CN106957369A - The recombinant vaccine for reducing marrow source inhibitory cells and modulability immunocyte is prepared and purposes - Google Patents

Abstract

The invention provides a kind of structure, preparation method and the purposes of the recombinant vaccine for reducing marrow source inhibitory cells and modulability immunocyte.Specifically, the invention provides a kind of Calcium Binding Protein Containing S100A8 and/or the recombinant protein of S100A9 structures, the recombinant protein has the structure from vaccine carrier albumen.The recombinant protein of the present invention effectively can produce immune response by excitating organism as the composition of bacterin preparation for calbindin S100A8 and/or S100A9 domain, reduce the marrow source inhibitory cells and modulability immunocyte level of tumor inducing, and on zoografting tumor model in show preferable therapeutic effect.

Description

Reduce marrow source inhibitory cells and modulability immunocyte recombinant vaccine prepare and Purposes

Technical field

The invention belongs to biological and field of medicaments, prepared by the vaccine of reduction marrow source inhibitory cells and modulability immunocyte Method and purposes.The marrow source inhibitory cells and modulability immunocyte that the present invention can reduce cancer induction press down in cancer immunity System, improves treatment of cancer effect.

Background technology

In tumor tissues and chronic sites of inflammation populated with marrow source precursor cell, immature granulocyte, immature huge The immature marrow source cell such as phagocyte and immature BMDC, is referred to as marrow with stronger immune suppression function Source suppresses cell (myeloid-derived suppressor cell, i.e. MDSC).MDSC is only accounted for always in healthy human peripheral blood The 2%~10% of cell number, with the physiological function for suppressing immune system attack health tissues.But in tumor model mouse and many Plant human cancer, the peripheral blood in patients such as adenocarcinoma of lung, the cancer of the esophagus, liver cancer, cancer of pancreas, thyroid tumors, meningioma, carcinoma of urinary bladder In, the ratio of marrow source inhibitory cells (MDSC) and modulability immunocyte, as cancer disease progression is stepped up can reach 20%~40%, even more high；Modulability immunocyte can promote body to occur immune tolerance phenomenon, contribute to tumour Immunologic escape.The marrow source inhibitory cells and modulability immunocyte of super quantity in cancer patient's body, are that limitation body is antitumor The key factor of immune response, it is suppressed that containment of the immune system to tumour cell, promotes tumour growth, differentiation, invasion and attack and turns Move, accelerate the development and transfer of cancer.

MDSC formation and/or reduction marrow source inhibitory cells are blocked, the immunosupress of tumor inducing can be mitigated, strengthened The effect for the treatment of of cancer.Have been reported that and show at present, an anti-vitamin A acid can promote mice with tumor MDSC to be divided into ripe DC in vivo, huge Phagocyte, granulocyte, so as to strengthen CD4⁺T cell, CD8⁺The anti-tumor immune response of T cell mediation, and tumor vaccine can be strengthened Effect.Srivastava etc. removes tumor-bearing mice peripheral blood marrow source inhibitory cells with antibody anti-Gr1 or anti-Ly6G, The activity of antigen presenting cell and the activity of NK, T effector cell are added, the IL-10 yield of cd8 t cell in tumour is reduced, Tumor angiogenesis factor (VEGF, CXCL2, CXCL5, and Angiopoietin1＆2) generation is reduced, anti-angiogenic life is promoted Into response (CXCL9 and CXCL10), the curative effect of tumor vaccine is finally enhanced.

SiRNA A20 (si-A20) the treatment tumor-bearing mices such as Shao, induction of MDSC apoptosis, improve T cell and control Therapeutic effect.2014, Hong Qin et al. developed a peptibody, can remove in tumor-bearing mice body two kinds of subgroups MDSC cells, with the effect for suppressing growth of tumour cell.Their research finds that the peptibody may act on MDSC cells S100A8 the and S100A9 albumen on surface, be subsequently found peptibody combination target spot be on MDSC S100A8 and/or S100A9。

These antibody and peptibody in oncotherapy specificity remove tumor inducing MDSC, it is necessary to frequently medication, Blood concentration change is frequent after medication, therefore in the urgent need to exploitation being capable of the easy curative drug of medication.

The content of the invention

It is an object of the invention to provide a kind of restructuring epidemic disease for carrying calbindin S100A8 and/or S100A9 domain Seedling, recombinant protein and composition (such as vaccine combination).

In the first aspect of the present invention, there is provided a kind of carrying S100A8 and/or S100A9 recombinant protein, the restructuring egg It is white that there is the domain from carrier protein.

In a preference, described carrier protein includes CTB (b subunit of cholera toxin), DTT (diphtheria toxin cross-film knots Structure domain), VLP, TT, PLY etc.

The second aspect of the present invention is there is provided a kind of polynucleotides, the weight described in described polynucleotide encoding first aspect Histone.

The third aspect of the present invention contains the multinuclear described in second aspect there is provided a kind of expression vector, the expression vector Thuja acid.

The fourth aspect of the present invention contains the table described in the third aspect there is provided a kind of host cell, described host cell Up to carrier, or it is integrated with genome the polynucleotides described in second aspect.

In a preference, described host includes Escherichia coli, yeast, Chinese hamster ovary celI, DC cells etc.

The fifth aspect of the present invention contains described in first aspect there is provided a kind of pharmaceutical composition, described pharmaceutical composition Recombinant protein, described in the expression vector described in polynucleotides or the third aspect or fourth aspect described in second aspect Host cell with and/or pharmaceutically acceptable carrier and/or auxiliary material.

In a preference, described composition is vaccine.

The sixth aspect of the present invention contains the weight described in first aspect there is provided a kind of vaccine combination, described composition The expression vector described in polynucleotides or the third aspect described in histone, second aspect or the host described in fourth aspect Cell, with and/or immunology on acceptable carrier and/or auxiliary material.

In a preference, described vaccine combination also contains adjuvant, and the adjuvant includes aluminum oxide, soap former times, quil A, muramyl dipeptide, mineral oil or vegetable oil, the adjuvant based on vesica, non-ionic block copolymer or deae dextran, cell The factor (including IL-1, IL-2, IFN-r, GM-CSF, IL-6, IL-12 and CpG)

The seventh aspect of the present invention there is provided the purposes of the S100A8 described in first aspect and/or S100A9 recombinant protein, (a) it is used to prepare the antibody for the S100A8 and/or S100A9；And/or (b) is used to prepare treatment and S100A8/ The medicine of disease related S100A9.

In a preference, described disease includes：Tumour, inflammation.

The eighth aspect of the present invention is there is provided a kind for the treatment of method, and the object to needs applies the restructuring described in first aspect The vaccine combination of pharmaceutical composition or the 6th aspect described in albumen, the 5th aspect.

In a preference, the mammal includes：Mouse, people etc.

In a preference, the C-terminal that the antigenic domains are connected to the carrier protein forms the fusion protein.

There is connection peptide in a preference, between the antigenic domains and the carrier protein.Preferably, the company It is 20 amino acid to connect peptide length.

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

Brief description of the drawings

Accompanying drawing 1 shows the antitumous effect of each immune combination in embodiment 1 and embodiment 2.Figure 1A shows implementation The tumor weight of each immune group mouse in example 1, as a result shows compared with carrier protein and adjuvant group, and CTB-S100A8 has notable Antitumous effect.Figure 1B be embodiment 2 in through be immunized after each group mouse tumor weight, as a result show CTB-S100A9 vaccines Compared with carrier protein and adjuvant group, the tumor protection effect with statistical significance.

Embodiment

The present invention is had found based on the structural framework of suitable carrier protein, in its C by in-depth study extensively End insertion MDSC specific antigens S100A8 and/or S100A9 domain, and suitable connection peptide length is adjusted, one can be made Class recombinant protein.The recombinant protein effectively excitating organism (such as mammal) can be not only answered the immune of the recombinant protein Answer, and can effectively produce the immune response for S100A8 the and/or S100A9 protein structure domains, including produce anti- Body, while the recombinant protein can also suppress the MDSC of tumor inducing, produces anti-tumor immune response.Complete this on this basis Invention.

Term

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

As used herein, term " antigen single domain " refers to the structure for intending other albumen that induced animal produces immune response Domain, the domain protein not referring to for carrier protein, carrier protein can cause the structure of immune response in itself Domain.Generally, antigenic domains refer to the domain that targeting is intended in immune response, preferably from the knot of mammal (such as people) albumen Structure domain, rather than from the carrier protein.

As used herein, term CTB refers to the B subunits of cholera toxin；MDSC refers to the inhibitory cells in medullary system source；S100A8 Refer to CalgranulinA albumen (also referred to as MRP8 albumen)；S100A9 refers to CalgranulinB albumen (also referred to as MRP14 albumen)；M- MDSC refers to the MDSC of cells of monocytic origin；G-MDSC refers to the MDSC in granulocyte source；Treg refers to regulatory T cells；MUC1 refers to Mucin 1 (MUC1).

Representational carrier protein

Choleratoxin B subunit (CTB)

Cholera toxin (cholera toxin) is the exotoxin (84kDa) of comma bacillus secretion, is made up of A, B subunit, is AB5 types.Choleratoxin B subunit (CTB) is the nontoxic part of cholera toxin, with good immunogenicity, through human trial It is proved to be the active ingredient of vaccine.The Ganglioside GM1 specificity that CTB can exist with most of mammalian cell surfaces is tied Close, stimulate body to produce mucous membrane IgA, strengthen antigenic mucosa immunity-inducing reaction.CTB have been used for new oral cholera vaccine, Adjuvant and protein carrier.

CTB is nontoxic, is made up of 2 sections of α screw elements and 6 sections of beta- pieces between core skeleton, structural detail by flexibility Ring region is connected.Five B subunits form Pentagram shape with the opposing parallel inside assembling of long α screw elements.Each subunit can Independent nerve node glycosides fat (GM1) acceptor combined on cell membrane.

CTB amino acid sequences (ABG56901) are as follows：

1 TPQNITDLCA EYHNTQIYTL NDKIFSYTES LAGKREMAII TFKNGAIFQV EVPGSQHIDS

60 QKKAIERMKD TLRIAYLTEA KVEKLCVWNN KTPHAIAAIS MAN

Composition and application process

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

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

The composition of the present invention includes pharmaceutical composition and vaccine combination.

The composition of the present invention can be monovalent (only containing a kind of recombinant protein or polynucleotides) or multivalence (containing a variety of recombinant proteins or polynucleotides).

The pharmaceutical composition or vaccine combination of the present invention can be prepared into various regular dosage forms, including (but do not limit In)：Injection, granula, tablet, pill, suppository, capsule, suspension, spray etc..

(1) pharmaceutical composition

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

Term " therapeutically effective amount " used herein refers to therapeutic agent treatment, alleviates or prevent the amount of target disease or situation, Or show the amount of detectable treatment or prevention effect.The effect can be detected for example, by antigen levels.Therapeutic effect Also the reduction of physical symptoms is included.For a certain object accurate effective dose depend on the object build and health status, The combination of therapeutic agent and/or therapeutic agent that the nature and extent of illness and selection are given.Therefore, preassigning accurately has Effect amount is useless.However, for certain given situation, the effective dose can be determined with normal experiment.

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

Pharmaceutical composition can also contain pharmaceutically acceptable carrier.Term " pharmaceutically acceptable carrier " refers to for controlling Treat the carrier of agent (recombinant protein of the invention) administration.The term refers to some such medicament carriers：Themselves is not induced The antibody being harmful to the individual for receiving said composition is produced, and does not have undue toxicity after administration.Suitable carrier can be big , be metabolized slow macromolecular, such as protein, polysaccharide, PLA (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 on pharmaceutically acceptable carrier or excipient.

The upper acceptable carrier of combination of traditional Chinese medicine may include liquid, such as water, salt solution, glycerine and ethanol.In addition, these are carried Complementary material, such as wetting agent or emulsifying agent, pH buffer substance are there is likely to be in body.Generally, composition can be made Injectable agent, such as liquid solution or suspension；It may also be fabricated which and be adapted to before the injection supplying solution or suspension, liquid excipient Solid form.Liposome is also included within the definition of pharmaceutically acceptable carrier.

(ii) vaccine combination

The vaccine (composition) of the present invention can be preventative (i.e. prevention disease) or curative (be controlled after illness Treat disease).

These vaccines include immunising antigen (including recombinant protein of the present invention), and generally with it is " pharmaceutically acceptable Carrier " is combined, and these carriers include itself not inducing any carrier for producing the antibody for being harmful to the individual for receiving said composition. Suitable carrier is typically big, is metabolized slow macromolecular, and such as protein, polysaccharide, PLA, polyglycolic acid, amino acid gather Compound, 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, lockjaw, 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 adjuvants And Freund Freund's incomplete adjuvants (IFA) (CFA)；(5) cell factor, such 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 stimulatory factor (M-CFS), TNF (TNF) etc.；(6) the detoxification variant of bacterial ADPribosylating toxin (such as E.coli LT LT)；And (7) Strengthen the other materials of composition effect as immunostimulant.

Vaccine combination including immunogenic composition is (for example, it may include antigen, pharmaceutically acceptable carrier And adjuvant), usually contain diluent, such as water, salt solution, glycerine, ethanol 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, bag Immunogenic polypeptide containing immunological effective amount, and above-mentioned other required components." immunological effective amount " refer to single dose or It is effective that a continuous agent part, which gives individual amount to treating or preventing,.The consumption can be according to the health status for treating individual With physiological status, treat individual classification (such as people), the ability of individual immunity system synthesis antibody, required degree of protection, Depending on assessment and other correlative factors of the preparation, treating physician of vaccine to medical conditions.It is expected that the consumption will be relatively In wide scope, it can be determined by normal experiment.

Generally, injectable agent, such as liquid solution or suspension can be made in vaccine combination or immunogenic composition；Also It can be made into and be adapted to supplying solution or suspension, the solid form of liquid excipient before the injection.Said preparation is also emulsifiable or is encapsulated in In liposome, to strengthen adjuvant effect.

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

(iii) method of administration and dosage

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

When as vaccine, the recombinant protein of the present invention can be directly applied to individual with known method.Generally use These vaccines are applied with conventional vaccine identical route of administration and/or simulation pathogenic infection path.

Giving the approach of pharmaceutical composition of the present invention or vaccine combination includes (but being not limited to)：Intramuscular, subcutaneous, skin Interior, intrapulmonary, intravenous, intranasal, by oral administration or other parenteral route of administration.It is possible if desired to combination medicine-feeding approach, or root It is adjusted according to disease event.Vaccine combination can be given with single dose or multiple dose, and can include give booster with Trigger and/or maintain immunity.The amount of recombinant protein vaccine, i.e. recombinant protein should be given with " effective dose " in selected administration Immune response is adequate to bring about in path, can effectively promote the disease for protecting host's resistance related.

Representational disease includes (but being not limited to)：Tumour etc..

The amount of selected recombinant protein in each vaccine dose part, be by can trigger protective immune response and without obvious Depending on the amount of side effect.Generally, after host cells infected, each dose of vaccine is enough containing about 1 μ g-1000mg, preferably 1 μ g-100mg, more preferably 10 μ g-50mg protein.Can use includes the IgG titers in the object of observation and the standard of other reactions Research method determines the optimum amount of specific vaccine.It can be determined the need for by monitoring the immunity level of vaccine offer Strengthen dosage.After the IgG titers in have evaluated serum, it may be necessary to from enhancing dose immunizations.Using adjuvant And/or immunostimulant can improve the immune response of the protein to the present invention.

Method for optimizing is to give immunogenic composition from parenteral (subcutaneously or intramuscularly) approach by injection.

In addition, the vaccine of the present invention can together be given with reference to other immunomodulators, or together with other therapeutic agents Give.

Main advantages of the present invention are：

(1) can effectively immune response of the excitating organism for S100A8 and/or S100A9 domain.

(2) the preparation low cost of S100A8 and/or S100A9 recombinant protein, convenient drug administration are carried.

(3) relative to the preparation with carrier protein chemical coupling, antigenic structure is definite, quality controllable, safer

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip Condition described in part, or according to the condition proposed by manufactory.

Tumor vaccine exploitation of the embodiment 1 using mouse S100A8 as target spot

Recent studies have shown that S100A8 play during tumor locus MDSC generation and recruitment it is very important Role.Show that MDSC not only secretes S100A8 in Sinha et al. parallel test done, it may have S100A8 bound site Point, these combine and may be by RAGE or carboxylic salts glycan, and activate the NF-kB signal paths in downstream.In addition, tumour Cell and MDSC secrete S100A8, and extracellular S100A8 albumen can also promote MDSC transfer.Tumor-bearing mice is given The treatment of carboxylic salts glycan sealer, can substantially reduce serum and stimulate the recruitment of MDSC in lymphoid organ, these research tables Bright, S100A8 plays very important role in MDSC recruitment.Therefore, targeting S100A8 is probably antitumor and anti- MDSC Critical policies.

Expression vector establishment of the step 1 using S100A8 as target spot

According to mouse S100A8 amino acid sequence (amino acid sequence P27005), according to e. coli codon preference Gene order is optimized, the gene after optimization is closed by Shanghai Jierui Biology Engineering Co., Ltd according to PCR synthetic methods Into, and load into pET28a carriers, obtain pET28a-S100A8 plasmids.Using pET28a-S100A8 as template, primer 1 is utilized And primer 2 enters performing PCR amplification, reaction system is 50 μ L, and condition is 95 DEG C of pre-degeneration 3min, 95 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 68 DEG C of extension 20s, carry out 35 circulations, last 68 DEG C of extensions 5min altogether.PCR primer carries out 1% agarose gel electrophoresis Detect (120V, 30min), obtain fragment of the size between 200bp to 300bp, the gene size 267bp phases with S100A8 Symbol, then cuts off the Ago-Gel residing for the band, is reclaimed according to the standard operation of Axygen gel reclaims kits.CTB Gene (gene order ABG56901) is synthesized by Shanghai Jierui Biology Engineering Co., Ltd according to PCR synthetic methods, and is filled It is loaded onto in pSJF2 carriers, forms pCTB2 plasmids.By pCTB2 plasmids, linearized with BamH I and BspE I restriction endonucleases, instead The μ L of system 50 are answered, condition is 37 DEG C, 4h.Digestion products carry out 1% agarose gel electrophoresis detection (120V, 35min), as a result show The pCTB2 plasmid sizes of linearization are about 4000bp-4200bp, meet expection, then coagulate the agarose residing for this fragment Glue is cut, and is reclaimed according to the standard operation of Axygen gel reclaims kits.

The S100A8 fragments that PCR is obtained, utilize the homology arm (homology arm of the pCTB2 plasmids inserted in its upstream and downstream primer It is following in primer 1 and primer 2 to draw broken line representation), it is integrated into linearization plasmid pCTB2 BamH 1 and BspE I digestions Between site.The μ L of reaction system 20, containing 2 μ L Exnase^TmII, 4 μ L 5X CE II buffer, linearisation pCTB2 plasmids 50ng (2 μ L), S100A8 fragments 100ng (5 μ L), 7 μ L ddH₂O, all components are softly mixed, 37 DEG C of incubation 30min, then It is immediately placed on 5min on ice standby.2 μ L reaction solutions are taken to be added in 50 μ L E.coli Top10 competent cells, ice bath 30min, 42 DEG C of heat shock 90s, it is immediately placed on 5min on ice and then adds 600 μ L LB non-resistant culture mediums, 37 DEG C, 180rpm shakes Recovery 1h is shaken, takes 300 μ L to be coated with LB solid plates (the μ g/mL of Amp 100), 37 DEG C are inverted overnight incubation and the training of picking monoclonal Support.Bacterium colony PCR identifications (PCR conditions, electrophoretic detection are as previously described) are carried out to the monoclonal of culture, in 200bp to 300bp Between may occur in which specific bright band, meet the theoretical sizes values 267bp of S100A8 genes, the monoclonal that will appear from above-mentioned band is sent Nanjing Genscript Biotechnology Co., Ltd. is sequenced.

So that correct CTB-S100Ag plasmids are sequenced as template, primer 5, primer 6 for primer enter performing PCR amplification, by CTB with Linker between S100A8 is mutated.The μ L of PCR reaction systems 50, condition is 95 DEG C of pre-degeneration 5min, 95 DEG C of denaturation 1min, 55 DEG C of annealing 1min, 68 DEG C of extension 5min, carry out 35 circulations, last 68 DEG C of extensions 10min altogether.PCR primer carries out 1% agar Sugared detected through gel electrophoresis (120V, 30min), the visible specific bright band at 3800-4000bp, by the fine jade residing for this bright band Sepharose is cut, and is reclaimed according to the standard operation of Axygen gel reclaims kits.DPN I enzymic digestion glue reclaims product is (anti- Answer the μ L DPN I enzymes of 50 μ of system L: 1, the μ L glue reclaim products of 5 μ L 10X NEB Buffer 4,34,10 μ L ddH₂O；37 DEG C incubate 120min is educated, 80 DEG C of denaturation 20min are standby).The μ L of digestive juice 2 are taken to convert 50 μ L E.coli Top10 competent cells, reactant System and condition are as it was previously stated, the Colony Culture grown on picking conversion rear plate is in 3mL LB (the μ g/mL of Amp 100) culture medium In, 37 DEG C, 180rpm shaking overnight incubations.It Jun Song Nanjing Genscript Biotechnology Co., Ltd. will be sequenced, as a result show overnight Linker between CTB and S100A8 has been increased to 20 amino acid residues, meets desired design.

This experiment each primer used is synthesized (such as following table) by Nanjing Genscript Biotechnology Co., Ltd..

Note：A lower stroke solid line represents that dotted line is drawn below BspE I restriction enzyme sites, homology arm to be represented in primer 1, lower stroke in primer 2 Solid line represents that dotted line is drawn below BamH1 restriction enzyme sites, homology arm to be represented.

It is prepared by fusion protein of the step 2 using S100A8 as target spot

Take pCTB2 and correct each 1 μ L of CTB-S100A8 plasmids are sequenced, heat shock method converts 50 μ L E.Coli TG1 impressions State cell (method for transformation refers to step 1), takes 300 μ L to be coated with LB solid plates (the μ g/mL of Amp 100), 37 DEG C of inversions were cultivated Night to monoclonal is grown, picking monoclonal to 25mL LB culture mediums (the μ g/mL of Amp 100), 37 DEG C, 180rpm shaking cultivated Night.1L M9 culture mediums (the μ g/mL of Amp 100), 28 DEG C, 200rpm shaking cultures will be forwarded to by 1: 500 (v/v) by bacterium overnight 30h, then adds 100 μ L 1mol/LIPTG and 100mL 10X TB culture mediums, 28 DEG C, 200rpm shaking cultures 65-70h. 8000rpm, room temperature centrifugation 5min collects thalline and weighed.

10mL sample-loading buffers (500mmol/L NaCl, 20mmol/L Tris, 20mmol/ are fully resuspended in per 1g thalline L Imidazole, pH 8.0), Ultrasonic Cell Disruptor crushes re-suspension liquid (work 3s, gap 5s, power 8%, ultrasonic 30min).4 DEG C, take supernatant standby after 12000rpm centrifugations 30min.Ni posts with equilibrium liquid (500mmol/L NaCl, 20mmol/L Tris, 20mmol/L Imidazole, pH 8.0) it is washed till after baseline, supernatant is reached with 2mL/min flow velocity loading, loading peak 2200mAu, washes miscellaneous liquid (500mmol/L NaCl, 20mmol/L Tris, 60mmol/L Imidazole, pH 8.0) and rinses 20 More than individual column volume until baseline punching is flat, then with eluent (500mmol/LNaCl, 20mmol/L Tris, 500mmol/L Imidazole, pH 8.0) elution, eluting peak is collected, its eluting peak can reach 1200mAu.G25 molecular sieves use 10mmol/L After PBS (pH 7.4) balances, eluting peak is crossed into G25 molecular sieves, collection flows through peak, flows through the mAu of peak about 200.

Take and flow through the μ L of peak 80 and dispense two pipes, be separately added into 10 μ L 5X denaturation reduction buffer solution and 5X denaturation is non-reduced slow Fliud flushing, is fully mixed, and the albumen sample for adding denaturation reduction buffer solution carries out boiling water bath 5min again, adds the non-reduced buffering of denaturation The sample of liquid is standby after then directly mixing.By after above-mentioned processing sample carry out SDS-PAGE electrophoresis detections, resolving gel concentration 12%, Voltage 120V, electrophoresis time about 80min, the preparation of protein adhesive and Running buffer, which are prepared, refers to Bio-Rad standard recipes. Electrophoresis result shows that CTB protein monomers about 20kDa, five that size about 66kDa can be formed under the conditions of denaturation is non-reducing gather Body, its purity of protein about 99% is analyzed by gel imaging system embedded software, meets the standard for carrying out zoopery.CTB- S100A8 protein monomer about 22~25kDa, its difference in size part be expression vector signal peptide length, similarly, its Pentamer can be formed in the state of denaturation is non-reduced, size about 110kDa, software analysis its purity of protein about 95%, after can be used for Continuous zoopery.

Protein concentration is detected according to the standard operation of BCA quantification kits, as a result shows that CTB and CTB-S100A8 albumen are dense Degree is respectively：1.8mg/mL, 1.5mg/mL.

The preparation of step 3 S100A8 single domain albumen

The μ L of pET28a-S100A8 plasmids 1 of synthesis are taken to add in 50 μ L E.coli BL21 (DE3) competent cells, heat The method of swashing conversion, operation is carried out with reference to step 1, takes the μ L of conversion fluid 300 to be coated with Amp flat boards (the μ g/mL of Amp 100), 37 DEG C were inverted Night cultivates to monoclonal and grown.Picking monoclonal is in 3mL LB (the μ g/mL of Amp 100) culture medium, 37 DEG C, 200rpm shaking trainings Support about 12h.Then 1L LB culture mediums (the μ g/mL of Amp 100) are forwarded to by 1: 500 (v/v), 37 DEG C, 200rpm shakings culture extremely OD₆₀₀For 0.6, addition 1mL 1mol/L IPTG, 20 DEG C, 180rpm shaking cultures 20h.8000rpm, room temperature centrifugation 5min are collected Thalline is simultaneously weighed.

20mL sample-loading buffers (500mmol/L NaCl, 20mmol/L Tris, 50mmol/ are fully resuspended in per 1g thalline L Imidazole, pH 8.0), Ultrasonic Cell Disruptor crushes re-suspension liquid (ultrasonic 3s, interval 5s, power 1%, working time 20min).Take supernatant standby after 4 DEG C of ultrasonication liquid, 12000rpm centrifugations 30min.Ni posts are with equilibrium liquid (500mmol/L NaCl, 20mmol/L Tris, 20mmol/L Imidazole, pH 8.0) it is washed till after baseline, supernatant is with 2mL/min flow velocity Loading, loading peak reaches 1500mAu, washes miscellaneous liquid (500mmol/L NaCl, 20mmol/L Tris, 50mmol/L Imidazole, pH 8.0) to rinse 25 column volumes flat to baseline punching, then with eluent (500mmol/L NaCl, 20mmol/ L Tris, 500mmol/L Imidazole, pH 8.0) elution, collect eluting peak, its eluting peak about 700mAu.G25 molecular sieves After being balanced using 10mmol/L PBS (pH 7.4), eluting peak is crossed into G25 molecular sieves, collection flows through peak, flows through peak about 90mAu. Take and flow through the μ L of peak 40, add 10 μ L 5X denaturation reduction buffer solutions and fully mix, then boiling water bath 5min carries out SDS-PAGE electricity Swimming detection, resolving gel concentration 12%, voltage 120V, electrophoresis time about 70min, the preparation of protein adhesive and Running buffer match somebody with somebody System refers to Bio-Rad standard recipes.Electrophoresis result shows, S100A8 albumen size about 12kDa, and software analysis its purity of protein is about For 96%, BCA quantitative displays, its protein concentration is about 1.1mg/mL, meets subsequent experimental basic demand.

The detection of step 4 animal immune and antiserum data

24 6 week old female BAl BIcs/SPF grades of C mouse are purchased from Chinese Academy of Sciences's Shanghai Experimental Animal Center, are randomly divided into 4 Group, and raised according to AAALAC guidelines.Each immune group is respectively：Alum adjuvant groups, S100A8 groups, CTB groups and CTB-S100A8 groups.50 μ of proteantigen (CTB, S100A8, CTB-S100A8) g//times, Al adjuvants 0.3mg//times, CpG 30 μ g//times, choose two points in mouse back backbone both sides after mixing and are subcutaneously injected respectively, the 14th day booster immunization Once, the 21st day mouse orbit is taken after blood, 37 DEG C of standing 2h, 4000rpm centrifugation 10min, takes supernatant serum standby.

S100A8 is diluted to concentration for 100ng/ with coating buffer solution (50mmol/L bicarbonate buffers, pH 9.6) 100 μ L, are then transferred on the orifice plate of polystyrene 96 and (add 100 μ L per hole), 4 DEG C of overnight incubations, with PBST (0.02mmol/L phosphorus Hydrochlorate, 0.15mol/L NaCl, 0.15% (v/v) Tween-20, pH 7.4) wash 6 times, wash every time 5 minutes.Add 300 μ L Confining liquid (being dissolved in PBST) containing 5% (m/v) skimmed milk power, 37 DEG C of incubations are closed for 2 hours.PBST is washed 6 times, and 5 are washed every time Minute.Then the antiserum of each immune group is diluted 100 times with above confining liquid, 100 μ l is added per hole, 4 DEG C of overnight incubations are used PBST is washed 6 times, is washed every time 5 minutes.Adding 100 μ L rabbit-anti mouse-IgG- horseradish peroxidases cross-linking agents, (confining liquid 1: 5000 is dilute Release), 37 DEG C are incubated 1 hour, are washed 12 times, washed every time 5 minutes with PBST.It is eventually adding 100 μ L substrate solution (10mL substrate solutions Tetramethyl benzidine containing 1mg (TMB), 0.0969g sodium citrates, 0.3496g Na₂HPO₄·12H₂O, 32 μ L0.75%H₂O₂)37 DEG C be incubated 15 minutes.Then 50 μ L 2mol/L H are added₂SO₄Terminating reaction.On ELIASA to each hole survey 450nm and Value at 630nm, calculates OD₄₅₀-OD₆₃₀。

Testing result shows that CTB-S100A8 immune groups antiserum is directed to S100A8 antibody OD₄₅₀-OD₆₃₀Value is respectively： 1.62、1.49、1.62、1.49、1.51、1.23.The value of CTB immune groups is 0.18,0.56,0.61,0.43,0.48,0.46. The OD of S100A8 immune groups₄₅₀-OD₆₃₀It is worth for 0.94,0.83,0.83,0.67,1.07,0.79.Divided by one-Way ANOVA Analysis, as a result shows that CTB-S100A8 groups can break through mouse autoimmune tolerance, with CTB immune groups (p ＜ 0.001) and S100A8 Immune group (p ＜ 0.001), which is compared, has significant difference, illustrates that CTB can effectively strengthen immune response of the body for S100A8.

Each immune group takes OD above₄₅₀-OD₆₃₀The pattern detection antibody titer that reading is worth between.S100A8 is diluted It is 100ng/100 μ L to concentration, is then transferred on the orifice plate of polystyrene 96 and (adds 100 μ L per hole), 4 DEG C of overnight incubations, washing, Closing is operated as more than.The antiserum following multiple of confining liquid doubling dilution：100x2⁰, 100x2¹..., 100x2⁹, 100x2¹⁰, the μ L of coating 100,4 DEG C of overnight incubations per hole.Secondary antibody is incubated, and washing and chromogenic reaction are operated as above, using double-wavelength method In reading data OD on ELIASA₄₅₀And OD₆₃₀, according to OD₄₅₀-OD₆₃₀Reading calculates each group antibody titer.As a result CTB- is shown S100A8 immune groups are that 25600, CTB immune groups and S100A8 immune groups are directed to the anti-of S100A8 for S100A8 antibody titer Body titre is respectively 3200,1600.

Structure and the antitumous effect research of step 5 tumor model

4T1 mouse mastopathy cells are purchased from Chinese science icm cell storehouse, and 5% is based on using RPMI-1640 cultures CO₂Cultivated in 37 DEG C of constant incubators.When cell fusion degree reaches 90%, digested through 0.25% pancreatin, collect cell in nothing In the nutrient solution of serum, gently shake, 1000rpm, centrifuge 5min, washed once, 10mmol/L PBS (pH 7.4) are resuspended, It is 10 to adjust cell concentration⁶.Counted through Trypan Blue, and with blood counting chamber, detection live cell fraction is more than 95%.

Mouse immune 2 times was simultaneously taken a blood sample at the 21st day, was inoculated with 3 × 10 in mouse oxter within the 25th day⁵Individual 4T1 tumour cells, and In the 28th day booster immunization once, dosage such as step 4.Go out after knurl every 3 days with vernier caliper measurement length of tumor and width.49th My god, put to death mouse and take tumour and blood sample, use electronic balance weighing tumor weight.According to formula：Inhibiting rate=(control Group average tumor weight-empirical average group tumor weight)/control group average tumor weight * 100% calculating inhibiting rates.All systems Count and statistical analysis is carried out using Graphpad Prism5.0 softwares, and chart.This experiment is using one-way ANOVA Inspection is analyzed the data significance of difference, and " * " represents p ＜ 0.05, and " * * " represent p ＜ 0.01, and " * * * " represent p ＜ 0.001.Measurement result as shown in figure 1, CTB-S100A8 immune group tumor weights be respectively 1.85g, 2.64g, 1.70g, 1.33g.The tumor weight of CTB immune groups is respectively 3.54g, 5.16g, 3.97g, 3.51g.The tumor weight of S100A8 immune groups For 2.54g, 3.70g, 1.77g, 3.20g.The tumor weight of CTB-S100A8 group mouse has significant difference compared with CTB groups (P ＜ 0.001), its tumor control rate is 54%.CTB-S100A8 immune groups are compared with S100A8 single domain immune groups, its knurl Though statistically there was no significant difference for body weight, tumor control rate still reaches 33%.Result above shows CTB-S100A8 With preferable antitumor activity.

Influences of step 6 CTB-S100A8 to the MDSC of tumor inducing

The 24th day after inoculated tumour, the disconnected neck of mouse is put to death, and alcohol-pickled 5 minutes, left abdomen skin is opened in super-clean bench Skin, careful separation hypodermis and abdominal muscles expose spleen, carefully lift spleen, cut off surrounding connective tissue, are put into Sheng In the capsule for having 10mmol/L PBS (pH 7.4), aseptic nipper is caught broken, and the cell suspension of acquisition is transferred into cell sieve filtering Remnant tissue is removed, the suspension 1500rpm centrifugation 5min after filtering abandon supernatant, add 2mL 10mmol/L PBS (pH 7.4) resuspended to wash twice, 1500rpm centrifugation 5min abandon supernatant, obtain the spleen cell of mouse, peripheral blood is then directly centrifuged. The spleen cell suspension and peripheral blood cells of acquisition are added to the erythrocyte cracked liquid of 3 times of cell volumes, gently piping and druming is mixed, room Anneal crack solution 2min.4 DEG C, 2000rpm centrifugation 5min abandon supernatant.Cell precipitation after centrifugation adds 3mL10mmol/L PBS (pH 7.4) resuspended, 2000rpm centrifugation 3min abandon supernatant, are repeated 1 times.

The above-mentioned cell prepared is resuspended with streaming buffer solution, 100 μ L cell suspensions are added in every streaming detection pipe, Often manage and add 1 μ L Fc γ R III/II antibody (being purchased from Shanghai You Ningwei bio tech ltd), it is soft to mix and in ice Upper incubation 10min, then often pipe add the above-mentioned PBS of 2mL wash twice, 1500rpm centrifugation 5min, abandon supernatant.Streaming buffer solution Re-suspended cell, respectively adds following fluorescent labeled antibody CD11b-FITC, Gr-1-APC, F4/80 (purchases in each flow cytometer detection pipe From Shanghai You Ningwei bio tech ltd) each 1 μ L, soft to mix, then each pipe lucifuge is incubated 30min, and often pipe adds 2mL Above-mentioned PBS is washed twice, 1500rpm centrifugation 5min, abandons supernatant.Finally often pipe adds 500 μ L streaming buffer solutions, fully resuspended thin Born of the same parents.Detected with flow cytometer.

The result of streaming is shown：After CTB-S100A8 protein immunization mouse, cells of monocytic origin in mouse peripheral blood MDSC (M-MDSC) content is respectively 12.7%, 6.5%, 13.2%, 7.3%.The analog value of CTB immune groups is respectively 13.5%th, 43.6%, 19.3%, 37.0%.The analog value of S100A8 immune groups is respectively 44.2%, 33.3%, 14.8%.With Alum adjuvants group and S100A8 single domain groups are compared, and CTB-S100A8 albumen is after immune through tumor inducing in human peripheral blood M-MDSC has significant inhibitory action (P ＜ 0.01).After CTB-S100A8 group immune mouses, granulocyte comes in mouse peripheral blood The MDSC (G-MDSC) in source content is respectively 70.5%, 79.5%, 78%, and total MDSC content is respectively in its spleen 52.8%th, 54%, 47.4%, without significant difference (P ＞ 0.05) compared with CTB, S100A8 and Alum adjuvant group.

Result above shows：CTB-S100A8 can significantly inhibit the quantity of M-MDSC in peripheral blood after immune.

Influences of step 7 CTB-S100A8 to the Treg of tumor inducing

The spleen and peripheral blood cells of 4T1 tumor-bearing mices are separated with reference to step 6, with streaming buffer solution re-suspended cell.Every 100 μ L cell suspensions are added in flow cytometer detection pipe, often manages and adds 1 μ L Fc γ R III/II antibody, it is soft to mix and on ice 10min is incubated, then the often pipe addition above-mentioned PBS of 2mL are washed twice, 1500rpm centrifugation 5min abandon supernatant.In the inspection of each streaming 1 μ L fluorescent labeled antibodies CD4-FITC, CD8-FITC (being purchased from Shanghai You Ningwei bio tech ltd) are added in test tube, respectively Pipe lucifuge is incubated 30min, repeated as above to wash twice.Then often pipe adds 1mL fixer, mixes, and lucifuge is incubated 50min, 2000rpm centrifuges 5min, and each pipe adds 2mL penetrating buffer solution, mixes, 2000rpm centrifugation 10min, removes supernatant.Added in pipe 1 μ L Foxp3-PE (are purchased from Shanghai You Ningwei bio tech ltd), and lucifuge is incubated 30min on ice.Then often pipe is added The above-mentioned PBS of 2mL are washed twice, and abandon supernatant.It is eventually adding 500 μ L streaming buffer solutions, abundant re-suspended cell.Upper machine testing.

The result of flow cytometer showed shows：After CTB-S100A8 immune mouses, the content of the Treg cells in mouse peripheral blood For 0.8%, 2.1%, 2.4%；The content of Treg cells is 0.4%, 0.5%, 0.4% in spleen.With CTB groups, S100A8 groups And Alum adjuvants group is compared to without significant difference (P ＞ 0.05), result above shows, CTB-S100A8 recombinant proteins it is anti- What function of tumor was realized not by the Treg cell quantities in spleen and peripheral blood are suppressed.

Tumor vaccine exploitation of the embodiment 2 using mouse S100A9 as target spot

S100A9 also plays very important role during tumor locus MDSC generation and recruitment.Before medullary system Body cell is to during dendritic cells or macrophage normal differentiation, and S100A9 expression is to lower, however, Cheng etc. The research of people shows that the cell factor of tumors secrete can promote the S100A9 tables that STAT-3 approach is relied in myeloid precursor Up to up-regulation, so as to suppress the recruitment that myeloid precursor produces MDSC to DC differentiation.The mouse of S100A9 gene knockouts with Wild mouse is compared, and MDSC recruitment and tumor growth rate are significantly reduced.On the other hand, S100A9 in Meloid progenitor Height expression also can result in MDSC recruitment in normal mouse.These researchs show that S100A9 plays the part of in MDSC recruitment Very important role.Therefore, targeting S100A9 is also likely to be antitumor and anti-MDSC Critical policies.

Expression vector establishment of the step 1 using S100A9 as target spot

According to mouse S100A9 amino acid sequence (amino acid sequence P31725), according to e. coli codon preference Gene order is optimized, the gene after optimization is closed by Shanghai Jierui Biology Engineering Co., Ltd according to PCR synthetic methods Into, and load into pET28a carriers, obtain pET28a-S100A9 plasmids.PET28a-S100A9 plasmids using synthesis is moulds Plate, performing PCR amplification is entered using primer 3 and primer 4, and reaction system is 50 μ L, and condition is 95 DEG C of pre-degeneration 3min, 95 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 68 DEG C of extension 30s, carry out 35 circulations, last 68 DEG C of extensions 5min altogether.PCR primer carries out 1% fine jade Sepharose electrophoresis detection (120V, 30min), obtains fragment of the size between 300bp to 400bp, the base with S100A9 Because size 336bp is consistent, then the Ago-Gel residing for the band is cut off, according to Axygen gel reclaims kit standards Operation is reclaimed.The preparation of linearisation pCTB2 plasmids is carried out according to the step 1 in embodiment 1.

The S100A9 fragments that PCR is obtained, utilize the homology arm (homology arm of the pCTB2 plasmids inserted in its upstream and downstream primer It is following in primer 3 and primer 4 to draw broken line representation), it is integrated into linearization plasmid pCTB2 BamH 1 and BspE I digestions Between site.The μ L of reaction system 20, containing 2 μ L Exnase^TmII, 4 μ L 5X CE II buffer, linearisation pCTB2 plasmids 60ng (2 μ L), S100A9 fragments 120ng (6 μ L), 6 μ L ddH₂O, all components are softly mixed, 37 DEG C of incubation 30min, then It is immediately placed on 5min on ice standby.2 μ L reaction solutions are taken to be added in 50 μ L E.coli Top10 competent cells, ice bath 30min, 42 DEG C of heat shock 90s, it is immediately placed on 5min on ice and then adds 600 μ L LB non-resistant culture mediums, 37 DEG C, 180rpm shakes Recovery 1h is shaken, takes 300 μ L nutrient solutions to be coated with LB solid plates (the μ g/mL of Amp 100), 37 DEG C are inverted overnight incubation and picking Dan Ke Grand culture.Bacterium colony PCR identifications (PCR conditions, electrophoretic detection are as described above) are carried out to the monoclonal of culture, arrived in 300bp Specific bright band is may occur in which between 400bp, meets the theoretical sizes values 336bp of S100A8 genes, will appear from the list of above-mentioned band Clone send Nanjing Genscript Biotechnology Co., Ltd. to be sequenced.

So that correct CTB-S100A9 plasmids are sequenced as template, primer 5, primer 6 for primer enter performing PCR amplification, by CTB with Linker between S100A9 is mutated.The μ L of reaction system 50, condition is 95 DEG C of pre-degeneration 5min, 95 DEG C of denaturation 1min, 55 DEG C annealing 1min, 68 DEG C extension 5min, altogether carry out 35 times circulation, it is last 68 DEG C extension 10min.PCR primer carries out 1% agarose Detected through gel electrophoresis (120V, 30min), the visible specific bright band at 3800-4000bp, by the agarose residing for this bright band Gel is cut, and is reclaimed according to the standard operation of Axygen gel reclaims kits.DPN I enzymic digestion glue reclaim product (reactants It is 50 μ L：1 μ L DPN I enzymes, the μ L glue reclaim products of 5 μ L 10X NEB Buffer 4,44；37 DEG C of incubation 120min, 80 DEG C of changes Property 20min is standby).The μ L of digestive juice 2 are taken to convert 50 μ L E.coli Top10 competent cells, ice bath 30min, 42 DEG C of heat shocks 90s, be immediately placed on 5min on ice then add 600 μ L LB non-resistant culture mediums, 37 DEG C, 180rpm shaking recovery 1h, take 300 The Colony Culture grown on μ L nutrient solutions coating LB solid plates (the μ g/mL of Amp 100), picking conversion rear plate is in 3mL LB In (the μ g/mL of Amp 100) culture medium, 37 DEG C, 180rpm shaking overnight incubations.Overnight the Nanjing Jin Sirui biotechnologies will be sent to have by bacterium Limit company is sequenced, and as a result shows that the Linker between CTB and S100A9 has been increased to 20 amino acid residues, meets expection and sets Meter.This experiment each primer used is synthesized (such as following table) by Nanjing Genscript Biotechnology Co., Ltd..

Note：A lower stroke solid line represents that dotted line is drawn below Bspe I restriction enzyme sites, homology arm to be represented in primer 3, lower stroke in primer 4 Solid line represents that dotted line is drawn below BamH1 restriction enzyme sites, homology arm to be represented.

It is prepared by fusion protein of the step 2 using S100A9 as target spot

The correct μ L of CTB-S100A9 plasmids 1 of sequencing are taken, heat shock method converts 50 μ L E.Coli TG1 competent cells and (turned The step 1 of change method reference implementation example 2), take 300 μ L to be coated with LB solid plates (the μ g/mL of Amp 100), 37 DEG C of inversion overnight incubations Grown to monoclonal, picking monoclonal to 25mL LB culture mediums (the μ g/mL of Amp 100), 37 DEG C, 180rpm shaking overnight incubations (about 12h).1L M9 culture mediums (the μ g/mL of Amp 100), 28 DEG C, 200rpm shaking trainings will be forwarded to by 1: 500 (v/v) by bacterium overnight 30h is supported, 120 μ L 1mol/L IPTG and 100mL 10X TB culture mediums, 28 DEG C, 200rpm shaking cultures 70h are then added. 8000rpm, room temperature centrifugation 5min collects thalline and weighed.

10mL sample-loading buffers (500mmol/L NaCl, 20mmol/L Tris, 20mmol/ are fully resuspended in per 1g thalline L Imidazole, pH 8.0), Ultrasonic Cell Disruptor crushes re-suspension liquid (work 3s, gap 5s, power 8%, ultrasonic 30min).4 DEG C, take supernatant standby after 12000rpm centrifugations 30min.Ni posts with equilibrium liquid (500mmol/L NaCl, 20mmol/L Tris, 20mmol/L Imidazole, pH 8.0) it is washed till after baseline, supernatant is reached with 2mL/min flow velocity loading, loading peak 2000mAu, washes miscellaneous liquid (500mmol/L NaCl, 20mmol/L Tris, 80mmol/L Imidazole, pH 8.0) and rinses 20 More than individual column volume until baseline punching is flat, then with eluent (500mmol/L NaCl, 20mmol/L Tris, 500mmol/L Imidazole, pH 8.0) elution, eluting peak is collected, its eluting peak can reach 600mAu.G25 molecular sieves use 10mmol/L After PBS (pH 7.4) balances, eluting peak is crossed into G25 molecular sieves, collection flows through peak, flows through peak about 80mAu.

Take and flow through the μ L of peak 80 and dispense two pipes, be separately added into 10 μ L 5X denaturation reduction buffer solution and 5X denaturation is non-reduced slow Fliud flushing, is fully mixed, and the albumen sample for adding denaturation reduction buffer solution carries out boiling water bath 5min again, adds the non-reduced buffering of denaturation The sample of liquid is standby after then directly mixing.By after above-mentioned processing sample carry out SDS-PAGE electrophoresis detections, resolving gel concentration 12%, Voltage 120V, electrophoresis time about 80min, the preparation of protein adhesive and Running buffer, which are prepared, refers to Bio-Rad standard recipes. Electrophoresis result shows that CTB-S100A9 protein monomer about 28~32kDa, its difference in size part is expression vector signal peptide Length, similarly, it can form a variety of aggressiveness forms including pentamer in the state of denaturation is non-reduced, minimum is more Aggressiveness size about 66kDa, maximum polymer size is more than 116kDa, and software analysis CTB-S100A9 purity of protein is about 91%, meet the standard of zoopery.

Protein concentration is detected according to the standard operation of BCA quantification kits, the protein concentration for as a result showing CTB-S100A9 is： 0.6mg/mL。

The preparation of step 3 S100A9 single domain albumen

The μ L of pET28a-S100A9 plasmids 1 of synthesis are taken to add in 50 μ L E.coli BL21 (DE3) competent cells, heat Step 1 in the method for swashing conversion, operation reference implementation example 2 is carried out, and takes the μ L of conversion fluid 300 to be coated with Amp flat boards (the μ g/mL of Amp 100), Incubated overnight to monoclonal is inverted for 37 DEG C to grow.Picking monoclonal is in 3mL LB (the μ g/mL of Amp 100) culture medium, 37 DEG C, 200rpm shaking cultures about 12h.Then 1LLB culture mediums (Amp100 μ g/mL), 37 DEG C, 200rpm are forwarded to by 1: 500 (v/v) Shaking is cultivated to OD₆₀₀For 0.6, addition 1mL 1mol/L IPTG, 20 DEG C, 180rpm shaking cultures 20h.8000rpm, room temperature from Heart 5min collects thalline and weighed.

20mL sample-loading buffers (500mmol/L NaCl, 20mmol/L Tris, 50mmol/ are fully resuspended in per 1g thalline L Imidazole, pH 8.0), Ultrasonic Cell Disruptor crushes re-suspension liquid (ultrasonic 3s, interval 5s, power 1%, working time 15min).Take supernatant standby after 4 DEG C of ultrasonication liquid, 12000rpm centrifugations 30min.Ni posts are with equilibrium liquid (500mmol/L NaCl, 20mmol/L Tris, 20mmol/L Imidazole, pH 8.0) it is washed till after baseline, supernatant is with 2.5mL/min stream Fast loading, loading peak reaches 1800mAu, washes miscellaneous liquid (500mmol/L NaCl, 20mmol/L Tris, 50mmol/L Imidazole, pH 8.0) to rinse 30 column volumes flat to baseline punching, then with eluent (500mmol/L NaCl, 20mmol/ L Tris, 500mmol/L Imidazole, pH 8.0) elution, collect eluting peak, its eluting peak about 900mAu.G25 molecular sieves After being balanced using 10mmol/L PBS (pH 7.4), eluting peak is crossed into G25 molecular sieves, collection flows through peak, flows through peak about 110mAu. Take and flow through the μ L of peak 40, add 10 μ L 5X denaturation reduction buffer solutions and fully mix, then boiling water bath 5min carries out SDS-PAGE electricity Swimming detection, resolving gel concentration 12%, voltage 120V, electrophoresis time about 70min, the preparation of protein adhesive and Running buffer match somebody with somebody System refers to Bio-Rad standard recipes.Electrophoresis result shows, S100A9 albumen size about 14kDa, and software analysis its purity of protein is about For 94%, BCA quantitative displays, its protein concentration is about 1.5mg/mL, meets subsequent experimental basic demand.

The detection of step 4 animal immune and antiserum data

24 6 week old female BAl BIcs/SPF grades of C mouse are purchased from Chinese Academy of Sciences's Shanghai Experimental Animal Center, are randomly divided into 6 Group, and raised according to AAALAC guidelines.Each immune group is respectively：Alum adjuvant groups, S100A9 groups, CTB groups and CTB-S100A9 groups.50 μ of proteantigen (CTB, S100A9, CTB-S100A9) g//times, Al adjuvants 0.3mg//times, CpG 30 μ g//times, choose two points in mouse back backbone both sides after mixing and are subcutaneously injected respectively, the 14th day booster immunization Once, the 21st day mouse orbit is taken after blood, 37 DEG C of standing 2h, 4000rpm centrifugation 10min, takes supernatant serum standby.

S100A9 is diluted to concentration for 100ng/ with coating buffer solution (50mmol/L bicarbonate buffers, pH 9.6) 100 μ L, are then transferred on the orifice plate of polystyrene 96 and (add 100 μ L per hole), 4 DEG C of overnight incubations, with PBST (0.02mmol/L phosphorus Hydrochlorate, 0.15mol/L NaCl, 0.15% (v/v) Tween-20, pH 7.4) wash 6 times, wash every time 5 minutes.Add 300 μ L Confining liquid (being dissolved in PBST) containing 5% (m/v) skimmed milk power, 37 DEG C of incubations are closed for 2 hours.PBST is washed 6 times, and 5 are washed every time Minute.Then the antiserum of each immune group is diluted 100 times with above confining liquid, 100 μ L is added per hole, 4 DEG C of overnight incubations are used PBST is washed 6 times, is washed every time 5 minutes.Adding 100 μ L rabbit-anti mouse-IgG- horseradish peroxidases cross-linking agents, (confining liquid 1: 5000 is dilute Release), 37 DEG C are incubated 1 hour, are washed 12 times, washed every time 5 minutes with PBST.It is eventually adding 100 μ L substrate solution (10mL substrate solutions Tetramethyl benzidine containing 1mg (TMB), 0.0969g sodium citrates, 0.3496 g Na₂HPO₄·12H₂O, 32 μ L 0.75%H₂O₂) 37 DEG C are incubated 15 minutes.Then 50 μ L 2mol/L H are added₂SO₄Terminating reaction.On ELIASA to each hole survey 450nm and Value at 630nm, calculates OD₄₅₀-OD₆₃₀。

Measurement result is shown：CTB-S100A9 immune groups antiserum is directed to S100A9 antibody OD₄₅₀-OD₆₃₀Value is respectively： 0.90、0.51、0.72、0.69、0.59、1.40.The value of CTB immune groups is 0.54,0.35,0.46,0.56,0.43,0.55. The OD of S100A9 immune groups₄₅₀-OD₆₃₀It is worth for 0.28,0.21,0.16,0.84,1.10,0.99.Divided by one-way ANOVA Analysis, as a result shows CTB-S100A9 immune groups compared with CTB immune groups and S100A9 immune groups, and its antiserum is for S100A9's OD₄₅₀-OD₆₃₀Value is higher, but statistically simultaneously there was no significant difference.CTB-S100A9 immune groups compared with Alum adjuvant groups then Significant difference (P ＜ 0.01).

Each immune group takes OD above₄₅₀-OD₆₃₀The pattern detection antibody titer that reading is worth between.S100A9 is diluted It is 100ng/100 μ L to concentration, is then transferred on the orifice plate of polystyrene 96 and (adds 100 μ L per hole), 4 DEG C of overnight incubations, washing, Closing is operated as more than.The antiserum following multiple of confining liquid doubling dilution：100x2⁰, 100x2¹..., 100x 2⁹, 100x 2¹⁰, the μ L of coating 100,4 DEG C of overnight incubations per hole.Secondary antibody is incubated, and washing and chromogenic reaction are operated as above, using double-wavelength method in enzyme Mark and data OD is read on instrument₄₅₀And OD₆₃₀, according to OD₄₅₀-OD₆₃₀Reading calculates each group antibody titer.As a result CTB- is shown S100A9, CTB and S100A9 immune group are 1600 for S100A9 antibody titer.

Structure and the antitumous effect research of step 5 tumor model

4T1 mouse mastopathy cells are purchased from Chinese science icm cell storehouse, and 5% is based on using RPMI-1640 cultures CO₂Cultivated in 37 DEG C of constant incubators.When cell fusion degree reaches 90%, digested through 0.25% pancreatin, collect cell in nothing In the nutrient solution of serum, gently shake, 1000rpm, centrifuge 5min, washed once, 10mmol/L PBS (pH 7.4) are resuspended, adjust Whole cell concentration is 10⁶.Counted through Trypan Blue, and with blood counting chamber, detection live cell fraction is more than 95%.

Mouse immune 2 times was simultaneously taken a blood sample at the 21st day, was inoculated with 3 × 10 in mouse oxter within the 25th day⁵Individual 4T1 tumour cells, and In the 28th day booster immunization once, dosage such as step 4.Go out after knurl every 3 days with vernier caliper measurement length of tumor and width.49th My god, put to death mouse and take tumour and blood sample, use electronic balance weighing tumor weight.According to formula：Inhibiting rate=(control Group average tumor weight-empirical average group tumor weight)/control group average tumor weight * 100% calculating inhibiting rates.All systems Count and statistical analysis is carried out using Graphpad Prism5.0 softwares, and chart.This experiment is using one-way ANOVA Inspection is analyzed the data significance of difference, and " * " represents p ＜ 0.05, and " * * " represent p ＜ 0.01, and " * * * " represent p ＜ 0.001.Measurement result as shown in fig. 1b, CTB-S100A9 immune group tumor weights be respectively 1.84g, 3.10g, 2.12g, 0.91g.The tumor weight of CTB immune groups is respectively 3.54g, 5.16g, 3.97g, 3.51g.The tumor weight of S100A9 immune groups For 4.31g, 2.80g.CTB-S100A9 tumor weight has significant difference (P ＜ 0.01), the suppression of its tumour compared with CTB groups Rate processed is 51%.CTB-S100A9 immune groups are compared with S100A9 single domain immune groups, though its tumor weight is statistically There was no significant difference, but tumor control rate still reaches 44%.Result above shows that CTB-S100A9 has preferable antitumor work Property.

Influences of step 6 CTB-S100A9 to the MDSC of tumor inducing

The 24th day after inoculated tumour, the disconnected neck of mouse is put to death, and alcohol-pickled 5 minutes, left abdomen skin is opened in super-clean bench Skin, careful separation hypodermis and abdominal muscles expose spleen, carefully lift spleen, cut off surrounding connective tissue, are put into Sheng In the capsule for having 10mmol/L PBS (pH 7.4), aseptic nipper is caught broken, and the cell suspension of acquisition is transferred into cell sieve filtering Remnant tissue is removed, the suspension 1500rpm centrifugation 5min after filtering abandon supernatant, add 2mL 10mmol/L PBS (pH 7.4) resuspended to wash twice, 1500rpm centrifugation 5min abandon supernatant, obtain the spleen cell of mouse, peripheral blood is then directly centrifuged. The spleen cell suspension and peripheral blood cells of acquisition are added to the erythrocyte cracked liquid of 3-5 times of cell volume, gently piping and druming is mixed, Lysis at room temperature 2min.4 DEG C, 2000rpm centrifugation 5min abandon supernatant, repeated the above steps once.Cell precipitation after centrifugation is added 3mL 10mmol/L PBS (pH 7.4) are resuspended, 2000rpm centrifugation 3min, abandon supernatant, are repeated 1 times.

The above-mentioned cell prepared is resuspended with streaming buffer solution, 100 μ L cell suspensions are added in every streaming detection pipe, Often manage and add 1 μ L Fc γ R III/II antibody, soft to mix and 10min is incubated on ice, then often pipe addition 2mL is above-mentioned PBS is washed twice, 1500rpm centrifugation 5min, abandons supernatant.Streaming buffer solution re-suspended cell, is added in each flow cytometer detection pipe Each 1 μ L of following fluorescent labeled antibody CD11b-FITC, Gr-1-APC, F4/80, soft to mix, then each pipe lucifuge is incubated 30min, the often pipe addition above-mentioned PBS of 2mL are washed twice, 1500rpm centrifugation 5min, abandon supernatant.Finally often pipe adds 500 μ L streamings Buffer solution, abundant re-suspended cell.Detected with flow cytometer.

Testing result result is shown：After CTB-S100A9 group immune mouses, G-MDSC (originate by granulocyte in mouse peripheral blood MDSC) content be about 48.1%, 20.4%, 22.0%；M-MDSC (MDSC of cells of monocytic origin) content is about 16.4%th, 43.0%, 37.9%, 34.2%.In CTB immune groups, in mouse peripheral blood G-MDSC content be about 44.2%, 70.9%th, 48.4%；M-MDSC content is about 13.5%, 43.6%, 19.3%, 37.0%.In S100A9 immune groups, mouse G-MDSC content is about 18.3%, 17.3%, 70.3% in peripheral blood；M-MDSC content is about 5.0%, 1.9%, 21.9%.For the mouse in Alum adjuvant groups, G-MDSC content is about 66.7%, 77.1%, 68.7% in its peripheral blood； M-MDSC content is about 24.8%, 11.5%, 22.7%.To the analysis shows of result above：Compared with Alum adjuvant groups, After S100A9 single domain protein immunization mouse, the G-MDSC (P ＜ 0.05) in its peripheral blood through tumor inducing can be significantly inhibited With M-MDSC (P ＜ 0.05), CTB-S100A9 can also significantly inhibit the G-MDSC (P ＜ 0.05) in peripheral blood, but to M-MDSC Do not make significant difference (P ＞ 0.05).

After CTB-S100A9 immune mouses, the content of total MDSC in mouse spleen account for cell total amount 50.6%, 58.0%th, 48.6%.Without significant difference (P ＞ 0.05) compared with CTB, S100A9 and Alum adjuvant group.

Analyze, after S100A9 single domain immune mouses, can significantly inhibit in its peripheral blood through tumour based on the above results The G-MDSC (P ＜ 0.05) and M-MDSC (P ＜ 0.05), CTB-S100A9 of induction are more likely to suppress the G-MDSC in peripheral blood (P ＜ 0.05), rather than M-MDSC.

Influences of step 7 CTB-S100A9 to the Treg of tumor inducing

The spleen and peripheral blood cells of 4T1 tumor-bearing mices are separated with reference to step 6, with streaming buffer solution re-suspended cell.Every 100 μ L cell suspensions are added in flow cytometer detection pipe, often manages and adds 1 μ L Fc γ R III/II antibody, it is soft to mix and on ice 10min is incubated, then the often pipe addition above-mentioned PBS of 2mL are washed twice, 1500rpm centrifugation 5min abandon supernatant.In the inspection of each streaming Each 1 μ L of fluorescent labeled antibody CD4-FITC, CD8-FITC are added in test tube, each pipe lucifuge is incubated 30min, washing two repeated as above It is secondary.Then often pipe adds 1mL fixer, mixes, and lucifuge is incubated 50min, and 2000rpm centrifugation 5min, each pipe adds the logical of 2mL Saturating buffer solution, is mixed, 2000rpm centrifugation 10min, removes supernatant.1 μ L Foxp3-PE are added in pipe, lucifuge is incubated 30min on ice. Then the often pipe addition above-mentioned PBS of 2mL are washed twice, and abandon supernatant.It is eventually adding 500 μ L streaming buffer solutions, abundant re-suspended cell.On Machine testing.

The result of streaming shows：After CTB-S100A9 immune mouses, Treg cells account for TCS in mouse peripheral blood Ratio is 0.4%, 0.4%, 0.7%.The respective value of CTB groups is 1.8%, 1.4%, 1.2%.The respective value of S100A9 groups is 0.7%th, 0.4%, 1.3%.The respective value of Alum adjuvant groups is 2.4%, 1.7%, 1.3%.Analysis shows S100A9 single structures Domain albumen (P ＜ 0.05) and CTB-S100A9 albumen (P ＜ 0.01) can be significantly inhibited in mouse peripheral blood through tumor inducing Treg cell quantities.For the ratio of Treg cells in mouse spleen, CTB-S100A9 groups are 0.2%, 0.3%, 0.2%. S100A9 groups are 0.4%, 0.3%.Without significant difference (P ＞ 0.05) compared with CTB and Alum adjuvant groups.

Analyzed based on the above results, S100A9 single domains albumen and CTB-S100A9 albumen can also be by notable Suppress the Treg cells through tumor inducing in mouse peripheral blood, so as to play its antitumous effect.

Double target spot vaccine constructions of the embodiment 3 based on S100A8 and MUC1

MUC1 (Mucin 1, MUC1) is the single chain polypeptide transmembrane glycoprotein of a huge high glycosylation.A variety of In tumour, such as breast cancer, cancer of pancreas, lung cancer, MUC1 shows significant high expression, and with serious prognosis mala. Meanwhile, the MUC1 glycosylations of tumor cell surface are incomplete, cause it to expose epitope hidden under normal circumstances.MUC1VNTR The PDTRP epitopes of area's core peptide are not limited by MHC, can be recognized by a variety of MUC1 antibody, by CTL cell recognitions and can also be killed Wound.Therefore, MUC1 is a kind of good anti-tumor target.Up to now, a variety of various forms of MUC1 vaccines are proved to MUC1 specificity antineoplastics cell and humoral immune response can be effectively induced on MUC1+ tumor-bearing mice, but clinical effectiveness is equal Not good, this is probably caused by being suppressed by tumour immunity.The suppression mechanism for having many inhibits tumor immune response, such as inhibition Receptor-ligand pair, immuno-suppressing cytokine, and tumour immunity suppress cell MDSC, Treg and TAM etc..Nearest research Show, block the signal transduction pathway of PD-1 and CTLA-4 mediations using monoclonal antibody is double and combine tumor cell vaccine and can have Effect recovers the function of tumour-specific CD8+T cells, strengthens the antitumor curative effect of vaccine.This shows to target inhibitive ability of immunity acceptor Or part can strengthen the immune response of tumor related antigen, and produce antitumous effect.But reduce by swelling that MDSC is mediated The immune response whether knurl immunosupress can strengthen tumor related antigen is also to be verified.A kind of target is devised in the present embodiment To MUC1 and S100A8 double target spot vaccines, the vaccine is expected to while suppressing the MDSC of tumor inducing, and strengthen MUC1 specificity Immune response.

The structure of 1 CTB-MUC1-S100A8 expression vectors

Using above-mentioned pCTB2 as template, CTB Q56-D59 amino acids are replaced with Muc1 sequences by primer 7, primer 8 for primer Row (MUC1 gene orders are following in primer 7 and primer 8 to draw double horizontal line marks), obtain recombinant C TB-Muc1 plasmids.Reactant It is 50 μ L, condition is 95 DEG C of pre-degenerations 5min, 95 DEG C of denaturation 1min, 55 DEG C of annealing 1min, 68 DEG C of extension 5min, is carried out 35 times altogether Circulation, last 68 DEG C of extensions 10min.PCR primer carries out 1% agarose gel electrophoresis detection (120 V, 35min), as a result shows In 3000bp to occurring specific bright band between 3500bp, the Ago-Gel residing for bright band is cut off, it is solidifying according to Axygen The operation of glue reclaim kit standard is reclaimed.DPN I enzymic digestion glue reclaim products, the μ L of reaction system 50, including 1 μ L DPN I enzymes, 5 The μ L glue reclaim products of μ L 10x NEB Buffer 4,34,10 μ L ddH₂O；37 DEG C of incubations 120min, 80 DEG C of denaturation 20min are standby With.The μ L of digestive juice 2 are taken, are added in 50 μ L E.coli Top10 competent cells, ice bath 30min, 42 DEG C of heat shock 90s, immediately It is placed in 5min on ice and then adds 600 μ L LB non-resistant culture mediums, 37 DEG C, 180rpm shaking recovery 1h takes 300 μ L to be coated with LB Solid plate (the μ g/mL of Amp 100), is inverted culture to monoclonal for 37 DEG C and grows.Picking Colony Culture is in 3mL LB (Amp 100 μ g/mL) in culture medium, 37 DEG C, 180rpm shaking overnight incubations.It will send Nanjing Jin Sirui biotechnologies limited public affairs by bacterium overnight Department's sequencing, as a result shows that MUC1 sequences have successfully replaced CTB Q56-D59 amino acids.

Correct CTB-MUC1 plasmids will be sequenced, will be linearized with BamH I and BspE I restriction endonucleases, the μ of reaction system 50 L, condition is 37 DEG C, 4h.Digestion products carry out 1% agarose gel electrophoresis detection (120V, 35min), as a result show linearisation CTB-MUC1 plasmid sizes be about 4000bp-4300bp, meet expection, then cut the Ago-Gel residing for this fragment Under, reclaimed according to the standard operation of Axygen gel reclaims kits.PET28a-S100A8 plasmids using synthesis are utilized as template Primer 1 and primer 2 enter performing PCR amplification, and reaction system and condition are identical with step 1 in embodiment 1.

The S100A8 fragments that PCR is obtained, the homology arm using the CTB-MUC1 plasmids inserted in its upstream and downstream primer is (homologous Arm is following in primer 1 and primer 2 to draw broken line representation), it is integrated into linearization plasmid CTB-MUC1 BamH 1 and BspE I restriction enzyme sites.The μ L of reaction system 20, containing 2 μ L Exnase^TmII, 4 μ L 5X CE II buffer, linearisation CTB-MUC1 matter Grain 50ng (2 μ L), S100A8 fragments 100ng (5 μ L), 7 μ L ddH₂O, all components are softly mixed, 37 DEG C of incubation 30min, so After to be immediately placed on 5min on ice standby.2 μ L reaction solutions are taken to be added in 50 μ L E.coli Top10 competent cells, ice bath 30min, 42 DEG C of heat shock 90s, it is immediately placed on 5min on ice and then adds 600 μ L LB non-resistant culture mediums, 37 DEG C, 180rpm shakes Recovery 1h is shaken, takes 300 μ L to be coated with LB solid plates (the μ g/mL of Amp 100), 37 DEG C are inverted overnight incubation and the training of picking monoclonal Support.Bacterium colony PCR identifications (PCR conditions, electrophoretic detection are as previously described) are carried out to the monoclonal of culture, in 200bp to 300bp Between may occur in which specific bright band, meet the theoretical sizes values 267bp of S100A8 genes, the monoclonal that will appear from above-mentioned band is sent Nanjing Genscript Biotechnology Co., Ltd. is sequenced.

So that correct CTB-MUC1-S100A8 plasmids are sequenced as template, primer 5, primer 6 enters performing PCR amplification for primer, will Linker between CTB-MUC1 and S100A8 is mutated.The μ L of PCR reaction systems 50, condition is 95 DEG C of pre-degeneration 5min, 95 DEG C denaturation 1min, 55 DEG C annealing 1min, 68 DEG C extension 5min, altogether carry out 35 times circulation, it is last 68 DEG C extension 10min.PCR primer 1% agarose gel electrophoresis detection (120V, 30min) is carried out, visible specific bright band, bright by this at 3800-4100bp Cut, and reclaimed according to the standard operation of Axygen gel reclaims kits with residing Ago-Gel.DPN I enzymic digestions glue is returned Receive product (the μ L DPN I enzymes of 50 μ of reaction system L: 1,5 μ L 10X NEB Buffer4,36 μ L glue reclaim products, 8 μ L ddH₂O； 37 DEG C of incubations 120min, 80 DEG C of denaturation 20min are standby).The μ L of digestive juice 2 are taken to convert 50 μ L E.coli Top10 competent cells, Reaction system and condition are as it was previously stated, the Colony Culture grown on picking conversion rear plate is in 3mL LB (the μ g/mL of Amp 100) In culture medium, 37 DEG C, 180rpm shaking overnight incubations.Jun Song Nanjing Genscript Biotechnology Co., Ltd. it will be sequenced overnight, knot Fruit shows that the Linker between CTB-MUC1 and S100A8 has been increased to 20 amino acid residues, meets desired design.This experiment Each primer used is synthesized (such as following table) by Nanjing Genscript Biotechnology Co., Ltd..

Note：A lower stroke solid line represents that dotted line is drawn below BspE I restriction enzyme sites, homology arm to be represented in primer 1, lower stroke in primer 2 Solid line represents that dotted line is drawn below BamH1 restriction enzyme sites, homology arm to be represented.Lower stroke of double solid line in primer 7 and primer 8 is insertion The gene order in MUC1VNTR areas.

The preparation of 2 CTB-MUC1-S100A8 fusion proteins

The correct μ L of CTB-MUC1-S100A8 plasmids 1 of sequencing are taken, it is thin that heat shock method converts 50 μ L E.Coli TG1 competence Born of the same parents' (step 1 of method for transformation reference implementation example 1), take 300 μ L to be coated with LB solid plates (the μ g/mL of Amp 100), 37 DEG C are inverted training Support and grown overnight to monoclonal, picking monoclonal to 25mLLB culture mediums (the μ g/mL of Amp 100), 37 DEG C, 180rpm shaking cultures Overnight.1L M9 culture mediums (the μ g/mL of Amp 100), 28 DEG C, 200rpm shaking cultures will be forwarded to by 1: 500 (v/v) by bacterium overnight 30h, then adds 150 μ L 1mol/L IPTG and 100mL 10X TB culture mediums, 28 DEG C, 200rpm shaking cultures 70h. 8000rpm, room temperature centrifugation 5min collects thalline and weighed.

10mL sample-loading buffers (500mmol/L NaCl, 20mmol/L Tris, 50mmol/ are fully resuspended in per 1g thalline L Imidazole, pH 8.0), Ultrasonic Cell Disruptor crushes re-suspension liquid (work 3s, gap 5s, power 8%, ultrasonic 30min).4 DEG C, take supernatant standby after 12000rpm centrifugations 30min.Ni posts with equilibrium liquid (500mmol/LNaCl, 20mmol/L Tris, 50mmol/L Imidazole, PH 8.0) it is washed till after baseline, supernatant is reached with 2mL/min flow velocity loading, loading peak 2100mAu, washes miscellaneous liquid (500mmol/L NaCl, 20mmol/L Tris, 80mmol/L Imidazole, pH 8.0) and rinses 25 Individual column volume is flat to baseline punching, then with eluent (500mmol/L NaCl, 20mmol/L Tris, 500mmol/L Imidazole, pH 8.0) elution, eluting peak is collected, its eluting peak can reach 400mAu.G25 molecular sieves use 10mmol/L After PBS (pH 7.4) balances, eluting peak is crossed into G25 molecular sieves, collection flows through peak, flows through peak about 30mAu.

Take and flow through the μ L of peak 80 and dispense two pipes, be separately added into 10 μ L 5X denaturation reduction buffer solution and 5X denaturation is non-reduced slow Fliud flushing, is fully mixed, and the albumen sample for adding denaturation reduction buffer solution carries out boiling water bath 5min again, adds the non-reduced buffering of denaturation The sample of liquid is standby after then directly mixing.By after above-mentioned processing sample carry out SDS-PAGE electrophoresis detections, resolving gel concentration 12%, Voltage 100V, electrophoresis time about 80min, the preparation of protein adhesive and Running buffer, which are prepared, refers to Bio-Rad standard recipes. Electrophoresis result shows, CTB-MUC1-S100A8 protein monomers about 25-30kDa, be denatured it is non-reducing under the conditions of can form five and gather The variforms such as body, its difference in size part is the length of expression vector signal peptide.Pass through gel imaging system embedded software point Its purity of protein about 98% is analysed, meets the standard for carrying out zoopery.It is dense according to BCA quantification kits standard operation detection albumen Degree, it is 1.4mg/mL as a result to show its protein concentration.

Double target spot vaccine constructions of the embodiment 4 based on S100A9 and MUC1

As described in example 3 above, the present embodiment constructs a kind of while targetting S100A9 and MUC1 double target spot vaccines, should Vaccine is expected to can be while suppress the MDSC of tumor inducing, and strengthens the specific immune responses of MUC1.

The structure of 1 CTB-MUC1-S100A9 expression vectors

Using pCTB2 as template, CTB Q56-D59 amino acids are replaced with Muc1 sequences by primer 7, primer 8 for primer, Obtain recombinant C TB-Muc1 plasmids.The μ L of reaction system 50, condition is 95 DEG C of pre-degeneration 5min, 95 DEG C of denaturation 1min, 55 DEG C of annealing 1min, 68 DEG C of extension 5min, carry out 35 circulations, last 68 DEG C of extensions 10min altogether.PCR primer carries out 1% Ago-Gel electricity Swimming detection (120V, 35min), is as a result shown in 3000bp to occurring specific bright band between 3500bp, by residing for bright band Ago-Gel is cut off, and is reclaimed according to the standard operation of Axygen gel reclaims kits.DPN I enzymic digestion glue reclaim products, instead Answer the μ L of system 50, including 1 μ L DPN I enzymes, 5 μ L 10x NEB Buffer4,34 μ L glue reclaim products, 10 μ L ddH₂O；37℃ 120min is incubated, 80 DEG C of denaturation 20min are standby.The μ L of digestive juice 2 are taken, are added in 50 μ L E.coli Top10 competent cells, Ice bath 30min, 42 DEG C of heat shock 90s, it is immediately placed on 5min on ice and then adds 600 μ L LB non-resistant culture mediums, 37 DEG C, 180rpm shakes recovery 1h, takes 300 μ L to be coated with LB solid plates (the μ g/mL of Amp 100), is inverted for 37 DEG C and cultivates long to monoclonal Go out.Picking Colony Culture is in 3mL LB (the μ g/mL of Amp 100) culture medium, 37 DEG C, 180rpm shaking overnight incubations.Incited somebody to action Ye Junsong Nanjing Genscript Biotechnology Co., Ltd. is sequenced, and as a result shows that MUC1 sequences have successfully replaced CTB Q56-D59 positions Amino acid.

Correct CTB-MUC1 plasmids will be sequenced, will be linearized with BamH I and BspE I restriction endonucleases, the μ of reaction system 50 L, condition is 37 DEG C, 4h.Digestion products carry out 1% agarose gel electrophoresis detection (120V, 35min), as a result show linearisation CTB-MUC1 plasmid sizes be about 4000bp-4300bp, meet expection, then cut the Ago-Gel residing for this fragment Under, reclaimed according to the standard operation of Axygen gel reclaims kits.PET28a-S100A9 plasmids using synthesis are utilized as template Primer 3 and primer 4 enter performing PCR amplification to obtain S100A9 fragments, and reaction system and condition are identical with step 1 in embodiment 2.

The S100A9 fragments that PCR is obtained, utilize the homology arm (homology arm of the pCTB2 plasmids inserted in its upstream and downstream primer It is following in primer 3 and primer 4 to draw broken line representation), it is integrated into linearization plasmid pCTB2 BamH 1 and BspE I enzymes Between enzyme site.The μ L of reaction system 20, containing 2 μ L Exnase^TmII, 4 μ L 5X CE II buffer, linearisation CTB-MUC1 matter Grain 50ng (2 μ L), S100A9 fragments 120ng (6 μ L), 6 μ L ddH₂O, all components are softly mixed, 37 DEG C of incubation 30min, so After to be immediately placed on 5min on ice standby.2 μ L reaction solutions are taken to be added in 50 μ L E.coli Top10 competent cells, ice bath 30min, 42 DEG C of heat shock 90s, it is immediately placed on 5min on ice and then adds 600 μ L LB non-resistant culture mediums, 37 DEG C, 180rpm shakes Recovery 1h is shaken, takes 300 μ L to be coated with LB solid plates (the μ g/mL of Amp 100), 37 DEG C are inverted overnight incubation and the training of picking monoclonal Support.Bacterium colony PCR identifications (PCR conditions, electrophoretic detection are as previously described) are carried out to the monoclonal of culture, in 300bp to 400bp Between may occur in which specific bright band, meet the theoretical sizes values 336bp of S100A9 genes, the monoclonal that will appear from above-mentioned band is sent Nanjing Genscript Biotechnology Co., Ltd. is sequenced.

So that correct CTB-MUC1-S100A9 plasmids are sequenced as template, primer 5, primer 6 enters performing PCR amplification for primer, will Linker between CTB-MUC1 and S100A9 is mutated.The μ L of PCR reaction systems 50, condition is 95 DEG C of pre-degeneration 5min, 95 DEG C denaturation 1min, 55 DEG C annealing 1min, 68 DEG C extension 5min, altogether carry out 35 times circulation, it is last 68 DEG C extension 10min.PCR primer 1% agarose gel electrophoresis detection (120V, 30min) is carried out, visible specific bright band, bright by this at 3800-4100bp Cut, and reclaimed according to the standard operation of Axygen gel reclaims kits with residing Ago-Gel.DPNI enzymic digestions glue is returned Receive product (the μ L DPNI enzymes of 50 μ of reaction system L: 1,5 μ L 10X NEB Buffer4,34 μ L glue reclaim products, 10 μ L ddH₂O； 37 DEG C of incubations 120min, 80 DEG C of denaturation 20min are standby).The μ L of digestive juice 2 are taken to convert 50 μ L E.coli Top10 competent cells, Reaction system and condition are as it was previously stated, the Colony Culture grown on picking conversion rear plate is in 3mL LB (the μ g/mL of Amp 100) In culture medium, 37 DEG C, 180rpm shaking overnight incubations.Jun Song Nanjing Genscript Biotechnology Co., Ltd. it will be sequenced overnight, knot Fruit shows that the Linker between CTB-MUC1 and S100A9 has been increased to 20 amino acid residues, meets desired design.This experiment Each primer used is synthesized (such as following table) by Nanjing Genscript Biotechnology Co., Ltd..

Note：A lower stroke solid line represents that dotted line is drawn below Bspe I restriction enzyme sites, homology arm to be represented in primer 3, lower stroke in primer 4 Solid line represents that dotted line is drawn below BamH1 restriction enzyme sites, homology arm to be represented.Lower stroke of double solid line in primer 7 and primer 8 is insertion The gene order in MUC1VNTR areas.

The preparation of 2 CTB-MUC1-S100A9 fusion proteins

The correct μ L of CTB-MUC1-S100A9 plasmids 1 of sequencing are taken, it is thin that heat shock method converts 50 μ L E.Coli TG1 competence Born of the same parents' (step 1 of method for transformation reference implementation example 4), take 300 μ L to be coated with LB solid plates (the μ g/mL of Amp 100), 37 DEG C are inverted training Support and grown overnight to monoclonal, picking monoclonal to 25mL LB culture mediums (the μ g/mL of Amp 100), 37 DEG C, 180rpm shaking trainings Support overnight.1LM9 culture mediums (the μ g/mL of Amp 100), 28 DEG C, 200rpm shaking cultures will be forwarded to by 1: 500 (v/v) by bacterium overnight 30h, then adds 150 μ L 1mol/L IPTG and 100mL 10X TB culture mediums, 28 DEG C, 200rpm shaking cultures 70h. 8000rpm, room temperature centrifugation 5min collects thalline and weighed.

10mL sample-loading buffers (500mmol/L NaCl, 20mmol/L Tris, 50mmol/ are fully resuspended in per 1g thalline L Imidazole, pH 8.0), Ultrasonic Cell Disruptor crushes re-suspension liquid (work 3s, gap 5s, power 8%, ultrasonic 30min).4 DEG C, take supernatant standby after 12000rpm centrifugations 30min.Ni posts with equilibrium liquid (500mmol/L NaCl, 20mmol/L Tris, 50mmol/L Imidazole, pH 8.0) it is washed till after baseline, supernatant is reached with 2mL/min flow velocity loading, loading peak 2000mAu, washes miscellaneous liquid (500mmol/L NaCl, 20mmol/L Tris, 80mmol/L Imidazole, pH 8.0) and rinses 25 Individual column volume is flat to baseline punching, then with eluent (500mmol/L NaCl, 20mmol/L Tris, 500mmol/L Imidazole, pH 8.0) elution, eluting peak is collected, its eluting peak can reach 500mAu.G25 molecular sieves use 10mmol/L After PBS (pH 7.4) balances, eluting peak is crossed into G25 molecular sieves, collection flows through peak, flows through peak about 40mAu.

Take and flow through the μ L of peak 40, add 10 μ L 5X denaturation reduction buffer solutions, fully mix and boiling water bath 5min, then carry out SDS-PAGE electrophoresis detections, resolving gel concentration 12%, voltage 120V, electrophoresis time about 80min, the preparation of protein adhesive and Running buffer, which are prepared, refers to Bio-Rad standard recipes.Electrophoresis result shows that CTB-MUC1-S100A9 protein monomers are about 25-30kDa, its purity of protein about 91% is analyzed by gel imaging system embedded software, meets the standard for carrying out zoopery. Protein concentration is detected according to the standard operation of BCA quantification kits, it is 0.6mg/mL as a result to show its protein concentration.

It should be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can be to present invention work Various changes or modification, these equivalent form of values equally fall within the application appended claims limited range.

Claims (10)

1. a kind of recombinant protein for including S100A8 and/or S100A9 domains, the recombinant protein also optionally includes carrier The domain of albumen.

2. recombinant protein as claimed in claim 1, it is characterised in that described carrier protein is pathogen protein, including disease Toxalbumin, bacterioprotein, I (chlamydia) protein, mycoplasma albumen, non-human animal's albumen or its combination.

3. a kind of polynucleotides, the recombinant protein described in described polynucleotide encoding claim 1.

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

5. a kind of host cell, described host cell contains the expression vector described in claim 4, or in genome it is whole Close the polynucleotides having the right described in requirement 3.

6. a kind of pharmaceutical composition, described composition contains the recombinant protein described in claim 1, described in claim 3 The host cell described in expression vector or claim 5 described in polynucleotides or claim 4, with and/or pharmaceutically Acceptable carrier and/or auxiliary material.

7. a kind of vaccine combination, described composition contains the recombinant protein described in claim 1 or 2, described in claim 3 Polynucleotides or claim 4 described in expression vector or the host cell described in claim 5, and in immunology Acceptable carrier and/or auxiliary material.

8. recombinant protein as claimed in claim 1 is for preparing in the antibody to S100A8 and/or S100A9 domains Purposes.

9. recombinant protein as claimed in claim 1 is in the medicine for preparing the disease related to S100A8 and/or S100A9 In purposes.

10. a kind of albumen for S100A8 and/or S100A9 antigenic domains, the antigenic domains albumen is dynamic from lactation S100A8 the and/or S100A9 albumen of thing, and the antigenic domains albumen and the restructuring egg of carrier protein of the present invention formation The same kind of mammal can be induced in vain produces the immune response for being directed to the low immunogenicity albumen.