CN105294857B - FIX-based epitope and application thereof - Google Patents

Abstract

The present invention discloses a FIX-based antigenic epitope and its application. Specifically, the present invention provides an antigenic epitope peptide, the 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 times the length of the full length of FIX -100%, and the length of the epitope peptide is 5-500 amino acids; and the recombinant protein formed by the epitope peptide and the carrier protein can induce the same species of the animal to produce an immune response against FIX. After the antigenic epitope peptide is fused with the carrier protein, a strong humoral immune response can be stimulated to generate an antibody specific for 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

Epitope and its application based on FIX

Technical field

The present invention relates to biomedicine fields, more particularly to a kind of epitope based on FIX and its application.

Background technique

FIX is the glycoprotein for participating in an important vitamin K of coagulation cascade reaction in blood coagulation system and relying on, it is in people Intracorporal shortage or deficiency will lead to hemophilia B.Lifelong infusion FIX preparation is the most important therapy approach of hemophilia B, that is, Say hemophilia B patient all the life all be unable to do without FIX treatment therefore obtain the hFIX of high security for hemophilia B patient It is very important.

The main source approach of FIX is FIX (pdhFIX) to be purified from human normal plasma and by technique for gene engineering system Standby recombination FIX (rhFIX).Traditional pdhFIX purification process includes calcium phosphate absorption method, aluminium hydroxide absorption method, ion exchange Method and heparin affinity purification etc..Since the concentration of hFIX in blood is very low, (hFIX concentration is about 5 μ g/ in human normal plasma ML), and the physicochemical properties of this kind of blood coagulating protein are again quite similar, and therefore, the hFIX purity of these conventional methods preparation is total Be it is very low, often containing FII, FVII, FX and PROTEIN C etc., since hemophilia people needs lifelong medication, inject repeatedly this The hFIX preparation of low-purity has the danger for increasing thrombosis and thromboembolism occurring.Although a smaller number of method can be made The higher hFIX of purity is obtained, but since its is at high cost or the rate of recovery is low, it can not be applied in the industrialized production of hFIX.? RhFIX develops aspect, has the report for having gone out active rhFIX by cow's milk system successful expression, but in its scale Aspect is isolated and purified, still fails to find effective method so far.The certain methods attempted, including ion-exchange chromatography, heparin Affinity chromatography or several method are used in series, and fail to obtain good as a result, being frequently not so that rhFIX inactivation is exactly Lead to that the rate of recovery is extremely low or purity is too low because step is various.Therefore, develop it is a kind of efficiently and be suitable for FIX (including PdhFIX and rhFIX) scale purifying method it is just most important and extremely urgent.

Immunoaffinity chromatography because of the potential ideal method that its high specific and High Purity efficiency are considered as purifying antigen, and Its successful key first consists in the anti-hFIX antibody that whether can be made that enough specificity are good and affinity is moderate.1984, Liebman etc. passes through preparation Ca²⁺The monoclonal antibody of dependent form purifies hFIX, as a result, just obtains through step purifying special Blood coagulation activity is up to the pure hFIX of 150IU/mg, and the rate of recovery is high, and the monoclonal antibody and the knot affinity of hFIX are more warm With just ensure that the activity of hFIX in this way.But the monoclonal antibody involves great expense, manufacturing cycle is very long, limit its Application in hFIX large-scale production.Polyclonal antibody can be used for the purifying of antigen, and prepare simply, but due to common Polyclonal antibody be the antibody mixture generated by multiple B epitopes, property is inhomogenous, and affinity is very strong, when it is used for When purifying, elution requirement difficulty or ease are grasped, and often due to excessively harsh elution requirement causes target protein to inactivate.Another party Face, mostly anti-specificity is often relatively low, when it is used to purify the rhFIX in cow's milk, it is likely that can be with ox endogenous Non-specific binding occurs for FIX (bFIX), reduces so as to cause the Product safety of purifying, therefore, it is not suitable for for purifying HFIX (including pdhFIX and rhFIX).Therefore, to solve the problems, such as that both antibody exist for purifying hFIX, it is necessary to look for To a kind of new anti-hFIX preparation method for antibody, the antibody that enough specificity are good and affinity is mild can be generated, thus The scale immunoaffinity purification for hFIX is enabled it to, high-purity is obtained and keeps the hFIX of blood coagulation activity.

As the antibody for purifying antigen, what it was identified is not entire antigen molecule, but the B table in antigen molecule Position.Studies have shown that the specificity of antibody is related to the specificity of antigen, and the specificity of antigen actually refers to the spy of epitope It is anisotropic.Therefore obtaining the epitope good, that affinity is moderate of specificity is the key that prepare antibody purification.

Summary of the invention

The purpose of the present invention is to provide a kind of epitope peptide based on FIX and its application, the epitope peptide and load Stronger humoral immune reaction can have been excited after body protein fusion, has generated the antibody that specificity is directed to FIX.

It is a further object of the present invention to provide the preparations of a kind of specific recognition and the antibody and FIX antibody of combination people FIX Method, what which can be used for people FIX isolates and purifies, detects and treats disease relevant to the epitope.

The first aspect of the present invention, provides a kind of epitope peptide, and the epitope peptide is originated from animal FIX and includes One or more epitopes,

And the epitope peptide length amino acid sequence is 2-100% (preferably, the antigen table of FIX overall length Position peptide amino acid sequence length is the 5-70% of corresponding low immunogenicity full length protein；It is highly preferred that the epitope peptide Length amino acid sequence is the 5-50% of corresponding low immunogenicity full length protein；Most preferably, the epitope peptide amino Acid sequence length is the 5-30% of corresponding low immunogenicity full length protein, such as 5%, 10%, 15%, 20%, 25%), and institute The length for stating epitope peptide is 5-500 amino acid；

And the recombinant protein that the epitope peptide and carrier protein are formed can induce the same kind of animal and produce The raw immune response for being directed to FIX.

Preferably, the epitope peptide length amino acid sequence is 3-57.It is highly preferred that the epitope peptide amino Acid sequence length is 5-17.Such as 5,6,7,8,9,10,11,12,13,14,15,16,17 amino acid.

In another preferred example, the animal is mammal.Such as, people, mouse, rabbit, ox or sheep.

In another preferred example, the epitope peptide includes people FIX full length sequence, mouse FIX full length sequence, rabbit FIX complete Long sequence, ox FIX full length sequence, sheep FIX full length sequence or its homologous sequence.

In another preferred example, the people FIX full length sequence is as shown in SEQ ID NO.:1.

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

In another preferred example, the ox FIX full length sequence is as shown in SEQ ID NO.:3.

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

(1)NAAINKY；

(2) amino acid sequence in (1) is formed by one or more replacing, missing or adding for amino acid residue , and there is the derivative polypeptide for inducing immune response function after merging with carrier protein.

In another preferred example, the carrier protein has at least one t cell epitope, and in the carrier protein At least one molecular surface amino acid residue area introduces the epitope peptide by splicing, replacement, and/or insertion.

In another preferred example, the carrier protein and the antigen peptide fragment are not from the same albumen, and described The immunogenicity of the epitope peptide can be enhanced in carrier protein.

In another preferred example, the carrier protein includes diphtheria toxin DT, the transmembrane domain DTT of diphtheria toxin, suddenly Random toxin B subunit (CTB), salmonella flagellin (FliC), pertussis toxin (PTX), tetanus toxin or above-mentioned egg White immunogenic fragments (the C segment of such as tetanus toxin), rotavirus VP 7, leishmanial heat shock protein, jejunum Campylobacter spp flagellin, Major Outer Membrane Protein of Chla mydia trachomatis, hemocyanin (Keyhole Limpet Hemocyanin, KLH), bovine serum albumin(BSA) (Bovine Serum Albumin, BSA), chicken ovalbumin (Ovalbumin, OVA), fiber egg Bai Yuan.

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

The second aspect of the present invention, provides a kind of fusion protein, and the fusion protein is such as first aspect present invention institute The epitope peptide stated is merged with carrier protein to be formed by.

In another preferred example, the carrier protein has at least one t cell epitope, and in the carrier protein At least one molecular surface amino acid residue area introduces the epitope peptide by splicing, replacement, and/or insertion.

In another preferred example, the carrier protein and the antigen peptide fragment are not from the same albumen, and described The immunogenicity of the epitope peptide can be enhanced in carrier protein.

In another preferred example, the carrier protein includes diphtheria toxin DT, the transmembrane domain DTT of diphtheria toxin, suddenly Random toxin B subunit (CTB), salmonella flagellin (FliC), pertussis toxin (PTX), tetanus toxin or above-mentioned egg White immunogenic fragments (the C segment of such as tetanus toxin), rotavirus VP 7, leishmanial heat shock protein, jejunum Campylobacter spp flagellin, Major Outer Membrane Protein of Chla mydia trachomatis, hemocyanin (Keyhole Limpet Hemocyanin, KLH), bovine serum albumin(BSA) (Bovine Serum Albumin, BSA), chicken ovalbumin (Ovalbumin, OVA), fiber egg Bai Yuan.

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

In another preferred example, the carrier protein is the transmembrane domain DTT of diphtheria toxin, and the loop Area includes 292-298,305-310 amino acids.In another preferred example, by the way that the epitope peptide is replaced the DDT 292-295 amino acids the fusion protein is made.

In another preferred example, the carrier protein is choleratoxin B subunit, and the area loop includes:

Choleratoxin B subunit (CTB), which can plant epitope, can plant epitope 29-38 amino acids

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

In another preferred example, there is link peptide between the epitope and the carrier protein.Preferably, the company Connecing peptide length is 3-30 amino acid.It is highly preferred that the link peptide length is 4-20 amino acid.Most preferably, the company Connecing peptide length is 7-17 amino acid.

In another preferred example, do not have link peptide between the epitope and the carrier protein.

In another preferred example, the 292-295 amino acids in the epitope peptide replacement DTT form the fusion Albumen.

In another preferred example, the fusion protein is selected from:

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

(b) shape by one or more replacing, missing or adding for amino acid residue respectively by each polypeptide in (a) At, and there is the polypeptide as derived from (a) for inducing immune response function.

The third aspect of the present invention provides a kind of antibody, the combination first aspect present invention institute of the antibody specificity Fusion protein described in the epitope peptide or second aspect of the present invention stated.

In another preferred example, the epitope peptide is NAAINKY.

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

The fourth aspect of the present invention provides a kind of isolated antigen antibody complex, wherein form the compound It is described anti-comprising fusion protein described in epitope peptide or second aspect of the present invention described in first aspect present invention in antigen Former epitope peptide forms epitope, is incorporated into the epitope to the antibody specificity.

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

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

The sixth aspect of the present invention, provides a kind of expression vector, and the expression vector contains fifth aspect present invention institute The polynucleotides stated.

The seventh aspect of the present invention, provides a kind of host cell, and the host cell contains sixth aspect present invention The expression vector, or polynucleotides described in fifth aspect present invention are integrated in genome.

In another preferred example, the host cell includes prokaryotic cell and eukaryocyte.

In another preferred example, the host cell includes Escherichia coli, yeast, Chinese hamster ovary celI, DC cell etc..

The eighth aspect of the present invention, provides a kind of pharmaceutical composition, and the composition contains first aspect present invention Fusion protein described in the epitope peptide, second aspect of the present invention, antibody described described in third aspect present invention, Expression vector described in the polynucleotides, sixth aspect present invention described in fourth aspect present invention or the present invention the 7th The host cell and pharmaceutically acceptable carrier and/or auxiliary material described in aspect.

In another preferred example, the composition is vaccine.

The ninth aspect of the present invention, provides a kind of vaccine composition, and the composition contains first aspect present invention Fusion protein described in the epitope peptide, second aspect of the present invention, antibody described described in third aspect present invention, Expression vector described in the polynucleotides, sixth aspect present invention described in fourth aspect present invention or the present invention the 7th Acceptable carrier and/or auxiliary material on the host cell and immunology described in aspect.

In another preferred example, the vaccine composition also contains adjuvant.

In another preferred example, the adjuvant includes aluminium oxide, saponin(e, quil A, muramyl dipeptide, mineral oil or plant Object oil, the adjuvant based on vesica, non-ionic block copolymer or deae dextran, cell factor (including IL-1, IL-2, IFN- R, GM-CSF, FIX, IL-12 and CpG).

The eleventh aspect of the present invention provides epitope peptide described in first aspect present invention, second party of the present invention It is fusion protein described in face, antibody described described in third aspect present invention, described more described in fourth aspect present invention The purposes of host cell described in expression vector described in nucleotide, sixth aspect present invention or seventh aspect present invention,

(a) it is used to prepare the antibody for the epitope；And/or (b) it is used to prepare treatment and the epitope The drug of relevant disease.

In another preferred example, the disease includes: cardiovascular disease, thrombus, apoplexy and infraction etc..

The twelveth aspect of the present invention provides described in antibody described in third aspect present invention or fourth aspect present invention Antigen antibody complex purposes, be used for

(1) FIX is isolated and purified；Or

(2) detection of non-diagnostic purpose FIX.

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

(1) fusion protein described in second aspect of the present invention is used to generate as antigen-immunized animal, and in animal body The antibody of anti-FIX；

(2) the FIX antibody is isolated and purified.

In another preferred example, the animal is mammal.

In another preferred example, the animal includes people, mouse, rabbit, ox or sheep.

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

(1) using the present invention the 13rd aspect described in method preparation people FIX antibody；

(2) using the antibody obtained in step (1), using the method Purification of Human FIX of affinity chromatography.

The fifteenth aspect of the present invention provides a kind for the treatment of method, applies first aspect present invention to the object of needs Fusion protein described in the epitope peptide, second aspect of the present invention, antibody described described in third aspect present invention, Expression vector described in the polynucleotides, sixth aspect present invention described in fourth aspect present invention or the present invention the 7th Host cell described in aspect, described in pharmaceutical composition or ninth aspect present invention described described in eighth aspect present invention Vaccine composition.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Detailed description of the invention

Fig. 1 shows the binding specificity of Western-blot detection purifying gained antibody in embodiment 3.

Fig. 2 shows antibody isothermal calorimetric titration measurement result in embodiment 3.

Specific embodiment

The present inventor is obtained a new class of FIX epitope, which is melted with carrier protein by extensive and in-depth research It closes, fusion protein obtained can obtain specific good, the moderate anti-FIX antibody of affinity as antigen-immunized animal, and should Antibody can identify pdhFIX and rhFIX, be the prepare with scale of the anti-hFIX antibody of high-purity, established solid Basis.

FIX

The amino acid sequence of people FIX is as follows:

MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEK CSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQF CKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQS FNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNV IRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYL RVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVS RYVNWIKEKTKLT(SEQ ID NO.:1)

The amino acid sequence of mouse FIX is as follows:

MKHLNTVMAESPALITIFLLGYLLSTECAVFLDRENATKILTRPKRYNSGKLEEFVRGNLERECIEER CSFEEAREVFENTEKTTEFWKQYVDGDQCESNPCLNGGICKDDISSYECWCQVGFEGRNCELDATCNIKNGRCKQF CKNSPDNKVICSCTEGYQLAEDQKSCEPTVPFPCGRASISYSSKKITRAETVFSNMDYENSTEAVFIQDDITDGAI LNNVTESSESLNDFTRVVGGENAKPGQIPWQVILNGEIEAFCGGAIINEKWIVTAAHCLKPGDKIEVVAGEYNIDK KEDTEQRRNVIRTIPHHQYNATINKYSHDIALLELDKPLILNSYVTPICVANREYTNIFLKFGSGYVSGWGKVFNK GRQASILQYLRVPLVDRATCLRSTTFTIYNNMFCAGYREGGKDSCEGDSGGPHVTEVEGTSFLTGIISWGEECAMK GKYGIYTKVSRYVNWIKEKTKLT(SEQ ID NO.:2)

The amino acid sequence of ox FIX is as follows:

YNSGKLEEFVRGNLERECKEEKCSFEEAREVFENTEKTTEFWKQYVDGDQCESNPCLNGGMCKDDINS YECWCQAGFEGTNCELDATCSIKNGRCKQFCKRDTDNKVVCSCTDGYRLAEDQKSCEPAVPFPCGRVSVSHISKKL TRAETIFSNTNYENSSEAEIIWDNVTQSNQSFDEFSRVVGGEDAERGQFPWQVLLHGEIAAFCGGSIVNEKWVVTA AHCIKPGVKITVVAGEHNTEKPEPTEQKRNVIRAIPYHSYNASINKYSHDIALLELDEPLELNSYVTPICIADRDY TNIFSKFGYGYVSGWGKVFNRGRSASILQYLKVPLVDRATCLRSTKFSIYSHMFCAGYHEGGKDSCQGDSGGPHVT EVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT(SEQ ID NO.:3)

Carrier protein

As used herein, term " carrier protein " refers to the egg in recombinant protein of the invention as protein structure skeleton It is white.In general, the carrier protein is the stronger albumen of immunogenicity, such as pathogen protein, representative example include (but It is not limited to): virus protein, bacterioprotein, I (chlamydia) protein, mycoplasma albumen etc..

As used herein, term " epitope (peptide) " refers to that quasi- induction animal generates one section of other albumen of immune response Peptide, the epitope not referring to for carrier protein, carrier protein itself can cause the peptide fragment of immune response.In general, anti- Former epitope refers to the peptide fragment of the quasi- targeting of immune response, preferably derives from one section of peptide of mammal (such as people) albumen, rather than comes From the carrier protein.

As used herein, term .pdb refers exclusively to tertiary protein structure data file, comes from Protein Data Bank (www.pdb.org)；

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

As used herein, term t cell epitope is also known as T cell antigen epitope, is that antigen molecule is thin by antigen presentation One section of peptide that enzymatic hydrolysis processing generates in born of the same parents, can be combined by major histocompatibility complex (MHC) molecule, be presented in cell table Face is combined by T cell receptor (TCR), activation T cell, including t helper cell epitope etc..

As used herein, term " low immunogenicity albumen " refers to that individually immune animal cannot cause enough immune responses Albumen.

As used herein, term " molecular surface amino acid residue area " or " surface amino groups acid residue area " refer to positioned at albumen Molecular surface amino acid residue composition region, it is preferable that " the molecular surface amino acid residue area " include the area loop, The area beta-tum, N-terminal or C-terminal.

Representative carrier protein

1. diphtheria toxin and its transmembrane domain

Diphtheria toxin (diphtheria toxin, DT) is the Bacterium diphtheriae for having infected beta bacteriophage The exotoxin that (Corynebacterium diphtheriae) is generated, is present in the DPT vaccine composition of clinical use.Peace Full property obtains the verifying of many years clinical use, rare serious adverse reaction, there is no caused allergic reaction by diphtheria composition at present Report.

Diphtheria toxin molecule is made of 535 amino acid residues, spatially relatively independent catalyst structure domain (1- 193AAs), transmembrane domain (205-378AAs) and receptor binding domains (386-535AAs) composition；Transmembrane domain and receptor knot Conjunction domain itself is non-toxic, and function is combined by cell surface receptor, catalyst structure domain transduction is entered intracellular.

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

GADDVVDSSK SFVMENFSSY HGTKPGYVDS IQKGIQKPKS GTQGNYDDDW KGFYSTDNKY

DAAGYSVDNE NPLSGKAGGV VKVTYPGLTK VLALKVDNAE TIKKELGLSL TEPLMEQVGT EEFIKRFGDGASRVVLSLPF AEGSSSVEYI NNWEQAKALS VELEINFETR GKRGQDAMYE YMAQACAGNR VRRSVGSSLSCINLDWDVIR DKTKTKIESL KEHGPIKNKM SESPNKTVSE EKAKQYLEEF HQTALEHPEL SELKTVTGTNPVFAGANYAA WAVNVAQVID SETADNLEKT TAALSILPGI GSVMGIADGA VHHNTEEIVA QSIALSSLMVAQAIPLVGEL VDIGFAAYNF VESIINLFQV VHNSYNRPAY SPGHKTQPFL HDGYAVSWNT VEDSIIRTGFQGESGHDIKI TAENTPLPIA GVLLPTIPGK LDVNKSKTHI SVNGRKIRMR CRAIDGDVTF CRPKSPVYVGNGVHANLHVA FHRSSSEKIH SNEISSDSIG VLGYQKTVDH TKVNSKLSLF FEIKS(SEQ ID No.:4)

In diphtheria toxin molecule there are 5 T- helper epitopes to be known by up to 80% or more people MHCclass II Not.The present inventor simulates the protein structure of diphtheria toxin, retains α spiral and β-pleated sheet element and T that stable structure needs Cell epitope can be replaced surface amino groups acid residue zone position 292-298, the 305-310 amino acids of implantation epitope.

Diphtheria toxin transmembrane domain (DTT) itself is nontoxic, mainly constitutes core skeleton, screw element by α screw element Between by flexibility ring region connect.

DTT amino acid sequence (1F0L.pdb:202-378) is as follows:

202INLDWDVIRD KTKTKIESLK EHGPIKNKMS ESPNKTVSEE KAKQYLEEFH QTALEHPELS

262ELKTVTGTNP VFAGANYAAW AVNVAQVIDS ETADNLEKTT AALSILPGIG SVMGIADGAV

322HHNTEEIVAQ SIALSSLMVA QAIPLVGELV DIGFAAYNFV ESIINLFQVV HNSYNRP(SEQ ID No.:5)

The present inventor simulates the protein structure of DTT, retains α spiral and β-pleated sheet element and T that stable structure needs Cell epitope can be replaced the position 292-298 of implantation epitope, 305-310 amino acids.

In preference of the invention, the peptide fragment that the quasi- target protein intervened of drug may be selected is transplanted on DTT, and is replaced The surface amino groups acid residue area of DTT, including ring region amino acid residue between α screw element.

It is of the invention studies have shown that diphtheria toxin transmembrane structure with after the peptide fragment integration from target protein, will not or it is basic On will not influence respective folding.In recombinant protein, DTT can after target protein peptide fragment is transplanted on DTT as peptide backbone To induce animal to generate the immune response for the target protein.Therefore DTT is a kind of most suitable peptide backbone.

2. choleratoxin B subunit (CTB)

Cholera toxin (cholera toxin) is the exotoxin (84kDa) of comma bacillus secretion, is made of A, B subunit, is AB5 type.Choleratoxin B subunit (CTB) is the nontoxic part of cholera toxin, has good immunogenicity, through human trial It is proved to be the effective component of vaccine.CTB can be with Ganglioside GM1 specificity knot existing for most of mammalian cell surfaces It closes, stimulation body generates mucous membrane IgA, reinforces antigenic mucosa immunity-inducing reaction.CTB have been used for new oral cholera vaccine, Adjuvant and protein carrier.

CTB is nontoxic, is made of core skeleton 2 sections of α screw elements and 6 sections of beta- pieces, by flexibility between structural detail Ring region connection.Five Asias B form Pentagram shape with the relatively parallel inwardly assembling of long α screw element.Each subunit can be independent Nerve node glycosides rouge (GM1) receptor on combination cell film.

CTB amino acid sequence (3CHB.pdb:1-103) is as follows:

1TPQNITDLCA EYHNTQIYTL NDKIFSYTES LAGKREMAII TFKNGAIFQV EVPGSQHIDS

60QKKAIERMKD TLRIAYLTEA KVEKLCVWNN KTPHAIAAIS MAN(SEQ ID No.:6)

CTB contains 2 t cell epitopes, and CTB-Th epitope 81-100 (81-KVEKLCVWNNKTPHAIAAIS-100) is advantage Epitope, CTB-Th epitope 31-50 (31-LAGKR EMAIITFKNGAIFQV-50) are weak tendency epitope, are distributed in subunit structure 4 sections of Beta- on pieces of core.

The present inventor simulates the protein structure of CTB, retains α spiral and β-pleated sheet element that stable structure needs, T Cell epitope, the position that can be replaced implantation epitope is 29-38 amino acids

3. salmonella flagellin (FliC)

Salmonella flagellin FliC (Phase1-C flagellin) is the filamentous composition of flagellum, with many animals Cell receptor TLR5 combine, excite the innate immune system response of animal, promote immature DC cell surface expression CD80 and CD86 secretes cytokine profiles and chemotactic factor (CF), plays in congenital immune response and specific specific immune response Stronger immunoadjuvant function is applied in multiple pathogen vaccines preparations as immunologic adjuvant composition.

FliC is made of 494 amino acid, and crystal structure is shown, this albumen inflection makes its N-terminal and C-terminal draw close to form shape such as The Greek alphabet gamma " Γ " of capitalization.

The amino acid sequence of FliC (1ucu.pdb:1-494) is as follows:

AQVINTNSLS LLTQNNLNKS QSALGTAIER LSSGLRINSA KDDAAGQAIA NRFTANIKGLTQASRNANDG ISIAQTTEGA LNEINNNLQR VRELAVQSAN STNSQSDLDS IQAEITQRLNEIDRVSGQTQ FNGVKVLAQD NTLTIQVGAN DGETIDIDLK QINSQTLGLD TLNVQQKYKVSDTAATVTGY ADTTIALDNS TFKASATGLG GTDQKIDGDL KFDDTTGKYY AKVTVTGGTGKDGYYEVSVD KTNGEVTLAG GATSPLTGGL PATATEDVKN VQVANADLTE AKAALTAAGVTGTASVVKMS YTDNNGKTID GGLAVKVGDD YYSATQNKDG SISINTTKYT ADDGTSKTALNKLGGADGKT EVVSIGGKTY AASKAEGHNF KAQPDLAEAA ATTTENPLQK IDAALAQVDTLRSDLGAVQN RFNSAITNLG NTVNNLTSAR SRIEDSDYAT EVSNMSRAQI LQQAGTSVLAQANQVPQNVL SLLR(SEQ ID No.:7)

The present inventor simulates the protein structure of FliC, is retaining the α spiral and β-pleated sheet element that stable structure needs In the case where, the position that can be replaced implantation epitope is 348-KYTADDGTSKTA-359 amino acid residue.

Composition and method of administration

The present invention also provides a kind of compositions, it contains: recombinant protein (i) of the invention or codified weight of the invention The polynucleotides of histone, and (ii) acceptable excipient or adjuvant pharmaceutically or in immunology.

In the present invention, term " containing " indicates that various composition can be applied to or be present in composition of the invention together. Therefore, term " mainly by ... form " and " consist of " were included in term " containing ".

Composition of the invention includes pharmaceutical composition and vaccine composition.

Composition of the invention can be a kind of (only containing recombinant protein or polynucleotides) of unit price, be also possible to multivalence (containing there are many recombinant protein or polynucleotides).

Pharmaceutical composition or vaccine composition of the invention can be prepared into various regular dosage forms, including (but and it is unlimited In): injection, granula, tablet, pill, suppository, capsule, suspension, spray etc..

(i) pharmaceutical composition

Pharmaceutical composition of the invention includes the recombinant protein or polynucleotides of the present invention of (or containing) therapeutically effective amount.

The amount that the term as used herein " therapeutically effective amount " refers to therapeutic agent treatment, alleviates or prevent target disease or situation, Or show the detectable amount for treating or preventing effect.The effect can be detected for example, by antigen levels.Therapeutic effect It also include the reduction of physical symptoms.For certain an object accurate effective quantity depend on the object figure and health status, The combination of therapeutic agent and/or therapeutic agent that the property and degree of illness and selection are given.Therefore, preassigning accurately has Effect amount is useless.However, can determine the effective quantity with routine experiment for the situation that Mr. Yu gives.

For the purposes of the present invention, effective dosage is to give individual about 0.001 mg/kg to 1000 mg/kgs, It is preferably about the recombinant protein of 0.01 mg/kg to 100 mg/kg weight.

Pharmaceutical composition can also contain pharmaceutically acceptable carrier.Term " pharmaceutically acceptable carrier " refers to for controlling Treat the carrier of agent (such as recombinant protein of the invention) administration.The term refers to medicament carriers some in this way: themselves is not induced It generates to receiving the harmful antibody of individual of the composition, and there is no excessive toxicity after being administered.Suitable carrier can be greatly , the slow macromolecular of metabolism, such as protein, polysaccharide, polylactic acid (polylactic acid), polyglycolic acid.These carriers It is well known to those of ordinary skill in the art.In Remington ' s Pharmaceutical Sciences (Mack Pub.Co., N.J.1991) in can find discussing fully about pharmaceutically acceptable carrier or excipient.

The upper acceptable carrier of combination of traditional Chinese medicine may include liquid, such as water, salt water, glycerol and ethyl alcohol.In addition, these are carried There is likely to be complementary substances, such as wetting agent or emulsifier, pH buffer substance in body.In general, composition can be made Injectable agent, such as liquid solution or suspension；It may also be fabricated which and be suitble to supplying solution or suspension, liquid excipient before the injection Solid form.Liposome is also included in the definition of pharmaceutically acceptable carrier.

(ii) vaccine composition

Vaccine (composition) of the invention can be preventative (i.e. prevention disease) or therapeutic (control after illness Treat disease).

These vaccines include immunising antigen (including recombinant protein of the present invention), and usually with it is " pharmaceutically acceptable Carrier " combination, these carriers include itself not inducing any carrier generated to the harmful antibody of individual for receiving the composition. Suitable carrier is usually big, the slow macromolecular of metabolism, as protein, polysaccharide, polylactic acid, polyglycolic acid, amino acid are poly- Close object, amino acid copolymer, lipid aggregates (such as oil droplet or liposome).These carriers are those of ordinary skill in the art institutes It is well known.In addition, these carriers can play immunostimulant (" adjuvant ").In addition, antigen can also be with bacterial toxoid (such as The toxoid of the pathogen such as diphtheria, tetanus, cholera, helicobacter pylori) coupling.

The preferred adjuvant of enhancing immune composition effect includes but is not limited to: (1) aluminium salt (alum), such as aluminium hydroxide, phosphorus Sour aluminium, aluminum sulfate etc.；(2) oil-in-water emulsion formula, for example, (a) MF59 (referring to WO90/14837), (b) SAF, and (c) Ribi^TMAdjuvant system (RAS) (Ribi Immunochem, Hamilton, MT), (3) saponin adjuvant；(4) Freund Freund's complete adjuvant (CFA) and Freund Freund's incomplete adjuvant (IFA)；(5) cell factor, as interleukin (such as IL-1, IL-2, IL-4, IL-5, FIX, IL-7, IL-12 etc.), interferon (such as interferon), macrophage colony stimulating factor (M-CFS), tumor necrosis factor (TNF) etc.；(6) the detoxification variant of bacterial ADPribosylating toxin (such as E.coli LT LT)；And (7) Enhance the other materials of composition effect as immunostimulant.

Including immunogenic composition vaccine composition (such as, it may include antigen, pharmaceutically acceptable carrier And adjuvant), usually contain diluent, such as water, salt water, glycerol, ethyl alcohol etc..In addition, auxiliary substances, such as wetting agent or emulsification Agent, pH buffer substance etc. may be present in this kind of carrier.

More specifically, the vaccine including immunogenic composition, the immunogenic polypeptide comprising immunological effective amount, And above-mentioned other required components." immunological effective amount ", which refers to, gives the amount of individual to treatment with single dose or continuous agent a part Or prevention is effective.The dosage can according to the health status and physiological status for treating individual, treat individual classification (such as People), the ability of individual immunity system synthesis antibody, required degree of protection, the preparation of vaccine, treating physician is to medical conditions Depending on assessment and other correlative factors.It is expected that the dosage is by relatively wide range, it can be by routine experiment come really It is fixed.

In general, injectable agent, such as liquid solution or suspension can be made for vaccine composition or immunogenic composition；Also It can be made into the solid form for being suitble to supplying solution or suspension, liquid excipient before the injection.Said preparation is also emulsifiable or is encapsulated in In liposome, to enhance adjuvant effect.

In addition, vaccine composition of the invention can be unit price or polyvaccine.

(iii) administration route and dosage

Once being made into the composition of the present invention, it can directly be given to object.Object to be treated can be mammal, Especially people.

When being used as vaccine, recombinant protein of the invention can be directly applied to individual by known method.It generallys use These vaccines are applied in administration method identical with conventional vaccine and/or simulation pathogenic infection path.

The approach for giving pharmaceutical composition or vaccine composition of the present invention includes (but being not limited to): intramuscular, subcutaneous, skin Interior, intrapulmonary, intravenous, intranasal, by oral administration or other parenteral route of administration.If desired, can be with combination medicine-feeding approach or root It is adjusted according to disease event.Vaccine composition can be given with single dose or multi-dose, and may include give booster with Cause and/or maintain immunity.

Recombinant protein vaccine should be given with " effective quantity ", i.e. the amount of recombinant protein is enough to draw in selected administration routes Immune response is sent out, can effectively promote that host is protected to resist relevant disease.

Representative disease includes (but being not limited to): autoimmune disease, tumour etc..

The amount of selected recombinant protein in each vaccine dose part, be by can cause protective immune response and without apparent Depending on the amount of side effect.In general, after infecting host cell, each dose of vaccine is enough containing about 1 μ g-1000mg, preferably 1 μ g-100mg, more preferably 10 μ g-50mg protein.The standard for including the IgG titers in observation object and other reactions can be used Research method determines the optimum amount of specific vaccine.It can be determined the need for by the immunity level of monitoring vaccine offer Enhance dosage.After having evaluated the IgG titers in serum, it may be necessary to select enhancing dose immunizations.Apply adjuvant And/or the immune response to protein of the invention just can be improved in immunostimulant.

Preferred method is to give immunogenic composition from parenteral (subcutaneously or intramuscularly) approach by injection.

In addition, vaccine of the invention can be given together in conjunction with other immunomodulators, or together with other therapeutic agents It gives.

Main advantages of the present invention are:

(1) comprising the fusion protein of the epitope of the invention based on FIX, being used as vaccine can effective exactor body acupuncture pair The immune response of FIX.

(2) preparation cost for carrying the recombinant protein of epitope of the invention is low, convenient drug administration.

(3) relative to the preparation with carrier protein chemical coupling, antigenic structure of the invention is definite, quality controllable, more pacifies Entirely.

(4) fusion protein immunization animal of the use comprising epitope peptide of the present invention, the antibody for FIX of preparation, High specificity, affinity is moderate, which is highly suitable for isolating and purifying for FIX.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and Number is calculated by weight.

Embodiment 1 designs B cell antigen epi-position and prepares fusion protein

In the present embodiment, the B epitope of 4 hFIX is devised, the essential information of these epitopes is shown in Tables 1 and 2.

The essential information of the B epitope for the hFIX that table 1 designs

The homology of the B epitope sequence for the hFIX that table 2 designs

3 epitope of table transplants result

1. prepared by epitope fusion protein

1.1 design of primers

Each FIX epitope gene sequence is as follows:

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

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

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

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

DTT gene order:

ataaatcttgattgggatgtcataagggataaaactaagacaaagatagagtctttgaaagagcatgg ccctatcaaaaataaaatgagcgaaagtcccaataaaacagtatctgaggaaaaagctaaacaatacctagaagaa tttcatcaaacggcattagagcatcctgaattgtcagaacttaaaaccgttactgggaccaatcctgtattcgctg gggctaactatgcggcgtgggcagtaaacgttgcgcaagttatcgatagcgaaacagctgataatttggaaaagac aactgctgctctttcgatacttcctggtatcggtagcgtaatgggcattgcagacggtgccgttcaccacaataca gaagagatagtggcacaatcaatagctttatcatctttaatggttgctcaagctattccattggtaggagagctag ttgatattggtttcgctgcatataattttgtagagagtattatcaatttatttcaagtagttcataattcgtataa tcgtccc(SEQ ID NO.:17)

Using the base sequence on the base sequence replacement DTT of the FIX epitope of recombinant PCR technology design, formed The alkali yl coding sequence of fusion protein.

Recombination primer used in recombinant PCR is as shown in table 5.

5 design of primers of table

1.2 vector construction

1) first round PCR

Using DTT plasmid as template, just with the reversed-D of primer DTT forward direction-A/DTT and each lower semisection of the reversed-B/ of each upper semisection First round PCR is carried out to-C, expands the upper semisection and lower semisection of each DTT-FIX epitope recombination respectively.

Reaction system is 5 μ 10 × KOD-Plus of L Buffer, 5 μ L dNTPs (2mmol/L), 2 μ L MgSO4 (25mmol/L), 1.5 μ L primer B or C, 1.5 μ L primer A or D, 1 μ L KOD-Plus (polymerase), 0.5 μ L DTT plasmid (mould Plate), it is mended with aseptic double-distilled water to 50 μ L.

Amplification program:

94 DEG C of initial denaturation 2min, 94 DEG C of denaturation 15s, 55 DEG C of annealing 30s, 68 DEG C of extension 2min, 34 circulations, 68 DEG C extend 10min.It 12 DEG C, Forever, saves.

Electrophoresis detection, all mutant fragments sizes are consistent with expection.

2) the second wheel PCR

First round PCR is tapped and recovered after lower semisection dilutes 15-20 times respectively on product, it then will by following reaction system Upper lower semisection mixing, amplifies DTT- epitope recombination complete sequence with the primer A and D at the both ends DTT.

Reaction system is 5 μ 10 × KOD-Plus of L Buffer, 5 μ L dNTPs (2mmol/L), 2 μ L MgSO₄ (25mmol/L), both ends primer each 1.5 μ L, 1 μ L KOD-Plus (polymerase), 0.5 μ L DTT plasmid (template), with sterile double Water is steamed to mend to 50 μ L.

DTT- epitope recombination is expanded with recombinant PCR mode, through electrophoresis detection, after first round PCR, is obtained respectively The genetic fragment of 200bp and 300bp or so shows that the segment up and down of each recombination expands success；And the second wheel mixing is spelled After connecing, all clip sizes are 500bp or so, in the same size with desired target fragment.

3) Hybrid connections product and carrier double digestion

Second wheel PCR product is through electrophoresis detection, after being tapped and recovered, with BamHI and Xho I double digestion.PGEX-6P-1 is carried The identical enzyme double digestion of body, digestion system are as follows:

By detection, carrier and target DNA can be attached by success double digestion.

4) connection and conversion

The double enzyme digestion product being tapped and recovered is connected with ligase.Linked system are as follows: 1 μ L pEGX-6P-1 carrier, 1 μ 10 × Ligation of L Buffer, 0.5 μ L T4DNA ligase, 1 μ L target DNA (10ng/ μ L) and 6.5 μ L ddH₂O。

Connection product is transferred in competent cell by 1:100DH5 α in proportion, ice bath 30min, 42 DEG C of water-bath heat shock 90S, It is transferred to the centrifuge tube of the culture medium of LB containing 1mL, 37 DEG C, 150rpm shaking table recovery 1h, 500 μ L painting plate is taken out and (contains ammonia benzyl mould Element), cultivate 20h or so in 37 DEG C of insulating boxs, picking positive single colonie, 37 DEG C of constant temperature incubations, OD=0.4 or so extracts recombination Plasmid.

Picking single colonie culture carries out bacterium solution PCR positive colony identification, and identified all bacterium solutions are positive colony, can It carries out that further verifying is sequenced.

5) it is sequenced

Identify correct positive colony through bacterium solution PCR, take the corresponding bacterium solution sequencing of 200 μ L, examining order by Nanjing gold this Auspicious biotech firm completes.

6) positive recombinant plasmid is extracted, BL21 (DE3) cell is converted.

The recombinant plasmid of correct positive colony bacterium solution is sequenced in extracting, is transferred in BL21 (DE3) cell again, and plasmid extracts Operation and conversion operation are same as above.

1.3 protein expressions and purifying

15ml centrifuge tube is taken, the LB culture medium with ampicillin of 3mL is added, picking is transformed into the positive of BL21 cell Clone is added thereto, 37 DEG C of constant temperature incubations, when OD=0.5 or so, is transferred to 1L (benzyl mould of ammonia containing one thousandth containing LB Element) triangular flask in shaking table culture, when OD=0.6 or so, millesimal 50mM IPTG, induction 5h or so is added, will induce Bacterium solution centrifugation, 12000g, 5min are resuspended with 10 times of volume PBS, after ultrasonication, are centrifuged (16000g, 20min) again, will be upper Clear liquid carries out affinity purification.

Protein purification: 5mLGST pillar, 10 times of volume 10mM PBS (pH7.5) balances, flow velocity 3ml/min are taken；It has balanced Afterwards, with the flow velocity loading of 1.5mL/min；After loading is complete, washed with 10mM PBS (pH7.5) with the flow velocity of 1.5-2.0mL/min it is miscellaneous, Until OD₂₈₀It is near close to 0 when stop；Destination protein is eluted with 50mM GSH eluent (pH8.0), collects destination protein, electrophoresis Detect its purity.

Electrophoresis detection is the result shows that purified molecular weight of albumen out is 45kDa or so, with DTT- epitope recombinant protein Albumen it is in the same size, therefore, required epitope fusion protein has successfully been made in we, through Image-lab software gray analysis, Purity reaches 90% or more, can be used to do animal immune experiment.

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

INLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGT NPVFAGANYAAWAVNVAQVIDSNAAINKYNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQ AIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRP(SEQ ID NO.:12)

The Activity determination of 2 fusion protein of embodiment

1 Epitope Identification

1.1. mouse immune

1) the female Bal b/c mouse of 42 4-5 week old is raised.

2) dosage of each albumen is got out according to the protein concentration and required immunizing dose surveyed, and with same volume Aluminum hydroxide adjuvant (8 times of dilution) is sufficiently mixed uniformly with it.

3) when mouse long to 6 weeks, the female Bal b/c mouse of 42 6 week old is divided into 6 groups, wherein 2 groups For negative control group, DTT-GST and aluminum hydroxide adjuvant are injected；Remaining 4 groups are experimental group, use corresponding epitope fusion protein respectively Every group of Bal b/c mouse is immunized, group is as follows:

9 mouse immune of table

4) it after antigen is ready to, is injected by above table, the method that initial immunity uses subcutaneous multi-point injection to be immunized Every mouse injects 50 μ g antigens and (in actual mechanical process, 2 points has been injected less than we of 100 μ L, greater than the note of 100 μ L Penetrate 3-4 point), and take blood to do negative control before immune.

5) immune for the first time to mix after two weeks with identical adjuvant with antigen and uniform, second of mouse immune of progress is exempted from Epidemic disease dosage is 50 μ g antigens/mouse, and injecting method is same as above.

6) immune for the second time secondary immune using same dosage and method progress third time after two weeks.

7) eyeball blood taking method is used to acquire mice serum after a week in second of booster immunization, 4000rpm, 4 DEG C, centrifugation obtains After obtaining serum, packing is stored in -80 DEG C of refrigerators, detects the specific antibody titres in serum with the method for ELISA, and further Do Western-Blot verifying analysis.

1.2ELISSA detects antibody production:

Using FIX standard items as envelope antigen, the serum after each group's doubling dilution is as primary antibody, and detection is directed to respectively The production of FIX antibody.Experimental design is as follows:

Table 10ELISA detection

Experimental implementation:

1) it is coated with

With CBS coating buffer by each antigen diluent to 0.1-1 μ g/mL, it is added 100 μ L into the reacting hole of each ELISA Plate, 4 DEG C be incubated overnight, then by board-washing machine washing wash 5 times, washed 3 minutes, after having washed with washing buffer (PBS-T) every time, general ELISA Plate back-off, gently pats dry liquid therein.

2) it closes

Every hole adds the confining liquid of 300 μ L, 37 DEG C of closing 2h, with cleaning solution board-washing 5 times, washing methods and upper identical, washing Gently button is dry after complete.

3) tested serum is made into 1:100 with antibody diluent respectively, 1:200,1:500, the dilution of 1:1000.Then by upper It states the every hole of table and corresponding 100 μ L of serum is respectively added, do 3 parallel multiple holes, ELISA Plate is buckled to after board-washing 5 times by 37 DEG C of incubation 2h, Gently pat dry liquid therein.

4) sheep anti-mouse igg-HRP antibody diluent is diluted 5000 times, the ELIAS secondary antibody 100 after the dilution is added in every hole μ L, 37 DEG C of incubation 1h are washed 5 times with cleaning solution, after ELISA Plate is buckled to, gently pat dry liquid therein.

5) substrate solution of 100 μ L Fresh, 37 DEG C of incubation 10-30min are respectively added into each reacting hole.Depending on color It terminates in due course.

6) terminate liquid of 50 μ L is added into each reacting hole, detects the light absorption value at its 450nm with microplate reader.

It is returned to zero with the light absorption value of blank control, if the OD to gaging hole₄₅₀Value is greater than or equal to 2.1 times of negative control hole, then It is defined as positive value, and by maximum serum diluting multiple corresponding under the value, is defined as the potency of specific antibody in serum.

The result shows that when extension rate is 100, negative control group DTT-GST and Al (OH)₃OD₄₅₀Value is 0.25 Left and right, and experimental group OD₄₅₀Peak is 0.92, the mice serum being immunized for FIX-2-DTT.Remaining group OD₄₅₀It is lower, do not surpass Cross 0.4.OD when serum diluting multiple increases, under each extension rate₄₅₀Value is diluting still using FIX-2-DTT group as highest When multiple is 1000, OD₄₅₀Value is still greater than the maximum OD of remaining component₄₅₀Value illustrates in the immune mice serum of FIX-2-DTT Produce anti-human FIX antibody, and not generate antibody or yield for FIX extremely low for remaining component.

For the antibody titer for further detecting FIX-2-DTT group, the multiple of FIX-2-DTT group serum is further increased, directly To OD₄₅₀Value is close to 0, or and OD of the negative control group under the extension rate₄₅₀Value is close.Measurement method and above-mentioned ELISA are complete It is exactly the same.When extension rate is 5000 times, the N/P value of FIX-2 group is greater than 2.1, and extension rate should when being greater than 5000 times Ratio is less than 2.1, and therefore, the antibody titer of measurement FIX-2-DDT group serum is 5000.

1.3.Western-blot antibody binding specificity is detected

Preparation of reagents:

Transfering buffering liquid: 25mM Tris base, 0.2M glycine, 20% methanol (pH8.3)

10mM PBS:140mMNaCl, 2.7mM KCl, 10mM Na₂HPO₄·10H₂O KH₂PO₄Adjust pH to 7.4.

Washing buffer (PBS-T): 10mM PBS adds 0.1%Tween-20, mixes.

Block buffer: add the skimmed milk power of 5% (w/v) into PBS-T.

Antibody diluent body: same to confining liquid.

Experimental design is as follows:

Table 11Western-blot detection

Experimental procedure:

1) sample is separated by electrophoresis

Identify glue quality, with 1 × loading buffer polishing, pre-staining Marker indicates transferring film efficiency and mark swimming Road.

2) transferring film (wet turn of slot type)

When electrophoresis closes to an end, 6 filter paper is first got out, and its clip and electroporation is in the same size, wait electrophoresis After, it is put in transfering buffering liquid and impregnates 10min or so, meanwhile, also glue is peeled, cuts the portion without target protein Point, it is put into the transfering buffering liquid and balances 10min or so.Immediately, clip and glue pvdf membrane of the same size, are first put into methanol 2min or so is impregnated, is then transferred in transfering buffering liquid and balances 8min or so.After the completion of balance, by cathode face (black side)-sea The assembled in sequence of the continuous positive pole-face (red face) of -3 layers of filter paper-gel--3 layers of pvdf membrane filter paper-sponge-shifts sandwich, and every layer adds After complete, make sure to keep in mind carefully to be rushed bubble with glass bar, to prevent short circuit.After installing sandwich, by it as (black side in transfer groove To black side), transfering buffering liquid is added, and whole device is placed in ice bath, plugs electrode, 300mA constant current, transferring film 1h.Or Transferring film stays overnight (10h or more) under 20V voltage.It after transferring film, cuts off the power, takes out hybond membrane.It will by preparatory point sample sequence Film is cut.

3) it is washed film 3 times, each 5min, room temperature, is shaken with 25mL PBS-T.

4) film is put in a plate, is added in the Block buffer of 25mL, room temperature shake be incubated for 1h, or 4 DEG C it is quiet It sets overnight.

5) it is washed 3 times with PBS-T, each 5min.

6) film of each cutting is respectively placed in a 10mL centrifuge tube, the serum after corresponding appropriate each dilution is added, It is incubated at room temperature 1h, is slowly shaken.

7) it is washed 3 times with PBS-T, each 5min.

8) by all films, as the secondary antibody after diluting in right amount in a plate, is added, (sheep anti-mouse igg-HRP dilutes together 5000 times), reaction is slowly shaken, 1h is incubated at room temperature.

9) it is washed 3 times with PBS-T, each 5min.

10) film is slightly washed again with deionized water, and is blotted the water on its surface with clean filter paper, it then will mixing Good luminous developing solution is carefully uniformly dripped on film, and colour developing 5min or so selects the suitable time for exposure, to be exposed into Picture.

The result shows that there is an apparent band, molecular weight 60KD or so, with FIX standard items molecule in positive controls Amount is consistent.In each group serum, only there is a band in same position in FIX-2-DTT group, and remaining group is without target stripe Occur.The result is consistent with ELISA result, illustrates to have produced the raw antibody for being directed to FIX really after mouse is immunized in FIX-2-DTT.

The above result shows that FIX-2 is the B epitope of FIX, it is transplanted to DTT_292-295After forming recombinant protein, have Good immunogenicity, immune mouse can generate the antibody for complete FIX.

3 Antibody preparation of embodiment

1.1 rabbits are immune

1) prepare 6 3-4 month big, male Japan large ear rabbit of weight 2kg or so, for FIX-2-DTT to be immunized Recombinant protein.

2) antigen emulsifies: getting out proteantigen according to required immunizing dose, the Freund's adjuvant of equivalent is added, uses emulsification instrument Sufficiently impulse mixing.Initial immunity uses Freund's complete adjuvant, and booster immunization uses Freund non-fully adjuvant.

3) antigen after every rabbit injection 1mg of initial immunity is emulsified.Using multi-point injection method, respectively in rabbit left neck Inject at 6 points, left front oxter injects at 6 points, 5 points of injection of left front shoulder back.Rabbit, auricular vein are fixed with apparatus for binding experimental rabbit before immune Blood 20-100 μ L preservation is taken to make negative control.

4) antigen after every rabbit injection 0.5mg of booster immunization is emulsified, equally takes multi-point injection method, respectively in rabbit 6 points of right neck injection, right preceding oxter inject at 6 points, 5 points of injection of right crop back.

5) identical adjuvant and antigen mix after two weeks, carry out secondary booster immunization, method is the same as step 4.

6) after a week in second of booster immunization, in the auricular vein of rabbit take L, 4000rpm, 4 DEG C of blood 20-100 μ from The heart obtains serum, detects the specific antibody titres in serum with the method for ELISA, to decide whether to continue booster immunization.

If 7) potency is not high, continue booster immunization, immunization method is identical as above-mentioned booster immunization, at most in total Immune 5 stoppings.

8) bloodletting: after immune, rabbit blood is acquired in such a way that arteria carotis takes blood, so that rabbit is lain on the back in behaviour first Make to fix on frame and by its head, then its four limbs is tied down with small cloth rope sling and is fixed up.Head is slightly lowerd one Point subtracts the hair of neck, then carries out skin degerming with alcohol swab to expose neck.After the completion of disinfection, with scalpel longitudinal direction It cuts forward neck portion skin and is about 10cm or so, subcutaneous connective tissue is separated, when the nutator of tracheae two sides completely reveals Afterwards, skin is separated with haemostatic clamp and is clamped, remove subcutaneous tissue, exposed muscle layer, separated with knife handle, that is, see that the neck of beating is dynamic Arteries and veins.Arteria carotis and vagus nerve removing are carefully about 5cm, blood vessel middle section is selected, the muscle around vascular wall is clamped with haemostatic clamp Film.Distal end is ligatured with silk thread, and proximal part is clamped with artery forceps, around alcohol cotton ball disinfection blood vessel, is cut with sterile scissors One V-notch.Taking long 2.5cm, diameter is one section of 1.6mm plastic tube, one end is cut into syringe needle sample inclined-plane, and neck is inserted at this end In artery, the other end is put into the sterile triangular flask of 200mL.It after procedure above is fully completed, unclamps haemostatic clamp and starts bloodletting, with dry Net reception container collection blood.The blood of collection is placed at room temperature for 2 hours or so, 4000rpm, 4 DEG C centrifugation 5min, in collection It is clear to obtain rabbit serum.

Rear rabbit antiserum titre is exempted from ELISA detection 3, the results showed that, it is special in the serum of all rabbits after being immunized three times Specific antibody titers reach 1:16000 or more, illustrate that every rabbit is all produced with FIX-2-DTT is immune for FIX's Target antibody, therefore, this also further demonstrates the B epitope that the FIX-2 that front is identified is strictly FIX.

1.2 antibody purification

1.2.1. prepared by antigen affinity media

1) Peptide systhesis:

One section of synthesis comprising there is the polypeptide (polypeptide sequence: CHNY of purpose epitopeNAAINKYNHD (SEQ ID NO.:27), Wherein C is the cysteine additionally added, as the conjugation sites with chromatography media；Dashed part is epitope sequences), this work By Shanghai, the biological Co., Ltd of bright benefit is completed.The Purity finally synthesized is up to 90%.

2) polypeptide is coupled:

The 10mg polypeptide synthesized is coupled with 2mg through the Sepharose4FF phase of cyanogen bromide-activated, affine Jie of polypeptide is made Matter.Coupling condition is 37 DEG C, 16h.Coupling reaction buffer system is 0.2M NaHCO₃(pH8.5)。

1.2.2. antibody purification

1) rabbit serum is diluted 5 times with the buffer of 10mM PBS pH7.0 stand-by (serum of all rabbits closes very much It is even)；

2) it takes out the above-mentioned polypeptide affinity media (2mL) prepared to be fitted into void column, with the 10mM PBS of 5 times of volumes (pH7.0) pillar is rinsed, until the liquid A of outflow₂₈₀Value is close to until zero；

3) serum of each rabbit after 10mM PBS dilutes well is taken out, flowing separately through polypeptide affinity column is purified, Loading flow control washes away unbonded non-specificity with 10mM PBS (pH7.0) after completion of the sample with 0.5mL/min or so Albumen, until efflux A₂₈₀Close to zero, whole receptions are flowed through；

4) it with the antibody of the Gly-HCl buffer elution of bound of pH3.0, collects the liquid being eluted out and (is receiving in advance Container in appropriate NaOH is added), adjust pH to neutrality immediately；

5) after being 7.0 with 10mM PBS (pH7.0) scouring media to efflux pH, solution will be flowed through and loading and washed again It is de-, it is repeated multiple times；Collect eluent；

6) antibody purity is detected by SDS-PAGE；

7) concentration of antibody is measured by BCA sizing technique；

8) antibody binding specificity is detected by Western-Blot.

Through polypeptide immunoaffinity chromatography, final purifying obtains the target antibody of 18mg specificity.

1.3Western-Blot detection

Respectively with ordinary milk skimmed milk, transgenosis milk skimmed milk (contains rhFIX in the skimmed milk, is obtained from Shanghai youngster Virgin hospital's genetic research institute, can refer to document, Huang is praised: goat-promotor of casein gene guidance transgenic mouse milk High efficient expression Human factor IX Acta Genetica Sinica 2002,29 (3): 206-211；Yan J-B etc.: Transgenic mice can express mutant human coagulation factor IX withhigher level of clotting activity.Biochemical genetics2006,44(7-8):347-358)。

HFIX standard items (are purchased from Enzyme Research Laboratories), and (PCC is purchased from prothrombin complex Magnificent blue biology) and ox FIX (being purchased from Enzyme Research Laboratories) as detection antigen, to purify resulting resist Body (mixes the antibody of above-mentioned purifying, and uniformly) as primary antibody, using goat anti-rabbit igg-HRP as secondary antibody, detects antibody Binding specificity, concrete operations are as follows:

1) sample is separated by electrophoresis: identification glue quality, hFIX standard items applied sample amount are 100ng, remaining sample applied sample amount is 15 μ L indicates transferring film efficiency and marker lane with pre-staining Marker.

2) transferring film (wet turn of slot type): glue is dipped in transfering buffering liquid and balances 10min.Size clip pvdf membrane according to glue With filter paper 6, be put into transfering buffering liquid and balance 10min.Saturation 1min such as need to be impregnated with pure methanol with pvdf membrane.Assembly transfer Sandwich: the positive pole-face (red face) of -3 layers of filter paper-gel of cathode face (black side)-sponge--3 layers of pvdf membrane filter paper-sponge -, often After layer is put well, bubble of rushing.Transfer groove is placed in ice bath, sandwich (black side is to black side) is put into, adds transfering buffering liquid, Plug electrode, 300mA constant current, 1h.Or 20V is shifted overnight (10h or more).It after transferring film, cuts off the power, takes out hybond membrane. Film is cut by preparatory point sample sequence.

3) it is washed film 3 times, each 5min, room temperature, is shaken with 25mL PBS-T.

4) film is put in the 1h in 25mL Block buffer, room temperature is shaken (or 4 spend night).

5) it is washed 3 times with PBS-T, each 5min.

6) film of each cutting is respectively placed in a 10mL centrifuge tube, primary antibody (1000 times of dilution), incubation at room temperature is added 1h slowly shakes.

7) it is washed 3 times with PBS-T, each 5min.

8) by all films, as the secondary antibody after diluting in right amount in a plate, is added, (goat anti-rabbit igg-HRP dilutes together 5000 times), reaction is slowly shaken, 1h is incubated at room temperature.

9) it is washed 3 times with PBS-T, each 5min.

10) film slightly being rinsed with deionized water, filter paper pastes angle suck dry moisture, and the also A and B that will shine is mixed in 1:1 ratio, It drips on film, 5min or so selects the suitable time for exposure, is exposed imaging.

Testing result such as Fig. 1, in figure: 1-5 detection antigen is followed successively by ordinary milk skimmed milk, transgenosis milk skimmed milk, FIX standard items, PCC and ox FIX.The applied sample amount of FIX standard items is 100ng, remaining antigen applied sample amount is 15 μ L.

As can be seen from Fig., No. 1 and No. 5 swimming lanes occur without any band, and No. 2-4 has band and there was only a band Occur.2 and 4 band molecular size ranges are about 55KD, close with the rFIX molecular weight in the FIX and transgene cow milk in PCC；3 The molecular weight about 61KD of number band, it is close with FIX standard items molecular weight.Result explanation, it is immune obtained by FIX-2-DTT Antibody can identify the hFIX in FIX standard items, PCC and transgene cow milk, and the egg in nonrecognition bFIX and common cow's milk It is white.That is, the antibody has good specificity, with non-FIX substance no cross reaction.Here, the standard items of hFIX point Son amount is bigger than the molecular weight of pdhFIX and rhFIX, this is because FIX is glycoprotein, the change of molecular weight range depends on it The height of sugar content, and hFIX standard items sugar content is compared to caused by pdhFIX and rhFIX higher.

1.4ITC200 measures affinity of antibody

1) preparation of samples: being first diluted to 0.1mg/mL with 1 × PBS for antibody, with identical PBS dialysis PCC, transgenic cow Milk skimmed milk and ordinary milk skimmed milk, and be concentrated, so that FIX molar concentration therein is at least antibody in reaction tank 10 times of molar concentration.Then titrated antibody is come as titrimetric substance using the sample after being concentrated.

2) water and methanol are changed: changing water and methanol, and buffer or deionized water are first centrifuged.

3) it is switched on and cleans: opening ITC200 and computer, click runs software ITC200With origin software, first will Temperature is set as in 25 DEG C of balances.ITC200 syringe and measuring cell are carefully cleaned according to prompt, clicks cell and syringe Wash starts to clean, and at least cleans 3 times, when cleaning, it is ensured that have in syringe needle into water, otherwise relaunder.

4) water droplet water preliminary experiment: after the completion of washing, first doing preliminary experiment with water droplet water, and 280 μ L water are added in sample cell.Sample-adding When, syringe sucks the reactant of about 380 μ L, and syringe is first inserted directly into bottom, and then side, which pushes, makes reactant drop in reaction Chi Zhong, side slowly above mention syringe, are during which sure not directly to extract syringe needle, in order to avoid into bubble.It will be more than pond face after dripping off Partial reactant sucks back again, checks in pond whether there is bubble, if so, blotting net rear sample-adding again.It is added in PCR pipe The water of 80 μ L, is put at load.Firstly, then moving on to syringe needle at the position rest, it is exhausted by syringe fill, then Syringe needle is moved on at load, adopting consecutive click chemical reaction OK, start loading, it is ensured that bubble will have been clapped in syringe needle, otherwise continue by Syringefill is until bubble drains.

6) run instrument: setting program and path, reaction temperature are set as 25 DEG C, carry out the isothermal titration reaction of 16 drops, Start by start, automatically into titration procedure after instrument balance a period of time.

7) data are analyzed: after the reaction was completed, obtaining the curve of energy variation, then pass through Origin ITC Version7.0One Site models fitting curve obtains binding constant (K_D), stoichiometry (N), enthalpy change (△ H) and Entropy Changes Thermodynamic parameters such as (△ S).

8) sample measures: being analyzed by preliminary experiment, it is ensured that after instrument state is normal, carry out the detection of sample to be tested, program With it is upper identical.The antibody-solutions of 280 μ L are added in reaction tank for the titrimetric substance of 40 μ L of sample introduction needle aspirate, carry out the isothermals of 16 drops Drop reaction.Data are analyzed and processed after the completion.

Above-mentioned purified anti-FIX antibody is injected in reaction tank, is injected separately into PCC, transgenosis milk separation in syringe needle Cream (containing rFIX) and non-transgenic milk separation cream carry out the reaction of isothermal calorimetric titration, and use Origin ITC Version7.0One Site model carries out curve simulation, and result is as shown in Fig. 2 and table 12, and Fig. 2 is, with turning for expression rFIX Gene milk separation cream titrated antibody carries out mould with Origin ITC version7.0One Site model as can be seen from Fig. It is quasi- to obtain preferable matched curve.And with PCC titrated antibody Origin ITC version7.0One Site model into Row simulation also obtains preferable matched curve；And when being fitted with ordinary milk skimmed milk titrated antibody with the model, out Existing miscue, i.e., can not be fitted.Illustrate the pdhFIX and transgenosis of native form in antibody obtained by the present embodiment and PCC Association reaction can occur for the rhFIX in milk, without association reaction occurs with other albumen in non-transgenic milk.Knot The Westerm-blot data for closing front illustrate the specificity of antibody preferably, and can identify the pdFIX of non denatured form with rFIX。

Binding constant, enthalpy change and the Entropy Changes that the anti-FIX antibody of table 12 is reacted from different antigen bindings

It is anti-to can be seen that anti-FIX antibody and PCC and transgenosis milk skimmed milk obtained by the present invention from the data in table The binding constant K answered_DRespectively 1.14 ± 0.34 × 10⁶(M^-1) and 4.69 ± 0.76 × 10⁶(M^-1), illustrate in 10mM PBS item Under part, the antibody and the affinity of pdFIX and rFIX compared with it is mild.Therefore, this is just to use antibody as affinity ligand Purifying FIX (including pdFIX and rFIX) is had laid a good foundation, this is because the affinity of it and pdFIX and rFIX compare It is relatively mild, when they are used for the immunoaffinity purification of FIX, i.e., will not image height affinity antibodies it is such, need harsh elution Condition, so as to cause destination protein inactivation；Will not be as low-affinity antibody, binding force is very weak, leads to purification efficiency Lowly.Therefore, they can either keep the activity of destination protein to be able to maintain preferable purification efficiency again, be for the immune of FIX The ideal ligand of affinity purification more.

From enthalpy change and the Entropy Changes number of all reactions it will be appreciated that the antibody and rFIX in the pdFIX and transgene cow milk in PCC Association reaction H < 0 △ and S < 0 △, illustrate, their predominant intermolecular forces are the complexs that hydrogen bond and Van der Waals force are formed System.

By above method, it has been made that a species specificity is good, the moderate anti-FIX antibody of affinity, this method is simple and efficient, Preparation cost is low, is the anti-FIX antibody of prepare with scale, for having laid a good foundation in the immunoaffinity chromatography of FIX.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Claims (10)

1. a kind of epitope peptide, which is characterized in that the epitope peptide is originated from animal FIX and includes one or more antigens Epitope,

The recombinant protein that the epitope peptide and carrier protein are formed can induce the same kind of animal and generate for FIX Immune response；

Wherein, the epitope peptide is NAAINKY.

2. a kind of fusion protein, the fusion protein is that epitope peptide as described in claim 1 merges institute with carrier protein It is formed, wherein the fusion protein is the polypeptide with amino acid sequence shown in SEQ ID NO.:12.

3. a kind of polynucleotides, described in epitope peptide described in the polynucleotide encoding claim 1 or claim 2 Fusion protein.

4. a kind of expression vector, the expression vector contains polynucleotides as claimed in claim 3.

5. a kind of host cell, the host cell contains expression vector as claimed in claim 4, or in genome it is whole Conjunction have the right to require 3 described in polynucleotides.

6. a kind of pharmaceutical composition, the composition contains described in epitope peptide described in claim 1, claim 2 Fusion protein, polynucleotides as claimed in claim 3, described in expression vector as claimed in claim 4 or claim 5 Host cell and pharmaceutically acceptable carrier and/or auxiliary material.

7. a kind of vaccine composition, the composition contains described in epitope peptide described in claim 1, claim 2 Fusion protein, polynucleotides as claimed in claim 3, described in expression vector as claimed in claim 4 or claim 5 Acceptable carrier and/or auxiliary material on host cell and immunology.

8. epitope peptide described in claim 1, fusion protein as claimed in claim 2, multicore glycosides as claimed in claim 3 The purposes of host cell described in expression vector sour, as claimed in claim 4 or claim 5,

(a) it is used to prepare the antibody for the epitope；And/or (b) be used to prepare treatment it is related to the epitope Disease drug.

9. a kind of method for preparing FIX antibody, spy be comprising steps of

(1) it uses fusion protein as claimed in claim 2 as antigen-immunized animal, and generates the anti-of anti-FIX in animal body Body；

(2) the FIX antibody is isolated and purified.

10. a kind of method for isolating and purifying people FIX, spy are, comprising steps of

(1) people FIX antibody is prepared using method as claimed in claim 9；

(2) using the antibody obtained in step (1), using the method Purification of Human FIX of affinity chromatography.