Cat major allergen Fel d 1 recombinant protein and application thereof

Abstract

The invention provides a cat major allergen Fed1 recombinant protein and an application thereof. The cat main allergen Fed1 recombinant protein has an amino acid sequence as shown in SEQ ID No.1, or is a protein which is obtained by substituting, deleting and / or adding one or more amino acids to the amino acid sequence as shown in SEQ ID No.1 and has the same activity with the amino acid sequence as shown in SEQ ID No.1. The recombinant protein provided by the invention can be used for preparing a detection reagent for detecting main anti-cat allergen IgE, and is used for detecting allergy such as cat hair.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant protein for preparing and detecting a major cat allergen Fel d 1 and an application thereof. Background Art

[0002] In the prior art, according to an epidemiological study on cat hair allergy in southern China, 5.81% of patients with suspected allergic diseases had a positive reaction to the allergen extracts detected by cat sIgE. At present, a variety of allergens have been found in extracts of cat dander, hair, saliva, sublingual glands, etc. Among them, Fel d 1 is the most important allergen, and more than 90% of patients who are allergic to cats have specific IgE antibodies against Fel d 1 in their serum. Fel d 1 is mainly secreted by the sublingual glands and sebaceous glands and is mainly present in saliva. When cats comb their hair, Fel d 1 will be transferred from saliva to hair. Dry saliva and dander will be widely dispersed into the environment in the form of fine airborne particles. These particles can be deposited in the respiratory tract and cause asthma symptoms.

[0003] At present, standardized cat dander allergens are often used in clinical practice to specifically diagnose dust mite allergy and perform desensitization treatment. However, due to the different sources of raw materials for making cat dander allergens, there are significant differences in the allergenic components in different extracts. At the same time, due to environmental influences, cat dander extracts may contain house dust mite allergens. Contaminated dust mite allergens may cause false positive results in skin tests of patients allergic to mites. How to prepare a recombinant cat dander allergen recombinant protein Fel d 1 antigen and immunoglobulin that can be used against cat dander allergens is of great significance. Summary of the invention

[0004] One object of the present invention is to provide a cat dander allergen FeI d 1 recombinant protein.

[0005] The second object of the present invention is to provide a gene encoding the cat dander allergen FeI d 1 recombinant protein.

[0006] The third object of the present invention is to provide the use of the protein in preparing a reagent for detecting anti-dandruff allergen IgE.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A cat dander allergen Fel d1 recombinant protein, the amino acid sequence of which is:

[0009] 1) the amino acid sequence shown in SEQ ID No.1; or

[0010] 2) Proteins having equivalent activity with substitution, deletion and / or addition of one or more amino acids in the amino acid sequence shown in SEQ ID No. 1.

[0011] The gene encoding the cat dander allergen FeI d1 recombinant protein as described above. Preferably, the nucleotide sequence is: (1) the nucleotide sequence shown in SEQ ID NO.2 of the sequence listing, or (2) the nucleotide sequence shown in SEQ ID No.2 which is substituted, deleted and / or added with one or more nucleotides, encoding the nucleotide sequence of the cat dander allergen recombinant protein.

[0012] Furthermore, the nucleotide sequence of the gene as described above is shown in SEQ ID No.3.

[0013] In addition, it should be understood that, considering the degeneracy of codons and the preference of codons in different species, those skilled in the art can use codons suitable for expression of a specific species as needed.

[0014] The gene of the present invention can be operably linked to an expression vector to obtain a recombinant expression vector capable of expressing the protein of the present invention, which can then be introduced into other bacterial species through transgenic methods well known in the art to enable them to have the ability to express the protein.

[0015] In one embodiment of the present invention, the nucleotide sequence shown in the synthetic SEQ ID No. 3 is inserted into the expression vector pET-22b to construct a recombinant expression vector pET-22b-fel d1 ch2+1, and finally transformed into the Escherichia coli expression host Arctic Express (DE3) so that it can be induced to express and obtain the cat dander allergen Fel d 1 recombinant protein.

[0016] Furthermore, the present invention also provides a detection reagent for detecting anti-cat dander allergen IgE, which comprises the cat dander allergen recombinant protein. The cat dander allergen recombinant protein can be coated on an ELISA plate to prepare an ELISA detection reagent, or coated on colloidal gold particles to prepare a colloidal gold detection paper, etc., an immunological detection reagent.

[0017] The beneficial effects of the present invention are:

[0018] The cat dander allergen recombinant protein provided by the present invention has the ability to specifically bind to anti-cat dander allergen antibodies and can be used for the detection of significantly increased immunoglobulin E (IgE) levels. It does not require expensive instruments and equipment, has a simple sample pretreatment process, and has very low technical requirements for analysts, and is suitable for on-site rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Electrophoresis diagram for enzyme digestion identification.

[0020] Figure 2 SDS-PAGE analysis was performed for protein expression identification.

[0021] Figure 3 SDS-PAGE analysis for protein purification.

[0022] Figure 4 For protein SDS-PAGE identification and analysis.

[0023] Figure 5 Western Blot analysis for protein identification

[0024] Figure 6 Four-parameter fitting standard curve. DETAILED DESCRIPTION

[0025] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the present invention all belong to the scope of the present invention.

[0026] Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the reagents used in the examples of the present invention are all analytically pure or above, and can be commercially available.

[0027] Example 1 Preparation of FeI d protein and immunization of mice

[0028] (I) Construction of plasmid

[0029] The full-length splicing primers were designed based on the PAS (PCR-based Accurate Synthesis) method, and protective bases were designed at both ends of the primers. The gene feld1ch2+1 (containing two chains of double-stranded dimers, chain1 and chain 2) was synthesized, and its sequence was shown in SEQ ID NO.3, which was connected between the NdeI-XhoI sites of the vector pET-22b. The obtained recombinant plasmid pET-22b-feld1ch2+1 was transferred into the Escherichia coli TOP10 cloning strain (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.), and the positive clones were selected for sequencing.

[0030] SEQ ID NO.3: GCCACTCAGCTTCCTTTCGGGCTTTGTTAGCAGCCG GATCTCAGTGGTGGTGGTGGTGGTG CTCGAGTCAATGGTGATGATGATGATGTTCCAGACACAGCGGGCTGGTATAGATTTTATCCAGCACGCTCA GTGCATTTTCTTTATCTTCTTCGGTCATTTTGGCATCAACGCAATTTTTCAGAATGCGTGCATTTTCCAGAACCAC CGGCAGTGCTTTATACTGTGCCACCTGTTCAACATATTCATCCGGGGTACCGGTCAGAAACAGATCCACATCACGT TTCACTGCCGGACAAATTTCGCGGCCCAGGGTATTCAGTTTCAGATCTTCCACGGTATTCTGCACTGCTTCGCCCA TGCAATCTTTGCTGCTACTAATGGTGGTCATAACCAGGCCATCCAGAACACGACTAATCAGGCCATTTTCCACATA GCAATCCTGAATCTTTTTCATGGCGGTACGTTCCGGTTCGGTTGCATTAACTTTGGTCAGACTCAGATCCAGCAGCAGTTCATTGCCATTGGCAACTGCAAAGAAAACATCATAGAAAATCGGGCAGGTTTCGGCCATCTTAACCATATG TATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTGTTATCCGCTCACAATTCCCCTATAGTGAGTCGTATTAATTTCGCGGGATCGAGATCTCGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAAGATCGGGCTCGCCACTTTCGGGCTCATGAGC.

[0031] 2. Plasmid digestion identification

[0032] The obtained recombinant plasmid pET-22b-Fel d 1ch2+1 plasmid was double-digested with restriction endonucleases NdeI and MluI. The digestion system was plasmid 3μL, endonuclease NdeI 0.25μL, endonuclease MluI 0.25μL, 10×Buffer 1.0μL, DDW up to 10μL. The digested product was subjected to agarose gel electrophoresis. The results are as follows: Figure 1 As shown in the figure; Marker: 250, 500, 750, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 8000, 12000bp, gene name: Feld 1ch2+1 (OD260 / OD280: 1.86) enzyme digestion identification: NdeI-MluI, M: Marker; Band 1: plasmid before enzyme digestion; Band 2: plasmid after enzyme digestion. Two bands are produced after enzyme digestion. According to the DNA molecular weight marker of the reference standard, the approximate sizes of the two bands are about 1000bp and 4000bp respectively. This shows that the target plasmid is divided into two fragments of different sizes after being cut by NdeI and MluI enzymes, and the band of about 1000bp is consistent with the target band.

[0033] (II) Inducible expression of recombinant protein

[0034] The positive recombinant plasmid pET-22b-fel d1 ch2+1 was transferred into the expression host Escherichia coli Arctic Express (DE3) competent cells. The specific operation is as follows:

[0035] (1) Add 1 μL of the positive recombinant plasmid into 100 μL of E. coli Arctic Express (DE3) competent cells and place on ice for 20 min.

[0036] (2) Heat shock at 42°C for 90 seconds, place on ice for 5 minutes, and add 600 μL LB culture medium.

[0037] (3) Shake at 37°C, 220 rpm for 1 h. After centrifugation, spread the entire volume on an LB plate containing 50 μg / mL Amp and culture inverted at 37°C overnight.

[0038] 2. Lactose-induced expression of pET22b-Fel d 1 vector fusion protein

[0039] (1) Pick a single clone from the transformation plate and inoculate it into a test tube containing 3 mL LB culture medium containing 50 μg / mL Amp, and shake at 37°C, 220 rpm, overnight.

[0040] (2) The next day, the cells were inoculated at a ratio of 1:100 into 30 mL of lactose autoinduction medium containing 50 μg / mL Amp and shaken at 37°C at 220 rpm until the OD600 of the cells reached 0.6-0.8.

[0041] (3) Take out 1 mL of culture, centrifuge at 10,000 rpm for 2 min at room temperature, discard the supernatant, and resuspend the bacterial pellet in 100 μL of 1× loading buffer.

[0042] (4) The remaining culture was shaken at 20°C and 220 rpm overnight to induce the expression of the fusion protein.

[0043] (5) Take out 1 mL of culture, centrifuge at 10,000 r / min at room temperature for 2 min, discard the supernatant, and resuspend the bacterial pellet with 100 μL 1× loading buffer. Centrifuge the remaining culture at 4,000 r / min for 10 min, discard the supernatant, and resuspend the bacterial pellet with PBS; after ultrasonically disrupting the resuspended liquid, take the supernatant and the pellet separately and add the loading buffer to resuspend.

[0044] (6) Perform 12% SDS-PAGE analysis and Coomassie Brilliant Blue staining to visualize the bands. The results are as follows: Figure 2 As shown, M in the figure is the protein molecular weight standard, and each band is 1: uninduced, 2: induced, 3: supernatant after induced disruption, 4: precipitate after induced disruption. The results show that the target protein is mainly present in the supernatant.

[0045] (III) Ni column affinity purification of fusion protein and result analysis

[0046] 1. Ni column purification

[0047] (1) Using a low-pressure chromatography system, the supernatant solution after ultrasonic disruption was loaded onto a Ni-IDA-Sepharose Cl-6B affinity chromatography column pre-equilibrated with Ni-IDA Binding-Buffer at a flow rate of 0.5 mL / min.

[0048] (2) Flush with Ni-IDA Binding-Buffer at a flow rate of 0.5 mL / min until the OD280 value of the effluent reaches the baseline.

[0049] (3) Wash with Ni-IDA Washing-Buffer (20 mM Tris-HCl, 30 mM imidazole, 0.15 M NaCl, pH 8.0) at a flow rate of 1 mL / min until the OD280 value of the effluent reaches the baseline.

[0050] (4) Elute the target protein with Ni-IDA Elution-Buffer (20 mM Tris-HCl, 250 mM imidazole, 0.15 M NaCl, pH 8.0) at a flow rate of 1 mL / min and collect the effluent.

[0051] (5) The collected protein solution was added to a dialysis bag and dialyzed with PBS overnight.

[0052] (6) 12% SDS-PAGE analysis

[0053] The target protein was obtained by Ni column affinity purification and analyzed by 12% SDS-PAGE. Figure 3 , 4 shown. Figure 3 M: protein molecular weight standard, 1: sample after crushing, 2: outflow, 3-5: elution. Figure 4 M: protein molecular weight standard, 1: 0.5 mg / ml BSA, 2: purified sample.

[0054] (7) Western Blot analysis

[0055] Take 5 μL of the target protein sample obtained by Ni column affinity purification and load it for Western Blot analysis. The primary antibody used was mouse anti-His antibody (1:1000 dilution), and incubated at 4°C overnight. The secondary antibody was goat anti-mouse secondary antibody (1:5000 dilution), and reacted at 37°C for 1 hour. The results are shown in Figure 5 As shown, where M: protein molecular weight standard, 1: purified sample. The results show that the target protein (containing His tag) obtained by Ni column affinity purification can specifically bind to the anti-His antibody.

[0056] The Feld 1 protein was obtained by Ni column purification. The amino acid sequence of the recombinant Feld protein is shown in SEQ ID NO.1, and the nucleotide sequence encoding the protein is shown in SEQ ID NO.2, which is the bases at positions 62-586 in SEQ ID NO.3.

[0057] SEQ ID NO.1: MVKMAETCPIFYDVFFAVANGNELLLDLSLTKVNATEPERTAMKKIQDCYVENGLISRVLDGLVMTTISSSKDCMGEAVQNTVEDLKLNTLGREICPAVKRDVDLFLTGTPDEYVEQVAQYKALPVVLENARILKNCVDAKMTEEDKENALSVLDKIYTSPLCLE。

[0058] SEQ ID NO.2: CTCGAGTCAATGGTGATGATGATGATGTTCCAGACACAGCGGGCTGGTATAGATTTTATCCAGCACGCTCAGTGCATTTTCTTTATCTTCTTCGGTCATTTTGGCATCAACGCAATTTTTCAGAATGCGTGCATTTTCCAGAACCACCGGCAGTGCTTTATACTGTGCCACCTGTTCAACATATTCATCCGGGGTACCGGTCAGAAACAGATCCACATCACGTTTCACTGCCGGACAAATTTCGCGGCCCAGGGTATTCAGTTTCAGATCTTCCACGGTATTCTGCACTGCTTCGCCCATGCAATCTTTGCTGCTACTAATGGTGGTCATAACCAGGCCATCCAGAACACGACTAATCAGGCCATTTTCCACATAGCAATCCTGAATCTTTTTCATGGCGGTACGTTCCGGTTCGGTTGCATTAACTTTGGTCAGACTCAGATCCAGCAGCAGTTCATTGCCATTGGCAACTGCAAAGAAAACATCATAGAAAATCGGGCAGGTTTCGGCCATCTTAACCATATG。

[0059] Example 2 ELISA experimental verification example:

[0060] In this example, the FeI d 1 recombinant protein described in Example 1 was used to construct an ELISA detection reagent for 11 positive serum samples (numbered 1-11) and 3 normal human serum samples (numbered 12-14). The standard operating procedures of indirect ELISA were followed to implement the verification and detect the level of specific IgE (sIgE) antibodies against the major cat allergen FeI d 1 in the serum. The serum samples used above were serum from patients who visited the outpatient department of the Department of Allergy (Allergy) Reaction of the Peking Union Medical College Hospital, Chinese Academy of Medical Sciences. The diagnosis results were confirmed by experienced clinicians based on medical history, skin test, and serum specific IgE (>0.35KUA / L was positive).

[0061] 1. ELISA experimental steps

[0062] 1) Coating: Coat with the FeI d 1 recombinant protein described in Example 1 at a coating amount of 2 μg / ml, 10 ml / plate. Prepare the coating antigen amount according to the actual usage; add 100 μl / well of coating antigen and coat overnight at 4°C.

[0063] 2) Washing: Wash the plate 5 times with TBST and pat dry on absorbent paper.

[0064] 3) Blocking: 300 μl / well of 0.5% BSA was used as blocking solution for blocking at 37°C for 2 h.

[0065] 4) Washing: Wash the plate 5 times with TBST and pat dry on absorbent paper.

[0066] 5) Add samples and standards: 100 μl / well serum samples and standards were added and incubated at 37°C for 30 min. Standard S9 was a serum sample (50 kUA / L) determined by the gold standard for clinical Fel d 1sIgE detection (ImmunoCAP Phadia 1000). S8-S1 were serum samples diluted with PBS buffer in equal multiples of S9, with concentrations of 25, 12.5, 6.25, 3.13, 1.57, 0.79, 0.40, and 0.20 KUA / L, respectively. S0 was PBS buffer.

[0067] 6) Washing: Wash the plate 5 times with TBST and pat dry on absorbent paper.

[0068] 7) Adding secondary antibody: Use mouse anti-human IgE Fc secondary antibody labeled with horseradish peroxidase (abcam, ab99806) at a volume ratio of 1:1000 between antibody and 0.5% BSA diluent, and incubate at 37°C for 1 hour.

[0069] 8) Washing: Wash the plate 5 times with TBST and pat dry on absorbent paper.

[0070] 9) Color development: 100 μl / well of TMB color development solution was added. After 10 min of color development, 100 μl / well of stop solution was added and the plate reader was used to read the results at 450 nm.

[0071] 2. Analysis of ELISA results

[0072] 1) Positive and negative judgment standard: According to the standard concentration and OD450nm four-parameter fitting standard curve ( Figure 6 ), substitute the OD450nm result of the sample to be tested into the back-calculated concentration, and if the test result is >0.35KUA / L, it is judged as positive.

[0073] 2) By calculation, as shown in Table 2. Using the FeI d 1 recombinant protein of Example 1 as the coating antigen to detect 11 positive sera and 3 normal human sera, the positive detection rate was 100% and the negative detection rate was 100%.

[0074] Table 2 Human serum sIgE test results

[0075]

[0076]

[0077] The results show that the FeI d recombinant protein prepared by the present invention has antigenicity and can be used to detect the immunoglobulin E (IgE) level specifically against the major cat allergen FeI d 1, and assist in the clinical diagnosis of cat hair allergy and disease typing.

Claims

1. A cat dander allergen Feld1 recombinant protein, characterized in that: Its amino acid sequence is: 1) the amino acid sequence shown in SEQ ID No.1; or 2) Proteins having equivalent activity with substitution, deletion and / or addition of one or more amino acids in the amino acid sequence shown in SEQ ID No.

1.

2. A gene encoding the cat dander allergen Fel d1 recombinant protein according to claim 1.

3. The gene as claimed in claim 2, whose nucleotide sequence is shown as SEQ ID No.

2.

4. The gene as claimed in claim 2, whose nucleotide sequence is shown as SEQ ID No.

3.

5. An expression vector containing the gene according to any one of claims 2 to 4.

6. A host cell transformed by the expression vector according to claim 5. The host cell according to claim 6 , which is Escherichia coli.

8. A detection reagent containing the protein according to claim 1.

9. Use of the protein according to claim 1 in the preparation of an IgE detection reagent for detecting anti-cat dander allergens.

10. The use according to claim 9, characterized in that The detection reagent is ELISA detection reagent or colloidal gold detection test paper.

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