivated vaccine and the FPV subunit vaccine to immunize horses;

(2) When the inhibition titer of FPV hemagglutination in horse serum is more than or equal to 1: 32, collecting hyperimmune blood; hemagglutination;

(3) Separating serum from hyperimmune blood, extracting IgG by using a n-caprylic acid-ammonium sulfate precipitation optimization method, and preparing immunoglobulin F (ab') 2.

The FPV inactivated vaccine is prepared by fully emulsifying a FPV-GXO1 strain and a white oil adjuvant according to the volume ratio of 1:2 (V/V).

The FPV subunit vaccine is prepared from FPV-VLPs, wherein the FPV-VLPs are encoded by an FPV VP2 gene which is subjected to insect cell codon preference optimization and fused with a signal peptide HBM, and are formed through expression in a baculovirus/insect cell expression system.

The FPV subunit vaccine is produced by the following operations of:

(1) Carrying out codon optimization on the FPV VP2 gene according to the codon preference of insect cells, and introducing an HBM signal peptide sequence into the N end of the FPV VP2 gene to obtain a codon optimized FPV VP2 gene;

(2) Connecting the optimized FPV VP2 gene into a transfer vector pFastBacl through directional cloning to construct a recombinant baculovirus transfer vector pFast VP2;

(3) Transforming the transfer vector in the step (2) into DH1OBac competent cells to prepare recombinant baculovirus DNA containing the optimized FPV VP2 gene;

(4) Transfecting the recombinant baculovirus DNA into insect sf9 cells to prepare recombinant baculovirus expressing VP2 protein;

(5) Infecting insect sf9 cells with recombinant baculovirus to express VP2 protein to obtain FPV-VLPs; (FPV virus-like particles);

(6) Collecting the supernatant of the cell culture solution containing FPV-VLPs, preliminarily purifying and concentrating the supernatant, and fully emulsifying the supernatant and ISA206 adjuvant according to the volume ratio of 1:1 (V/V) to prepare the FPV subunit vaccine.

The optimized FPVVP2 gene has a base sequence in the sequence table SEQ.ID.No.1, and the VP2 protein has an amino acid sequence in the sequence table SEQ.ID.No.2.

The step (1) comprises the following steps: injecting 5 mL of the FPV inactivated vaccine into the neck of each horse for the first basic immunization, and injecting 5 mL of the FPV inactivated vaccine into the neck of each horse in the same manner for the second basic immunization at intervals of 14 days; injecting 6 mL of the FPV subunit vaccine into each horse for the first boosting immunization 21 days after the completion of the basic immunization, then performing the next boosting immunization at intervals of 4 days for the next boosting immunization, wherein the boosting immunization is carried out for 4 times, the immunization dose of the FPV VLPs vaccine is increased by 1.5 mL each time; and carrying out the 5th boosting immunization if the FPV hemagglutination inhibition titer is lower than 1:32.

The step (3) is carried out as follows:

(1) Separating hyperimmune serum, diluting the serum by 2-5 times by a 0.06M acetate buffer solution with a pH value of 4.5, adjusting the pH value of the solution to 4.0-4.5, adding 25 pL of n-caprylic acid into each milliliter of diluent, slowly adding the n-caprylic acid under the condition of magnetic stirring, stirring for 30 min at a room temperature, centrifuging for 20-30 min at 4 °C and 10000 rpm, and collecting supernatant;

(2) Taking supernatant, adding PBS with the equal volume, adjusting the pH to 7.2 7.5, slowly adding ammonium sulfate powder according to the proportion of 0.227g ammonium sulfate per milliliter of solution under the condition of magnetic stirring, stirring at room temperature for 30 min, standing overnight at 4 °C, centrifuging at 4 DEG C and 5000 rpm for 15-30 min, and collecting precipitate;

(3) Dissolving the precipitate with PBS to restore the original serum volume, slowly adding saturated ammonium sulfate solution to ensure that the ammonium sulfate saturation in the solution is 33.3%, standing at the room temperature for 30-60 min, centrifuging at 5000 rpm for 15-30 min, collecting the precipitate, dissolving the precipitate with PBS, dialyzing overnight, and centrifuging to remove the precipitate to obtain refined IgG;

(4) Measuring the content of IgG, adjusting the pH of IgG solution to 3.2-3.5, adding pepsin of 10-20 mu g for per milligram of IgG; digesting at 30-37 °C for 1-3 h, collecting an enzyme digestion solution for Protein A+G column purification, collecting an effluent, and filtering to remove bacteria to obtain immunoglobulin F(ab')2.

Aiming at the status quo that the heterologous feline panleukopenia virus therapeutic antibody is insufficient, the inventor designs and prepares the feline panleukopenia virus therapeutic antibody by taking the FPV inactivated vaccine and the FPV subunit vaccine as immunogens to immunize horses. Based on this, a corresponding preparation method is established and comprises the following steps of: creatively inoculating healthy horses with two different types of FPV immunogens according to a certain process, taking the FPV inactivated vaccine as a basic immunogen, taking the FPV subunit vaccine as a booster immunogen, collecting hyperimmune blood after generating a high-potency antibody, carrying out purification by using IgG, cutting an Fe' fragment, and preparing high-activity immunoglobulin F (ab') 2, namely the horse anti-FPV specific immunoglobulin F (ab') 2. The FPV inactivated vaccine is prepared based on a FPV GXO1 strain (FPV clinical isolate GXO1), and the FPV subunit vaccine is prepared on the basis of FPV virus-like particles formed by a FPV-VP2 gene in a baculovirus/insect cell expression system by connecting a signal peptide HBM with an N-terminal end and subjected to insect cell codon preference optimization. The method can be used for preparing a large amount of horse-derived anti-FPV hyperimmune serum, and extracting and purifying the high-potency F (ab') 2 antibody. As shown in experiments, the feline panleukopenia virus therapeutic antibody prepared by the method disclosed by the invention not only has high potency, but also has exact effects in preventing and treating feline panleukopenia virus. It can be applied to emergent prevention and treatment of feline panleukopenia virus and has important practical significance and wide application prospect.

DESCRIPTION OF THE INVENTION

I. Preparation of FPV Inactivated Vaccine

The step comprises: synchronously inoculating an FPV-GXO strain into FK81 cells of cat kidney passage cells according to a volume of 1/10, standing in a C02 incubator at 37 °C for 24 hours, discarding a growth solution, replacing the growth solution by a maintenance solution for continuous culture, observing cytopathic effects day by day, collecting the cells when 75% of the cells have the cytopathic effects, repeatedly freezing and thawing the cells for 3 times, centrifuging at 4 °C and 12,000 rpm for 15 minutes, removing cell fragments, collecting a supernate, measuring a TCID5/0.1 mL value till it is more than or equal to 5.0 and measuring a hemagglutination titer till it is more than or equal to 1:210, adding formaldehyde till the final concentration reaches 0.1% after adding thymosin till the final concentration reaches 0.3 mg/mL, inactivating at 37 °C for 24h, shaking once at intervals of 4h, and then adding 4% Tween-80 as the water phase, mixing Span-80 and No. 10 white oil at a volume ratio (V/V) of 3:47 as the oil phase, and mixing the water phase and the oil phase at a volume ratio of 1:2 for oil-coating-water stirring and emulsification to prepare an FPV oil-emulsion inactivated vaccine.

II. Preparation of FPV Subunit Vaccine

The FPV VP2 gene was subjected to insect cell codon preference optimization, and the HBM signal peptide sequence was introduced into its N-terminal and then artificially synthesized to obtain the codon-optimized FPV VP2 gene (SEQ.ID.No.1); it was cloned into a baculovirus transfer carrier pFastBacl to construct recombinant plasmidpFastBac VP2. The recombinant plasmid was transformed into competent cells of escherichia coli DH10Bac, diluted by 100 folds, and then spread on a SOB plate coated with tetracycline (10 pg/mL), kanamycin (50 tg/mL), gentamicin (7 tg/mL), beta-galactoside (100 ptg/mL) and IPTG (40 tg/mL) in SOB plates. After 72 hours of culture, white colonies were picked and re-inoculated in a new SOB plate. Incubation was conducted at 37 DEG C overnight. White mono-clones were picked to an LB culture solution containing (50 pg /mL) kanamycin, (7 tg /mL) gentamicin, and (10 pg /mL) tetracycline in LB medium, and cultured at 37 °C for 8-12 h. Then, recombinant bacmid Bac-VP2 was extracted. A M13 primer was used for identification, and thus a recombinant bacmid containing the target gene was obtained. With Lipofectamine* 3000, the recombinant bacmid identified to be correct was transfected into Sf9 cells at 27 °C for 4-5 days and the recombinant baculovirus was collected. The recombinant baculovirus was used to infect the Sf9 cells according to an inoculum size of 10%, and was fixed with 80% pre-chilled acetone after the cells were subjected to cytopathic effects. The recombinant baculovirus expression product was subjected to IFA identification using FITC fluorescently labelled goat anti mouse IgG as the primary antibody; the recombinant baculovirus cultures were collected for electron microscopy observation; after IFA and electron microscopy observations, by which the expression of VP2 protein (SEQ.ID.No.2) was confirmed and after formation of virus-like particles, mass culture was conducted for collection of cells and their culture supernatants, repeated freezing and thawing was conducted for 3 times at -20-20 °C, centrifugation was conducted at 4 °C and 1200rpm for 15min, the supernatant was collected, the protein was precipitated with saturated (NH4)2SO 4, and the baculovirus was inactivated with Triton X-100 and tributyl phosphate (TBP). The dialysis bag is used for preliminary purification of the protein. The hemagglutination titer was measured and adjusted to 1:29, and finally the ISA 206 adjuvant was added for complete emulsification according to the antigen/adjuvant volume ratio of 1:1 prepare a recombinant FPV subunit vaccine.

III. Preparation of F (ab')2

(1) Immunization inoculation procedures are divided into two stages: basal immunization and booster immunization.

The method comprises the following steps of: injecting 5 mL of the FPV inactivated vaccine into the neck of each horse for the first basic immunization, and injecting 5 mL of the FPV inactivated vaccine into the neck of each horse in the same manner for the second basic immunization at intervals of 14 days; injecting 6 mL of the FPV subunit vaccine into each horse for the first boosting immunization 21 days after the completion of the basic immunization, then performing the next boosting immunization at intervals of 4 days for the next boosting immunization, wherein the boosting immunization is carried out for 4 times, the immunization dose of the FPV VLPs vaccine is increased by 1.5 mL each time; and carrying out the 5' boosting immunization if the FPV hemagglutination inhibition titer is lower than 1:32.

(2) When FPV hemagglutination inhibition titer in the horse serum is more than or equal to 1:32: collecting horse hyperimmune blood, standing at a room temperature for 1 2 h, centrifuging at 4 °C and 10000 rpmm for 5 min, separating serum, diluting the serum by 4 times with a 0.06M acetate buffer solution with the pH value of 4.0, adjusting the pH value of the solution to 4.5, adding 25 pL of n-octanoic acid into each ml of the diluent, slowly adding n-octanoic acid under the condition of magnetic stirring, stirring at the room temperature for 30 min, centrifuging at 4 °C and 10000 rpm for 30 minutes, and collecting supernatant;

(3) Taking supernatant, adding PBS with the equal volume, adjusting the pH to 7.5, slowly adding ammonium sulfate powder according to the proportion of 0.227g ammonium sulfate per milliliter of solution under the condition of magnetic stirring, stirring at room temperature for 30 min, standing overnight at 4 °C, centrifuging at 4 °C and 5000 rpm for 20 min, and collecting precipitate;

(4) Dissolving the precipitate with PBS to restore the original serum volume, slowly adding saturated ammonium sulfate solution to ensure that the ammonium sulfate saturation in the solution is 33.3%, standing at the room temperature for 40 min, centrifuging at 5000 rpm for 20 min, and collecting the precipitate;

(5) Dissolving the precipitate with PBS, loading into a dialysis bag, dialyzing in PBS for 48 h, replacing PBS for 3 times, centrifuging at 5000 rpm for 20 min to remove the precipitate and obtain refined IgG;

(6) measuring the content of IgG, adjusting the pH of IgG solution to 3.5, adding pepsin of 20 mu g for per milligram of IgG; digesting at 37 °C for 2 h, collecting an enzyme digestion solution for Protein A+G column purification, collecting an effluent, and filtering to remove bacteria to obtain immunoglobulin F(ab')2.

IV. Measurement of Titer of F(ab')2 Antibody

When the concentration of F(ab')2 was adjusted to 10 mg/mL, the hemagglutination inhibition test was carried out. The F (ab')2 prepared can effectively inhibit FPV virus solution, and the hemagglutination inhibition titer was as high as 1:256.

V. Clinical Therapeutic Effect of V, F (ab') 2 Antibody

12 cats infected with FPV at the age of 4 months were randomly divided into 3 groups, 4 in each group. The F(ab')2 antibody prepared according to the present invention was used in the first group, the feline panleukopenia virus monoclonal antibody injection sold in the market was used in the second group, and normal saline was used in the third group. In the first group, 4 mL of F(ab')2 antibody was injected intramuscularly each time for 3 days; in the second group, 2 mL of commercial monoclonal antibody was injected for 3 days; in the third group, 4 mL of normal saline was injected by the same method; in addition, each group of cats were given adjuvant therapies such as anti inflammation, anti-emetic, and hemostasis at the same time of antibody injection. The treatment was observed and recorded.

As shown in results (Table 1), those silk cats were generally subject to mental depression, somnolence, emesis, diarrhea and the like before treatment, and the mental states of the cats were significantly improved and the clinical symptoms were significantly alleviated after the treatment. Clinical symptoms of 75% of the cats disappeared after 7 days of treatment, while the cat injected with normal saline died 7 days after the attack. The results show that the prepared F(ab')2 antibody is similar in effects to the monoclonal antibody in the treatment of feline panleukopenia virus, and has a good distemper treatment effect.

Table 1 Clinical therapeutic effects of F(ab')2

Group Experimental Before Symptoms Cured Cure Cure Animals (1) and after number rate time treatment

Mental Body Emesis Diarrhea (1) (%) (d) state temperature °C symptom symptom

1 4 Before Mental 40.20 Intermittent Blood-like 3 75% 6 treatment depression vomiting faeces

After Good 38.20 Asymptomatic Asymptomatic treatment mental state

2 4 Before Low in 40.10 Intermittent Blood-like 3 75% 7 treatment spirit vomiting faeces

After Good 38.60 Asymptomatic Asymptomatic treatment mental state

3 4 Before Dispirited 40.15 Intermittent Blood-like 0 0% treatment vomiting faeces

After Death treatment

VI. Experiment on Clinical Preventive Effect of F(ab')2 Antibody

Twelve healthy 4-month-old cats were divided into groups according to the above mentioned grouping mode for prevention experiments. The experimental group and the control group were injected intramuscularly with 3 mL of F(ab')2 antibody, 2 mL of monoclonal antibody and 3 mL of normal saline for 3 days in succession through the same manner. On the 5th day, all the tested cats were injected with 1 mL of FPV cell culture solution for artificial challenging, and their incidence situations were observed. Detection of FPV in faeces, increase of body temperature, diarrhea and vomiting were used as criteria for the diagnosis of the disease attack.

As shown in the results, the incidence rate of 4 cats which suffered the attack in the normal saline injection group was 100%. The incidence rates in the injection groups of F(ab')2 antibody and monoclonal antibody were 50%, which was significantly lower than that in normal saline control group. The result shows that the prepared F(ab')2 antibody has good effect on preventing feline panleukopenia virus compared with the monoclonal antibody.

Table 2 Clinical Preventive Effects of F(ab')2

Group Experimental Prophylactic Incidence Incidence Protection Animals (1) method number (1) (%) Rate (%)

1 4 Immunoglobulin 2 50% 50%

F(ab')2

2 4 Monoclonal 2 50% 50% antibodies

3 4 Normal saline 4 100% 0%

Claims

1. A feline panleukopenia virus therapeutic antibody, wherein the antibody is prepared by immunizing horses with a FPV inactivated vaccine and a FPV subunit vaccine as immunogens.

2. The feline panleukopenia virus therapeutic antibody according to claim 1, wherein the feline panleukopenia virus therapeutic antibody is prepared by sequentially immunizing horses with the FPV inactivated vaccine as a basic immunogen and the FPV subunit vaccine as a booster immunogen.

3. The feline panleukopenia virus therapeutic antibody according to claim 2, wherein the FPV inactivated vaccine is prepared based on a FPV-GXO1 strain; and the FPV subunit vaccine is prepared on the basis of FPV virus-like particles formed by a FPV VP2 gene in a baculovirus/insect cell expression system by connecting a signal peptide HBM with an N-terminal end and subjected to insect cell codon preference optimization.

4. A preparation method of the feline panleukopenia virus therapeutic antibody according to claim 3, comprising the steps of:

(2) When the inhibition titer of FPV hemagglutination in horse serum is more than or equal to 1: 32, collecting hyperimmune blood;

(3) Separating serum from hyperimmune blood, extracting IgG by using an n caprylic acid-ammonium sulfate precipitation optimization method, and preparing immunoglobulin F(ab')2.

5. The preparation method of the feline panleukopenia virus therapeutic antibody according to claim 4, wherein the FPV inactivated vaccine is prepared by fully emulsifying a FPV-GXO1 strain and a white oil adjuvant.

6. The preparation method of the feline panleukopenia virus therapeutic antibody according to claim 4, wherein the FPV subunit vaccine is prepared from FPV-VLPs, wherein the FPV-VLPs are encoded by an FPV VP2 gene which is subjected to insect cell codon preference optimization and fused with a signal peptide HBM, and are formed through expression in a baculovirus/insect cell expression system.

7. The preparation method of the feline panleukopenia virus therapeutic antibody according to claim 6, wherein the FPV subunit vaccine is produced by the following operations of:

(2) Connecting the optimized FPV VP2 gene into a transfer vector pFastBacI through directional cloning to construct a recombinant baculovirus transfer vector pFast VP2;

(5) Infecting insect sf9 cells with recombinant baculovirus to express VP2 protein to obtain FPV-VLPs; and

(6) Collecting the supernatant of the cell culture solution containing FPV-VLPs, preliminarily purifying and concentrating the supernatant, and fully emulsifying the supernatant and ISA206 adjuvant according to the volume ratio of 1:1 to prepare the FPV subunit vaccine.

8. The preparation method of the feline panleukopenia virus therapeutic antibody according to claim 7, wherein the optimized FPVVP2 gene has a base sequence in the sequence table SEQ.ID.No.1, and the VP2 protein has an amino acid sequence in the sequence table SEQ.ID.No.2.

9. The preparation method of the feline panleukopenia virus therapeutic antibody according to claim 4, wherein the step (1) comprises the following steps: injecting 5 mL of the FPV inactivated vaccine into the neck of each horse for the first basic immunization, and injecting 5 mL of the FPV inactivated vaccine into the neck of each horse in the same manner for the second basic immunization at intervals of 14 days; injecting 6 mL of the FPV subunit vaccine into each horse for the first boosting immunization 21 days after the completion of the basic immunization, then performing the next boosting immunization at intervals of 4 days for the next boosting immunization, wherein the boosting immunization is carried out for 4 times, the immunization dose of the FPV VLPs vaccine is increased by 1.5 mL each time; and carrying out the 5th

boosting immunization if the FPV hemagglutination inhibition titer is lower than 1:32.

10. The preparation method of the feline panleukopenia virus therapeutic antibody according to claim 4, wherein the step (3) is carried out as follows:

(1) Separating hyperimmune serum, diluting the serum by 2-5 times by a 0.06M acetate buffer solution with a pH value of 4.5, adjusting the pH value of the solution to 4.0-4.5, adding 25 pL of n-caprylic acid into each milliliter of diluent, slowly adding the n-caprylic acid under the condition of magnetic stirring, stirring for 30min at a room temperature, centrifuging for 20-30 min at 4 °C and 10000 rpm, and collecting supernatant;

(2) Taking supernatant, adding PBS with the equal volume, adjusting the pH to 7.2 7.5, slowly adding ammonium sulfate powder according to the proportion of 0.227g ammonium sulfate per milliliter of solution under the condition of magnetic stirring, stirring at room temperature for 30 min, standing overnight at 4 °C, centrifuging at 4 °C and 5000 rpm for 15-30 min, and collecting precipitate;

(4) Measuring the content of IgG, adjusting the pH of IgG solution to 3.2-3.5, adding pepsin of 10-20 pg for per milligram of IgG; digesting at 30-37 °C for 1-3 h, collecting an enzyme digestion solution for Protein A+G column purification, collecting an effluent, and filtering to remove bacteria to obtain immunoglobulin F(ab')2.