CN110563844A

CN110563844A - Polyclonal antibody against canine interleukin 31 receptor and application thereof

Info

Publication number: CN110563844A
Application number: CN201910831713.5A
Authority: CN
Inventors: 丁一; 丁明星; 曲燕燕
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2019-09-04
Filing date: 2019-09-04
Publication date: 2019-12-13

Abstract

The invention relates to the field of biological immunity, in particular to a polyclonal antibody for resisting a canine interleukin 31 receptor and application thereof. A preparation method of polyclonal antibody of anti-canine interleukin 31 receptor comprises the following steps: coupling the anti-canine interleukin 31 receptor with BSA carrier protein, then taking blood of immune animals to prepare antiserum, and finally purifying the antiserum to obtain the polyclonal antibody of the anti-canine interleukin 31 receptor. The application of the polyclonal antibody against the canine interleukin 31 receptor and the application of the polyclonal antibody against the canine interleukin 31 receptor in the canine atopic dermatitis injection. The polyclonal antibody against the canine interleukin 31 receptor can treat the canine atopic dermatitis and inhibit pruritus and inflammation.

Description

Polyclonal antibody against canine interleukin 31 receptor and application thereof

Technical Field

The invention relates to the field of biological immunity, in particular to a polyclonal antibody for resisting a canine interleukin 31 receptor and application thereof.

Background

Atopic Dermatitis (AD) is a common chronic inflammatory skin disease characterized by infiltration of the skin with T cells, eosinophils, and mast cells. The intense itching, skin inflammation and other atopic symptoms of AD reduce the quality of life of pets and their owners. Pruritus is a basic characteristic and a major symptom of dog atopic dermatitis, and seriously affects the quality of life of dogs and their owners.

Cytokines are the most important factors mediating the irritation of itching and inflammation. Recent studies have found that IL-31 has been identified as an important cytokine in the induction of itch in dogs. Interleukin-31 (IL-31) is a four-helix bundle cytokine belonging to the IL-6 cytokine family, which signals through a heterodimeric receptor composed of the oncostatin M receptor (OSMR) and the IL-31 receptor alpha (IL-31RA) subunit. The JAK enzymes are attached to the intracellular domain of the IL-31 receptor and are present in various tissues of the body, including the skin and the peripheral and central nervous systems. The cytokine IL-31 which promotes pruritus and inflammation is combined with a receptor, activates an intracellular JAK-STAT pathway, generates more bioactive substances which mediate pruritus and inflammation, and leads to the vicious circle of 'pruritus-scratch-inflammation'. Based on the existing study on the relationship between IL-31 signaling and AD pruritus, IL-31 signal blocking is considered as an effective target for treating atopic dermatitis. We have therefore prepared a polyclonal antibody that specifically binds to IL-31RA and inhibits its signalling, to treat pruritus due to atopic dermatitis.

At present, glucocorticoid and antiallergic drugs such as dexamethasone, mometasone furoate cream and loratadine are used for treating dog atopic dermatitis, but due to the chronic and long-term property of AD, a plurality of side effects are caused. Short-term hormone use can cause side effects such as polydipsia, diuresis, polyphagia, behavior change and the like, and can further influence the liver function of animals and interfere the endocrine system. Long-term use of hormones can cause systemic irreversible damage. Antihistamines are of little or no help in the treatment of acute onset canine atopic dermatitis, and antihistamines are not safe and harmless. Immunosuppressive agents such as cyclosporin have a slow onset of action, and it takes about 4 to 6 weeks to show clinical effects. Has no significant benefit for acute pruritus or atopic dermatitis in acute attack stage, and has adverse effects such as emesis.

disclosure of Invention

In order to solve the above problems, it is an object of the present invention to provide a polyclonal antibody against canine interleukin 31 receptor and use thereof, capable of treating canine atopic dermatitis, inhibiting itching and inflammation thereof.

In order to achieve the purpose, the antigen of the polyclonal antibody is the anti-canine interleukin 31 receptor, the base sequence of the anti-canine interleukin 31 receptor is shown as SEQ ID No.1, and the amino acid sequence of the anti-canine interleukin 31 receptor is shown as SEQ ID No. 2.

A preparation method of polyclonal antibody of anti-canine interleukin 31 receptor comprises the following steps: the anti-canine interleukin 31 receptor of claim 1 is coupled with BSA carrier protein, and then the immune animal is subjected to blood sampling to prepare antiserum, and finally the antiserum is purified to obtain the polyclonal antibody against the canine interleukin 31 receptor.

the application of the polyclonal antibody against the canine interleukin 31 receptor and the application of the polyclonal antibody against the canine interleukin 31 receptor in the preparation of the injection for the canine atopic dermatitis.

An injection for canine atopic dermatitis comprising the polyclonal antibody against canine interleukin 31 receptor according to claim 1.

Drawings

FIG. 1 is an SDS-PAGE detection of polyclonal antibodies against canine interleukin 31 receptor;

FIG. 2 is a western blot assay of polyclonal antibodies against the canine interleukin 31 receptor;

Lane 1 is a sample of IL-31RA polyclonal antibody, and lane 2 is blank negative serum

FIG. 3 is a graph of the increase of immune serum for detecting IL-31RA protein by indirect ELISA;

FIG. 4 is a graph of antibody titers of canine anti-IL-31 RA protein immune sera measured by indirect ELISA;

FIG. 5 is a graph comparing the itch scores of test dogs with control dogs;

FIG. 6 is a graph comparing skin thickness of test dogs to control dogs;

FIG. 7 is a histological section of skin tissue before treatment of dogs;

Fig. 8 is a histological section of skin tissue after treatment of dogs.

Detailed Description

For a better understanding of the present invention, reference will now be made in detail to the present invention, examples of which are illustrated in the accompanying drawings.

EXAMPLE 1 obtaining of antigenic proteins

The IL-31RA short peptide coupled with BSA is used as the antigen protein, and the small peptide/hapten must be coupled with carrier protein (KLH, BSA, Ova) to obtain high-efficiency antibody. In this embodiment, BSA carrier protein is cloned into plasmid by genetic engineering technology, and the BSA carrier protein is obtained after expression of transformed escherichia coli, and the specific cloning and purification process is as follows:

(1) first, the IL-31RA ectomembrane sequence gene synthesized by the same company was obtained. The cloned IL-31RA gene has a nucleotide sequence shown in SEQ ID No.1, and SEQ ID No.2 is a corresponding amino acid sequence.

(2) Cloning the obtained IL-31RA gene to a pet42b vector, transforming a recombinant plasmid into escherichia coli BL21(DE), coating an agar plate containing kanamycin, growing at 37 ℃, selecting a single colony to inoculate in a liquid culture medium containing kanamycin after the colony grows out, and growing at 37 ℃ and 200rpm overnight; the next day, inoculating the strain into a fresh LB culture medium containing kanamycin at a ratio of 1:100, and growing the strain at 37 ℃ until the OD value is between 0.6 and 0.9; 1M IPTG (isopropyl-. beta. -D thiogalactopyranoside) was added at a final concentration of 0.5mM, and the mixture was induced at 37 ℃ and 200rpm for 6 hours; centrifuging at 5000rpm and 4 deg.C for 10min, repeatedly freezing and thawing the thallus for three times, and ultrasonically crushing; centrifuging at 1000rpm and 4 ℃ for 10 minutes; dissolving the inclusion body with pH8.0, 0.02M PBS (sulfate buffer), 6M guanidine hydrochloride buffer, centrifuging at 1000rpm and 4 ℃ for 10 minutes; the Ni-metal affinity column was equilibrated with buffer solutions of pH8.0, 0.02M PBS and 8M urea, the solubilized inclusion bodies were passed through the column, IL-31RA was adsorbed, washed with buffer solutions containing 0.1M NaCl, pH8.0, 0.02M PBS and 8M urea to remove impure proteins, eluted with buffer solutions containing 0.5M NaCl, pH8.0, 0.02M PBS and 8M urea to obtain canine IL-31RA protein, diluted to 1mg/ml with buffer solutions of pH8.0, 0.02M PBS and 8M urea, and stored at-20 ℃.

(3) Preparation of BSA-coupled IL-31RA short peptides

Coupling of BSA to sulfo-SMCC

(taking 20mg of coupled polypeptide as an example, scaling the volume of all reagents according to the actual coupling amount of an experiment in the same ratio) weighing 10mg of BSA (Pierce), and dissolving in 1ml of ultrapure water to prepare a [10mg/ml BSA solution ]; weighing 5mg of Sulfo-SMCC, and dissolving in 1ml of ultrapure water to prepare a [5mg/ml Sulfo-SMCC solution ]; mixing the two solutions in equal volume, reacting at room temperature (25 ℃) for 60 minutes, and uniformly stirring slowly by using a magnetic stirrer for reaction; and after the reaction is finished, filling the reaction solution into a 10kD dialysis bag, dialyzing in a PBS (pH7.2) solution to remove excessive sulfoc-SMCC, and changing the solution every 3 hours for at least 3-4 times to obtain the activated BSA carrier.

3.2. Conjugation of polypeptides

Weighing 20mg of polypeptide, dissolving in 5ml of cross-linking buffer solution to prepare 4mg/ml of polypeptide solution; mixing the activated BSA carrier with a 4mg/ml polypeptide solution at room temperature of 25 ℃ for 4 hours; finally, the free unconjugated polypeptide was removed by dialysis in PBS (pH7.2) using a 10kD dialysis bag, and the solution was changed at least 4 times. Stirring and dialyzing on a magnetic stirrer every 2 hours, adjusting to a proper concentration, subpackaging into small tubes, and storing at-20 ℃ to obtain the BSA coupled IL-31RA short peptide with the amino acid sequence shown as SEQ ID No. 3.

EXAMPLE 2 preparation of IL-31RA polyclonal antibody

(1) Preparation of antigen for immunization: the BSA coupled IL-31RA protein obtained in example 1 was used as an antigen, and 200. mu.g of the antigen for immunization and a mixture of an equal amount of a water-soluble adjuvant were injected subcutaneously in five portions with 200. mu.L of suction.

(2) Immunization procedure: breeding a female 10kg beagle dog (purchased from Hubei Yisincerity Biotechnology Co., Ltd.) for several weeks, and then inoculating the beagle dog with 5-10 inoculating points subcutaneously; the booster was then given every 15 days for a total of 4 boosts.

(3) Blood collection: 2ml of blood is collected by vein before the first immunization, and negative control is carried out. Blood was collected 1 time before each booster and 100ml of blood was collected in jugular vein 10 days after the 5 th booster.

(4) Separating serum: the antibody was purified by Protein A affinity chromatography. Then dialyzed to obtain a solution containing a polyclonal antibody against canine IL-31RA, which was stored at 4 ℃ for a short period and at-20 ℃ for a long period.

Example 3 ELISA Titer detection of IL-31RA polyclonal antibody

The ELISA titer detection of the IL-31RA polyclonal antibody comprises the following steps:

(1) The antigen was coated with coating buffer CBS (0.05M carbonate buffer, pH9.6) at a concentration of 1. mu.g/ml, 100. mu.L/well was added to the microplate, and the microplate was sealed with a cover film and coated overnight at 4 ℃.

(2) The plates were spun off, the plates were patted dry, and 50. mu.L of blocking solution (coating buffer CBS containing 2% BSA) was added to each well for 2h at room temperature.

(3) The plate was washed 3 times with washing solution PBST (PBS containing 0.05% Tween-20, pH7.4), and the hyperimmune positive serum and blank negative serum to be tested were diluted 1: 2-fold in a dilution of antibody (PBS containing 1% BSA, pH7.4) in succession starting from 1:500, 100. mu.L/well, and incubated at 37 ℃ for 1 hour.

(4) the plate was washed 3 times with washing solution, 100. mu.L of secondary antibody working solution (HRP-labeled rabbit anti-canine IgG diluted with antibody diluent 1: 3000) was added to each well, and incubated at 37 ℃ for 1 h.

(5) Taking out, patting to dry, washing the plate for 3 times by using a washing solution, adding 100 mu L of single-component TMB substrate into each hole, and incubating for 10min at room temperature.

(6) Add 50. mu.L of stop buffer to each well.

(7) Absorbance at 450nm was read on a microplate reader.

(8) As shown in FIG. 3 and FIG. 4, the antibody titer was measured to be 1:1024000, indicating that the polyclonal antibody prepared by the present invention has a strong specific reaction with IL-31 RA.

Example 4 detection of binding reaction of polyclonal antibody and antigen protein by Western Blot immunoblotting experiment

(1) The BSA coupled IL-31RA protein obtained in example 1 was applied to 10. mu.L of the sample at a concentration of 1mg/ml, and subjected to 10% SDS-PAGE. The gel protein bands were transferred to nitrocellulose membranes in a conventional manner.

(2) Western Blot immunoblotting using the polyclonal antibody obtained in example 2 as a primary antibody and enzyme-labeled rabbit anti-canine IgG as a secondary antibody showed specific bands as shown in FIGS. 1 and 2, in FIG. 2, a lane 1 shows that BSA-conjugated IL-31RA binds to the polyclonal antibody obtained in example 2, and a lane 2 shows that BSA-conjugated IL-31RA binds to negative serum. The polyclonal antibody prepared by the invention has stronger specific reaction with the IL-31RA coupled with BSA.

EXAMPLE 5 polyclonal antibodies against the canine Interleukin 31 receptor (IL-31RA) for the treatment of canine atopic dermatitis

(1) Dogs with atopic dermatitis were injected intravenously with 1ml/kg of polyclonal antibody against canine interleukin 31 receptor (IL-31 RA). The control group was injected with physiological saline.

(2) The 4 times per day were selected and their itch scores (manifestation of itch behavior: licking/chewing of paws, flank and/or anal area, grabbing flank or neck, grabbing floor, shaking head and rapid movement of buttocks over the cage floor, etc.) were recorded periodically, and a "yes/no" classification decision was made every 1 minute. The cumulative number of times a "yes" is made during each observation period is the itch score.

(3) the results are shown in fig. 5, which indicates that the itch score of the test group dogs was significantly lower than that of the control group dogs.

(4) the skin thickness was measured before and after treatment of the dogs and the results are shown in figure 6.

(5) Tissue sections were prepared from skin tissue before and after dog treatment, and the results are shown in fig. 7 and 8. In combination with FIG. 7 and FIG. 8, in which the black arrows indicate eosinophils, it can be seen that there is a significant reduction in eosinophils after treatment.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

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Claims

1. The polyclonal antibody for resisting the canine interleukin 31 receptor is characterized in that the antigen of the polyclonal antibody is the canine interleukin 31 receptor, the base sequence of the canine interleukin 31 receptor is shown as SEQ ID No.1, and the amino acid sequence of the canine interleukin 31 receptor is shown as SEQ ID No. 2.

2. A method for preparing a polyclonal antibody against canine interleukin 31 receptor, comprising the steps of: the anti-canine interleukin 31 receptor of claim 1 is coupled with BSA carrier protein, and then the immune animal is subjected to blood sampling to prepare antiserum, and finally the antiserum is purified to obtain the polyclonal antibody against the canine interleukin 31 receptor.

3. The application of the polyclonal antibody against the canine interleukin 31 receptor is characterized in that the polyclonal antibody against the canine interleukin 31 receptor is applied to the canine atopic dermatitis injection.

4. An injection for canine atopic dermatitis comprising the polyclonal antibody against canine interleukin 31 receptor according to claim 1.