A combined antigen fragment of cat allergens Fel d1 and Fel d4, as well as its preparation method and application

Abstract

The present invention belongs to the field of biotechnology and specifically relates to a combined antigenic fragment of the cat allergens Fel d1 and Fel d4, as well as a preparation method and application. The fragment is composed of multiple antigenic determinants of Fel d1 chain I, Fel d1 chain II, and Fel d4, connected by a GGGGSGGGGS flexible linker or an EAAAKEAAAK rigid linker. The amino acid sequence is shown in SEQ ID NO:4. The combined antigenic fragment of the cat allergens Fel d1 and Fel d4, as well as its preparation method and application, has the following advantages: the antigenic epitopes of the amino acid sequences of Fel d1 chain I, chain II, and Fel d4 are predicted, extracted, and combined with a linker tag to ensure that the expressed recombinant protein antigenic epitopes are fully exposed, allowing all antigenic epitopes to generate corresponding antibodies to obtain the combined antigenic fragment. The recombinant antigen and egg yolk antibody are then prepared into a liposome sustained-release system and added to cat food to block cat allergens in the cat's mouth, reducing allergic reactions in cat owners.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a combined antigen fragment of cat allergens FeI d1 and FeI d4, a preparation method and an application thereof. Background Art

[0002] About 10% to 20% of adults in the world's population are allergic to cats to varying degrees. Cat allergens, as common indoor inhaled allergens, have become the third leading cause of human respiratory allergies. The World Health Organization and the International Society of Immunology jointly identified cat allergens as secretory globulin Fel d1, serum albumin Fel d2, cystatin Fel d3, lipocalin Fel4 and Fel 7, immunoglobulins Fel d5 and Fel d6, and Latherin-like protein Fel d8. Fel d1 is the most important cat allergen, and 96% of cat allergy patients have Fel d1-specific antibodies. Fel d4 is a lipid transport protein and is the second most common cat allergen. About 63% of cat allergy patients have Fel d4-specific antibodies.

[0003] Cat allergens Fel d1 and Fel d4 are mainly found in sebaceous glands, squamous glands and squamous epithelial cells, as well as salivary glands, anal glands and lacrimal glands. They are transferred to the fur through cats licking and combing their fur, and are eventually stored on the skin and fur. They are spread through the air and trigger human immune responses.

[0004] All current cat allergens are expressed in prokaryotes. However, under natural conditions, the complete cat allergens Feld1 and Feld4 are expressed in mammalian cells. When a prokaryotic expression system is used, the obtained antigens may have incomplete exposure of the antigen epitopes due to their different spatial structures from those under natural conditions, thereby causing the antibodies produced by the antigen to have lower activity.

[0005] In current research, all cat allergens are expressed in prokaryotes. However, under natural conditions, the complete cat allergens Fel d1 and Fel d4 are expressed in mammalian cells. Therefore, when using a prokaryotic expression system, the resulting antigen may have a different spatial conformation than that under natural conditions, causing the antigen epitope to change from a functional epitope to a hidden epitope (the epitope located on the surface of the antigen molecule is easily recognized by the corresponding lymphocytes, that is, it is accessible and can directly initiate an immune response, so it is called a functional epitope. The epitope present in the antigen molecule has no function of directly triggering an immune response and is called a hidden epitope). It is hidden inside the antigen molecule, thereby causing the antibody produced by the antigen to have a low titer. In this patent, the epitopes of the antigen are analyzed and intercepted, and combined with a linker tag to ensure that the epitopes of the expressed recombinant protein are fully exposed, so that all epitopes can produce corresponding antibodies. Summary of the Invention

[0006] In response to the shortcomings of the existing technology, the present invention provides a combined antigen fragment of cat allergens Fel d1 and Fel d4, as well as a preparation method and application. The epitopes of the antigens are analyzed and intercepted, and then combined with a connection tag to ensure that the antigen epitopes of the expressed recombinant protein are fully exposed, so that all antigen epitopes can produce corresponding antibodies.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A combined antigenic fragment of cat allergens Fel d1 and Fel d4 is composed of a combination of Fel d1Ⅰ chain, Fel d1Ⅱ chain and multiple antigenic determinants of Feld4. The antigenic determinants are connected by a GGGGSGGGGS flexible linker or an EAAAKEAAAK rigid linker. Its amino acid sequence is shown in SEQ ID NO:4.

[0009] Furthermore, the gene sequence of the combined antigen fragment is shown in SEQ ID NO:5.

[0010] A recombinant vector or recombinant bacterium containing the above-mentioned combined antigen fragment gene sequence.

[0011] A method for preparing a combined antigenic fragment of cat allergens Fel d1 and Fel d4 comprises introducing the gene sequence shown in SEQ ID NO: 5 into Escherichia coli to obtain a recombinant Escherichia coli strain containing the gene sequence, and expressing and purifying the combined antigenic fragment of cat allergens Fel d1 and Fel d4.

[0012] The invention discloses an application of a combined antigen fragment of cat allergens FeI d1 and FeI d4, wherein the combined antigen fragment is used for preparing polyclonal antibodies against cat allergens FeI d1 and FeI d4.

[0013] Furthermore, the combined antigen fragments were used to immunize laying hens, and the egg yolks were collected and purified to obtain egg yolk antibodies IgY against cat allergens Fel d1 and Fel d4.

[0014] Furthermore, the egg yolk is diluted with water to extract egg yolk antibodies, the steps comprising:

[0015] The egg yolk and water were mixed evenly at a volume ratio of 1:6-8, the pH value was adjusted to 5.0-5.2, and the mixture was placed in a 4°C refrigerator for overnight. The next day, the mixture was centrifuged at 6000 rpm for 20 minutes, the precipitate was discarded, and the supernatant was lyophilized into powder to obtain polyclonal antibody powder.

[0016] Use of a combined antigen fragment of cat allergens Fel d1 and Fel d4 in the preparation of an antibody composition, wherein the antibody composition comprises the above-mentioned egg yolk antibody IgY, lecithin, and a sucrose aqueous solution, and has a water-in-oil-in-water structure;

[0017] The inner aqueous phase is a PBS solution of egg yolk antibody IgY.

[0018] The outer aqueous phase is a 10% sucrose aqueous solution.

[0019] The oil phase is lecithin.

[0020] Furthermore, the preparation method of the antibody composition is:

[0021] (1) Prepare the oil phase: dissolve lecithin in anhydrous ethanol at 37°C in a water bath to a lecithin concentration of 20 mg / ml.

[0022] (2) Preparation of the internal aqueous phase: Dissolve the polyclonal antibody powder in PBS to a concentration of 1.65 mg / ml;

[0023] (3) Preparation of external aqueous phase: Prepare 10% sucrose aqueous solution;

[0024] (4) Preparation of water-in-oil: The aqueous phase in (2) was slowly added dropwise to the oil phase in (1), and sheared at 6000 rpm for 2 min using a high-speed shearing machine to form a water-in-oil structure to obtain a liposome solution containing polyclonal antibodies. The volume ratio of the aqueous phase to the oil phase was 4-5:1;

[0025] (5) Preparation of water-in-oil-in-water emulsion: The liposome solution containing the polyclonal antibody obtained in (4) was added as a dispersed phase to the external aqueous phase of (3), and sheared at 6000 rpm for 2 min to form a water-in-oil-in-water structure to obtain a W1 / O / W2 emulsion of sucrose-lecithin-polyclonal antibody, with the volume ratio of the liposome solution to the external aqueous phase being 3-4:1;

[0026] The S / N value of the polyclonal antibody in the obtained W1 / O / W2 emulsion of sucrose-lecithin-polyclonal antibody is 10-20, preferably 16.

[0027] The invention relates to an application of a combined antigen fragment of cat allergens Fel d1 and Fel d4 in the preparation of wet cat food. The wet cat food contains the above-mentioned antibody composition, and the addition amount is 30%-50% of the total weight of the cat food, preferably 45%.

[0028] The present invention provides a combined antigen fragment of cat allergens Fel d1 and Fel d4, as well as a preparation method and application thereof. The present invention predicts the antigen epitopes of the amino acid sequence of chain I and chain II of Fel d1 and Fel d4, intercepts them, and combines them with a linker tag to ensure that the antigen epitopes of the expressed recombinant protein are fully exposed, so that all antigen epitopes can generate corresponding antibodies to obtain a combined antigen fragment, and then prepares the recombinant antigen and egg yolk antibody. After being prepared into a liposome sustained-release system and added to cat food, the cat allergens in the cat's mouth are blocked, thereby reducing allergic reactions in cat owners.

[0029] In addition, Fel d4 also functions as a lipid transport protein. After binding to Fel d4 in saliva, Fel d4 egg yolk antibodies neutralize its activity, allowing the liposomes to better maintain their original shape. Therefore, in this application, Fel d4 egg yolk antibodies can not only act as a key allergen antibody, but also serve as a key component to significantly enhance the sustained-release function of egg yolk antibody liposomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is an epitope analysis diagram of the amino acid sequence of the Fel d1Ⅰ chain of the embodiment of the present invention;

[0031] Figure 2 This is an epitope analysis diagram of the amino acid sequence of the II chain of Fel d1 according to an embodiment of the present invention;

[0032] Figure 3 This is an epitope analysis diagram of the amino acid sequence of the FeI d4 chain of the embodiment of the present invention;

[0033] Figure 4 This is a diagram showing the SDS-PAGE results of the recombinant E. coli strain containing the combined antigen fragment gene after expression according to an embodiment of the present invention;

[0034] Figure 5 This is a diagram showing the SDS-PAGE results after purification of the combined antigen fragments according to an embodiment of the present invention;

[0035] Figure 6 This is a graph showing the S / N results of ELISA testing of egg yolk antibodies at different times according to an embodiment of the present invention;

[0036] Figure 7 This is a graph showing the S / N results of ELISA testing of egg yolk antibodies against different concentrations of PFel d1A4 in an embodiment of the present invention;

[0037] Figure 8 This is a graph showing the S / N results of ELISA testing of egg yolk antibodies at different concentrations of FeI d 1 in an embodiment of the present invention;

[0038] Figure 9This is a graph showing the S / N results of ELISA testing of egg yolk antibodies against different concentrations of FeI d 4 in an embodiment of the present invention;

[0039] Figure 10 : This is the S / N graph of the egg yolk antibody and the egg yolk antibody after the W1 / O / W2 emulsion is mixed with saliva in vitro according to the embodiment of the present invention;

[0040] Figure 11 This is a graph showing the S / N of egg yolk antibodies in the saliva of cats fed with cat food containing different antibodies at different times according to an embodiment of the present invention;

[0041] Figure 12 The flow cytometric images of volunteer 1 before and after the experiment in the present invention are shown on the left, before the experiment, and after the experiment on the right.

[0042] Figure 13 The flow cytometry images of volunteer 2 before and after the experiment in the present invention are shown on the left, before the experiment, and after the experiment on the right. DETAILED DESCRIPTION

[0043] The specific implementation is further described below with reference to the accompanying drawings.

[0044] The reagents and instruments in the following examples are all conventional experimental reagents and instruments.

[0045] Example 1:

[0046] A combined antigenic fragment of cat allergens Fel d1 and Fel d4,

[0047] 1. Obtaining the gene sequence of the combined antigen fragment of cat allergens Fel d1 and Fel d4

[0048] DNAStar software was used to analyze and predict the hydrophilicity, surface accessibility, antigenicity, flexibility, and secondary structure of the amino acid sequences of chain I (SEQ ID NO.1), chain II (SEQ ID NO.2), and Fel d4 (SEQ ID NO.3). At the same time, Bepipred LinearEpitope Prediction 2.0 was used to predict the antigenic epitopes of the amino acid sequences of chain I, chain II, and Fel d4, and the amino acid sequence of the combined antigenic fragment of cat allergens Fel d1 and Fel d4 was obtained as SEQ ID NO:4: PDEYVEQVAQYNALPV GGGGSGGGGS DAKMTEEDKEN GGGGSGGGGSTKVNATEPERTA MK EAAAK CYVENGLISRVLDG EAAAK NEYCMGEAVQNTVEDLKLN GGGGSGGGGS EEEN VVRSNIDI GGGGSGGGGS DLTEVDRCLQARGSEVAQDS, GGGGSGGGGS is a flexible linker, EAAAKEAAAK is a rigid linker

[0049] FeI d1I chain, SEQ ID NO: 1:

[0050] VRRSPSTLPYCCGHSRDCEICPAVKRDVDLFLTGTPDEYVEQVAQYNALPVVLENARILKNCVDAKMTEEDKENALSVLDKIYTSPLC

[0051] The results of antigen epitope analysis of the amino acid sequence of Fel d1Ⅰ chain are shown in Figure 1 ;

[0052] FeI d1 II chain, SEQ ID NO: 2:

[0053] MRGALLVLALLVTQALGVKMAETCPIFYDVFFAVANGNELLLDLSLTKVNATEPERTA MKKIQDCYVENGLISRVLDGLVMIAINEYCMGEAVQNTVEDLKLNTLGR

[0054] The results of antigen epitope analysis of the amino acid sequence of Fel d1 II chain are shown in Figure 2 ;

[0055] FeI d4, SEQ ID NO: 3:

[0056] MKLLLLCLGLILVCAHEEENVVRSNIDISKISGEWYSILLASDVKEKIEENGSMRVFVEHIKALDNSSLSFVFHTKENGKCTEIFLVADKTKDGVYTVVYDGYNVFSIVETVYDEYILLHLLNFDKTRPFQLVEFYAREPDVSQKLKEKFVKYCQEHGIVNILDLTEVDRCLQARGSEVAQDSSVE

[0057] The results of antigenic epitope analysis of the amino acid sequence of Fel d4 are shown in Figure 3 ;

[0058] The amino acid sequence of the combined antigen fragment of cat allergens Fel d1 and Fel d4, SEQ ID NO. 4, was optimized for E. coli to obtain the gene sequence SEQ ID NO: 5:

[0059] CCCGATGAATATGTAGAGCAGGTTGCTCAATACAACGCGCTGCCGGTTGGTGGCGGTGG

[0060] CTCCGGTGGCGGTGGCAGCGACGCGAAGATGACCGAGGAAGACAAAGAAAACGGTGG

[0061] CGGCGGTTCTGGTGGCGGAGGTTCGACGAAGGTAAATGCCACTGAGCCGGAACGCACC

[0062] GCAATGAAAGAGGCTGCTGCGAAGTGCTATGTGGAAAACGGCCTGATCAGCCGTGTTCT

[0063] GGATGGTGAAGCGGCGGCGAAAAACGAATACTGCATGGGTGAAGCCGTTCAAAACACC

[0064] GTTGAGGACCTTAAGCTGAATGGTGGCGGCGGTAGCGGCGGTGGCGGTTCTGAGGAAG

[0065] AGAACTGGTGCGCAGCAATATTGATATCGGTGGCGGCGGTAGCGGTGGTGGCGGCTCC

[0066] GACTTGACCGAGGTCGATCGTTGTTTGCAGGCACGTGGTTCCGAGGTGGCTCAGGATAG

[0067] C

[0068] 2. Obtaining a recombinant Escherichia coli strain containing the gene sequence of the combined antigen fragment of cat allergens Fel d1 and Fel d4

[0069] The gene sequence SEQ ID NO.5 was sent to GenScript Biotech Co., Ltd. for gene synthesis and to obtain a recombinant Escherichia coli strain containing the gene sequence. At the same time, recombinant Escherichia coli strains containing the Fel d1Ⅰ chain encoding gene, the Fel d1Ⅱ chain encoding gene, and the Fel d4 encoding gene were prepared respectively.

[0070] 3. Expression and purification of combined antigenic fragments of cat allergens Fel d1 and Fel d4

[0071] (1) Induced expression and SDS-PAGE analysis and identification

[0072] The recombinant E. coli strain containing the gene sequence of SEQ ID NO.5 was inoculated into LB medium containing 50 μg / mL kanamycin sulfate. 600 When the pH value was 0.5-0.8, IPTG was added to the culture medium at a final concentration of 0.2 mM, and then the culture medium was placed at 15°C and 37°C for 16 h to induce expression, and then the bacteria were collected.

[0073] The whole bacteria were lysed by ultrasonication in 50 mM Tris (pH 8.0), 300 mM NaCl, 20 mM Imidazole containing 1% Triton X-100, 1 mM DTT, and 1 mM PMSF. The supernatant and precipitate were analyzed by SDS-PAGE. The results are shown in Figure 4 1: supernatant of whole-cell lysis after induction and expression at 15°C; 2: supernatant of whole-cell lysis after induction and expression at 36°C; 3: precipitate of whole-cell lysis after induction and expression at 36°C. Therefore, the combined antigen fragment of cat allergens Fel d1 and Fel d4 is dissolved in the supernatant.

[0074] (2) Affinity chromatography purification in the supernatant:

[0075] The supernatant of the bacterial cells after ultrasonic lysis was purified by Ni-IDA affinity chromatography column equilibrated with 50mM Tris (pH 8.0), 300mM NaCl, 20mM Imidazole buffer, and the target protein in the eluate was collected and analyzed by SDS-PAGE. Figure 5 : R is the target protein with a molecular weight of approximately 16 KDa, which is consistent with the theoretical size.

[0076] The eluate was collected and dialyzed into 1×PBS (pH 7.4). After dialysis, it was placed in a vacuum freeze dryer and freeze-dried to obtain a combined antigen fragment of cat allergens Fel d1 and Fel d4 (recombinant antigen PFel d1A4), which was redissolved in 1×PBS (pH 7.4).

[0077] Recombinant Escherichia coli strains containing the gene encoding Fel d1Ⅰ chain, Fel d1Ⅱ chain and Fel d4 were simultaneously expressed and purified to obtain recombinant antigens Fel d1-Ⅰ, recombinant antigen Fel d1-Ⅱ and recombinant antigen Fel d4.

[0078] 4. Preparation of polyclonal antibody IgY against recombinant antigen PFel d1A4

[0079] (1) Vaccine preparation

[0080] The purified recombinant antigen PFel d1A4 was slowly added to an equal volume of aluminum adjuvant at a concentration of 2 mg / ml to form the PFel d1A4 aluminum adsorption vaccine.

[0081] The purified Fel d1 I chain protein and Fel d1 II chain protein were first mixed with each other at a concentration of 2 mg / ml, and then slowly added to an equal volume of aluminum adjuvant to form a Fel d1 aluminum adsorption vaccine.

[0082] The purified FeI d4 protein was slowly added to an equal volume of aluminum adjuvant at a concentration of 2 mg / ml to form FeI d4 aluminum adsorption vaccine.

[0083] (2) Immunity

[0084] The PFel d1A4 aluminum vaccine group, Fel d1 aluminum vaccine group, and Fel d4 aluminum vaccine group were set up. On the 0th day, 7th day, 21st day and every 14 days thereafter, 1 ml of the dose was injected into the breast or wing base of each chicken according to the corresponding group. A blank control group without treatment was set up.

[0085] A batch of eggs before immunization were collected, and eggs from the immunization group and the control group were collected every day after the first immunization began. Yolk antibodies were extracted by water dilution method. The yolk and water were mixed uniformly at a volume ratio of 1:8, the pH value was adjusted to 5.0-5.2, and the mixture was placed in a 4°C refrigerator for overnight. The next day, the mixture was centrifuged at a speed of 6000 r / min for 20 minutes, the precipitate was discarded, and the supernatant was freeze-dried into powder to obtain yolk antibody powder.

[0086] ELISA was performed every 7 days. Three eggs were selected from a batch of eggs collected before immunization, mixed and then the yolk antibodies were extracted as negative control samples. 312 .

[0087] (3) ELISA detection of antibody titer

[0088] Preparation of coated plates: Dilute FeI d1I chain protein, FeI d1II chain protein, and FeI d4 protein with pH 9.6 sodium carbonate buffer to form an antigen coating solution with a final concentration of 500 ng / ml FeI d1I chain protein, 500 ng / ml FeI d1II chain protein, and 1000 ng / ml FeI d4 protein. Incubate overnight at 4°C, block with skim milk at 37°C for 1 hour, and vacuum dry.

[0089] Dissolve 1g of egg yolk antibody freeze-dried powder in 300ml of PBS, add 100ul to each well, and take 10mgN 312 The negative control sample egg yolk antibody freeze-dried powder was dissolved in 3 ml of PBS, and 100 μl of sample was added to both wells as negative wells. After incubation at 37°C for 1 hour, the wells were washed three times with 300 μl of PBST per well, and 100 μl of HRP-labeled mouse anti-chicken IgY was added to each well. After incubation at 37°C for 1 hour, the wells were washed three times with 300 μl of PBST per well, and 100 μl of single-component TMB substrate was added to each well. After incubation at 37°C for 15 minutes, the wells were terminated with 100 μl of 2 M sulfuric acid per well, and the OD was detected by microplate reader. 450 The OD value of the sample well is counted as the S value, and the average OD value of the negative well is counted as the N value. The S / N value is calculated and recorded.

[0090] (4) Experimental results:

[0091] Figure 6 The S / N results of ELISA testing of egg yolk antibodies at different times are shown;

[0092] The results show that egg yolk antibody titers peaked between days 21 and 28 in all groups, with the highest antibody levels in the Fel d1A4 group, the lowest in the Fel d1 group, and somewhere in between in the Fel d4 group. Based on these results, eggs aged 28 days and older were selected for preparing egg yolk antibodies for subsequent products.

[0093] It can be demonstrated herein that the recombinant antigen PFel d1A4 with fully exposed antigenic epitopes can effectively enhance its immunogenicity as an antigen or the reactivity of the antibodies generated thereby, compared to the complete recombinantly expressed Fel d1 protein and Fel d4 protein, especially compared to the Fel d1 protein, and can effectively increase antibody production or antibody activity.

[0094] 5. Preparation of specific egg yolk antibody IgY

[0095] (1) Preparation

[0096] Egg yolk containing polyclonal antibodies to the cat allergen recombinant antigen PFel d1A4 was collected, and the egg yolk antibodies were extracted by a water dilution method. The egg yolk and water were uniformly mixed in a volume ratio of 1:8, the pH value was adjusted to 5.0-5.2, and the mixture was placed in a 4°C refrigerator for overnight. The next day, the mixture was centrifuged at 6000 r / min for 20 minutes, the precipitate was discarded, and the supernatant was lyophilized into a powder to obtain PFeld1A4 egg yolk antibody powder. Fel d1 and Fel d4 egg yolk antibody powders were also prepared simultaneously.

[0097] (2) ELISA was used to detect the antibody titer.

[0098] Preparation of coated plates: Dilute FeI d1I chain protein, FeI d1II chain protein, and FeI d4 protein with pH 9.6 sodium carbonate buffer to form an antigen coating solution with a final concentration of 500 ng / ml FeI d1I chain protein, 500 ng / ml FeI d1II chain protein, and 1000 ng / ml FeI d4 protein. Incubate overnight at 4°C, block with skim milk at 37°C for 1 hour, and vacuum dry.

[0099] Take 1g of egg yolk antibody freeze-dried powder and dissolve it in different volumes of PBS to different concentrations. Add 100ul to each well. Take 10mgN 312 The negative control sample egg yolk antibody freeze-dried powder was dissolved in 3 ml of PBS, and 100 μl of sample was added to both wells as negative wells. After incubation at 37°C for 1 hour, the wells were washed three times with 300 μl of PBST per well, and 100 μl of HRP-labeled mouse anti-chicken IgY was added to each well. After incubation at 37°C for 1 hour, the wells were washed three times with 300 μl of PBST per well, and 100 μl of single-component TMB substrate was added to each well. After incubation at 37°C for 15 minutes, the wells were terminated with 100 μl of 2 M sulfuric acid per well, and the OD was detected by microplate reader. 450 The OD value of the sample well is counted as the S value, and the average OD value of the negative well is counted as the N value. The S / N value is calculated and recorded.

[0100] (3) Experimental results:

[0101] Table 1 ELISA detection of PFel d1A4 egg yolk antibody gradient dilution and OD value

[0102]

[0103] Table 2 ELISA detection of Fel d1 egg yolk antibody gradient dilution and OD value

[0104]

[0105] Table 3 ELISA detection of Fel d4 egg yolk antibody gradient dilution and OD value

[0106]

[0107] Figure 7-9 The figure shows the S / N results of ELISA testing of different concentrations of egg yolk antibodies;

[0108] The final concentrations of the three antibodies are controlled to have an S / N value between 10 and 20, preferably about 16. For the egg yolk antibody prepared in this example, when the S / N value is between 10 and 20, the corresponding concentrations are PFel d1A4: 1-1.2 mg / ml; Fel d1: 3.7-3.9 mg / ml; and Fel d4: 1.4-1.6 mg / ml.

[0109] VI. Preparation of Sucrose-Lecithin-Polyclonal Antibody W1 / O / W2 Emulsion

[0110] (1) Preparation method

[0111] Oil phase: Dissolve lecithin in anhydrous ethanol at 37 degrees Celsius to a concentration of 20 mg / ml.

[0112] Internal aqueous phase: PFel d1A4 polyclonal antibody powder was dissolved in PBS to a concentration of 1.65 mg / ml

[0113] External aqueous phase: prepare 10% sucrose aqueous solution.

[0114] Water-in-oil: The aqueous phase was slowly added dropwise to the oil phase, and the mixture was sheared at 6000 rpm for 2 minutes to form a water-in-oil structure to obtain a lecithin-polyclonal antibody liposome solution with a volume ratio of 5:1 between the aqueous phase and the oil phase.

[0115] Water-in-oil-in-water: The above-mentioned liposome solution containing polyclonal antibodies was added as the dispersed phase to the external aqueous phase and sheared at 6000 rpm for 2 min to form a water-in-oil-in-water structure to obtain a W1 / O / W2 emulsion of sucrose-lecithin-polyclonal antibody with a volume ratio of liposomes to external aqueous phase of 4:1.

[0116] The concentration of egg yolk antibody PFel d1A4 in W1 / O / W2 emulsion was 1-1.2 mg / ml.

[0117] In order to extend the storage time of the sucrose-lecithin-polyclonal antibody W1 / O / W2 emulsion, a small amount of preservatives can be optionally added.

[0118] (2) Detection method

[0119] Preparation of control samples: Simultaneously prepare a W1 / O / W2 emulsion containing Fel d1 egg yolk antibody at a concentration of 5.7 mg / ml and a W1 / O / W2 emulsion containing Fel d4 egg yolk antibody at a concentration of 2.25 mg / ml. The egg yolk antibody concentrations in the W1 / O / W2 emulsion are: Fel d1: 3.7-3.9 mg / ml; Fel d4: 1.4-1.6 mg / ml

[0120] Also, prepare untreated 3.7-3.9 mg / ml Fel d1 egg yolk antibody solution, 1.4-1.6 mg / ml Fel d4 egg yolk antibody solution, and 1-1.2 mg / ml Fel d1A4 egg yolk antibody solution.

[0121] Blank group: water

[0122] After the cats were deprived of water and food for 2 hours, their saliva was collected.

[0123] Detection method: All the above samples were mixed with an equal volume of saliva, placed in a 37°C environment with a magnetic stirrer at 20 rpm. Every 20 minutes, 1 ml of the mixture was centrifuged at 25°C and 10,000 rpm for 30 minutes. The supernatant was collected and the IgY content was detected by ELISA.

[0124] ELISA test:

[0125] Preparation of coated plates: Dilute FeI d1I chain protein, FeI d1II chain protein, and FeI d4 protein with pH 9.6 sodium carbonate buffer to form an antigen coating solution with a final concentration of 500 ng / ml FeI d1I chain protein, 500 ng / ml FeI d1II chain protein, and 1000 ng / ml FeI d4 protein. Incubate overnight at 4°C, block with skim milk at 37°C for 1 hour, and vacuum dry.

[0126] The sample was centrifuged at 25°C and 10000 r / min for 30 min, the supernatant was collected, 100 μl of sample was added to each well, and 10 mg of N was taken. 312 The negative control sample egg yolk antibody lyophilized powder was dissolved in 3 ml of PBS, and 100 ul of sample was added to both wells as negative wells. After incubation at 37°C for 1 hour, the wells were washed three times with 300 ul of PBST per well, and 100 ul of HRP-labeled mouse anti-chicken IgY was added to each well. After incubation at 37°C for 1 hour, the wells were washed three times with 300 ul of PBST per well. 100 ul of single-component TMB substrate was added to each well and incubated at 37°C for 15 minutes. The cells were terminated with 100 ul of 2 M sulfuric acid per well. The OD450 was detected by a microplate reader. The OD value of the sample well was calculated as the S value, and the average OD value of the negative well was calculated as the N value. The S / N value was calculated and recorded.

[0127] (3) Experimental results:

[0128] Figure 10 The S / N graphs of egg yolk antibodies and egg yolk antibodies after W1 / O / W2 emulsion was mixed with saliva in vitro;

[0129] For the three egg yolk antibodies that were not prepared as W1 / O / W2 emulsions, the antibody concentrations all reached their lowest point around the 40th minute. However, after being prepared as W1 / O / W2 emulsions, the time points at which the antibody concentrations reached their lowest point were significantly different. The antibody concentration of Fel d1 egg yolk antibody liposomes reached its lowest point at the 60th minute, after which it began to slowly increase. The antibody concentration of PFel d1A4 egg yolk antibody liposomes reached its lowest point around the 160th minute, and the antibody concentration of Fel d4 egg yolk antibody liposomes reached its lowest point around the 120th minute.

[0130] According to the experimental results, the concentration of Fel d1 in saliva may be lower than that of Fel d4. However, after the free yolk antibodies outside the liposomes were consumed quickly, the release rate of Fel d1 yolk antibody liposomes was significantly different from that of Fel d4 and PFel d1A4 yolk antibody liposomes. Therefore, Fel d4 yolk antibody may play a key role in this process.

[0131] Fel d4 is a lipid transport protein, and studies have found that some of these proteins can spontaneously bind to lipids in aqueous solutions. This may explain the significant differences in the sustained-release rates of the three liposomes. Fel d4 egg yolk antibodies bind to Fel d4 in saliva, neutralizing its activity and allowing the liposomes to better maintain their original shape. Therefore, in this application, Fel d4 egg yolk antibodies not only serve as a key allergen antibody but also serve as a key component, significantly enhancing the sustained-release function of the egg yolk antibody liposomes.

[0132] 7. Preparation of functional cat food

[0133] (1) Add a W1 / O / W2 emulsion of sucrose-lecithin-polyclonal antibody (PFel d1A4) into wet cat food or paste cat food at a content of 30%-50%, preferably 45%.

[0134] (2) Detecting changes in antibodies in cats' oral cavity

[0135] Preparation of control samples: cat food containing sucrose-lecithin-polyclonal antibody (Fel d1), cat food containing sucrose-lecithin-polyclonal antibody (Fel d4), cat food containing egg yolk antibody Fel d1, cat food containing egg yolk antibody Feld4, and cat food containing egg yolk antibody PFel d1A4 were prepared simultaneously.

[0136] The blank group was a mixture of water and cat food with the same content.

[0137] Detection steps: 50g experimental animals were fed, and 200 μl of saliva was collected before feeding and every 20 minutes after feeding. After centrifugation at 25°C and 10,000 rpm for 30 minutes, the supernatant was collected and the IGY content was detected by ELISA.

[0138] ELISA test:

[0139] Coating plate preparation: Dilute FeI d1I chain protein, FeI d1II chain protein, and FeI d4 protein with pH 9.6 sodium carbonate buffer to form an antigen coating solution with a final concentration of 500 ng / ml FeI d1I chain protein, 500 ng / ml FeI d1II chain protein, and 1000 ng / ml FeI d4 protein. Incubate overnight at 4°C, block with skim milk at 37°C for 1 hour, and vacuum dry.

[0140] The sample was centrifuged at 25°C and 10000 r / min for 30 min, the supernatant was collected, 100 μl of sample was added to each well, and 10 mg of N was taken. 312 The negative control sample egg yolk antibody lyophilized powder was dissolved in 3 ml of PBS, and 100 ul of sample was added to both wells as negative wells. After incubation at 37°C for 1 hour, the wells were washed three times with 300 ul of PBST per well, and 100 ul of HRP-labeled mouse anti-chicken IgY was added to each well. After incubation at 37°C for 1 hour, the wells were washed three times with 300 ul of PBST per well. 100 ul of single-component TMB substrate was added to each well and incubated at 37°C for 15 minutes. The cells were terminated with 100 ul of 2 M sulfuric acid per well. The OD450 was detected by a microplate reader. The OD value of the sample well was calculated as the S value, and the average OD value of the negative well was calculated as the N value. The S / N value was calculated and recorded.

[0141] Experimental results:

[0142] Figure 11 The S / N graph of egg yolk antibodies in cat saliva at different times after being fed with different cat foods;

[0143] The three egg yolk antibodies that were not processed into liposomes all reached antibody-negative levels between 40 and 60 minutes (the S / N value was lower than the highest S / N value of the blank group). However, after being processed into liposomes, similar to the in vitro experiments, there were significant differences in the time points at which different egg yolk antibody liposomes reached antibody-negative levels. The Fel d1 egg yolk antibody liposomes reached antibody-negative levels at 120 minutes, the PFel d1A4 egg yolk antibody liposomes reached antibody-negative levels at 200 minutes, and the Fel d4 egg yolk antibody liposomes reached antibody-negative levels at 180 minutes.

[0144] The results showed that after liposomes containing PFel d1A4 egg yolk antibodies were added to cat food, the residence time of IgY antibodies in the oral cavity was significantly enhanced. At the same time, it also proved again that the presence of Fel d4 antibodies can significantly enhance the sustained-release effect of liposomes. In addition, the results presented by the PFel d1A4 antibody group showed that its effect in producing Fel d4 antibodies with enhanced sustained-release effect was better than that of antibodies prepared by prokaryotic expression of the full-length Fel d4 antigen. The reason may be related to the more complete exposure of the Fel d4 antigen epitope in PFel d1A4.

[0145] (3) Flow cytometry to detect inflammatory changes in human blood

[0146] Volunteers who live in cat-owning households and have cat allergy symptoms were selected, and peripheral blood lymphocytes were collected from the volunteers. The CD4 positive cell count was detected by flow cytometry. The volunteers were divided into two parallel experimental groups and fed with cat food supplemented with sucrose-lecithin-polyclonal antibody (PFel d1A4) W1 / O / W2 emulsion. The volunteers were required to consume no less than 50g each time and no less than 3 times a day, and maintain normal interaction with their pet cats. One month after the experiment, the volunteers' peripheral blood lymphocytes were collected after feeding the pet cat for 2 hours and interacting with it without shielding (such as masks and gloves) for 2 hours. The changes in CD4 positive cells were analyzed by flow cytometry.

[0147] Experimental results:

[0148] Figure 12 The flow cytometry images of the first group of volunteers before and after the experiment are shown on the left, before the experiment, and after the experiment on the right.

[0149] The results showed that in the first experimental group, the CD4 positivity rate dropped from 63.4% to 56.5%, and the CD4 / CD8 ratio dropped from 2.2 to 2.06. At the same time, the subjective feeling of allergic rhinitis symptoms in this group of volunteers was significantly alleviated.

[0150] Figure 13 The flow cytometry images of the second group of volunteers before and after the experiment are shown on the left, before the experiment, and after the experiment on the right.

[0151] In the second experimental group, the CD4 positivity rate dropped from 69.4% to 65.3%, and the CD4 / CD8 ratio dropped from 2.7 to 2.4. At the same time, the volunteers in this group subjectively felt that their allergic symptoms were significantly alleviated after feeding pet cats for two weeks. By the time of the final test, the allergic symptoms had basically completely disappeared.

[0152] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made based on the essence of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A combined antigenic fragment of cat allergens FeI d1 and FeI d4, characterized by: The amino acid sequence of the combined antigen fragment is shown in SEQ ID NO:

4.

2. A gene expressing the combined antigen fragment of cat allergens FeI d 1 and FeI d 4 according to claim 1, characterized in that: The gene of the combined antigen fragment is shown in SEQ ID NO:

5.

3. A recombinant vector or recombinant bacterium containing the combined antigen fragment gene as claimed in claim 2.

4. A method for preparing the combined antigenic fragment of the cat allergens FeI d 1 and FeI d 4 as claimed in claim 1, characterized in that: The gene shown in SEQ ID NO: 5 was introduced into Escherichia coli to obtain a recombinant Escherichia coli strain containing the gene, and the combined antigen fragment of cat allergens FeI d1 and FeI d4 was obtained by expression and purification.

5. A use of the combined antigen fragment of the cat allergens FeI d 1 and FeI d 4 as claimed in claim 1, characterized in that: The combined antigen fragments are used to prepare polyclonal antibodies against cat allergens FeI d 1 and FeI d 4.

6. Use of the combined antigen fragment of cat allergens FeI d 1 and FeI d 4 according to claim 1 in the preparation of egg yolk antibody IgY, characterized in that: The combined antigen fragments were used to immunize laying hens, and the egg yolks were collected and purified to obtain egg yolk antibodies IgY against cat allergens Fel d1 and Fel d4.

7. Use of the combined antigen fragment of cat allergens FeI d 1 and FeI d 4 according to claim 6 in the preparation of egg yolk antibody IgY, characterized in that: The egg yolk antibody is extracted by a water dilution method, the steps comprising: Mix egg yolk and water in a volume ratio of 1:6-8, adjust the pH to 5.0-5.2, and place in a 4°C refrigerator overnight. The next day, centrifuge at 6000 r / min for 20 minutes, discard the precipitate, and take the supernatant. The supernatant is lyophilized into powder to obtain polyclonal antibody powder.

8. Use of a combined antigen fragment of cat allergens FeI d 1 and FeI d 4 in the preparation of an antibody composition, characterized in that: The antibody composition is composed of the egg yolk antibody IgY according to any one of claims 6-7, lecithin, and a sucrose aqueous solution, and has a water-in-oil-in-water structure; The inner aqueous phase is a PBS solution of egg yolk antibody IgY. The outer aqueous phase is a 10% sucrose aqueous solution. The oil phase is lecithin.

9. Use of the combined antigen fragment of cat allergens FeI d 1 and FeI d 4 in preparing an antibody composition according to claim 8, characterized in that: The method for preparing the antibody composition is as follows: (1) Prepare the oil phase: dissolve lecithin in anhydrous ethanol at 37°C in a water bath to a lecithin concentration of 20 mg / ml. (2) Prepare the inner aqueous phase: Dissolve the polyclonal antibody powder in PBS to a concentration of 1.65 mg / ml. (3) Preparation of external aqueous phase: prepare 10% sucrose aqueous solution; (4) Preparation of water-in-oil: The aqueous phase in (2) was slowly added dropwise to the oil phase in (1), and sheared at 6000 rpm for 2 min using a high-speed shearing machine to form a water-in-oil structure to obtain a liposome solution containing polyclonal antibodies. The volume ratio of the aqueous phase to the oil phase was 4-5:

1. (5) Preparation of water-in-oil-in-water emulsion: The liposome solution containing the polyclonal antibody obtained in (4) was added as the dispersed phase to the external aqueous phase of (3), and sheared at 6000 rpm for 2 min to form a water-in-oil-in-water structure to obtain a sucrose-lecithin-polyclonal antibody W1 / O / W2 emulsion, with the volume ratio of the liposome solution to the external aqueous phase being 3-4:1; The S / N value of the polyclonal antibody in the obtained W1 / O / W2 emulsion of sucrose-lecithin-polyclonal antibody was 10-20.

10. Use of the combined antigen fragment of cat allergens FeI d 1 and FeI d 4 in preparing an antibody composition according to claim 9, characterized in that: The S / N value of the polyclonal antibody was 16.

11. Use of a combined antigenic fragment of cat allergens FeI d1 and FeI d4 in the preparation of wet cat food, characterized in that: The wet cat food contains the antibody composition according to any one of claims 8 to 10, and the added amount is 30% to 50% of the total weight of the cat food.

12. Use of the combined antigen fragment of cat allergens FeI d 1 and FeI d 4 in the preparation of wet cat food according to claim 11, characterized in that: The added amount is 45% of the total weight of the cat food.

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