CN113521312A - Nasal cavity stimulating reagent based on recombinant dust mite Der f2 protein - Google Patents

Nasal cavity stimulating reagent based on recombinant dust mite Der f2 protein Download PDF

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CN113521312A
CN113521312A CN202110794266.8A CN202110794266A CN113521312A CN 113521312 A CN113521312 A CN 113521312A CN 202110794266 A CN202110794266 A CN 202110794266A CN 113521312 A CN113521312 A CN 113521312A
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dust mite
nasal cavity
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徐枫
张利能
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Zhongshan Hospital Fudan University
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Abstract

The invention relates to a nasal cavity stimulating reagent based on recombinant dust mite Der f2 protein, and belongs to the technical field of biological medicines. The invention provides an application of recombinant dust mite Der f 2-based protein in preparation of an allergen nasal cavity excitation reagent; and an application of the recombinant dust mite Der f 2-based protein in preparing a diagnostic reagent for diagnosing allergic rhinitis. The agent comprises spray, drop or injection; the present invention demonstrates that it can cause degranulation of mast cells in vivo by providing key allergenic peptide fragments of recombinant dust mite proteins. The protein is applied to a nasal cavity excitation experiment to check whether the nasal mucosa has corresponding IgE, so that the diagnosis can be made definitely. The recombinant protein can be prepared into standard concentration, meets the requirement of concentration gradient, and ensures the safety and accuracy of the nasal cavity excitation test.

Description

Nasal cavity stimulating reagent based on recombinant dust mite Der f2 protein
Technical Field
The invention relates to a nasal cavity stimulating reagent based on recombinant dust mite Der f2 protein, and belongs to the technical field of biological medicines.
Background
Allergic rhinitis is a common disease with the gradually rising morbidity, the specific diagnosis needs to be triggered by an allergen nasal cavity, a nasal mucosa triggering test (NPT) is the currently internationally accepted gold standard for diagnosing the allergic rhinitis allergen, and the NPT is widely applied to the evaluation of the effectiveness of a therapeutic medicament and the research of the pathophysiological mechanism of the nose caused by the stimulation of the allergen. Dust mites are common respiratory allergens in south china. The allergen currently has no standardized nasal cavity stimulating agent. Therefore, there is a great need in the art to obtain a dust mite-like respiratory allergen for use in allergen nasal cavity challenge tests.
Disclosure of Invention
The invention aims to solve the technical problem of how to obtain a respiratory tract allergen of a dust mite species for an allergen nasal cavity excitation test.
In order to solve the problems, the technical scheme adopted by the invention is to provide an application of recombinant dust mite Der f2 protein in preparation of an allergen nasal cavity excitation reagent.
The invention provides an application of recombinant dust mite Der f 2-based protein in preparation of a diagnostic reagent for diagnosing allergic rhinitis.
Preferably, the agent comprises a spray, drops or injection.
Compared with the prior art, the invention has the following beneficial effects:
the present invention demonstrates that it can cause degranulation of mast cells in vivo by providing key allergenic peptide fragments of recombinant dust mite proteins. The protein is applied to a nasal cavity excitation experiment to check whether the nasal mucosa has corresponding IgE, so that the diagnosis can be made definitely. The recombinant protein can be prepared into standard concentration, meets the requirement of concentration gradient, and ensures the safety and accuracy of the nasal cavity excitation test. Thus obtaining a respiratory allergen of a dust mite species for use in allergen nasal cavity challenge tests.
Drawings
FIG. 1 is a gel electrophoresis pattern of purified Der f2 protein.
FIG. 2 is a Der f2 specific IgE standard curve.
FIG. 3 is a flow chart of animal experiment sensitization and excitation.
FIG. 4 shows eosinophil infiltration in the observation of nasal mucosal epithelium of a mouse model.
Wherein a is a photo micrograph of a hematoxylin-eosin stained section of nasal mucosa of a control guinea pig; b is a light micrograph of a hematoxylin-eosin stained section of the nasal mucosa of an experimental guinea pig; panel c is a photomicrograph of control guinea pig lung tissue hematoxylin-eosin stained sections; d is a light micrograph of a hematoxylin-eosin stained section of lung tissue of an experimental guinea pig;
Detailed Description
In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings:
the invention provides an application of recombinant dust mite Der f 2-based protein in preparation of an allergen nasal cavity excitation reagent.
The invention provides an application of recombinant dust mite Der f 2-based protein in preparation of a diagnostic reagent for diagnosing allergic rhinitis. The agent comprises spray, drop or injection.
Examples
1. Materials and methods
1.1. Material
1.1.1. Serum of dust mite allergic patients 26 patients with allergic rhinitis received common allergen skin prick test, wherein 24 patients with positive dust mite prick reaction and 2 patients with negative dust mite reaction. 5mLl venous blood was drawn, and serum was isolated and stored at-80 ℃. Der f2 specific IgE positive serum disks (panel) were from the company PlasmaLab International and GRER LABORATORIES, INC.
1.1.2. Clinical prick test materials: the dust mite allergen pricking liquid and the pricking needle are Allergopharma products.
BCA protein quantification kit was purchased from Pierce, Inc.
1.1.4. Purified wild type Der f2 was expressed prokaryotic in dust mite protein.
ELISA reagents and material coated reagents: 0.05M carbonate buffer (pH 9.0). Washing liquid: 1PBS, Tween-20 to 0.02% concentration was added. Sealing liquid: a solution containing 3% BSA was prepared with the washing solution. HRP-labeled anti-human IgE antibody, TMB substrate solution, and 2N H2SO4 stop solution were purchased from KPL corporation. 96-well microplate-wells were purchased from Nunc. Other reagents and commodities are imported, subpackaged or domestic analytically pure. The microplate reader is a product of Bio-Tek company.
1.2. Experimental methods
Der f2 prokaryotic expression and purification
And designing a primer according to the sequence of the second component of the dermatophagoides farinae allergen protein. The upstream and downstream primer sequences are added with EcoR I and BamH I sites respectively to facilitate connection with a pBV220 vector. And (3) amplifying the coding sequence by using cDNA of the second component of the dermatophagoides farinae allergen protein as a template and using a PCR method. And (3) cutting the adhesive tape where the specific PCR strip is located after agarose gel electrophoresis of the PCR product, and extracting the PCR product according to the adhesive recovery kit. The PCR product recovered from the gel was ligated with the T-vector. And transforming the ligation product into DH5 alpha competent cells, selecting white colony shake bacteria through blue-white spot screening, slightly extracting plasmids, performing double enzyme digestion on the recombinant T vector by EcoRI + BamH I, and connecting the Der f2DNA fragment with the pBV220 plasmid. The ligation product is transformed into DH5 alpha, and the transformed bacteria are subjected to sequencing detection. BL21 strain is transformed by recombinant plasmid to induce expression.
Inoculating the engineering bacteria mother liquor into 2YT culture solution according to the volume ratio of 1%, culturing until OD600 is 0.8, and inducing and culturing for 6 h. The bacterial pellet is suspended in 50mL bacterial lysate; after ultrasonic bacteria breaking, centrifuging at 12000rpm at 4 ℃ for 30min, and removing supernatant. After washing the inclusion body precipitate, the inclusion body precipitate was dissolved in a denaturing solution. The Der f2 was purified and renatured on-line using an Amersham FPLC system. Protein peaks were collected at 1mL per tube by A280 assay. After silver staining by Tricine-SDS-PAGE, the peak of Der f2 protein was combined.
Transferring the Der f2 protein elution solution to an Amicon Ultra-15 ultrafiltration tube, and centrifuging at 5000rpm and 4 ℃ for 30 min; the resulting mixture was replaced 4 times with 12mL of ultrafiltration replacement solution. Finally, the mixture is concentrated to 1mL, and is subpackaged and stored at the temperature of 80 ℃ below zero after the quantification of the BCA protein.
1.2.2. Skin prick test
Common allergens were pricked on the forearm of each patient, along with histamine and 0.9% sodium chloride solution, as positive and negative controls, respectively, according to the protocol of the Allergopharma kit. The reaction was observed 20min after prick and judged positive when the diameter of the skin mound was greater than that of the skin mound pricked with histamine.
ELISA-coated proteins were diluted to a concentration of 60ng.mL-1 in coating solution, 100. mu.L was added to each well, and coated overnight at 4 ℃. The washing was performed 3 times for 5min each time with washing solution. And (3) sealing: add 300. mu.L of blocking solution to each well and block overnight at 4 ℃. Reaction: mu.L of serum or 5-fold diluted serum was added to each well and reacted at 37 ℃ for 2 hours. The washing solution was used for 4 washes, each for 5 min. HRP-labeled anti-antibody reaction: mu.L of 1 diluted HRP-anti-human IgE at 3000 was added to each well and reacted at 37 ℃ for 2 hr. The washing solution was used for 4 washes, each for 5 min. Substrate coloration: mu.L of substrate solution was added to each well, and after reaction at 37 ℃ for 15min, 100. mu.L of stop solution was added. Reading: OD570-OD450 values were read at 570nm and 450nm wavelengths.
1.2.4 sensitization and challenge experiments:
guinea pigs were injected intraperitoneally on days 1 and 8 with 200 μ g wt Der f2 protein and 2.5mg aluminum hydroxide gel (al (oh)3 gel) in a total volume of 0.5 mL. Control guinea pigs were injected intraperitoneally with saline (N.S.) and 2.5mg of Al (OH)3 gel in a total volume of 0.5 mL. On day 15, successful sensitization was confirmed by skin reactions at the injection site 30 minutes after challenge. Animals with no obvious erythema and edema response were excluded from the experimental group.
Sensitized guinea pigs were placed in a clear acrylic chamber and challenged on days 16 and 23 by exposure to a wt-derf2 aerosol (4mL of 1mg/mL saline) generated by an ultrasonic nebulizer for 30 minutes. The aerosol was delivered to the room by an air pump (300 mL/min). Control guinea pigs were nebulized with saline.
Guinea pigs were challenged intensively by bilateral nasal instillation of (II) wt Der f 220. mu.L (adsorbed on Al (OH)3 gel at a concentration of 20. mu.g protein/0.5 mg Al (OH)3 gel per nostril) [12] on days 19, 26 and 30. To prevent rapid elimination of antigen by ciliary body movement, the nasal mucosal surface was anesthetized by inhalation of a mixture of 4% lidocaine hydrochloride solution for 2 minutes prior to each sensitization. The control group was applied to the nose with physiological saline plus alumina gel. The flow chart for sensitization and challenge is shown in figure 3.
2. Results
Construction of pBV220-wt Der f2 recombinant plasmid
Wt Der f2 and mu Der f2 were inserted into the vectors pBV220, respectively, to construct pBV220-wt Der f2 and pBV220-mu Der f2 recombinant plasmids. Cleavage with EcoR I and BamH I yielded a fragment of the same size as the Der f2 gene. After DNA sequencing verification, the result shows that the DNA sequence is consistent with the sequence of NCBI.
2.2. Expression and purification of recombinant protein of Dermatophagoides farinae Der f2
The wt Der f2 protein formed inclusion bodies under induction conditions at 42 ℃. After the thalli are subjected to ultrasonic disruption, the inclusion bodies are fully washed to remove soluble protein and membrane protein. And (2) dissolving the inclusion bodies in a denaturing solution, carrying out Sephacryl S-100HR column chromatography separation and renaturation, removing impure proteins in the inclusion bodies by using a molecular sieve, and simultaneously correctly folding the wt Der f2 protein through continuous adsorption-dissociation of a filler medium to obtain the renatured wt Der f2 protein.
2.3. Linear range and sensitivity of detection method
A standard curve was obtained with PlasmaLab dust mite Derf2 allergen specific IgE standard serum (1.66IU.mL-1- -100IU.mL-1) (FIG. 2). The measured OD value and the Der f 2-specific IgE concentration were subjected to regression analysis, and the value of the regression coefficient R2 was 0.9965. According to the ELISA sensitivity calculation method, the standard solution of 0IU.mL-1 is tested for 10 times, the mean value (x) and the standard deviation(s) of the optical density of the zero dose point are obtained, the value of x +2s is subjected to interpolation operation, and the sensitivity value of 0.73IU.mL is obtained-1。
2.4 repeatability of detection method-Intra-and inter-batch CV%
With low value (4 IU.mL)-1) Median value (12 IU.mL)-1) And high value serum (22iu. ml)-1) An intra-batch and an inter-batch repeatability experiment were performed. Each of the experiments was conducted 10 times in each of the lots, and the coefficient of variation CV values were as shown in Table 1.
TABLE 1 repetitive IgE ELISA detection method for Dermatophagoides farinae allergen Der f2 specificity
Figure BDA0003162291840000051
2.5 detection of clinical serum samples
In 26 patients with allergic rhinitis, 24 patients showed positive prick reaction of dust mite, and 2 patients showed negative prick reaction. The concentration of Der f2 specific IgE in the serum of 24 patients who have positive prick reaction is analyzed by an ELISA kit using wild Der f2 as a coating material, and the serum is divided into positive and negative according to the international sIgE classification standard. As a result, 22 positive samples were obtained, and the positive rate was 91.7% (Table 2).
TABLE 2 comparison of skin prick test and Der f2 specific IgE ELISA detection method
Figure BDA0003162291840000052
2.6 guinea pig experimental nasal symptom assessment:
nasal symptoms were evaluated independently on days 19 and 26 by two observers blinded to the experimental group. After 10 minutes of acclimation, the guinea pigs were observed for sneezing, nasal rubbing and frequency of rhinorrhea events for more than 10 minutes. The score criteria for each symptom were 0-3 points (table 3). When the score exceeded 5 points, the allergic rhinitis model was considered successful.
Figure BDA0003162291840000053
Figure BDA0003162291840000061
2.7 detection of eosinophils in nasal mucosa and lung tissue:
as shown in FIG. 4, photomicrographs of hematoxylin-eosin stained sections of nasal mucosa (panels a and b) or lung tissue (panels c and d) of control guinea pigs (panels a and c) or experimental guinea pigs (panels b and d).
Guinea pigs were sacrificed 2h after the last intranasal stimulation. Nasal and lung tissues were taken, fixed by slow aeration with formalin buffer and then paraffin embedded. Tissue sections (2-4 μm thick) were stained with hematoxylin and eosin solution. Histopathological evaluation was performed on randomized sections to detect eosinophil (magnification x 400) infiltration in nasal mucosa and lung tissue. The experimental results show that: the allergic rhinitis model was successful.
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

Claims (3)

1. An application of recombinant dust mite Der f2 protein in preparing allergen nasal cavity exciting reagent.
2. An application of recombinant dust mite Der f2 protein in preparing a diagnostic reagent for diagnosing allergic rhinitis.
3. The use of a recombinant dust mite Der f 2-based protein according to claim 1 for the preparation of an allergen nasal cavity challenge reagent, wherein: the agent comprises spray, drop or injection.
CN202110794266.8A 2021-07-14 2021-07-14 Nasal cavity stimulating reagent based on recombinant dust mite Der f2 protein Pending CN113521312A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114504659A (en) * 2022-01-29 2022-05-17 叶菁 Nasal cavity excitation kit and preparation method and application thereof
CN117298297A (en) * 2023-09-26 2023-12-29 广州医科大学附属第一医院（广州呼吸中心） Dust mite nose excitation kit and preparation method and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
M. YASUE ET AL: "Effects of oral hyposensitization with recombinant Der f2 on immediate airway constriction in a murine allergic model", 《EUR RESPIR J》 *
肖浩 等: "鼻腔黏膜激发试验的临床应用及研究进展", 《中国耳鼻咽喉头颈外科》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114504659A (en) * 2022-01-29 2022-05-17 叶菁 Nasal cavity excitation kit and preparation method and application thereof
CN114504659B (en) * 2022-01-29 2024-04-02 叶菁 Nasal cavity excitation kit and preparation method and application thereof
CN117298297A (en) * 2023-09-26 2023-12-29 广州医科大学附属第一医院（广州呼吸中心） Dust mite nose excitation kit and preparation method and application thereof

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