CN-122011184-A - Egg yolk antibody for resisting canine interleukin and preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses an anti-canine interleukin egg yolk antibody, and a preparation method and application thereof. The yolk antibody is prepared by taking an immunocompetent epitope peptide of canine interleukin-31 as an immunogen, wherein the epitope peptide is obtained by predicting a key epitope of IL-31 through ANTIGENIC tools and optimizing in series, and a recombinant expression vector is constructed after a coding gene is optimized by an escherichia coli codon, and the recombinant expression vector is induced to express and purified to obtain a high-purity antigen. After basic immunity and twice boosting immunity, egg yolk antibody is extracted and purified from egg yolk, and ELISA is superior to that of commercial antibody prepared with canine IL-31 protein. The yolk antibody can specifically bind with canine IL-31, obviously reduce the histamine level in the serum of itchy dogs, effectively relieve the symptoms of atopic dermatitis related itching, provide a high-efficiency and safe biological therapeutic preparation for canine IL-31 mediated itching diseases, and have wide clinical application prospects.

Inventors

• MENG XIANWEI
• ZHANG ZHENGGUO
• CHEN YAN
• SHANG DIANMING
• LI PENG

Assignees

• 长春市中保牧生物科技有限公司

Dates

Publication Date

20260512

Application Date

20260228

Claims (6)

1. 1. The yolk antibody for resisting the canine interleukin is characterized in that the yolk antibody is prepared by taking canine interleukin-31 epitope peptide as an immunogen, and the amino acid sequence of the epitope peptide is shown as SEQ ID NO. 2.
2. 2. A nucleic acid molecule encoding the epitope peptide of claim 1, wherein the nucleotide sequence is set forth in SEQ ID No. 3.
3. 3. A recombinant expression vector comprising the nucleic acid molecule of claim 2, wherein the expression vector is a pET-28a (+) vector digested with BamHI and XhoI, and the recombinant expression vector is designated pET-28a-IL-31-P-His.
4. 4. A recombinant engineering bacterium, which is characterized by comprising the recombinant expression vector of claim 3, wherein the engineering bacterium is escherichia coli BL21 (DE 3).
5. 5. The method for preparing the egg yolk antibody against canine interleukin according to claim 1, which is characterized by comprising the following steps: (1) Connecting the nucleic acid molecule of claim 2 with a pET-28a (+) vector to construct a recombinant expression vector, transforming escherichia coli BL21 (DE 3), and purifying by Ni2+ -NTA affinity chromatography after induced expression to obtain recombinant IL-31-P epitope peptide with the purity of more than or equal to 96%; (2) Taking the recombinant IL-31-P epitope peptide prepared in the step (1) as an immunogen, emulsifying with an oil phase to prepare a vaccine, and performing basic immunization and two boosting immunization on the laying hen, wherein the basic immunization and the boosting immunization dose are 1 mL/dose, and the two boosting immunization are 14 days apart; (3) The extraction and purification of egg yolk antibody, which is to collect the eggs of immunized chickens, separate and dilute the egg yolk, carry out PEG-6000 gradient precipitation and crude extraction, and then carry out dialysis and purification to obtain the egg yolk antibody for resisting the interleukin of dogs.
6. 6. Use of the egg yolk antibody against canine interleukin of claim 1 in one or more of the following: (1) Preparing a biological agent for neutralizing canine interleukin-31; (2) Preparing a medicament for reducing serum histamine levels in dogs; (3) Preparing a medicament for treating canine interleukin-31 mediated atopic dermatitis; (4) Preparing the medicine for treating the canine pruritus disease.

Description

Egg yolk antibody for resisting canine interleukin and preparation method and application thereof Technical Field The invention relates to a method, belongs to the technical field of biological medicine, in particular to preparation and application of an antibody medicament, and particularly relates to a yolk antibody for canine interleukin-31 and a preparation method thereof, and application of the antibody in treating IL-31 mediated pruritus diseases such as canine atopic dermatitis. Background With the rapid development of pet raising industry, the incidence of canine skin diseases is rising year by year, wherein atopic dermatitis is one of the most common chronic inflammatory skin diseases, the core symptom of which is severe pruritus, the quality of life of dogs is seriously affected, and great trouble is brought to pet owners. The research shows that the canine interleukin-31 (IL-31) is a key cytokine for mediating canine skin itch, and is a core pathogenic factor of canine atopic dermatitis and other itch diseases by combining with receptors on the surfaces of skin keratinocytes and mast cells, activating inflammatory signal paths, promoting the release of itch-causing mediators such as histamine and the like, and inducing itch perception and cutaneous inflammation injury. Thus, the development of agents that specifically block canine IL-31 biological activity has become a critical break-through for the treatment of such diseases. At present, the treatment means for the canine pruritus disease mainly comprises glucocorticoid, antihistamine, immunosuppressant and the like, but the medicaments have obvious limitations that the long-term use of the glucocorticoid can easily cause side effects of obesity, osteoporosis, immunosuppression and the like of dogs, the antihistamine has limited curative effect on IL-31 mediated pruritus, and the traditional immunosuppressant has the problems of wide action targets, insufficient safety and the like. In recent years, egg yolk antibodies (IgY) have been receiving a great deal of attention in the field of animal disease treatment due to their advantages of low preparation cost, weak immunogenicity, strong specificity, etc. The egg yolk antibody is a specific antibody produced in egg yolk after hen is immunized by specific antigen, has the characteristics of simple preparation process, mass production, no residual risk and the like, and has been applied to the prevention and treatment of various animal diseases. However, the existing antibody preparation technology for canine IL-31 still has the defects that the traditional method mostly adopts complete IL-31 protein as an immunogen, but the complete protein has the problems of low titer, low specificity and the like of the prepared antibody caused by masking antigen epitopes, unbalanced immunogenicity and the like, and part of the technology adopts a prokaryotic expression system to prepare the immunogen, but does not carry out accurate screening and optimization on antigen epitopes or carry out codon optimization to adapt to the expression system, so that the antigen expression quantity is low, the solubility is poor and the preparation efficiency and the activity of the antibody are further influenced. In addition, the existing antibody has the problems of limited treatment effect, non-lasting relieving of pruritus symptoms and the like in clinical application, and is difficult to meet clinical treatment requirements. Therefore, the anti-canine IL-31 egg yolk antibody with accurate epitope, high antibody titer and remarkable treatment effect is developed, and has important significance for solving the clinical treatment problem of canine pruritus disease. Disclosure of Invention Aiming at the problems of large side effect, low titer, limited treatment effect and the like of the existing anti-IL-31 antibody of the traditional medicament for treating the canine pruritus disease, the invention provides the anti-canine interleukin yolk antibody and the preparation method and application thereof, and the key epitope of canine IL-31 is precisely screened, the antigen preparation process is optimized, the high-titer and high-specificity yolk antibody is obtained, and the safe and effective treatment of the canine IL-31 mediated pruritus disease is realized. In order to achieve the above object, the present invention provides the following technical solutions: The invention provides a canine interleukin-31 (IL-31) epitope peptide, the amino acid sequence of which is shown as SEQ ID NO. 2. The invention provides a yolk antibody for resisting canine interleukin, which is prepared by taking canine interleukin-31 (IL-31) epitope peptide as an immunogen, wherein the amino acid sequence of the epitope peptide is shown as SEQ ID NO. 2. The invention provides a nucleic acid molecule for encoding the canine interleukin-31 (IL-31) epitope peptide in the technical scheme, and the nucleotide sequence is shown as SEQ ID NO. 3. The in