CN107298717A - A kind of heat endurance protein（Polypeptide）And preparation method thereof - Google Patents
A kind of heat endurance protein（Polypeptide）And preparation method thereof Download PDFInfo
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- CN107298717A CN107298717A CN201710643209.3A CN201710643209A CN107298717A CN 107298717 A CN107298717 A CN 107298717A CN 201710643209 A CN201710643209 A CN 201710643209A CN 107298717 A CN107298717 A CN 107298717A
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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
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
- C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Abstract
The invention discloses a kind of heat endurance protein (polypeptide) and preparation method thereof, more particularly to a kind of protein (polypeptide) having from mineralization ability, containing that can induce the polypeptide of inorganic matter mineralising on described protein (polypeptide), the polypeptide is introduced using technique for gene engineering.Heat endurance protein (polypeptide) prepared by the present invention, which is characterized in that, to form one layer of amorphous calcium phosphate outside it while protein (polypeptide) itself is formed a certain size nano particle by biomimetic mineralization.Protein (polypeptide) molecule can be wrapped in inside by amorphous calcium phosphate, so as to improve the heat endurance of protein (polypeptide), and can resist the degradation of protease.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of heat endurance protein (polypeptide) and preparation method thereof.
Background technology
At present, protein (polypeptide) class medicine has high activity because of it, and high specific, hypotoxicity and biological function are clear and definite
It is widely used in clinic etc. advantage.Protein (polypeptide) class medicine can be divided into cell factor, enzyme, antibody, vaccine etc..With
Protein (polypeptide) group and the development of molecular biology, protein (polypeptide) class medicine applied to various disease treatments
Development and application turn into Bio-pharmaceutical Industry focus.
Compared with micromolecular compound medicine, protein (polypeptide) drug molecule is big, usually thousands of or tens of thousands of or even ten
Tens of thousands of dalton.The function of protein (polypeptide) is made up of a variety of functional groups, but these functional groups are very unstable, easily same
A variety of degradation reactions occur in one phase, cause protein inactivation, drug effect disappears.This unstability of protein (polypeptide)
It significantly limit the clinical practice of this kind of medicine.Influence protein (polypeptide) factors of instability a lot, wherein temperature is most
One of common cause.Easy recurring structure stretches protein (polypeptide) molecule under the action of heat, makes to be embedded in intramolecule originally
Hydrophobic region be exposed to solvent among and finally inactivate.Because the activity of protein (polypeptide) is very sensitive to temperature, therefore,
A metastable low temperature environment is required for during the production of protein (polypeptide) medicine, transport, preservation.However, cold
The standing charges of chain are fairly expensive, especially lacking the developing country of reliable cold chain infrastructure and poverty-stricken area, protein
The Cord blood of (polypeptide) class medicine largely limits it in these regional uses.Therefore, protein is improved (many
Peptide) heat endurance have higher clinic and economic value.
At present, improving the method for the heat endurance of protein (polypeptide) medicine mainly has following several：(1) protein is added
(polypeptide) stabilizer, most simple and effective additive is the salt of ionic compound, and it is (many that the combination of ion can increase protein
Peptide) heat endurance, salt by with protein (polypeptide) non-specific binding, slow down the denaturation of protein (polypeptide).(2)
Chemical modification, is primarily referred to as being chemically modified protein medicaments using dressing agent, to change their biological distribution behavior
And solubility behavior, so that strengthen the physics of medicine, chemistry and biological stability.(3) protein (polypeptide) engineering, by chemistry,
New gene is modified gene or synthesized to physics, molecular biology method, existing protein (polypeptide) is carried out highly special
The method of one transformation, so as to play a part of strengthening protein (polypeptide) stability.(4) in liquid condition, mineralising skill is utilized
Art directly forms calcium phosphate mineralized shell to improve the stability of protein (polypeptide) in protein surface.Above strategy is improving egg
There are some in the application of white matter (polypeptide) medicine to limit, such as method (1) (2) (3) generally requires the processing procedure, simultaneously of complexity
And be of limited application, method (3) can not still produce good protecting effect and may change the property of protein (polypeptide)
Matter, influences the effect of protein (polypeptide) medicine.It is substantial amounts of to calcium that the strategy of method (4) directly mineralising needs protein surface to exist
The group of ion high adsorption, and this method is only applicable to individual protein.Using biomimetic mineralization on protein (polypeptide) surface
It is a kind of new, with applications well prospect strategy come the heat endurance for improving vaccine to introduce mineral shell.Before this,
There is applicant to apply for the correlation technique of " a kind of genetic engineering thermal-stability vaccine and preparation method thereof ", its Application No.
201310278495.X, applying date 2013.07.02, the technology are mainly used in the making of virus particle vaccine, but are not suitable for
The stability improvement of soluble large biological molecule, because virus is a granulin molecule in itself, therefore can pass through surface expression
Mineralising peptide and form mineralising shell, and shla molecule can not be directly in surface mineralising, so that the technology can not improve solubility
The stability of macromolecular, and the amorphous calcium phosphate formed by the technology can not be stabilized in liquid solution, it is impossible to make
Protein (polypeptide) obtains heat endurance.In order to improve protein (polypeptide) heat endurance, so that protein (polypeptide) class medicine
Thing has wider application, and the present invention proposes a kind of method for combining chemical modification technology by technique for gene engineering,
Polypeptide of the fusion with induction inorganic matter mineralization ability in protein (polypeptide) medicine, it is wrapped by forming nano particle
In mineral matter, so as to improve protein (polypeptide) heat endurance, and make it while having the property that proteolytic degradation is acted on.
The content of the invention
Problem to be solved by this invention is to provide a kind of heat endurance protein (polypeptide) and its method prepared, simultaneously
The albumen has the degradation of antiprotease, and protein (polypeptide) prepared by the present invention is that itself forms mineralising nano particle simultaneously
And the heat endurance protein (polypeptide) of one layer of amorphous calcium phosphate shell is wrapped up on surface.Because the present invention is existed using genetic engineering
Biomineralization peptide is introduced on protein (polypeptide), so as to break the limitation of protein (polypeptide) itself mineralising, this method is fitted
For almost all of protein (polypeptide), the extensive use of protein product is greatly facilitated in this.
The present invention is adopted the following technical scheme that：
Biomineralization peptide, institute are connected with a kind of protein (polypeptide) with heat endurance, the protein (polypeptide)
The polypeptide stated has the ability of induction inorganic matter mineralising, and described polypeptide is introduced by technique for gene engineering.
The induction inorganic matter mineralization ability refers to that the polypeptide connected on protein (polypeptide) has stronger chelating inorganic
The ability of thing ion, can promote protein (polypeptide) to be enriched with inorganic matter ion in the relatively low solution of mineral concentration, make egg
White matter (polypeptide) forms a certain size nano particle.
Described inorganic matter is calcium mineral.Described calcium mineral refers to that its main cation constituent is Ca2+Ion
Mineral, described calcium mineral may be selected from calcium phosphate, calcium carbonate, calcium oxalate any one or it is any a variety of.Described
Inorganic matter can be by adding Ca in protein (polypeptide) solution2+Ion is (for example：Calcium chloride) and acid ion (described acid
Radical ion be selected from phosphate anion, carbanion, oxalate denominationby any one or it is any a variety of, contain for example, can add
Have the soluble-salt of above-mentioned acid ion) form mineral and obtain, preferably, the present invention adds the formation of phosphate radical mineralising without fixed
Shape calcium phosphate.
A kind of described protein (polypeptide) with heat endurance, adds magnesium ion, with stable amorphous in mineralising
The stability of calcium phosphate in the solution.The amorphous of mineralising can be made by adding magnesium ion when protein (polypeptide) chelates calcium ion
Calcium phosphate is not easily formed crystallization in the solution, so that amorphous calcium phosphate is more stablized.
Preferably, described polypeptide includes one of sequence as follows：
CGPKDVVVGVPGGQDGPC；
DSSEEKFLRRIGRFG；
SVKRGTSVGMKPSPRP；
RWRLEGTDDKEEPESQRRIGRFG。
Further, prepared the invention provides one kind with the method from mineralization protein matter (polypeptide), including following step
Suddenly：
(1) base of encoding human mineralising peptide is added on encoding proteins matter (polypeptide) base sequence, obtains protein
The recombinant clone of (polypeptide)；
(2) protein (polypeptide) recombinant clone that step (2) is obtained is expressed in bacterium, obtains having from mineralization ability
Protein expression bacterium.
(3) protein (polypeptide) expression and purification technology is utilized, what is purified has the protein from mineralization ability (many
Peptide).
Preferably, described polypeptide includes one of sequence as follows：
CGPKDVVVGVPGGQDGPC；
DSSEEKFLRRIGRFG；
SVKRGTSVGMKPSPRP；
RWRLEGTDDKEEPESQRRIGRFG
Further, the present invention provides a kind of mineralization methods of the protein (polypeptide) with from mineralization ability, including such as
Lower step：
(1) initial system is configured：Added in 1ml systems with protein (polypeptide) 100-600ng from mineralization ability.
CaCl is added into protein (polypeptide) solution2、Mgcl2(Na)2HPO4/NaH2PO4。
(2) by the system in 4 DEG C, PH>Magnetic bead Stirring 4h under the conditions of 6.0, preparing has heat-staple mineralising egg
In vain.
Preferably, Ca in described original system2+Ion concentration is 5-10 μm of ol/ml；Preferred Ca2+Ion is dense
Spend for 7-9 μm of ol/ml.
Preferably, Mg in described original system2+Ion concentration is 5-10 μm of ol/m；Preferred Mg2+Ion concentration
For 7-9 μm of ol/ml.
Preferably, PO in described original system4 3-Ion concentration is 3-10 μm of ol/ml.Preferred PO4 3-Ion is dense
Spend for 5-7 μm of ol/ml.
It is furthermore preferred that protein (polypeptide) and Ca in described original system2+Ion, Mg2+Ion and PO4 3-Ion
Ratio is 100-600ng：7-9μmol：7-9μmol：5-7μmol
Preferably, the pH value in the present invention is 6-9, preferred pH value is 6.8-7.8.
The purpose of the step (1) is to make Ca2+Ion is fully enriched with protein (polypeptide), adds Mg2+Ion is to make
Part magnesium ion is inlayed in the middle of calcium ion, stable amorphous calcium phosphate is formed on albumen when adding phosphate anion.
The incubation process of the step (2) is to make the uniform nano particle of protein (polypeptide) formation, and makes albumen
The amorphous calcium phosphate of one layer of mineralising is formed in matter (polypeptide).
Preferably, the incubation temperature of the step (2) is at 0 DEG C -10 DEG C, the incubation temperature of the preferred step (2)
Spend for 4 DEG C.
Preferably, the incubation time of the step (2) is 2-4h, the incubation time of the preferred step (2) is
4h。
The present invention is possible to induce the polypeptide of inorganic matter mineralising to be incorporated on protein (polypeptide) by technique for gene engineering,
The mineralising peptide being introduced into can be enriched with inorganic matter ion from solution and then induce inorganic matter mineralising, make protein (polypeptide) formation big
Small homogeneous mineralising nano particle.Present invention introduces mineralising peptide almost all of protein (polypeptide) can be enable to possess from mineralising
Power, with universality.
The method that the present invention prepares heat endurance protein (polypeptide) is simple, with low cost.The protein that mineralising is obtained is (more
Peptide) there is advantages below：By introducing biomineralization peptide on protein (polypeptide), form protein (polypeptide) itself mineralising
In its outer one layer of amorphous calcium phosphate parcel of formation while a certain size nano particle, this method has been broken to particulate vector
Limitation, it is possible to increase the stability of soluble large molecule, have in terms of the stability of protein (polypeptide) medicine it is very big should
Use prospect.Mineralising improves the settleability of protein (polypeptide) so that protein (polypeptide) can be enriched with constant speed centrifugal condition.
One layer of amorphous calcium phosphate shell has been wrapped up on mineralization protein matter (polypeptide) surface, and amorphous calcium phosphate can slow down protein (polypeptide)
Internal and extraneous hydrogen bond is exchanged, so that the inactivation of the protein that slows down (polypeptide), while the parcel of amorphous calcium phosphate can be reduced
The activated centre of protein (polypeptide) digestive ferment obtains binding site with protein (polypeptide), so that the digestion to protease has one
Fixed resistivity, effectively improves the stability of protein (polypeptide).The protein (polypeptide) prepared according to this method can be
Room temperature storage still has protein (polypeptide) activity in more than one week.When protein (polypeptide) molecule is as vaccine, mineralising
Mineralising nano particle can effectively strengthen the antigen submission of vaccine protein in protein (polypeptide) vaccine, promote humoral immunity and cell
It is immune, strengthen protective effect of the vaccine protein to host, and in the absence of the situation that immune local tubercle occurs, compared with the effect of aluminium adjuvant
Should be more preferable.By means of the invention it is possible to effectively improve the heat endurance of protein (polypeptide), reduce in protein (polypeptide) medicine
Expenditure in refrigeration, promotes the extensive use of protein (polypeptide) medicine, in the life of Novel thermostable protein (polypeptide)
There is higher practical value in production and preparation.
Brief description of the drawings
Fig. 1 is that 1,2,3,4,5 bands in the electrophoretogram of construction recombination plasmid, wherein Fig. 1 are Δ Ply, Δ Ply- respectively
PA44、ΔPly-NW、ΔPly-N6、ΔPly-W6。
Fig. 2 is Δ Ply, Δ respectively for 1,2,3,4,5 bands in expression and recombinant protein after purification, wherein Fig. 2
Ply-PA44、ΔPly-NW、ΔPly-N6、ΔPly-W6。
Fig. 3 is the electron microscope of mineralization protein.Wherein Fig. 3 A are Δ Ply-PA44；3B is Δ Ply-PA44-Ca；3C is Δ
Ply-PA44-CaP-Mg2+；3D is Δ Ply-NW-CaP；3E is Δ Ply-W6-CaP；3F is Δ Ply-N6-CaP.
Fig. 4 is the XPS analysis of mineralization protein, wherein A:ΔA146Ply-PA44；B:ΔA146Ply-NW；C:Δ
A146Ply-N6； D:ΔA146Ply-W6；E:ΔA146Ply-PA44@CaP；F:ΔA146Ply-NW@CaP；G:Δ
A146Ply-N6@CaP； H:ΔA146Ply-W6@CaP
Fig. 5 is the protease K digesting stability analysis of protein.1st, 2,3 bands are that Proteinase K handles 0 minute, 4,5,6
Band is that Proteinase K is handled 1 minute, and 7,8,9 bands are that Proteinase K is handled 5 minutes, and 10,11,12 bands are Proteinase K processing
10 minutes；Band 1,4,7,10 is that Δ Ply-PA44,2,5,8,11 are that Δ Ply-PA44-CaP, 3,6,9,12 are Δ Ply-
PA44-CaP-Mg2+
Fig. 6 is the thermal stability analysis of protein.1st, 2,3 bands are that room temperature is placed 0 week, and 4,5,6 bands are that room temperature puts 1
Week, 7,8,9 bands are that Proteinase K handles room temperature placement in 5 minutes 2 weeks, and 10,11,12 bands are that room temperature is placed 3 weeks；Band 1,4,
7th, 10 be that room temperature is placed 4 weeks.Band 1,4,7,10 is Δ Ply-PA44-CaP-Mg2+, 2,5,8,11 be Δ Ply-PA44-CaP,
3rd, 6,9,12 be Δ Ply-PA44.
Fig. 7 is anti-Δ A146Ply IgG antibody Potency Analysis after subcutaneous inoculation.
Fig. 8 be subcutaneous inoculation mouse after stimulate the amount of splenocyte secrete cytokines to analyze again.
Embodiment
Embodiment 1, the preparation with the protein from mineralization ability
The embodiment to remove the pneumolysin Δ Ply albumen of 146 amino acids as model skeleton, first with
PET28a plasmids are carrier, build pET28a (+)-Δ Ply-PA44, pET28a (+)-Δ Ply-NW, pET28a (+)-Δ
Ply-N6, pET28a (+)-Δ Ply-W6 recombinant expression plasmids, are then transformed into Escherichia coli by recombinant expression plasmid, and big
Express, identify and purify in enterobacteria
(1) mineralising peptide is connected on Δ Ply albumen
1st, recombinant expression plasmid is built
(1) material：
Plasmid pET28a (+) be purchased from Novagen companies, PCR Prime Star high-fidelities enzymes, dNTPs, Buffer,
MgCl2Purchased from the precious biotech company in Dalian, PTC-200PCR instrument is Perkin Elmer products, and RG-3000 is Corbett
Research products.
(2) the design synthesis of primer：
Using streptococcus pneumonia TIGR4 genomic DNAs template, with reference to its complete sequence (GeneBank numbering AE005672),
Primer is designed using premier5.0, Sheng Gong companies synthesize by Shanghai.
Upper polypeptide PA44, NW, N6, W6 are connected in Δ Ply fragments, and corresponding recombinant protein is named as Δ Ply-
PA44、
ΔPly-NW、ΔPly-N6、ΔPly-W6.The corresponding fusion DNA vaccine primer of polypeptide PA44, NW, N6, W6 is：
(3) PCR amplifying target genes：
The amplification of Δ Ply-PA44 genes
System：
Condition：98℃10s、55℃15s、72℃100s、30cycle；72 DEG C of 10min, 1 time
Using above-mentioned condition, corresponding target gene has been amplified
(4) structure of prokaryotic expression carrier
PCR primer reclaims the kit explanation provided by Roche and carried out, and plasmid pET28a (+) presses Omega mini-scale plasmids DNA
Extraction agent box explanation is carried out, and then carries out carrier DNA and exogenous DNA carries out double digestion and recovery, finally connect recovery product,
Coupled reaction system is as follows：
Coupled reaction system 1：
The μ l of Δ Ply-PA44 fragments 6；
The μ l of pET28a (+) DNA large fragments 2；
Connect the μ l of buffer I 1；
The μ l of T4 ligases 1
Cumulative volume：10μl
Reaction condition：Set to 0 in .5mlEP pipes, 22 DEG C, 10min
Connection product is converted into E. coli DH5 α competent cells：
Take E.coli DH5 α bacterium streak inoculation LB flat boards
37 DEG C are incubated overnight (12-14h)
Picking single bacterium colony, is inoculated in 3ml LB
37 DEG C of 200rpm stay overnight (12-14h), take 100 μ l to add 2mlLB37 DEG C, 300rpm 3h
1.5ml bacterium solutions are taken to add in ice precooling EP pipes, 4000rpm after ice bath 10min, 5min collects thalline
0.1mMCacl2150 the μ l, 9000rpm after resuspension, 2min for adding precooling collect thalline
The 0.1mMCacl2150 μ l of precooling are added, are resuspended
Add 10 μ l coupled reaction products, after mixing, ice-water bath 30min
42 DEG C of thermal shock 5min, ice-water bath 2min
Add 800ulLB culture mediums, 37 DEG C, 100rpm 1h recoveries
200 μ l bacterium solutions are taken to be coated on LK flat boards
37 DEG C of incubations, 13h
Picking single bacterium colony carries out the increasing dientification of bacteria
(5) screening and identification of pET28a (+)-Δ Ply-PA44 recons
The card of picking 10 receives chloramphenicol resistance bacterium colony, puts respectively in 2ml LK (LB of the Kana containing 50ug/ml) culture medium,
180rpm 3h increase bacterium, bacterium solution PCR identifications；Choose 3 probable positive bacterium colonies and increase bacterium, being sent to the prosperous biotechnology in Chengdu Qing Ke Chinese catalpas has
Limit company makees two-way sequencing.
As a result see accompanying drawing 1, obtain single PCR bands；PCR primer sequencing result is compared with expected sequence to fit like a glove.
Result above confirms that target gene fragment is correctly inserted into expression vector.
2nd, expression, identification and purifying of prokaryotic expression pET28a (+)-Δ Ply-PA44 recombinant plasmids in Escherichia coli
(1) recombinant plasmid pET28a (+)-Δ Ply-PA44 is converted into Host Strains BL21 (DH5 α)
(2) IPTG induces Δ Ply-PA44 great expression
(3) purifying of recombinant protein：After carrying out ultrasonic bacteria breaking, bacteria breaking liquid supernatant is taken to be used to purify；4 DEG C, 10000rpm ×
10min, supernatant collects filtrate stand-by with 0.45 μm of membrane filtration.
Affinity chromatography purification：Inhale 2ml 50% Ni2+- NTA resin suspensions are slow with 20ml ultrasonications in chromatographic column
Fliud flushing is balanced；Suction out the Ni after balance2+- NTA resin suspensions are fully mixed with above-mentioned filtrate, ice bath 1h, therebetween at interval of
5min is gently mixed once；Suspension is transferred in chromatographic column, allows liquid to flow out naturally, balance columns bed；Different imidazole concentrations are carried out
Gradient elution, collects SDS-PAGE identifications are carried out after eluent respectively.
(4) PBS ultrafiltration removes imidazoles.
(5) quantitative (the Bradford detection methods) of recombinant protein
A. inhale the 20mg/ml μ l of standard items bovine serum albumin solution 6 and dilute 40 times extremely with 0.15mmo1/L NaCl
0.5mg/ml, adds 10 μ l, 20 μ l, 30 μ l, 40 μ l BSA solution, then with 0.15mmol/ in two groups of each 4 test tubes respectively
L NaCl supply cumulative volume for 200 μ l, while taking a test tube only to add 200 μ l 0.15mmol/LNaCl to be used as zeroing；
B. the μ l of purified product 20 are taken, the μ l of cumulative volume 200 are also complemented to 0.15mmol/LNaCl；
C. often pipe adds Coomassie brilliant G-250 dye liquor 2ml, and vibration is stored at room temperature 30min after mixing；
D. inA590 values are read in-Series600 all-wave length spectrophotometers, standard curve is drawn by instrument automatically
And calculate sample protein content.
As a result show：Show through SDS-PAGE and graphical analysis, recombinant protein purity is up to more than 90%, through Bradford
Method measures protein concentration result after purifying dialysis and sees accompanying drawing 1, obtains single PCR bands；PCR primer sequencing result and expected sequence
Row comparison fits like a glove.
Result above confirms that target gene fragment is correctly inserted into expression vector.Δ Ply-PA44 is 8.81ug/ul
According to the similar step of step (1), polypeptide NW, N6, W6 are connected on Δ Ply albumen, successfully building has certainly
Protein Δ Ply-NW, Δ Ply-N6, the Δ Ply-W6 of mineralization ability, protein concentration after measuring purifying dialysis through Bradford methods
Δ Ply-NW is that 4.8ug/ul, Δ Ply-N6 are that 2.4ug/ul, Δ Ply-W6 are 3.4ug/ul.(result is shown in accompanying drawing 1, accompanying drawing 2)
(2) biomineralization peptide is connected on DnaJ albumen
(1) recombinant expression plasmid
According to the similar step of step (1), polypeptide PA44, NW, N6, W6 are connected on DnaJ albumen, and will be corresponding
Recombinant protein is named as DnaJ-PA44, DnaJ-NW, DnaJ-N6, DnaJ-W6.
(2) by recombinant plasmid transformed to Escherichia coli
The same step (1) of method
(3) expression and purification of recombinant proteins DnaJ-PA44, DnaJ-NW, DnaJ-N6, DnaJ-W6.The same step (1) of method
Success is built with 3-protein d naJ-PA44, DnaJ-NW, DnaJ-N6, DnaJ-W6 from mineralization ability.Through Bradford methods
It is that 2.4ug/ul, DnaJ-NW are that 4.1ug/ul, DnaJ-N6 are 2.1ug/ to measure protein concentration DnaJ-PA44 after purifying dialysis
Ul, DnaJ-W6 are 1.4ug/ul.
Embodiment 2, recombinant protein Δ Ply-PA44 calcium phosphate are analyzed from mineralising and biological characteristic
(1) mineralising of recombinant protein
CaCl is added in Δ Ply-PA44 protein solutions (adjustment protein concentration is 100ng/ml)2, make Ca2+Ion is dense
Degree reaches 5 μm of ol/ml, adds Mgcl2Ion, makes Mg2+Ion concentration is 5 μm of ol/ml；Finally plus (Na)2HPO4/NaH2PO4
Make PO4 3-Ion concentration is 3 μm of ol/ml, by the system under the conditions of 4 DEG C, PH=6.5 magnetic bead Stirring 2h, preparation has
Heat-staple mineralization protein.
In above-mentioned preparation process, protein, Ca in initial system2+Ion, Mg2+Ion and PO4 3-The ratio of ion is
100(ng):5(umol):5(umol):3(umol)。
(2) biological characteristic is analyzed
(1) mineralization protein Δ Ply-PA44 is centrifuged into (5000g；5min) precipitated and supernatant, precipitation 40ulPBS weights
It is outstanding, 10ul 5 × loading buffer boiling water boiling 10min are added in the precipitation being resuspended respectively to 40ul supernatants 40ul, are taken
10ul samples are in the loading hole of Page glue.Voltage：The 80V times：30min voltages：The 120V times:90min, uses Coomassie brilliant blue
Dye liquor dyeing 3min (microwave stove heat 3min), is finally decolourized to stay overnight with Coomassie brilliant blue destainer.As a result show, mineralization protein
Supernatant in be not detected by protein.
(2) by protein solution Δ Ply-PA44, mineralization protein Δ Ply-PA44@CaP, mineralization protein Δ Ply-PA44@
CaP-Mg2+Solution dilutes 50 times, electron microscopic observation result.As a result accompanying drawing 3A, 3B, 3C are seen.As a result the albumen for not adding mineralising peptide is shown
Matter can not form uniform nano particle, and calcium phosphate mineralized protein can form a certain size nano particle, but put
Put and easily crystallized after a period of time, the protein that mineralising adds magnesium ion stable can be formed uniform mineralising nano particle and
It can stablize in the solution long period of time.
(3) mineralization protein matter solution Δ Ply-PA44@CaP drops are fully dried on slide, X-ray energy spectrum detection
Mineralization protein matter surface-element.As a result accompanying drawing 4 is seen, mineralization protein surface is able to detect that the elements such as calcium ion.
(3) stability analysis of mineralization protein matter
(1) the digestion resistance analysis of mineralization protein confrontation Proteinase K
By mineralization protein Δ A146Ply-PA44@CaP, Δ A146Ply-PA44@CaP-Mg2+The albumen Δ of non-mineralising
A146Ply-PA44 handles 0min, 1min, 5min with Proteinase K respectively, after 10min, added into 40ul samples 10ul 5 ×
Loading buffer boiling water boiling 10min, take 10ul samples in the loading hole of Page glue respectively.Voltage：The 80V times：30min
Voltage：The 120V times:90min, dyes 3min (microwave stove heat 3min) with Coomassie brilliant blue dye liquor, finally uses Coomassie brilliant blue
Destainer decolourizes to stay overnight.As a result accompanying drawing 5 is seen.As a result show, after Protease Treatment 10min, the albumen of non-mineralising can not
Detect, but mineralization protein Δ A146Ply-PA44@CaP, Δ A146Ply-PA44@CaP-Mg2+Remain able to be detected.
(2) the thermally-stabilised analysis of mineralization protein matter
By mineralization protein Δ A146Ply-PA44@CaP, Δ A146Ply-PA44@CaP-Mg2+The albumen Δ of non-mineralising
After A146Ply-PA44 room temperatures are placed one week, two weeks, three weeks, 5 × loadingbuffer of 10ul boilings are added into 40ul samples
Water boils 10min, and 10ul samples are taken respectively in the loading hole of Page glue.Voltage：The 80V times：30min voltages：The 120V times:
90min, dyes 3min (microwave stove heat 3min) with Coomassie brilliant blue dye liquor, was finally decolourized with Coomassie brilliant blue destainer
Night.As a result accompanying drawing 6 is seen.As a result show room temperature place 2 weeks after the albumen of non-mineralising can not detect, and but mineralization protein Δ
A146Ply-PA44@CaP, Δ A146Ply-PA44@CaP-Mg2+Remain able to be detected.
Embodiment 3：Recombinant protein Δ Ply-NW calcium phosphate is analyzed from mineralising and biological characteristic
(1) mineralising of recombinant protein
CaCl is added in Δ Ply-PA44 protein solutions (adjustment protein concentration is 200ng/ml)2, make Ca2+Ion is dense
Degree reaches 7 μm of ol/ml, adds Mgcl2Ion, makes Mg2+Ion concentration is 7 μm of ol/ml；Finally plus (Na)2HPO4/NaH2PO4
Make PO4 3-Ion concentration is 5 μm of ol/ml, by the system under the conditions of 4 DEG C, PH=6.5 magnetic bead Stirring 2h, preparation has
Heat-staple mineralization protein.
In above-mentioned preparation process, protein, Ca in initial system2+Ion, Mg2+Ion and PO4 3-The ratio of ion is
200(ng):7(umol):7(umol):5(umol)。
(1) biological characteristic is analyzed
(1) mineralization protein Δ Ply-NW is centrifuged into (5000g；5min) precipitated and supernatant, precipitation 40ulPBS weights
It is outstanding,
10ul 5 × loadingbuffer boiling water boiling 10min are added in the precipitation being resuspended respectively to 40ul supernatants 40ul,
10ul samples are taken in the loading hole of Page glue.Voltage：The 80V times：30min voltages：The 120V times:90min, uses coomassie
Light blue dye liquor dyeing 3min (microwave stove heat 3min), is finally decolourized to stay overnight with Coomassie brilliant blue destainer.As a result show, mineralising
Protein is not detected by the supernatant of albumen.
(2) by protein solution Δ Ply-NW, mineralization protein Δ Ply-NW@CaP solution dilutes 50 times, electron microscopic observation knot
Really.As a result accompanying drawing 3D is seen, mineralization protein can form a certain size mineralized particles.
(3) mineralization protein matter solution Δ Ply-NW drops are fully dried on slide, X-ray energy spectrum detection mineralising egg
White matter surface-element.As a result accompanying drawing 4 is seen, mineralization protein surface is able to detect that the elements such as calcium ion.
(3) stability analysis of mineralization protein matter
(1) the digestion resistance analysis of mineralization protein confrontation Proteinase K
Mineralization protein Δ A146Ply-NW@CaP, the albumen Δ A146Ply-NW of mineralising are handled with Proteinase K respectively
After 0min, 1min, 5min, 10min, 10ul 5 × loading buffer boiling water boiling 10min are added into 40ul samples, respectively
10ul samples are taken in the loading hole of Page glue.Voltage：The 80V times：30min voltages：The 120V times:90min, it is bright with coomassie
Blue dye liquor dyeing 3min (microwave stove heat 3min), is finally decolourized to stay overnight with Coomassie brilliant blue destainer.As a result show, mineralising egg
White Δ A146Ply-NW@CaP have the digestion of certain resistance protease.
(2) the thermally-stabilised analysis of mineralization protein matter
Mineralization protein Δ A146Ply-NW@CaP, and non-mineralising albumen Δ A146Ply-NW room temperatures are placed into one week, two
Week, after three weeks, into 40ul samples add 10ul 5 × loading buffer boiling water boiling 10min, take respectively 10ul samples in
In the loading hole of Page glue.Voltage：The 80V times：30min voltages：The 120V times:90min, is dyed with Coomassie brilliant blue dye liquor
3min (microwave stove heat 3min), is finally decolourized to stay overnight with Coomassie brilliant blue destainer.As a result show, mineralization protein Δ
A146Ply-NW@CaP have certain heat endurance.
Embodiment 4：Recombinant protein Δ Ply-W6 calcium phosphate is from mineralising
(1) mineralising of recombinant protein
CaCl is added in Δ Ply-NW protein solutions (adjustment protein concentration is 300ng/ml)2, make Ca2+Ion concentration
9 μm of ol/ml are reached, Mgcl is added2Ion, makes Mg2+Ion concentration is 9 μm of ol/ml；Finally plus (Na)2HPO4/NaH2PO4
Make PO4 3-Ion concentration is 7 μm of ol/ml, by the system under the conditions of 4 DEG C, PH=8.0 magnetic bead Stirring 4h, prepare tool
There is heat-staple mineralization protein.
In above-mentioned preparation process, protein, Ca in initial system2+Ion, Mg2+Ion and PO4 3-The ratio of ion is
300(ng):9(umol):9(umol):7(umol)。
(2) biological characteristic is analyzed
(1) mineralization protein Δ Ply-W6 is centrifuged into (5000g；5min) precipitated and supernatant, precipitation 40ulPBS weights
It is outstanding, 10ul 5 × loadingbuffer boiling water boiling 10min are added in the precipitation being resuspended respectively to 40ul supernatants 40ul, are taken
10ul samples are in the loading hole of Page glue.Voltage：The 80V times：30min voltages：The 120V times:90min, it is bright with coomassie
Blue dye liquor dyeing 3min (microwave stove heat 3min), is finally decolourized to stay overnight with Coomassie brilliant blue destainer.As a result show, mineralising egg
Protein is not detected by white supernatant.
(2) by protein solution Δ Ply-W6, mineralization protein Δ Ply-W6@CaP solution dilutes 50 times, electron microscopic observation knot
Really.As a result accompanying drawing 3E is seen, mineralization protein can form a certain size mineralized particles.
(3) mineralization protein matter solution Δ Ply-W6 drops are fully dried on slide, X-ray energy spectrum detection mineralising egg
White matter surface-element.As a result accompanying drawing 4 is seen, mineralization protein surface is able to detect that the elements such as calcium ion.
(3) stability analysis of mineralization protein matter
(1) the digestion resistance analysis of mineralization protein confrontation Proteinase K
Mineralization protein Δ A146Ply-W6@CaP, the albumen Δ A146Ply-W6 of mineralising are handled with Proteinase K respectively
After 0min, 1min, 5min, 10min, 10ul 5 × loading buffer boiling water boiling 10min are added into 40ul samples, point
10ul samples are not taken in the loading hole of Page glue.Voltage：The 80V times：30min voltages：The 120V times:90min, with examining horse
This light blue dye liquor dyeing 3min (microwave stove heat 3min), is finally decolourized to stay overnight with Coomassie brilliant blue destainer.As a result show, ore deposit
Changing albumen Δ A146Ply-W6@CaP has the digestion of certain resistance protease.
(2) the thermally-stabilised analysis of mineralization protein matter
Mineralization protein Δ A146Ply-W6@CaP, and non-mineralising albumen Δ A146Ply-W6 room temperatures are placed into one week, two
Week, after three weeks, into 40ul samples add 10ul 5 × loadingbuffer boiling water boiling 10min, take respectively 10ul samples in
In the loading hole of Page glue.Voltage：The 80V times：30min voltages：The 120V times:90min, is dyed with Coomassie brilliant blue dye liquor
3min (microwave stove heat 3min), is finally decolourized to stay overnight with Coomassie brilliant blue destainer.As a result show, mineralization protein Δ
A146Ply-W6@CaP have certain heat endurance.
Embodiment 5：Recombinant protein Δ Ply-N6 calcium phosphate is from mineralising
(1) mineralising of recombinant protein
CaCl is added in Δ Ply-N6 protein solutions (adjustment protein concentration is 300ng/ml)2, make Ca2+Ion concentration
10 μm of ol/ml are reached, Mgcl is added2Ion, makes Mg2+Ion concentration is 10 μm of ol/ml；Finally plus (Na)2HPO4/
NaH2PO4Make PO4 3-Ion concentration is 7 μm of ol/ml, by the system under the conditions of 4 DEG C, PH=9.0 magnetic bead Stirring 4h, system
It is standby that there is heat-staple mineralization protein.
In above-mentioned preparation process, protein, Ca in initial system2+Ion, Mg2+Ion and PO4 3-The ratio of ion is
300(ng):10(umol):10(umol):7(umol)。
(2) biological characteristic is analyzed
(1) mineralization protein Δ Ply-N6 is centrifuged into (5000g；5min) precipitated and supernatant, precipitation 40ulPBS weights
It is outstanding, 10ul 5 × loadingbuffer boiling water boiling 10min are added in the precipitation being resuspended respectively to 40ul supernatants 40ul, are taken
10ul samples are in the loading hole of Page glue.Voltage：The 80V times：30min voltages：The 120V times:90min, it is bright with coomassie
Blue dye liquor dyeing 3min (microwave stove heat 3min), is finally decolourized to stay overnight with Coomassie brilliant blue destainer.As a result show, mineralising egg
Protein is not detected by white supernatant.
(2) by protein solution Δ Ply-N6, mineralization protein Δ Ply-N6@CaP solution dilutes 50 times, electron microscopic observation knot
Really.As a result accompanying drawing 3F is seen, mineralization protein can form a certain size mineralized particles.
(3) mineralization protein matter solution Δ Ply-N6 drops are fully dried on slide, X-ray energy spectrum detection mineralising egg
White matter surface-element.As a result accompanying drawing 4 is seen, mineralization protein surface is able to detect that the elements such as calcium ion.
(3) stability analysis of mineralization protein matter
(1) the digestion resistance analysis of mineralization protein confrontation Proteinase K
Mineralization protein Δ A146Ply-N6@CaP, the albumen Δ A146Ply-N6 of mineralising are handled with Proteinase K respectively
After 0min, 1min, 5min, 10min, 10ul 5*loading buffer boiling water boiling 10min are added into 40ul samples, respectively
10ul samples are taken in the loading hole of Page glue.Voltage：The 80V times：30min voltages：The 120V times:90min, uses coomassie
Light blue dye liquor dyeing 3min (microwave stove heat 3min), is finally decolourized to stay overnight with Coomassie brilliant blue destainer.As a result show, mineralising
Albumen Δ A146Ply-N6@CaP have the digestion of certain resistance protease.
(2) the thermally-stabilised analysis of mineralization protein matter
Mineralization protein Δ A146Ply-N6@CaP, and non-mineralising albumen Δ A146Ply-N6 room temperatures are placed into one week, two
Week, after three weeks, into 40ul samples add 10ul 5 × loadingbuffer boiling water boiling 10min, take respectively 10ul samples in
In the loading hole of Page glue.Voltage：The 80V times：30min voltages：The 120V times:90min, is dyed with Coomassie brilliant blue dye liquor
3min (microwave stove heat 3min), is finally decolourized to stay overnight with Coomassie brilliant blue destainer.As a result show, mineralization protein Δ
A146Ply-N6@CaP have certain heat endurance.
Embodiment 6：Recombinant protein DnaJ-PA44 calcium phosphate is from mineralising
CaCl is added in DnaJ-PA44 protein solutions (adjustment protein concentration is 300ng/ml)2, make Ca2+Ion is dense
Degree reaches 9 μm of ol/ml, adds Mgcl2Ion, makes Mg2+Ion concentration is 9 μm of ol/ml；Finally plus (Na)2HPO4/NaH2PO4
Make PO4 3-Ion concentration is 7 μm of ol/ml, by the system under the conditions of 4 DEG C, PH=9.0 magnetic bead Stirring 4h, preparation has
Heat-staple mineralization protein DnaJ-PA44@CaP.
In above-mentioned preparation process, protein, Ca in initial system2+Ion, Mg2+Ion and PO4 3-The ratio of ion is
300(ng):9(umol):9(umol):7(umol)。
(2) biological characteristic is analyzed
(1) mineralization protein DnaJ-PA44 is centrifuged into (5000g；5min) precipitated and supernatant, precipitation 40ulPBS weights
It is outstanding, 10ul 5 × loading buffer boiling water boiling 10min are added in the precipitation being resuspended respectively to 40ul supernatants 40ul, are taken
10ul samples are in the loading hole of Page glue.Voltage：The 80V times：30min voltages：The 120V times:90min, it is bright with coomassie
Blue dye liquor dyeing 3min (microwave stove heat 3min), is finally decolourized to stay overnight with Coomassie brilliant blue destainer.
As a result show, protein is not detected by the supernatant of mineralization protein.
(2) mineralization protein DnaJ-PA44@CaP solution is diluted into 50 times, electron microscopic observation result.
(3) mineralization protein matter solution D naJ-PA44@CaP drops are fully dried on slide, X-ray energy spectrum detection ore deposit
Change protein surface element.
(3) stability analysis of mineralization protein matter
(1) the digestion resistance analysis of mineralization protein confrontation Proteinase K
Mineralization protein DnaJ-PA44@CaP and the protein D naJ-PA44 of non-mineralising are handled into 0min with Proteinase K respectively,
After 1min, 5min, 10min, 10ul 5 × loading buffer boiling water boiling 10min are added into 40ul samples, are taken respectively
10ul samples are in the loading hole of Page glue.Voltage：The 80V times：30min voltages：The 120V times:90min, it is bright with coomassie
Blue dye liquor dyeing 3min (microwave stove heat 3min), is finally decolourized to stay overnight with Coomassie brilliant blue destainer.As a result show, mineralising egg
White DnaJ-PA44@CaP have the digestion of certain resistance protease.
(2) the thermally-stabilised analysis of mineralization protein matter
The protein D naJ-PA44 room temperatures of mineralization protein DnaJ-PA44@CaP and non-mineralising are placed one week, two weeks, three weeks
Afterwards, 10ul 5 × loading buffer boiling water boiling 10min are added into 40ul samples, 10ul samples are taken respectively in Page glue
Loading hole in.Voltage：The 80V times：30min voltages：The 120V times:90min, dyes 3min (micro- with Coomassie brilliant blue dye liquor
Ripple stove heat 3min), finally decolourized to stay overnight with Coomassie brilliant blue destainer.As a result show, mineralization protein DnaJ-PA44@CaP tools
There is certain heat endurance.
Embodiment 7, recombinant protein Δ Ply-PA44 calcium oxalate are from mineralising
CaCl is added in Δ Ply-PA44 protein solutions (adjustment protein concentration is 300ng/ml)2, make Ca2+Ion is dense
Degree reaches 9 μm of ol/ml, adds Mgcl2Ion, makes Mg2+Ion concentration is 9 μm of ol/ml；Finally plus oxalate, oxalate is made
Ion concentration is 7 μm of ol/ml, by the system under the conditions of 4 DEG C, PH=8.0 magnetic bead Stirring 4h, preparing has heat steady
Fixed mineralization protein.
In above-mentioned preparation process, protein, Ca in initial system2+Ion, Mg2+The ratio of ion and oxalate denominationby
For 300 (ng):9(umol):9(umol):7(umol).
(2) biological characteristic is analyzed
(1) mineralization protein Δ Ply-PA44 is centrifuged into (5000g；5min) precipitated and supernatant, precipitation 40ulPBS weights
It is outstanding, 10ul 5*loadingbuffer boiling water boiling 10min are added in the precipitation being resuspended respectively to 40ul supernatants 40ul, 10ul is taken
Sample is in the loading hole of Page glue.Voltage：The 80V times：30min voltages：The 120V times:90min, uses Coomassie brilliant blue dye liquor
3min (microwave stove heat 3min) is dyed, is finally decolourized to stay overnight with Coomassie brilliant blue destainer.As a result show, mineralization protein it is upper
A small amount of protein is detected in clear liquid, mineralising is incomplete.
(2) by protein solution Δ Ply-PA44, mineralization protein Δ Ply-PA44@CaC2O4Solution dilutes 50 times, and Electronic Speculum is seen
Examine result.As a result show that mineralization protein can form a certain size nano particle.
(3) mineralization protein matter solution is dripped on slide, fully dried, X-ray energy spectrum detection mineralization protein matter surface
Contain the elements such as calcium ion.
Embodiment 8, recombinant protein Δ Ply-PA44 calcium carbonate are from mineralising
CaCl is added in Δ Ply-PA44 protein solutions (adjustment protein concentration is 300ng/ml)2, make Ca2+Ion is dense
Degree reaches 9 μm of ol/ml, adds Mgcl2Ion, makes Mg2+Ion concentration is 9 μm of ol/ml；Finally plus oxalate, carbonate is made
Ion concentration is 7 μm of ol/ml, by the system under the conditions of 4 DEG C, PH=8.0 magnetic bead Stirring 4h, prepare have it is thermally-stabilised
Mineralization protein.
In above-mentioned preparation process, protein, Ca in initial system2+Ion, Mg2+The ratio of ion and carbanion
For 300 (ng):9(umol):9(umol):7(umol).
(2) biological characteristic is analyzed
(1) mineralization protein Δ Ply-PA44 is centrifuged into (5000g；5min) precipitated and supernatant, precipitation 40ulPBS weights
It is outstanding, 10ul 5 × loadingbuffer boiling water boiling 10min are added in the precipitation being resuspended respectively to 40ul supernatants 40ul, are taken
10ul samples are in the loading hole of Page glue.Voltage：The 80V times：30min voltages：The 120V times:90min, it is bright with coomassie
Blue dye liquor dyeing 3min (microwave stove heat 3min), is finally decolourized to stay overnight with Coomassie brilliant blue destainer.As a result show, mineralising egg
Protein is not detected by white supernatant.
(2) by protein solution Δ Ply-PA44, mineralization protein Δ Ply-PA44@CaCO3Solution dilutes 50 times, and Electronic Speculum is seen
Examine result.As a result show that mineralization protein can form a certain size nano particle.
(3) mineralization protein matter solution is dripped on slide, fully dried, X-ray energy spectrum detects the elements such as calcium ion.
When embodiment 9, protein are vaccine, the Analysis of Immunogenicity of mineralization protein vaccine：
(1) the antibody titer detection after the mineralising nano particle of vaccine protein in serum
By vaccine protein matter Δ A146Ply-PA44@CaP, Δ A146Ply-NW@CaP, the Δ A146Ply-N6@of mineralising
CaP, Δ A146Ply-W6@CaP, are tested as follows：
Take 6-8 week old, female C57BL/6 mouse to be randomly divided into 11 groups, the anesthesia of 1.5% yellow Jackets, each group respectively according to
Mouse is immunized according to following reagent and dose subcutaneous：
| PBS | 100 μ l/ are only | - |
| ΔA146Ply | 100 μ l/ are only | 18 μ g/ are only |
| Δ A146Ply+ aluminium adjuvants | 100 μ l/ are only | 18 μ g/ are only |
| ΔA146Ply-PA44 | 100 μ l/ are only | 18 μ g/ are only |
| ΔA146Ply-NW | 100 μ l/ are only | 18 μ g/ are only |
| ΔA146Ply-N6 | 100 μ l/ are only | 18 μ g/ are only |
| ΔA146Ply-W6 | 100 μ l/ are only | 18 μ g/ are only |
| ΔA146Ply-PA44@CaP | 100 μ l/ are only | 18 μ g/ are only |
| ΔA146Ply-NW@CaP | 100 μ l/ are only | 18 μ g/ are only |
| ΔA146Ply-N6@CaP | 100 μ l/ are only | 18 μ g/ are only |
| ΔA146Ply-W6@CaP | 100 μ l/ are only | 18 μ g/ are only |
It is immunized 3 times altogether, every minor tick 14 days.It is percutaneous using the soluble protein and mineralising nano particle of same protein dosage
Mouse is immunized down, final immunization takes mice serum to detect anti-Δ A146Ply IgG (Fig. 7) after 7 days.As a result show, with Δ
A146Ply immune groups are compared, and the anti-Δ A146Ply IgG antibody potency that mineralising nano particle immune group is produced is dramatically increased, to the greatest extent
The antibody titer of pipe mineralising nano particle immune group does not have significant difference with the Δ A146Ply immune groups containing aluminium adjuvant, but still
Obvious increased trend can be observed.The result shows, compared to soluble protein and for the soluble protein containing aluminium adjuvant,
Mineralising nano particle can produce stronger humoral immune response with inducing mouse.As a result accompanying drawing 7 is seen
(2) detection of splenocyte factor level after mineralising nano particle is immunized
In order to determine the effect of cellullar immunologic response, mouse is ibid immunized, final immunization is collected splenocyte and adopted after 7 days
Stimulated again with immunizing antigen 72 hours, the concentration of cell factor IFN-γ in supernatant, IL-4 and IL-17A is detected by ELISA
(Fig. 8).As a result show：The splenocyte of all mineralising nano particle immune groups secretes the IFN-γ of higher level, IL-4 and
IL-17A, and the IL-4 and IL-17A of the secretion of all mineralising nano particle immune groups are all remarkably higher than soluble protein group；With containing
When the Δ A146Ply immune groups of aluminium adjuvant are compared, three kinds of biomineralization protein nano particle immune group (Δ A146Ply-PA44@
CaP, Δ A146Ply-NW@CaP, Δ A146Ply-W6@CaP) IL-4 secretory volume significantly increase.These Notes of Key Data mineralisings
Nano particle can induce the Th1/Th2/Th17 type cellullar immunologic responses of host, and it can induce stronger Th2/ compared with soluble protein
Th17 type cellullar immunologic responses, and stronger Th2 type cellullar immunologic responses are can induce compared with Al adjuvants, as a result see accompanying drawing 8.
Result above shows that calcium phosphate mineralized albumen can effectively improve the heat endurance of protein, when protein is vaccine
When, while the immunogenicity of protein vaccine can be effectively improved.
Claims (7)
1. a kind of heat endurance protein (polypeptide), it is characterised in that：Biomineralization peptide is connected with the protein (polypeptide),
Described biomineralization peptide has the ability of induction inorganic matter mineralising, and described biomineralization peptide is drawn by technique for gene engineering
Enter.
2. heat endurance protein (polypeptide) according to claim 1, it is characterised in that：Described inorganic matter is calcic ore deposit
Thing.
3. heat endurance protein (polypeptide) according to claim 2, it is characterised in that：Described calcium mineral is selected from phosphorus
Sour calcium, calcium oxalate, calcium carbonate.
4. heat endurance protein (polypeptide) according to claim 1, it is characterised in that：Magnesium ion is added in mineralising.
5. according to claim 1 have heat endurance protein (polypeptide), it is characterised in that：Described biomineralization peptide
Include one of sequence as follows：
CGPKDVVVGVPGGQDGPC；
DSSEEKFLRRIGRFG；
SVKRGTSVGMKPSPRP；
RWRLEGTDDKEEPESQRRIGRFG。
6. heat endurance protein (polypeptide) according to claim 1, in the protein (polypeptide) that said process is obtained
Add CaCl2、Mgcl2(Na)2HPO4/NaH2PO4。
7. the preparation method of the heat endurance protein (polypeptide) according to claim any one of 1-6, it is characterised in that：Institute
The heat endurance protein (polypeptide) stated, its preparation method is comprised the steps of：
(1) concentration of protein (polypeptide) is 100-600ng/ml in the original system；
(2) Ca is added in above-mentioned protein (polypeptide) solution2+, Ca in described original system2+Ion concentration is 5-10 μm of ol/
ml；
(3) Mg is added in above-mentioned protein (polypeptide) solution2+Mg in ion, described original system2+Ion concentration is 5-10 μ
mol/m；
(4) PO is added in above-mentioned protein (polypeptide) solution4 3-PO in ion, described original system4 3-Ion concentration is 5-7
μmol/ml。
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| CN114736310A (en) * | 2022-06-10 | 2022-07-12 | 北京华夏兴洋生物科技有限公司 | Protein and biological material for producing circovirus type 2 virus-like particles and application |
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| CA2674029A1 (en) * | 2006-12-28 | 2008-12-04 | University Of Maryland Biotechnology Institute | Metal coated virus-based nanoelectrodes and method of assembling of same |
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| CN110859824A (en) * | 2019-11-25 | 2020-03-06 | 重庆医科大学 | Method for preparing heat-stable vaccine based on calcium phosphate mineralization |
| CN114736310A (en) * | 2022-06-10 | 2022-07-12 | 北京华夏兴洋生物科技有限公司 | Protein and biological material for producing circovirus type 2 virus-like particles and application |
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Application publication date: 20171027 |