CN104744581A

CN104744581A - Toxoplasma gondii TgVP1 extracellular region antigen polypeptide, anti-toxoplasma gondii TgVP1 polyclonal antibody and application of polyclonal antibody

Info

Publication number: CN104744581A
Application number: CN201510093445.3A
Authority: CN
Inventors: 郝文波; 罗树红; 肖斌
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2015-03-02
Filing date: 2015-03-02
Publication date: 2015-07-01
Anticipated expiration: 2035-03-02
Also published as: CN104744581B

Abstract

The invention discloses a toxoplasma gondii TgVP1 extracellular region antigen polypeptide, an anti-toxoplasma gondii TgVP1 polyclonal antibody and the application of the polyclonal antibody. The amino acid sequence of the toxoplasma gondii TgVP1 extracellular region antigen polypeptide is as shown in SEQ ID NO: 1. A New Zealand rabbit is immunized by the toxoplasma gondii TgVP1 extracellular region antigen polypeptide as an immunogen so that the anti-toxoplasma gondii TgVP1 polyclonal antibody can be obtained; an identification result indicates that the polyclonal antibody is capable of specifically identifying the toxoplasma gondii TgVP1 protein and can be applied to the detection of the toxoplasma gondii TgVP1 protein in tests such as ELISA, Western blot and immunofluorescence; a powerful tool is provided for the fundamental research of the protein functions and the research of the protein as a potential anti-toxoplasma gondii drug target; in short, the polyclonal antibody is wide in application prospect.

Description

The polyclonal antibody of a kind of toxoplasma protein TgVP1 intracellular region antigenic peptide, resisting toxoplasmosis TgVP1 and application thereof

Technical field

The present invention relates to genetically engineered field, be specifically related to a kind of TgVP1 born of the same parents Inner district antigenic peptide, the polyclonal antibody of resisting toxoplasmosis TgVP1 and application thereof.

Background technology

V-H+-PPase is a kind of proton pump of uniqueness, all exists in multiple animals and plants.It can be hydrolyzed the phosphoric anhydride bonds of inorganic pyrophosphate, and utilizes the energy transhipment proton discharged, and produces the electrochemical gradient of cross-film, plays and effect that proton pump ATP enzyme is similar.V-H+-Ppase can make the energy efficient of storage be converted into H+ and/or electric transferring film gradient, for multiple different intracellular transport process.V-PPases is found to be present in plant and photosynthetic bacterium the earliest, and recent research shows that trypanosome, leishmania, toxoplasma gondii, plasmodium falciparum also exist this enzyme.It is worth noting, in these parasitic animal hosts, all there is not this enzyme, therefore V-PPases has important researching value as the drug target that parasitosis is potential.The V-PPases of toxoplasma gondii is mainly distributed in acid calcium body and plasma membrane, and the difference according to structure and function can be divided into two types, VP1 and VP2.TgVP1 needs K+ to activate its biologic activity.When toxoplasma gondii invasion cell, TgVP1 forms cyclic structure and is distributed in toxoplasma gondii outer rim, and moves in polypide along with the intrusion of toxoplasma gondii.When toxoplasma gondii completes invasive procedure, TgVP1 comes back to the top of toxoplasma gondii.TgVP1 contains 17 membrane-spanning domains, and N end has signal peptide sequence, and TgVP1 can be instructed to enter the Secretory Pathway of toxoplasma gondii, but concrete mechanism of action is still not clear.There are some researches show that toxoplasma gondii PPase Activity can suppress toxoplasma gondii to copy intracellular.Therefore, TgVP1 has the possibility as potential toxoplasmosis clinical diagnosis and therapeutic targets, the antibody tool of preparation TgVP1 is of great significance, and can lay the foundation, simultaneously for its feasibility as disease marker provides experimental data support for the research of this protein function.

Summary of the invention

The object of the invention is to overcome the deficiencies in the prior art, a kind of polyclonal antibody of resisting toxoplasmosis TgVP1 albumen that can be used for ELISA, western-blot, Immunofluorescence test is provided.

Another object of the present invention is to provide a kind of toxoplasma gondii TgVP1 born of the same parents Inner district antigenic peptide.

Another object of the present invention is to provide the application of the polyclonal antibody of above-mentioned resisting toxoplasmosis TgVP1 albumen.

The present invention is achieved through the following technical solutions above-mentioned purpose:

A kind of toxoplasma gondii TgVP1 born of the same parents Inner district antigenic peptide, aminoacid sequence is as shown in SEQ ID NO:1.

A polyclonal antibody of anti-TgVP1 is that immune animal is prepared from aforementioned TgVP1 antigenic peptide for immunogen.

The preparation method of the polyclonal antibody of above-mentioned anti-TgVP1, step is as follows: the blue carrier protein KLH of the keyhole blood that TgVP1 antigenic peptide and maleinamide activate, as immunogen and adjuvant mixed immunity new zealand rabbit after desalting and purifying, period carries out more than twice immunity, after ELISA detection serum antibody titer reaches 1:100000, gather rabbit anteserum, after purifying, ELISA and western blot identify, obtain the polyclonal antibody of anti-TgVP1.

The polypeptide antigen of chemosynthesis is small molecules, itself is difficult to the antigenicity had, and can only produce very weak immune response by induced animal, it is very important for being thus cross-linked with carrier proteins.Carrier proteins contains a lot of epitope, can stimulate t helper cell, and then elicit B cell reaction.Have multiple for the carrier proteins crosslinked with polypeptide, the carrier wherein the most generally used is keyhole limpet hemocyanin (keyhole limpet hemacyanin, KLH), bovine serum albumin (bovine serum albumin, BSA), ovalbumin (ovalbumin, OVA) and bovine thyroglobulin (bovine thyroglobulin, THY).KLH has higher antigenicity, and after crosslinked with TgVP1 antigenic peptide of the present invention, immunogenicity is the strongest, and therefore the present invention selects KLH as coupling carrier albumen.BSA is also commonly used to as peptide carrier, but antibody that the method is produced also exists certain limitation in application because BSA is often used as the blocker of detection experiment.

The application of polyclonal antibody in the toxoplasma gondii TgVP1 protein measurement assays such as ELISA, western-blot, immunofluorescence of toxoplasma gondii TgVP1.

Compared with prior art, the present invention has following beneficial effect:

(1) secondary structure, immunogenicity, hydrophilic and hydrophobic, surperficial accessibility, membrane-spanning domain etc. of the present invention to toxoplasma gondii TgVP1 protein amino acid sequence is analyzed, and determines that a suitable Duan Bao Inner district peptide sequence carries out synthetic; By the polypeptide of synthesis and the carrier mcKLH coupling that activates of maleoyl imido, this coupled product is carried out immunize New Zealand rabbit after desalting column purifying; Rabbit anteserum ELISA method antagonist through repeatedly immunity is tired and is detected, and tires after reaching ideal value and collects immunize rabbit serum, and with Protein G-protein purification column antibody purification; Purified antibodies is carried out to the qualifications such as ELISA, Western bot.Qualification result shows that this polyclonal antibody can specific recognition toxoplasma gondii TgVP1 albumen;

(2) experiment such as ELISA, Western bot, immunofluorescence dyeing that this antibody purification is used for carrying out toxoplasma gondii by the present invention proves that it can identify natural TgVP1 albumen.The polyclonal antibody of this TgVP1 is that the function studying toxoplasma gondii TgVP1 is further laid a good foundation, for verifying that it provides strong instrument as potential resisting toxoplasmosis medicine target spot.

Accompanying drawing explanation

Fig. 1 is SDS-PAGE electrophoresis (12%) detected result of polyclonal antibody after protein G gel column purifying of anti-TgVP1.Swimming lane M: albumen Marker; Swimming lane 1,2: the polyclonal antibody of the anti-TgVP1 after purifying.

Fig. 2 is the western-blot qualification result of the polyclonal antibody of anti-TgVP1.Swimming lane 1: loading is OFTu cytolytic proteins; Swimming lane 2: loading is toxoplasma tachyzoite crack protein; Swimming lane M: albumen Marker.

Embodiment

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments only for explaining the present invention, and be not used in and limit the scope of the invention.Under the prerequisite not deviating from technical scheme of the present invention, any change that those of ordinary skill in the art made for the present invention easily realize all will fall within right of the present invention.

Embodiment 1 prepares toxoplasma gondii TgVP1 antigenic peptide

1. the design of toxoplasma gondii TgVP1 polypeptide and synthesis: obtain toxoplasma gondii TgVP1 protein sequence, containing 816 amino acid according to the toxoplasma gondii TgVP1 sequence (accession number: EPT25031.1) on GenBank.

2., with DNAstar software analysis TgVP1 protein characteristic, analytical results is in table 1, and the molecular weight of this albumen is 85296 dalton, and iso-electric point is 4.83, is acidic protein.

Table 1

Analysis project (Analysis)	Whole protein (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
Iso-electric point (Isoelectric Point)	4.83
		Electric charge (Charge at pH 7)	-16.1

3. with the membrane-spanning domain of TMHMM software analysis TgVP1 albumen, IEDB software analysis TgVP1 protein immunogenic, hydrophilic and hydrophobic and surperficial accessibility, result shows that toxoplasma gondii TgVP1 protein 29 2-320aa has 29 amino acid antigenicities, wetting ability and surperficial accessibility comparatively strong, and this peptide section is positioned at TgVP1 albumen born of the same parents Inner district.

4.TgVP1 antigenic peptide screens: through above-mentioned analysis, and final screening peptide sequence is: YTKAADVGADLSGKNEYGMSEDDPRNPAC (292aa-320aa, SEQ ID NO:1).

The preparation of embodiment 2TgVP1 antigenic peptide and and carrier protein couplet

1. Peptide systhesis

TgVP1 antigenic peptide: YTKAADVGADLSGKNEYGMSEDDPRNPAC is synthesized by gill biochemical corp, Shanghai.Peptide C end is a halfcystine, is convenient to and carrier protein couplet.

2. polypeptide and carrier protein couplet

A. dilute the mcKLH of a pipe maleimide activation with 200 μ L ultrapure waters, be diluted to the solution of 10mg/mL.(note: above-mentioned solution is that pearl opal is translucent, must not vibrate or heat, otherwise mcKLH can be caused to precipitate.)

B. the haptens (i.e. TgVP1 antigenic peptide) containing sulfydryl is dissolved with the coupling buffer being equivalent to solution 1.0 ~ 2.5 times of volumes in step a.Such as: 2mg haptens 200 ~ 500 μ L coupling buffers are dissolved, joins mcKLH.If polypeptide is Yi Rong, can join in mcKLH suspension with solid.(note: if haptens is not easily molten, can add DMSO increases dissolving.DMSO in coupling lysate concentration lower than 30%, otherwise carrier proteins volatility).

C. polypeptide, mcKLH solution is mixed immediately, then at room temperature reaction 2h.

3. coupled product purifying

Purifying conjugate is carried out by gel chromatography desalting column.If conjugate was injected within one week, purify with PBS.If conjugate is frozen, purify with purifying buffering salt, if form precipitation when coupling, centrifugal, collect supernatant, retain precipitation.Only purifying supernatant.The conjugate of purifying and precipitation are combined.

A. dissolve one bottle of purifying buffering salt, add the ultrapure water that 60mL is degassed, 4 DEG C of preservations.

B. to take away the lid of T&B of desalting column, storage liquid is discharged.One desalting column can purifying 0.5mL sample.

C. the purification buffer of 3 ~ 5 times of column volumes (15 ~ 25mL) is used to rinse pillar.

D. the peptide carrier mixture of 0.5mL is directly added post center.Add the purification buffer of 0.5mL, in separator tube, collect each peak.

E. under 280nm, absorbancy is measured to determine which part is containing conjugate.What detect at first absorption peak will be hapten conjugation thing.Mix all parts containing conjugate.

F. after the part containing conjugate occurs, continue to add damping fluid in post, collect the haptens not having coupling.

G. by conjugate filtration sterilization, Preservation in sterile condition is at-80 DEG C.

Result: coomassie brilliant blue records protein concentration and content after coupling, often pipe 250 μ g packing ,-80 DEG C of preservations.

The preparation of embodiment 3 anti-TgVP1 polypeptide rabbit polyclonal antibody and purifying

1. animal immune

Coupled product immunity two new zealand male rabbits, body weight 2 ~ 2.5kg.Freund's complete adjuvant or Freund's incomplete adjuvant available from Sigma.

Immune programme for children, in table 2, first dilutes 100 μ g antigens with PBS, and to 1.25mL, with the mixing of equal-volume adjuvant, vortex vibration 100min, detects emulsification result, get an antigen and drip in waterborne, insoluble solution diffusion in a minute.Immunity vestibule venous blood sampling, 4 DEG C leave standstill after get serum ,-80 DEG C of preservations, make negative control sera.

The concrete immune programme for children of table 2

Antibody titer ELISA detects

(1) experiment reagent

A. phosphate buffered saline buffer (PBS) (10 × concentrated): sodium-chlor (NaCl) 50g, Repone K (KCl) 1.25g, potassium primary phosphate (KH ₂pO ₄) 1.25g, Sodium phosphate dibasic (Na ₂hPO ₄12H ₂o) 18.1g, distilled water adds to 1000mL, adjusts pH to 7.2 with 1MHCl.

B. confining liquid: normal calf serum 10mL, 1 × PBS (not containing tween) 90mL.

C. lavation buffer solution: tween 20 (Tween 20) 0.2mL, 1 × PBS 1000mL.

D. substrate colour developing A liquid: sodium-acetate (CH ₃cOONa) 13.6g, citric acid (C ₆h ₈o ₇h ₂o) 1.6g, hydrogen peroxide (H ₂o ₂30%) 0.3mL, distilled water adds to 500mL.

E. substrate colour developing B liquid: disodium ethylene diamine tetraacetate (EDTA-Na ₂) 0.2g, citric acid (C ₆h ₈o ₇h ₂o) 0.95g, glycerine (C ₃h ₈o ₃) 50mL, tetramethyl benzidine (TMB) 0.2g, distilled water adds to 500mL.

F. stop buffer (2M H ₂sO ₄): get dense H ₂sO ₄27.62mL, slowly joins in the distilled water of 473mL, mixes.

G. antigen coated liquid (0.1M carbonate buffer solution): pH 9.6, sodium carbonate (Na ₂cO ₃) 1.59g, sodium bicarbonate (NaHCO ₃) 2.93g, sodium azide (NaN ₃) 0.2g, distilled water adds to 1000mL.

H. the goat anti-mouse igg (commercialization) of horseradish peroxidase-labeled.

(2) experimental procedure

A. antigen coated: pure polypeptide antigen final concentration is generally 1 ~ 2 μ g/mL, get 100 μ L add in each hole of polystyrene enzyme joint inspection drafting board with after coating buffer dilution, 4 DEG C spend the night after, wash liquid 3 times.Advise that 4 DEG C of bags are spent the night.

B. close: every hole adds 200 μ L or fills it up with confining liquid, 4 DEG C are spent the night or 37 DEG C after two hours, wash 3 times, pat dry.Put 4 DEG C of Refrigerator stores for subsequent use.

C.ELISA detects

1. add testing sample: separation of serum after rabbit ear vein blood sampling, use PBS doubling dilution, 50 ~ 100 μ L/ holes are loaded onto bag by good enzyme plate, and meanwhile, choosing the front rabbit anteserum of immunity is respectively negative control, hatches 30min for 37 DEG C, washs 3 times, pat dry.

2. add two to resist: according to the selection extension rate of tiring of ELIAS secondary antibody, 100 μ L/ holes, hatch 30min for 37 DEG C, wash 3 times, pat dry.

3. develop the color: add the substrate colour developing each 80 μ L/ holes of A, B liquid, 37 DEG C of colour developing 15min.

4. stop: add stop buffer 80 μ L/ hole.

5. reading: measure each hole OD value with 450nm Single wavelength, is limited to be greater than 2.5 with the ratio (P/N) of negative control hole OD value, as being judged as the positive or determining the stagnation point of tiring.

Rabbit anteserum is collected:

Adopt carotid artery depletion method, non-anti-freezing rabbit whole blood hatches 2h at 37 DEG C after collecting.

2. antibody purification

(1) saturated ammonium sulphate method

A. saturated ammonium sulphate solution (SAS) is prepared: by 767g (NH ₄) ₂sO ₄slowly be added to while stirring in 1L distilled water.Sulfuric acid pH7.0 is transferred to ammoniacal liquor or sulfuric acid.This namely saturation ratio be the ammoniumsulphate soln (4.1mol/L, 25 DEG C) of 100%.

B. precipitate

1. the centrifugal 30min of sample (serum) 20 000 × g, removing cell debris;

2. supernatant liquor is retained and measurement volumes;

3. slowly add isopyknic SAS while stirring in supernatant liquor, final concentration is 1:1;

4. solution is placed on stirring 6 hours or stirring on magnetic stirring apparatus to spend the night (4 DEG C), protein is fully precipitated.

C. dialyse

1. the centrifugal 30min of protein soln 10 000 × g (4 DEG C).Abandon supernatant and retain precipitation;

2. precipitation is dissolved on a small quantity in (10 ~ 20mL) PBS-0.2g/L sodium azide.Put into dialysis tubing after resolution of precipitate to PBS-0.2g/L sodium azide dialysis 24 ~ 48 hours (4 DEG C), change dialysis buffer liquid once every 3 ~ 6 hours, thoroughly to remove sulfate of ammoniac;

3. dialyzate is centrifugal, measures protein content in supernatant liquor.

(2) Protein G affinity purification

Required reagent and specific installation: 1.0mol/L Tris (pH8.0); 100mmol/L Tris (pH8.0); 10mmol/LTris (pH8.0); 50mmol/L glycine (pH3.0); Column chromatography instrument.

Operation steps:

A. the 1.0mol/L Tris (pH8.0) adding 1/10 volume adjusts the pH value to 8.0 containing antibody samples (serum, tissue culture supernatant or ascites).

B. antibody-solutions is passed through albumin A or Protein G microballoon post.In these posts, the wet microballoon of every milliliter can in conjunction with about 10 ~ 20mL antibody (1 albumin A or Protein G microballoon molecule be in conjunction with 2 molecular antibodies).About volume of record dress post microballoon.Because the volume of microballoon post determines the amount using cleaning and elution buffer.

C. the 100mmol/L Tris (pH8.0) of 10 times of column volumes is used to wash microballoon.

D. the 10mmo1/L Tris (pH8.0) of 10 times of column volumes is used to wash microballoon.

E. use 50mmol/L glycine (pH3.0) wash-out microballoon post, add the damping fluid of about 1/2 column volume, gradation adds at every turn.Collect elutriant with the test tube of the 1mol/L Tris (pH8.0) containing 1/10 column volume, each pipe is slowly shaken up, makes its pH value return to neutrality.

F. the collection tube containing antibody is mixed, survey total protein with SDS-PAGE and coomassie brilliant blue staining.

Result: through SDS-PAGE protein electrophoresis qualification (Fig. 1) after antibody purification, it is 0.4mg/mL that ultraviolet spectrophotometer measures antibody concentration.Purified antibodies is stored in-20 DEG C, in 0.01M PBS damping fluid, containing 40% glycerine and 0.5%BSA in solution.

Embodiment 4: the qualification of the polyclonal antibody of anti-TgVP1

1. the ELISA qualification of the polyclonal antibody of anti-TgVP1

With the TgVP1 polypeptide of synthesis for detectable antigens, coated elisa plate, using the not immune rabbit anteserum of 1:2000 dilution as negative control, by the antibody doubling dilution after rabbit anteserum and purifying, application ELISA method detects, and is judged as the positive to be greater than 2.1 with the ratio of negative serum, calculates monoclonal antibody and tires.

As a result, the tiring of anti-TgVP1 antibody of purifying reaches 1:128000 (table 3).

The anti-TgVP1 antibody titer (k=1000) of table 3

Extension rate	1:2k	1:4k	1:8k	1:16k	1:32k	1:64k	1:128k	1:256k	1:512k	Negative control	PBS
												OD value	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 qualification of the polyclonal antibody of anti-TgVP1

Collect toxoplasma tachyzoite and OFTu cell respectively, toxoplasma gondii crack protein and OFTu crack protein will be made after its cracking, by loading after the cracking of 2 × SDS lysis buffer, carry out 10%SDS-PAGE electrophoresis, with reference to the western blotting method described in " molecular cloning ", electrotransfer condition is 100V electrophoresis 1h, confining liquid is the TBST containing 5% skim-milk, primary antibodie is the polyclonal antibody (final concentration 0.1 μ g/mL) of the anti-TgVP1 in the present invention, two resist the sheep anti-mouse igg antibody for horseradish peroxidase-labeled, immunoblotting assay is carried out by ECL Color Appearance System.

Result: the polyclonal antibody of the anti-TgVP1 in the present invention can detect the protein band about 85KD size in toxoplasma gondii lysate, conforms to (Fig. 2) with toxoplasma gondii TgVP1 molecular weight of albumen size.

3. the identified by immunofluorescence of the polyclonal antibody of anti-TgVP1

Prepare toxoplasma gondii smear, 4% paraformaldehyde fixes 15min, drips 0.25%TritonX100, incubated at room 10min, makes cytolemma have perviousness, so that antibody enters.PBS washs three times, and the PBST room temperature containing 1%BSA closes 2h.PBS washs 3 times, then uses polyclonal antibody (PBST 1:500 dilutes) the incubated at room 1.5h of the anti-TgVP1 in the present invention.PBS washs 3 times, goat anti-rabbit igg (PBST 1:1000 dilutes) the incubated at room 1h marked with Alexa Fluor 546 (red fluorescence).DAPI dyeing (blueness) 10min after PBS washing.After PBS develops a film, fluorescence microscopy Microscopic observation.Make negative control with normal rabbit serum simultaneously.

Result: all observe red fluorescence in the toxoplasma gondii tenuigenin of the polyclonal antibody dyeing of the anti-TgVP1 in the present invention, prove the TgVP1 albumen that anti-TgVP1 antibody identifiable design in the present invention is natural.

Claims

1. a toxoplasma gondii TgVP1 born of the same parents Inner district antigenic peptide, is characterized in that its aminoacid sequence is as shown in SEQ ID NO:1.

2. a polyclonal antibody of resisting toxoplasmosis TgVP1, it is characterized in that immune animal is prepared from so that described in claim 1, toxoplasma gondii TgVP1 antigenic peptide is for immunogen.

3. the preparation method of the polyclonal antibody of resisting toxoplasmosis TgVP1 described in claim 2, it is characterized in that step is as follows: the blue carrier protein KLH of keyhole blood toxoplasma gondii TgVP1 born of the same parents Inner district's antigenic peptide and maleinamide described in claim 1 activated, as immunogen and adjuvant mixed immunity new zealand rabbit after desalting and purifying, period carries out more than twice immunity, after ELISA detection serum antibody titer reaches 1:100000, gather rabbit anteserum, after purifying, ELISA and western blot identify, obtain the polyclonal antibody of resisting toxoplasmosis TgVP1.

4. the application of polyclonal antibody in toxoplasma gondii TgVP1 Protein Detection of resisting toxoplasmosis TgVP1 described in claim 2.