CN106478777B - Staphylococcus aureus FnBPA A albumen mimic epitope peptide, mimic epitope peptide combinations and its application with immune protective - Google Patents
Staphylococcus aureus FnBPA A albumen mimic epitope peptide, mimic epitope peptide combinations and its application with immune protective Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/085—Staphylococcus
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- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B30/00—Methods of screening libraries
- C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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Abstract
The present invention relates to mimic epitope peptide, mimic epitope peptide combinations and its application of the staphylococcus aureus FnBPA A albumen with immune protective.The amino acid sequence of 2 immune protective mimic epitope peptides provided by the invention is respectively such as SEQ ID NO:1 and SEQ ID NO:Shown in 2, and mimic epitope peptide combinations are then by SEQ ID NO:1 and SEQ ID NO:2 kinds of polypeptides composition shown in 2, and SEQ ID NO:Polypeptide shown in 1 and SEQ ID NO:The quality proportioning of polypeptide shown in 2 is 2:1.The experiment of experimental animal immune protectiveness shows; 2 mimic epitope peptides of the present invention can stimulate body to produce high-caliber specific antibody; with a certain degree of immune protective, and the immune protective effect that mimic epitope peptide combinations are infected staphylococcus aureus is better than FnBPA A holoproteins.Therefore, mimic epitope peptide of the invention and combinations thereof can be used as active ingredient, for the exploitation of staphylococcus aureus polyepitope vaccines, prevent mastadenitis of cow caused by staphylococcus aureus.
Description
Technical field
The present invention relates to epitope peptide, more particularly to the staphylococcus aureus FnBPA-A eggs with immune protective
White mimic epitope peptide, mimic epitope peptide combinations and its application in staphylococcus aureus polyepitope vaccines are prepared.
Background technology
Staphylococcus aureus (Staphylococcus aureus, S.aureus) is a kind of important Amphixenosis
It opportunistic pathogen, can not only cause a variety of diseases of the mankind, and often cause clinical type and recessive mastadenitis of cow.For a long time, due to beast
Doctor is clinically unreasonable to use antibiotic, and induced animal source property methicillin-resistant staphylococcus aureus bacterial strain continuously emerges, no
It is very difficult to only result in medical treatment mastadenitis of cow, brings serious harm to dairy, and influence the product of milk and milk productses
Matter and safety and human health.Therefore, it is immune pre- to select suitable target antigen development of new S. aureus vaccines
The research emphasis of anti-mastadenitis of cow.
However, how to obtain suitable vaccine target antigen and its epitope development of new staphylococcus aureus polyepitope vaccines
It is always then the technical barrier that those skilled in the art make great efforts to solve.
The content of the invention
An object of the present invention is to provide a kind of staphylococcus aureus FnBPA-A albumen with immune protective
Mimic epitope peptide.
It is as follows to reach one of above-mentioned purpose, the technical solution adopted by the present invention：
A kind of staphylococcus aureus FnBPA-A albumen mimic epitope peptides with immune protective, the mimic epitope
Peptide is the small peptide being made up of 12 amino acid residues, its amino acid sequence such as SEQ ID NO:1 and SEQ ID NO:Shown in 2：
SEQ ID NO:1：His-Thr-Glu-Gln-Gly-Thr-Leu-Phe-Leu-Lys-Met-Pro；
SEQ ID NO:2：Ser-Tyr-Phe-Asp-Ala-Leu-Glu-Arg-Met-Leu-Pro-Gly.
Further, encoding amino acid sequence such as SEQ ID NO:The nucleotide sequence SEQ of 1 mimic epitope peptide
ID NO:Shown in 3, encoding amino acid sequence such as SEQ ID NO:The nucleotide sequence SEQ ID NO of 2 mimic epitope peptide:
Shown in 4：
SEQ ID NO:3：cat acg gag cag ggg act ttg ttt ttg aag atg ccg；
SEQ ID NO:4：agt tat ttt gat gcg ctt gag agg atg ttg ccg ggg.
It should be noted that described mimic epitope peptide can use conventional method artificial synthesized, gene work can also be utilized
Journey technique construction recombinant expression plasmid, induced expression obtain.Therefore, recombinant expression plasmid containing described nucleotide sequence with
And the host cell containing the recombinant expression plasmid falls within the innovative part of the present invention.
The second object of the present invention is to provide a kind of containing the foregoing staphylococcus aureus with immune protective
The composition of FnBPA-A albumen mimic epitope peptides, said composition is by SEQ ID NO:Polypeptide shown in 1 and SEQ ID NO:Shown in 2
Polypeptide presses 2:1 mass ratio combination obtains.
It is corresponding with the second object of the present invention, present invention also offers it is a kind of containing foregoing with immune protective
The mimic epitope tandem polypeptide of staphylococcus aureus FnBPA-A albumen mimic epitope peptides, the mimic epitope tandem polypeptide are using base
Because engineering technology is by SEQ ID NO:Polypeptide shown in 1 and SEQ ID NO:Polypeptide shown in 2 carries out expressing in series acquisition, series system
For：SEQ ID NO:Polypeptide shown in 1-alanine alanine tyrosine joint-SEQ ID NO:The ammonia of polypeptide-alanine third shown in 1
Sour tyrosine joint-SEQ ID NO:Polypeptide shown in 2.
Further, the nucleotide sequence such as SEQ ID NO of foregoing immune protectiveness mimic epitope tandem polypeptide are encoded:5 institutes
Show：
SEQ ID NO:5：cat acg gag cag ggg act ttg ttt ttg aag atg ccg gcc gcc
tac cat acg gag cag ggg act ttg ttt ttg aag atg ccg gcc gcc tac agt tat ttt
gat gcg ctt gag agg atg ttg ccg ggg。
The third object of the present invention is to provide a kind of foregoing staphylococcus aureus FnBPA-A eggs with immune protective
White application of the mimic epitope peptide in staphylococcus aureus polyepitope vaccines are prepared.
Likewise, present invention also offers a kind of foregoing staphylococcus aureus FnBPA-A eggs with immune protective
White application of the mimic epitope peptide combinations in staphylococcus aureus polyepitope vaccines are prepared.
Present invention also offers a kind of foregoing staphylococcus aureus FnBPA-A albumen with immune protective to simulate table
The mimic epitope tandem polypeptide of position peptide, the application in staphylococcus aureus polyepitope vaccines are prepared.
The fourth object of the present invention is to provide a kind of staphylococcus aureus polyepitope vaccines, contains in the polyepitope vaccines
Staphylococcus aureus FnBPA-A albumen mimic epitope peptides with immune protective, so as to which the polyepitope vaccines can be effective
Prevent mastadenitis of cow caused by staphylococcus aureus.
Finally, present invention also offers a kind of staphylococcus aureus FnBPA-A albumen simulation with immune protective
The screening technique of epitope peptide, comprises the following steps：
S1, using rFnBPA-A purifying proteins as immunogene, prepare rabbit-anti FnBPA-A antibody；The rabbit-anti FnBPA-A antibody
Preparation process be：The rFnBPA-A purifying proteins and Tween-80 that concentration is 1mg/mL are mixed as aqueous phase, add department
This white oil and the concussion emulsification repeatedly in vortex oscillator, the rFnBPA-A purifying proteins, Tween-80, the body for taking charge of this white oil
Product is than being 1:0.042:3.126；Then the immunogene that the neck in experimental rabbit, dorsal sc multi-point injection have emulsified, immunizing agent
Measure as 1mg/ only, one exempt from after carry out within the 14th day two exempting from, two exempt from dosage and immunization route is same exempts from, two exempt from after carry out three within the 10th day
Exempt from, three exempt from no immunologic adjuvant, three exempt from dosage and immunization route exempt from one it is identical；32 days collection rabbit blood and isolated after exempting from three
Rabbit anteserum is to obtain the rabbit-anti FnBPA-A antibody；
S2, the rabbit-anti FnBPA-A antibody purifying：Using in rabbit anteserum described in ammonium sulfate graded precipitation coarse extraction
IgG, using Protein G affinity columns IgG purifications to obtain the rabbit-anti FnBPA-A antibody of purifying after desalination of dialysing；
S3, using the rabbit-anti FnBPA-A antibody of the purifying affine elutriation, elutriation are carried out to phage random dodecapeptide storehouse
The storage capacity of the phage random dodecapeptide storehouse kit used is 2.7 × 109, titre be 1.5 × 1013/μL、
Escherichia coli ER2537 are the Host Strains in peptide storehouse；The panning process is：With 0.1mol/L, pH8.6 NaHCO3
Rabbit-anti FnBPA-A antibody after purification is diluted to 100 μ g/mL by solution, and coating enzyme mark hole, 4 DEG C overnight；Next day, washed with TBST
Wash liquid to wash 6 times, add 5%BSA confining liquids, 37 DEG C are closed 1h, contain 0.5%Tween-20 in the TBST cleaning solutions；It
After abandon confining liquid, by original peptide storehouse, TBST cleaning solutions, negative serum after purification with 10:195:195 volume ratios mix after according to
100 μ L/ holes add enzyme mark hole, gently shake 1h；Liquid in enzyme mark hole is abandoned again, is used after being washed 6 times using TBST cleaning solutions
The bacteriophage that 0.2mol/L, pH2.2 glycine-HCl elution buffers elution are combined with rabbit-anti FnBPA-A antibody specificities,
And the Tris-HCl buffer solutions for adding 1mol/L, pH9.1 neutralize；Elution buffer described in 5 μ L is taken to carry out phage titre measure,
Remaining Phage Infection Host Strains E.coli ER2738 are expanded, and for next round elutriation, carry out four-wheel elutriation altogether to obtain
The higher specific bacteriophage positive colony of affinity；The rabbit-anti FnBPA-A reduced after purification by wheel in four-wheel panning process resists
Body diluted concentration, the concentration by Tween-20 in the wheel increase TBST cleaning solutions, wherein described in fourth round elutriation after purification
Rabbit-anti FnBPA-A antibody diluted concentration be 50 μ g/mL, described in fourth round elutriation in TBST cleaning solutions Tween-20 concentration
Increase to 0.5%；
S4, the specific bacteriophage positive colony DNA sequencing and epitope peptide analysis：Carried using phage DNA is single-stranded
Take kit to extract the single stranded DNA of the specific bacteriophage positive colony, and DNA sequencing carried out to the single stranded DNA sample,
According to the exogenous gene sequence carried in the single stranded DNA sample, the FnBPA-A albumen moulds for being showed in phage surface are derived
Intend Epitope peptide sequences.
Beneficial effects of the present invention are as follows：
1) fibronectin binding protein A (Fibronectin-binding proteins A, FnBPA) is nearly all gold
It is highly conserved caused by staphylococcus aureus clinical separation strain to stick fibroin, the albumen can be present in blood plasma, body fluid and
Fibrin-specific in extracellular matrix combines, so that bacterium is effectively attached in host tissue, causes infection.
Research shows that FnBPA protein structures include tetra- functional areas of A, B, C, D, and wherein A areas are not only the main work of adhesion of the albumen
Energy domain, can specifically bind with fibrin, be played a significant role in the starting stage that infection is established, and FnBPA-A
Antibody can resist infection of staphylococcus aureus caused by protein induced body.Therefore, FnBPA-A is to develop Staphylococcus aureus
One of preferable target antigen of bacteria vaccine.
The invention have selected staphylococcus aureus FnBPA-A albumen mimic epitope peptide as the foregoing skill of solution
The point of penetration of art problem, good basis is provided for the final technical problem that solves.
2) staphylococcus aureus virulence factor is numerous and mechanism of causing a disease is complicated, and vaccine is prepared with single protective antigens,
The immanoprotection action that it is induced is limited, and this just needs multiple protective antigens chimeric expressions preparing the chimeric epidemic disease of subunit
Seedling.However, due to expression vector finite capacity so that the antigen levels of structure subunit chimeric are restricted, even gone out
The problems such as existing chimeric protein expression quantity is low or does not express.However, epitope (epitope) is the critical segment that antigen plays a role,
The built-up polyepitope vaccines of protective epitope's series connection of a variety of antigens can either be excluded that nothing is immunized in whole protein molecular
Composition or immune tolerance composition are closed, induces the more effective protective immune response of more complete albumen, and can overcomes expression vector
To the capacity limit of a variety of antigen chimeric expressions.
Therefore, the invention provides simulate table containing the staphylococcus aureus FnBPA-A albumen with immune protective
The mimic epitope tandem polypeptide of position peptide, so as to further improve the immanoprotection action of vaccine
3) primary link for preparing polyepitope vaccines is to identify epitope or mimic epitope with immune protective, and is bitten
The effective means that phage display technique provides for research epitope.The principle of display technique of bacteriophage Screening and Identification epitope is with pure
The monoclonal or polyclonal antibody of change are target molecule, biopanning phage random peptide library, you can filter out specific antibody
Binding peptide, by the binding peptide compared with native antigen sequence, discovery wherein has plenty of identical with native antigen sequence
Linear epitope；Other are then to be differed with native antigen sequence or incomplete same but intimate mimic epitope peptide.
Therefore, the present invention has carried out staphylococcus aureus FnBPA- using the experiment of display technique of bacteriophage combination animal immune protectiveness
The screening of the immune protective mimic epitope peptide of A albumen and appraisal, prevent the golden yellow of mastadenitis of cow for further exploitation
Color staphylococcus polyepitope vaccines lay the foundation.
4) to further illustrate advantages of the present invention, now the technological approaches and technique effect of the present invention are described as follows：
First, the rFnBPA-A albumen (preparation of this laboratory) of present invention purifying is immunogene, prepares rabbit-anti rFnBPA-
A protein antibodies.Antibody is purified by saturated ammonium sulfate fractional precipitation combination Protein G affinity columns.Through nucleic acid
The concentration that protein assay measures antibody purification is about 2.5mg/mL, and it is about 95% that SDS-PAGE, which analyzes its purity, indirect ELISA
Method detects its potency more than 1:838860800.The antibody purification meets to be used as target molecule elutriation phage random dodecapeptide storehouse
Condition.
Then, the present invention is carried out affine using the anti-rFnBPA-A antibody purified as target molecule to phage random dodecapeptide storehouse
Elutriation.Reduce antibody coating concentration (being down to 50 μ g/mL from 100 μ g/mL) by wheel in affine panning process and be gradually increased and wash
Tween-20 concentration (increasing to 0.5% from 0.1%) in de- liquid, 8 bacteriophage sun are obtained by 4 wheel elutriations and ELISA identifications
Property clone.Sequencing shows that 8 bacteriophage positive colonies show that 6 kinds of 12 different peptide sequences (are respectively designated as altogether with sequence analysis
P1, P2, P3, P4, P5, P6), due to this 6 displaying Epitope peptide sequences with FnBPA-A protein sequences without primary structure homology
(Score<50) mimic epitope that they are staphylococcus aureus FnBPA-A albumen, is determined, and it is further by peptide ELISA
FnBPA-A albumen and anti-rFnBPA-A antibody generation specific binding can be simulated instead really by demonstrating this 6 mimic epitope peptides
Should.
The present invention further passes through 6 mimic epitopes of indirect ELISA method and immune protective testing inspection mouse immune
Serum antibody generation is horizontal after peptide and attacks immune protective rate after poison, as a result finds that 6 mimic epitope peptides can stimulate body to produce
The specific antibody of varying level, but only mimic epitope peptide P1 and P2 has a certain degree of immune protective, and they are to exempting from
The immune protective rate of epidemic disease mouse is respectively 25% and 50%, and P1 and P2 presses 2:The peptide composition of 1 mass ratio mixing is to Huang
The immune protective effect of color staphy lococcus infection mouse is higher than FnBPA-A holoproteins, and immune protective rate is up to 75%.Therefore, originally
Immune protective mimic epitope peptide P1 and P2 in invention and combinations thereof can be used as active ingredient, be further used for multi-epitope epidemic disease
The exploitation of seedling, has a good application prospect.
Brief description of the drawings
Checkings of Fig. 1 peptides ELISA to mimic epitope peptide.
Fig. 1 shows the average OD of 6 mimic epitope peptides and anti-rFnBPA-A antibody responses492nmValue is all higher than itself and negative blood
The average OD of clearance response492nmValue 2.1 times, and with the average OD of His tag antibody reacting holes492nmIt is cloudy with rabbit that value is respectively less than it
Property the average OD in seroreaction hole492nm2.1 times of value.Illustrate that these mimic epitope peptides can simulate FnBPA-A albumen, with resisting
Specific binding reaction occurs for FnBPA-A antibody.
Embodiment
For the ease of understanding, the present invention will be described in detail by specific embodiment and accompanying drawing below.Need spy
Not, it is noted that these describe the description being merely exemplary, and it is not meant to limit the scope of the invention.
Experimental method described in the description below, it is conventional method unless otherwise specified；The reagent in experimental method
And biomaterial, unless otherwise specified, commercially obtain.
Percentage composition in the description below, it is weight/mass percentage composition unless otherwise instructed.
Ratio in the description below, it is volume ratio unless otherwise instructed.
Embodiment 1：The preparation and purification of rabbit-anti FnBPA-A protein antibodies
1.1 Antibody preparation
By the rFnBPA-A purifying proteins (preparation of this laboratory) and 42 μ LTween-80 mixing works that 1mL concentration is 1mg/mL
For aqueous phase, 3126 μ L this white oil of department and the concussion emulsification repeatedly in vortex oscillator are added.Neck, dorsal sc in experimental rabbit
The immunogene that multi-point injection has emulsified, immunizing dose are 1mg/.One, which exempts from progress two in latter 14th day, exempts from, immunizing dose and immune way
Footpath is same to exempt from.Two, which exempt from progress three in latter 10th day, exempts from, and three are not added with immunologic adjuvant, immunizing dose and immunization route when exempting from exempts from phase with one
Together.Before immune and latter 32nd day collection rabbit blood, separation serum is immunized, using 20 μ g/mL rFnBP-A protein liquids as coating
Antigen, 1:5000 goat-anti rabbit HRP-IgG are secondary antibody, and immune serum moderate resistance FnBPA-A antibody is detected using indirect ELISA method
Potency is 1:209715200.
1.2 antibody purification
IgG in immune rabbit anteserum is slightly carried using saturated ammonium sulfate precipitation classification, after desalination of dialysing, reused
Protein G affinity column IgG purifications.The concentration that antibody purification is measured through nucleic acid-protein analyzer is about 2.5mg/mL,
It is about 95% that SDS-PAGE, which analyzes its purity, and indirect ELISA method detects its potency more than 1:838860800.
Conclusion：The rabbit-anti FnBPA-A antibody of preparation and purification can be used as target molecule elutriation bacteriophage dodecapeptide storehouse.
Example 2：The epitope screening of FnBPA-A albumen and identification
2.1 carry out elutriation with rabbit-anti FnBPA-A antibody purifications to phage random dodecapeptide storehouse
Phage random dodecapeptide storehouse kit is purchased from NEB companies, and wherein storage capacity is 2.7 × 109Individual transformant, titre
For 1.5 × 1013Pfu/ μ L, Escherichia coli ER2537 are the Host Strains in peptide storehouse.Washed in a pan by kit specification
Choosing, simplified process are as follows:With 0.1mol/L pH8.6NaHCO3Rabbit-anti FnBPA-A antibody after purification is diluted to 100 μ by solution
G/mL, 96 orifice plates are coated with, 4 DEG C overnight.Next day, washed 6 times with TBST solution (TBS+0.5%Tween-20), add 5%BSA,
37 DEG C of closing 1h.Confining liquid is abandoned, by 10 μ L original peptides storehouses (1.0 × 1011PFU), the feminine gender of 195 μ L TBST and 195 μ L after purification
Serum adds enzyme mark hole after mixing, and 100 μ L/ holes, gently shakes 1h.Abandoning liquid in hole, TBST is washed 6 times, with pH2.2,
The bacteriophage that the elution of 0.2mol/L glycine-HCl elution buffers is combined with anti-FnBPA-A antibody specificities, and add 1mol/
L pH9.1Tris-HCl buffer solutions neutralize.5 μ L eluents are taken to carry out phage titre measure, remaining Phage Infection E.coli
ER2738 is expanded, and for next round elutriation, carries out four-wheel elutriation altogether.In order to obtain the specific bacteriophage of high-affinity,
Antibody concentration (being down to 50 μ g/mL from 100 μ g/mL) is reduced by wheel in four-wheel panning process, increases Tween-20 in cleaning solution
Concentration (increasing to 0.5% from 0.1%), as a result find, the input and output ratio of bacteriophage raises by wheel, be followed successively by 1.8 ×
10-5、1.9×10-4、6.5×10-4With 8.9 × 10-3, specific bacteriophage, which is cloned, to be enriched with, and the richness of elutriation is taken turns in the 2nd, 3 and 4
Collection multiple respectively reaches 11,36 and 494 times.
The 2.2 bacteriophage positive clone identifications combined with anti-FnBPA-A antibody specificities
The random 25 phage clones progress sandwich ELISA Preliminary Identification selected after fourth round elutriation.It is with concentration
100 μ g/mL rabbit-anti FnBPA-A antibody coated elisa plates, 4 DEG C overnight.Next day abandons coating buffer, adds 5%BSA, 37 DEG C of closings
1h；Confining liquid is outwelled, is washed 6 times with TBST solution.Add phage clone to be checked, 108PFU/ holes, each clone do 3 weights
It is multiple, 37 DEG C of effect 2h, set blank control (replacing phage clone to be checked with PBS) and negative control (to add former peptide storehouse in experiment
In M13 bacteriophages).TBST is washed 6 times, adds 1:5000HRP marks the anti-M13 antibody of mouse, 37 DEG C of effect 1h.Hereafter, by washing
Wash, develop the color, terminate, determine OD492nmConventional method operation.As a result there is the average OD of 18 clones to be checked492nmValue is more than original
The average OD of peptide storehouse bacteriophage negative control492nm2.1 times of value, it was initially believed that they are bacteriophage positive colony.
18 primary dcreening operation bacteriophage positive colonies are further identified using Competitive assays ELISA method.It is 100 μ i.e. with concentration
G/mL rabbit-anti FnBPA-A antibody coated elisa plates, each one row's enzyme mark hole of clone's coating to be identified, 4 DEG C overnight.Next day abandons bag
By liquid, closing, washing are same as above.Respectively by various concentrations (12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL, 100 μ g/mL and 200 μ g/
ML competitor (including rFnBPA-A albumen and His label proteins)) respectively with primary dcreening operation positive phage clones (5.0 ×
109PFU) isometric mixing, mixed liquor is taken to be added separately in antibody coating hole, 100 μ L/ holes, 37 DEG C of effect 1h, TBST washings 6
Secondary, remaining steps are same as above.Done simultaneously in experiment and do not add the primary dcreening operation positive phage clones control of competitor (to be replaced with PBS competing
Strive thing to mix in equal volume with phage clone).Inhibiting rate, inhibiting rate (%)=(do not suppress OD are calculated according to formula492nmValue-suppression
OD after system492nmValue)/do not suppress OD492nmValue × 100%.As a result as shown in table 1, with the increase of rFnBPA-A protein concentrations,
Its Competitive assays rate to C2, C8, C10, C11, C15, C19, C21 and C238 primary dcreening operation phage clones gradually increases.When
When the concentration of rFnBPA-A albumen is 200 μ g/mL, 50% is all higher than to the Competitive assays rate of 8 phage clones, and it is identical dense
The free His label proteins competitor of degree is below 20% to the inhibiting rate of above-mentioned 8 phage clones, illustrates this 8 phagocytosis
Body clone is the bacteriophage positive colony combined with rFnBPA-A antibody specificities.
Inhibiting rate of the competition albumen of the various concentrations of table 1 to phage clone
The epitope peptide analysis of 2.3 bacteriophage positive colony DNA sequencings and its surface display
Use the list of the single-stranded extracts kit of phage DNA (being purchased from Omega companies of the U.S.) extraction positive phage clones
Chain DNA, and entrust Shanghai Invitrogen Bioisystech Co., Ltd that single stranded DNA sample is sequenced.According to positive bacteriophage
The exogenous gene sequence carried in single stranded DNA, derive the Epitope peptide sequences for being showed in phage surface.As a result as shown in table 2,
8 bacteriophage positive colonies show 6 kinds of 12 different peptide sequences altogether, wherein clone C8, C11 and C21 show identical peptide sequence (life
Entitled P3), in addition 5 clone (C19, C23, C2, C10, C15) show respectively different peptide sequences (be respectively designated as P1, P2,
P4、P5、P6).The FnBPA-A protein sequences that 6 displaying Epitope peptide sequences are included with GenBank respectively are compared,
From table 3, they are with FnBPA-A albumen without primary structure homology (Score<50), show that this 6 displayed polypeptides are
The mimic epitope of FnBPA-A albumen.
The bacteriophage positive colony of table 2 shows peptide sequence
Table 3 shows the comparison result of peptide sequence and FnBPA-A protein sequences
The peptide ELISA checkings of 2.4 mimic epitope peptides
Shanghai Tao Pu Technology Co., Ltd. is entrusted to synthesize the simulation table of above-mentioned 6 FnBPA-A albumen using solid-phase synthesis
Position peptide (it is required that its purity is more than 98%), is verified using peptide ELISA to it.Simplified process is as follows:It is 40 μ g/mL with concentration
Synthesis mimic epitope peptide coating enzyme mark hole, 4 DEG C of coatings are overnight.Coating buffer is abandoned, adds 5%BSA confining liquids, 37 DEG C are closed 2h,
0.5%PBST is washed 6 times.Add 1:The rabbit-anti FnBPA-A antibody of 200 times of dilutions, while do negative serum control and 1:200
The rabbit-anti His label protein antibody controls of dilution again, 37 DEG C are reacted 1h, and 0.5%PBST is washed 6 times, adds 1:5000HRP- sheep
Anti-rabbit, 37 DEG C are incubated 1h, and 0.5%PBST is washed 6 times.Add o-phenylene diamine substrate solution, room temperature lucifuge effect 10min, add and terminate
Liquid, determine OD492nmValue.If the OD in anti-FnBPA-A antibody responses hole492nmValue be more than with 2.1 times of negative serum reacting hole, simultaneously
Rabbit-anti His label protein antibody responses hole OD492nmValue is less than 2.1 times of negative serum, then sentences peptide ELISA reactions for the positive, i.e.,
FnBPA-A antibody can identify mimic epitope.As a result as shown in figure 1,6 mimic epitope peptides and rFnBPA-A antibody responses it is flat
Equal OD492nmValue is all higher than its average OD with negative serum reaction492nmValue 2.1 times, and with His tag antibody reacting holes
Average OD492nmValue is respectively less than itself and the average OD of rabbit negative serum reacting hole492nm2.1 times of value.Illustrate these mimic epitope peptides with
The association reaction of rFnBPA-A antibody is specific, and they simulate the immune anti-of staphylococcus aureus FnBPA-A albumen
Ying Xing.
Conclusion：The mimic epitope of 6 FnBPA-A albumen is screened and identified using display technique of bacteriophage.
Example 3：The immunocompetence detection of FnBPA-A albumen mimic epitopes
The immune and immunogenicity of mimic epitope peptide of 3.1 experiment mices detects
Body weight is that 18-20g kunming mouse is randomly divided into 12 groups, every group 20.1st group is rFnBPA-A protein immunizations
Group；2nd group is bacteriophage M13 immune groups；3-8 groups are respectively that C2, C8, C10, C15, C19 and C23 positive phage clones are immunized
Group；9-11 groups are that row do not mix group to mimic epitope peptide P1 on year-on-year basis with P2；12nd group is saline control group.RFnBPA-A albumen
RFnBPA-A albumen after the injection Freund's adjuvant emulsification of immune group mouse, 30 μ g/ are only；Positive phage clones immune group mouse is noted
Penetrate phage clone, 1 × 1012Pfu/ is only；Mimic epitope peptide mixing group mouse injection Freund's adjuvant emulsification after hybrid peptide (P1 with
P2 difference in mass ratio 1:1、1:2 and 2:1 proportioning), 30 μ g/ are only；Control group mice then injecting normal saline, 100 μ L/ are only.It is first
Exempt from rear each booster immunization of 14d and 24d once, immunizing dose is exempted from head.34d takes a blood sample from eyeball of mouse veniplex after immune, and 5
Only/group, serum is separated, detecting each immune group antibody using indirect ELISA produces level.As a result as shown in table 4,6 of displaying
Mimic epitope peptide can stimulate body to produce specific antibody, show that they have immunogenicity, and with P1 and P2 immunogene
Property is preferable.
Antibody titer after table 4 is immune in each immune group mice serums of 34d
The immune protective detection of 3.2 mimic epitope peptides
35d carries out challenge test to each experimental mice after immune.I.e. with minimum lethal dose (6.25 × 106CFU/mL)
The immune mouse of staphylococcus aureus WWGSP-30 separation strains intraperitoneal injection, 15/group, 0.5mL/ only, while not to be immunized
Murine Model of Intraperitoneal Infection equivalent bacterium solution is as control.Observed 7 days after injection, record mouse survival situation, Computation immunity protective rate：
[immune protective rate=(the 1- immune groups death rate/control group death rate) × 100%].Cut open inspection is carried out to dead mouse, observes it
Pathological change, and sterile take liver separation of bacterial again.As a result as shown in table 5, bacteriophage M13 immune groups, positive bacteriophage
The mouse for cloning C2, C8, C10 and C15 immune group is all dead；Positive phage clones C19 (displaying mimic epitope peptide P1) and
The immune protective rate of C23 (displaying mimic epitope peptide P2) immune group mouse is respectively 25% and 50%；Mimic epitope peptide P1 and P2
With 1:1、1:2 and 2:1 mass is respectively 31.25%, 18.75% and 75% than the immune protective rate of mixed immunity group mouse.Extremely
It is that eye has extract or bleeding, nose and pawl point bleeding to die mouse to observe lesion；Cut open inspection lesion is pulmonary hemorrhage, quality becomes fragile,
Spleen necrosis, seroperitoneum；Gold is separated to again from liver using staphylococcus aureus Baird-Paker Selective agar mediums
Staphylococcus aureus.
The immune protective of the mimic epitope peptide of table 5
Conclusion：The simulation of 6 FnBPA-A albumen in table 2 is determined by the antibody test after animal immune and challenge test
Epitope peptide is respectively provided with immunogenicity, but only P1 and P2 is the mimic epitope peptide with immune protective, and P1 and P2 are in mass ratio
2:The immune protective effect of 1 composition is significantly better than single immune protective mimic epitope peptide and FnBPA-A albumen.
Claims (4)
1. a kind of composition of the staphylococcus aureus FnBPA-A albumen mimic epitope peptides with immune protective, its feature
It is：Said composition is by SEQ ID NO:Polypeptide shown in 1 and SEQ ID NO:Polypeptide shown in 2 presses 2:1 mass ratio combination obtains；
The SEQ ID NO:1 and SEQ ID NO:2 is as follows：
SEQ ID NO:1：His-Thr-Glu-Gln-Gly-Thr-Leu-Phe-Leu-Lys-Met-Pro；
SEQ ID NO:2：Ser-Tyr-Phe-Asp-Ala-Leu-Glu-Arg-Met-Leu-Pro-Gly.
2. the staphylococcus aureus FnBPA-A albumen mimic epitope peptides according to claim 1 with immune protective
Composition, it is characterised in that：Encoding amino acid sequence such as SEQ ID NO:The nucleotides sequence of polypeptide shown in 1 is classified as SEQ ID
NO:3, encoding amino acid sequence such as SEQ ID NO:The nucleotides sequence of polypeptide shown in 2 is classified as SEQ ID NO:4：
SEQ ID NO:3：cat acg gag cag ggg act ttg ttt ttg aag atg ccg；
SEQ ID NO:4：agt tat ttt gat gcg ctt gag agg atg ttg ccg ggg.
3. the group of the staphylococcus aureus FnBPA-A albumen mimic epitope peptides with immune protective described in claim 1
Application of the compound in staphylococcus aureus polyepitope vaccines are prepared.
4. a kind of staphylococcus aureus polyepitope vaccines, it is characterised in that containing described in claim 1 in the polyepitope vaccines
Composition.
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