CN-122005776-A - Method for improving antigen immunogenicity

Abstract

The invention relates to the field of animal immunity, in particular to a method for improving the immunogenicity of an antigen. The invention covalently couples the antigen with high homology with BCG vaccine to form antigen-BCG vaccine complex, and immunity is realized by intravenous injection. The method is simple, fusion expression of the protein or polypeptide and other proteins or coupling of a special co-stimulatory sequence are not needed, and the coupling method and the injection way are both mature technology in the industry. With reference to the method of the invention, the immunogenicity of the antigen with high homology can be obviously improved.

Inventors

• LI GUILIN
• HOU PENGYUN
• SHI RUIQI
• DAI CHUNYING
• ZHAO QIAOHUI
• FU GUANGYU
• WU XUEWEI
• YANG ZENGLI

Assignees

• 郑州伊美诺生物技术有限公司

Dates

Publication Date

20260512

Application Date

20260228

Claims (10)

1. 1. The method for improving the immunogenicity of the high-homology antigen for non-diagnosis and treatment is characterized by comprising the steps of covalently coupling the high-homology antigen and the BCG vaccine to obtain an antigen-BCG vaccine complex, and inoculating the antigen-BCG vaccine complex into an animal to be immunized to obtain an antigen with improved immunogenicity; the amino acid sequence homology of the high homology antigen and the corresponding protein or the corresponding polypeptide of the animal to be immunized is more than 30 percent.
2. 2. The method of claim 1, wherein the covalent coupling is based on a carbodiimide type condensing agent.
3. 3. The method of claim 2, comprising the steps of: Mixing a buffer solution containing the high-homology antigen with a buffer solution containing BCG vaccine, and then mixing the buffer solution with the carbodiimide condensing agent for reaction to obtain a reaction product; dialyzing the reaction product to obtain the antigen-BCG vaccine complex; and (3) mixing the antigen-BCG vaccine complex, normal saline and an immune adjuvant, emulsifying, and inoculating the animal to be immunized to obtain the antigen with improved immunogenicity.
4. 4. A method according to claim 2 or claim 3 wherein the carbodiimide condensing agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
5. 5. The method of any one of claims 1 to 4, wherein the means of inoculation is intravenous injection.
6. 6. The method of claim 5, wherein the intravenous injection is a tail vein injection.
7. 7. The method of any one of claims 1 to 6, wherein the highly homologous antigen is INHA- βa and the animal to be immunized is a mouse.
8. 8. The method of claim 7, wherein the amino acid sequence of INHA- βa is set forth in SEQ ID No. 1.
9. 9. A method for producing an antibody, characterized in that it comprises inoculating said animal to be immunized according to the method of any one of claims 1 to 8, obtaining an immunized animal, and isolating said antibody from said immunized animal.
10. 10. A method for preparing a hybridoma cell, comprising inoculating said animal to be immunized according to the method of any one of claims 1 to 8, obtaining an immunized animal, and preparing said hybridoma cell from said immunized animal.

Description

Method for improving antigen immunogenicity Technical Field The invention relates to the field of animal immunity, in particular to a method for improving the immunogenicity of antigens with high homology. Background Antigen (Ag) refers to all substances that induce an immune response in the body. I.e., a substance that is specifically recognized and bound by antigen receptors (TCR/BCR) on the surface of T/B lymphocytes, activates T/B cells, proliferates and differentiates them, generates immune response products (sensitized lymphocytes or antibodies), and specifically binds to the corresponding products in vivo and in vitro. Conventional immunoadjuvants such as Freund's adjuvant, whose components include liquid paraffin, lanolin, BCG or inactivated Mycobacterium tuberculosis, are widely used in animal immunization experiments, and induce a strong immune response to most immunogens (including soluble proteins, viruses, cells, parasites, polysaccharides, etc.). However, certain protein or polypeptide antigens have very high homology of amino acid sequences with corresponding proteins or polypeptides of animals to be immunized, generally more than 30%, and even up to 90% -100%. For these antigens with higher amino acid sequence homology, it is difficult for conventional immunoadjuvants to effectively induce a strong specific immune response in immunized animals, mainly because of the poor immunogenicity of the antigen. Traditional methods of enhancing the immunogenicity of antigens include optimization of adjuvants. As in patent CN1343482a, the bacillus calmette-guerin vaccine is injected into the animal to be immunized before or simultaneously with the vaccine immunization, and the bacillus calmette-guerin vaccine is used for replacing the traditional medicine immunopotentiator, so that the immunization effect of the vaccine injection is effectively enhanced. However, the method only verifies that when animal vaccines such as swine fever vaccines and chicken coccidiosis vaccines are used as immunogens, the immunogenicity improving effect on high-homology antigens is unknown on young pigs with 20-40 days collar or young chickens with 7-14 days collar. Or by conjugating poorly immunogenic antigens to foreign macromolecules used as carriers to increase the immunogenicity of these antigens. As in patent CN115484977a, a protein or polypeptide is conjugated to a saccharide to form a saccharide-protein/polypeptide antigen conjugate, which has increased immunogenicity compared to the unconjugated protein/polypeptide antigen, preferably the saccharide species is streptococcus pneumoniae capsular polysaccharide. Because of the weak immunogenicity of saccharide substances, few methods for conjugating antigens with weak immunogenicity with saccharide species with weak immunogenicity to improve the immunogenicity of antigens have been reported, and the practical application effect of the method is still to be observed. Disclosure of Invention In view of the above, the invention provides a method for improving the immunogenicity of the antigen with high homology, and the method provided by the invention can obviously improve the immunogenicity of the antigen with high homology relative to unconjugated antigen, unconjugated antigen and BCG vaccine or antigen injected by a non-special way. In order to achieve the above object, the present invention provides the following technical solutions: The invention provides a method for improving the immunogenicity of a high-homology antigen, which comprises the steps of covalently coupling the high-homology antigen with bacillus calmette-guerin to obtain an antigen-bacillus calmette-guerin complex, and inoculating the antigen-bacillus calmette-guerin complex into an animal to be immunized to achieve the aim of improving the immunogenicity of the high-homology antigen; The amino acid sequence homology of the high homology antigen and the corresponding protein or the corresponding polypeptide of the animal to be immunized is 30%, 40% or more than 50%. In some embodiments of the present invention, said bacillus calmette-guerin of the above method refers to live bacterins made from attenuated bacillus calmette-guerin suspensions. In some embodiments of the invention, the covalent coupling of the above-described method is based on a carbodiimide-type condensing agent to effect covalent coupling; The carbodiimide condensing agent is coupled through carboxyl-amino, and can be at least one of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC.HCl), N ' -Dicyclohexylcarbodiimide (DCC), N ' -Diisopropylcarbodiimide (DIC) and N-cyclohexyl-N ' - (2-morpholinoethyl) carbodiimide methyl p-toluenesulfonate (CMC); the carbodiimide type condensing agent can also be coupled through amino-mercapto, and can be 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC). In some embodiments of the present invention, the mass ratio of the antigen with high homology to t