CN105713096B - A kind of preparation and application of the ATT fusion protein preventing infection of staphylococcus aureus - Google Patents
A kind of preparation and application of the ATT fusion protein preventing infection of staphylococcus aureus Download PDFInfo
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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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
- C07K14/40—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Candida
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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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Abstract
It is 9 amino acid for being connected to Candida albicans Als3T cell epitope with Linker before the amino acid sequence of staphylococcus aureus TRAP fusion protein, the amino acid sequence of fusion protein is as shown in SEQ ID No.2 the present invention relates to a kind of ATT fusion protein.The invention further relates to the preparation methods and application of the gene of coding ATT fusion protein, ATT fusion protein.ATT fusion protein immunization Mice Inoculated of the invention, and carry out immune detection and challenge test, the result shows that ATT fusion protein can induce body to generate stronger immune response and anti-Staphylococcus aureus infection effect, it is the prevention ideal vaccine candidate antigen of infection of staphylococcus aureus.
Description
Technical field
The invention belongs to genetic engineering field, it is related to a kind of ATT fusion protein for preventing infection of staphylococcus aureus
Preparation and application.
Background technique
Staphylococcus aureus (Staphylococcus aureus, S.aureus) is generally existing in a kind of nature
Gram-positive pathogenic bacterium, be to cause mankind hospital and the most commonly seen reason of Nosocomial Infections, seriously endanger human health.
S.aureus is also the main pathogen of mastitis for milk cows, causes huge economic losses to milk industry.In the past to staphylococcus aureus
Infection mainly uses antibiotic treatment, but being widely used with antibiotic, a large amount of drug-fast bacterias occurs, makes antibacterials curative effect
It is bad.More and more people recognize that vaccine is the first choice for preventing infection of staphylococcus aureus.Studies have shown that anti-golden yellow Portugal
In grape coccus course of infection, cellullar immunologic response especially Th1 and Th17 cell plays key effect, can adjust enhancing CD8+
T cell and B cell reaction by congenital immunity recruiting cells to inflammation part and enhance its to the killing of S.aureus, removing
Ability.
Staphylococcus aureus TRAP (target of RNAIII-activating protein) has anti-oxidation stress
Effect can prevent DNA of bacteria gene mutation because of caused by oxidation, and TRAP can induce body and generate good immune guarantor
Shield effect.Als3 is the adhesion factor of Candida albicans, body can be induced to generate strong cellullar immunologic response, to golden yellow
Staphylococcus has cross immunity effect.Further research confirms, can there is a t cell epitope in Als3 albumen
Body is induced to generate good Th1 and Th17 cellullar immunologic response.In order to further enhance TRAP induction body generation resist it is golden yellow
Color aureus immunity protective effect, more effectively prevents infection of staphylococcus aureus, it may be considered that by Staphylococcus aureus
T cell epitope in bacterium TRAP albumen and Als3 albumen carries out amalgamation and expression, to obtain better immunization.
Summary of the invention
The first object of the present invention is to provide a kind of ATT fusion protein, by S. aureus TRAP protein and Als3
T cell epitope in albumen carries out amalgamation and expression and forms, to obtain better immunization.
The second object of the present invention is to provide the encoding gene of above-mentioned ATT fusion protein.
The third object of the present invention is to provide the preparation method of above-mentioned ATT fusion protein.
The fourth object of the present invention is to provide the application of above-mentioned ATT fusion protein.
The present invention is achieved through the following technical solutions:
One, a kind of ATT fusion protein is used before the amino acid sequence of staphylococcus aureus TRAP fusion protein
Linker is connected to 9 amino acid of Candida albicans Als3T cell epitope, the amino acid sequence of fusion protein such as SEQ ID
Shown in No.2.
Two, the gene of above-mentioned ATT fusion protein is encoded, nucleotide sequence is as shown in SEQ ID No.1.
Three, the preparation method of above-mentioned ATT fusion protein, be above-mentioned gene cloning is carried out into prokaryotic cell it is heterologous
Expression, purifies to obtain fusion protein.
Four, above-mentioned ATT 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 with staphylococcus aureus
Genome is template, is expanded by PCR method and obtains trap genetic fragment.Design primer later includes Candida albicans in primer
The gene and Linker of the t cell epitope of bacterium Als3.The fusion for obtaining Trap and Als3T cell epitope is expanded by PCR method
Gene, and the gene is inserted into expression vector pET-28a, Escherichia coli are converted, can get high level table after IPTG is induced
The soluble fusion protein ATT reached.
The present invention further provides above-mentioned fusion proteins to prepare the application in S. aureus vaccines.Specifically:
The 6 continuous His residue energy and Ni contained using the fusion protein N-terminal2+The characteristic that column combines selects MagneHisTM albumen
Purification system is purified, and fusion protein after purification is mixed with vaccine immune mouse with Freund's adjuvant, is then carried out
Immunogenicity and immanoprotection action research.
Good effect by adopting the above technical scheme: the present invention chooses the t cell epitope and TRAP protein fusion of Als3, system
It is standby and immunogenicity research to be carried out to it at recombination ATT fusion protein, it as a result confirms the t cell epitope of Als3 and TRAP egg
It can induce body after white fusion and generate preferable immanoprotection action, be to prepare the ideal candidate of S. aureus vaccines to resist
There is better immunogenicity and induction more body to generate strong immanoprotection action for original, i.e., fusion protein of the invention.
Detailed description of the invention
Fig. 1 is ATT fusion protein amplification method schematic diagram；
Fig. 2 is ATT, the PCR product electrophoretic analysis result of TRAP.Wherein, 1 be ATT PCR product, 558bp；2 be TRAP
PCR product as a result, 498bp；M is DNA Marker DL8000；
Fig. 3 is the enzyme of recombinant plasmid ATT-pET28a, TRAP-pET28a and eAls-pET28a expression vector recombinant plasmid
Cut identification and PCR qualification result.A figure is ATT-pET28a digestion identification and PCR identification；B figure is TRAP-pET28a digestion identification
It is identified with PCR；C figure is eAls-pET28a digestion identification.Wherein, the M in each figure is DNA Marker DL8000；A figure and B
In figure 1 be recombinant plasmid qualification result；2 be the digestion qualification result of recombinant plasmid；1 reflects in C figure for the digestion of recombinant plasmid
Determine result；
Fig. 4 is recombination ATT, TRAP and eAls protein expression and purification result.Figure A is ATT；Figure B is TRAP；Scheming C is
eAls.Wherein, 1 in each figure is the pET-28a not induced；2 be the pET-28a after IPTG induction；3 be the recombination not induced
Bacterium；4 be the recombinant bacterium after IPTG induction；5 be protein purification result；M is protein Marker；
Fig. 5 is to attack malicious result (n=10) after mouse is immunized in recombinant protein ATT, TRAP, eAls+TRAP and eAls.Wherein,
Figure A is Newman plants of experimental animal S.aureus and attacks survival number after poison；Figure B is that 46 plants of experimental animal S.aureus Wood is attacked
Survival number after poison；
Fig. 6 is the analysis result that ATT, TRAP, eAls+TRAP and eAls bis- exempts from IgG subclass in rear mice serum.
Fig. 7 is the secretion level that ATT, TRAP, eAls+TRAP and eAls bis- exempts from IFN-γ in rear Mouse spleen cells.
Fig. 8 is that ATT, TRAP, eAls+TRAP and eAls bis- exempts from IL-4, IL-10, IL-17A in rear Mouse spleen cells
Secretion level.
Specific embodiment
The source of biomaterial in the present invention:
1, staphylococcus aureus type strain Newman: the immunogenicity of staphylococcus aureus 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 be also disclosed in patent " staphylococcus aureus IsdBid-TRAP fusion protein
In preparation and application ", the patent No.: 201110231144.4, applying date 2011.08.12, publication date 2011.12.14 and patent
In " staphylococcus aureus ITC fusion protein and preparation method and application ", the patent No.: 201310480520.2, the applying date
2013.10.15 in publication date 2014.02.12；
2, recombinant plasmid pET32a-TRAP: 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；
3, all primers are designed, designed and the raw work biotechnology in Shanghai are entrusted to take Co., Ltd's synthesis.
Below with reference to specific embodiment, the following further describes the technical solution of the present invention, but should not be construed as to this
The limitation of invention:
Embodiment 1
This example demonstrates that the preparation of ATT fusion protein and corresponding experiment GAP-associated protein GAP.
1, the design and synthesis of primer
Referring to the staphylococcus aureus trap gene order delivered on NCBI, using Oligo6.67 and DNAStar software
2 pairs of PCR primers are designed, the upstream and downstream primer of amplification ATT albumen is F1, R1；Expand TRAP albumen upstream and downstream primer be F2,
R2.Specific expanding fragment length, number, primer location and sequence are shown in Table 1 and Fig. 1.For further orientation clone, in upstream
5 ' end of primer introduces BamH I, introduces Hind IIII in 3 ' end of downstream primer, and underscore represents restriction enzyme site,
Wave represents Linker (GSGSGSGS).Above-mentioned primer takes Co., Ltd by the raw work biotechnology in Shanghai and synthesizes.
1 PCR primer of table and Linker design
2, the culture of TRAP-pET32a recombinant bacterium and plasmid extract
It takes -70 DEG C of glycerol frozen to save bacterium DH5 α-pET32a-TRAP recombinant bacteriums, is being contained with connecing collarium and picking bacterium solution
It crosses on the LB solid medium of Amp+, after 37 DEG C are incubated overnight, the bacterium of next day sterile working picking median size, neat in edge
It falls, aseptic inoculation is in 4mL LB/Amp+ fluid nutrient medium, 37 DEG C of shake culture 12h of 180rpm/min.Sterile working is drawn
For 2ml bacterium solution in 2.0tube pipe, 12000rpm/min is centrifuged 2min, abandons net supernatant, and plasmid extracts experiment reagent box (GT
Plasmid miniprep purification kit) it extracts, by specification carries out.Take 2-5 μ l collection liquid solidifying in 1% agar
It is verified in gel electrophoresis.
3, target gene expands
PCR reaction system and step carry out in accordance with the following methods: PCR pipe being placed on ice, successively to specifications successively
After following reagent is added, PCR amplification is carried out.
The amplification of ATT fusion protein gene fraction:
Using pET32a-TRAP recombinant plasmid as template, PCR amplification is carried out by primer of F1, R1 respectively, reaction condition is such as
Under: after 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 45s, 58 DEG C of annealing 40s, 72 DEG C of extension 40s carry out 30 and recycle, and last 72 DEG C
Extend 8min, 4 DEG C of PCR product preservations.
The amplification of trap genetic fragment:
Using pET32a-TRAP recombinant plasmid as template, PCR amplification is carried out by primer of F2, R2 respectively, reaction condition is such as
Under: after 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 45s, 56 DEG C of annealing 40s, 72 DEG C of extension 40s carry out 30 and recycle, and last 72 DEG C
Extend 8min, 4 DEG C of PCR product preservations.
Respectively take 3 μ L pcr amplification products, verified by 1% agarose gel electrophoresis, electrophoresis result in 558bp and
Occur electrophoresis band at 498bp respectively, it is in the same size with expected amplified production target fragment, as shown in Figure 2.
4, PCR product recycling and purifying
Target fragment is purified and returned with AxyPrep gel reclaims kit (DNA Gel Extraction Kit)
It receives, concrete operation step carries out to specifications.It takes 2 μ l to recycle sample, is verified by 1% agarose gel electrophoresis, out
Product after the recovery is saved backup in -20 DEG C after existing purpose band.
5, the connection conversion of PCR product and pMD18-T carrier
According to pMD18-T Vector kit specification, successively it is added in 2 1.5ml centrifuge tubes and respectively autotomys glue purification
3 μ l, pMD 18-T Vector of genetic fragment product, 2 μ l, Ligation Solution I, 5 μ l.Centrifuge tube is set after mixing
In 16 DEG C of dry-type thermostats, the Connection Time is set as 2h.Then, connection product full dose is added in competent cell, is mixed
It is even, 30min is placed on ice, then 42 DEG C of heat shock 90s, be careful not to shake, quickly pipe is transferred in ice bath, keep cell cold
But 2min, is added the 800 μ L LB culture mediums for being preheating to 37 DEG C of antibiotic-frees in advance immediately, and 37 DEG C of air tables mildly vibrate training
1h is supported, culture brief centrifugation takes 200 μ L supernatants, is spread evenly across containing 50 μ g/mLAmp+LB plate on, to liquid quilt
It is inverted plate after absorption, is incubated overnight in 37 DEG C of incubators.
6, the identification of recombinant plasmid
ATT-pMD18T and TRAP-pMD18T recombinant plasmid by following system carry out double digestion identification (30 μ l of recombinant plasmid,
10 × Buffer, 5 μ l, BamH I, 2.5 μ l, Hind III, 2.5 μ l, ddH2O total system, 50 μ l).37 DEG C of water-bath 3h after mixing.
Take 0.5 μ l of recombinant plasmid as template, remaining reagent, reaction system and reaction condition respectively refer to respective purpose PCR in 1.5 and expand
Increasing system.5 μ l loadings are taken, are verified in 1% agarose gel electrophoresis.As a result as shown in figure 3, there is purpose base in desired location
Because of band and carrier segments band, it will identify that correct plasmid as template, carries out PCR verifying, 1% fine jade by the primer of design
There is purpose band and in the same size with target gene after double digestion in rouge gel electrophoresis result.It will identify that correct positive strain is sent
Beijing Jin Weizhi company carries out gene sequencing, by the standard sequence delivered in sequencing result BLSAT method and GenBank into
Row compares, and sequencing result is compared with staphylococcus aureus TRAP sequence on NCBI and the sequence of experimental design, homology
Reach 100%.
7, eAls albumen pronucleus cloning vector constructs
The Als3T cell epitope sequence delivered is obtained, one section of sequence is designed by this Als3T cell epitope sequence and repeats 6
It is secondary, one section of flexibility Linker (GSGSGSGS) is added between every two Als3T cell epitope sequence and in first Als3T cell table
5 ' ends of bit sequence and 3 ' ends of the last one Als3T cell epitope sequence respectively add the same Linker of the preceding paragraph convenient for expressing,
The fragment expression product is known as eAls.In 5 ' addition BamH I restriction endonuclease sites of this section of sequence, 3 ' end addition Hind
III restriction endonuclease sites.This section of sequence is synthesized and be connected to by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd
On pUC57 carrier.
8, recombinant protein Prokaryotic expression vector construction
PET-28a plasmid is subjected to double digestion with restriction endonuclease BamH I and Hind III, is purified/is returned with DNA
Kit is received to be recycled.Recombinant clone plasmid pMD-18T-ATT, pMD-18T-TRAP and pUC57-eAls are used respectively simultaneously
Restriction endonuclease BamH I and Hind III carries out double digestion, purified with DNA/QIAquick Gel Extraction Kit recycles each target gene
Segment.In the PCR reaction tube that 3 sterilize, respectively by each target gene fragment and carrier pET-28a double digestion segment,
The lower connection of Solution I effect overnight, is then converted by preceding method, and PCR and digestion detection determine target fragment just
Really insertion.It will identify that correct plasmid is transferred in BL21 (DE3) competent cell, serve the sequencing of Hai Sheng work bio-engineering corporation.
Obtained sequence is compared with Blast program with the standard sequence that oneself delivers in GenBank.The recombination of positive strain will be accredited as
Clone designation is ATT-pET28a, TRAP-pET28a, eAls-pET28a.
9, the inducing expression of recombinant protein
Recombinant plasmid BL21 (DE3) expression bacterial strain containing ATT-pET28a, TRAP-pET28a, eAls-pET28a is connect
Kind is in the 50mL LB liquid medium containing Kan+, 37 DEG C of cultures to OD600When value is 0.6, IPTG is added to final concentration of
1mmol/L continues violent shaken cultivation, takes 2ml bacterium solution in 4h, 12000r/min is centrifuged 1min, outwells supernatant, adds in precipitating
Enter 40 μ l ddH2O, 10 μ l 5 × SDS-PAGE loading buffer, piping and druming mixes, boils 5min, take 10 μ l 10%
SDS-PAGE is separated by electrophoresis.Gel is taken out after electrophoresis, is placed in coomassie brilliant blue R_250 dyeing liquor, on decolorization swinging table
1h is dyed, dyeing liquor is discarded, after eluting gel with distilled water, destainer is added, decolourize 1h on shaking table, disappears to blue background
It when mistake, purpose band are clear, is rinsed with clear water, photograph saves.
10, 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.As a result as shown in figure 4, by with white matter molecular weight Marker and induction
For 0h compared to pair, the destination protein size after induction is respectively 24KD (ATT), 22KD (TRAP) and 18KD (eAls), and after purification
Destination protein it is in the same size.
Embodiment 2
This example demonstrates that ATT, Trap and eAls protein immunization Contrast on effect.
1, animal immune and challenge test
Take health, female, SPF grade C57/B6 mouse 100 of identical week old, be divided into S.aureus Newman with
S.aureus Wood46 attacks malicious group.Carry out attacking poison after mouse ATT protein immunization, at the same with TRAP albumen, eALS albumen,
EAls albumen is used as with TRAP albumen mixing (eAls+TRAP) and PBS and compares, and every group 10.Purify each experiment histone 1mg/
Ml is emulsified with the ratio of 1:1 and not formula Freund's complete adjuvant, every 200 μ l mouse leg muscle injecting immunes；After first immunisation
21d emulsifies albumen and incomplete Freund's adjuvant with 1:1 ratio, and leg muscle injects booster immunization.
This experiment takes S.aureus Newman, and Wood46 reference culture carries out attacking poison.After taking the bacterium solution diluted to exempt to two
The mouse peritoneal of 14d attacks poison, and records mouse state and mortality daily, continues two weeks, dissects under germ-free condition dead
Mouse simultaneously separates germ, and analyzing whether it is experiment and attacking toadstool leads to dead mouse.
This experiment is that model carries out challenge viral dosage using SPF grades of C57/B6 mouse, and specific grouping is shown in Table 2.
2 mouse of table attacks malicious model grouping
The 14d staphylococcus aureus Newman after booster immunization, Wood46 bacterial strain is with absolute lethal dose (golden yellow Portugal
Newman plants of grape coccus with 8.0 × 108CFU/mouse and staphylococcus aureus Wood46 bacterial strain are with 6.0 × 108CFU/mouse
Dosage) carry out mouse peritoneal and attack poison, attack observation experiment dead mouse situation after poison and record data, be shown in Table 3.Attack malicious Newman
The immune protective rate of strain and Wood46 plants of ATT are 80%.
3 chamber group mouse model of table attacks malicious result
2, antibody level
IgG subclass in isolated serum sample is detected with indirect ELISA.Large-scale purification experimental group albumin A TT, TRAP,
EAls 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；The diluted secondary antibody of PBS, 37 DEG C of incubation 1h, washing is added in every hole；100 μ L are added in every hole
TMB developing solution, the sulfuric acid that 50 μ L 2M are added in every hole after color development at room temperature 10min terminate reaction, survey OD450nm light absorption value.Analysis knot
Fruit.It is the positive when sample OD450 value >=(+3 times of standard variances of OD450 value of negative serum).
After each experimental group antigen has been coated with elisa plate with 1 μ g/well, exempts from 14d serum for two and make by 1:100 dilution proportion
For primary antibody, as secondary antibody after being diluted with the sheep anti-mouse igg 1 of HRP label, IgG2a, IgG2b, IgG3 using 1:5000, using indirect
ELISA method detects antibody level, microplate reader OD450Lower reading numerical values, the results showed that recombinant protein A TT antibody titer highest is high
In Trap and eAls experimental group (Fig. 6).
3, cytokines measurement
INF- γ content is detected with Elispot method.IL-4, IL-10, IL-17 content are detected with ELISA method.By thin
The operating procedure of intracellular cytokine detection kit carries out.
The lymphocyte IFN-γ secretion for measuring each experiment immunization group and control group mice using ELISPOT method is horizontal
(Fig. 7).In recombinant protein A TT experimental group INF- γ content be higher than TRAP experimental group and mixing group, significant difference (P < 0.05),
It is extremely significant (p < 0.01) with other each group comparing differences.
It is measured in each experiment recombinant protein immune group and control immune group Mouse spleen cells supernatant using ELISA method
IL-4, IL-10, IL-17 secretion level (Fig. 8).Acquired results are after t- is examined, ATT immune group mouse spleen lymphocyte supernatant
IL-4, IL-10 secretory volume are higher than TRAP experimental group (P < 0.01) in liquid；In IL-17A secretory volume, except mixing group is slightly above ATT
Group, ATT group and TRAP experimental group comparing difference are significant (p < 0.05), extremely significant (P < 0.01) with other each group comparing differences.
The above result shows that the immune effect of ATT 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 ATT fusion protein is to be connected before the amino acid sequence of staphylococcus aureus TRAP fusion protein with link peptide
9 amino acid of Candida albicans Als3 t cell epitope, the amino acid sequence of ATT fusion protein such as SEQ ID No. 2 are met
It is shown.
2. encoding the gene of ATT fusion protein described in claim 1, nucleotide sequence is as shown in SEQ ID No. 1.
3. the preparation method of ATT fusion protein described in claim 1 is by gene cloning as stated in claim 2 to original
Heterogenous expression is carried out in nucleus, purifies to obtain fusion protein.
4. ATT fusion protein described in claim 1 is preparing the application in S. aureus vaccines.
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| CN105713096A (en) | 2016-06-29 |
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