CN105294857A - FIX-based epitope and application thereof - Google Patents

Abstract

The invention discloses a FIX-based antigenic epitope and its application. Specifically, the present invention provides an antigenic epitope peptide, which is derived from animal FIX and contains one or more antigenic epitopes, and the amino acid sequence length of the antigenic epitope peptide is 2 times the full length of FIX. -100%, and the length of the antigenic epitope peptide is 5-500 amino acids; and the recombinant protein formed by the antigenic epitope peptide and carrier protein can induce the same kind of animal to produce an immune response against FIX. The fusion of the antigenic epitope peptide and the carrier protein can stimulate a strong humoral immune response and produce specific antibodies against FIX. The invention also discloses an antibody that specifically recognizes and binds to human FIX and a preparation method of the FIX antibody. The antibody can be used for the separation and purification of human FIX, detection and treatment of diseases related to the antigenic epitope.

Description

FIX-based epitope and its application

technical field

The invention relates to the field of biomedicine, in particular to a FIX-based antigenic epitope and its application.

Background technique

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.

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 healthy human plasma is about 5 μg/mL), and the physicochemical properties of this type of coagulation protein are very similar, therefore, the purity of hFIX prepared by these traditional methods is always very low, often Contains FII, FVII, FX and protein C, etc. Since hemophiliacs need life-long medication, repeated injections of this low-purity hFIX preparation will increase the risk of thrombosis and thromboembolism. Although a few methods can produce hFIX with higher purity, they cannot be applied to the industrial production of hFIX due to their high cost or low recovery rate. In the development of rhFIX, it has been reported that the active rhFIX has been successfully expressed through the milk system, but in terms of its large-scale separation and purification, no effective method has been found so far. Some methods tried, including ion-exchange chromatography, heparin affinity chromatography, or the use of several methods in series, failed to achieve good results, often either inactivating rhFIX or resulting in extremely low recovery or purity due to numerous steps. too low. Therefore, it is very important and urgent to develop an efficient and suitable method for large-scale purification of FIX (including pdhFIX and rhFIX).

Immunoaffinity chromatography is considered as a potentially ideal method for purifying antigens due to its high specificity and high purification efficiency, and the key to its success lies in whether it can produce sufficient anti-hFIX antibodies with good specificity and moderate affinity. In 1984, Liebman et al. purified hFIX by preparing a Ca ²⁺ dependent monoclonal antibody. As a result, after one-step purification, pure hFIX with a specific coagulation activity of 150 IU/mg was obtained, with a high recovery rate, and the monoclonal antibody was compatible with hFIX The binding affinity is relatively mild, which ensures the activity of hFIX. However, the monoclonal antibody is expensive and has a long preparation period, 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 grasp, 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). Therefore, in order to solve the problem that these two antibodies are used to purify hFIX, it is necessary to find a new anti-hFIX antibody preparation method, so that it can produce enough antibodies with good specificity and mild affinity, so that it can be used for Large-scale immunoaffinity purification of hFIX to obtain hFIX with high purity and maintaining coagulation activity.

As an antibody used to purify antigen, what it recognizes is not the whole antigen molecule, but the B epitope in the antigen molecule. Studies have shown that the specificity of antibodies is related to the specificity of antigens, and the specificity of antigens actually refers to the specificity of epitopes. Therefore, obtaining epitopes with good specificity and moderate affinity is the key to preparing purified antibodies.

Contents of the invention

The purpose of the present invention is to provide a FIX-based antigenic epitope peptide and its application. After the antigenic epitope peptide is fused with a carrier protein, it can stimulate a strong humoral immune response and produce specific antibodies against FIX.

Another object of the present invention is to provide an antibody that specifically recognizes and binds to human FIX and a method for preparing the FIX antibody. The antibody can be used for the separation, purification, detection and treatment of diseases related to the epitope of human FIX.

The first aspect of the present invention provides an antigenic epitope peptide derived from animal FIX and comprising one or more antigenic epitopes,

And the amino acid sequence length of the antigenic epitope peptide is 2-100% of the full length of FIX (preferably, the amino acid sequence length of the antigenic epitope peptide is 5-70% of the full length of the corresponding low immunogenic protein; more preferably Preferably, the amino acid sequence length of the antigenic epitope peptide is 5-50% of the full length of the corresponding low immunogenic protein; most preferably, the amino acid sequence length of the antigenic epitope peptide is the full length of the corresponding low immunogenic protein 5-30%, such as 5%, 10%, 15%, 20%, 25%), and the length of the antigenic epitope peptide is 5-500 amino acids;

And the recombinant protein formed by the epitope peptide and the carrier protein can induce the same kind of animal to produce an immune response against FIX.

Preferably, the length of the amino acid sequence of the antigenic epitope peptide is 3-57. More preferably, the length of the amino acid sequence of the antigenic epitope peptide is 5-17. Such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 amino acids.

In another preferred example, the animal is a mammal. For example, human, mouse, rabbit, cow or sheep.

In another preferred example, the antigenic epitope peptide includes the full-length sequence of human FIX, the full-length sequence of mouse FIX, the full-length sequence of rabbit FIX, the full-length sequence of bovine FIX, the full-length sequence of sheep FIX or homologous sequences thereof.

In another preferred example, the full-length sequence of human FIX is shown in SEQ ID NO.:1.

In another preferred example, the full-length sequence of the mouse FIX is shown in SEQ ID NO.:2.

In another preferred example, the full-length sequence of bovine FIX is shown in SEQ ID NO.:3.

In another preferred example, the antigenic epitope peptide is selected from the following group:

(1)NAAINKY;

(2) A derivative polypeptide formed by substituting, deleting or adding one or more amino acid residues to the amino acid sequence in (1), and having the function of inducing an immune response after being fused with a carrier protein.

In another preferred example, the carrier protein has at least one T cell epitope, and the antigen epitope is introduced into at least one molecular surface amino acid residue region of the carrier protein by splicing, replacement, and/or insertion. Bit peptide.

In another preferred example, the carrier protein and the antigen peptide are not from the same protein, and the carrier protein can enhance the immunogenicity of the epitope peptide.

In another preferred example, the carrier protein includes diphtheria toxin DT, transmembrane domain DTT of diphtheria toxin, cholera toxin B subunit (CTB), Salmonella flagellin (FliC), pertussis toxin (PTX), tetanus toxin , or immunogenic fragments of the above proteins (such as the C fragment of tetanus toxin), rotavirus VP7, heat shock protein of Leishmania, Campylobacter jejuni flagellin, Chlamydia trachomatis major outer membrane protein, hemocyanin ( KeyholeLimpetHemocyanin, KLH), bovine serum albumin (BovineSerumAlbumin, BSA), chicken ovalbumin (Ovalbumin, OVA), fibrinogen.

In another preferred example, the "molecular surface amino acid residue region" includes loop region, beta-tum region, N-terminal or C-terminal.

The second aspect of the present invention provides a fusion protein formed by fusing the epitope peptide described in the first aspect of the present invention with a carrier protein.

In another preferred example, the carrier protein has at least one T cell epitope, and the antigen epitope is introduced into at least one molecular surface amino acid residue region of the carrier protein by splicing, replacement, and/or insertion. Bit peptide.

In another preferred example, the carrier protein and the antigen peptide are not from the same protein, and the carrier protein can enhance the immunogenicity of the epitope peptide.

In another preferred example, the carrier protein includes diphtheria toxin DT, transmembrane domain DTT of diphtheria toxin, cholera toxin B subunit (CTB), Salmonella flagellin (FliC), pertussis toxin (PTX), tetanus toxin , or immunogenic fragments of the above proteins (such as the C fragment of tetanus toxin), rotavirus VP7, heat shock protein of Leishmania, Campylobacter jejuni flagellin, Chlamydia trachomatis major outer membrane protein, hemocyanin ( KeyholeLimpetHemocyanin, KLH), bovine serum albumin (BovineSerumAlbumin, BSA), chicken ovalbumin (Ovalbumin, OVA), fibrinogen.

In another preferred example, the "molecular surface amino acid residue region" includes loop region, beta-tum region, N-terminal or C-terminal.

In another preferred example, the carrier protein is the transmembrane domain DTT of diphtheria toxin, and the loop region includes amino acids 292-298 and 305-310. In another preferred example, the fusion protein is prepared by replacing the 292-295th amino acid of the DDT with the antigenic epitope peptide.

In another preferred example, the carrier protein is cholera toxin B subunit, and the loop region includes:

Cholera toxin B subunit (CTB) implantable epitope Implantable epitope 29-38 amino acids

In another preferred example, the antigen epitope peptide is connected to the C-terminal and/or N-terminal of the carrier protein to form the fusion protein.

In another preferred example, there is a connecting peptide between the antigen epitope and the carrier protein. Preferably, the connecting peptide is 3-30 amino acids in length. More preferably, the connecting peptide is 4-20 amino acids in length. Most preferably, the connecting peptide is 7-17 amino acids in length.

In another preferred example, there is no connecting peptide between the antigen epitope and the carrier protein.

In another preferred example, the antigen epitope peptide replaces amino acids 292-295 in DTT to form the fusion protein.

In another preference, the fusion protein is selected from:

(a) a polypeptide having the amino acid sequence shown in SEQ ID NO.:12;

(b) A polypeptide derived from (a) that is formed by substituting, deleting or adding one or more amino acid residues to each polypeptide in (a), and has the function of inducing an immune response.

The third aspect of the present invention provides an antibody that specifically binds to the epitope peptide described in the first aspect of the present invention or the fusion protein described in the second aspect of the present invention.

In another preferred example, the antigenic epitope peptide is NAAINKY.

In another preferred example, the amino acid sequence of the fusion protein is shown in SEQ ID NO.:12.

The fourth aspect of the present invention provides an isolated antigen-antibody complex, wherein the antigen forming the complex contains the epitope peptide described in the first aspect of the present invention or the epitope peptide described in the second aspect of the present invention. In a fusion protein, the epitope peptide forms an epitope, and the antibody specifically binds to the epitope.

In another preferred example, the amino acid sequence of the antigenic epitope is NAAINKY.

The fifth aspect of the present invention provides a polynucleotide encoding the epitope peptide described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention or the third aspect of the present invention Aspects of the antibody.

The sixth aspect of the present invention provides an expression vector containing the polynucleotide described in the fifth aspect of the present invention.

The seventh aspect of the present invention provides a host cell containing the expression vector of the sixth aspect of the present invention, or the polynucleotide of the fifth aspect of the present invention integrated in the genome.

In another preferred example, the host cells include prokaryotic cells and eukaryotic cells.

In another preferred example, the host cells include Escherichia coli, yeast, CHO cells, DC cells and the like.

The eighth aspect of the present invention provides a pharmaceutical composition, which contains the antigenic epitope peptide described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, the third aspect of the present invention The antibody described in the fourth aspect of the present invention, the polynucleotide described in the fourth aspect of the present invention, the expression vector described in the sixth aspect of the present invention or the host cell described in the seventh aspect of the present invention, and pharmaceutical acceptable carriers and/or excipients.

In another preferred embodiment, the composition is a vaccine.

The ninth aspect of the present invention provides a vaccine composition, which contains the antigenic epitope peptide described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, and the vaccine composition described in the third aspect of the present invention. The antibody described in the fourth aspect of the present invention, the polynucleotide described in the fourth aspect of the present invention, the expression vector described in the sixth aspect of the present invention or the host cell described in the seventh aspect of the present invention, and immune Pharmaceutically acceptable carriers and/or excipients.

In another preferred example, the vaccine composition further contains an adjuvant.

In another preferred example, the adjuvant includes alumina, saponin, quilA, muramyl dipeptide, mineral oil or vegetable oil, vesicle-based adjuvant, non-ionic block copolymer or DEAE dextran, Cytokines (including IL-1, IL-2, IFN-r, GM-CSF, FIX, IL-12, and CpG).

The eleventh aspect of the present invention provides the antigenic epitope peptide described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, the antibody described in the third aspect of the present invention, and the antibody described in the third aspect of the present invention. Use of the polynucleotide described in the fourth aspect, the expression vector described in the sixth aspect of the present invention, or the host cell described in the seventh aspect of the present invention,

(a) for preparing antibodies against the antigenic epitopes; and/or (b) for preparing medicaments for treating diseases related to the antigenic epitopes.

In another preferred example, the diseases include: cardiovascular disease, thrombosis, stroke and infarction, etc.

The twelfth aspect of the present invention provides the use of the antibody described in the third aspect of the present invention or the antigen-antibody complex described in the fourth aspect of the present invention, for

(1) Separation and purification of FIX; or

(2) Detection of FIX for non-diagnostic purposes.

A thirteenth aspect of the present invention provides a method for preparing a FIX antibody, comprising the steps of:

(1) using the fusion protein described in the second aspect of the present invention as an antigen to immunize animals, and produce anti-FIX antibodies in animals;

(2) Separating and purifying the FIX antibody.

In another preferred example, the animal is a mammal.

In another preferred example, the animal includes human, mouse, rabbit, cow or sheep.

The fourteenth aspect of the present invention provides a method for isolating and purifying human FIX, comprising the steps of:

(1) using the method described in the thirteenth aspect of the present invention to prepare human FIX antibody;

(2) Using the antibody obtained in step (1), purify human FIX by affinity chromatography.

The fifteenth aspect of the present invention provides a treatment method, administering the antigenic epitope peptide described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, and the epitope peptide described in the third aspect of the present invention to a subject in need. The antibody described above, the polynucleotide described in the fourth aspect of the present invention, the expression vector described in the sixth aspect of the present invention or the host cell described in the seventh aspect of the present invention, the eighth aspect of the present invention The pharmaceutical composition described above or the vaccine composition described in the ninth aspect of the present invention.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1 shows the binding specificity of the purified antibodies detected by Western-blot in Example 3.

FIG. 2 shows the results of antibody isothermal calorimetric titration measurements in Example 3.

detailed description

Through extensive and in-depth research, the inventors obtained a new class of FIX epitope, fused the epitope with carrier protein, and used the fusion protein as an antigen to immunize animals to obtain anti-FIX antibodies with good specificity and moderate affinity , and the antibody can recognize both pdhFIX and rhFIX, laying a solid foundation for the large-scale preparation of high-purity anti-hFIX antibodies.

FIX

The amino acid sequence of human FIX is as follows:

MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT(SEQIDNO.:1)

The amino acid sequence of mouse FIX is as follows:

MKHLNTVMAESPALITIFLLGYLLSTECAVFLDRENATKILTRPKRYNSGKLEEFVRGNLERECIEERCSFEEAREVFENTEKTTEFWKQYVDGDQCESNPCLNGGICKDDISSYECWCQVGFEGRNCELDATCNIKNGRCKQFCKNSPDNKVICSCTEGYQLAEDQKSCEPTVPFPCGRASISYSSKKITRAETVFSNMDYENSTEAVFIQDDITDGAILNNVTESSESLNDFTRVVGGENAKPGQIPWQVILNGEIEAFCGGAIINEKWIVTAAHCLKPGDKIEVVAGEYNIDKKEDTEQRRNVIRTIPHHQYNATINKYSHDIALLELDKPLILNSYVTPICVANREYTNIFLKFGSGYVSGWGKVFNKGRQASILQYLRVPLVDRATCLRSTTFTIYNNMFCAGYREGGKDSCEGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT(SEQIDNO.:2)

The amino acid sequence of bovine FIX is as follows:

YNSGKLEEFVRGNLERECKEEKCSFEEAREVFENTEKTTEFWKQYVDGDQCESNPCLNGGMCKDDINSYECWCQAGFEGTNCELDATCSIKNGRCKQFCKRDTDNKVVCSCTDGYRLAEDQKSCEPAVPFPCGRVSVSHISKKLTRAETIFSNTNYENSSEAEIIWDNVTQSNQSFDEFSRVVGGEDAERGQFPWQVLLHGEIAAFCGGSIVNEKWVVTAAHCIKPGVKITVVAGEHNTEKPEPTEQKRNVIRAIPYHSYNASINKYSHDIALLELDEPLELNSYVTPICIADRDYTNIFSKFGYGYVSGWGKVFNRGRSASILQYLKVPLVDRATCLRSTKFSIYSHMFCAGYHEGGKDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT(SEQIDNO.:3)

carrier protein

As used herein, the term "carrier protein" refers to a protein serving as a protein structural backbone in the recombinant protein of the present invention. Usually, the carrier protein is a protein with strong immunogenicity, such as a pathogen protein, representative examples include (but not limited to): viral protein, bacterial protein, chlamydia protein, mycoplasma protein and the like.

As used herein, the term "antigenic epitope (peptide)" refers to a peptide of other proteins that is intended to induce an immune response in animals. The epitope, relative to the carrier protein, does not refer to the peptide of the carrier protein itself that can cause an immune response. Generally, an antigenic epitope refers to a peptide to be targeted by an immune response, preferably a peptide derived from a mammalian (eg, human) protein rather than the carrier protein.

As used herein, the term .pdb refers specifically to protein tertiary structure data files, from ProteinDataBank (www.pdb.org);

As used herein, the term DTT refers to the transmembrane domain of diphtheria toxin;

As used herein, the term T cell epitope, also known as T cell antigen epitope, is a peptide produced by enzymatic processing of antigen molecules in antigen-presenting cells, which can be combined and presented by major histocompatibility complex (MHC) molecules It is bound by the T cell receptor (TCR) on the cell surface to activate T cells, including T helper cell epitopes.

As used herein, the term "low immunogenic protein" refers to a protein that does not elicit an adequate immune response in an animal alone.

As used herein, the term "molecular surface amino acid residue region" or "surface amino acid residue region" refers to a region consisting of amino acid residues located on the surface of a protein molecule. Preferably, the "molecular surface amino acid residue region" includes loop region, beta-tum region, N-terminal or C-terminal.

representative carrier protein

1. Diphtheria toxin and its transmembrane domain

Diphtheria toxin (diphtheriatoxin, DT) is an exotoxin produced by Corynebacterium diphtheriae infected with beta bacteriophage, and exists in the clinically used DPT vaccine components. The safety has been verified by clinical use for many years, and serious adverse reactions are rare, and there is no report of allergic reactions caused by diphtheria components.

The diphtheria toxin molecule consists of 535 amino acid residues, and is composed of relatively independent catalytic domains (1-193AAs), transmembrane domains (205-378AAs) and receptor binding domains (386-535AAs); the transmembrane domains And the receptor binding domain itself is non-toxic, and its function is to transduce the catalytic domain into the cell through the binding of the cell surface receptor.

The diphtheria toxin amino acid sequence (P00588, DTX_CORBE) is as follows:

GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKY

DAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS(SEQIDNo.:4)

There are five T-helper cell epitopes in the diphtheria toxin molecule that can be recognized by more than 80% of human MHC class Ⅱ. The inventors simulated the protein structure of diphtheria toxin, retained the α-helix and β-sheet elements and T cell epitope required for structural stability, and could be replaced and implanted in the surface amino acid residue region of the antigen epitope at positions 292-298, 305- 310th amino acid.

Diphtheria toxin transmembrane domain (DTT) itself is non-toxic, mainly composed of α-helical elements to form the core skeleton, and the helical elements are connected by flexible loop regions.

The amino acid sequence of DTT (1F0L.pdb:202–378) is shown below:

202INLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELS

262ELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAV

322HHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRP (SEQ ID No.: 5)

The inventors simulated the protein structure of DTT, retained the α-helix and β-fold elements and T cell epitope required for structural stability, and could be replaced and implanted in amino acids 292-298 and 305-310 of the antigenic epitope.

In a preferred example of the present invention, the peptide segment of the target protein to be intervened by the drug can be selected and grafted onto DTT, and replace the amino acid residue region on the surface of DTT, including the amino acid residues in the loop region between α-helical elements.

The research of the present invention shows that the integration of the diphtheria toxin transmembrane structure and the peptide segment from the target protein will not or basically not affect the respective folding. In the recombinant protein, DTT is used as the protein skeleton, and after the target protein peptide is grafted onto DTT, it can induce animals to produce an immune response against the target protein. Therefore DTT is a very suitable protein backbone.

2. Cholera toxin B subunit (CTB)

Cholera toxin (choleratoxin) is an exotoxin (84kDa) secreted by Vibrio cholerae, which is composed of A and B subunits and is of type AB5. Cholera toxin B subunit (CTB) is a non-toxic part of cholera toxin, which has good immunogenicity and has been proved to be an effective component of vaccines by human experiments. CTB can specifically bind to the ganglioside GM1 present on the surface of most mammalian cells, stimulate the body to produce mucosal IgA, and enhance antigenicity to induce mucosal immune response. CTB has been used in novel oral cholera vaccines, adjuvants, and protein carriers.

CTB is non-toxic, and consists of two α-helical elements and six beta-sheets to form the core skeleton, and the structural elements are connected by flexible loop regions. The five B subunits assemble relatively parallel inwardly with long α-helical elements, forming a five-pointed star shape. Each subunit can independently bind neuroganglioside (GM1) receptors on the cell membrane.

The CTB amino acid sequence (3CHB.pdb: 1–103) is shown below:

1TPQNITDLCAEYHNTQIYTLNDKIFSYTESLAGKREMAIITFKNGAIFQVEVPGSQHIDS

60QKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN (SEQ ID No.: 6)

CTB contains two T cell epitopes, CTB-Th epitope 81-100 (81-KVEKLCVWNNKTPHAIAAIS-100) is the dominant epitope, CTB-Th epitope 31-50 (31-LAGKREMAIITFKNGAIFQV-50) is the weak epitope, the distribution On the 4-segment Beta-sheet at the core of the subunit structure.

The inventors simulated the protein structure of CTB, retained the α-helix and β-sheet elements required for structural stability, and the T cell epitope, which can be replaced and implanted in the 29-38 amino acid position of the antigen epitope

3. Salmonella flagellin (FliC)

Salmonella flagellin FliC (Phase1-Cflagellin) is the filamentous component of the flagella. It binds to a variety of animal cell receptors TLR5, stimulates the animal's innate immune system response, promotes the expression of CD80 and CD86 on the surface of immature DC cells, and secretes a variety of Cytokines and chemokines play a strong immune adjuvant role in the innate immune response and specific specific immune response. As immune adjuvant components, they have been used in many pathogen vaccine preparations.

FliC is composed of 494 amino acids. The crystal structure shows that the N-terminus and C-terminus of this protein are folded back to form a shape like the uppercase Greek letter gamma "Γ".

The amino acid sequence of FliC (lucu.pdb: 1–494) is shown below:

AQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKYKVSDTAATVTGYADTTIALDNSTFKASATGLGGTDQKIDGDLKFDDTTGKYYAKVTVTGGTGKDGYYEVSVDKTNGEVTLAGGATSPLTGGLPATATEDVKNVQVANADLTEAKAALTAAGVTGTASVVKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKDGSISINTTKYTADDGTSKTALNKLGGADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR(SEQIDNo.:7)

The inventors simulated the protein structure of FliC, and in the case of retaining the α-helix and β-sheet elements required for structural stability, the position that can be replaced and implanted with the antigenic epitope is 348-KYTADDGTSKTA-359 amino acid residues.

Composition and method of application

The present invention also provides a composition, which contains: (i) the recombinant protein of the present invention or the polynucleotide encoding the recombinant protein of the present invention, and (ii) a pharmaceutically or immunologically acceptable excipient or adjuvants.

In the present invention, the term "comprising" means that various components can be applied together or present in the composition of the present invention. Accordingly, the terms "consisting essentially of" and "consisting of" are included in the term "comprising".

Compositions of the present invention include pharmaceutical compositions and vaccine compositions.

The composition of the present invention may be monovalent (containing only one recombinant protein or polynucleotide) or multivalent (containing multiple recombinant proteins or polynucleotides).

The pharmaceutical composition or vaccine composition of the present invention can be prepared into various conventional dosage forms, including (but not limited to): injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays and the like.

(i) Pharmaceutical composition

The pharmaceutical composition of the present invention comprises (or contains) a therapeutically effective amount of the recombinant protein or polynucleotide of the present invention.

As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or exhibits a detectable therapeutic or preventive effect. This effect can be detected, for example, by antigen levels. A therapeutic effect also includes a reduction in physical symptoms. The precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the disorder, and the therapeutic agents and/or combination of therapeutic agents chosen for administration. Therefore, it is not useful to prespecify an exact effective amount. However, the effective amount can be determined by routine experimentation for a given situation.

For the purposes of the present invention, an effective dose is about 0.001 mg/kg to 1000 mg/kg, preferably about 0.01 mg/kg to 100 mg/kg body weight of the recombinant protein administered to an individual.

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent such as the recombinant protein of the present invention. The term refers to pharmaceutical carriers which do not, by themselves, induce the production of antibodies deleterious to the individual receiving the composition and which are not unduly toxic upon administration. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, and the like. These vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Pharmaceutically acceptable carriers in compositions can include liquids such as water, saline, glycerol and ethanol. In addition, there may also be auxiliary substances in these carriers, such as wetting agents or emulsifying agents, pH buffering substances and the like. Typically, the compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. Liposomes are also included within the definition of pharmaceutically acceptable carriers.

(ii) Vaccine composition

The vaccines (compositions) of the present invention can be prophylactic (ie, prevent disease) or therapeutic (ie, treat disease after disease).

These vaccines comprise immunizing antigens, including recombinant proteins of the invention, and are usually combined with a "pharmaceutically acceptable carrier," which includes any carrier that does not, by itself, induce antibodies deleterious to the individual receiving the composition. Suitable carriers are usually large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and the like. These vectors are well known to those of ordinary skill in the art. In addition, these carriers can act as immunostimulants ("adjuvants"). In addition, the antigen can also be coupled with bacterial toxoids (such as toxoids of diphtheria, tetanus, cholera, Helicobacter pylori and other pathogens).

Preferred adjuvants for enhancing the effect of immune compositions include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations, for example, (a) MF59 (see WO90/14837), (b) SAF, and (c) Ribi ^™ Adjuvant System (RAS) (RibiImmunochem, Hamilton, MT), (3) saponin adjuvant; (4) Freund's complete adjuvant (CFA ) and Freund's incomplete adjuvant (IFA); (5) cytokines, such as interleukins (such as IL-1, IL-2, IL-4, IL-5, FIX, IL-7, IL-12, etc.), interference (such as gamma interferon), macrophage colony-stimulating factor (M-CFS), tumor necrosis factor (TNF), etc.; Toxic variants; and (7) other substances that act as immunostimulants to enhance the effect of the composition.

Vaccine compositions, including immunogenic compositions (eg, may include antigens, pharmaceutically acceptable carriers, and adjuvants), generally contain diluents such as water, saline, glycerol, ethanol, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

More specifically, vaccines, including immunogenic compositions, comprise an immunologically effective amount of an immunogenic polypeptide, and other desirable components as described above. An "immunologically effective amount" refers to an amount administered to a subject as a single dose or a fraction of consecutive doses that is effective for treatment or prophylaxis. The amount can be based on the health and physiological condition of the individual to be treated, the class of the individual to be treated (e.g., human), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating physician's assessment of the medical condition, and other related factors. This amount is expected to lie within a relatively wide range and can be determined by routine experimentation.

Generally, vaccine or immunogenic compositions are prepared as injectables, such as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The formulation can also be emulsified or encapsulated in liposomes for enhanced adjuvant effect.

Furthermore, the vaccine compositions of the present invention may be monovalent or multivalent vaccines.

(iii) Route of Administration and Dosage

Once formulated, the compositions of the invention can be administered directly to a subject. The subject to be treated may be a mammal, especially a human.

When used as a vaccine, the recombinant protein of the present invention can be directly administered to an individual using a known method. These vaccines are usually administered by the same route of administration as conventional vaccines and/or by simulating pathogen infection routes.

The routes of administering the pharmaceutical composition or vaccine composition of the present invention include (but are not limited to): intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, oral or other parenteral routes of administration. Routes of administration can be combined, if desired, or adjusted according to disease conditions. Vaccine compositions may be administered in single or multiple doses, and may include administration of booster doses to elicit and/or maintain immunity.

The recombinant protein vaccine should be administered in an "effective amount", that is, the amount of the recombinant protein is sufficient to trigger an immune response in the selected route of administration, and can effectively promote the protection of the host against related diseases.

Representative diseases include (but are not limited to): autoimmune diseases, tumors, and the like.

The amount of recombinant protein used in each vaccine dose is determined by the amount that can elicit an immune protective response without obvious side effects. Typically, each dose of the vaccine is sufficient to contain about 1 μg-1000 mg, preferably 1 μg-100 mg, more preferably 10 μg-50 mg of protein after infection of the host cells. The optimal amount for a particular vaccine can be determined using standard research methods, including observation of antibody titers and other responses in subjects. The need for a booster dose can be determined by monitoring the level of immunity provided by the vaccine. After assessment of antibody titers in sera, a booster dose of immunization may be required. Administration of adjuvants and/or immunostimulants can enhance the immune response to the proteins of the invention.

A preferred method is to administer the immunogenic composition by injection from the parenteral (subcutaneous or intramuscular) route.

In addition, the vaccines of the invention can be administered in conjunction with other immunomodulators, or with other therapeutic agents.

The main advantages of the present invention are:

(1) The fusion protein comprising the FIX-based epitope of the present invention can be used as a vaccine to effectively stimulate the body's immune response against FIX.

(2) The preparation cost of the recombinant protein carrying the antigenic epitope of the present invention is low and the administration is convenient.

(3) Compared with the preparation chemically coupled with carrier protein, the antigen of the present invention has definite structure, controllable quality and is safer.

(4) Using the fusion protein containing the antigen epitope peptide of the present invention to immunize animals, the antibody against FIX prepared has strong specificity and moderate affinity, and the antibody is very suitable for the separation and purification of FIX.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, generally follow conventional conditions such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions suggested by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Example 1 Design of B cell antigen epitope and preparation of fusion protein

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.

Table 1 Basic information of the designed hFIX B epitope

Homology of the B epitope sequence of hFIX designed in Table 2

Table 3 Epitope grafting results

1. Epitope fusion protein preparation

1.1 Primer design

Each FIX epitope gene sequence is as follows:

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

FIX-2: AATGCAGCTATTAATAAGTAC (SEQ ID NO.: 14)

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

FIX-4: GTTGAAACTGGTGTTAAAATT (SEQ ID NO.: 16)

DTT gene sequence:

ataaatcttgattgggatgtcataagggataaaactaagacaaagatagagtctttgaaagagcatggccctatcaaaaataaaatgagcgaaagtcccaataaaacagtatctgaggaaaaagctaaacaatacctagaagaatttcatcaaacggcattagagcatcctgaattgtcagaacttaaaaccgttactgggaccaatcctgtattcgctggggctaactatgcggcgtgggcagtaaacgttgcgcaagttatcgatagcgaaacagctgataatttggaaaagacaactgctgctctttcgatacttcctggtatcggtagcgtaatgggcattgcagacggtgccgttcaccacaatacagaagagatagtggcacaatcaatagctttatcatctttaatggttgctcaagctattccattggtaggagagctagttgatattggtttcgctgcatataattttgtagagagtattatcaatttatttcaagtagttcataattcgtataatcgtccc(SEQIDNO.:17)

The base sequence on the DTT is replaced with the base sequence of the designed FIX antigen epitope by recombinant PCR technology to form the base coding sequence of the fusion protein.

The recombination primers used in the recombination PCR are shown in Table 5.

Table 5 Primer design

1.2 Vector construction

1) The first round of PCR

Using the DTT plasmid as a template, use the primers DTT forward-A/DTT reverse-D and each upper half reverse-B/each lower half forward-C to perform the first round of PCR to amplify each DTT-FIX respectively The upper and lower half of the epitope recombined gene.

The reaction system was 5 μL 10×KOD-PlusBuffer, 5 μL dNTPs (2 mmol/L), 2 μL MgSO4 (25 mmol/L), 1.5 μL primer B or C, 1.5 μL primer A or D, 1 μL KOD-Plus (polymerase), 0.5 μL DTT plasmid (template ), filled to 50 μL with sterile double distilled water.

Amplification procedure:

Pre-denaturation at 94°C for 2min, denaturation at 94°C for 15s, annealing at 55°C for 30s, extension at 68°C for 2min, 34 cycles, extension at 68°C for 10min. 12°C, Forever, save.

Electrophoresis detection showed that the sizes of all mutant fragments were consistent with expectations.

2) The second round of PCR

Dilute the upper and lower half of the product recovered from the first round of PCR tapping by 15-20 times respectively, then mix the upper and lower half according to the following reaction system, and use primers A and D at both ends of DTT to amplify the complete sequence of the DTT-epitope recombinant gene.

The reaction system was 5 μL of 10×KOD-PlusBuffer, 5 μL of dNTPs (2 mmol/L), 2 μL of MgSO ₄ (25 mmol/L), 1.5 μL of primers at both ends, 1 μL of KOD-Plus (polymerase), 0.5 μL of DTT plasmid (template), and sterile double Make up to 50 μL with distilled water.

The DTT-epitope recombinant gene was amplified by recombinant PCR, and after the first round of PCR, gene fragments of about 200bp and 300bp were respectively obtained after the first round of PCR, indicating that the upper and lower fragments of each recombinant gene were successfully amplified; while the second round After hybrid splicing, all the fragments are about 500bp in size, which is consistent with the expected target fragment size.

3) Mixed ligation product and vector double digestion

The second-round PCR product was detected by electrophoresis, and after being recovered by tapping the gel, it was double-digested with BamHI and XhoI. Double digest the pGEX-6P-1 vector with the same enzyme, and the digestion system is as follows:

Through detection, both the vector and the target DNA have been successfully double-digested and can be ligated.

4) Connection and transformation

The double-digested products recovered from rubber tapping were ligated with ligase. The ligation system is: 1 μL pEGX-6P-1 vector, 1 μL 10×LigationBuffer, 0.5 μL T4 DNA ligase, 1 μL target DNA (10 ng/μL) and 6.5 μL ddH ₂ O.

Transfer the ligation product into competent cells at a ratio of 1:100 DH5α, ice-bath for 30 min, heat shock in a water bath at 42°C for 90 seconds, transfer to a centrifuge tube containing 1 mL of LB medium, revive at 37°C for 1 h on a shaker at 150 rpm, and take out 500 μL to spread on a plate ( containing ampicillin), cultivated in a 37°C incubator for about 20 hours, picked a positive single colony, cultured at a constant temperature of 37°C, and the recombinant plasmid was extracted at about OD=0.4.

Pick a single colony for culture, and carry out the PCR positive colony identification of the bacterial liquid. After identification, all the bacterial liquids are positive clones, which can be further verified by sequencing.

5) Sequencing

The correct positive clones were identified by bacterial liquid PCR, and 200 μL of the corresponding bacterial liquid was taken for sequencing. The sequencing work was completed by Nanjing GenScript.

6) Extract positive recombinant plasmids and transform them into BL21(DE3) cells.

Extract the recombinant plasmid of the positive clone bacterial liquid with correct sequencing, and retransfer it into BL21 (DE3) cells, and the plasmid extraction and transformation operations are the same as above.

1.3 Protein expression and purification

Take a 15ml centrifuge tube, add 3mL of LB medium containing ampicillin, pick the positive clones transformed into BL21 cells into it, and culture at a constant temperature at 37°C. When OD=0.5, transfer it to 1L LB containing ampicillin 1/1 ampicillin) in a shaker flask, when OD=0.6, add 1/1000 of 50mMIPTG, induce for about 5h, centrifuge the induced bacteria solution, 12000g, 5min, resuspend with 10 times the volume of PBS, After sonication, it was centrifuged again (16000g, 20min), and the supernatant was subjected to affinity purification.

Protein purification: take a 5mL GST column, equilibrate with 10 times the volume of 10mMPBS (pH7.5), flow rate 3ml/min; after equilibration, load the sample at a flow rate of 1.5mL/min; Wash the impurities at a flow rate of 1.5-2.0mL/min until the OD ₂₈₀ is close to 0; use 50mMGSH eluent (pH8.0) to elute the target protein, collect the target protein, and check its purity by electrophoresis.

The results of electrophoresis detection showed that the molecular weight of the purified protein was about 45kDa, which was consistent with the size of the DTT-epitope recombinant protein. Therefore, we successfully produced the required epitope fusion protein. After grayscale analysis by Image-lab software, The purity reaches more than 90%, which can be used for animal immune experiments.

The amino acid sequence of the FIX-2-DTT recombinant protein (fusion protein) designed in this embodiment is as follows:

INLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDS NAAINKY NLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRP (SEQ ID NO.:12)

Activity detection of embodiment 2 fusion protein

1 Epitope identification

1.1. Mice immunization

1) Feed 42 female Balb/c mice aged 4-5 weeks.

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).

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

Table 9 Mouse Immunization

4) After the antigen is ready, inject it according to the above table. The initial immunization adopts the method of subcutaneous multi-point injection immunization. Each mouse is injected with 50 μg antigen (in the actual operation process, we injected 2 points for those less than 100 μL, and for injections larger than 100 μL 3-4 points), and blood was taken before immunization as a negative control.

5) Two weeks after the first immunization, the same adjuvant was mixed with the antigen and homogenized, and the second mouse immunization was carried out. The immunization dose was 50 μg antigen/mouse, and the injection method was the same as above.

6) Two weeks after the second immunization, the same dose and method were used for the third immunization.

7) One week after the second booster immunization, the mouse serum was collected by eyeball blood sampling method, 4000rpm, 4°C, centrifuged to obtain the serum, and stored in a -80°C refrigerator, and the specific antibody in the serum was detected by ELISA method Titer, and further Western-Blot validation analysis.

1.2 ELISSA detection of antibody production:

The FIX standard was used as the coating antigen, and the serum diluted by each group was used as the primary antibody to detect the production of FIX antibody. The experimental design is as follows:

Table 10 ELISA detection

Experimental operation:

1) coated

Dilute each antigen to 0.1-1 μg/mL with CBS coating solution, add 100 μL to the reaction well of each microtiter plate, incubate overnight at 4 °C, and then wash 5 times with a plate washer, each time with washing buffer ( PBS-T) was washed for 3 minutes. After washing, the microtiter plate was turned upside down, and the liquid in it was patted gently.

2) closed

Add 300 μL of blocking solution to each well, block at 37°C for 2 hours, wash the plate 5 times with the washing solution, the washing method is the same as above, and gently blot dry after washing.

3) Dilute the tested serum with antibody diluent at 1:100, 1:200, 1:500, and 1:1000 respectively. Then add 100 μL of the corresponding serum to each well according to the above table, make 3 parallel wells, incubate at 37°C for 2 hours, wash the plate 5 times, turn the microplate upside down, and gently pat dry the liquid in it.

4) Dilute goat anti-mouse IgG-HRP 5000 times with antibody diluent, add 100 μL of the diluted enzyme-labeled secondary antibody to each well, incubate at 37°C for 1 hour, wash 5 times with washing solution, and then turn the enzyme-labeled plate upside down. Gently pat dry any liquid in it.

5) Add 100 μL of freshly prepared substrate solution to each reaction well, and incubate at 37° C. for 10-30 min. Timely termination depending on the color.

6) Add 50 μL of stop solution to each reaction well, and detect its absorbance at 450 nm with a microplate reader.

The absorbance value of the blank control was adjusted to zero. If the OD ₄₅₀ value of the well to be tested was greater than or equal to 2.1 times that of the negative control well, it was defined as a positive value, and the corresponding maximum serum dilution factor under this value was defined as the specificity in the serum. Antibody titers.

The results showed that when the dilution factor was 100, the OD ₄₅₀ values of DTT-GST and Al(OH) ₃ in the negative control group were both about 0.25, while the highest value of OD ₄₅₀ in the experimental group was 0.92, which was a small effect of FIX-2-DTT immunization. mouse serum. The OD ₄₅₀ of the rest groups were all low, no more than 0.4. When the serum dilution multiples increased, the OD ₄₅₀ value at each dilution multiple was still the highest in the FIX-2-DTT group, and when the dilution multiple was 1000, its OD ₄₅₀ value was still greater than the maximum OD ₄₅₀ value of the other components, indicating that Anti-human FIX antibodies were produced in the serum of mice immunized with FIX-2-DTT, while the other components did not produce antibodies against FIX or produced very low amounts.

In order to further detect the antibody titer of the FIX-2-DTT group, further increase the multiple of the serum of the FIX-2-DTT group until the OD ₄₅₀ value is close to 0, or close to the OD ₄₅₀ value of the negative control group at this dilution multiple. The measurement method is exactly the same as the above ELISA. When the dilution factor is 5000 times, the N/P value of the FIX-2 group is greater than 2.1, and when the dilution factor is greater than 5000 times, the ratio is less than 2.1. Therefore, the antibody titer of the serum in the FIX-2-DDT group is determined to be 5000 .

1.3. Western-blot detection of antibody binding specificity

Reagent preparation:

Transfer buffer: 25mM Trisbase, 0.2M glycine, 20% methanol (pH8.3)

10mMPBS: 140mM NaCl, 2.7mM KCl, 10mMNa ₂ HPO ₄ ·10H ₂ O Adjust the pH to 7.4 with KH ₂ PO ₄ .

Washing buffer (PBS-T): 10mMPBS, add 0.1% Tween-20, mix well.

Blocking buffer: Add 5% (w/v) non-fat dry milk to PBS-T.

Antibody diluent: same as blocking solution.

The experimental design is as follows:

Table 11 Western-blot detection

Experimental steps:

1) Separation of samples by electrophoresis

To identify whether the gel is good or bad, fill it up with 1×loadingbuffer, pre-stained Marker to indicate the transfer efficiency and mark the lane.

2) Film transfer (slot wet transfer)

When the electrophoresis is about to end, prepare 6 pieces of filter paper and cut them to the same size as the electroporator. After the electrophoresis is over, soak them in the transfer buffer for about 10 minutes. At the same time, peel off the gel and cut them into pieces. Remove the part that does not contain the target protein, and put it into the transfer buffer to equilibrate for about 10 minutes. Immediately, cut out a PVDF membrane with the same size as the gel, soak it in methanol for about 2 minutes, and then transfer it to the transfer buffer to equilibrate for about 8 minutes. After the balance is completed, assemble the transfer sandwich in the order of the negative side (black side)-sponge-3 layers of filter paper-gel-PVDF membrane-3 layers of filter paper-sponge-positive side (red side). After adding each layer, remember to use Carefully drive off air bubbles with the glass rod to prevent short circuits. After the sandwich is installed, place it in the transfer tank (black side to black side), add transfer buffer, place the whole device in an ice bath, plug in the electrode, 300mA constant current, and transfer to the membrane for 1h. Or transmembrane overnight (more than 10h) under 20V voltage. After transferring the membrane, cut off the power supply and take out the hybridization membrane. Cut the membrane according to the pre-dotted sequence.

3) Wash the membrane 3 times with 25mL PBS-T, 5min each time, shake at room temperature.

4) Put the membrane in a plate, add 25mL of blocking buffer, incubate with shaking at room temperature for 1 hour, or stand overnight at 4°C.

5) Wash 3 times with PBS-T, 5 min each time.

6) Put each cut membrane into a 10mL centrifuge tube, add corresponding appropriate amount of each diluted serum, incubate at room temperature for 1 hour, and shake slowly.

7) Wash 3 times with PBS-T, 5 min each time.

8) Put all the membranes together in a plate, add an appropriate amount of diluted secondary antibody (goat anti-mouse IgG-HRP, diluted 5000 times), shake the reaction slowly, and incubate at room temperature for 1 hour.

9) Wash 3 times with PBS-T, 5 min each time.

10) Wash the membrane with deionized water for a while, and dry the water on its surface with a clean filter paper, then carefully and evenly drop the mixed luminescence and color development solution on the membrane, and develop color for about 5 minutes. Exposure time for exposure imaging.

The results showed that an obvious band appeared in the positive control group, with a molecular weight of about 60KD, which was consistent with the molecular weight of the FIX standard. Among the sera of each group, only one band appeared at the same position in the FIX-2-DTT group, while no target band appeared in the other groups. This result is consistent with the ELISA result, indicating that the FIX-2-DTT immunized mice did produce antibodies against FIX.

The above results show that FIX-2 is the B epitope of FIX, and after transplanting it into DTT _292-295 to form a recombinant protein, it has good immunogenicity, and the immunized mice can produce antibodies against intact FIX.

Example 3 Antibody Preparation

1.1 Rabbit Immunization

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.

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.

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.

4) Booster immunization. Each rabbit was injected with 0.5 mg of the emulsified antigen. The multi-point injection method was also adopted, 6 points were injected in the right neck of the rabbit, 6 points were injected in the right front armpit, and 5 points were injected in the right front shoulder.

5) After two weeks, the same adjuvant is mixed with the antigen, and a second booster immunization is carried out, and the method is the same as step 4.

6) One week after the second booster immunization, take 20-100 μL of blood from the ear vein of the rabbit, centrifuge at 4000 rpm, 4°C to obtain serum, and use ELISA to detect the specific antibody titer in the serum to decide whether to continue Boost immunity.

7) If the titer is not high, proceed to booster immunization, the immunization method is the same as the above-mentioned booster immunization, and stop immunization for a total of 5 times at most.

8) Bloodletting: After the immunization, the blood of the rabbits was collected by taking blood from the carotid artery. First, the rabbits were placed on the operating stand and their heads were fixed, and then their limbs were tied and fixed with small cloth ropes. The head is lowered slightly to expose the neck, the neck hair is shaved, and the skin is disinfected with alcohol swabs. After the disinfection is completed, use a scalpel to cut the skin of the front neck longitudinally about 10 cm long, and separate the subcutaneous connective tissue. When the sternocleidomastoid muscles on both sides of the trachea are completely exposed, use hemostats to clamp the skin separately, and peel off the subcutaneous tissue. The tissue, exposing the muscle layer, is separated with a knife handle, and the pulsating carotid artery can be seen. Carefully strip the carotid artery and vagus nerve about 5 cm long, select the middle part of the blood vessel, and clamp the fascia around the blood vessel wall with a hemostat. The distal end was ligated with silk thread, the proximal end was clamped with arterial forceps, alcohol cotton balls were used to disinfect around the blood vessels, and a V-shaped notch was cut with sterile scissors. Take a section of plastic tube with a length of 2.5cm and a diameter of 1.6mm, cut one end into a needle-like bevel, insert this end into the carotid artery, and put the other end into a 200mL sterile Erlenmeyer flask. After all the above procedures are completed, loosen the hemostat to start bleeding, and collect the blood with a clean receiving container. The collected blood was left at room temperature for about 2 hours, centrifuged at 4000 rpm at 4°C for 5 minutes, and the supernatant was collected to obtain rabbit serum.

Antiserum titers of rabbits after three immunizations were tested by ELISA. The results showed that after three immunizations, the specific antibody titers in all rabbits’ serum reached 1:16000 or above, indicating that every rabbit immunized with FIX-2-DTT produced The target antibody against FIX was obtained, therefore, this also further verified that the FIX-2 identified above is indeed a B epitope of FIX.

1.2 Antibody purification

1.2.1. Antigen affinity medium preparation

1) Polypeptide synthesis:

Synthesize a polypeptide containing a target epitope (polypeptide sequence: CHNY NAAINKY NHD (SEQ ID NO.: 27), wherein C is an additional cysteine, as a coupling site with the chromatographic medium; the underlined part is the table Bit sequence), this work was completed by Shanghai Shengli Biological Co., Ltd. The purity of the final synthesized polypeptide is up to 90%.

2) Peptide coupling:

10 mg of the synthesized peptide was coupled with 2 mg of Sepharose4FF activated by cyanogen bromide to prepare a peptide affinity medium. Coupling conditions are 37°C, 16h. The coupling reaction buffer system is 0.2M NaHCO ₃ (pH8.5).

1.2.2. Antibody purification

1) Dilute the rabbit serum 5 times with 10mMPBSpH7.0 buffer solution for use (the serum of all rabbits is well mixed);

2) Take out the prepared peptide affinity medium (2mL) and put it into an empty column, wash the column with 5 times the volume of 10mMPBS (pH7.0) until the A ₂₈₀ value of the effluent liquid is close to zero;

3) Take out the serum of each rabbit that has been diluted with 10mMPBS, and flow through the peptide affinity column for purification. The flow rate of the sample is controlled at about 0.5mL/min. For unbound non-specific proteins, until the effluent A ₂₈₀ is close to zero, all the flow-through is received;

4) Elute the bound antibody with Gly-HCl buffer at pH 3.0, collect the eluted liquid (add an appropriate amount of NaOH to the receiving container in advance), and immediately adjust the pH to neutral;

5) Rinse the medium with 10mMPBS (pH 7.0) until the pH of the effluent is 7.0, reload the flow-through solution and elute, repeating several times; collect the eluate;

6) Detection of antibody purity by SDS-PAGE;

7) Determination of antibody concentration by BCA quantitative method;

8) Detection of antibody binding specificity by Western-Blot.

After polypeptide immunoaffinity chromatography, 18 mg of specific target antibody was finally purified.

1.3 Western-Blot detection

Ordinary milk skim milk, transgenic milk skim milk (the skim milk contains rhFIX, obtained from the Institute of Genetics, Shanghai Children's Hospital, can refer to literature, Huang Zan et al.: Goat-casein gene promoter-directed transgenic mouse milk is highly efficient Express human coagulation factor IX. Acta Genetics 2002,29(3):206-211; YanJ-B et al: TransgenicmicecanexpressmutanthumancoagulationfactorIXwithhigherlevelofclottingactivity.Biochemicalgenetics2006,44(7-8):347-358).

hFIX standard (purchased from EnzymeResearch Laboratories), prothrombin complex (PCC, purchased from Hualan Biology) and bovine FIX (purchased from EnzymeResearch Laboratories) were used as detection antigens, and the purified antibodies were used as primary antibodies (the above purified antibodies were mixed , and evenly), using goat anti-rabbit IgG-HRP as the secondary antibody to detect the binding specificity of the antibody, the specific operations are as follows:

1) Separation of samples by electrophoresis: To identify whether the gel is good or bad, the sample volume of hFIX standard substance is 100ng, and the sample volume of other samples is 15μL, and the pre-stained marker is used to indicate the transfer efficiency and mark the lane.

2) Membrane transfer (slot wet transfer): soak the gel in the transfer buffer and equilibrate for 10 minutes. According to the size of the gel, cut out 6 pieces of PVDF membrane and filter paper, and put them into the transfer buffer to equilibrate for 10 minutes. If PVDF membrane is used, soak and saturate with pure methanol for 1min. Assembly transfer sandwich: Negative side (black side)-sponge-3 layers of filter paper-gel-PVDF membrane-3 layers of filter paper-sponge-positive side (red side), after each layer is placed, remove the air bubbles. Place the transfer tank in an ice bath, put in the sandwich (black side to black side), add transfer buffer, insert electrodes, 300mA constant current, 1h. Or transfer 20V overnight (more than 10h). After transferring the membrane, cut off the power supply and take out the hybridization membrane. Cut the membrane according to the pre-sampling sequence.

3) Wash the membrane 3 times with 25mL PBS-T, 5min each time, shake at room temperature.

4) Place the membrane in 25 mL of blocking buffer for 1 hour at room temperature and shake (or overnight at 4).

5) Wash 3 times with PBS-T, 5 min each time.

6) Put each cut membrane into a 10mL centrifuge tube, add primary antibody (diluted 1000 times), incubate at room temperature for 1 hour, and shake slowly.

7) Wash 3 times with PBS-T, 5 min each time.

8) Put all the membranes together in a plate, add an appropriate amount of diluted secondary antibody (goat anti-rabbit IgG-HRP, diluted 5000 times), shake the reaction slowly, and incubate at room temperature for 1 hour.

9) Wash 3 times with PBS-T, 5 min each time.

10) Rinse the membrane with deionized water for a while, stick the corner of the filter paper to absorb the water, mix the luminescent agent A and B at a ratio of 1:1, drop it on the membrane, choose an appropriate exposure time for about 5 minutes, and perform exposure imaging.

The detection results are shown in Figure 1. In the figure: 1-5 detection antigens are ordinary milk skim milk, transgenic milk skim milk, FIX standard, PCC and bovine FIX. The loading volume of FIX standard was 100ng, and the loading volume of other antigens was 15μL.

It can be seen from the figure that there are no bands in lanes 1 and 5, and there are bands in lanes 2-4, and only one band appears in each. Bands 2 and 4 have a molecular weight of about 55KD, which is similar to the molecular weight of FIX in PCC and rFIX in transgenic milk; the molecular weight of band 3 is about 61KD, which is close to the molecular weight of FIX standard. The results indicated that the antibodies prepared by FIX-2-DTT immunization could recognize hFIX in standard FIX, PCC and transgenic milk, but not bFIX and proteins in normal milk. That is to say, the antibody has good specificity and no cross-reaction with non-FIX substances. Here, the molecular weight of the hFIX standard product is larger than that of pdhFIX and rhFIX. This is because FIX is a glycoprotein, and its molecular weight variation range depends on its sugar content, while the sugar content of the hFIX standard product is higher than that of pdhFIX and rhFIX. due to high.

1.4 ITC200 Determination of Antibody Affinity

1) Sample preparation: Dilute the antibody to 0.1 mg/mL with 1×PBS, dialyze PCC, transgenic milk skim milk and ordinary milk skim milk with the same PBS, and concentrate them so that the molar concentration of FIX in them is at least 10 times the molar concentration of antibody in the reaction pool. The concentrated sample is then used as a titrant to titrate the antibody.

2) Change water and methanol: Change water and methanol, and centrifuge the buffer or deionized water first.

3) Startup and cleaning: Turn on the ITC200 and the computer, click to run the software ITC200Withorigin software, and first set the temperature to 25°C for balance. Follow the prompts to carefully clean the ITC200 syringe and measuring tank, click cellandsyringewash to start cleaning, and wash at least 3 times. When cleaning, make sure that there is water in the needle, otherwise wash again.

4) Pre-experiment with water drops: after washing, perform a pre-test with water drops, and add 280 μL of water to the sample pool. When adding the sample, the syringe sucks about 380 μL of the reactant. The syringe is first inserted directly into the bottom, and then pushed to make the reactant drop into the reaction pool, while slowly lifting the syringe. During this period, do not pull out the needle directly to avoid air bubbles . After dripping, re-absorb the reactant that exceeds the surface of the pool, check whether there are air bubbles in the pool, if so, re-absorb and add the sample. Add 80 μL of water to the PCR tube and place it at the load. First, then move the needle to the rest position, press syringefill to exhaust, then move the needle to the load position, click OK continuously, and start sample loading, make sure that the air bubbles in the needle are all shot, otherwise continue to press syringefill until the air bubbles are exhausted.

6) Run the instrument: set the program and path, set the reaction temperature to 25°C, perform an isothermal titration reaction of 16 drops, press start to start, and the instrument will automatically enter the titration program after a period of equilibrium.

7) Data analysis: After the reaction is completed, the energy change curve is obtained, and the curve is fitted by the OriginITCversion7.0OneSite model to obtain the binding constant (K _D ), stoichiometry (N), enthalpy change (△H) and entropy change (△ S) and other thermodynamic parameters.

8) Sample determination: after pre-experimental analysis to ensure that the instrument is in normal condition, test the sample to be tested. The procedure is the same as above. The injection needle draws 40 μL of titrant, and 280 μL of antibody solution is added to the reaction pool to carry out 16 drops of isothermal titration reaction. After completion, analyze and process the data.

Inject the above-mentioned purified anti-FIX antibody into the reaction pool, inject PCC, transgenic milk skim milk (including rFIX) and non-transgenic milk skim milk into the needle for isothermal calorimetric titration reaction, and use the OriginITCversion7.0OneSite model for curve simulation, The results are shown in Figure 2 and Table 12. Figure 2 shows that the antibody was titrated with transgenic milk skim milk expressing rFIX. It can be seen from the figure that a better fitting curve was obtained by simulation with the OriginITCversion7.0OneSite model. In addition, a good fitting curve was obtained by using the OriginITCversion7.0OneSite model to simulate the antibody titration with PCC; however, when the antibody titration with ordinary milk and skim milk was fitted with this model, an error message appeared, that is, it could not be fitted. It shows that the antibody prepared in this example can bind to the natural form of pdhFIX in PCC and rhFIX in transgenic milk, but not to other proteins in non-transgenic milk. Combined with the previous Western-blot data, it shows that the specificity of the antibody is better, and it can recognize the non-denatured forms of pdFIX and rFIX.

Table 12 Binding constant, enthalpy change and entropy change of anti-FIX antibody binding reaction with different antigens

As can be seen from the data in the table, the binding constants K _D of the anti-FIX antibody prepared in the present invention to react with PCC and transgenic milk skim milk are 1.14±0.34×10 ⁶ (M ⁻¹ ) and 4.69±0.76×10 respectively. ⁶ (M ^-1 ), indicating that the affinity of this antibody to pdFIX and rFIX is moderate under the condition of 10mMPBS. Therefore, this lays a good foundation for the use of antibodies as affinity ligands to purify FIX (including pdFIX and rFIX), because its affinity to pdFIX and rFIX is relatively mild, and when they are used for the immunoaffinity of FIX When purification and purification, it will not require harsh elution conditions like high-affinity antibodies, which will lead to the inactivation of the target protein; nor will it be like low-affinity antibodies, the binding force is very weak, resulting in low purification efficiency. Therefore, they can not only maintain the activity of the target protein but also maintain better purification efficiency, and are ideal ligands for immunoaffinity purification of FIX.

From the enthalpy change and entropy change data of all reactions, it is known that △H<0 and △S<0 of the binding reaction between the antibody and pdFIX in PCC and rFIX in transgenic milk, indicating that their main forces are hydrogen bonds and Composite systems formed by van der Waals forces.

Through the above method, an anti-FIX antibody with good specificity and moderate affinity has been prepared. The method is simple and quick, and the preparation cost is low. Foundation.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (15)

1. an antigenic epitope peptide, is characterized in that, described antigenic epitope peptide is derived from animal FIX and comprises one or more antigenic epitopes, And the amino acid sequence length of the antigenic epitope peptide is 2-100% of the full length of FIX, and the length of the antigenic epitope peptide is 5-500 amino acids; And the recombinant protein formed by the epitope peptide and the carrier protein can induce the same kind of animal to produce an immune response against FIX. 2. The antigenic epitope peptide as claimed in claim 1, wherein, the antigenic epitope peptide is selected from the group consisting of: (1)NAAINKY; (2) A derivative polypeptide formed by substituting, deleting or adding one or more amino acid residues to the amino acid sequence in (1), and having the function of inducing an immune response after being fused with a carrier protein. 3. A fusion protein, which is formed by fusing the epitope peptide according to claim 1 with a carrier protein. 4. The fusion protein according to claim 3, wherein the antigenic epitope peptide replaces the 292-295th amino acid in DTT to form the fusion protein; preferably, the fusion protein is selected from: (a) a polypeptide having the amino acid sequence shown in SEQ ID NO.:12; (b) A polypeptide derived from (a) that is formed by substituting, deleting or adding one or more amino acid residues to each polypeptide in (a), and has the function of inducing an immune response. 5. An antibody, characterized in that the antibody specifically binds to the epitope peptide of claim 1 or the fusion protein of claim 3. 6. An isolated antigen-antibody complex, characterized in that the antigen forming the complex comprises the antigenic epitope peptide according to claim 1 or the fusion protein according to claim 3, and the antigenic epitope peptide An epitope is formed to which the antibody specifically binds. 7. A polynucleotide encoding the epitope peptide of claim 1, the fusion protein of claim 3 or the antibody of claim 5. 8. An expression vector comprising the polynucleotide according to claim 7. 9. A host cell, which contains the expression vector according to claim 8, or has the polynucleotide according to claim 7 integrated in the genome. 10. A pharmaceutical composition, said composition containing the epitope peptide of claim 1, the fusion protein of claim 3, the antibody of claim 5, and the polynucleoside of claim 7 acid, the expression vector of claim 8 or the host cell of claim 9, and pharmaceutically acceptable carriers and/or adjuvants. 11. A vaccine composition, said composition containing the antigenic epitope peptide according to claim 1, the fusion protein according to claim 3, the polynucleotide according to claim 7, the polynucleotide according to claim 8 The expression vector or the host cell according to claim 9, and an immunologically acceptable carrier and/or adjuvant. 12. The epitope peptide of claim 1, the fusion protein of claim 3, the polynucleotide of claim 7, the expression vector of claim 8 or the host cell of claim 9 use, (a) for preparing antibodies against the antigenic epitopes; and/or (b) for preparing medicaments for treating diseases related to the antigenic epitopes. 13. The antibody of claim 5 or the purposes of the antigen-antibody complex of claim 6, for (1) Separation and purification of FIX; or (2) Detection of FIX for non-diagnostic purposes. 14. A method for preparing FIX antibody, characterized in that it comprises steps: (1) using the fusion protein according to claim 3 as an antigen to immunize animals, and to produce anti-FIX antibodies in animals; (2) Separating and purifying the FIX antibody. 15. A method for separating and purifying human FIX, characterized in that it comprises the steps of: (1) using the method described in claim 14 to prepare human FIX antibody; (2) Using the antibody obtained in step (1), purify human FIX by affinity chromatography.