A Fel D1 and Fel D4 fusion protein and its application

Abstract

The present invention discloses a fusion protein of Fel D1 and Fel D4 and its application. The present invention obtains the fusion protein of Fel D1 and Fel D4 by prokaryotic expression. The fusion expressed protein greatly simplifies the operation procedures in terms of inducing protein expression, purification, concentration and immunization, and reduces the economic cost. The present invention provides a method for preparing egg yolk antibodies. The egg yolk antibodies obtained by immunizing laying hens with fusion proteins can react with Fel D1 and Fel D4 respectively, and the titer is higher than that of a single antigen protein immunization group and a combined immunization group. The present invention also provides a method for preparing a functional cat food. Egg yolk powder is added to the cat food in a certain proportion. After feeding for a period of time, the content of Fel D1 and Fel D4 in the cat's hair and saliva is significantly reduced, and the cat food prepared by the fusion protein egg yolk antibody can resist Fel D1 and Fel D4 at the same time, and the blocking effect is significantly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and specifically relates to a Fel D1 and Fel D4 fusion protein and application thereof. Background Art

[0002] About 10% to 20% of the world's population is allergic to cats, and allergic symptoms include sneezing, runny nose, itchy skin and watery eyes, and in severe cases can cause anaphylactic shock. Frequent cleaning and long-term use of anti-allergic drugs can reduce or prevent allergic reactions, and repeated injections of increasing doses of crude cat dander extracts may also help desensitize patients with cat allergies, but these methods are time-consuming and labor-intensive.

[0003] When the body first contacts the allergen, the body's immune cells process, handle and record the allergen. When the body contacts the same allergen again, the specific IgE on the surface of mast cells and basophils is activated, releasing active mediators such as histamine, which act on tissues and organs such as the skin, respiratory tract and digestive tract, causing capillary dilation, mucus secretion and smooth muscle contraction, ultimately leading to allergic reactions such as rhinitis, asthma, and even anaphylactic shock. Therefore, blocking the interaction between allergens and IgE is a key link in preventing and treating allergic reactions.

[0004] With the development of molecular biology technology, the molecular structures of many allergens have been determined. Eight cat allergen molecules have been recorded in the allergen database by the World Health Organization (WHO) and the International Immunology Society (IUIS). Most of these allergens are distributed in saliva and dander, and are spread to the hair and the environment when cats lick their hair or dander fall off. Felis catus (domestic cat) allergen 1 (Fel D1) is the cat allergen with the highest sensitizing activity, which can cause allergic reactions in about 93% of cat allergy patients. The frequency and sensitizing activity of Fel D4 recognized by human IgE are second only to Fel D1, and can cause allergic reactions in about 63% of cat allergy patients. Therefore, Fel D1 and Fel D4 are the focus of allergy prevention for patients with cat allergies. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0006] The invention provides a Fel D1 and Fel D4 fusion protein: CH1 and CH2 gene fragments of the Fel D1 gene are connected through a flexible linker to obtain a Fel D1 sequence, wherein the CH1 and CH2 gene fragments are sequences after the signal peptide is removed; the Fel D4 sequence is a sequence after the signal peptide is removed from the Fel D4 gene; and the Fel D1 and Fel D4 sequences are connected through a rigid linker, and then connected together with the gene sequence of a tag protein to an expression vector to obtain a recombinant plasmid, which is transformed into a prokaryotic expression strain, and the recombinant protein is induced to express and purified to obtain the Fel D1 and Fel D4 fusion protein.

[0007] As a preferred embodiment of the fusion protein of Fel D1 and Fel D4 of the present invention: the expression vector comprises pET-3a, pET-11a, pET-12a, pET-14b, pET-15b, pET-16b, pET-17b, pET-19b, pET-20b, pET-21a, pET-22b, pET-23a, pET-24a, pET-25b, pET-26b, pET-27b, pET-28a, pET-29a, pET-30a, pET-31b, pET-32a, pET- 33b, pET-34b, pET-35b, pET-37b, pET-39b, pET-40b, pET-41a, pET-42a, pET-43.1a, pET-44a, pET-45b, pET-47b, pET One of -48b, pET-49b, pET-50b, pET-51b, pET-52b, pETDuet-1, pACYCDuet-1, pCDFDuet-1, pRSFDuet-1, and pColaDuet-1.

[0008] As a preferred embodiment of the Fel D1 and Fel D4 fusion protein of the present invention: the tag protein includes one of polyhistidine (His), glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), streptavidin (SA), polyhistidine (Poly-His), small molecule ubiquitin-like modifier protein (SUMO), and hemagglutinin tag (HA).

[0009] As a preferred embodiment of the Fel D1 and Fel D4 fusion protein of the present invention: the prokaryotic expression strain includes one of BL21, cBL21, BL21 (DE3), BL21codonplus (DE3), BL21 Star (DE3), B834 (DE3), Tuner (DE3), Rosetta (DE3), Rosetta2 (DE3), Arctic Express (DE3), Origami (DE3), Origami2 (DE3), OrigamiB (DE3), SHuffle T7, ER2566, OverExpressC43 (DE3), BLR (DE3), HMS174 (DE3), GT115, MG1655, and NovaBlue (DE3).

[0010] As a preferred embodiment of the Fel D1 and Fel D4 fusion protein of the present invention: the induced expression comprises induced expression by 0.5-1 mM IPTG.

[0011] As a preferred embodiment of the Fel D1 and Fel D4 fusion protein of the present invention: the purification includes purifying the recombinant protein by affinity chromatography.

[0012] As a preferred embodiment of the Fel D1 and Fel D4 fusion protein of the present invention: the amino acid sequence of the Fel D1 and Fel D4 fusion protein is shown in SEQ ID NO:1.

[0013] The present invention also provides the use of the Fel D1 and Fel D4 fusion protein in immunizing laying hens to prepare yolk antibodies.

[0014] Preferably, the amount of egg yolk added to the cat food is 1-10wt%.

[0015] The present invention also provides the use of the Fel D1 and Fel D4 fusion protein in preparing a cat food additive: immunizing laying hens with the Fel D1 and Fel D4 fusion protein to obtain egg yolk containing yolk antibodies, and adding the egg yolk to cat food as a cat food additive.

[0016] Beneficial effects of the present invention: The present invention obtains Fel D1 & Fel D4 fusion protein by prokaryotic expression. The fusion expressed protein greatly simplifies the operation procedures in terms of inducing protein expression, purification, concentration and immunization, and reduces the economic cost. The present invention provides a method for preparing egg yolk antibodies. The egg yolk antibodies obtained by immunizing laying hens with Fel D1 & Fel D4 fusion protein can react with Fel D1 and Fel D4 respectively, and the titer is higher than that of the single antigen protein immunization group and the combined immunization group. The present invention also provides a method for preparing functional cat food. Egg yolk powder is added to cat food in a certain proportion. After feeding for a period of time, the content of Fel D1 and Fel D4 in cat hair and saliva is significantly reduced, and the cat food prepared by the fusion protein Fel D1 & Fel D4 egg yolk antibody can resist Fel D1 and Fel D4 at the same time, and the blocking effect is significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following briefly introduces the drawings required for describing the embodiment, wherein:

[0018] Figure 1 For PCR amplification of DNA fragments of Fel D1 (A), Fel D4 (B), and Fel D1&Fel D4 (C), the product sizes were 551 bp (including homology arms), 553 bp (including homology arms), and 1095 bp (including homology arms), respectively.

[0019] Figure 2 The expression of Fel D1 (A), Fel D4 (B) and Fel D1&Fel D4 (C) proteins was induced by IPTG.

[0020] Figure 3 The changes in yolk antibody titer after laying hens were immunized with recombinant proteins Fel D1, Fel D4, Fel D1+Fel D4 and Fel D1&Fel D4.

[0021] Figure 4 To study the changes in Fel D1 and Fel D4 levels in the saliva and hair of cats after feeding functional cat food. DETAILED DESCRIPTION

[0022] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0023] The present invention obtains recombinant fusion proteins: Fel D1 & Fel D4 by prokaryotic expression, and the yolk antibodies obtained after immunizing high-yielding laying hens can be used to develop functional cat food that specifically blocks cat allergens Fel D1 and Fel D4. The yolk antibodies obtained after immunizing high-yielding laying hens with the recombinant fusion proteins Fel D1 & Fel D4 can be used to develop functional cat food that specifically blocks cat allergens Fel D1 and Fel D4.

[0024] On one hand, the present invention provides a method for expressing cat allergens Fel D1, Fel D4 and Fel D1 & Fel D4 fusion proteins, wherein the Fel D1 gene, the Fel D4 gene, and the Fel D1 and Fel D4 tandem gene are connected to an expression vector, transformed into an expression strain, and induced to express. The detailed method comprises the following steps:

[0025] 1) The CH1 and CH2 gene fragments constituting Fel D1 were amplified from cat skin cDNA using PCR technology. The CH2 and CH1 gene fragments were concatenated with a flexible amino acid linker using overlap extension PCR technology to obtain the Fel D1 gene sequence (551 bp, including homology arms) ( Figure 1 A); The codon-optimized Fel D4 gene sequence (553 bp, including homology arms) was obtained by artificial synthesis ( Figure 1 B); Fel D1 and Fel D4 were concatenated with a rigid linker by overlap extension PCR technology to obtain the concatenated gene sequence of Fel D1 & Fel D4 (1095 bp, including homology arms) ( Figure 1 C).

[0026] 2) The pET-28a plasmid was linearized by Xho I and Nco I. The gene sequences of FelD1, Fel D4 and Fel D1&Fel D4 were connected to the linearized pET28a vector by homologous recombination technology to obtain recombinant plasmids pET28a-Fel D1, pET28a-Fel D4 and pET28a-Fel D1&Fel D4.

[0027] 3) The above recombinant plasmid vectors were transformed into Rosetta (DE3) expression strains by chemical transformation, and soluble recombinant proteins were obtained by induction with 0.5 mM IPTG (isopropylthiogalactoside) ( Figure 2 ).

[0028] 4) The above recombinant protein contains a His tag. The recombinant protein was purified using nickel column affinity chromatography, and the total protein was separated by SDS-PAGE gel and purified and identified by Coomassie brilliant blue staining.

[0029] The present invention also provides a method for preparing yolk antibodies by immunizing hens with the above-mentioned Fel D1, Fel D4 and Fel D1&Fel D4 recombinant proteins, comprising the following steps:

[0030] The above-mentioned Fel D1, Fel D4 and Fel D1&Fel D4 recombinant proteins were diluted with sterile PBS, and 200 μg of the recombinant protein was mixed with the immune adjuvant at a volume ratio of 1:1. High-yielding laying hens were immunized by subcutaneous injection in the neck. A blank control group (NC), Fel D1 group, Fel D4 group, Fel D1+Fel D4 group (Fel D1 protein and Fel D4 protein were mixed at a mass ratio of 1:1 for combined immunization) and Fel D1&Fel D4 (fusion protein) group were set.

[0031] Eggs from each group were collected, and the yolk antibody titer of each group at a specific time was determined by Elisa method. The laying hens in Fel D1 and Fel D4 groups produced corresponding yolk antibodies, and the hens in Fel D1+Fel D4 group and Fel D1&Fel D4 group immunized with histones produced yolk antibodies against both Fel D1 and Fel D4. The yolk antibody titer produced by laying hens in Fel D1&Fel D4 fusion protein group was significantly higher than that in single immunization group and combined immunization group ( Figure 3 ).

[0032] The present invention also provides a method for preparing functional cat food using the egg yolk antibody, comprising the following steps: uniformly mixing the egg yolk of the egg and maltodextrin of equal mass, drying at low temperature and crushing to obtain egg yolk powder.

[0033] The above-mentioned egg yolk powder was mixed into conventional cat food at a mass ratio of 5% to obtain functional cat food, which was divided into a blank control group (NC group), a group with anti-Fel D1 egg yolk antibody added (Fel D1 group), a group with anti-Fel D4 egg yolk antibody added (Fel D4 group), a group with anti-Fel D1 and anti-Fel D4 egg yolk antibody added (Fel D1+Fel D4 group) and a group with anti-Fel D1&Fel D4 fusion protein egg yolk antibody added.

[0034] After feeding the functional cat food to adult cats for a period of time, the content of Fel D1 and Fel D4 in the saliva and hair of the cats was detected by Elisa method. The corresponding content of Fel D1 and Fel D4 in the saliva and hair of the cats in the Fel D1 group and the Fel D4 group decreased significantly. The content of Fel D1 and Fel D4 in the saliva and hair of the cats in the Fel D1+Fel D4 group and the Fel D1&Fel D4 group decreased. At the same time, the content of Fel D1 and Fel D4 in the saliva and hair of the cats in the Fel D1&Fel D4 group decreased most significantly ( Figure 4 ).

[0035] Embodiment 1:

[0036] Expression method of cat allergens Fel D1, Fel D4 and their fusion protein Fel D1 & Fel D4:

[0037] 1) Construction of recombinant plasmid:

[0038] Total RNA was extracted from cat skin tissue using Trizol, reverse transcribed into cDNA, and then the corresponding fragments were amplified using primers at both ends of the CH1 and CH2 genes. CH2 and CH1 were connected using overlap extension PCR technology to obtain the Fel D1 gene fragment. Fel D4 gene fragment information was downloaded from the NCBI database and synthesized by Shanghai Shenggong Biotechnology Co., Ltd. after codon optimization. Fel D1 and Fel D4 fragments were connected by overlap extension PCR technology to obtain the Fel D1 & Fel D4 fusion fragment.

[0039] The pET28a plasmid was linearized using two fast-cutting enzymes, XhoI and EcoRI, and the linearized pET28a vector was connected with the Fel D1 fragment, the Fel D4 fragment, and the Fel D1 & Fel D4 fusion fragment respectively by homologous recombination.

[0040] The amino acid sequence of the recombinant Fel D1 protein is (SEQ ID NO: 2):

[0041]

[0042] The amino acid sequence of the recombinant Fel D4 protein is (SEQ ID NO: 3):

[0043]

[0044] The amino acid sequence of the recombinant Fel D1 & Fel D4 fusion protein is (SEQ ID NO: 1):

[0045]

[0046] 2) IPTG-induced protein expression and protein purification:

[0047] The three recombinant plasmids (pET28a-Fel D1, pET28a-Fel D4, and pET28a-Fel D1&Fel D4) were transformed into the Rosetta (DE3) expression strain by chemical transformation, and the positive strains after transformation were screened by kanamycin LB solid medium.

[0048] After inducing the expression of recombinant protein with 0.5 mM IPTG at 16°C for 24 h, the bacterial precipitate was obtained by centrifugation and the bacteria were resuspended in PBS. The bacteria were disrupted by cell ultrasonic disruptor, and the bacterial homogenate, homogenate supernatant (the supernatant after homogenate centrifugation), and homogenate precipitate (the precipitate after homogenate centrifugation) were collected. Protein loading buffer (4×) was added to the obtained protein sample and boiled at 100°C for 10 min to prepare SDS-PAGE gel protein sample. After SDS-PAGE gel electrophoresis, the protein sample was stained with Coomassie Brilliant Blue staining solution to observe the expression effect of the recombinant protein.

[0049] The recombinant protein (with His protein tag) was purified by nickel column affinity chromatography. The process was to wash the column with deionized water, balance the column with Lysis buffer, pass the sample through the column (repeat once), balance the column again, wash the column with 50 mM imidazole buffer (remove non-specific binding impurities), wash the column with 100 / 200 / 500 mM imidazole buffer (elute the target protein), wash the column with deionized water, and wash the column with 20% ethanol. All the liquids used to wash the column were filtered through a 0.22 μM pore size filter. The imidazole buffer formula was 20 mM NaH2PO4, 500 mM NaCl, and imidazole of the corresponding substance concentration, pH = 8.6. The purified protein sample was placed in an ultrafiltration tube and centrifuged and concentrated.

[0050] Embodiment 2:

[0051] Method for producing yolk antibodies by immunizing laying hens with the recombinant protein in Example 1:

[0052] 1) Immunization of laying hens

[0053] The recombinant antigen proteins of Fel D1, Fel D4, Fel D1+Fel D4 (Fel D1 and Fel D4 proteins were mixed at a mass ratio of 1:1 for joint immunization) and Fel D1&Fel D4 (fusion protein) purified and concentrated in Example 1 were diluted with sterile PBS. Specifically, each group of proteins was diluted with sterile PBS to a concentration of 400 μg / mL, mixed with an immune adjuvant at a volume ratio of 1:1, and 1 mL of the mixed solution (i.e., the antigen protein content in the mixed solution was 200 μg) was taken, and laying hens (n ​​= 6, 200-250 days old) at the peak of egg laying were immunized by subcutaneous injection in the neck. The specific immunization procedure is shown in Table 1.

[0054] Table 1

[0055]

[0056] 2) Yolk antibody titer determination

[0057] After the first immunization, the 0th, 1st, 2nd, 3rd, 4th, 5th, 6th, and 7th eggs of each group were collected, the egg surface was cleaned, the egg white and yolk were separated, and the titer of the yolk antibody in each group was determined by Elisa. The specific steps are as follows:

[0058] ① Cleaning and disinfection: First use clean water to clean the egg shell to remove surface dirt, and then use 75% alcohol to disinfect the egg shell.

[0059] ② Yolk separation: Open the eggs under sterile conditions and carefully transfer the yolk to a sterilized container.

[0060] ③ Yolk dilution: Dilute the egg yolk in a 2-fold ratio with sterilized PBS.

[0061] ④ Antigen coating: Recombinant proteins Fel D1 and Fel D4 were used as antigens, diluted to 10 μg / mL with coating solution, and added to the ELISA plate at 100 μL / well for overnight at 4°C. The antigen coating solution was carbonate buffer (PH=9.6).

[0062] ⑤ Blocking: discard the coating solution, wash with washing solution for 3 times, pat the ELISA plate dry, add 200 μL of blocking solution to each well, and incubate at 37℃ for 2 hours. The washing solution is PBST (0.5% Tween-20 in PBS, pH=7.4), and the blocking solution is 5% skim milk powder solution (solvent is PBST).

[0063] ⑥ Primary antibody incubation: discard the blocking solution, wash with washing solution 3 times, pat the ELISA plate dry, add the above diluted egg yolk to the ELISA plate, 100 μL / well, incubate at 37℃ for 1 h. The primary antibody diluent is PBS buffer containing 1% skim milk powder, pH=7.4.

[0064] ⑦ Enzyme-labeled secondary antibody incubation: discard the primary antibody diluent, wash with washing solution 3 times, pat the ELISA plate dry, add mouse anti-chicken IgY-HRP antibody, 100 μL / well, incubate at 37℃ for 1 h. The secondary antibody diluent is PBS buffer containing 1% skim milk powder, pH=7.4.

[0065] ⑧ Color development reaction: discard the secondary antibody diluent in the ELISA plate, wash with washing solution for 3 times, pat the ELISA plate dry, add 100 μL of color development solution to each well, and incubate at 37°C in the dark for 10 min.

[0066] ⑨ Collect signals: Add 100 μL of stop solution to each well and incubate at 37°C in the dark for 10 min. Use a microplate reader to measure the absorbance of each well at 450 nm. The OD450 nm of the sample well is recorded as P, and the OD 450 nm of the negative well is recorded as N. When P / N ≥ 2.1, the well is recorded as a positive well, and the maximum dilution factor of the positive well is recorded as the antibody titer.

[0067] Embodiment 3:

[0068] Method for preparing functional cat food using the egg yolk antibody obtained in Example 2:

[0069] 1) Preparation of egg yolk powder and functional cat food

[0070] Collect the eggs of each group of 28-49 days in Example 2, separate the egg white and the egg yolk. Mix the egg yolk and maltodextrin in equal amounts, dry at 45°C, and crush the dried mixture to obtain egg yolk powder. Mix the egg yolk powder into conventional cat food at a mass ratio of 5% to obtain the functional cat food.

[0071] 2) Effect testing of functional cat food

[0072] Forty adult cats were randomly divided into NC group, Fel D1 group, Fel D4 group, Fel D1+Fel D4 group and Fel D1&FelD4 group (n=5), and fed with functional cat food supplemented with corresponding egg yolk antibodies (egg yolk powder). Saliva and hair of cats in each group were collected at week 0, week 1, week 2, week 4 and week 8, and the contents of Fel D1 and Fel D4 in them were detected by Elisa method.

[0073] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A Fel D1 and Fel D4 fusion protein, characterized in that: The CH1 and CH2 gene fragments of the Fel D1 gene are connected through a flexible linker to obtain the sequence of Fel D1, wherein the CH1 and CH2 gene fragments are sequences after the signal peptide is removed; the sequence of Fel D4 is the sequence of the Fel D4 gene after the signal peptide is removed; after the sequences of Fel D1 and Fel D4 are connected through a rigid linker, they are connected together with the gene sequence of the tag protein to an expression vector to obtain a recombinant plasmid, which is transformed into a prokaryotic expression strain, and the recombinant protein is induced to express and purified to obtain a fusion protein of Fel D1 and Fel D4; the amino acid sequence of the fusion protein of Fel D1 and Fel D4 is shown in SEQ ID NO:

1.

2. The Fel D1 and Fel D4 fusion protein according to claim 1, characterized in that: The expression vector includes pET-28a.

3. The Fel D1 and Fel D4 fusion protein according to claim 1 or 2, characterized in that: The tag protein includes polyhistidine His.

4. The Fel D1 and Fel D4 fusion protein according to claim 1 or 2, characterized in that: The prokaryotic expression strains include Rosetta DE3.

5. The Fel D1 and Fel D4 fusion protein according to claim 1 or 2, characterized in that: The induced expression includes inducing expression by 0.5-1 mM IPTG.

6. The Fel D1 and Fel D4 fusion protein according to claim 1 or 2, characterized in that: The purification includes purification of the recombinant protein by affinity chromatography.

7. Use of the Fel D1 and Fel D4 fusion protein according to claim 1 in immunizing laying hens to prepare yolk antibodies.

8. Use of the Fel D1 and Fel D4 fusion protein according to claim 1 in preparing a cat food additive, characterized in that: The Fel D1 and Fel D4 fusion proteins are used to immunize laying hens to obtain egg yolks containing yolk antibodies, and the egg yolks are added to cat food as cat food additives.

9. The use according to claim 8, characterized in that: The amount of the egg yolk added to the cat food is 1-10wt%.

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