CN104744581B - A kind of toxoplasma protein TgVP1 intracellular regions antigen polypeptide, the polyclonal antibody of resisting toxoplasmosis TgVP1 and its application - Google Patents
A kind of toxoplasma protein TgVP1 intracellular regions antigen polypeptide, the polyclonal antibody of resisting toxoplasmosis TgVP1 and its application Download PDFInfo
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- CN104744581B CN104744581B CN201510093445.3A CN201510093445A CN104744581B CN 104744581 B CN104744581 B CN 104744581B CN 201510093445 A CN201510093445 A CN 201510093445A CN 104744581 B CN104744581 B CN 104744581B
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- C—CHEMISTRY; METALLURGY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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Abstract
The invention discloses a kind of areas toxoplasma protein TgVP1 born of the same parents Inner antigen polypeptide, the polyclonal antibody of resisting toxoplasmosis TgVP1 and its applications.The amino acid sequence such as SEQ ID NO of the areas toxoplasma TgVP1 born of the same parents Inner of the present invention antigen polypeptide:Shown in 1.The polyclonal antibody of resisting toxoplasmosis TgVP1 has been prepared using the toxoplasma TgVP1 antigen polypeptides as immunogen immune new zealand rabbit, qualification result shows that the polyclonal antibody can specific recognition toxoplasma TgVP1 albumen, it can be used for the detection of toxoplasma TgVP1 albumen in the experiments such as ELISA, Western blot, immunofluorescence, powerful is provided for the basic research of the protein function and its research as potential resisting toxoplasmosis medicine target spot, is had a extensive future.
Description
Technical field
The present invention relates to genetic engineering fields, and in particular to a kind of areas TgVP1 born of the same parents Inner antigen polypeptide, resisting toxoplasmosis TgVP1
Polyclonal antibody and its application.
Background technology
V-H+-PPase is a kind of unique proton pump, is all existed in a variety of animals and plants.It can hydrolyze inorganic burnt phosphorus
The phosphoric anhydride bonds of hydrochlorate, and discharged energy is utilized to transport proton, generate the electrochemical gradient of cross-film, performance and proton pump
The similar effect of ATP enzyme.V-H+-Ppase can make the energy efficient of storage be converted into H+ and/or electric transferring film gradient, for a variety of
Different intracellular transport processes.V-PPases is found to be present in plant and photosynthetic bacteria earliest, recent studies suggest that cone
There is also this enzymes for worm, Leishmania, toxoplasma, plasmodium falciparum.It is worth noting that in the animal place of these parasites
This enzyme is not present in master, therefore V-PPases has important research valence as the potential drug target of parasitic disease
Value.The V-PPases of toxoplasma is distributed mainly on acid calcium body and plasma membrane, can be divided into two types according to the difference of structure and function
Type, VP1 and VP2.TgVP1 needs K+ to activate its biological activity.When toxoplasma invades cell, TgVP1 forms cyclic structure
It is distributed in toxoplasma outer rim, and as the intrusion of toxoplasma is in worm vivo migration.When toxoplasma completes invasive procedure, TgVP1
Come back to the top of toxoplasma.TgVP1 contains 17 membrane-spanning domains, and N-terminal has signal peptide sequence, TgVP1 can be instructed to enter
The secretory pathway of toxoplasma, but specific mechanism of action is still not clear.Some researches show that toxoplasma PPase Activities can inhibit bow
The duplication of shape worm in the cell.Therefore, TgVP1 has the possibility as potential toxoplasmosis clinical diagnosis and therapeutic targets, system
The antibody of standby TgVP1 has a very important significance, and can be that the research of the protein function lay the foundation, while being it as disease
The feasibility of sick marker provides experimental data and supports.
Invention content
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of to be used for ELISA, western-blot, exempt from
The polyclonal antibody of the resisting toxoplasmosis TgVP1 albumen of epidemic disease fluoroscopic examination.
Another object of the present invention is to provide a kind of areas toxoplasma TgVP1 born of the same parents Inner antigen polypeptide.
It is yet another object of the invention to provide the applications of the polyclonal antibody of above-mentioned resisting toxoplasmosis TgVP1 albumen.
Above-mentioned purpose that the invention is realized by the following technical scheme：
A kind of areas toxoplasma TgVP1 born of the same parents Inner antigen polypeptide, amino acid sequence such as SEQ ID NO:Shown in 1.
A kind of polyclonal antibody of anti-TgVP1, be using aforementioned TgVP1 antigen polypeptides as immunogene, be immunized animal prepare and
At.
The preparation method of the polyclonal antibody of above-mentioned anti-TgVP1, steps are as follows：TgVP1 antigen polypeptides are lived with maleic amide
The keyhole blood indigo plant carrier protein KLH of change, as immunogene and adjuvant mixed immunity new zealand rabbit after desalting and purifying, during which
It is immunized more than twice, waits for that ELISA detects serum antibody titer up to 1:After 100000, rabbit anteserum is acquired, by purifying,
After ELISA and western blot identifications, the polyclonal antibody of anti-TgVP1 is obtained.
Chemically synthesized polypeptide antigen is small molecule, itself is difficult the antigenicity having had, and animal can only be induced to generate very
Weak immune response, thus it is critically important to be crosslinked with carrier protein.Carrier protein contains many epitopes, can stimulate
T helper cell, and then induce B cell reaction.For with the crosslinked carrier protein of polypeptide there are many, wherein the load most generally used
Body is keyhole limpet hemocyanin (keyhole limpet hemacyanin, KLH), bovine serum albumin(BSA) (bovine serum
Albumin, BSA), ovalbumin (ovalbumin, OVA) and bovine thyroglobulin (bovine thyroglobulin,
THY).KLH has higher antigenicity, most strong with immunogenicity after the TgVP1 antigen polypeptides crosslinking of the present invention, therefore the present invention
Select KLH as coupling carrier albumen.BSA is also often used as peptide carrier, but since BSA is often used as detection experiment
Blocking agent and make this method production antibody application on there is certain limitations.
The polyclonal antibody of toxoplasma TgVP1 is in the toxoplasmas TgVP1 albumen such as ELISA, western-blot, immunofluorescence
Application in test experience.
Compared with prior art, the invention has the advantages that：
(1) present invention is to the secondary structure of toxoplasma TgVP1 protein amino acid sequences, immunogenicity, hydrophilic and hydrophobic, surface
Accessibility, membrane-spanning domain etc. are analyzed, and it is artificial synthesized to determine that the suitable areas Duan Bao Inner peptide sequence carries out；By the polypeptide of synthesis with
The carrier mcKLH couplings of maleimide ammonia activation, carry out desalting column by this coupled product and new zealand rabbit are immunized after purification；Through excessive
Secondary immune rabbit anteserum is detected antibody titer with ELISA method, and potency collects immune rabbit anteserum after reaching ideal value, is used in combination
Protein G-protein purification column antibody purifications；The identifications such as ELISA, Western bot are carried out to purified antibodies.Qualification result
Show that the polyclonal antibody can specific recognition toxoplasma TgVP1 albumen；
(2) this antibody purification is used to carry out ELISA, Western bot, the immunofluorescence dyeing etc. of toxoplasma by the present invention
Experiments have shown that it can identify natural TgVP1 albumen.The polyclonal antibody of this TgVP1 is further to study toxoplasma TgVP1's
Function is laid a good foundation, and strong tool is provided as potential resisting toxoplasmosis medicine target spot to verify it.
Description of the drawings
Fig. 1 is that the polyclonal antibody of anti-TgVP1 is examined through the SDS-PAGE electrophoresis (12%) of protein G gel columns after purification
Survey result.Swimming lane M：Albumen Marker；Swimming lane 1,2：The polyclonal antibody of anti-TgVP1 after purification.
Fig. 2 is the western-blot qualification results of the polyclonal antibody of anti-TgVP1.Swimming lane 1：Loading is that OFTu cells are split
Solve albumen；Swimming lane 2：Loading is toxoplasma tachyzoite crack protein；Swimming lane M：Albumen Marker.
Specific implementation mode
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are only used for explaining this hair
It is bright, rather than limit the scope of the invention.Under the premise of without departing substantially from technical scheme of the present invention, made for the present invention
Field those of ordinary skill any change easy to implement is fallen within scope of the presently claimed invention.
Embodiment 1 prepares toxoplasma TgVP1 antigen polypeptides
1. the design and synthesis of toxoplasma TgVP1 polypeptides：According to the toxoplasma TgVP1 sequence (accession number on GenBank：
EPT25031.1 toxoplasma TgVP1 protein sequences) are obtained, 816 amino acid are contained.
2. analyzing TgVP1 protein characteristics with DNAstar softwares, analysis result is shown in Table 1, and the molecular weight of the albumen is 85296
Dalton, isoelectric point 4.83 are acidic protein.
Table 1
| Analysis project (Analysis) | Holoprotein (Whole Protein) |
| Molecular weight (Molecular Weight) | 85.296kDa |
| Length (Length) | 816aa |
| 1microgram= | 11.724pMoles |
| Molar extinction coefficient (Molar Extinction coefficient) | 95770 |
| 1A (280)= | 1.10mg/mL |
| Isoelectric point (Isoelectric Point) | 4.83 |
| Charge (Charge at pH 7) | -16.1 |
3. with the membrane-spanning domain of TMHMM softwares analysis TgVP1 albumen, IEDB softwares analyze TgVP1 protein immunogenics, close and distant
Aqueous and surface accessibility, the results showed that toxoplasma TgVP1 protein 29s 2-320aa have 29 amino acid antigenicities, hydrophily and
Surface accessibility is stronger, which is located at the areas TgVP1 albumen born of the same parents Inner.
4.TgVP1 antigen polypeptides screen：By above-mentioned analysis, finally screening polypeptide sequence is：
YTKAADVGADLSGKNEYGMSEDDPRNPAC (292aa-320aa, SEQ ID NO:1).
Embodiment 2TgVP1 antigen polypeptides prepare and and carrier protein couplet
1. Peptide systhesis
TgVP1 antigen polypeptides：YTKAADVGADLSGKNEYGMSEDDPRNPAC is synthesized by Shanghai gill biochemical corp.It is more
Peptide C end is a cysteine, is convenient for and carrier protein couplet.
2. polypeptide and carrier protein couplet
A. the mcKLH that a pipe maleimide activation is diluted with 200 μ L ultra-pure waters, is diluted to the solution of 10mg/mL.(note：
Above-mentioned solution is translucent in blue and white, must not vibrate or heat, otherwise mcKLH can be caused to precipitate.)
B. the haptens containing sulfydryl is dissolved (i.e. with the coupling buffer for being equivalent to 1.0~2.5 times of volumes of solution in step a
TgVP1 antigen polypeptides).Such as：200~500 μ L coupling buffers of 2mg haptens are dissolved, mcKLH is added to.If more
Peptide is readily soluble, can be added to solid in mcKLH suspensions.(note：If haptens is less soluble, DMSO increases can be added
Dissolving.DMSO concentration in being coupled lysate is less than 30%, otherwise carrier protein mutability).
C. mixing polypeptide, mcKLH solution immediately, then in room temperature reaction 2h.
3. coupled product purifies
Conjugate is purified by gel chromatography desalting column.If conjugate is injected within one week, purified with PBS.Such as
Fruit conjugate freezes, and is purified with purifying buffer salt, if forming precipitation in coupling, supernatant is collected in centrifugation, retains precipitation.Only
Purify supernatant.The conjugate of purifying and precipitation are combined.
A. one bottle of purifying buffer salt is dissolved, the ultra-pure water of 60mL degassings, 4 DEG C of preservations are added.
B. take away desalting column top and bottom lid, make storage liquid be discharged.One desalting column can purify 0.5mL samples.
C. pillar is rinsed with the purification buffer of 3~5 times of column volumes (15~25mL).
D. the peptide carrier mixture of 0.5mL is directly added into column center.The purification buffer of 0.5mL is added, is detaching
Each peak is collected in pipe.
E. absorbance is measured at 280nm to determine which partly contains conjugate.It is incited somebody to action what first absorption peak detected
It is hapten conjugation object.Mix all parts containing conjugate.
F. after the part containing conjugate occurs, continue that buffer solution is added into column, collect the haptens not being coupled.
G. by conjugate filtration sterilization, -80 DEG C are sterilely maintained within.
As a result:Coomassie brilliant blue measures albumen concentration and content after coupling, often 250 μ g packing of pipe, -80 DEG C of preservations.
3 anti-TgVP1 polypeptides rabbit polyclonal antibody of embodiment prepares and purifying
1. animal immune
Two new zealand male rabbits, 2~2.5kg of weight is immunized in coupled product.Freund's complete adjuvant or incomplete Freund's adjuvant
Purchased from Sigma companies.
Immune programme is shown in Table 2, first dilutes 100 μ g antigens with PBS, until 1.25mL, with isometric adjuvant mixing, vortex oscillation
100min, detection emulsification is as a result, take drop antigen drop in waterborne, insoluble diffusion in one minute.Immune vestibule venous blood sampling, 4 DEG C
Serum, -80 DEG C of preservations is taken to make negative control sera after standing.
2 specific immune programme of table
Antibody titer ELISA detections
(1) experiment reagent
A. phosphate buffer (PBS) (10 × concentration)：Sodium chloride (NaCl) 50g, potassium chloride (KCl) 1.25g, di(2-ethylhexyl)phosphate
Hydrogen potassium (KH2PO4) 1.25g, disodium hydrogen phosphate (Na2HPO4·12H2O) 18.1g, distilled water add to 1000mL, with 1MHCl tune pH
To 7.2.
B. confining liquid：Normal calf serum 10mL, 1 × PBS (being free of tween) 90mL.
C. washing buffer：Tween-20 (Tween 20) 0.2mL, 1 × PBS 1000mL.
D. substrate colour developing A liquid：Sodium acetate (CH3COONa) 13.6g, citric acid (C6H8O7·H2O) 1.6g, hydrogen peroxide
(H2O230%) 0.3mL, distilled water add to 500mL.
E. substrate colour developing B liquid：Disodium ethylene diamine tetraacetate (EDTA-Na2) 0.2g, citric acid (C6H8O7·H2O) 0.95g,
Glycerine (C3H8O3) 50mL, tetramethyl benzidine (TMB) 0.2g, distilled water adds to 500mL.
F. terminate liquid (2M H2SO4)：Take dense H2SO427.62mL is added slowly in the distilled water of 473mL, and mixing is
It can.
G. antigen coat liquid (0.1M carbonate buffer solutions)：PH 9.6, sodium carbonate (Na2CO3) 1.59g, sodium bicarbonate
(NaHCO3) 2.93g, Sodium azide (NaN3) 0.2g, distilled water adds to 1000mL.
H. the goat anti-mouse igg (commercialization) of horseradish peroxidase-labeled.
(2) experimental procedure
A. antigen coat：Pure polypeptide antigen final concentration is generally 1~2 μ g/mL, and 100 μ L is taken to be added after being diluted with coating buffer
In each hole of the enzyme-linked detection plate of polystyrene, after 4 DEG C are stayed overnight, wash liquid 3 times.It is recommended that 4 DEG C of coatings are overnight.
B. it closes：Add 200 μ L or fill it up with confining liquid per hole, 4 DEG C overnight or 37 DEG C after two hours, washs 3 times, pats dry.Set 4
DEG C refrigerator saves backup.
C.ELISA is detected
1. plus sample to be tested：Serum is detached after rabbit ear vein blood sampling, with PBS doubling dilutions, 50~100 holes μ L/ are loaded onto
In the ELISA Plate being coated with, meanwhile, it is negative control to choose immune preceding rabbit anteserum respectively, and 37 DEG C of incubation 30min are washed 3 times, clapped
It is dry.
2. plus secondary antibody：Extension rate, 100 holes μ L/, 37 DEG C of incubation 30min, washing 3 are selected according to the potency of ELIAS secondary antibody
It is secondary, it pats dry.
3. developing the color：Each holes 80 μ L/ of substrate colour developing A, B liquid, 37 DEG C of colour developing 15min are added.
4. terminating：80 holes μ L/ of terminate liquid are added.
5. reading：Each hole OD values are measured with 450nm Single wavelengths, to be more than 2.5 with the ratio (P/N) of negative control hole OD values
It is limited, as the critical point for being judged as positive or determining potency.
Rabbit anteserum is collected：
Using arteria carotis depletion method, non-anti-freezing rabbit whole blood is incubated 2h at 37 DEG C after collecting.
2. antibody purification
(1) saturated ammonium sulphate method
A. saturated ammonium sulfate solution (SAS) is prepared：By 767g (NH4)2SO4It is slowly added in 1L distilled water while stirring.With
Ammonium hydroxide or sulfuric acid are transferred to sulfuric acid pH7.0.This ammonium sulfate (4.1mol/L, 25 DEG C) that i.e. saturation degree is 100%.
B. it precipitates
1. sample (serum) 20 000 × g centrifuges 30min, cell fragment is removed；
2. retaining supernatant and measuring volume；
3. being slowly added into while stirring in isometric SAS to supernatant, final concentration of 1:1；
It stirs 6 hours or is stirred overnight (4 DEG C) 4. solution is placed on magnetic stirring apparatus, protein is made fully to precipitate.
C. it dialyses
1. protein solution 10 000 × g centrifugation 30min (4 DEG C).It abandons supernatant and retains precipitation；
2. precipitation is dissolved in a small amount of (10~20mL) PBS-0.2g/L Sodium azides.It is put into bag filter pair after precipitation dissolving
PBS-0.2g/L Sodium azides are dialysed 24~48 hours (4 DEG C), and it is primary to change elution buffer every 3~6 hours, thoroughly to remove desulfuration
Sour ammonia；
3. dialyzate centrifuges, protein content in supernatant is measured.
(2) Protein G affinity purification
Required reagent and special installation：1.0mol/L Tris(pH8.0)；100mmol/L Tris(pH8.0)；10mmol/
L Tris(pH8.0)；50mmol/L glycine (pH3.0)；Column chromatography instrument.
Operating procedure：
A. the 1.0mol/L Tris (pH8.0) that 1/10 volume is added adjust (serum, tissue culture supernatant containing antibody samples
Or ascites) pH value to 8.0.
B. antibody-solutions are passed through into albumin A or Protein G microballoon column.In these columns, every milliliter of wet microballoon can be in conjunction with about
10~20mL antibody (1 albumin A or Protein G microballoon molecule combine 2 molecular antibodies).The about volume of record dress column microballoon.Cause
The amount using cleaning and elution buffer is determined for the volume of microballoon column.
C. microballoon is washed with the 100mmol/L Tris (pH8.0) of 10 times of column volumes.
D. microballoon is washed with the 10mmo1/L Tris (pH8.0) of 10 times of column volumes.
E. it uses 50mmol/L glycine (pH3.0) to elute microballoon column, the buffer solution of about 1/2 column volume is added every time, by several times
It is added.Eluent is collected with the test tube of the 1mol/L Tris (pH8.0) containing 1/10 column volume, each pipe is slowly shaken up, its pH is made
Value is restored to neutrality.
F. the collecting pipe containing antibody is mixed, total protein is surveyed with SDS-PAGE and coomassie brilliant blue staining.
As a result:(Fig. 1) is identified through SDS-PAGE protein electrophoresis, ultraviolet specrophotometer measures antibody concentration after antibody purification
For 0.4mg/mL.Purified antibodies are stored in -20 DEG C, and in 0.01M PBS buffer solution, 40% glycerine and 0.5% are contained in solution
BSA。
Embodiment 4：The identification of the polyclonal antibody of anti-TgVP1
1. the ELISA of the polyclonal antibody of anti-TgVP1 is identified
It is detection antigen, coated elisa plate, with 1 with the TgVP1 polypeptides of synthesis：2000 diluted not immune rabbit anteserum conducts
Negative control, antibody doubling dilution by rabbit anteserum and after purification, is detected using ELISA method, with the ratio with negative serum
It is judged as the positive more than 2.1, calculates monoclonal antibody potency.
As a result, the potency of the anti-TgVP1 antibody of purifying reaches 1:128000 (tables 3).
3 anti-TgVP1 antibody titers (k=1000) of table
| Extension rate | 1:2k | 1:4k | 1:8k | 1:16k | 1:32k | 1:64k | 1:128k | 1:256k | 1:512k | Negative control | PBS |
| OD values | 2.267 | 2.029 | 1.933 | 1.501 | 0.905 | 0.768 | 0.453 | 0.220 | 0.142 | 0.127 | 0.048 |
2. the Western-Blot of the polyclonal antibody of anti-TgVP1 is identified
Toxoplasma tachyzoite and OFTu cells are collected respectively, and toxoplasma crack protein and OFTu cracking are made after being cracked
Albumen, loading after being cracked with 2 × SDS lysis buffers carry out 10%SDS-PAGE electrophoresis, reference《Molecular cloning》Described in
Western blotting method, electrotransfer condition are 100V electrophoresis 1h, and confining liquid is the TBST containing 5% skimmed milk power, and primary antibody is the present invention
In anti-TgVP1 polyclonal antibody (0.1 μ g/mL of final concentration), secondary antibody is that the sheep anti-mouse igg of horseradish peroxidase-labeled is anti-
Body carries out immunoblotting assay by ECL Color Appearance Systems.
As a result：The polyclonal antibody of anti-TgVP1 in the present invention can detect that 85KD sizes are left in toxoplasma lysate
Right protein band, is consistent (Fig. 2) with toxoplasma TgVP1 molecular weight of albumen sizes.
3. the identified by immunofluorescence of the polyclonal antibody of anti-TgVP1
Toxoplasma smear is prepared, 4% paraformaldehyde fixes 15min, and 0.25%TritonX100, incubation at room temperature is added dropwise
10min makes cell membrane have permeability, so that antibody enters.PBS is washed three times, and the PBST room temperatures containing 1%BSA close 2h.
PBS is washed 3 times, then uses the polyclonal antibody (PBST 1 of the anti-TgVP1 in the present invention:500 dilutions) incubation at room temperature 1.5h.
PBS is washed 3 times, the goat anti-rabbit igg (PBST 1 marked with Alexa Fluor 546 (red fluorescence):1000 dilutions) room temperature incubates
Educate 1h.DAPI dyes (blue) 10min after PBS washings.After PBS develops a film, fluorescence microscopy is under the microscope.Use normal rabbit serum simultaneously
Make negative control.
As a result：Red is observed in the toxoplasma cytoplasm of polyclonal antibody dyeing through the anti-TgVP1 in the present invention
Fluorescence, it was demonstrated that the anti-TgVP1 antibody in the present invention can recognize that natural TgVP1 albumen.
Claims (2)
1. a kind of polyclonal antibody of resisting toxoplasmosis TgVP1, it is characterised in that be to be with toxoplasma TgVP1 intracellular region antigen polypeptides
Immunogene, immune animal are prepared, the amino acid sequence such as SEQ ID NO of the toxoplasma TgVP1 intracellular region antigen polypeptides:
Shown in 1.
2. the preparation method of the polyclonal antibody of resisting toxoplasmosis TgVP1 described in claim 1, it is characterised in that steps are as follows：It will
The keyhole blood indigo plant carrier protein KLH of toxoplasma TgVP1 born of the same parents Inner area's antigen polypeptides and maleic amide activation is pure through desalination
It as immunogene and adjuvant mixed immunity new zealand rabbit after change, is during which immunized more than twice, waits for that ELISA detects serum antibody
Potency is up to 1:After 100000, rabbit anteserum is acquired, after purifying, ELISA and western blot identifications, obtains resisting toxoplasmosis
The polyclonal antibody of TgVP1.
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| Title |
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| Characterization of a novel organelle in Toxoplasma gondii with similar composition and function to the plant vacuole;Kildare Miranda et al.;《Mol Microbiol》;20100601;第76卷(第6期);1358-1375 * |
| 伯氏疟原虫酸性钙体的分离纯化及其蛋白质组学研究;程莎莎;《中国优秀硕士论文全文数据库（医药卫生科技辑）》;20150115;E059-228 * |
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