CN105384800B - Staphylococcus aureus TAF fusion protein preparation method and applications - Google Patents
Staphylococcus aureus TAF fusion protein preparation method and applications Download PDFInfo
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- CN105384800B CN105384800B CN201510889714.7A CN201510889714A CN105384800B CN 105384800 B CN105384800 B CN 105384800B CN 201510889714 A CN201510889714 A CN 201510889714A CN 105384800 B CN105384800 B CN 105384800B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
- C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
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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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- 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
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
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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
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Abstract
The present invention relates to a kind of staphylococcus aureus FnBPA immunodominance segments, and provide the encoding gene of the immunodominance segment.The invention further relates to a kind of fusion proteins containing staphylococcus aureus FnBPA immunodominance segment, it is to be formed by the TRAP albumen of staphylococcus aureus, alpha hemolysin immunodominance segment and staphylococcus aureus FnBPA immunodominance segment composition, and provide encoding gene, preparation method and the purposes of the fusion protein.Using TAF fusion protein of the invention to mouse immune challenge test, shows that TAF fusion protein has preferable immune protective effect, be the ideal vaccine antigen of staphylococcus aureus, there is important value in terms of the development and application of new generation vaccine.
Description
Technical field
The invention belongs to genetic engineering fields, specifically, being related to a kind of staphylococcus aureus TAF fusion protein preparation
Method and its application.
Background technique
Staphylococcus aureus (Staphylococcus aureus, S.aureus) can cause mankind's multi-infection, and
A kind of important pathogenic bacteria of mastitis for milk cows.As a large amount of appearance of antibody-resistant bacterium and antibiotic curative effect are gradually lost, vaccine immunity is connect
Kind becomes the optimal selection of prevention S.aureus infection.Staphylococcus aureus generates a large amount of virulence factor, can be by viscous
Attached, immune evasion and the aggressive factors such as toxin and enzyme of generation are caused a disease, and therefore, effective S.aureus vaccine should be directed to
More antigen vaccines of the above link are blocked simultaneously.
Staphylococcus aureus FnBPA is important adhesion molecule, and has good antigenicity.Trap has anti-oxidant
Stress, and can induce good immune protective effect.Alpha hemolysin is the important exotoxin of S.aureus, and is caused
The major virulent factor of pneumonia has good immunogenicity.Up to the present, in relation to FnBPA, Trap and Hla fusion protein
Immunogenicity and immanoprotection action have not been reported.
Summary of the invention
The first object of the present invention is to provide a kind of staphylococcus aureus FnBPA immunodominance segment and encodes the advantage
The gene of segment.
The second object of the present invention is to provide the fusion egg containing above-mentioned staphylococcus aureus FnBPA immunodominance segment
Gene that is white and encoding the fusion protein.
The third object of the present invention is to provide the preparation method of above-mentioned fusion protein.
The fourth object of the present invention is to provide the purposes of above-mentioned fusion protein.
The present invention is achieved through the following technical solutions:
One, a kind of staphylococcus aureus FnBPA immunodominance segment, including 301-430 in FnBPA albumen n-quadrant
Two segments of 801-874 amino acids of FnBPAr10-11, amino acid sequence in amino acid and the region FnBPA albumen r1-11
As shown in SEQ ID No.2.
Further, the gene of above-mentioned immunodominance segment is encoded, nucleotide sequence is as shown in SEQ ID No.1.
Further, above-mentioned staphylococcus aureus FnBPA immunodominance segment is in preparation staphylococcus aureus epidemic disease
Application in seedling.
Two, a kind of staphylococcus aureus TAF fusion protein is TRAP albumen, alpha hemolysis by staphylococcus aureus
Plain immunodominance segment and staphylococcus aureus FnBPA immunodominance segment composition form, amino acid sequence such as SEQ ID
Shown in No.4.
Further, the gene of above-mentioned fusion protein is encoded, nucleotide sequence is as shown in SEQ ID No.3.
Three, the preparation method of above-mentioned fusion protein, it is heterologous including being carried out with above-mentioned gene cloning into prokaryotic cell
Expression, and purify the fusion protein.
Four, above-mentioned fusion protein is preparing the application in S. aureus vaccines.
The purpose of the present invention can also be further achieved by the following technical measures.
First using staphylococcus aureus gene group as template, is expanded and obtained in FnBPA albumen n-quadrant by PCR method
301-430 amino acids (FnBPA301-430aa) genetic fragment and the region FnBPA albumen r1-11 in FnBPAr10-11 801-
874 amino acids (FnBPA801-874aa) genetic fragment, then manually peptide linker by FnBPA301-430aaAnd FnBPA801-874aa
Fusion is named as FL, and is studied by immanoprotection action, selects and determined FnBPA immunodominance area (FL).It is basic herein
On, design primer is expanded the fusion for obtaining Trap, HlaD and FL by Overlap extension PCR method, and the gene is inserted into
To expression vector pET-32a, Escherichia coli are converted, can get the soluble fusion protein of high level expression after IPTG is induced
TAF。
The 6 continuous His residue energy and Ni contained using the fusion protein N-terminal2+The characteristic that column combines is selected
MagneHisTM protein purification system is purified, and fusion protein after purification is mixed with vaccine immunity with Freund's adjuvant
Then mouse carries out immunogenicity and immanoprotection action research.
Good effect by adopting the above technical scheme: after vaccine immune mouse made from TAF fusion protein of the invention, inspection
It is horizontal to survey humoral and cellular immune response response, and carries out attacking poison, as a result confirms that the amalgamation protein vaccine has preferable immunoprotection
Property effect, be to prepare the ideal candidate antigens of S. aureus vaccines, i.e., fusion protein of the invention have preferably exempts from
Epidemic focus and immanoprotection action；In addition, TAF fusion protein of the invention not only further improves anti-Staphylococcus aureus
The protective effect of infection, and preparation process is simplified, there is important value in terms of the development and application of new generation vaccine.
Detailed description of the invention
Fig. 1 is FnBPA301-430aa, the PCR product electrophoretic analysis result of FnBPA801-874aa, fl.Wherein, A is
The PCR product of FnBPA301-430aa, 390bp；B is the PCR product of FnBPA801-874aa as a result, 222bp；C is the PCR of fl
Product is as a result, 657bp；Wherein in each figure, M is DNA Marker DL5000；1 is negative control；2 be PCR product result；
Fig. 2 is recombinant plasmid FnBPA301-430aa-pET32a, FnBPA801-874aa-pET32a and fl-pET32a table
Digestion identification and PCR qualification result up to carrier recombinant plasmid.A figure is FnBPA301-430aa-pET32a digestion identification and PCR
Identification；B figure is FnBPA801-874aa-pET32a digestion identification and PCR identification；C figure is fl-pET32a digestion identification and PCR
Identification.Wherein, the M in each figure is DNA Marker DL5000；1 is the qualification result of recombinant plasmid；2 be recombinant plasmid
Digestion qualification result；
Fig. 3 is recombination FnPBA301-430aa, FnBPA801-874aa and FL protein expression and purification result.Scheming A is
FnBPA301-430aa；Figure B is FnBPA801-874aa；Figure C is FL.Wherein, 1 in each figure is the recombinant bacterium not induced；
2-6 is the recombinant bacterium that IPTG induces 1-5h respectively；7 be protein purification result；M is protein Marker；
Fig. 4 is to attack malicious result (n=after mouse is immunized in recombinant protein FnBPA301-430aa, FnBPA801-874aa and FL
10).Wherein, figure A attacks survival number after poison for Newman plants of experimental animal S.aureus；Figure B is experimental animal S.aureus
46 plants of Wood are attacked survival number after poison；Figure C is HLJ23-1 plants of experimental animal S.aureus and attacks survival number after poison；
Fig. 5 is the PCR product electrophoretic analysis result of HlaD, Trap and fl.Wherein A is the PCR product of HlaD, 459bp；B
For Trap PCR product as a result, 498bp；C is the PCR product of fl as a result, 657bp；M is DNA Marker DL5000；
Fig. 6 is TAF gene PCR amplification procedure result.Wherein, A is Trap+linker gene PCR amplified production；B is
Linker+HlaD+linker gene PCR amplified production；C is Trap+linker+HlaD+linker gene PCR amplified production；D
For Linker+fl gene PCR amplified production；E is Trap+Linker+HlaD+Linker+fl (TAF) gene amplification product；
Fig. 7 is recombinant plasmid Trap-pET32a, HlaD-pET32a, TAF-pET32a double digestion qualification result；
Fig. 8 is that the SDS-PAGE of recombinant protein Trap, HlaD, FL, TAF analyze result.Wherein, 1 swimming lane is protein molecular
Measure Marker；2,6,10,14 swimming lanes are pET32a empty vector control；3-5 swimming lane is respectively before recombinant protein FL is induced, after induction
And albumen after purification；49.1KD；7-9 swimming lane be respectively recombinant protein HlaD induction before, after induction and albumen after purification；
36.8KD；11-13 swimming lane be respectively Trap protein induced before, after induction and after purification albumen；38.2KD；15-17 swimming lane difference
For before recombinant protein TAF induction, after induction and albumen after purification；82.4KD；
Fig. 9 attacks poison for lung and organizes immune mouse lung bacterial content (Newman)；
Figure 10 is Trap-HlaD-FL (TAF) fusion protein amplification method.
Specific embodiment
The source of biomaterial in the present invention:
1, staphylococcus aureus: Newman plants, Wood46 plants of staphylococcus aureus and HLJ23-1, golden yellow grape
The immunogenicity of coccus agglutination factor A, Feng Hao, Liu Lefeng, Chi Jiaqi, Wang Ning, the intercalation graceful, Tong Chunyu of Lee, horse principal column, Zhu Zhanbo,
Cui Yudong, bioengineering journal, 2009,25 (8): 1180-1186；In addition, the ecological patent is also disclosed in patent " golden yellow Portugal
In the preparation and application of grape coccus IsdBid-TRAP fusion protein ", the patent No.: 201110231144.4, the applying date
2011.08.12, publication date 2011.12.14 and patent " staphylococcus aureus ITC fusion protein and preparation method and application "
In, the patent No.: in 201310480520.2, applying date 2013.10.15, publication date 2014.02.12；
2, recombinant plasmid FnBPA-pET32a: staphylococcus aureus Trap, FnBPA and alpha hemolysin fusion protein immunization
Originality research, Liu Daolong, Master's thesis, conferring unit: Heilongjiang Bayi Agricultural Reclamation University, tutor: Cui Yudong professor, publication date
Phase: 2014；
3, recombinant plasmid Trap-pET32a: staphylococcus aureus IsdB3 and TRAP fusion protein immunization protective effect is ground
Study carefully, Wang Ning, Master's thesis, conferring unit: Heilongjiang Bayi Agricultural Reclamation University, tutor: Cui Yudong professor, publication date: 2009；
In addition, the ecological patent is also disclosed in patent " preparation and application of staphylococcus aureus IsdBid-TRAP fusion protein ",
The patent No.: in 201110231144.4, applying date 2011.08.12, publication date 2011.12.14；
4, recombinant plasmid HlaD-pET28a: the new generation vaccine of prevention staphylococcus alpha hemolysin pneumonia, Dai Jian, master's opinion
Text, conferring unit: Heilongjiang Bayi Agricultural Reclamation University, tutor: Cui Yudong professor, publication date: 2013；
5, all primers are designed, designed and Shanghai Sangon Biological Engineering Technology And Service Co., Ltd are entrusted to synthesize.
Below with reference to embodiment and test example, the following further describes the technical solution of the present invention, but should not be construed as pair
Limitation of the invention:
Embodiment 1
This example demonstrates that staphylococcus aureus FnBPA immunodominance segment (FL) is selected and is determined.
1, referring to the FnBPA gene order delivered, the data of acquisition and the epitope analysis of online software are studied by the base
Because sequence is divided into two sections, 301-430 amino acids in FnBPA albumen n-quadrant are taken to be named as FnBPA301-430aa, design is up and down
Trip primer is F1, R1；FnBPAr10-11 (801-874aa) in the region FnBPAr1-11 is taken to be named as FnBPA801-874aa, if
Meter upstream and downstream primer is F2, R2；Artificial peptide fragment rigidity Linker (LAEAAAKEAAAKAAA) is designed by FnBPA801-874aa
It is merged with FnBPA301-430aa, is named as FL, connection F3, F10 upstream and downstream primer difference are designed using overlap PCR method
For P2, P3；Specific Primers of coding, Primer location, Primer sequence and expanding fragment length are shown in
Table 1；Underscore represents the restriction enzyme site introduced, and upstream introduces BamH I, and downstream introduces Hind III.
1 PCR primer of table and Linker design
2, the culture of FnBPA-pET32a recombinant bacterium and plasmid extract
The recombinant bacterium containing FnBPA-pET32a recombinant plasmid for taking -70 DEG C of preservations is containing ammonia benzyl resistance (Amp+) LB
It crosses in solid agar plate, 37 DEG C of overnight incubations, next day sterile working picking single colonie is added cultivates containing Amp+ resistance LB
In base, 37 DEG C of culture 16h of 180rpm/min or so, sterile working takes 2ml bacterium solution to be added in 2.0tube pipe, 12000rpm/
Min is centrifuged 1min, abandons net supernatant.Plasmid extracts experiment reagent box (GT Plasmid miniprep purification
Kit it) extracts, by specification carries out.2-5 μ l is taken to verify into 1% agargel electrophoresis the plasmid of extraction.
3, target gene expands
PCR reaction is carried out according to following scheme.Micro-reaction pipe is placed on ice, is sequentially added to specifications, it is (sterile
DdH2O14.8 μ l, 10 × PCR buffer 2 μ l, 1 μ l of oligonucleotides, 1 μ l, Template DNA of upstream and downstream primer (20 μM)
(200ng/ μ l) 1 μ l, DNA polyase (5U/ μ l) 0.2 μ l is used for PCR amplification.
(1) amplification of FnBPA301-430aa, FnBPA801-874aa genetic fragment
Using FnBPA-pET32a plasmid as template DNA, using F1, R1 as primer amplification FnBPA301-430aa gene, instead
Answer condition as follows: after 94 DEG C of initial denaturation 10min, 95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 60s carry out 30 and follow
Ring, last 72 DEG C of extensions l0min, 4 DEG C of preservations after PCR product verifying.
(2) amplification of fl genetic fragment
Using the PCR product of FnBPA301-430aa after purification as template, using F1, P2 as primer amplification fragment upstream FL1, instead
Answer in condition annealing temperature be 60 DEG C remaining with FnBPA301-430aa gene amplification method；It is purified with FnBPA801-874aa
PCR product is template afterwards, and respectively with P3, R2 is primer amplification downstream FL2 segment, in reaction condition annealing temperature be 59 DEG C remaining
With FnBPA301-430aa gene amplification method；With FL1 after purification, the PCR product of FL2 is template (according to DNA concentration with 1:1
Template is added in ratio), it is that upstream and downstream primer expands target fragment FL with F1, R2, gradient exploration annealing temperature is in reaction condition
54 DEG C remaining with f3 gene amplification method, PCR product carries out the piece that FL mesh is recycled after 1% agarose gel electrophoresis is verified
Section is that upstream and downstream primer carries out amplification purification again, PCR product with F1, R2 with target fragment FL gene template after the recovery
4 DEG C of preservations after the verifying of 1% agarose gel electrophoresis.
PCR product is verified with 1% agarose gel electrophoresis, and electrophoresis result is distinguished at 390bp, 222bp and 657bp
There is electrophoresis band, it is in the same size with expected amplified production target fragment, as shown in Figure 1.
4, PCR product recycling and purifying
After pcr amplification product in Ago-Gel electrophoresis, cut under ultraviolet lamp solidifying containing target DNA fragment
Glue recycles with DNA gel QIAquick Gel Extraction Kit (DNA Gel Extraction Kit) and purifies DNA, and detailed process is according to explanation
Book carries out.
5, the connection conversion of PCR product and pMD18-T carrier
In PCR micro-reaction pipe, according to pMD18-T Vector kit kit specification, pMD18-T is sequentially added
0.5 μ l of Vector, after purified 4.5 μ l, Solution I of PCR product, 5.0 μ l is mixed, 16 DEG C of water-bath connections are overnight.So
Afterwards, connection product full dose is added in competent cell, mixes, places 30min on ice, then 42 DEG C of heat shock 90s, pay attention to
It not shake, quickly pipe is transferred in ice bath, make the cooling 2min of cell, be added be preheating to 37 DEG C of antibiotic-frees in advance immediately
800 μ L LB culture mediums, the mild shaken cultivation 1h of 37 DEG C of air tables, culture brief centrifugation take 200 μ L supernatants, uniformly apply
It is distributed in containing 50 μ g/mL Amp+LB plate on, plate is inverted after liquid is absorbed, is incubated overnight in 37 DEG C of incubators.
6, the identification of recombinant plasmid
FnBPA301-430aa-pMD18T, FnBPA801-874aa-pMD18T and fl-pMD18T recombinant plasmid are by following
System progress double digestion identification (30 5 μ l, BamH I of μ l, 10 × Buffer, 2.5 μ l, Hind III of recombinant plasmid, 2.5 μ l,
50 μ l of ddH2O total system).37 DEG C of water-bath 3h after mixing take 5 μ l loadings, verify in 1% agarose gel electrophoresis.It will identify just
True positive strain send Beijing Jin Weizhi company to carry out gene sequencing, will send out in sequencing result BLSAT method and GenBank
The standard sequence of table is compared.
7, recombinant protein Prokaryotic expression vector construction
PET-32a plasmid is subjected to double digestion with restriction endonuclease BamHI and Hind III, is purified/is returned with DNA
Kit is received to be recycled.Simultaneously by recombinant clone plasmid FnBPA301-430aa-pMD18, FnBPA801-874aa-pMD18,
Fl-pMD18 carries out double digestion with restriction endonuclease BamHI and Hind III respectively, purified with DNA/QIAquick Gel Extraction Kit returns
Receive target gene FnBPA301-430aa, FnBPA801-874aa and fl segment.In the PCR reaction tube that 3 sterilize, respectively will
Target gene FnBPA301-430aa, FnBPA801-874aa and fl genetic fragment and carrier pET-32a double digestion segment, in T4
The lower connection of Ligase effect overnight, is then converted by preceding method, is sent out after being analyzed after digestion by 1% agargel electrophoresis
It is existing, there is target gene band and carrier segments band in desired location；Correct plasmid will be identified as template, by setting
The primer of meter carries out PCR verifying, 1% agargel electrophoresis result occur purpose band and with target gene size one after double digestion
It causes, as shown in Figure 2.It will identify that correct plasmid is transferred in Rosetta (DE3) competent cell, serve the raw work bioengineering in sea
Company's sequencing.Obtained sequence is compared with Blast program with the standard sequence that oneself delivers in GenBank, and homology reaches
100%.The recombinant clone for being accredited as positive strain is named as FnBPA301-430aa-pET32a, FnBPA801-874aa-
pET32a、fl-pET32a。
8, the inducing expression of recombinant protein
By the recombinant plasmid containing FnBPA301-430aa-pET32a, FnBPA801-874aa-pET32a, fl-pET32a
Rosetta (DE3) expresses strain inoculated in the 50mL LB liquid medium containing Amp+, 37 DEG C of cultures to OD600Value is 0.6
When, IPTG to final concentration of 1mmol/L is added, continues violent shaken cultivation, takes 2mL bacterium solution in 1h, 2h, 3h, 4h respectively,
12000r/min is centrifuged 1min, outwells supernatant, 90 1 × sample-loading buffers of μ L and 10 μ L 1M DTT are added in precipitating, blows and beats
It mixes, boils 5min, 10 μ L is taken to be separated by electrophoresis in 10% SDS-PAGE.Gel is taken out after electrophoresis, and it is bright to be placed in coomassie
In blue R-250 dyeing liquor, 1h being dyed on decolorization swinging table, discards dyeing liquor, after eluting gel with distilled water, destainer is added,
Decolourize 1h on shaking table, when blue background disappears, purpose band is clear, is rinsed with clear water, photograph saves.
By with protein molecular weight Marker and induction 0h compared with pair, the destination protein size after induction is respectively
39.3KD (FnBPA301-430aa), 33.1KD (FnBPA801-874aa) and 49.1KD (FL), with destination protein after purification
It is in the same size, as shown in Figure 3.
9, the purifying of recombinant protein
Recombinant bacteria culture grows to A600Value is 0.6-0.8, and IPTG to final concentration 1mM/L is added, and continues shaken cultivation
The A of 4-5h measurement induction bacterium600Value.Then, with the MagneHis of Promega companyTMProtein purification kit is purified, pure
Albumen carries out purity verifying by SDS-PAGE after change.
10, animal immune and challenge viral dosage
Purifying protein and isometric Freund's adjuvant are emulsified, immunogene is prepared.Take 150 Healthy females, 6 weeks
Age, 16~18g of weight SPF grade ICR mouse be randomly divided into 5 groups, every group of 30 (FnBPA group, FnBPA301-430aaImmune group,
FnBPA801-874aaImmune group, FL experimental group, Mock group).Wherein it is further divided into 3 groups for every group, every group 10, respectively
S.aureus Newman, Wood46, HLJ23-1 attack malicious group；Separately take 25 Healthy females, 6 week old, 16~18g of weight SPF
Grade blab/c mouse is randomly divided into 5 groups of (FnBPA group, FnBPA301-430aa immune groups, FnBPA801-874aa immune group, FL
Group, Mock group).Every mouse uses 100 μ g recombinant protein antigen of intramuscular injection, and control group injects isometric adjuvant and PBS
Mixture, after first immunisation 21d carry out second it is immune, 14d carries out abdominal cavity and attacks poison after secondary immunity, daily to observe the dead of mouse
Situation is died, the record time is 10d.
Each experiment immunization group uses staphylococcus aureus Newman, Wood46, HLJ23-1 bacterial strain after booster immunization 14d
It carries out attacking poison with absolute lethal dose, attack observation experiment dead mouse situation after poison and records data.The results show that FL experimental group pair
The immune protective rate for attacking toxic bacterial strain is respectively 60% (Newman plants), 60% (Wood46 plants) and 80% (HLJ23-1 plants),
FnBPA301-430aa experimental group attack against each other toxic bacterial strain immune protective rate be respectively 40% (Newman plants), 30% (Wood46
Strain) and 40% (HLJ23-1 plants), the attack against each other immune protective rate of toxic bacterial strain of FnBPA801-874aa experimental group is respectively 50%
(Newman plants), 50% (Wood46 plants) and 50% (HLJ23-1 plants), FnBPA experimental group are attacked against each other the immune protective rate of toxic bacterial strain
Respectively 80% (Newman plants), 70% (Wood46 plants) and 80% (HLJ23-1 plants), as shown in Figure 4.
It is described above, FL have with immunogenicity similar in FnBPA and immanoprotection action, be immunodominance segment.
Embodiment 2
This example demonstrates that the preparation of staphylococcus aureus TAF fusion protein.
1, according to this experiment obtain FL and this laboratory early period obtain albumen HlaD and Trap, gene order just
Under the premise of really, according to the gene order delivered, using Primer5 software Design primers and artificial peptide fragment Linker.Specifically
It is shown in Table shown in 2 and amplification method schematic diagram (Figure 10).Underscore is restriction enzyme site in table, and primer italic is Linker.Primer
PT, RT expand destination protein Trap, and primer PH, RH expand destination protein HlaD, and primer PF, RF expand destination protein FL.
The design scheme of table 2 primer and linker
2, the amplification of Trap, HlaD and FL target gene
PCR amplification method: micro-reaction pipe is put under low temperature, and agents useful for same is added according to specification, (sterile
Each (30 μM) 1.5 μ l, Template of ddH2O12.5 μ l, 10 × PCR buffer 2 μ l, 1 μ l of oligonucleotides, upstream and downstream primer
1 μ l of DNA (200ng/ μ l), 0.5 μ l of archaeal dna polymerase (5U/ μ l) carry out PCR amplification.The amplification of Trap gene: with Trap-
PET32a is template, and PT1 and RT2 are upstream and downstream primer；Reaction condition is as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 55
DEG C annealing 30s, 72 DEG C of extensions 60s, 30 recycle；72 DEG C of extension 10min.The amplification of HlaD gene: it is saved with laboratory
HlaD-pET28a is template, and PH1 and RH2 are upstream and downstream primer；Reaction condition is the same as Trap gene PCR amplification condition.Fl gene
Amplification: slightly, see aforementioned.1% agarose gel electrophoresis of PCR amplification result product is verified.Electrophoresis result is in 459bp, 498bp
With occur electrophoresis band at 657bp respectively, it is in the same size with expected amplified production target fragment, as shown in Figure 5.
3, TAF gene design scheme
Using overlapping primers PCR method.Specifically:
(1) using Trap gene after purification as template, using PT1 and P4 as upstream and downstream primer.Reaction condition is as follows: 94 DEG C pre-
It is denaturalized 5min；94 denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 60s and 30 circulations, 72 DEG C all extend 10min.Product passes through
1% Ago-Gel carries out electrophoresis verifying, obtains size 543bp target fragment A.
(2) using HlaD gene after purification as template, using P5 and P6 as upstream and downstream primer.Reaction condition is as follows: 94 DEG C pre-
It is denaturalized 5min；94 denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 60s and 30 circulations, 72 DEG C all extend 10min.Product passes through
The verifying of 1% agarose gel electrophoresis, this obtains the target fragment B of size 549bp.
(3) using the A of recovery purifying, 1 B gene segment as template, PT1 and P6 are upstream and downstream primer.Reaction condition is as follows: 94
DEG C initial denaturation 5min；94 denaturation 30s, 66 DEG C of annealing 30s, 72 DEG C of extension 60s and 25 circulations, 72 DEG C all extend 10min.It produces
Object is verified by 1% agarose gel electrophoresis.Using recovery product as template, PT1 and P6 are that upstream and downstream primer carries out PCR again.
Reaction condition is as follows: 94 DEG C of initial denaturation 5min；94 denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 60s and 30 circulations, 72 DEG C complete
Portion extends 10min.Product is verified by 1% agarose gel electrophoresis, obtains the target fragment C of size 1047bp.
(4) using the FL genetic fragment after recovery purifying as template, using P7 and RF2 as upstream and downstream primer.94 DEG C of initial denaturations
5min；Reaction condition is as follows: 94 denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 60s and 30 circulations, 72 DEG C all extend
10min.Product is verified by 1% agarose gel electrophoresis, obtains the target fragment D of size 702bp.
(5) using C, D genetic fragment of recovery purifying as template, PT1 and RF2 are upstream and downstream primer.Reaction condition is as follows: 94
DEG C initial denaturation 5min；94 denaturation 30s, 68 DEG C of annealing 30s, 72 DEG C of extension 60s and 25 circulations, 72 DEG C all extend 10min.Product
It is verified by 1% agarose gel electrophoresis.Using recovery product as template, PT1 and RF2 are that upstream and downstream primer carries out PCR again.Instead
Answer condition as follows: 94 DEG C of initial denaturation 5min；94 denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 60s and 30 circulations, 72 DEG C of wholes
Extend 10min.Product is verified by 1% agarose gel electrophoresis, obtains the target fragment E of expected size 1704bp, is this reality
Test TAF target gene fragment.
As a result as shown in Figure 6.
4, the building, identification and expression of fusion TAF prokaryotic expression carrier
Specific method is shown in embodiment one.
Correct double digestion target fragment will be sequenced and carry out gene recycling, recovery product is the same as III pair of enzyme of BanmH I and Hind
The expression vector pET32a cut is attached conversion culture, extracts single colonie, and amplification cultivation extracts plasmid, plasmid BanmH I
There is the purpose band of expected size in 1% gel electrophoresis analysis with III digestion verification of Hind, carrier size is 5900bp,
As shown in Figure 7.
Take the correct plasmid of identification to be transformed into expression bacterium Rosetta (DE3), cultivated in Amp+ resistance LB culture medium to
Behind OD6000.4~0.6, IPTG to final concentration 1mM is added, 5h bacterium solution after taking induction preceding and induction carries out SDS-PAGE after processing
Verifying.Purify destination protein by ni-sepharose purification method, SDS-PAGE analyzed, as the result is shown 49.1KD (FL),
It is identical as expected size that band occur in 36.8KD (HlaD), 38.2KD (Trap), 82.4KD (TAF), as shown in Figure 8.Albumen point
Son amount Marker red stripes size is 72KD.
Embodiment 3
This example demonstrates that TAF, Trap, HlaD and FL protein immunization Contrast on effect.
1, animal immune and challenge test
SPF grade ICR mouse 180 of healthy, female, identical week old are taken, abdominal cavity group and lung's group are divided into.Wherein abdominal cavity
Group is divided into Trap experimental group, FL experimental group, TAF experimental group, mixed protein group (Mixture group) and Mock group, and every group 20；Lung
Portion's group is divided into HlaD experimental group, TAF experimental group, mixed protein group (Mixture group) and Mock group, and every group 20.Purify each reality
Histone 1 mg/ml is tested, is emulsified with the ratio of 1:1 and not formula Freund's complete adjuvant, every 200 μ l mouse leg muscles injection is exempted from
Epidemic disease；21d after first immunisation emulsifies albumen and incomplete Freund's adjuvant with 1:1 ratio, and leg muscle injection is reinforced exempting from
Epidemic disease.
This experiment takes source of people S.aureus Newman and ox source S.aureus HLJ23-1 reference culture to carry out attacking poison.It takes
The mouse peritoneal that the bacterium solution diluted exempts from rear 14d to two attacks poison and lung fills lung and attacks poison, and records mouse state and dead daily
Number is died, continues one week, dead mouse is dissected under germ-free condition and separates germ, analyzing whether it is experiment and attacking toadstool leads to mouse
It is dead.
It is specifically grouped using SPF grades of ICR mouse for model when test and is shown in Table 3.
The grouping of 3 abdominal cavity group mouse model of table
The 14d staphylococcus aureus Newman after booster immunization, HLJ23-1 bacterial strain are (golden yellow with absolute lethal dose
Newman plants of staphylococcus with 1.5 × 1010CFU/mouse and staphylococcus aureus HLJ23-1 separation strains with 1.0 ×
1010The dosage of CFU/mouse) it carries out mouse peritoneal and attacks poison, it attacks observation experiment dead mouse situation after poison and records data, be shown in Table
4.Attacking malicious Newman plants of TAF immune protective rate is 90%, and attacking malicious HLJ23-1 plants of TAF immune protective rate is 80%.
4 chamber group mouse model of table attacks malicious result
Mouse lung attack poison group use Newman plants of staphylococcus aureus with 5 × 108CFU/mouse and HLJ23-1
Square separation strains are with 4 × 108The dosage of CFU/mouse carries out, and attacks observation experiment dead mouse situation after poison and records data, is shown in Table
5.Attacking malicious Newman plants of TAF immune protective rate is 100%, and attacking malicious HLJ23-1 plants of TAF immune protective rate is 90%.
5 lung's group mouse model of table attacks malicious result
2, antibody level
IgG content in isolated serum sample is detected with indirect ELISA.Large-scale purification tests histone TAF, HlaD, FL,
Trap is spare.
For envelope antigen with 10 μ g/mL concentration, every 100 μ L of hole is coated with 96 hole elisa Plates, 37 DEG C two hours, then use PBST
Washing.Every hole adds the PBST confining liquid of 100 μ L5% skimmed milks, 37 DEG C of closing 1h, washing；Every hole is added that PBS is diluted to be checked to exempt from
Epidemic disease serum, 37 DEG C of incubation 1h, washing；Every hole addition diluted Goat anti-Mouse IgG-Peroxidase secondary antibody of PBS, 37
DEG C be incubated for 1h, washing；100 μ L TMB developing solutions are added in every hole, and the sulfuric acid end of 50 μ L 2M is added in every hole after color development at room temperature 10min
It only reacts, surveys OD450nm light absorption value.Analyze result.When sample OD450 value >=(+3 times of standard sides of OD450 value of negative serum
Difference) when be the positive.
The result shows that recombinant protein TAF antibody titer highest, is higher than Trap, HlaD and FL experimental group.
Each experimental group Serum Antibody IgG potency ratio of table 6
3, cytokines measurement
IL-4, INF- γ content are detected with Elispot method.IL-2, IL-17 content are detected with ELISA method.Press cell
The operating procedure of factor detection reagent box carries out.
Lymphocyte IL-4, the IFN-γ secretion of each experiment immunization group and control group mice are measured using ELISPOT method
Horizontal (table 7).IL-4, INF- γ content are higher than other each experimental group significant difference (P < in recombinant protein TAF experimental group
0.05), more significant (p < 0.01) with control group comparing difference.
Each experimental group cell factor IL-4, the INF- γ content of table 7
It is measured in each experiment recombinant protein immune group and control immune group Mouse spleen cells supernatant using ELISA method
IL-2, IL-17 secretion level (table 8).Acquired results show recombinant protein immune group mouse spleen lymphocyte supernatant through t- inspection
IL-2, IL-17 secretory volume are significant (P < 0.05) with other each experimental group comparing differences in liquid, and difference is relatively aobvious compared with the control group
It writes (p < 0.01).
8 IL-2, IL-17 content analysis of table
4, mouse lung bacterium bearing capacity is tested
The pathological relation between mouse symptoms of pneumonia and pathogen is detected, after experiment mice attacks malicious a period of time (for 24 hours),
Disconnected neck is put to death, its lung is taken in gnotobasis, claims its weight, and the sterile PBS of 2ml is added with 1mg ratio, is added after being fully ground
For 7ml sterile PBS buffer to 10ml, 200 μ l supernatants carry out continuous doubling dilution, after dilution in TSA culture medium coated plate
It counts, records colony count in each experimental group and control group, and analyze data result.
As a result: recombinant protein TAF experimental group lung bearing capacity is minimum, and HlaD immune group is secondly, control group is minimum, such as Fig. 9
It is shown.
The above result shows that the immune effect of TAF amalgamation protein vaccine of the present invention individually or is blended in one better than each albumen
It plays immune immune effect, is to prepare the ideal candidate antigens of S. aureus vaccines, i.e., fusion protein tool of the invention
There are better immunogenicity and immanoprotection action.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (4)
1. a kind of staphylococcus aureus TAF fusion protein, it is characterised in that: be by staphylococcus aureus TRAP albumen,
Alpha hemolysin immunodominance segment and staphylococcus aureus FnBPA immunodominance segment composition form, and amino acid sequence is such as
Shown in SEQ ID No.4.
2. encoding the gene of fusion protein described in claim 1, nucleotide sequence is as shown in SEQ ID No.3.
3. the preparation method of fusion protein described in claim 1, including with the gene cloning described in claim 2 to protokaryon
Heterogenous expression is carried out in cell, and purifies the fusion protein.
4. fusion protein described in claim 1 is preparing the application in S. aureus vaccines.
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| CN1953991A (en) * | 2004-05-21 | 2007-04-25 | 惠氏公司 | Altered fibronectin-binding protein of staphylococcus aureus |
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| CN103570835A (en) * | 2013-10-15 | 2014-02-12 | 黑龙江八一农垦大学 | Staphylococcus aureus ITC (Inverse Transition Cycling) fusion protein as well as preparation method and application thereof |
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| EP0397633B1 (en) * | 1989-05-11 | 1995-08-02 | Alfa-Laval Agri International Ab | Fibronectin binding protein as well as its preparation |
| CN1953991A (en) * | 2004-05-21 | 2007-04-25 | 惠氏公司 | Altered fibronectin-binding protein of staphylococcus aureus |
| CN102304185A (en) * | 2011-09-02 | 2012-01-04 | 黑龙江八一农垦大学 | Fusion protein surface immunogenic protein-target of RNA III activating protein (SIP-TRAP) for preventing bovine mastitis and preparation method and use thereof |
| CN103570835A (en) * | 2013-10-15 | 2014-02-12 | 黑龙江八一农垦大学 | Staphylococcus aureus ITC (Inverse Transition Cycling) fusion protein as well as preparation method and application thereof |
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