CN105198973B - Staphylococcus protein A Z structural domain derivative and application thereof - Google Patents

Abstract

The invention discloses a staphylococcus protein A Z structural domain derivative and application thereof. Specifically, the invention provides a Z domain derivative of staphylococcal protein A, which has the capacity of binding IgG, the Z domain derivative is formed by amino acid mutation of Z domain; and KZFY/KZ is less than or equal to 10 percent, wherein KZFY is the binding constant of the Z structural domain derivative and IgG, and KZ is the binding constant of the Z structural domain and IgG; and immunizing an animal with the Z domain derivative induces an immune response in the animal against staphylococcal protein A. The ZFY derivative is coupled with carrier protein DTT to obtain a protein which is convenient to express and purify and can effectively stimulate an immune system to generate a protein A antibody.

Description

A kind of staphylococcal protein A Z domain derivative and its application

Technical field

The invention belongs to biomedicine fields, specifically, the present invention relates to a kind of derivative of staphylococcal protein A and It is applied.

Background technique

Staphylococcus aureus (Staphylococcus aureus, hereinafter referred to as S. aureus L-forms) is that hospital and community obtain One of the main pathogenic bacteria of sexuality dye, can cause mankind's pyogenic infection, Skin and soft tissue infection, necrotizing pneumonia, septicopyemia The diseases such as disease, bacteremia, osteomyelitis and endocarditis.Currently, having the S. aureus L-forms of multiple antibiotic resistance, come to treatment zone The rapid sprawling of great difficulty, especially methicillin resistant strains is so that rely on merely antibiotic treatment Staphylococcus aureus The related disease that bacterium causes becomes more and more unreliable.Immunotherapy method can be by activating or enhancing the siberian crabapple of body System, and antibiotic synergism, and can reduce and select pressure caused by antibiotic, delay the generation of drug-fast bacteria, is expected to solve S. aureus L-forms infect this thorny problem.

Staphylococcal protein A (abbreviation albumin A, SPA) is primarily present in S. aureus L-forms, about 90% or more S. aureus L-forms bacterium Contain this ingredient in strain, is one of important virulence factor of S. aureus L-forms, has as good S. aureus L-forms vaccine candidate component Potentiality.Albumin A functional areas are highly conserved, E, D, A, B, C-structure domain containing 5 concatenated high homologies (Uhlen M, Guss B,Nilsson B,et al.Complete sequence of the staphylococcal gene encoding Albumin A .A gene evolved through multiple duplications [J] .J Biol Chem.1984,259 (3):1695-1702).For the resistance to alkali ability for improving albumin A, Gly with the stronger B structure domain Residue positions 29 of IgG binding force Mutagenesis is directed into Ala, new construction domain is named as Z, and (Meng Zhaohui, Lin Bing, Xie Yuehui wait .Fc receptor seq binding protein (SPA- SPG) gene cloning and expression and its application Progress in Biochemistry and Biophysics .2004,31 (2) in IgG purifying: 146- 149.).Simultaneously because Fc sections of IgG and albumin A cause it that can compete with phagocyte there are stronger affinity, body is reduced Specific proteins A antibody is generated, causes pathogen in the infection and recurrence of human body.It thus studies a kind of weaker with IgG binding force Albumin A derivative, for S. aureus L-forms vaccine exploitation have important meaning.

Summary of the invention

The purpose of the present invention is to provide a kind of and weaker albumin A derivatives and application thereof of IgG binding force.

Another object of the present invention is to provide a kind of fusion proteins comprising above-mentioned albumin A derivative.

The first aspect of the present invention provides a kind of Z domain derivative of staphylococcal protein A, has and combines IgG Ability, in which:

(1) the Z domain derivative is formed by the amino acid mutation of Z structural domain；

(2)K_ZFY/K_Z≤ 10%, wherein K_ZFYFor the binding constant of the Z domain derivative and IgG, K_ZFor Z structural domain With the binding constant of IgG；With

(3) animal can be induced by, which being immunized after animal using the Z domain derivative, generates for staphylococcal protein A Immune response.

In another preferred example, the K_ZFY/K_Z≤ 1%, preferably≤0.1%, more preferably≤0.01%.

In another preferred example, the amino acid sequence of the Z structural domain is as shown in SEQ ID NO.:1.

In another preferred example, the amino acid number being mutated in the Z structural domain is 1-10, and preferably 1-5 is a, more It is goodly 1-3.

In another preferred example, the 13rd Phe and/or the 14th Tyr in Z structural domain occurs for the mutation.

In another preferred example, the 13rd Phe of the Z structural domain sports Gly and/or the 14th Tyr and sports Gly。

In another preferred example, the amino acid sequence of the Z domain derivative is as shown in SEQ ID NO.:2.

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 Z domain derivative stated is merged with carrier protein to be formed by, and the fusion protein can induce body generation and be directed to The immune response of staphylococcal protein A.

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 Z domain derivative 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 DTT.

In another preferred example, the Z domain derivative is connected to the C-terminal and/or N-terminal shape of the carrier protein At the fusion protein.

In another preferred example, there is link peptide between the Z domain derivative and the carrier protein；Preferably, The link peptide length is 3-30 amino acid；It is highly preferred that the link peptide length is 4-20 amino acid；Most preferably, The link peptide length is 7-17 amino acid.

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

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

(a) polypeptide with amino acid sequence shown in SEQ ID NO.:20 or 21；

(b) polypeptide in (a) is formed by one or more replacing, missing or adding for amino acid residue, and had There is the polypeptide as derived from (a) for inducing immune response function.

The third aspect of the present invention provides a kind of polynucleotides, the polynucleotide encoding first aspect present invention Fusion protein described in the Z domain derivative or second aspect of the present invention.

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

The fifth aspect of the present invention, provides a kind of host cell, and the host cell contains fourth aspect present invention The expression vector, or polynucleotides described described in third 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 sixth aspect of the present invention, provides a kind of pharmaceutical composition, and the composition contains first aspect present invention Fusion protein described in the Z domain derivative, second aspect of the present invention, multicore glycosides described in third aspect present invention Host cell described in expression vector described in acid, fourth aspect present invention or fifth aspect present invention, and pharmaceutically may be used The carrier and/or auxiliary material of receiving.

In another preferred example, the composition is vaccine.

The seventh aspect of the present invention, provides a kind of vaccine composition, and the composition contains first aspect present invention Fusion protein described in the Z domain derivative, second aspect of the present invention, multicore glycosides described in third aspect present invention On host cell described in expression vector described in acid, fourth aspect present invention or fifth aspect present invention and immunology Acceptable carrier and/or auxiliary material.

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, IL-6, IL-12 and CpG).

The eighth aspect of the present invention provides Z domain derivative, second party of the present invention described in first aspect present invention Fusion protein described in face, expression vector described in polynucleotides, fourth aspect present invention described in third aspect present invention or The purposes of host cell described in person's fifth aspect present invention,

(a) it is used to prepare the antibody for staphylococcal protein A；And/or (b) it is used to prepare treatment and staphy lococcus infection The drug of relevant disease.

In another preferred example, the disease includes: the suppurative sense of the mankind caused by infection of staphylococcus aureus Diseases such as dye, Skin and soft tissue infection, necrotizing pneumonia, pyemia, bacteremia, osteomyelitis and endocarditis etc..

The ninth aspect of the present invention provides a kind for the treatment of method, applies first aspect present invention institute to the object of needs Fusion protein described in the Z domain derivative stated, second aspect of the present invention, polynucleotides described in third aspect present invention, The pharmaceutical composition described in expression vector sixth aspect present invention described in fourth aspect present invention or the present invention the 7th Vaccine composition described in aspect.

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 isothermal calorimetric titration amount curve that human IgG is reacted with Z, ZFY albumen.

Fig. 2 shows mouse resisting anteserum titration result.

Fig. 3 shows performance of Elisa detection control group F (ab ') 2 with specificity F (ab ') 2 in conjunction with SPA.

Specific embodiment

The present inventor passes through extensive and in-depth research, it has unexpectedly been found that will be in the stronger Z structural domain of IgG binding force 13,14 Phe, Tyr, two critical amino acid residues sport Gly, i.e. ZFY derivative, can make ZFY energy in conjunction with IgG It is reduced to -61.68kcal/mol, only the 10.69% of original interaction force, and ZFY albumen is constructed by PCR mutating technology Expression system.ZFY derivative and carrier protein DTT are coupled simultaneously, obtained a kind of albumen for facilitating expression and purification, the albumen Immune system can be effectively excited to generate protein A antibody.

The present invention provides fusion protein and its applications that can effectively excite immune system to generate protein A antibody.It is described to melt Hop protein includes the Z structural derivative of staphylococcal protein A, and the Z structural derivative includes (1) by the amino acid of Z structural domain Mutation is formed；(2)K_ZFY/K_Z≤ 10%, wherein K_ZFYFor the binding constant of the Z domain derivative and IgG, K_ZFor Z structure Domain and the binding constant with IgG；(3) the animal generation can be induced for the immune of staphylococcal protein A after animal is immunized Reaction.

It is a further object of the present invention to provide the vaccine being made of the composition comprising the fusion protein, the composition Containing Z domain derivative, fusion protein, polynucleotides, expression vector or host cell of the present invention, and it is immune Acceptable carrier and/or auxiliary material on.

It is a further object of the present invention to provide a kind of methods for treating disease, particularly for disease caused by staphylococcus. This method is by giving to the patient with the disease as caused by staphy lococcus infection by comprising fusion protein of the invention The vaccine of composition composition, the disease is selected from the group: mankind's pyogenic infection, Skin and soft tissue infection, necrotizing pneumonia, Pyemia, bacteremia, osteomyelitis and endocarditis etc..

Protein A domain Z amino acid sequence are as follows:

VDNKFNKEQQNAFYEILHLPNLTEEQRAAFIQSLKDDPSQSAELLAEAKKLNDAQAPK(SEQ ID NO.:1)

ZFY derivative amino acid sequence are as follows:

VDNKFNKEQQNAGGEILHLPNLTEEQRAAFIQSLKDDPSQSAELLAEAKKLNDAQAPK(SEQ ID NO.:2)

By PCR mutating technology, it is derivative to get ZFY that the 13rd, 14 in Z structural domain Phe, Tyr are sported into Gly Object.

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 " Z domain derivative (peptide) " refers to that quasi- induction animal generates other albumen of immune response One section of peptide, the epitope for carrier protein not referring to carrier protein itself can cause immune response peptide fragment. In general, Z domain derivative refers to the peptide fragment of the quasi- targeting of immune response, one section of mammal (such as people) albumen is preferably derived from Peptide, rather than 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 Z domain derivative, is that antigen molecule is in by antigen One section of peptide that enzymatic hydrolysis processing generates in delivery cell, can be combined by major histocompatibility complex (MHC) molecule, be presented thin Cellular surface 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 TEPLMEQVGTEEFIKRFGDG ASRVVLSLPF AEGSSSVEYI NNWEQAKALS VELEINFETR GKRGQDAMYE YMAQACAGNRVRRSVGSSLS CINLDWDVIR DKTKTKIESL KEHGPIKNKM SESPNKTVSE EKAKQYLEEF HQTALEHPELSELKTVTGTN PVFAGANYAA WAVNVAQVID SETADNLEKT TAALSILPGI GSVMGIADGA VHHNTEEIVAQSIALSSLMV AQAIPLVGEL VDIGFAAYNF VESIINLFQV VHNSYNRPAY SPGHKTQPFL HDGYAVSWNTVEDSIIRTGF QGESGHDIKI TAENTPLPIA GVLLPTIPGK LDVNKSKTHI SVNGRKIRMR CRAIDGDVTFCRPKSPVYVG NGVHANLHVA FHRSSSEKIH SNEISSDSIG VLGYQKTVDH TKVNSKLSLF FEIKS

(SEQ ID No.:3)

In diphtheria toxin molecule there are 5 T- helper epitopes can be by up to 80% or more people MHC class II Identification.Diphtheria toxin transmembrane domain (DTT) is mainly made of core skeleton α screw element, by flexibility between screw element Ring region connection.

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.:4)

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.:5)

3. salmonella flagellin (FliC)

Salmonella flagellin FliC (Phase 1-C flagellin) is the filamentous composition of flagellum, is moved with a variety of Object cell receptor TLR5 is combined, and excites the innate immune system response of animal, and immature DC cell surface is promoted to express CD80 And CD86, cytokine profiles and chemotactic factor (CF) are secreted, is sent out in congenital immune response and specific specific immune response Stronger immunoadjuvant function is waved, 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 NRFTANIKGL

TQASRNANDG ISIAQTTEGA LNEINNNLQR VRELAVQSAN STNSQSDLDS IQAEITQRLN

EIDRVSGQTQ FNGVKVLAQD NTLTIQVGAN DGETIDIDLK QINSQTLGLD TLNVQQKYKV

SDTAATVTGY ADTTIALDNS TFKASATGLG GTDQKIDGDL KFDDTTGKYY AKVTVTGGTG

KDGYYEVSVD KTNGEVTLAG GATSPLTGGL PATATEDVKN VQVANADLTE AKAALTAAGV

TGTASVVKMS YTDNNGKTID GGLAVKVGDD YYSATQNKDG SISINTTKYT ADDGTSKTAL

NKLGGADGKT EVVSIGGKTY AASKAEGHNF KAQPDLAEAA ATTTENPLQK IDAALAQVDT

LRSDLGAVQN RFNSAITNLG NTVNNLTSAR SRIEDSDYAT EVSNMSRAQI LQQAGTSVLA

QANQVPQNVL SLLR(SEQ ID NO.:6)

4. pneumolysin (Ply)

Pneumolysin (Pneumolysin, Ply) is the main protein antigen of streptococcus pneumonia, is present in thin It in cytoplasm, is released by autolysis, therefore vaccine can carry out mucosal immunity by oral or inhalation route, be a kind of Ideal vaccine carrier candidate albumen.

Ply is made of 471 amino acid, molecular weight 53kDa, contain 4 Structure and function domains, amino acid sequence (P0C2J9, TACY_STRPN) as follows:

MANKAVNDFI LAMNYDKKKL LTHQGESIEN RFIKEGNQLP DEFVVIERKK RSLSTNTSDI

SVTATNDSRL YPGALLVVDE TLLENNPTLL AVDRAPMTYS IDLPGLASSD SFLQVEDPSN

SSVRGAVNDL LAKWHQDYGQ VNNVPARMQY EKITAHSMEQ LKVKFGSDFE KTGNSLDIDF

NSVHSGEKQI QIVNFKQIYY TVSVDAVKNP GDVFQDTVTV EDLKQRGISA ERPLVYISSV

AYGRQVYLKL ETTSKSDEVE AAFEALIKGV KVAPQTEWKQ ILDNTEVKAV ILGGDPSSGA

RVVTGKVDMV EDLIQEGSRF TADHPGLPIS YTTSFLRDNV VATFQNSTDY VETKVTAYRN

GDLLLDHSGA YVAQYYITWN ELSYDHQGKE VLTPKAWDRN GQDLTAHFTT SIPLKGNVRN

LSVKIRECTG LAWEWWRTVY EKTDLPLVRK RTISIWGTTL YPQVEDKVEN D(SEQ ID NO.: 7)

5. pertussis toxin (pertussis toxin, PTX)

Pertussis toxin (pertussis toxin, PTX) is the virulence factor generated by Bordetella pertussis, can cause hundred The numerous clinical symptoms of day cough, are main immunogene, and various acellular pertussis vaccines (Acelluar pertussis Vaccine, APV) it is unique common to ingredient.PTX is the A-B type toxin being made of 6 subunits (S1-S5), and wherein S1 is sub- Unit is main functionality A subunit, has a variety of toxicity.B subunit is made of S2-S4 and S3-S4 dimer, and function is knot It is the preferred vector albumen of vaccine design of the present invention together in eukaryocyte surface receptor.

The amino acid sequence of each subunit of PTXB is as follows.

PTXB S1(P04977,TOX1_BORPE)

DDPPATVYRY DSRPPEDVFQ NGFTAWGNND NVLDHLTGRS CQVGSSNSAF VSTSSSRRYT

EVYLEHRMQE AVEAERAGRG TGHFIGYIYE VRADNNFYGA ASSYFEYVDT YGDNAGRILA

GALATYQSEY LAHRRIPPEN IRRVTRVYHN GITGETTTTE YSNARYVSQQ TRANPNPYTS

RRSVASIVGT LVRMAPVIGA CMARQAESSE AMAAWSERAG EAMVLVYYES IAYSF(SEQ ID NO.:8)

PTXB S2(P04978,TOX2_BORPE)

STPGIVIPPQ EQITQHGGPY GRCANKTRAL TVAELRGSGD LQEYLRHVTR GWSIFALYDG

TYLGGEYGGV IKDGTPGGAF DLKTTFCIMT TRNTGQPATD HYYSNVTATR LLSSTNSRLC

AVFVRSGQPV IGACTSPYDG KYWSMYSRLR KMLYLIYVAG ISVRVHVSKE EQYYDYEDAT

FETYALTGIS ICNPGSSLC(SEQ ID NO.:9)

PTXB S3(P04979,TOX3_BORPE)

VAPGIVIPPK ALFTQQGGAY GRCPNGTRAL TVAELRGNAE LQTYLRQITP GWSIYGLYDG

TYLGQAYGGI IKDAPPGAGF IYRETFCITT IYKTGQPAAD HYYSKVTATR LLASTNSRLC

AVFVRDGQSV IGACASPYEG RYRDMYDALR RLLYMIYMSG LAVRVHVSKE EQYYDYEDAT

FQTYALTGIS LCNPAASIC(SEQ ID NO.:10)

PTXB S4 (P0A3R5, TOX4_BORPE):

DVPYVLVKTN MVVTSVAMKP YEVTPTRMLV CGIAAKLGAA ASSPDAHVPF CFGKDLKRPG

SSPMEVMLRA VFMQQRPLRM FLGPKQLTFE GKPALELIRM VECSGKQDCP(SEQ ID NO.:11)

PTXB S5(P04981,TOX5_BORPE)

GLPTHLYKNF TVQELALKLK GKNQEFCLTA FMSGRSLVRA CLSDAGHEHD TWFDTMLGFA

ISAYALKSRI ALTVEDSPYP GTPGDLLELQ ICPLNGYCE(SEQ ID NO.:12)

6. tetanus toxin (Tetanus toxin, TT)

TT toxicity is extremely strong, and being generated by clostridium tetani is made of the single polypeptide chain of a treaty 150kD, can be cut into Two heavy chains and light chain connected by disulfide bond, heavy chain then include the structural domain of two 50Dk, and N-terminal is migration structural domain, Ion channel is formed in lipid bilayer；The C-terminal of heavy chain is gangliosides structural domain, also known as C segment (TThC), It is played a very important role in conjunction with target cell membrane and during lps molecule enters cholinomimetic neuron.TT heavy chain It (TTh) is currently preferred epiposition vaccine carrier.

TTh amino acid sequence (1YYN.pdb) is as follows:

EDIDVILKKS TILNLDINND IISDISGFNS SVITYPDAQL VPGINGKAIH LVNNESSEVI

VHKAMDIEYN DMFNNFTVSF WLRVPKVSAS HLEQYGTNEY SIISSMKKHS LSIGSGWSVS

LKGNNLIWTL KDSAGEVRQI TFRDLPDKFN AYLANKWVFI TITNDRLSSA NLYINGVLMG

SAEITGLGAI REDNNITLKL DRCNNNNQYV SIDKFRIFCK ALNPKEIEKL YTSYLSITFL

RDFWGNPLRY DTEYYLIPVA SSSKDVQLKN ITDYMYLTNA PSYTNGKLNI YYRRLYNGLK

FIIKRYTPNN EIDSFVKSGD FIKLYVSYNN NEHIVGYPKD GNAFNNLDRI LRVGYNAPGI

PLYKKMEAVK LRDLKTYSVQ LKLYDDKNAS LGLVGTHNGQ IGNDPNRDIL IASNWYFNHL

KDKILGCDWY FVPTDEGWTN D(SEQ ID NO.:13)

Fusion protein

As used herein, term " fusion protein " refers to that Z domain derivative of the present invention merges institute with carrier protein It is formed, and the fusion protein can induce the recombinant protein for the immune response that body is generated for staphylococcal protein A. In addition, the term further includes that can induce body to generate be directed to the immune response of staphylococcal protein A, SEQ ID NO:2 The variant form of sequence.These variant forms include (but being not limited to): 1-3 (usually 1-2, more preferably 1) amino Acid missing, insertion and/or substitution, and C-terminal and/or N-terminal addition or lack it is one or several (usually 3 with It is interior, be more preferably within 1 within preferably 2) amino acid.For example, in the art, use is similar in performance When amino acid is replaced, the function of protein is not usually changed.For another example, in C-terminal and/or N-terminal addition or missing One or several amino acid will not generally also change the structure and function of protein.In addition, the term further includes monomer and more The polypeptide of the present invention of dimer form.The term further includes linear and nonlinear polypeptide (such as cyclic peptide).

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..

(1) 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 WO 90/14837), (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, IL-6, 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) present invention has unexpectedly discovered a kind of derivative ZEY of albumin A Z structural domain, the combination of the derivative and IgG Power is moderate, can effectively excite body to generate the immune response for albumin A as vaccine, it is demonstrated experimentally that obtained by ZFY immune group Sero-fast potency can be higher by obtained one to two orders of magnitude of sero-fast potency of native protein A direct immunization.

(2) ZEY is merged to the fusion protein of preparation, the obtained sero-fast potency of DTT-ZFY immune group with DTT carrier Two to three orders of magnitude of the obtained antiserum titre of native protein A direct immunization can be higher by.

(3) preparation cost of ZEY and DTT-ZFY recombinant protein of the invention is low, convenient drug administration.

(4) relative to the preparation with carrier protein chemical coupling, fusion protein antigenic structure of the invention is definite, quality can Control, it is safer.

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.

The building of 1 protein carrier of embodiment

According to sumo gene in GeneBank, (small molecule ubiquitin sample modifies albumen, the fusion mark as recombinant protein expression Label), the sequence of the open reading frame of the T domain gene mRNA of DT and protein A gene mRNA, design primer passes through over-lap PCR Principle connect the T structural domain of DT after sumo gene, be then re-introduced into one of structural domain Z of albumin A, and by structure Two mutation, F13G/Y14G are introduced in the Z of domain, and is introducing restriction enzyme site NdeI and BamHI end to end, and introduce protection base, even Enter PET28.It is synthesized according to primer by Nanjing Genscript Biotechnology Co., Ltd..The sumo-DTT- specifically designed of the invention Shown in the following SEQ ID NO:7 of the chain amino acids sequence of ZFY；Its DNA sequence dna is as shown in SEQ ID NO:8；And it designs simultaneously Synthetic primer is as follows: P1:SEQ ID NO:14；P2:SEQ ID NO:15；P3:SEQ ID NO:16；P4:SEQ ID NO:17； P5:SEQ ID NO:18；P6:SEQ ID NO:19.

The chain amino acids sequence of DTT-ZFY:

INLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGT NPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIP LVGELVDIGFAAYNFVESIINLFQVVHNSYNRPQAPKVDAKFDVDNKFNKEQQNAGGEILHLPNLTEEQRAAFIQS LKDDPSQSAELLAEAKKLNDAQAPK(SEQ ID NO.:20)

The chain amino acids sequence of sumo-DTT-ZFY:

GSTADNKFINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELS ELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSS LMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPQAPKVDAKFDVDNKFNKEQQNAGGEILHLPNLTEE QRAAFIQSLKDDPSQSAELLAEAKKLNDAQAPKC(SEQ ID NO.:21)

The nucleic acid sequence of sumo-DTT-ZFY:

GGTAGCACTGCTGACAACAAATTCATAAATCTTGATTGGGATGTCATAAGGGATAAAACTAAGACAAA GATAGAGTCTTTGAAAGAGCATGGCCCTATCAAAAATAAAATGAGCGAAAGTCCCAATAAAACAGTATCTGAGGAA AAAGCTAAACAATACCTAGAAGAATTTCATCAAACGGCATTAGAGCATCCTGAATTGTCAGAACTTAAAACCGTTA CTGGGACCAATCCTGTATTCGCTGGGGCTAACTATGCGGCGTGGGCAGTAAACGTTGCGCAAGTTATCGATAGCGA AACAGCTGATAATTTGGAAAAGACAACTGCTGCTCTTTCGATACTTCCTGGTATCGGTAGCGTAATGGGCATTGCA GACGGTGCCGTTCACCACAATACAGAAGAGATAGTGGCACAATCAATAGCTTTATCATCTTTAATGGTTGCTCAAG CTATTCCATTGGTAGGAGAGCTAGTTGATATTGGTTTCGCTGCATATAATTTTGTAGAGAGTATTATCAATTTATT TCAAGTAGTTCATAATTCGTATAATCGTCCCCAAGCACCAAAAGTTGATGCGAAATTCGATGTGGATAACAAATTC AACAAAGAACAACAAAATGCGGGAGGTGAAATCTTACATTTACCTAACTTAACTGAAGAACAACGCGCTGCTTTCA TCCAAAGCCTTAAAGACGATCCTTCACAAAGCGCTGAACTTTTAGCAGAAGCTAAAAAGCTAAATGATGCACAAGC ACCAAAATGC(SEQ ID NO.:22)

First round PCR:PCR reaction system (50 μ L) is as follows: 10 × KOD-Plus Buffer, 5 μ L, dNTPs (2mmol/ L)5μL,MgSO₄(25mmol/L) 2 μ L, sumo forward direction (10 μm of ol/L) i.e. P11.5 μ L, sumo reversed (10 μm of ol/L) is i.e. 1 μ L, sumo plasmid of P21.5 μ L, KOD-Plus, 0.5 μ L, aseptic double-distilled water are mended to 50 μ L.

Second wheel PCR:PCR reaction system (50 μ L) is as follows: 10 × KOD-Plus Buffer 50.5 μ L, dNTPs (2mmol/L)50.5μL,MgSO₄(25mmol/L) 20.5 μ L, DTT forward direction (10 μm of ol/L) i.e. P31.50.5 μ L, DTT is reversed 10.5 μ L, DTT-T plasmid 0.50.5 μ L of (10 μm of ol/L) i.e. P41.50.5 μ L, KOD-Plus, aseptic double-distilled water are mended to 500.5 μ L。

Third round PCR:PCR reaction system (50 μ L) is as follows: 10 × KOD-Plus Buffer 50.5 μ L, dNTPs (2mmol/L)50.5μL,MgSO₄(25mmol/L) 20.5 μ L, SPA-Z forward direction (10 μm of ol/L) i.e. P51.50.5 μ L, SPA-Z is anti- To (10 μm of ol/L) i.e. P61.50.5 μ L, KOD-Plus 10.5 μ L, Z (F13G/Y14G) plasmid 0.50.5 μ L, aseptic double-distilled water It mends to 500.5 μ L.

94 DEG C of denaturation 2min；94 DEG C of 15s, 55 DEG C of 30s, 68 DEG C of 2min, 35 circulations；68℃ 10min.2 μ L are taken to expand Increase production object and carry out electrophoresis, purpose band occurs after Goldview dyeing observation, remaining 5 μ L is put into spare in 4 DEG C of refrigerators.

Gel recycles above-mentioned first round PCR product

The sumo-DTT-ZFY segment needed by three-wheel PCR synthesis.The first round respectively using P1/P2 as primer, to contain The PET28-sumo plasmid (Tiangeng biochemical technology Co., Ltd) of sumo segment (SEQ ID NO:10) is template, expands sumo piece Section；Using P3/P4 as primer, with containing DTT segment (SEQ ID NO:11) PET28-DTT (Tiangeng biochemical technology Co., Ltd, Plasmid extraction kit) it is template, expand DTT segment；Using P5/P6 as primer, with artificial synthesized ZFY sequence (SEQ ID NO:9) it is template, expands ZFY segment.Second wheel is using two product sumo segments and DTT the segment mixing of the first round as mould Plate expands sumo-DTT segment using P1/P4 as primer.Third round is using P1/P6 as primer, with sumo-DTT segment and ZFY segment For template, final product sumo-DTT-ZFY segment is amplified.

Agarose gel electrophoresis recycles PCR product.Digestion carrier pET28a is distinguished with restriction enzyme NdeI and BamHI And finally obtained sumo-DTT-ZFY segment obtains digestion products with cleaning reagent and recycling digestion 8 hours at 37 DEG C, uses The connection of T4 ligase, connects 12 hours, obtains the connection liquid containing connection product by 22 DEG C.Connection 5 μ l of liquid is drawn with liquid-transfering gun to add In the bacillus coli DH 5 alpha competent bacteria for entering 50 μ l, 20 minutes are stood on ice.42 DEG C heat shock 90 seconds, be immediately placed in ice 5 minutes；The LB culture solution of 600 μ l37 DEG C preheating is added.It 37 DEG C, 220rpm, shakes 1 hour, is all coated on after centrifugation containing 50 μ The LB plate of g/ml kanamycins is inverted overnight incubation in 37 DEG C of constant incubators.Monoclonal on random picking plate is to containing In the test tube of the 3ml LB culture solution of 30 μ g/ml kanamycins, 37 DEG C of 220rpm shakings overnight, take 1 μ l to carry out bacterium colony PCR, fine jade Sepharose electroresis appraisal.The positive strain culturing of bacterium colony PCR identification is stayed overnight, plasmid, sequencing identification are extracted.Positive colony by The sequencing of Jin Sirui sequencing company, sequencing result are consistent with desired design.

Sequencing, which is drawn, with liquid-transfering gun identifies that correct 5 μ l of plasmid is added in the e. coli bl21 competent bacteria of 50 μ l, 20 minutes are stood on ice.42 DEG C heat shock 90 seconds, be immediately placed in ice 5 minutes；The LB culture of 600 μ l37 DEG C preheating is added Liquid.It 37 DEG C, 220rpm, shakes 1 hour, the LB plate containing 50 μ g/ml kanamycins, 37 DEG C of constant temperature trainings is all coated on after centrifugation It supports in case and is inverted overnight incubation.Monoclonal on random picking plate is to the 3ml LB culture solution for containing 30 μ g/ml kanamycins In test tube, 37 DEG C of 220rpm shakings overnight, draw the bacterium solution of 800 μ l and 80% glycerol of 200 μ l are added, be placed on -80 DEG C of refrigerators The glycerol stock that middle freezen protective obtains.

It is prepared by the expression identification and scale of 2 albumen of embodiment

Protein expression

Escherichia coli single colonie containing recombinant expression plasmid sumo-DTT-ZFY fusion protein is inoculated into 5mL LB training Support base, 37 DEG C of overnight incubations.Then it is diluted to 1:50 containing in 30ng/L kanamycins, LB culture medium 50mL, culture about 4 is small When make its OD600 value be about 0.6, add IPTG to final concentration of 1mM, 37 DEG C induce 4 hours when take out sample preparation.

The culture for taking 50mL to induce places 30min on ice, and 12000rpm is centrifuged 8min, collects thallus, abandons supernatant；It is heavy It forms sediment and breaks up resuspension with 1 × PBS (5mL/100mL culture) plus isometric ice-cold sterile water；The lysozyme of 300 μ g/mL is added, On ice after jog 30min, sets -70 DEG C and freeze 12h；Protease inhibitors is added after sample is melted with flowing water, in ice bath Ultrasonication (200W power), interval is broken, is often crushed 5s, is spaced 5s, is crushed total 8min repeatedly, (can observe under the microscope Broken results).Mixture is centrifuged 12min in 4 DEG C of revolving speeds with 12,000g；Take mixing, supernatant, 4 DEG C of precipitating preservation spare.

The mixing of albumen, supernatant are taken, precipitating quantitative determines protein content with Coomassie Brilliant Blue respectively and is diluted to certain It takes 200uL that 5 × loading of 50uL buffer is added after concentration, boils 5min, 12,000rpm is centrifuged 8min and adds after taking out cooling Sample.The 20 above-mentioned mixed liquors of μ L are taken with microsyringe, by buffer, sample is carefully added to gel spill sample cell bottom Portion.

The positive and negative anodes of electrophoresis apparatus are connected, open electrophoresis apparatus switch, with initial voltage be 45V when current strength carry out Current stabilization electrophoresis, the pressure stabilizing electrophoresis when voltage reaches 65V stop electrophoresis when Bromophenol Blue dye is away from silica gel frame 0.2cm, close electricity Source.After electrophoresis, gel mold is removed, short glass plate is pried open with scalpel, cuts separating gel.1h is fixed with fixer, then 3h is dyed with dyeing liquor, is finally decolourized with destainer, until protein band is clear.

Picking converts the single colonie for having plasmid, and 50 μ l are inoculated in the LB liquid medium of 50ml containing kanamycin, 37 DEG C, 180rpm, 12 hours, as primary seed solution.Primary seed solution 10ml is taken, 1000ml containing kanamycin is inoculated in LB liquid medium in, 37 DEG C, 150rpm, 3 hours, when OD600 reaches 0.6~0.8 be added IPTG to final concentration 1mM, 37 DEG C, 150rpm, 5 hours.4000rpm is centrifuged 30 minutes, discards supernatant fermentation liquid, retains bacterial sediment, and weighing obtains thallus 3g.Bacterial sediment it will be resuspended with 1mL PBS (pH=7.4) and washed once that thalline were collected by centrifugation after induction, volume 60mL is added Ultrasonication after PBS (pH=7.4) is resuspended, 15min are centrifuged after pulse is broken with 50mL centrifuge tube, after 15000rpm, 40min, Supernatant carefully is taken, 4 DEG C of storages are spare.

Protein purification

His column (5ml column volume) is taken, is balanced with sample-loading buffer (PBS, 0.14M NaCl), flow velocity 2ml/ minutes, supernatant Loading flow velocity 1.5ml/ minutes, collects when ultraviolet absorption peak to appear and flows through liquid, after completion of the sample with washing buffer (PBS, 0.14M NaCl) wash off impurity, flow velocity 2ml/ minutes, when UV absorption baseline is to 0, start gradient elution, elution buffer (PBS, 0.14M NaCl, 500mM imidazoles) elutes purpose egg, and continuous gradient elutes (0-100%, 100ml), works as ultraviolet absorption peak Eluent, 4ml/ pipe are collected when appearance.Each pipe eluate concentration of SDS-PAGE electrophoresis detection.

According to electrophoresis detection as a result, taking the biggish elution liquid pipe of purity higher concentration, it is packed into the bag filter of 3500Da, 4 In DEG C refrigerator in PBS dialysed overnight, ULP enzyme then is added into dialysis product, 25 DEG C, digestion 2 hours, then will be resulting Digestion products loading crosses the His column (5ml column volume) that PBS has been balanced, and iterative cycles loading 2 hours, flow velocity 2ml/ minutes, collects It flows through, is concentrated with the super filter tube of 10kD, obtains the PBS solution of final DTT-ZFY albumen.Albumen is diluted to 1mg/ after quantitative ML, electrophoresis detection result is as expected, and purity reaches 90% or more.

The experiment of 3 isothermal titration of embodiment

By using isothermal titration calorimetric method, the Z structural domain and ZFY mutant and people and rabbit igg of measurement native protein A Binding constant variation.Structural domain ZFY if necessary to verify mutation hardly has association reaction with IgG, reacts energy by it Amount variation can intuitively reflect.If the two is no during mixing to there is apparent energy variation, it may be considered that There is no combining between ZFY albumen and IgG, the point mutation transformation for Z structural domain is successful；And if the two was mixing There are apparent energy variations in journey, then may infer that ZFY albumen still can be reacted with IgG, such point mutation does not have effect Or not exclusively.It is tested by isothermal titration, whether the transformation that can effectively verify the Z structural domain of albumin A in vitro succeeds.

Testing result is as shown in Figure 1.Fig. 1 shows the isothermal calorimetric titration amount curve that human IgG is reacted with Z, ZFY albumen. Figure 1A is the isothermal calorimetric titration amount curve that human IgG is reacted with Z albumen；Figure 1B is the isothermal amount that human IgG is reacted with ZFY albumen Pyrotitration amount curve；

Enthalpy change, Entropy Changes, the binding constant of 1 human IgG of table and Z, ZFY association reaction

Generate enthalpy change factor mainly in conjunction with heat absorption and heat release in the process, and generate Entropy Changes because being known as hydrophobic work With the folding or other conformation changes etc. in, cohesive process.The thermodynamic function of binding force type and mechanism between molecule Variation can be such that enthalpy change and Entropy Changes reduces in regularity variation, hydrogen bond and Van der Waals force, i.e., H < 0 △ and S < 0 △, 1,2 result of table are said Bright, the main function between people, rabbit igg and Z, ZFY albumen is since hydrogen bond and Van der Waals force form compound system.In heating power On, the key data for evaluating intermolecular interaction intensity is binding constant (Ka), according to Thermodynamics Formulas △ G=△ H-T △ S, △ G=-RTlnKa can extrapolate the Ka entirely reacted by the thermodynamic data of table 1,2.The combination of ZFY and human IgG is normal Number is reduced to 4.71, and the single domain Z albumen of native protein A and human IgG be there are significant reaction, and K value is 2.66 × 10⁶, only It is the 1.5 × 10 of former binding constant^-6；The binding constant of ZFY and rabbit igg is reduced to 1.2 × 10⁴, only former binding constant 0.39%；Demonstrating ZFY is a kind of and the weak albumin A derivative of IgG binding force.And pass through animal immune, measurement antibody drop Degree, it was demonstrated that ZFY is a kind of albumin A derivative that can be significantly improved body and generate specific antibody.

The detection of 4 immunocompetence of embodiment

Fusion protein immunization mouse

In order to verify DTT-ZFY can inducing antibodies, we are first with PBS blank control, SPA, DTT, ZFY, Normal BALB/c mouse is immunized in DTT-ZFY immunizing antigen, by antigen diluent at 30ug/mL after, it is mixed in 1:1 ratio and aluminium adjuvant It is even.The subcutaneous multi-point injection of back part is mixed, the potency of antibody is detected after two weeks booster immunizations, such booster immunization 2 times.

Immunizing rabbit with fusion protein

In order to verify DTT-ZFY can inducing antibodies, normal screech owl man is immunized first with DTT-ZFY in we Rabbit, by antigen diluent at 1mg/mL after, mixed in 1:1 ratio and aluminium adjuvant.The subcutaneous multi-point injection of back part is mixed, was added every two weeks It is strong immune, the potency of antibody is detected after such booster immunization 2 times.

Mouse ELISA detects potency

It is 1 μ g/ml that albumen ZFY sample, which is diluted to protein content, with sodium bicarbonate coating buffer.In each polyphenyl In the reacting hole of vinyl plate plus 100ul, the i.e. hole 100ng/ is coated with, and 4 DEG C overnight.Next day abandons solution in hole, uses washing buffer Liquid (1XPBST) is washed 6 times, 3 minutes every time (referred to as washing, similarly hereinafter).3%BSA solution 100ul is added, sets 37 DEG C of constant-temperature incubations 2 Hour.It is washed out, washing is same as above step.It is parallel with sample progress to do blank well, Positive control wells and negative control hole simultaneously Reaction and same treatment.In each reacting hole, the serum of each group prepared before being added as primary antibody, concentration uses 1 respectively × PBS is diluted to 1/100,1/200,1/400,1/800,1/1600,1/3200,1/6400,1/12800,1/25600,1/ 51200,1/102400,1/204800,1/409600 and 1/819200,0.1ml is added in every hole, and 37 DEG C are incubated for 2 hours, washing. It is washed out, pats dry, 100ul is added as the sheep anti mouse-IgG- horseradish peroxidase cross-linking agent of secondary antibody in every hole, and (1:5000 is dilute Release), 37 DEG C are incubated for 1 hour, washes clean, and pat dry.It is added the tmb substrate solution 100ul now prepared in each reacting hole, 37 DEG C be incubated for 20min.2M sulfuric acid solution 50ul is added in each reacting hole.OD value is measured at microplate reader 450nm wavelength immediately, and Record reading.

The potency of ZFY structural domain is directed to by ELISA measurement antiserum, such as Fig. 2 aluminium adjuvant group (Alum) and DTT immune group Antiserum in, be below 10 for the potency of ZFY structural domain³；In the antiserum of SPA immune group, for the effect of ZFY structural domain Valence is 10³；In ZFY and the antiserum of DTT-ZFY immune group, 10 are above for the potency of ZFY structural domain⁴.Wherein, ZFY is immune Potency in the antiserum of group for ZFY structural domain is significantly higher than SPA immune group (P < 0.05)；The anti-blood of DTT-ZFY immune group Potency in clear for ZFY structural domain is significantly higher than ZFY immune group (P < 0.05).The obtained sero-fast effect of ZFY immune group Valence can be higher by one to two orders of magnitude of the obtained sero-fast potency of native protein A direct immunization, illustrate the knot of ZFY Structure mutation plays really avoids Z structural domain from being combined by force with antibody Fc section, causes immune response (i.e. specific antibody to improve Generate) effect；And the obtained sero-fast potency of DTT-ZFY immune group can be immunized with ZFY it is obtained sero-fast An order of magnitude of potency illustrates that DTT can actually improve the effect of immunostimulation, generates more antibody.

Rabbit ELISA detects potency

It is 1 μ g/ml that albumen ZFY sample, which is diluted to protein content, with sodium bicarbonate coating buffer.In each polyphenyl In the reacting hole of vinyl plate plus 100ul, the i.e. hole 100ng/ is coated with, and 4 DEG C overnight.Next day abandons solution in hole, uses washing buffer Liquid (1XPBST) is washed 6 times, 3 minutes every time (referred to as washing, similarly hereinafter).3%BSA solution 100ul is added, sets 37 DEG C of constant-temperature incubations 2 Hour.It is washed out, washing is same as above step.It is parallel with sample progress to do blank well, Positive control wells and negative control hole simultaneously Reaction and same treatment.In each reacting hole, the serum of each group prepared before being added as primary antibody, concentration uses 1 respectively × PBS is diluted to 1/100,1/200,1/400,1/800,1/1600,1/3200,1/6400,1/12800,1/25600,1/ 51200,1/102400,1/204800,1/409600 and 1/819200,0.1ml is added in every hole, and 37 DEG C are incubated for 2 hours, washing. It is washed out, pats dry, 100ul is added as the chicken anti-rabbit-IgG- horseradish peroxidase cross-linking agent of secondary antibody in every hole, and (1:5000 is dilute Release), 37 DEG C are incubated for 1 hour, washes clean, and pat dry.It is added the tmb substrate solution 100ul now prepared in each reacting hole, 37 DEG C be incubated for 20min.2M sulfuric acid solution 50ul is added in each reacting hole.OD value is measured at microplate reader 450nm wavelength immediately, and Record reading.

Immune effect is similar to the result in mouse immune experiment, and rabbit passes through the protein immunization of DTT-Zfy, Ke Yixian Write the yield for enhancing immune animal antigen.Gained reaches 409600 for the antiserum titre of albumen ZFY.

Antibody specificity assessment after immune

Since albumin A mainly passes through the Fc section of antibody in conjunction with antibody, and the Immune discrimination function on antibody mainly passes through Fab section embodies, so the specific antibody that the present inventor's conception is directed to ZFY by passing through the affine column purification of ZFY albumen first, so Antibody molecule is cut with pepsin afterwards, isolated Fab and Fc section obtain finally by native protein A by Fc section removals The Fab of purifying, is reacted by ELISA, and can verify purified Fab in conjunction with albumin A.If Fab and albumin A exist obvious Reaction, then can prove the immune specific antibody for ZFY generated of DTT-ZFY also can specific recognition Fab, thus Illustrate that DTT-ZFY has the potential ability as vaccine.

The preparation process of ZFY antigen column:

By recombinant protein ZFY solution, in 4 DEG C of constant temperature refrigerators, it is placed in 1L0.2M sodium bicarbonate solution and stirs dialysis, every 4 Hour replacement dialyzate, replaces 4 times, solution system is replaced into 0.2M sodium bicarbonate solution.Finally obtained with BCA standard measure Recombination ZFY protein solution after dialysis is concentrated, 6000rpm, 4 DEG C then according to its actual concentrations with the ultrafiltration concentration pipe of 3KD, It is concentrated or is added suitable 0.2M sodium bicarbonate solution, controlling final protein concentration is 3mg/ml.

It is low to measure about 5ml, 3000rpm, 4 DEG C of the activated medium of 1,6-, bis- iodohexane being kept in dark place in 4 DEG C of refrigerators Temperature centrifugation 10 minutes goes the ethyl alcohol on upper layer to save liquid, 0.2M sodium bicarbonate solution 5ml is added, is protected from light in 4 DEG C of refrigerators after resuspension Overnight stand 12 hours, the volume after taking out measuring medium sufficient standing precipitating was 3ml.Dispense 15ml reaction tube (reaction liquid Product should be the 1/3 to 1/2 or so of reaction flask volume) each 1.5ml, the solution of 6ml ZFY containing albumen is added, and (0.2M sodium bicarbonate is molten Liquid system, 18mg albumen) and the anhydrous sodium sulfate of 2.7g is added, it mixes and dissolves it sufficiently.After packaging is fully sealed, it is put into 37 DEG C of constant incubator rotations, middling speed rotation, 16 hours.Medium is taken out, gravity column is filled, with 2ml deionized water or phosphoric acid buffer Liquid (1 × PBS, 0.14M NaCl) cleans 3 times, connects coupling liquid.Pillar is washed with the 0.1M NaAc-HAc pH3.0 of 2ml, then is used The 0.2M Gly-NaOH pH11.0 of 1ml washs pillar, and 4 DEG C of pillar preservations are then washed with water.

Family's rabbit igg digestion time is tested, IgG digestion 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 are distinguished Hour, 24 hours, 48 hours, non-reduced SDS-PAGE electroresis appraisal, the final optimal conditions for determining pepsin digestion rabbit-anti For pepsin: the mass ratio 1:20 of rabbit anteserum IgG, 37 DEG C water-bath digestion 8 hours.

The purifying of control group F (ab ') 2

The purifying electroresis appraisal for preparing adjuvant group rabbit anteserum IgG shows: by the bionical affinity chromatography purifying of albumin A, obtaining Rabbit igg albumen to purity 90% or more, in SDS-PAGE electroresis appraisal, for clearly the heavy chain band of 55KD or so and rabbit Resist the light chain bands of 27KD of distinctive disperse or so.

It prepares the identification of adjuvant group rabbit anteserum IgG pepsin restriction enzyme digestion and electrophoresis to show in pepsin: the matter of rabbit anteserum IgG Amount is than 1:20, and 37 DEG C after water-bath digestion 8 hours, the digestion of adjuvant group rabbit igg is complete, no complete IgG molecule residual.

Prepare adjuvant group rabbit F (ab ') 2 and purify electroresis appraisal and show: having finally obtained the band of 110kD or so, size with The size of the F (ab ') 2 recorded in document is consistent, and purity meets the requirement further detected, completes control group F (ab ') 2 preparation.

The purifying of specific F (ab ') 2

The purifying electroresis appraisal of DTT-ZFY antigen immune group rabbit anteserum IgG shows: containing day by being coupled on medium The single structure ZFY mutant of right albumin A, has obtained purer specific antibody.In SDS-PAGE electroresis appraisal, for clearly The light chain bands of the 27KD of the distinctive disperse of heavy chain band and rabbit-anti of 55KD or so or so.

Preparation DTT-ZFY antigen immune group rabbit anteserum IgG pepsin restriction enzyme digestion and electrophoresis qualification result shows in stomach cardia Enzyme: the mass ratio 1:20 of rabbit anteserum IgG, 37 DEG C after water-bath digestion 8 hours, the digestion of DTT-ZFY antigen immune group rabbit igg is complete Entirely, no complete IgG molecule residual.

It prepares DTT-ZFY antigen immune group rabbit F (ab ') 2 and purifies electroresis appraisal the result shows that having finally obtained 110kD or so Band, the size of the F (ab ') 2 recorded in size and document is consistent, and purity meets the requirement further detected, completes The preparation of specific F (ab ') 2.

Calculate albumen yield: 1ml antiserum obtains 0.15mg specific IgG after antigen column purification, by digestion, Repurity obtains the specific F (ab ') 2 of 0.05mg.

Specific 2 immunocompetence of F (ab ') detection

It is detected by Elisa, compares performance of the control group F (ab ') 2 with specificity F (ab ') 2 in conjunction with SPA, experiment knot Fruit is as shown in Figure 3: with adjuvant group F (ab ') 2 no matter under great concentration all compared with albumin A is without significant reaction, 31.25ng The rabbit F (ab ') 2 of DTT-ZFY antigen immune group as primary antibody can generate significant between the coating protein SPA of 100ng Reaction, so as to measure higher absorbance value, i.e., with the immunocompetence specifically bound with albumin A.DTT-ZFY Immunocompetence verification experimental verification of the rabbit F (ab ') 2 of antigen immune group in conjunction with protein A, antigen is immune after monomer point mutation Afterwards, rabbit can produce the high titre specific antibody for albumin A, and antibody is made by the F (ab ') 2 that digestion purifying is prepared into For primary antibody, coating protein A can be combined, it was demonstrated that the immune rabbit-anti generated of recombinant protein can be specific in conjunction with albumin A.And it is this The specific antibody of high titre has the potential autoimmune response for causing as vaccine and being directed to albumin A, so as to cause for gold The follow-up immunization of staphylococcus aureus pathogen reacts and immune attack, effectively inhibits infection of staphylococcus aureus or and antibiosis The element caused infection for the treatment of staphylococcus aureus together.

It discusses

The present invention is by PCR mutating technology, by two critical sites in Z structural domain in conjunction with IgG, the 13rd, 14 Phe, Tyr transform Gly as.Discovery Studio analysis shows that, mutant ZFY and IgG binding force reduces 515.57kcal/mol, the 10.69% of only former interaction force；Albumen ZFY after building is mutated, measures, ZFY through ITC 4.71 are reduced to the binding constant of human IgG, and the single domain Z albumen of native protein A is with human IgG that there are significant reaction, K values It is 2.66 × 10⁶, the 1.5 × 10 of only former binding constant^-6；The binding constant of ZFY and rabbit igg is reduced to 1.2 × 10⁴, only The 0.39% of former binding constant；Demonstrating ZFY is a kind of and the weak albumin A derivative of IgG binding force, and is exempted from by animal Epidemic disease measures antibody titer, it was demonstrated that ZFY is a kind of albumin A derivative that can be significantly improved body and generate specific antibody.

ZFY mutant and carrier protein DTT are coupled, a kind of albumen for facilitating expression and purification has been obtained, after animal is immunized It can effectively excite animal immune system to generate protein A antibody, establish reason to study pathogenesis and its vaccine development of S. aureus L-forms By basis.

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 (9)

1. a kind of Z domain derivative of staphylococcal protein A, which is characterized in that the amino acid sequence of the Z domain derivative Column are as shown in SEQ ID NO.:2.

2. a kind of fusion protein, which is characterized in that the fusion protein be Z domain derivative as described in claim 1 with Carrier protein fusion is formed by, and the fusion protein can induce body and generate for the immune of staphylococcal protein A Reaction.

3. fusion protein as claimed in claim 2, which is characterized in that the carrier protein is DTT；The fusion protein are as follows:

(a) polypeptide with amino acid sequence shown in SEQ ID NO.:20 or 21.

4. a kind of polynucleotides, which is characterized in that Z domain derivative described in the polynucleotide encoding claim 1 Or fusion protein as claimed in claim 2.

5. a kind of expression vector, which is characterized in that the expression vector contains polynucleotides as claimed in claim 4.

6. a kind of host cell, which is characterized in that the host cell contains expression vector described in claim 5, or Polynucleotides as claimed in claim 4 are integrated in genome.

7. a kind of pharmaceutical composition, the composition contains Z domain derivative described in claim 1, claim 2 institute Described in the fusion protein stated, polynucleotides as claimed in claim 4, expression vector or claim 6 described in claim 5 Host cell and pharmaceutically acceptable carrier and/or auxiliary material.

8. a kind of vaccine composition, the composition contains Z domain derivative described in claim 1, claim 2 institute Described in the fusion protein stated, polynucleotides as claimed in claim 3, expression vector or claim 6 described in claim 5 Host cell and immunology on acceptable carrier and/or auxiliary material.

9. Z domain derivative described in claim 1, fusion protein as claimed in claim 2, as claimed in claim 4 more The purposes of expression vector described in nucleotide, claim 5 or host cell as claimed in claim 6,

(a) it is used to prepare the antibody for staphylococcal protein A；And/or (b) it is used to prepare treatment staphy lococcus infection disease Drug.