CN106478777A - 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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Abstract
The present invention relates to the mimic epitope peptide of staphylococcus aureus FnBPA A albumen with immune protective, mimic epitope peptide combinations and its application.The amino acid sequence of 2 immune protective mimic epitope peptides that the present invention is provided is respectively 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 polypeptide compositions 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 test 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 mimic epitope peptide combinations are better than FnBPA A holoprotein to the immune protective effect that staphylococcus aureus infect.Therefore, mimic epitope peptide of the present invention and combinations thereof can be as active ingredient, for the exploitation of staphylococcus aureus polyepitope vaccines, the mastadenitis of cow for preventing staphylococcus aureus to cause.
Description
Technical field
The present invention relates to epitope peptide, the staphylococcus aureus FnBPA-A egg more particularly to 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
Opportunistic pathogen, can not only cause the multiple diseases of the mankind, and often cause clinical type and recessive mastadenitis of cow.For a long time, due to beast
Clinically unreasonable use antibiotic is cured, induced animal source property methicillin-resistant staphylococcus aureus bacterial strain continuously emerges, no
Only result in medical treatment mastadenitis of cow very difficult, serious harm is brought to dairy, and affect the product of milk and milk productses
Matter and safety and human health.Therefore, it is that immunity is 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 epi-position development of new staphylococcus aureus polyepitope vaccines
Technical barrier that those skilled in the art made great efforts solve then is always.
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.
For reaching above-mentioned purpose, one of the technical solution used in the present invention is as follows：
A kind of staphylococcus aureus FnBPA-A albumen mimic epitope peptide 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-Lys-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 be artificial synthesized using conventional method, it is also possible to using gene work
Journey technique construction recombinant expression plasmid, abduction delivering are obtained.Therefore, the 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 that offer is a kind of containing the aforementioned staphylococcus aureus with immune protective
The composition of FnBPA-A albumen mimic epitope peptide, 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 is obtained.
Corresponding with the second object of the present invention, present invention also offers a kind of containing aforementioned with immune protective
The mimic epitope tandem polypeptide of staphylococcus aureus FnBPA-A albumen mimic epitope peptide, the mimic epitope tandem polypeptide is to adopt 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 third ammonia of polypeptide-alanine 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 is 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 aforementioned staphylococcus aureus FnBPA-A egg with immune protective
Application of the white mimic epitope peptide in staphylococcus aureus polyepitope vaccines are prepared.
Likewise, a kind of present invention also offers aforementioned staphylococcus aureus FnBPA-A egg with immune protective
Application of the white mimic epitope peptide combinations in staphylococcus aureus polyepitope vaccines are prepared.
Present invention also offers a kind of aforementioned staphylococcus aureus FnBPA-A albumen simulation table with immune protective
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 peptide with immune protective, can be effective so as to the polyepitope vaccines
The mastadenitis of cow that prevention staphylococcus aureus causes.
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, with rFnBPA-A purifying protein as immunogene, prepare the anti-FnBPA-A antibody of rabbit；The anti-FnBPA-A antibody of the rabbit
Preparation process be：Concentration is that the rFnBPA-A purifying protein of 1mg/mL and Tween-80 are mixed as water phase, adds department
The simultaneously concussion emulsification repeatedly in vortex oscillator of this white oil, the rFnBPA-A purifying protein, Tween-80, takes charge of the body of this white oil
Product is than being 1:0.042:3.126；Then the immunogene that neck in experimental rabbit, dorsal sc multi-point injection are emulsified, immunizing agent
Measure as 1mg/ only, one exempt from after carry out two within the 14th day and exempt 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 nothing immunologic adjuvant, three exempt from dosage and immunization route exempt from one identical；Exempt from latter 32 days collection rabbit blood isolated in three
Rabbit anteserum is to obtain the anti-FnBPA-A antibody of the rabbit；
The purifying of the anti-FnBPA-A antibody of S2, the rabbit：Slightly extracted in the rabbit anteserum using ammonium sulfate graded precipitation
IgG, obtains the anti-FnBPA-A antibody of rabbit of purifying after desalination of dialysing using Protein G affinity column IgG purification；
S3, affine elutriation, elutriation are carried out to phage random dodecapeptide storehouse using the anti-FnBPA-A antibody of rabbit of the purifying
The storage capacity of the phage random dodecapeptide storehouse kit for using is 2.7 × 109, titre be 1.5 × 1013/μL、
Escherichia coli ER2537 is the Host Strains in peptide storehouse；The panning process is：NaHCO3 with 0.1mol/L, pH8.6
Anti- for rabbit after purification FnBPA-A antibody is diluted to 100 μ g/mL by solution, is coated enzyme mark hole, and 4 DEG C overnight；Next day, washed with TBST
Wash liquid to wash 6 times, 5%BSA confining liquid is added, 37 DEG C of closing 1h, containing 0.5%Tween-20 in the TBST cleaning solution；It
After abandon confining liquid, by original peptide storehouse, TBST cleaning solution, 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；Abandon enzyme mark in the hole liquid again, washed using TBST cleaning solution and use after 6 times
Glycine-HCl the elution buffer of 0.2mol/L, pH2.2 elutes the bacteriophage combined by FnBPA-A antibody specificity anti-with rabbit,
And add the Tris-HCl buffer solution of 1mol/L, pH9.1 to neutralize；Taking elution buffer described in 5 μ L carries out phage titre measure,
Remaining Phage Infection Host Strains E.coli ER2738 is expanded, and for next round elutriation, carries out four-wheel elutriation altogether to obtain
The higher specific bacteriophage positive colony of affinity；Anti- by the anti-FnBPA-A of reduction rabbit after purification is taken turns in four-wheel panning process
Body diluted concentration, the concentration by Tween-20 in the wheel increase TBST cleaning solution, wherein described in fourth round elutriation after purification
The anti-FnBPA-A antibody diluted concentration of rabbit be 50 μ g/mL, described in fourth round elutriation in TBST cleaning solution Tween-20 concentration
Increase to 0.5%；
S4, the specific bacteriophage positive colony DNA sequencing and epi-position peptide analysis：Carry using phage DNA is single-stranded
The single stranded DNA that kit extracts the specific bacteriophage positive colony is taken, and DNA sequencing is 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 mould for being showed in phage surface is 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
Staphylococcus aureus clinical separation strain produce highly conserved stick fibroin, the albumen can be present in blood plasma, body fluid and
Fibrin-specific in extracellular matrix is combined, so that bacterium is effectively attached in host tissue, causes infection to occur.
Research shows that FnBPA protein structure includes tetra- functional areas of A, B, C, D, and wherein A area is not only the main work of adhesion of the albumen
Energy domain, can be specifically bound with fibrin, and the starting stage that sets up in infection plays a significant role, and FnBPA-A
The antibody that protein induced body is produced can resist infection of staphylococcus aureus.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 solving aforementioned skill
The point of penetration of art problem, is finally to solve technical problem to provide good basis.
2) staphylococcus aureus virulence factor is numerous and mechanism of causing a disease is complicated, prepares vaccine with single protective antigens,
The immanoprotection action induced by which is limited, and this is accomplished by for 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 for building subunit's chimeric are restricted, even go out
The problems such as existing chimeric protein expression is low or does not express.However, epi-position (epitope) is the critical segment that antigen plays a role,
Polyepitope vaccines built-up for protective epitope's series connection of multiple antigens can either be excluded immunity nothing in whole protein molecular
Composition or immune tolerance composition is closed, the more effective protective immune response of more complete albumen is induced, expression vector can be overcome again
Capacity limit to multiple antigen chimeric expressions.
Therefore, the invention provides simulating 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 epi-position or the mimic epitope with immune protective, and bites
Phage display technique is to study the effective means that epi-position is provided.The principle of display technique of bacteriophage Screening and Identification epi-position be with pure
The monoclonal of change or polyclonal antibody are target molecule, biopanning phage random peptide library, you can filter out specific antibody
Binding peptide, the binding peptide is compared with native antigen sequence, and discovery wherein has plenty of identical with native antigen sequence
Linear epitope；Other are then to differ with native antigen sequence or incomplete same but intimate mimic epitope peptide.
Therefore, the present invention has carried out staphylococcus aureus FnBPA- using display technique of bacteriophage with reference to the test of animal immune protectiveness
The screening of the immune protective mimic epitope peptide of A albumen and appraisal, are the golden yellow of exploitation prevention mastadenitis of cow further
Color staphylococcus polyepitope vaccines lay the foundation.
4) for further illustrating advantages of the present invention, now the technological approaches and technique effect to the present invention is described as follows：
First, the rFnBPA-A albumen (prepared by this laboratory) of present invention purifying is immunogene, prepares the anti-rFnBPA- of rabbit
A protein antibodies.Purified with reference to Protein G affinity column antagonist by saturated ammonium sulfate fractional precipitation.Through nucleic acid
Protein assay measures the concentration of antibody purification and is about 2.5mg/mL, SDS-PAGE and analyzes its purity and is about 95%, indirect ELISA
Method detects its potency more than 1:838860800.The antibody purification is met as target molecule elutriation phage random dodecapeptide storehouse
Condition.
Then, the present invention is carried out affine with the anti-rFnBPA-A antibody for purifying as target molecule to phage random dodecapeptide storehouse
Elutriation.Be coated concentration (50 μ g/mL being down to from 100 μ g/mL) and be gradually increased by wheel reduction antibody in affine panning process wash
Tween-20 concentration (increasing to 0.5% from 0.1%) in de- liquid, obtains 8 bacteriophage sun through 4 wheel elutriations and ELISA identification
Property clone.With sequence analysis, sequencing shows that 8 bacteriophage positive colonies show that 6 kinds of 12 different peptide sequences (are respectively designated as altogether
P1, P2, P3, P4, P5, P6), as this 6 show Epitope peptide sequences with FnBPA-A protein sequence all nothing primary structure homology
(Score<50) their mimic epitopes for staphylococcus aureus FnBPA-A albumen, are determined, and further by peptide ELISA
Demonstrate this 6 mimic epitope peptides and can simulate really FnBPA-A albumen with anti-rFnBPA-A antibody generation specific binding instead
Should.
The present invention passes through 6 mimic epitopes of indirect ELISA method and immune protective testing inspection mouse immune further
Serum antibody generation level and immune protective rate after poison is attacked after peptide, as a result find that 6 mimic epitope peptides can all stimulate body to produce
The specific antibody of varying level, but only mimic epitope peptide P1 and P2 have 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 holoprotein, 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 further used for multi-epitope epidemic disease as active ingredient
The exploitation of seedling, has a good application prospect.
Description of the drawings
Checking of Fig. 1 peptide ELISA to mimic epitope peptide.
Fig. 1 shows the average OD of 6 mimic epitope peptides and anti-rFnBPA-A antibody response492nmValue is all higher than which with negative blood
The average OD of clearance response492nm2.1 times of value, and the average OD with His tag antibody reacting hole492nmIt is cloudy with rabbit that value is respectively less than which
The property average OD in seroreaction hole492nm2.1 times of value.Illustrate that these mimic epitope peptides can all simulate FnBPA-A albumen, and anti-
There is specific binding reaction in FnBPA-A antibody.
Specific embodiment
For the ease of understanding, below the present invention will be described in detail by specific embodiment and accompanying drawing.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 technique described in the description below, if no special instructions, is conventional method；The reagent in experimental technique
And biomaterial, if no special instructions, all commercially obtain.
Percentage composition in the description below, if no special instructions, is weight/mass percentage composition.
Ratio in the description below, if no special instructions, is volume ratio.
Embodiment 1：The preparation of the anti-FnBPA-A protein antibodies of rabbit and purifying
1.1 Antibody preparation
By 1mL concentration for 1mg/mL rFnBPA-A purifying protein (this laboratory prepare) and 42 μ L Tween-80 mixing
As water phase, 3126 μ L this white oil of department are added and concussion emulsification repeatedly in vortex oscillator.Neck in experimental rabbit, back skin
The immunogene that lower multi-point injection is emulsified, immunizing dose are 1mg/.One exempt from after carry out two within the 14th day and exempt from, immunizing dose and immunity
Approach is same to be exempted from.Two exempt from after carry out three within the 10th day and exempt from, three are not added with immunologic adjuvant when exempting from, immunizing dose and immunization route exempt from phase with one
With.32nd day collection rabbit blood before the immunity and after immunity, serum is separated, with 20 μ g/mL rFnBP-A protein liquids as being coated
Antigen, 1:5000 goat-anti rabbit HRP-IgG are anti-for two, using anti-FnBPA-A antibody in indirect ELISA method detection immune serum
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, is reused
Protein G affinity column IgG purification.The concentration for antibody purification being measured through nucleic acid-protein analyzer is about 2.5mg/mL,
SDS-PAGE analyzes its purity and is about 95%, and indirect ELISA method detects its potency more than 1:838860800.
Conclusion：Preparing can be used as target molecule elutriation bacteriophage dodecapeptide storehouse with the anti-FnBPA-A antibody of the rabbit of purifying.
Example 2：The epitope screening of FnBPA-A albumen and identification
2.1 carry out elutriation with the anti-FnBPA-A antibody purification of rabbit to phage random dodecapeptide storehouse
Phage random dodecapeptide storehouse kit is purchased from NEB company, and wherein storage capacity is 2.7 × 109Individual transformant, titre
For 1.5 × 1013Pfu/ μ L, Escherichia coli ER2537 is the Host Strains in peptide storehouse.Washed in a pan by kit specification
Choosing, simplified process are as follows:With 0.1mol/L pH8.6 NaHCO3Anti- for rabbit after purification FnBPA-A antibody is diluted to 100 by solution
μ g/mL, is coated 96 orifice plates, and 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 peptide storehouses (1.0 × 1011PFU), 195 μ L TBST and 195 μ L are after purification
Addition enzyme mark hole after negative serum mixing, 100 μ L/ holes, gently shake 1h.Abandon in the hole liquid, TBST is washed 6 times, with pH2.2,
The bacteriophage combined with anti-FnBPA-A antibody specificity by 0.2mol/L glycine-HCl elution buffer wash-out, and add 1mol/
L pH9.1Tris-HCl buffer solution is neutralized.Taking 5 μ L eluents carries 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,
Reduce AC (50 μ g/mL being down to from 100 μ g/mL) by wheel in four-wheel panning process, increase Tween-20 in cleaning solution
Concentration (increasing to 0.5% from 0.1%), as a result find, the input and output ratio of bacteriophage is raised by wheel, be followed successively by 1.8 ×
10-5、1.9×10-4、6.5×10-4With 8.9 × 10-3, specific bacteriophage clone 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 identification combined with anti-FnBPA-A antibody specificity
Selecting 25 phage clones after fourth round elutriation at random carries out sandwich ELISA Preliminary Identification.With concentration it is
The anti-FnBPA-A antibody coated elisa plate of the rabbit of 100 μ g/mL, 4 DEG C overnight.Next day abandons and is coated liquid, adds 5%BSA, 37 DEG C of closings
1h；Confining liquid is outwelled, and 6 times is washed with TBST solution.Add phage clone to be checked, 1083 weights are done in PFU/ hole, each clone
Multiple, 37 DEG C of effect 2h, blank (replacing phage clone to be checked with PBS) and negative control (plus former peptide storehouse is set in experiment
In M13 bacteriophage).TBST is washed 6 times, adds 1:The anti-M13 antibody of 5000HRP mark 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 identified further using Competitive assays ELISA method.It is 100 μ i.e. with concentration
The anti-FnBPA-A antibody coated elisa plate of the rabbit of g/mL, each clone to be identified are coated row's enzyme mark hole, and 4 DEG C overnight.Next day abandons bag
By liquid, close, wash ibid.Respectively by variable 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 protein)) respectively with primary dcreening operation positive phage clones (5.0 ×
109PFU) equal-volume mixing, takes mixed liquor and is added separately to antibody and be coated in hole, 100 μ L/ holes, 37 DEG C of effect 1h, TBST washings 6
Secondary, remaining steps are ibid.The primary dcreening operation positive phage clones control of competitor is not added (to be replaced with PBS competing while doing in test
Strive thing to mix with phage clone equal-volume).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 concentration,
Its Competitive assays rate to 8 primary dcreening operation phage clones of C2, C8, C10, C11, C15, C19, C21 and C23 is gradually increased.When
When the concentration of rFnBPA-A albumen is 200 μ g/mL, the Competitive assays rate to 8 phage clones is all higher than 50%, and identical dense
The free His label protein competitor of degree is below 20% to the inhibiting rate of above-mentioned 8 phage clones, and this 8 phagocytosis are described
Body clone is the bacteriophage positive colony combined with rFnBPA-A antibody specificity.
The competition inhibiting rate of the albumen to phage clone of 1 variable concentrations of table
2.3 bacteriophage positive colony DNA sequencings and its epi-position peptide analysis of surface display
The list of positive phage clones is extracted using the single-stranded extracts kit of phage DNA (purchased from Omega company of the U.S.)
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, derives 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 peptide sequence (life identical with C21 displaying
Entitled P3), in addition 5 clones (C19, C23, C2, C10, C15) show respectively different peptide sequence (be respectively designated as P1, P2,
P4、P5、P6).The FnBPA-A protein sequence that 6 displaying Epitope peptide sequences are included with GenBank respectively compares,
From table 3, they are with FnBPA-A albumen all nothing primary structure homology (Score<50), show that this 6 displayed polypeptides are
The mimic epitope of FnBPA-A albumen.
2 bacteriophage positive colony of table shows peptide sequence
Table 3 shows the comparison result of peptide sequence and FnBPA-A protein sequence
The peptide ELISA checking 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 (requires its purity more than 98%), which is verified using peptide ELISA.Simplified process is as follows:It is 40 μ g/mL with concentration
Synthesis mimic epitope peptide be coated enzyme mark hole, 4 DEG C are coated overnight.Abandon and liquid be coated, 5%BSA confining liquid is added, 37 DEG C are closed 2h,
0.5%PBST is washed 6 times.Add 1:The anti-FnBPA-A antibody of rabbit of 200 times of dilutions, while do negative serum control and 1:200
The anti-His label protein antibody control of rabbit of times dilution, 37 DEG C of reactions 1h, 0.5%PBST are washed 6 times, add 1:5000HRP- sheep
Anti-rabbit, 37 DEG C of incubations 1h, 0.5%PBST wash 6 times.Plus o-phenylene diamine substrate solution, room temperature lucifuge effect 10min, add and terminate
Liquid, determines OD492nmValue.If the OD in anti-FnBPA-A antibody response hole492nmValue is more than and 2.1 times of negative serum reacting hole, while
The anti-His label protein antibody response hole OD of rabbit492nmLess than 2.1 times of negative serum, then it is the positive to sentence peptide ELISA reaction to value, i.e.,
FnBPA-A antibody is capable of identify that mimic epitope.As a result as shown in figure 1,6 mimic epitope peptides are flat with rFnBPA-A antibody response
All OD492nmValue is all higher than its average OD with negative serum reaction492nmValue 2.1 times, and with His tag antibody reacting hole
Average OD492nmValue is respectively less than its OD average with 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 the immunity that they simulate staphylococcus aureus FnBPA-A albumen is anti-
Ying Xing.
Conclusion：The mimic epitope of 6 FnBPA-A albumen is screened and is identified using display technique of bacteriophage.
Example 3：The immunocompetence detection of FnBPA-A albumen mimic epitope
The immunity of 3.1 experiment mices is detected with the immunogenicity of mimic epitope peptide
Body weight is randomly divided into 12 groups for the kunming mouse of 18-20g, per group 20.1st group is rFnBPA-A protein immunization
Group；2nd group is bacteriophage M13 immune group；3-8 group is respectively the immunity of C2, C8, C10, C15, C19 and C23 positive phage clones
Group；9-11 group for mimic epitope peptide P1 and P2 not on year-on-year basis row mix group；12nd group is saline control group.RFnBPA-A albumen
RFnBPA-A albumen after the emulsification of immune group injected in mice Freund's adjuvant, 30 μ g/ are only；Positive phage clones immune group mouse is noted
Phage clone is penetrated, 1 × 1012Pfu/ is only；Mimic epitope peptide mixing group injected in mice 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.First
Exempt from each booster immunization of rear 14d and 24d once, immunizing dose is exempted from head.After immunity, 34d is taken a blood sample from eyeball of mouse veniplex, and 5
Only/group, separates serum, detects that each immune group antibody produces level using indirect ELISA.As a result as shown in table 4,6 of displaying
Mimic epitope peptide can all stimulate body to produce specific antibody, show that they have immunogenicity, and the immunogene with P1 and P2
Property is preferable.
Antibody titer after 4 immunity of table in each immune group mice serum of 34d
The immune protective detection of 3.2 mimic epitope peptides
After immunity, 35d carries out challenge test to each experimental mice.I.e. with minimum lethal dose (6.25 × 106CFU/mL)
Staphylococcus aureus WWGSP-30 separation strains lumbar injection immune mouse, 15/group, 0.5mL/ only, while with not immunity
Murine Model of Intraperitoneal Infection equivalent bacterium solution is used as control.Observe 7 days after injection, record mouse survival situation, Computation immunity protective rate：
[immune protective rate=(the 1- immune group death rate/control group death rate) × 100%].Cut open inspection is carried out to dead mouse, observes which
Pathological change, and aseptic take liver separation of bacterial again.As a result as shown in table 5, bacteriophage M13 immune group, positive bacteriophage
The mouse of clone's C2, C8, C10 and C15 immune group is all dead；Positive phage clones C19 (show mimic epitope peptide P1) and
The immune protective rate of C23 (showing 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
The mouse that dies observes pathology has extract or bleeding, nose and pawl point bleeding for eye；Cut open inspection pathology is pulmonary hemorrhage, quality becomes fragile,
Spleen necrosis, seroperitoneum；Gold is separated to from liver again using staphylococcus aureus Baird-Paker Selective agar medium
Staphylococcus aureus.
The immune protective of 5 mimic epitope peptide of table
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 are 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 (10)
1. a kind of staphylococcus aureus FnBPA-A albumen mimic epitope peptide with immune protective, it is characterised in that：Described
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:2
Shown：
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-Lys-Pro-Gly.
2. the staphylococcus aureus FnBPA-A albumen mimic epitope peptide with immune protective according to claim 1,
It is characterized in that：Encoding amino acid sequence such as SEQ ID NO:The nucleotide sequence SEQ ID NO of 1 mimic epitope peptide:3
Shown, 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.
3. a kind of staphylococcus aureus FnBPA-A albumen simulation with immune protective containing described in claim 1
The composition of epitope peptide, it is characterised in that：Said composition is by SEQ ID NO:Polypeptide shown in 1 and SEQ ID NO:Polypeptide shown in 2
By 2:1 mass ratio combination is obtained.
4. a kind of staphylococcus aureus FnBPA-A albumen simulation with immune protective containing described in claim 1
The mimic epitope tandem polypeptide of epitope peptide, it is characterised in that：The mimic epitope tandem polypeptide be using technique for gene engineering by SEQ ID
NO:Polypeptide shown in 1 and SEQ ID NO:Polypeptide shown in 2 carries out expressing in series acquisition, and series system is：SEQ ID NO:Shown in 1
Polypeptide-alanine alanine tyrosine joint-SEQ ID NO:Polypeptide shown in 1-alanine alanine tyrosine joint-SEQ ID
NO:Polypeptide shown in 2.
5. the staphylococcus aureus FnBPA-A albumen mimic epitope peptide with immune protective according to claim 4
Composition, it is characterised in that：The nucleotide sequence such as SEQ ID NO of coding foregoing immune protectiveness mimic epitope tandem polypeptide:5
Shown：
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.
6. the staphylococcus aureus FnBPA-A albumen mimic epitope peptide with immune protective described in claim 1 is in system
Application in standby staphylococcus aureus polyepitope vaccines.
7. the staphylococcus aureus FnBPA-A albumen mimic epitope peptide combination with immune protective described in claim 3
Application of the thing in staphylococcus aureus polyepitope vaccines are prepared.
8. the mould of the staphylococcus aureus FnBPA-A albumen mimic epitope peptide with immune protective described in claim 4
Intend epi-position tandem polypeptide, the application in staphylococcus aureus polyepitope vaccines are prepared.
9. a kind of staphylococcus aureus polyepitope vaccines, it is characterised in that containing described in claim 1 in the polyepitope vaccines
The staphylococcus aureus FnBPA-A albumen mimic epitope peptide with immune protective.
10. a kind of staphylococcus aureus FnBPA-A albumen with immune protective as claimed in claim 1 or 2 is simulated
The screening technique of epitope peptide, comprises the following steps：
S1, with rFnBPA-A purifying protein as immunogene, prepare the anti-FnBPA-A antibody of rabbit；The system of the anti-FnBPA-A antibody of the rabbit
For process it is：Concentration is that the rFnBPA-A purifying protein of 1mg/mL and Tween-80 are mixed as water phase, adds department's raw white
Oil simultaneously concussion emulsification repeatedly in vortex oscillator, the rFnBPA-A purifying protein, Tween-80, takes charge of the volume ratio of this white oil
For 1:0.042:3.126；Then the immunogene that neck in experimental rabbit, dorsal sc multi-point injection are emulsified, immunizing dose is
1mg/ only, one exempt from after carry out two within the 14th day and exempt from, two exempt from dosage and immunization route is same exempts from, two exempt from after carry out three within the 10th day and exempt from, three
Exempt from nothing immunologic adjuvant, three exempt from dosage and immunization route exempt from one identical；Exempt from latter 32 days collection rabbit blood isolated rabbit blood in three
Clearly to obtain the anti-FnBPA-A antibody of the rabbit；
The purifying of the anti-FnBPA-A antibody of S2, the rabbit：IgG in the rabbit anteserum is slightly extracted using ammonium sulfate graded precipitation,
The anti-FnBPA-A antibody of rabbit of purifying is obtained after desalination of dialysing using Protein G affinity column IgG purification；
S3, affine elutriation is carried out to phage random dodecapeptide storehouse using the anti-FnBPA-A antibody of rabbit of the purifying, elutriation is used
The phage random dodecapeptide storehouse kit storage capacity be 2.7 × 109, titre be 1.5 × 1013/μL、
Escherichia coli ER2537 is the Host Strains in peptide storehouse；The panning process is：NaHCO3 with 0.1mol/L, pH8.6
Anti- for rabbit after purification FnBPA-A antibody is diluted to 100 μ g/mL by solution, is coated enzyme mark hole, and 4 DEG C overnight；Next day, washed with TBST
Wash liquid to wash 6 times, 5%BSA confining liquid is added, 37 DEG C are closed 1h, contain 0.5%Tween-20 in the TBST cleaning solution, it
After abandon confining liquid, by original peptide storehouse, TBST cleaning solution, 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；Abandon enzyme mark in the hole liquid again, washed using TBST cleaning solution and use after 6 times
Glycine-HCl the elution buffer of 0.2mol/L, pH2.2 elutes the bacteriophage combined by FnBPA-A antibody specificity anti-with rabbit,
And add the Tris-HCl buffer solution of 1mol/L, pH9.1 to neutralize；Taking elution buffer described in 5 μ L carries out phage titre measure,
Remaining Phage Infection Host Strains E.coli ER2738 is expanded, and for next round elutriation, carries out four-wheel elutriation altogether to obtain
The higher specific bacteriophage positive colony of affinity；Anti- by the anti-FnBPA-A of reduction rabbit after purification is taken turns in four-wheel panning process
Body diluted concentration, the concentration by Tween-20 in the wheel increase TBST cleaning solution, wherein described in fourth round elutriation after purification
The anti-FnBPA-A antibody diluted concentration of rabbit be 50 μ g/mL, described in fourth round elutriation in TBST cleaning solution Tween-20 concentration
Increase to 0.5wt%；
S4, the specific bacteriophage positive colony DNA sequencing and epi-position peptide analysis：Examination is extracted using phage DNA is single-stranded
Agent box extracts the single stranded DNA of the specific bacteriophage positive colony, and carries out DNA sequencing to the single stranded DNA sample, according to
The exogenous gene sequence carried in the single stranded DNA sample, derives the FnBPA-A albumen simulation table for being showed in phage surface
Position peptide sequence.
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| CN111387133A (en) * | 2020-02-25 | 2020-07-10 | 中国农业大学 | Method for establishing model for inhibiting mouse mastitis by using Klebsiella pneumoniae bacteriophage |
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| CN111387133A (en) * | 2020-02-25 | 2020-07-10 | 中国农业大学 | Method for establishing model for inhibiting mouse mastitis by using Klebsiella pneumoniae bacteriophage |
| CN111387133B (en) * | 2020-02-25 | 2021-04-16 | 中国农业大学 | Method for establishing model for inhibiting mouse mastitis by using Klebsiella pneumoniae bacteriophage |
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