CN104693293A - B cell immunodominance epitope peptide of staphylococcus aureus enterotoxin B and preparation method and application thereof - Google Patents

B cell immunodominance epitope peptide of staphylococcus aureus enterotoxin B and preparation method and application thereof Download PDF

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CN104693293A
CN104693293A CN201510143971.6A CN201510143971A CN104693293A CN 104693293 A CN104693293 A CN 104693293A CN 201510143971 A CN201510143971 A CN 201510143971A CN 104693293 A CN104693293 A CN 104693293A
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seb
peptide
epitope peptide
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吴超
赵�卓
邹全明
李滨
孙合强
陈立
章金勇
魏姗姗
胡健
曾浩
王逸麟
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Third Military Medical University TMMU
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Abstract

The invention relates to a B cell immunodominance epitope peptide of staphylococcus aureus enterotoxin B and a preparation method and application thereof, and particularly provides the B cell immunodominance epitope peptide of the staphylococcus aureus enterotoxin. The amino acid sequence of the B cell immunodominance epitope peptide is shown in SEQ ID NO:42, and the amino acid sequence of core epitope corresponding to the B cell immunodominance epitope peptide is shown in SEQ ID NO:69. According to the B cell immunodominance epitope peptide of the staphylococcus aureus enterotoxin B and the preparation method and application thereof, an overlapping peptide combined titrimetric method is utilized to identify the B cell immunodominance epitope peptide of the staphylococcus aureus enterotoxin B, the screening method is simple, convenient, rapid and accurate, and no omission exists. The immunodominance epitope peptide does not contain unnecessary or even harmful parts, so that using risks of the prepared vaccine is reduced, a larger vaccine application value is achieved, and the B cell immunodominance epitope peptide of the staphylococcus aureus enterotoxin B and the preparation method and application thereof can be used for preparing the epitope vaccine or the preventive vaccine of the staphylococcus aureus enterotoxin B.

Description

B cell immunodominant epitope peptide of SEB and its preparation method and application
Technical field
The present invention relates to field of biological pharmacy, particularly relate to from three B cell immunodominant epitopes of SEB, the method identifying this B cell immunodominant epitope and its application in technical field of pharmaceutical biotechnology.
Background technology
Streptococcus aureus (Staphylococcus aureus, S.aureus), as the representative of gram-positive microorganism, is the pathogenic coccus that a class extensively exists at nature, is also a kind of important pathogenic bacteria causing hospital infection and Nosocomial Infections.Investigation shows, in the U.S., skin and soft tissue infection modal pathogenic bacteria in community's is S.aureus (accounting for 75%); In Japan, in pustulosis S.aureus cause epidermolysis useless fellow disease account for 92%; In Africa, in myositis purulenta tropica pathogenic agent, S.aureus accounts for 55% ~ 72%; In China, the No.1 pathogenic bacterium of infective endocarditis are S.aureus (accounting for 31% ~ 34%).In addition, S.aureus infects with acute, suppurative for feature, and local can cause the pyogenic infection of skin and soft tissue etc., does not prolongedly heal; Whole body can cause severe infections and the complication such as osteomyelitis, septic arthritis, endocarditis, pneumonia, pyemia, and mortality ratio is up to 20%.Meanwhile, the extracellular toxin of S. aureus L-forms also can cause the whole body lethal infections such as food poisoning, scalded skin syndrome and toxic shock syndrome.
Streptococcus aureus nutritional requirement is not high, well-grown on ordinary culture medium, grows better in the substratum containing blood and glucose, aerobic or amphimicrobian.28 DEG C ~ 38 DEG C all can grow, and optimum temperuture is 37 DEG C, and pH is 4.8 ~ 9.4, and the suitableeest is 7.4.Salt tolerance is strong, all can grow containing in 10% ~ 15% sodium-chlor substratum.The bacterium colony that blood fat flat board is formed is comparatively large, and periphery of bacterial colonies forms obvious all-transparent zone of hemolysis (beta hemolysis), and hemolytic strain has more pathogenic.According to bacteriophage typing, streptococcus aureus can be divided into 5 groups of 26 types.Bacteriophage typing follows the trail of contagium when epidemiology survey and the pass between research bacterial type and kinds of Diseases is fastened all significant.Enterotoxin type food poisoning is caused by III and IV group of streptococcus aureus, the II group of bacterium velocity ratio I of resistance is produced to microbiotic and IV group slowly a lot.What cause hospital infection severe epidemic is 52 in I group, 52A, 80 and 81 type bacterial strains.Causing herpetic is often II group of 71 type with bacterial strain that is exfoliative dermatitis.
Streptococcus aureus Heat stable Enterotoxin (Staphylococcal enterotoxin; SE); be cause that human foods is poisoning, the major cause of systemic inflammatory response and septic shock, be also that S. aureus vaccines studies one of most important protective antigen.SE is divided into the multiple-types such as SEA, SEB and SEC because of serotype difference SE, and the clinical MRSA strain isolated of every strain can secrete 1-2 kind enterotoxin, and wherein SEB, SEC and TSST-1 are the abundantest.Every strain streptococcus aureus can produce the enterotoxin of more than a type or amphitypy.In recent years find, cause the Methicillin-resistant Staphylococcus aureus of hospital infection, more than 80% produces enterotoxin B (Staphylococcalenterotoxin B, SEB).Individual hospitals strain isolated almost 100% produces SEB.
S. aureus L-forms SEB belongs to superantigen, does not need the processing treatment of antigen presenting cell, with complete protein molecule directly with the α at MHC class Ⅱmolecule 1the β chain V district of structural domain and TCR combines, thus stimulates T cell activation propagation, discharges a large amount of cytokine as IL-2, IFN-γ and TNF-α.The case fatality rate of the toxic shock syndrome caused by S. aureus L-forms SEB can reach 50%, and the S. aureus L-forms SEB concurrent with influenza infects its mortality ratio can reach more than 90%, and therefore, the S. aureus L-forms SEB in aerosol is a kind of potential chemical and biological weapons.After SEB enters body, the combination of its superantigen causes mononuclearcell to activate, and causes release of cytokines widely, and the TNF-α of early stage TCR-α β T cell release plays important effect to death.
Confirm through Blast comparison, SEB is sequence preservative in each bacterial strain of MRSA, Stability Analysis of Structures.Research in mouse model confirms, SEB has humoral response advantage more better than other antigen, and the people SEB monoclonal antibody of synthesis can prevent MRSA to infect common toxic shock syndrome.But SEB is originally as toxin, and there is potential safety hazard for human body, therefore, the mutant SEB losing toxicity is MRSA vaccine better protecting antigen.At present, SEB mutant vaccine is used for the poisoning and anti-MRSA of SEB and infects and succeed in animal model.
Proteantigen plays its function mainly through epi-position to embody specificity.Prepare vaccine with the Dominant Epitopes in SEB epi-position, be conducive to removing unfavorable composition, strengthen immune effect.Therefore, the qualification of the protective epitope of immunodominance response in SEB antigen is the important prerequisite promoting and optimize based on the S.aureus vaccine design of SEB antigen.Multiple immunodominant epitopes of screening SEB, carry out series connection and obtain multi-epitope polypeptide vaccine and can excite more effective immunne response by the epi-position of each antigen.The B cell epi-position of current known SEB has 5, and screening method used is Bioinformatics Prediction and monoclonal antibody screening mainly.Not high by the B cell epi-position accuracy rate of Bioinformatics Prediction antigen, the epi-position doped only is confined to the peptide section of length-specific, unpredictable immunodominant epitope.Research also confirms, the result that the B cell epi-position of experiment gained and bioinformatics software are predicted is also inconsistent.By the B cell epi-position complex steps of monoclonal antibody screening antigen, the test period is long, the antigen B cell epi-position identified not entirely, also fubaritic immunodominant epitope.Therefore, set up and a kind ofly screen accurately and effectively and identify that the method for B cell immunodominant epitope seems particularly important.
Summary of the invention
The invention provides a kind of B cell immunodominant epitope peptide of SEB, its aminoacid sequence is as shown in SEQ ID NO:42.The aminoacid sequence of the core epitope of its correspondence is respectively as shown in SEQID NO:69.
The invention provides a kind of method identifying the B cell immunodominant epitope peptide of described SEB, it mainly comprises step:
1) antiserum(antisera) is obtained with the mutant immune mouse of SEB;
2) step moves overlap 18 peptide that synthesis covers SEB full length sequence;
3) by step 1) the antiserum(antisera) integrating step 2 of gained) the B cell immunodominant epitope peptide of overlap 18 peptide screening SEB of gained;
4) by step 3) the B cell immunodominant epitope peptide that sieves and keyhole limpet hemocyanin coupling, gained conjugate is used for animal immune, adaptive immune advantage peptide antiserum(antisera); The neutralising capacity that described immunodominant epitope antiserum(antisera) carries out SEB detects, and identifies that described B cell immunodominant epitope peptide is targetted B-cell immunodominant epitope peptide.
Further, above-mentioned authentication method comprises step:
5) for step 3) the B cell immunodominant epitope peptide sequence of gained, blocks 2 amino acid respectively successively from N-end and C-end, synthesizes the truncated peptide corresponding to each B cell immunodominant epitope peptide;
6) by step 4) the described immunodominance peptide antiserum(antisera) integrating step 5 that obtains) described in truncated peptide, determine the minimum epi-position of described B cell immunodominant epitope peptide;
7) by step 6) the minimum epi-position of B cell immunodominant epitope peptide of sieving and keyhole limpet hemocyanin albumen coupling, gained conjugate is used for animal immune, adaptive immune Dominant Epitopes antiserum(antisera);
8) by step 7) the described immunodominant epitope antiserum(antisera) that the obtains neutralising capacity that carries out SEB detects;
9) by bioinformatics method determining step 6) conservative property of minimum epi-position in each bacterial strain of streptococcus aureus of determined B cell immunodominant epitope peptide, and its position in SEB protein three-dimensional structure clear and definite.
The B cell immunodominant epitope peptide of described SEB has high immunogenicity, can cause strong immune response; With the antiserum(antisera) that described immunodominant epitope immune animal is obtained, all there is the effect with SEB toxin in specificity.Described immunodominant epitope not containing unnecessary even harmful part, thus reduces the application risk of the vaccine prepared by it, has larger vaccine using value, can be used for preparing SEB epiposition vaccine and/or vaccine.
The antiserum(antisera) that the present invention infects by obtaining prevention S.aureus (streptococcus aureus) by the immune BALB/c mouse of SEB mutant (rSEB), has gone out B cell three immunodominant epitope peptides of SEB in conjunction with 42 the overlapping peptide Stepwise Screenings covering SEB total length.With immunodominant epitope peptide-KLH (keyhole limpet hemocyanin) conjugate immune animal, acquisition can strongly in conjunction with the epitope peptide specific antisera of SEB toxin.Then blocking overlapping peptide and this epitope peptide antiserum(antisera) screens further with covering three immunodominant epitope peptides multiple respectively, specify that core sequence and the Dominant Epitopes of this immunodominant epitope peptide.In vitro, three immunodominant epitopes of qualification all can neutralize the superantigen ability of natural SEB toxin.Screening and identification method accuracy rate provided by the present invention is high, can avoid Lou sieving and sieving phenomenon by mistake, the immundominance of epitope and immune subdominance can be distinguished, and the epitope filtered out all can bring out high-level Dominant Epitopes antiserum(antisera) in animal body, and with the function of SEB toxin superantigen ability during this antiserum(antisera) has, making to prepare efficient, low toxicity, high security the vaccine based on SEB thus becomes possibility.
The present invention covers the immunodominance B cell epitope peptide of many groups overlapping peptide in conjunction with ELISA method screening SEB of antigen protein total length, overlapping peptide to carry out clearly this Dominant Epitopes peptide core sequence in conjunction with ELISA method is blocked further with what cover Dominant Epitopes peptide total length, method is fast easy, epi-position, without omission, obtains three immunodominant epitopes of the B cell of SEB.This immunodominant epitope all has stronger immunogenicity, and with this Dominant Epitopes immune animal, immunological reagent, without irrelevant composition or objectionable constituent, can obtain high-caliber Dominant Epitopes antiserum(antisera), and, this antiserum(antisera) can strongly in and the superantigen ability of SEB toxin.The vaccine being activeconstituents with this Dominant Epitopes has better prevention than SEB whole protein mutant vaccine and neutralizes the effect of the SEB toxin brought out, due to Dominant Epitopes SEB 97-112and SEB 207-222sequence preservative in multiple S.aureus bacterial strain, points out it to infect other S.aureus and also has a good application prospect.
For above and other objects of the present invention, feature and advantage can be become apparent, preferred embodiment cited below particularly, and coordinate accompanying drawing, be described in detail below.
Accompanying drawing explanation
Fig. 1 is the screening of the immunodominant epitope peptide of SEB.
Fig. 2 is that the immunodominant epitope peptide antiserum(antisera) of SEB and the combination of natural SEB detect.
Fig. 3 Ai to Fig. 3 Cii is that the minimum epi-position of the immunodominant epitope of SEB is determined; Wherein
Fig. 3 Ai is SEB 97-112minimum epi-position determine;
Fig. 3 Bi is SEB 207-222minimum epi-position determine;
Fig. 3 Ci is SEB 247-257minimum epi-position determine;
Fig. 3 Aii, Fig. 3 Bii and Fig. 3 Cii is subdominant epitope peptide SEB respectively 31-48, SEB 133-150and SEB 193-210core sequence.
Fig. 4 A to Fig. 4 D is that the minimum epi-position antiserum(antisera) of immunodominance of SEB is to the detection of the neutralising capacity of this toxin; Wherein
Fig. 4 A is the T cell propagation that the minimum epi-position antiserum(antisera) of immunodominance has neutralized SEB induction;
Fig. 4 B is the secretion that the minimum epi-position antiserum(antisera) of immunodominance has blocked the IL-2 of SEB induction;
Fig. 4 C is the secretion that the minimum epi-position antiserum(antisera) of immunodominance has blocked the IFN-γ of SEB induction;
Fig. 4 D is the secretion that the minimum epi-position antiserum(antisera) of immunodominance has blocked the TNF-α of SEB induction.
Fig. 5 A to Fig. 5 B is the immunodominant epitope conservative Analysis of SEB and the location in SEB whole protein three-dimensional structure thereof; Wherein
Fig. 5 A is the immunodominant epitope conservative Analysis of SEB;
Fig. 5 B is the location of immunodominant epitope conservative Analysis in SEB whole protein three-dimensional structure of SEB.
Embodiment
Below in conjunction with specific examples; further elaboration the present invention; be understood that; these examples are only for illustration of the present invention instead of limitation of the present invention; to the simple modifications of preparation method of the present invention under concept thereof of the present invention, and all the scope of protection of present invention is belonged to the utilization of epitope peptide of the present invention.
Thinking of the present invention is as follows: (Mutant Preparation reference: Oral Vaccine Formulations Stimulate Mucosaland Systemic Antibody Responses against Staphylococcal Enterotoxin B in a PigletModel.Clinical and Vaccine Immunology based on the SEB mutant that this teaching and research room has prepared, 2010 (17) 8:1163-1169), take BALB/c mouse as animal model, prepare at the mosaic epitopes vaccine BALB/c mouse animal model to be studied MRSA.The present invention's step moves synthesis method, has synthesized 42 overlapping peptides covering SEB total length.Thereafter, with immunodominant epitope peptide-KLH (keyhole limpet hemocyanin) conjugate immune animal, obtain Dominant Epitopes peptide specific antiserum(antisera).For each immunodominant epitope peptide sequence, block 2 amino acid respectively successively from N-end and C-end, synthesis covers the truncated peptide of the correspondence of each immunodominant epitope peptide total length.With the truncated peptide of Dominant Epitopes peptide specific antiserum(antisera) in conjunction with its correspondence, the core sequence of screening Dominant Epitopes peptide, and verify this core epitope further by antiserum(antisera) volumetry.With this core epitope-KLH (keyhole limpet hemocyanin) conjugate immune animal, obtain Dominant Epitopes specific antisera.The superantigen activity of SEB toxin is blocked with Dominant Epitopes specific antisera.Finally, by the conservative property of bioinformatics method determination Dominant Epitopes in each bacterial strain of streptococcus aureus, and its position in SEB protein three-dimensional structure clear and definite, analyze its infect at the different staphylococcus aureus strains of prevention caused by the poisoning versatility of SEB.
The preparation of material:
1. SEB mutant is applicant's preparation.
2. laboratory animal: BALB/c mouse: the 6-8 week tinkling of pieces of jade, SPF level, female, body weight 18-22g, purchased from Third Military Medical University's Animal House.
3. improvement on synthesis: synthesized by Shanghai Qiang Yao company, polypeptide dry powder divalence sulfoxide (DMSO) is dissolved into the concentration of 0.5mg/mL ,-70 DEG C of preservations.Face the used time according to working concentration phosphate buffered saline buffer (PBS) dilution.
4. not formula adjuvant: not formula Freund's complete adjuvant: paraffin oil (1 part)+lanolin (1 part)+inactivated vaccine 1, not formula Freund's incomplete adjuvant: paraffin oil (1 part)+lanolin (1 part)
5.PBS damping fluid (PH7.2) Na2HPO414mmol, NaH2PO46mmol, NaCl 9g, adding distil water is to 1L
The preparation of 6.ELISA reagent
1) bag is by diluent 0.05mol/L PH9.6 carbonate buffer solution: Na2CO31.6g, NaHCO32.9g, NaN30.2g, adding distil water is to 1000ml.
2) Sample dilution/antibody diluent: Tween-20 is dissolved in volume ratio in PBS is 0.5 ‰ 3) confining liquid: add 1%BSA (mass volume ratio) and Tween200.5 ‰ (volume volume ratio) in PBS
3) it is 0.5 ‰ that washings: Tween-20 is dissolved in volume ratio in PBS
4) tmb substrate liquid: Chinese Tian Gen company
7.BSA (bSA): Chinese BIOSHARP company.
8.SDS-PAGE albumen sample-loading buffer (5 ×): Chinese green skies company.
9. pre-dyed Marker: Chinese MBI company.
10. skim-milk: Chinese doctor's moral company.
The flat Tissue Culture Plate of the 11.96 removable enzyme plate in hole and 96 holes: Corning company of the U.S..
12.IPTG: Chinese Ding Guo company.
13. dimethyl sulfoxide (DMSO) (DMSO): Sigma company.
14. 3h-thymidine: Nuclear Medicine of southwest hospital of Third Military Medical University.
15.IL-2, IFN-γ and TNF-α. cytokine detection kits (Dakewe company)
16. mouse lymphocyte parting liquids: Chinese Tianjin Ho ocean company.
17.R-1640 cell culture fluid and foetal calf serum: Hyclone company.
18.HRP-goat anti-mouse igg: company of Chinese Zhong Shan Golden Bridge.
embodiment 1step moves the overlapping synthesis containing 18 amino acid short peptides
SEB is SEB, and between each bacterial strain, sequence is conservative, total length 261 amino acid, and wherein, 1-27 amino acid is signal peptide.The overlapping peptide of recombination to construct is 18 peptides between 1-261, derives from MRSA252 international standard bacterial strain.It is the SEB protein sequence (numbering Q6GFN0) retrieving streptococcus aureus MRSA252 source in UniProt albumen database, from No. 1 amino acid, synthesize step move overlapping 18 amino acid short peptides (assisting synthesis by Shanghai Qiang Yao company), totally 42, purity is all greater than 90%.Synthetic peptide information is in table 1.The peptide section DMSO of synthesis is dissolved to the storage concentration of 0.5mg/mL ,-70 DEG C of preservations after packing.
UniProt protein database search network address: http://www.uniprot.org/uniprot/P57839
The aminoacid sequence following (totally 261 amino acid) of the 1-260 position of SEB:
MKLFAFIFICVKSCSLLFMLNGNPKPEQLNKASEFTGLMDNMRYLYDDKHVSEINIKAQEKFLQHDLLFKINGSKIDGSKILKTEFNNNSLSDKYKNKNIDLFGTNYYYQCYFSADNMELNDGRLIEKTCMYGGVTEHDGNQIDKNNSTDNSHNILIKVFENERNSLSFDIPTNKKNITAQEIDYKVRNYLLKHKNLYEFNSSPYETGYIKFIEGNGHSFWYDMMPESGEKFYPTKYLLIYNDNKTVESKSINVEVHLTKK
Table 1: the overlap 18 peptide amino acid sequence information (being corresponding in turn to the SEQ ID No:1-42 in sequence table) covering SEB full length sequence
embodiment 2the screening of antigen dominant epitope peptide and qualification
1., under Alum adjuvant is assisted, the provide protection analysis of antigen immune and antiserum(antisera) are collected
(1) recombinant mutant SEB is prepared and qualification:
According to bibliographical information, three amino acid sites (L45R in sudden change SEB, Y89A, Y94A), select pGEX6p-2 carrier, clonal expression recombinant SEB albumen (rSEB), after rSEB albumen GST column purification (purity of protein is greater than 90%), identifies through Western Blot.(mutational site reference: Oral VaccineFormulations Stimulate Mucosal and Systemic Antibody Responses againstStaphylococcal Enterotoxin B in a Piglet Model.Clinical and Vaccine Immunology, 2010 (17) 8:1163-1169)
(2) animal immune:
The immunity of Alum adjuvant treated animal adopts intramuscular injection approach, and the immunity of freund's adjuvant treated animal adopts subcutaneous inoculation approach.Respectively the auxiliary lower immune 6-8 female BAl BIc/c mouse in age in week of different adjuvant, grouping and immunizing dose as shown in the table.Respectively at 0 day, the 14th day, immunity in the 28th day, after final immunization the 7th day, tail venous blood sampling, after separation of serum, aseptic subpackaged be stored in-80 DEG C for subsequent use.This experiment is divided into following three groups, often organize 10 mouse, proteantigen consumption be 100 μ g/ time/: 1. rSEB+AlPO 4; 2. PBS+AlPO 4; 3. PBS
(3) antiserum(antisera) is collected:
After BALB/c mouse vaccine final immunization the 7th day, putting to death through plucking after eyeball gets blood, collecting antiserum(antisera).In systemic infection model, observe the survival condition of mouse, survival mice is put to death through plucking after eyeball gets blood, collects the serum of survival mice, aseptic subpackaged be stored in-80 DEG C for subsequent use.
2. screen the immunodominance B cell epi-position in antigen
(1) Linear B Cell Epitopes of the ELISA method screening antigen of overlapping peptide:
Indirect elisa method.Peptide and recombinant mutant SEB albumen bag are 5 μ g/mL by concentration, 4 DEG C of bags are spent the night, SEB antiserum(antisera) 1:500 dilutes, every hole adds by the SEB antiserum(antisera) 100 μ L diluted, 37 DEG C of incubation 1.5h, PBS-T washs 3 times, dry, every hole adds the HRP-goat anti-mouse igg 100 μ L that the PBS-T containing 0.5%BSA dilutes with 1:3000, 37 DEG C, incubation 1h, PBS washs 3 times, dry, every hole adds TMB100 μ L, lucifuge normal temperature hatches 15min, every hole adds TMB nitrite ion 100 μ L, stop buffer 100 μ L is added after lucifuge 15min, measure the OD value at 450nm place.
Result: three the immunodominant epitope peptide SEB filtering out antigen 97 – 114, SEB 205-222and SEB 247-261, three subdominant epitope peptide SEB 31-48, SEB 133-150and SEB 193-210.
3. identify the B cell linearity advantage epitope peptide of antigen
(1) animal immune and antiserum(antisera) are collected:
Animal immune adopts subcutaneous inoculation approach.Respectively the auxiliary lower immune 6-8 female BAl BIc/c mouse in age in week of different adjuvant, respectively at 0 day, the 14th day, immunity in the 28th day.After final immunization the 7th day, tail venous blood sampling, separation of serum, aseptic subpackaged be stored in-80 DEG C for subsequent use.This experiment is divided into following several groups, often organize 10 mouse, proteantigen consumption be 100 μ g/ time/:
①SEB 97–114+CFA/IFA；②SEB 205-222+CFA/IFA；③SEB 247-261+CFA/IFA④PBS+CFA/IFA；
(2) the B cell linearity advantage epitope peptide Immunity identification of antigen:
Indirect elisa method.With SEB whole protein bag quilt, concentration is 5 μ g/mL, and 4 DEG C of bags are spent the night.Epitope peptide specific antisera dilutes with 1:000,1:2000,1:4000 respectively, every hole adds by the antiserum(antisera) 100 μ L diluted, 37 DEG C of incubation 1.5h, PBS-T washs 3 times, dry, every hole adds the HRP-goat anti-mouse igg 100 μ L that the PBS-T containing 0.5%BSA dilutes with 1:3000,37 DEG C, incubation 1h, PBS wash 3 times, dry, every hole adds TMB100 μ L, and lucifuge normal temperature hatches 15mi n, and every hole adds TMB nitrite ion 100 μ L, add stop buffer 100 μ L after lucifuge 15min, measure the OD value at 450nm place.Specifically please refer to Fig. 1.
Result: three immunodominant epitope peptide specific antiserum(antisera) Anti-SEB 97 – 114, Anti-SEB 205-222and Anti-SEB 247-261strongly in conjunction with SEB natural toxin, compared with Normal Mouse Serum binding ability, remarkable significant difference can be had.
embodiment 3step moves the synthesis of the corresponding truncated peptide of overlapping immunodominance peptide
For the immunodominant epitope peptide sequence that embodiment 2 is screened, block 2 amino acid respectively successively from N-end and C-end, synthesis covers the truncated peptide of the correspondence of each immunodominant epitope peptide total length; The overlapping peptide of recombination to construct is three groups of truncated peptides, often the corresponding immunodominant epitope peptide of group, and often in group, adjacent truncated peptide differs 2 amino acid, and synthesis purity is all greater than 90% (assisting synthesis by Shanghai Qiang Yao company).Synthesis truncated peptide information is in table 3.The peptide section DMSO of synthesis is dissolved to the storage concentration of 0.5mg/mL ,-70 DEG C of preservations after packing.
The corresponding truncated peptide amino acid sequence information of immunodominance peptide is as following table 3, and its peptide numbers the SEQ ID:43-73 in corresponding sequence table respectively
Peptide is numbered Peptide fragment position Sequence (N → C) Original purity (%)
(NO：43)D-1 99-114aa NIDLFGTNYYYQCYFS 0.9464
(NO：44)D-2 101-114aa DLFGTNYYYQCYFS 0.9134
(NO：45)D-3 103-114aa FGTNYYYQCYFS 0.9232
(NO：46)D-4 105-114aa TNYYYQCYFS 0.911
(NO：47)D-5 107-114aa YYYQCYFS 0.9091
(NO：48)D-6 97-112aa NKNIDLFGTNYYYQCY 0.9377
(NO：49)D-7 97-110aa NKNIDLFGTNYYYQ 0.9804
(NO：50)D-8 97-108aa NKNIDLFGTNYY 0.9756
(NO：51)D-9 97-106aa NKNIDLFGTN 0.9715
(NO：52)D-10 97-104aa NKNIDLFG 0.9892
(NO：53)D-11 98-103aa KNKNIDLF 0.9552
(NO：54)D-12 207-222aa TGYIKFIEGNGHSFWY 0.9611
(NO：55)D-13 209-222aa YIKFIEGNGHSFWY 0.9806
(NO：56)D-14 211-222aa KFIEGNGHSFWY 0.9585
(NO：57)D-15 213-222aa IEGNGHSFWY 0.9743
(NO：58)D-16 215-222aa GNGHSFWY 0.9046
(NO：59)D-17 205-220aa YETGYIKFIEGNGHSF 0.9757
(NO：60)D-18 205-218aa YETGYIKFIEGNGH 0.9775
(NO：61)D-19 205-216aa YETGYIKFIEGN 0.9016
(NO：62)D-20 205-214aa YETGYIKFIE 0.9888
(NO：63)D-21 205-212aa YETGYIKF 0.9981
(NO：64)D-22 249-261aa SKSINVEVHLTKK 0.9869
(NO：65)D-23 251-261aa SINVEVHLTKK 0.9416
(NO：66)D-24 253-261aa NVEVHLTKK 0.9981
(NO：67)D-25 255-261aa EVHLTKK 0.9985
(NO：68)D-26 247-259aa VESKSINVEVHLT 0.9243
(NO：69)D-27 247-257aa VESKSINVEVH 0.9891
(NO：70)D-28 247-255aa VESKSINVE 0.9833
(NO：71)D-29 247-253aa VESKSIN 0.9887
(NO：72)D-30 252-261aa INVEVHLTKK 0.9924
(NO：73)D-31 110-119aa QCYFSADNME 0.9117
embodiment 4the screening of antigen dominant epitope core sequence and qualification
1. the screening of antigen dominant epitope core sequence
Indirect elisa method.Truncated peptide and total length Dominant Epitopes peptide bag are 5 μ g/mL by concentration, 4 DEG C of bags are spent the night, SEB antiserum(antisera) 1:500 dilutes, every hole adds by the SEB antiserum(antisera) 100 μ L diluted, 37 DEG C of incubation 2h, PBS-T washs 3 times, dry, every hole adds the HRP-goat anti-mouse igg 100 μ L that the PBS-T containing 0.5%BSA dilutes with 1:3000, 37 DEG C, incubation 1h, PBS washs 3 times, dry, every hole adds TMB100 μ L, lucifuge normal temperature hatches 15mi n, every hole adds TMB nitrite ion 100 μ L, stop buffer 100 μ L is added after lucifuge 15min, measure the OD value at 450nm place, specifically please refer to Fig. 3.
Result: at SEB 97 – 114in group truncated peptide, SEB 97 – 112, SEB 97 – 114, SEB 97 – 110, SEB 99 – 114, SEB 101 – 114and SEB 97 – 112compare other truncated peptides and SEB 97 – 114sero-fast binding ability stronger (please refer to Fig. 3 Ai); At SEB 205 – 222in group truncated peptide, SEB 205 – 222, SEB 207 – 222, SEB 209 – 222, SEB 211 – 222, SEB 205 – 220, SEB 205 – 218and SEB 205 – 216compare other truncated peptides and SEB 205 – 222sero-fast binding ability stronger (please refer to Fig. 3 Bi); At SEB 247 – 261in group truncated peptide, SEB 247 – 261, SEB 249 – 261, SEB 252 – 261, SEB 251 – 261, SEB 247 – 259and SEB 247 – 257compare other truncated peptides and SEB 247 – 261sero-fast binding ability stronger (please refer to Fig. 3 Ci).
2. the determining further of antigen dominant epitope core sequence
Choose step often to organize the stronger truncated peptide of response and carry out bag quilt, wrap and be 5 μ g/mL by concentration, carry out epitope peptide antiserum(antisera) serum titration.According to the indirect elisa method of upper step, set multiple antiserum(antisera) extent of dilution, from 1:200,1:400,1:800,1:1600,1:3200,1:6400,1:12800 to 1:256000, other experimental techniques are constant, measure the OD value at 450nm place.
Result: identify the truncated peptide that often group response is the strongest: SEB 97 – 112, SEB 207-222and SEB 247-257.
Please refer to Fig. 3 Aii, Fig. 3 Bii and Fig. 3 Cii, is three Dominant Epitopes peptide SEB respectively 97 – 114, SEB 205-222and SEB 247-261core sequence.
embodiment 5the sero-fast capability analysis that neutralizes a toxin of Dominant Epitopes
1. animal immune and antiserum(antisera) are collected
Animal immune adopts subcutaneous inoculation approach.Respectively the auxiliary lower immune 6-8 female BAl BIc/c mouse in age in week of different adjuvant, grouping and immunizing dose as shown in the table.Respectively at 0 day, the 14th day, immunity in the 28th day.After final immunization the 7th day, tail venous blood sampling, separation of serum, aseptic subpackaged be stored in-80 DEG C for subsequent use.This experiment is divided into following several groups, often organize 10 mouse, proteantigen consumption be 100 μ g/ time/:
①SEB 97–112+CFA/IFA；②SEB 207-222+CFA/IFA；③SEB 247-257+CFA/IFA；④PBS+CFA/IFA；
2. the sero-fast neutralising capacity analysis of Dominant Epitopes
(1) mouse lymphocyte is separated: aseptic BALB/c mouse spleen of getting grinds, erythrocyte splitting (1 spleen+7mL), 15min; Resuspended, mend to 15mL centrifugal; 500g, centrifugal 10min; 15mL physiological saline washes 1 time; 15mL physiological saline is resuspended, point 2 pipes, is separated (mouse lymphocyte parting liquid specification sheets, Tianjin Ho ocean company) by Ficoll-Paque method gradient centrifugation; Get tunica albuginea confluent monolayer cells, physiological saline is mended to 15mL; Centrifugal, 2000rpm/min, 15min; 15mL physiological saline washes 2 times; 10mL physiological saline re-suspended cell; Get in 10 μ l to 90 μ l physiological saline, calculate the minicell number of living; With normal saline dilution mouse lymphocyte to final concentration 10 7/ mL, for subsequent use.
(2) test with SEB toxin in Dominant Epitopes antiserum(antisera): with 0.22uM frit Dominant Epitopes antiserum(antisera), and detect antiserum(antisera) IgG concentration, by complete 1640 adjustment antiserum(antisera) IgG concentration, on the 96 flat Tissue Culture Plates in hole, select IgG concentration to be the antiserum(antisera) 50 μ l of 100 μ g/ml with concentration to be respectively the SEB solution 50 μ l of 50ng/mL to mix, hatch 1h at 37 degree; Select the Normal Mouse Serum of equal IgG concentration to compare, select untreated hole to make blank background and detect, each condition repeats six holes; Every hole add 100 μ l be separated mouse lymphocyte (cell concn is 10 6/ 100 μ l/ holes), cell cultivates 48h at 37 degree of cell culture incubators; Each condition gets three hole 1uCi 3h-thymidine stimulate, then cultivate 12h ( 3h-thymidine incorporation methods); Detect CPM value (having been assisted by Nuclear Medicine of southwestern hospital, stimulation index StimulationIndex (SI)=each experiment disposal hole average CPM values/untreated hole average CPM values); Under each condition, other three holes also continue to cultivate 12h, and collecting cell culture supernatant, with IL-2, IFN-γ and TNF-α. and cytokine detection kits detects (being undertaken by test kit operation instructions)
Result: often organize 100 μ g/ml minimum epi-position antiserum(antisera) IgG (Anti-SEB 97-112serum IgG, Anti-SEB 207-222serum IgG and Anti-SEB 247-257serum IgG) can completely in and 50ng/ml SEB toxin-induced T cell propagation and splenocyte cytokine IL-2, IFN-γ and TNF-α secretion (please refer to Fig. 4 A to Fig. 4 D, Fig. 4 A be the minimum epi-position antiserum(antisera) of immunodominance neutralized SEB induction T cell propagation; Fig. 4 B is the secretion that the minimum epi-position antiserum(antisera) of immunodominance has blocked the IL-2 of SEB induction; Fig. 4 C is the secretion that the minimum epi-position antiserum(antisera) of immunodominance has blocked the IFN-γ of SEB induction; Fig. 4 D is the secretion that the minimum epi-position antiserum(antisera) of immunodominance has blocked the TNF-α of SEB induction: the icon about Fig. 4 A to Fig. 4 D illustrates: Medium alone (substratum blank); SEB alone (positive control); SEB+Anti-SEB 97-114(antiserum(antisera) Anti-SEB 97-114igG is to the neutralization of SEB toxin ability); SEB+Anti-SEB 207-222(antiserum(antisera) Anti-SEB 207-222igG is to the neutralization of SEB toxin ability); SEB+Anti-SEB 247-257(antiserum(antisera) Anti-SEB 247-257igG is to the neutralization of SEB toxin ability); SEB+mixedantisera, (the antiserum(antisera) Anti-SEB of equivalent 97-114igG, Anti-SEB 207-222igG and Anti-SEB 247-257igG mixed antiserum IgG is to the neutralization of SEB toxin ability); SEB+normal sera (Normal Mouse Serum IgG is to the neutralization of SEB toxin ability).
embodiment 6the bioinformatic analysis of antigen immune Dominant Epitopes space structure
1. Dominant Epitopes sequence conservation analyze: retrieve in UniProt albumen database different staphylococcus aureus strains source SEB protein sequence (UniProt protein database search network address: http:// www.uniprot.org/uniprot/Q6GFN0), NCBI website (network address: http:// blast.ncbi.nlm.nih.gov/Blast.cgi) above input the protein sequence looked into, carry out BLAST sequence alignment; The sequence being positioned at 97-112aa, 207-222aa and 247-257aa position is selected to analyze.
Result: three immunodominant epitope SEB 97 – 112and SEB 207-222sequence preservative (conservative rate is greater than 85%) in different staphylococcus aureus strains, and SEB 247-257sequence not conservative (please refer to Fig. 5 A) in different staphylococcus aureus strains.
2. Dominant Epitopes is located in SEB antigen three-dimensional structure: SEB crystalline structure pdb data are downloaded by Pubmedprotein data base database, by Papageorgiou, A.C. people is waited to resolve (Papageorgiou AC in 1998, Tranter HS, Acharya KR:Crystal structure of microbialsuperantigen staphylococcal enterotoxin B at 1.5A resolution:implications forsuperantigen recognition by MHC class II molec μ les and T-cell receptors.J Mol Biol1998, 277 (1): 61-79.), with PyMOL 1.1program software analysis and the position of clear and definite antigen dominant epitope in antigen three-dimensional structure.
Result: SEB in three immunodominant epitopes 97 – 112be positioned at the Loop district of antigenic structure inside, and SEB 207-222and SEB 247-257be positioned at the β lamella (please refer to Fig. 5 B) of antigenic structure outside.
Although the present invention discloses as above with preferred embodiment; so itself and be not used to limit the present invention; any person of ordinary skill in the field; without departing from the spirit and scope of the invention; when doing a little change and improvement, the protection domain of therefore the present invention is when being as the criterion depending on the claim person of defining.

Claims (6)

1. a B cell immunodominant epitope peptide for SEB, its aminoacid sequence is as shown in SEQ ID NO:42.
2. the B cell immunodominant epitope peptide of SEB according to claim 1, is characterized in that, the aminoacid sequence of the core epitope of described B cell immunodominant epitope peptide is as shown in SEQ IDNO:69.
3. identify a method for the B cell immunodominant epitope peptide of SEB according to claim 1, it is characterized in that, comprise step:
1) antiserum(antisera) is obtained with the mutant immune mouse of SEB;
2) step moves overlap 18 peptide that synthesis covers SEB full length sequence;
3) by step 1) the antiserum(antisera) integrating step 2 of gained) the B cell immunodominant epitope peptide of overlap 18 peptide screening SEB of gained;
4) by step 3) the B cell immunodominant epitope peptide that sieves and keyhole limpet hemocyanin coupling, gained conjugate is used for animal immune, adaptive immune advantage peptide antiserum(antisera); The neutralising capacity that described immunodominant epitope antiserum(antisera) carries out SEB detects, and identifies that described B cell immunodominant epitope peptide is targetted B-cell immunodominant epitope peptide.
4. method according to claim 3, is characterized in that, comprises step further:
5) for step 3) the B cell immunodominant epitope peptide sequence of gained, blocks 2 amino acid respectively successively from N-end and C-end, synthesizes the truncated peptide corresponding to each B cell immunodominant epitope peptide;
6) by step 4) the described immunodominance peptide antiserum(antisera) integrating step 5 that obtains) described in truncated peptide, determine the minimum epi-position of described B cell immunodominant epitope peptide;
7) by step 6) the minimum epi-position of B cell immunodominant epitope peptide of sieving and keyhole limpet hemocyanin albumen coupling, gained conjugate is used for animal immune, adaptive immune Dominant Epitopes antiserum(antisera);
8) by step 7) the described immunodominant epitope antiserum(antisera) that the obtains neutralising capacity that carries out SEB detects;
9) by bioinformatics method determining step 6) conservative property of minimum epi-position in each bacterial strain of streptococcus aureus of determined B cell immunodominant epitope peptide, and its position in SEB protein three-dimensional structure clear and definite.
5. the B cell immunodominant epitope peptide of SEB according to claim 1 is preparing the application in SEB epiposition vaccine and/or vaccine.
6. SEB epiposition vaccine and/or a vaccine, is characterized in that, the B cell immunodominant epitope peptide containing SEB according to claim 1.
CN201510143971.6A 2014-01-23 2014-01-23 B cell immunodominance epitope peptide of staphylococcus aureus enterotoxin B and preparation method and application thereof Pending CN104693293A (en)

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