Cat allergen Fel d1 yolk antibody and its preparation method and application

Abstract

This application discloses a feline allergen Fel d1 egg yolk antibody and its preparation method and application, comprising: obtaining PCR amplification products through PCR amplification; constructing a recombinant plasmid by ligating the PCR amplification products with pET-28a(+); transforming the recombinant plasmid into Rosetta competent cells to obtain transformants; obtaining recombinant antigens by extracting proteins from the transformants; obtaining a subunit vaccine by mixing and emulsifying the recombinant antigens with white oil adjuvant; administering the subunit vaccine to laying hens to obtain immunized egg yolk liquid; adding Tris-HCl buffer and polyethylene glycol to the egg yolk liquid and centrifuging to collect the protein aqueous phase; filtering the protein aqueous phase to obtain a filtrate, adding polyethylene glycol, mixing, centrifuging to collect the precipitate; mixing the precipitate with PBS buffer, adding ammonium sulfate, and centrifuging to obtain the egg yolk antibody. This invention solves the problem that the safety and economy of existing feline allergy treatment methods cannot be simultaneously achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of cat allergen antibodies, and in particular to a cat allergen Fel d1 egg yolk antibody and a preparation method and application thereof. Background Art

[0002] Cat allergy is one of the most common allergic diseases worldwide, affecting approximately 10% to 20% of the population. The main allergen of cat allergy is Fel d1, a protein secreted by the salivary glands and sebaceous glands of cats and transmitted through the cat's fur. Fel d1 has strong stability and adhesion, can exist in the environment for a long time, and can cause allergic reactions even in an environment without cats.

[0003] Currently, treatments for cat allergies mainly include avoidance therapy, drug therapy, immunotherapy and other therapies.

[0004] Avoidance therapy: Avoid contact with cats and their hair, dander, etc. But this is not realistic for many cat lovers, and even if you don't have a cat, Fel d1 is widely present in the environment and is difficult to avoid completely.

[0005] Drug therapy: Use antihistamines, corticosteroids, etc. to relieve allergic symptoms. This method can only temporarily control symptoms, cannot cure allergies, and long-term use may cause side effects.

[0006] Immunotherapy: Through repeated injections of gradually increasing doses of allergen extracts, the body gradually tolerates the allergens. This method has good efficacy, but the treatment course is long, the cost is high, and there are certain risks.

[0007] Other treatments: such as using air purifiers, inhaled corticosteroids, etc., can reduce allergens in the environment, but the effects are limited.

[0008] In recent years, some progress has been made in the research on Fel d1, such as the development of recombinant Fel d1 protein for diagnosis and immunotherapy. However, there is still a lack of a safe, effective, convenient and economical method for preventing and treating cat allergies. Summary of the invention

[0009] In order to overcome the defects of the prior art, a cat allergen Fel d1 yolk antibody and a preparation method and application thereof are provided to solve the problem that the safety and economy of the existing cat allergy treatment methods cannot be taken into account at the same time.

[0010] To achieve the above object, a method for preparing cat allergen Fel d1 yolk antibody is provided, comprising the following steps:

[0011] A PCR amplification product is obtained by PCR amplification using the nucleotide sequence shown in SEQ ID NO.1 as an upstream primer and the nucleotide sequence shown in SEQ ID NO.2 as a downstream primer;

[0012] Connecting the PCR amplification product with pET-28a(+) to construct a recombinant plasmid;

[0013] Transforming the recombinant plasmid into Rosetta competent cells to obtain transformants;

[0014] A recombinant antigen is obtained by extracting the protein from the transformant, wherein the amino acid sequence of the recombinant antigen is shown in SEQ ID NO.3;

[0015] The recombinant antigen is mixed and emulsified with a white oil adjuvant to obtain a subunit vaccine;

[0016] administering the subunit vaccine to laying hens to obtain immune eggs;

[0017] Obtaining egg yolk liquid from the immune eggs;

[0018] Adding Tris-HCL buffer and polyethylene glycol to the egg yolk solution and centrifuging to collect the protein aqueous phase;

[0019] Filtering the protein aqueous phase to obtain a filtrate, adding polyethylene glycol, mixing, centrifuging, and collecting a precipitate;

[0020] The precipitate was mixed with PBS buffer, and then ammonium sulfate was added and centrifuged to prepare cat allergen Fel d 1 egg yolk antibody.

[0021] Furthermore, the recombinant antigen is purified Fel d1 protein with a concentration of 1 mg / mL.

[0022] Furthermore, the step of obtaining the recombinant antigen by extracting the protein in the transformant comprises:

[0023] Picking a monoclonal colony of the transformant and inoculating it into LB culture medium containing kanamycin resistance and culturing it until the OD600 value of the bacterial solution reaches 0.6-0.8;

[0024] Adding IPTG inducer to the bacterial solution to induce expression;

[0025] Collect the bacterial cells from the bacterial solution after induced expression;

[0026] The bacterial cells are added to PBS buffer, and the recombinant antigen is obtained through multiple resuspension, ultrasonic disruption, centrifugation and purification processes.

[0027] Furthermore, the step of emulsifying the recombinant antigen and the white oil adjuvant to obtain the subunit vaccine comprises:

[0028] mixing the recombinant antigen with Tween to obtain an aqueous phase;

[0029] The oil phase is obtained by mixing white oil, sphenanthene and aluminum stearate;

[0030] Mixing the oil phase and the water phase and emulsifying to obtain an emulsion;

[0031] The emulsion is centrifuged to obtain the subunit vaccine.

[0032] Furthermore, the step of inoculating the subunit vaccine into laying hens to obtain immune eggs includes inoculating the subunit vaccine into laying hens through three immunizations, so that the Feld1 yolk antibody titer in the yolk liquid of the immune eggs is higher than 1:64.

[0033] The invention provides a cat allergen Fel d1 yolk antibody. The cat allergen Fel d1 yolk antibody is prepared by adopting a cat allergen Fel d1 yolk antibody preparation method.

[0034] The invention provides an application of cat allergen Fel d1 egg yolk antibody. The cat allergen Fel d1 egg yolk antibody is mixed with a lyophilizer and freeze-dried to obtain a lyophilized powder.

[0035] Furthermore, the lyophilized agent includes a protective agent, a filler, an antioxidant and a buffer;

[0036] The protective agent includes one or more combinations of trehalose, sucrose, β-dextrin, dextran or polyvinyl pyrrolidone;

[0037] The filler includes one or more combinations of glycine, sodium glutamate, sorbitol, mannitol or gelatin;

[0038] The antioxidant includes one or more combinations of vitamin E, vitamin C or protein hydrolysate.

[0039] Furthermore, the mass ratio of the protective agent, the filler and the antioxidant in the lyophilized agent is 40-80:6-10:1-2.

[0040] The beneficial effects of the present invention are that the method for preparing the cat allergen Fel d1 egg yolk antibody of the present invention controls the coordination of each step by designing a new method for preparing the Fel d1 egg yolk antibody, including adopting a specific immunization program and an extraction method, the operation is simple, the IgY titer in the yolk of the immunized laying hen can be maintained at a high level, the antibody titer may be maintained for a longer time, the purity, clarity and fat removal rate of the obtained egg yolk antibody are significantly improved, the titer is high, the specificity is high, the pertinence is stronger, the protection rate is high, the prevention and treatment costs are low, the method can be used to prevent cat allergies, and the method has a more practical application prospect.

[0041] The present invention designs and implements a complete and innovative method for preparing Fel d1 egg yolk antibodies, which not only includes the preparation of recombinant antigens, but also optimizes and innovates the construction of subunit vaccines, immunization procedures and antibody purification, and finally obtains high-titer and high-specificity Fel d1 egg yolk antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Other features, objects and advantages of the present application will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

[0043] Figure 1 This is an SDS-PAGE identification diagram of the cat allergen FeI d 1 protein according to an embodiment of the present invention.

[0044] Figure 2 This is a graph showing the titer test results of the cat allergen Fel d 1 yolk antibody according to an embodiment of the present invention.

[0045] Figure 3 This is a graph showing the effect of temperature on cat allergen Fel d 1 yolk antibodies according to an embodiment of the present invention.

[0046] Figure 4 This is a graph showing the effect of pH on cat allergen Fel d 1 yolk antibodies according to an embodiment of the present invention. DETAILED DESCRIPTION

[0047] The present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the relevant invention, rather than to limit the invention. It is also necessary to explain that, for ease of description, only the parts related to the invention are shown in the accompanying drawings.

[0048] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0049] The present invention provides a method for preparing cat allergen Fel d1 yolk antibody, comprising the following steps:

[0050] S1. Using the nucleotide sequence shown in SEQ ID NO.1 as an upstream primer and the nucleotide sequence shown in SEQ ID NO.2 as a downstream primer, PCR amplification is performed to obtain a PCR amplification product.

[0051] In this embodiment, during PCR amplification, a pair of PCR primers was designed using the molecular biology primer design software SnapGene 7.1.2, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, specifically:

[0052] AGTGGTGGTGGTGGTGGTGCTCGAGACACAGCGGGCTGGTGT A.

[0053] The nucleotide sequence of the downstream primer is shown in SEQ ID NO.2, specifically:

[0054] GGACAGCAAATGGGTCGCGGATCCATGGTAAAAATGGCTGAA.

[0055] The PCR reaction system (25 μL) is:

[0056] Template DNA: 1 μL (concentration 50 ng / μL);

[0057] Upstream primer: 1 μL (10 μM, final concentration 0.4 μM);

[0058] Downstream primer: 1 μL (10 μM, final concentration 0.4 μM);

[0059] dNTP mixture: 0.5 μL (10 mM, 2.5 mM of each base);

[0060] Taq enzyme or high-fidelity enzyme: 0.5 μL (usually 2.5 U / μL, final concentration about 0.05 U / μL);

[0061] 10×Buffer (with Mg 2+ ): 2.5 μL;

[0062] ddH2O: 18.5μL.

[0063] PCR reaction conditions:

[0064] 1. Pre-denaturation: 95℃, 3min.

[0065] 2. Loop 30 times:

[0066] Denaturation: 95°C, 20 s;

[0067] Annealing: 58°C, 20 s;

[0068] Extension: 72°C, 30 s.

[0069] 3. Final extension: 72°C, 5 min.

[0070] S2. Connect the PCR amplification product with pET-28a(+) to construct a recombinant plasmid.

[0071] S3. Transform the recombinant plasmid into Rosetta competent cells to obtain transformants.

[0072] S4. Obtain a recombinant antigen by extracting the protein from the transformant. The amino acid sequence of the recombinant antigen is shown in SEQ ID NO.3.

[0073] Specifically, step S4, the step of obtaining the recombinant antigen by extracting the protein in the transformant, comprises:

[0074] S41. Pick a monoclonal colony of the transformant and inoculate it into LB culture medium containing kanamycin resistance and culture it until the OD600 value of the bacterial solution reaches 0.6-0.8.

[0075] S42. Add IPTG inducer to the bacterial solution to induce expression.

[0076] S43, collecting the bacterial cells from the bacterial solution after induced expression.

[0077] S44. Add the bacteria into PBS buffer (phosphate buffered saline), and obtain the recombinant antigen through multiple resuspension, ultrasonic disruption, centrifugation and purification.

[0078] In this example, the amino acid sequence of the recombinant antigen is shown in SEQ ID NO.3, specifically:

[0079] MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSMVKMAETCPIFYDVFFAVANGNELLLDLSLTKVNATEPERTAMKKIQDCYVENGLISRVLDGLVMTTISSSKDCMGEAVQNTVEDLKLNTLGREICPAVKRDVDLFLTGTPDEYVEQVAQYKALPVVLENARILKNCVDAKMTEEDKENALSLLDKIYTSPLCLEHHHHHH.

[0080] As a preferred embodiment, the recombinant antigen is purified Fel d 1 protein with a concentration of 1 mg / mL.

[0081] In this example, the monoclonal colony was inoculated into LB (Luria-Bertani) culture medium (LB liquid culture medium is the preparation (1 L) of LB liquid culture medium) pre-prepared with kanamycin resistance for culture. According to the culture conditions, the monoclonal colony was inoculated into LB culture medium for culture, and when the OD600 value of the bacterial solution reached 0.6 to 0.8, IPTG inducer was added to induce expression.

[0082] When the bacterial solution reaches the preset concentration after induction expression, stop culturing. Transfer the optimized synthetic plasmid into Rosetta competent cells, pick monoclonal colonies, inoculate them into LB culture medium containing kanamycin resistance for overnight culture, and transfer them into new LB culture medium containing kanamycin resistance at a ratio of 1:100 the next day, and culture them at 37°C and 220r / min on a shaking table for 3-4h. When the OD600 value of the bacterial solution reaches 0.6-0.8, take 1ml of the bacterial solution as a control, and add IPTG inducer to the remaining bacterial solution, with a final working concentration of 0.6mmol / L.

[0083] The cultured bacteria are collected and purified and pre-treated to obtain the allergen Fel d1 sample containing the target protein, specifically including:

[0084] The bacterial solution after induced expression is centrifuged to obtain the bacterial cells and the supernatant is discarded;

[0085] Add buffer to the discarded supernatant, and use a pipette to blow the bacteria several times to resuspend them. Ultrasonicate the reselected bacterial solution until the bacterial solution becomes clear and transparent.

[0086] After the ultrasound, the bacterial solution was centrifuged at 12000 rpm for 10 min, and the supernatant was collected to obtain the allergen Fel d1 sample containing the target protein.

[0087] The specific treatment of multiple resuspension, ultrasonic disruption and centrifugation of the bacteria includes:

[0088] After culturing in a shaking incubator at 25°C and 200 r / min for 15 h, the cells were collected by centrifugation at 8000 rpm for 5 min. The supernatant was discarded and the bacterial precipitate was added to the buffer solution. The cells were resuspended by blowing up and down with a pipette several times. The resuspended bacterial solution was ultrasonically disrupted and kept on ice throughout the operation. Ultrasonication was performed for 5 s, paused for 3 s, and the power was 50 W. The bacterial solution was determined to be clear and transparent. After the ultrasonication, the bacterial solution was centrifuged at 12000 rpm for 10 min, and the supernatant and precipitate were collected to prepare the sample.

[0089] The expression level of protein in cat allergen Fel d1 samples was analyzed by SDS-PAGE electrophoresis. Figure 1 shown.

[0090] The cat allergen Fel d1 sample is expanded and cultured, induced to express, and a certain amount of purified antigen is obtained for vaccine preparation.

[0091] The purified Fel d1 protein obtained after purification. The use of purified Fel d1 protein can further accelerate the cycle of obtaining high-immunity eggs, the IgY titer in the yolk of immunized laying hens can be maintained at a high level, and the antibody titer can be maintained for a longer time.

[0092] S5. The recombinant antigen is mixed and emulsified with white oil adjuvant to obtain a subunit vaccine.

[0093] Specifically, step S5, the step of emulsifying the recombinant antigen and the white oil adjuvant to obtain the subunit vaccine comprises:

[0094] S51, mixing the recombinant antigen with Tween to obtain an aqueous phase, wherein the Tween is Tween-80.

[0095] S52, mixing white oil, Siben and aluminum stearate to obtain an oil phase. The white oil is injection white oil. The mass ratio of the white oil to Siben is 94:6.

[0096] S53, mixing the oil phase and the water phase and emulsifying to obtain an emulsion, wherein the volume ratio of the oil phase to the water phase is 2:1.

[0097] The stirring speed of the emulsification treatment was 10000 r / min, stirring for 3 min, stopping for 5 min, and repeating this process 4 times.

[0098] In this embodiment, the recombinant antigen and Tween-80 are mixed in a mass ratio of 96:4, and the mixture is fully shaken to dissolve Tween-80 completely and evenly to obtain an aqueous phase. 6% span80, 2% aluminum stearate and 92% white oil are mixed and stirred evenly and sterilized in a pressure cooker. The resulting light yellow solution is the oil phase, which is placed in a 4°C refrigerator for cooling and standby use. The oil phase is placed in an emulsion cylinder, and a slow rotation and stirring is set. At the same time, the aqueous phase is slowly added. The oil phase and the aqueous phase are mixed in a ratio of 2:1. After the addition, the mixture is stirred at 10000r / min for 3min to fully emulsify. After emulsification, 10ml is added to a centrifuge tube and centrifuged at 3000r / min for 15min. The aqueous phase precipitated at the bottom of the tube is ≤0.5ml, thereby obtaining a Fel d1 subunit vaccine.

[0099] S54. Centrifuge the emulsion to obtain the subunit vaccine.

[0100] S6. Inoculate the subunit vaccine into laying hens to obtain immune eggs.

[0101] Specifically, step S6, the step of inoculating the subunit vaccine into laying hens to obtain immune eggs, comprises inoculating the subunit vaccine into laying hens through three immunizations, so that the titer of Fel d1 yolk antibody in the yolk liquid of the immune eggs is higher than 1:64.

[0102] In this embodiment, healthy egg-laying hens were selected, isolated and raised for observation, and immunized with subunit vaccines. The specific immunization procedure included:

[0103] Basic immunization: Vaccinate 1-2 mL of subunit vaccine subcutaneously in the neck or into the chest muscle of each healthy laying hen.

[0104] Secondary immunization: The second vaccination is given 15 to 21 days after the primary immunization, with 1 to 2 mL of subunit vaccine injected subcutaneously in the neck or into the chest muscle.

[0105] Three immunizations: The third vaccination is carried out 15 to 21 days after the second vaccination, and 1.5 to 2.5 mL of subunit vaccine is inoculated subcutaneously in the neck or in the chest muscle. The recombinant protein concentration in the cat allergen Fel d1 subunit vaccine is not less than 0.8 mg / ml.

[0106] After three immunizations, when the titer of Fel d1 yolk antibody in the egg yolk fluid was not less than 1:64, the immunized eggs were collected.

[0107] As a preferred embodiment, healthy egg-laying hens include Hy-Line brown laying hens or Roman brown laying hens, aged 5 to 6 months. After the third immunization, according to the titer of Fel d1 yolk antibody, 1.5 to 2.5 mL of Fel d1 subunit vaccine is inoculated subcutaneously in the neck or in the chest muscle once again every month. 9 immunized eggs are randomly collected, and the immunized eggs are collected when the titer of Fel d1 yolk antibody in the egg yolk liquid is not less than 1:128.

[0108] In this embodiment, 5-month-old healthy Hailan brown laying hens were selected, isolated and raised for observation for one week, and Fel d1 subunit vaccine was vaccinated when the performance was normal. The injection site was the chest muscle, 1.5 ml for basic immunization, 1.5 ml for secondary immunization at an interval of 15 days, and 2.0 ml for tertiary immunization at an interval of 15 days. 14 days after the three immunizations, 9 sample eggs were drawn, and the titer of cat allergen Fel d1 specific antibody in the egg yolk liquid was determined. The titer was ≥1:128, and high-immunity eggs were collected. The collected high-immunity eggs were placed in a 4°C environment for no more than 7 days.

[0109] S7. Obtain egg yolk liquid from immune eggs.

[0110] The immune eggs are disinfected, the egg yolk liquid is separated and collected, and the egg yolk liquid is inactivated.

[0111] Specifically, the collected immune eggs are immersed in a 0.1% to 0.2% chlorhexidine aqueous solution at 40 to 45° C. for 20 to 30 minutes for disinfection. The eggs are then immersed in a water bath above 95° C. for disinfection for 5 to 10 seconds, taken out and dried, mechanically beaten to fully remove the egg white, germinal disc and frenulum, and the egg yolk liquid is collected.

[0112] As a preferred embodiment, the obtained egg yolk liquid is inactivated at 65-68° C. for 10-20 minutes.

[0113] In this embodiment, the collected high-immune eggs are immersed in a 0.1% chlorhexidine aqueous solution at 42°C for 15 minutes for immersion disinfection. Subsequently, the high-immune eggs are immersed in a water bath above 95°C for 5 seconds for disinfection, and then taken out and dried. The eggs are mechanically beaten to fully remove the egg white, embryonic disc and frenulum, and the egg yolk liquid is collected. The collected egg yolk liquid is added to a pasteurization tank, an appropriate amount of injection water is added, and after stirring and mixing, it is heated to 65°C and kept warm for 15 minutes for inactivation. After the inactivation is completed, the water bath is cooled to below 30°C.

[0114] S8. Add Tris-HCL buffer and polyethylene glycol to the egg yolk solution and collect the protein aqueous phase by centrifugation.

[0115] S9. Filter the protein aqueous phase to obtain a filtrate, add polyethylene glycol, mix, and centrifuge to collect the precipitate.

[0116] S10. Mix the precipitate with PBS buffer, add ammonium sulfate and centrifuge to obtain cat allergen Fel d1 yolk antibody.

[0117] The Feld1 yolk antibody was extracted from the yolk of the immune chicken eggs using polyethylene glycol combined with ammonium sulfate precipitation.

[0118] The polyethylene glycol combined with ammonium sulfate precipitation method comprises: mixing egg yolk liquid with Tris-HCL buffer, adding polyethylene glycol, centrifugally collecting the protein aqueous phase and filtering to obtain a filtrate, mixing the filtrate with polyethylene glycol, centrifugally collecting the precipitate, mixing the precipitate with PBS buffer, adding ammonium sulfate, centrifugally collecting the precipitate, and obtaining cat allergen Fel d1 egg yolk antibody.

[0119] Specifically, in this embodiment, according to a volume ratio of 1:2, 2 volumes of Tris-HCL buffer (pH7.6) are added to 1 part of egg yolk liquid, and the mixture is shaken vigorously for 30 seconds. Then, 3.5% (W / V) of polyethylene glycol (PEG-6000) is added, and the PEG-6000 is fully dissolved by shaking, and the mixture is allowed to stand overnight at 4°C, and centrifuged at 12000r / min at 4°C for 30 minutes to obtain a protein aqueous phase. The protein aqueous phase is filtered with a polypropylene 750B filter cloth, and then filtered with a column filter until clarified to obtain a filtrate. 8.5% (W / V) of PEG-6000 is added to the filtrate, and the mixture is shaken and mixed thoroughly, and the mixture is subjected to 25°C for 30 minutes. The mixture is centrifuged at 12000r / min at 4°C for 30 minutes, and the supernatant is discarded to obtain a precipitate. The precipitate is mixed with a PBS buffer to obtain an egg yolk antibody solution. Then gradually add ammonium sulfate to the egg yolk antibody solution dissolved in PBS, adding at a saturation of 50%-80% (W / V) to achieve salting out and precipitation of the protein. Ammonium sulfate needs to be added slowly in batches, stirring gently while adding to ensure that the ammonium sulfate is completely dissolved. After adding, let it stand at room temperature for 30 minutes to allow the antibody to fully precipitate. After centrifugation at 4°C and 12000r / min for 30 minutes, collect the precipitate and discard the supernatant. Dissolve the precipitate with PBS (pH 7.4) (the dissolution ratio is 1mL PBS for every 10mL egg yolk stock solution) to obtain a high-purity cat allergen Fel d1 egg yolk antibody solution.

[0120] In some embodiments, the obtained cat allergen Fel d 1 egg yolk antibody is mixed with a stabilizer and sterilized by filtration using a microporous filter membrane. The stabilizer is composed of the following ingredients: 5% (W / V) sucrose, 0.5% (W / V) bovine serum albumin (BSA), 0.05% (W / V) ascorbic acid, and phosphate buffered saline (PBS, pH 7.4), prepared to a final volume.

[0121] In this embodiment, after the egg yolk antibody is extracted, a stabilizer is added, mixed evenly, filtered and sterilized with a 0.2 μm microporous filter membrane, and aseptically packaged to obtain the refined egg yolk antibody.

[0122] The method for preparing cat allergen Fel d1 egg yolk antibody of the present invention designs a completely new method for preparing Fel d1 egg yolk antibody, controls the coordination of various steps, includes adopting a specific immunization program and an extraction method, is simple to operate, can maintain a relatively high level of IgY titer in the yolk of immunized laying hens, and the antibody titer may be maintained for a longer time. The purity, clarity and fat removal rate of the obtained egg yolk antibody are significantly improved, the titer is high, the specificity is high, the pertinence is stronger, the protection rate is high, the prevention and treatment costs are low, the method can be used to prevent cat allergies, and has a more practical application prospect.

[0123] The invention provides a cat allergen Fel d1 egg yolk antibody, which is prepared by adopting the above-mentioned cat allergen Fel d1 egg yolk antibody preparation method.

[0124] The present invention also provides an application of cat allergen Fel d1 egg yolk antibody, which comprises mixing the cat allergen Fel d1 egg yolk antibody with a lyophilizer and freeze-drying the mixture to obtain a lyophilized powder.

[0125] Lyophilized agents include protective agents, bulking agents, antioxidants and buffers.

[0126] The protective agent includes one or more combinations of trehalose, sucrose, β-dextrin, dextran or polyvinyl pyrrolidone.

[0127] The filler includes one or more combinations of glycine, sodium glutamate, sorbitol, mannitol or gelatin.

[0128] The antioxidant includes one or more combinations of vitamin E, vitamin C or protein hydrolysate.

[0129] The mass ratio of the protective agent, the filler and the antioxidant in the lyophilized agent is (40-80):(6-10):(1-2).

[0130] The volume ratio of cat allergen Fel d1 egg yolk antibody to lyophilized agent is (5-10):1.

[0131] Specifically, the freeze drying process includes: setting the temperature to -3 to -1°C, pre-freezing for 0.5 to 1 hour, then cooling to -40 to -50°C for 1 to 2 hours, vacuuming the freeze dryer to 0.1 to 0.3 mBar, heating to -30 to -20°C for 20 to 30 hours; heating to -20 to -10°C for 10 to 20 hours; heating to 0 to 10°C for 10 to 20 hours. The model of the freeze dryer is LYO-20SE, and its manufacturer is Beijing Boyikang Laboratory Equipment Co., Ltd. (Boyikang).

[0132] In this embodiment, 20 parts by mass of trehalose, 13 parts by mass of β-cyclodextrin, 7 parts by mass of dextran, 3.8 parts by mass of sodium glutamate, 2.2 parts by mass of sorbitol, 0.3 parts by mass of vitamin C, and 0.7 parts by mass of soy peptone were weighed according to the formula ratio and dissolved in deionized water, and the mixture was sterilized under high pressure at 120° C. for 30 minutes to obtain a lyophilized agent.

[0133] The cat allergen Fel d1 refined egg yolk antibody solution and the lyophilizer were thoroughly mixed at a volume ratio of 5:1, and a buffer solution was added to adjust the pH to 7 to obtain a sample before lyophilization.

[0134] The sample before lyophilization was placed in a freeze dryer, set at -2°C, pre-frozen for 1 hour, then cooled to -50°C and maintained for 1 hour, the freeze dryer was vacuumed to 0.1 mBar, heated to -30°C and maintained for 20 hours; heated to -10°C and maintained for 20 hours; heated to 0°C and maintained for 20 hours. The purified egg yolk antibody lyophilized powder was obtained.

[0135] In this embodiment, eggs were collected 14 days after the three immunizations, and the antibody titer was determined to be ≥1:128. In the later stage, maintenance immunity was carried out according to the antibody titer, and booster vaccination was performed once every 2 months, 2.0 ml per chicken. In this embodiment, immune eggs were collected 14 to 240 days after the three immunizations. During the egg collection period, the average egg production rate of Hy-Line brown laying hens was 79.1. The neutralization titer of the lyophilized powder of the cat allergen Fel d1 yolk antibody prepared in this embodiment was detected by indirect ELISA. The dilution method was 0.5 g of lyophilized yolk antibody dissolved in 1 ml of PBBS buffer, and 2 replicates were performed. The results are as follows: Figure 2 , Figure 3 , Figure 4 As shown, the antibody titer was 1:128000.

[0136] See also Figure 3 As shown, stable at 37℃~40℃: At 37℃ and 40℃, the titer values ​​are relatively close and the standard deviation is small, indicating that the titer is relatively stable. High level at 40℃~70℃: Between 40℃~70℃, although the titer has a downward trend, it still maintains a high level. After exceeding 70℃, the titer drops sharply and approaches the detection limit at 90℃, indicating that the antibody activity is almost completely lost.

[0137] See also Figure 4 As shown, pH 4-9 is stable: within the pH 4-9 range, the titer value is relatively stable, with a small change range. Although the titer at pH 3 decreases, it still remains at a high level. pH>9 decreases significantly: the titers at pH 10 and 11 decrease significantly. pH>11 activity is basically lost: the titer at pH 12 is close to the detection limit, indicating that the antibody activity is basically lost.

[0138] The invention adopts a polyethylene glycol combined with ammonium sulfate precipitation method and optimizes it under specific conditions to obtain a high-purity, high-titer and high-specificity Fel d1 egg yolk antibody.

[0139] The present invention constructs a complete technical route for preparing Fel d1 egg yolk antibody, covering the entire process from preparation of recombinant antigen (codon optimization), construction of subunit vaccine, immune program design to extraction and purification of egg yolk antibody and preparation of freeze-dried powder.

[0140] Specifically, the codon optimization step is to change the original DNA sequence to SEQ ID NO.4, specifically:

[0141] ATGGTCAAGATGGCGGAAACTTGCCCCATTTTTTATGACGTCTTTTTTGCGGTGGCCAATGGAAATGAATTACTGTTGGACTTGTCCCTCACAAAAGTCAATGCTACTGAACCAGAGAGAACAGCCATGAAAAAAATCCAGGATTGCTACGTGGAGAACGGACTCATATCCAGGGTCTTGGATGGACTAGTCATGACAACCATCAGCTCCAGCAAAGATTGCATGGGTGAAGCAGTTCAGAACACC GTAGAAGATCTCAAGCTGAACACTTTGGGGAGAGAAATTTGCCCAGCCGTGAAGAGGGATGTTGACCTATTCCTGACGGGAACCCCCGACGAATATGTTGAGCAAGTGGCACAATACAAAGCACTACCTGTAGTATTGGAAAATGCCAGAATACTGAAGAACTGCGTTGATGCAAAAATGACAGAAGAGGATAAGGAGAATGCTCTCAGCTTGCTGGACAAAATATACACAAGTCCTCTGTGTTAA.

[0142] Escherichia coli was chosen because different hosts have specific preferences for codon usage.

[0143] Select the restriction enzyme cutting sites that need to be avoided and enter the restriction enzyme cutting sites such as XhoI, BamHI, etc. that need to be avoided so that the subsequent cloning operation will not be interrupted.

[0144] Adjust codon usage frequency: replace with codons preferred by the host.

[0145] Increase GC content: Improve sequence stability (e.g. the GC content of the original sequence was 44.92%, which increased to 50% after optimization).

[0146] Removal of repetitive sequences: Avoid transcription or DNA synthesis problems caused by long repetitive regions.

[0147] Preserve coding identity: Ensure that the optimized sequence still encodes the same protein.

[0148] After the optimization is completed, the optimized codon sequence is shown in SEQ ID NO.5, specifically:

[0149] ATGGTAAAAATGGCTGAAACATGTCCCATATTCTACGACGTGTTTTTCGCCGTAGCGAACGGTAATGAACTGTTGTTGGACCTGTCCCTGACGAAAGTTAACGCTACCGAGCCGGAACGTACC GCAATGAAGAAGATCCAGGATTGCTATGTGGAGAACGGCCTGATTAGCCGCGTGCTGGATGGCCTGGTTATGACGACCATTAGCTCTAGCAAGGACTGTATGGGTGAGGCTGTGCAGAATACC GTTGAGGATCTTAAGCTGAATACCCTGGGTCGTGAAATCTGCCCGGCAGTCAAACGCGATGTGGACTTGTTTCTGACCGGCACCCCAGATGAGTACGTGGAGCAAGTTGCCCAATATAAAGCG CTGCCGGTTGTTTTAGAAAACGCGCGTATTTTGAAGAACTGCGTCGACGCGAAAATGACTGAGGAAGATAAAGAAAACGCGCTCTCGCTGTTGGACAAGATCTACACCAGCCCGCTGTGTTAA.

[0150] The GC content of the original DNA sequence was increased from 44.92% to 50.00% of the optimized DNA sequence, which enhanced the expression adaptability of the sequence in Escherichia coli, ensured that there were no restriction enzyme sites or other potential problems in the optimized sequence, and ensured the smoothness of the experimental operation.

[0151] Through codon optimization, efficient and soluble expression of the target gene in Escherichia coli was achieved, which not only solved the problem of protein easy formation of inclusion bodies in traditional prokaryotic expression systems, but also significantly improved the protein expression efficiency.

[0152] The present invention aims to prepare high titer and high specificity egg yolk antibodies against cat allergen Fel d1, which requires a complete and well-designed preparation process.

[0153] The present invention designs and implements a complete and innovative method for preparing Fel d1 egg yolk antibodies, which not only includes the preparation of recombinant antigens, but also optimizes and innovates the construction of subunit vaccines, immunization procedures and antibody purification, and finally obtains high-titer and high-specificity Fel d1 egg yolk antibodies.

[0154] The above description is only a preferred embodiment of the present application and an explanation of the technical principles used. Those skilled in the art should understand that the scope of the invention involved in the present application is not limited to the technical solution formed by a specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the inventive concept. For example, the above features are replaced with (but not limited to) technical features with similar functions disclosed in the present application.

Claims

1. A method for preparing cat allergen Feld1 yolk antibody, characterized in that: The following steps are involved: A PCR amplification product is obtained by PCR amplification using the nucleotide sequence shown in SEQ ID NO.1 as an upstream primer and the nucleotide sequence shown in SEQ ID NO.2 as a downstream primer; Connecting the PCR amplification product with pET-28a(+) to construct a recombinant plasmid; Transforming the recombinant plasmid into Rosetta competent cells to obtain transformants; A recombinant antigen is obtained by extracting the protein from the transformant, wherein the amino acid sequence of the recombinant antigen is shown in SEQ ID NO.3; The recombinant antigen is mixed and emulsified with a white oil adjuvant to obtain a subunit vaccine; administering the subunit vaccine to laying hens to obtain immune eggs; Obtaining egg yolk liquid from the immune eggs; Adding Tris-HCL buffer and polyethylene glycol to the egg yolk solution and centrifuging to collect the protein aqueous phase; Filtering the protein aqueous phase to obtain a filtrate, adding polyethylene glycol, mixing, centrifuging, and collecting a precipitate; The precipitate was mixed with PBS buffer, and then ammonium sulfate was added and centrifuged to prepare cat allergen Feld1 yolk antibody.

2. The method for preparing cat allergen Feld1 yolk antibody according to claim 1, characterized in that: The recombinant antigen is purified Fel d1 protein with a concentration of 1 mg / mL.

3. The method for preparing cat allergen Feld1 yolk antibody according to claim 2, characterized in that: The step of obtaining the recombinant antigen by extracting the protein in the transformant comprises: Picking a monoclonal colony of the transformant and inoculating it into LB culture medium containing kanamycin resistance and culturing it until the OD600 value of the bacterial solution reaches 0.6-0.8; Adding IPTG inducer to the bacterial solution to induce expression; Collect the bacterial cells from the bacterial solution after induced expression; The bacterial cells are added to PBS buffer, and the recombinant antigen is obtained through multiple resuspension, ultrasonic disruption, centrifugation and purification processes.

4. The method for preparing cat allergen Feld1 yolk antibody according to claim 1, characterized in that: The step of mixing and emulsifying the recombinant antigen and the white oil adjuvant to obtain the subunit vaccine comprises: mixing the recombinant antigen with Tween to obtain an aqueous phase; The oil phase is obtained by mixing white oil, sphenanthene and aluminum stearate; Mixing the oil phase and the water phase and emulsifying to obtain an emulsion; The emulsion is centrifuged to obtain the subunit vaccine.

5. The method for preparing cat allergen Feld1 yolk antibody according to claim 1, characterized in that: The step of inoculating the subunit vaccine into laying hens to obtain immune eggs comprises inoculating the subunit vaccine into laying hens through three immunizations, so that the Feld1 yolk antibody titer in the yolk fluid of the immune eggs is higher than 1:

64.

6. A cat allergen Feld1 yolk antibody, characterized in that: The cat allergen Feld1 yolk antibody is prepared by the method for preparing the cat allergen Feld1 yolk antibody according to any one of claims 1 to 5.

7. A use of the cat allergen Feld1 yolk antibody as claimed in claim 6, characterized in that: The cat allergen Fel d1 egg yolk antibody is mixed with a lyophilizing agent and freeze-dried to obtain a lyophilized powder.

8. The use of cat allergen Feld1 egg yolk antibody according to claim 7, characterized in that: The lyophilized agent comprises a protective agent, a filler, an antioxidant and a buffer; The protective agent includes one or more combinations of trehalose, sucrose, β-dextrin, dextran or polyvinyl pyrrolidone; The filler includes one or more combinations of glycine, sodium glutamate, sorbitol, mannitol or gelatin; The antioxidant includes one or more combinations of vitamin E, vitamin C or protein hydrolysate.

9. The use of cat allergen Feld1 yolk antibody according to claim 8, characterized in that: The mass ratio of the protective agent, the filler and the antioxidant in the lyophilized agent is 40-80:6-10:1-2.

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