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Antigen epitope based on fix and its application

An antigen epitope and antibody technology, applied in the field of biomedicine, can solve problems such as limited application, low specificity of polyclonal antibodies, and high cost of monoclonal antibodies

Active Publication Date: 2019-02-19
玥特农生物科技河北有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the monoclonal antibody is expensive and the preparation cycle is very long, which limits its application in the large-scale production of hFIX
Polyclonal antibodies can be used for the purification of antigens, and the preparation is simple, but because ordinary polyclonal antibodies are antibody mixtures produced by multiple B epitopes, the properties are not uniform, and the affinity is very strong. When it is used for purification, The elution conditions are difficult to master, and the target protein is often inactivated due to too harsh elution conditions
On the other hand, the specificity of the polyclonal antibody is often relatively low. When it is used to purify rhFIX in milk, it is likely to bind non-specifically to bovine endogenous FIX (bFIX), resulting in a reduced safety of the purified product. , therefore, it is not suitable for purifying hFIX (including pdhFIX and rhFIX)

Method used

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  • Antigen epitope based on fix and its application
  • Antigen epitope based on fix and its application
  • Antigen epitope based on fix and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0153] Example 1 Design of B cell antigen epitopes and preparation of fusion proteins

[0154] In this example, four B epitopes of hFIX were designed, and the basic information of these epitopes is shown in Table 1 and Table 2.

[0155] Table 1 Basic information of the designed hFIX B epitope

[0156]

[0157]

[0158] Homology of the B epitope sequence of hFIX designed in Table 2

[0159]

[0160] Table 3 Epitope grafting results

[0161]

[0162] 1. Epitope fusion protein preparation

[0163] 1.1 Primer design

[0164] Each FIX epitope gene sequence is as follows:

[0165] FIX-1: ATTGAGGAGACAGAACAT (SEQ ID NO.: 13)

[0166] FIX-2: AATGC AGCTATTAAT AAGTAC (SEQ ID NO.: 14)

[0167] FIX-3: CTCCACAAAGGGAGATCA (SEQ ID NO.: 15)

[0168] FIX-4: GTTGAA ACTGGTGTTA AAATT (SEQ ID NO.: 16)

[0169] DTT gene sequence:

[0170] ataaatcttgattgggatgtcataagggataaaactaagacaaagatagagtctttgaaagagcatggccctatcaaaaataaaatgagcgaaagtcccaataaaacagtatctgaggaaaaagctaaacaatacctagaag...

Embodiment 2

[0204] Activity detection of embodiment 2 fusion protein

[0205] 1 Epitope identification

[0206] 1.1. Mice immunization

[0207] 1) Feed 42 female Bal b / c mice aged 4-5 weeks.

[0208] 2) Prepare the amount of each protein according to the measured protein concentration and the required immunization dose, and mix it thoroughly with the same volume of aluminum hydroxide adjuvant (diluted 8 times).

[0209] 3) When the mice grew to 6 weeks, 42 6-week-old female Bal b / c mice were equally divided into 6 groups, wherein 2 groups were negative control groups and injected with DTT-GST and aluminum hydroxide adjuvant; The remaining 4 groups are the experimental groups, and the Bal b / c mice of each group are immunized with the corresponding epitope fusion protein respectively, and the groups are as follows:

[0210] Table 9 Mouse Immunization

[0211]

[0212] 4) After the antigen is ready, inject it according to the above table. The initial immunization adopts the method of su...

Embodiment 3

[0258] Example 3 Antibody Preparation

[0259] 1.1 Rabbit Immunization

[0260] 1) Prepare 6 male Japanese big-eared white rabbits aged 3-4 months and weighing about 2kg for immunization with FIX-2-DTT recombinant protein.

[0261]2) Antigen emulsification: Prepare the protein antigen according to the required immunization dose, add an equal amount of Freund's adjuvant, and fully shock and mix with an emulsifier. Freund's complete adjuvant was used for the initial immunization, and Freund's incomplete adjuvant was used for the booster immunization.

[0262] 3) For the initial immunization, each rabbit was injected with 1 mg of the emulsified antigen. Using the multi-point injection method, inject 6 points in the left neck of the rabbit, 6 points in the left front armpit, and 5 points in the left front shoulder. Before immunization, rabbits were fixed with an experimental rabbit fixer, and 20-100 μL of blood was collected from the ear vein and stored as a negative control. ...

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Abstract

The present invention discloses an FIX-based epitope and application thereof. In particular, the present invention provides an antigen peptide. The antigen peptide derive from animal FIX and comprises one or more epitopes. The length of an antigen peptide amino acid sequence is 2%-100% of the full length of the FIX, and the length of the antigen peptide is 5-500 amino acids; and recombinant protein formed by the antigen peptide and carrier protein can induce the animals of the same species to produce an immune response against the FIX. The fusion of the antigen peptide and the carrier protein can stimulate a strong humoral immune response, and produce specific antibodies against the FIX. The present invention further discloses specific identification, and a preparation method for FIX antibodies combined with human FIX antibodies. The antibodies prepared by the preparation method provided by the invention can be used for human FIX separation and purification, detection and treatment of diseases associated with epitopes.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a FIX-based antigenic epitope and its application. Background technique [0002] FIX is an important vitamin K-dependent glycoprotein involved in the blood coagulation cascade reaction in the blood coagulation system, and its deficiency or deficiency in the human body will lead to hemophilia B. Lifelong infusion of FIX preparations is the main treatment for hemophilia B, that is to say, hemophilia B patients cannot do without FIX treatment for life. very important. [0003] The main sources of FIX are the purification of FIX (pdhFIX) from healthy human plasma and the preparation of recombinant FIX (rhFIX) by genetic engineering techniques. Traditional pdhFIX purification methods include calcium phosphate adsorption, aluminum hydroxide adsorption, ion exchange, and heparin affinity purification. Because the concentration of hFIX in the blood is very low (the concentration of hFIX in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/745C07K19/00C07K16/36C12N15/12C12N15/62C12N15/13C12N15/70C12N1/21A61K39/00A61K39/395A61K47/42A61K48/00A61P7/02G01N33/68
Inventor 李荣秀杜进
Owner 玥特农生物科技河北有限责任公司
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