CN104745536A - Monoclonal antibody hybridoma 3A6 and monoclonal antibody for toxoplasma protein TgVPI - Google Patents
Monoclonal antibody hybridoma 3A6 and monoclonal antibody for toxoplasma protein TgVPI Download PDFInfo
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- CN104745536A CN104745536A CN201510093458.0A CN201510093458A CN104745536A CN 104745536 A CN104745536 A CN 104745536A CN 201510093458 A CN201510093458 A CN 201510093458A CN 104745536 A CN104745536 A CN 104745536A
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Abstract
The invention discloses a monoclonal antibody hybridoma 3A6 and a monoclonal antibody for toxoplasma protein TgVPI. The monoclonal antibody hybridoma 3A6 for toxoplasma protein TgVPI is collected at China Center For Type Culture Collection on December 16, 2014 with a CCTCC NO:C2014231. According to the invention, a mouse is immunized with toxoplasma protein TgVPI synthetic peptide and a hybridoma cell strain 3A6 with resistance to toxoplasma protein TgVPI is obtained through a hybridoma technology. The monoclonal antibody secreted by the hybridoma cell strain 3A6 is capable of specifically identifying the protein TgVPI and therefore can be applied to identification of the protein TgVPI in a test and has an application value.
Description
Technical field
The present invention relates to cell engineering field, be specifically related to toxoplasma protein TgVP1 monoclonal antibody hybridoma cell strain 3A6 and monoclonal antibody.
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 monoclonal antibody that can be used for the resisting toxoplasmosis albumen TgVP1 of ELISA, western-blot and Immunofluorescence test is provided.
To achieve these goals, the present invention adopts following technical scheme:
The antibody of detection toxoplasma protein TgVP1 of the present invention obtains as antigen immune BALB/c mouse with synthesis TgVP1 antigenic peptide, and the hybridoma cell strain 3A6 producing this antibody is obtained with cell-fusion techniques, the antibody of hybridoma secretion is that IgG1 is positive, and light chain is κ type.Described resisting toxoplasmosis albumen TgVP1 monoclonal antibody hybridoma 3A6, be preserved in China typical culture collection center on December 16th, 2014, preserving number is CCTCC NO:C2014231.
In the present invention, the aminoacid sequence of toxoplasma protein TgVP1 is EPT25031.1, as shown in SEQ ID NO:1.
In the present invention, the antigen polypeptide sequence of toxoplasma protein TgVP1 is as shown in SEQ ID NO:2.
Described preserving number is the preparation method of the hybridoma cell strain 3A6 of CCTCC NO:C2014231: the blue carrier protein KLH of the keyhole blood that TgVP1 antigenic peptide and maleinamide activate, as immunogen and adjuvant mixed immunity BALB/c mouse after desalting and purifying, period carries out more than twice immunity, gets serum titer and is greater than 1:10
5bALB/c mouse splenocyte and SP2/0 myeloma cell merge with 50%PEG-4000 according to a conventional method; With the HAT RPMI-1640 Screening of Media fused cell containing 20% calf serum, with TgVP1 antigenic peptide bag by elisa plate, carry out ELISA screening; Through repeatedly limiting dilution, finally obtain the hybridoma cell strain of the anti-TgVP1 monoclonal antibody of stably excreting, be labeled as 3A6.Apply this strain of hybridoma with 1 × 10
6/ amount only injects the BALB/c female mice abdominal cavity in paraffin pretreated 8-10 age in week, extracts ascites when mouse web portion expands after breeding observing 10-14 days.Adopt affinity chromatography Protein G Sepharose FastFlow monoclonal antibody purification, identify the purity of monoclonal antibody with SDS-PAGE, purity reaches more than 90%.
Compared with prior art, the present invention has following beneficial effect:
The present invention by elisa plate, detects the activity of antibody purification by ELISA method with the TgVP1 antigenic peptide bag of synthesis, and carries out Western-blot with this antibody purification to the toxoplasma protein of purifying and prove that this antibody can identify TgVP1 albumen.
The 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 monoclonal antibody of this strain of hybridoma secretion lays the foundation, simultaneously for its feasibility as disease marker provides experimental data support for studying TgVP1 function further.
The synthesis TgVP1 antigenic peptide of its immunogen of monoclonal antibody of the present invention KLH that has been coupling, total length 29 amino acid.Monoclonal antibody of the present invention can also be applied to cellular immunofluorescence and detect unmodified natural TgVP1 albumen except being applied to ELISA and Western detection sex change TgVP1 albumen.There is no pin toxoplasma gondii TgVP1 specific antibody both at home and abroad at present, and antibody of the present invention can not only be combined with metaprotein, can also be combined with the native protein with higher structure, thus can the experiments such as immunofluorescence be applied to.The present invention is directed to TgVP1 to synthesize the monoclonal antibody prepared of small peptide and have higher specificity and sensitivity to detection TgVP1 albumen, the application of this antibody provides experimental data support using for the functional study of TgVP1 and its feasibility as disease marker.
Accompanying drawing explanation
Fig. 1 be monoclonal antibody 3A6 of the present invention to the result of Toxoplasma immune trace, its molecular weight in conjunction with band is about 85kDa.Swimming lane 1: loading is toxoplasma tachyzoite crack protein; Swimming lane 2: loading is OFTu cytolytic proteins; Swimming lane M: albumen Marker.
Embodiment
The following example is intended to illustrate instead of limit the present invention.
The Preparation and identification of embodiment 1 monoclonal antibody of the present invention
1, the preparation of monoclonal antibody 3A6
1) design of toxoplasma gondii TgVP1 antigenic peptide, preparation and carrier conjugation
Toxoplasma gondii TgVP1 protein sequence is obtained, containing 816 amino acid according to the toxoplasma gondii TgVP1 sequence (accession number: EPT25031.1) on GenBank.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 stronger.Through above-mentioned analysis, final screening peptide sequence is: 292aa-320aa, SEQ ID NO:2.This small peptide (TgVP1-P1) and C hold the small peptide (TgVP1-P1-KLH) of the blue carrier proteins KLH of coupling keyhole blood to be synthesized by gill biochemical corp, Shanghai.
2) immune mouse
With synthetic peptide TgVP1-P1-KLH immunity 6-8 female BAl BIc/c mouse in age in week.
First time immunity: get after synthetic peptide TgVP1-P1-KLH and isopyknic Bentonite adjuvant mix, with the amount intraperitoneal injection BALB/c mouse of every 50 μ g/500 μ L;
Second time immunity: after 2 weeks, gets after synthetic peptide TgVP1-P1-KLH and isopyknic Bentonite adjuvant mix, with the amount intraperitoneal injection BALB/c mouse of every 50 μ g/500 μ L;
Third time immunity: again after 2 weeks, gets after synthetic peptide TgVP1-P1-KLH and isopyknic Bentonite adjuvant mix, with the amount abdominal injection BALB/c mouse of every 50 μ g/500 μ L; After immune 10 days of third time, mouse tail vein gets blood, and with synthetic peptide TgVP1-P1 bag quilt, ELISA detects serum titer, and getting tires is greater than 1:10
5mouse boosting cell and myeloma cell merge;
Bentonite adjuvant commodity in use product.
3) immune serum titration
Adopt indirect elisa method to measure immune serum to tire.Get 50 μ g synthetic peptide TgVP1-P1 and be dissolved in 10ml 0.05M pH9.6 carbonate buffer solution, wrap by micro-96 orifice plates of polystyrene, 100 μ l/ holes, 4 DEG C are spent the night.PBS (containing 0.05% (V/V) Tween-20) is used to wash plate three times, with 10mM PBS containing 1%BSA confining liquid 100 μ l/ hole, 37 DEG C of closed 2h, PBS (containing 0.05% (V/V) Tween-20) is used to wash plate three times, third time immunity mouse tail vein blood sampling in latter 10 days, mouse immune serum carries out 10 with containing 1%BSA 10mM PBS
-2~ 10
-8doubly dilution, add 96 orifice plates, after 100 37 DEG C, μ l/ hole 1h, PBS (containing 0.05% (V/V) Tween-20) wash plate three times, add 1:10000 and doubly dilute horseradish peroxidase-labeled goat anti-mouse igg (Sigma, INC.), 100 37 DEG C, μ l/ hole 30min, the same wash plate after, TMB develops the color, 100 μ l/ holes, room temperature lucifuge 10min, adds 50 μ l/ hole 2M H
2sO
2termination reaction, surveys 450nm absorption value, and before immunity, mice serum is as negative control, must judge tiring of immune serum with measured value and control value than>=2.1 for positive.
4) preparation of hybridoma
Get serum titer and be greater than 1:10
5mouse, merge first 3 days, get after synthetic peptide TgVP1-P1 and isopyknic PBS mixes, carry out booster immunization with the amount abdominal injection BALB/c mouse to be fused of every only 50 μ g/500 μ L.Asepticly get mouse spleen, make splenocyte suspension to mix in the ratio of 1:1 with the murine myeloma cell strain SP2/0 of logarithmic phase, the centrifugal 5min of 1000 × g room temperature, abandon supernatant, flick bottom centrifuge tube with finger, make precipitation loose, centrifuge tube is placed in 37 DEG C of water-baths, by the 50% polyoxyethylene glycol (PEG at 37 DEG C of water bath heat preservations, MW4000, Sigma) add in centrifuge tube with dropper is one after another drop of, drip while shake centrifuge tube, drip off in 1min, drip off rear standing 2min, the serum-free 1640 substratum 1ml of 37 DEG C of preheatings is added every 1 minute, 2ml, 3ml, 4ml, 5ml and 10ml stops the effect of polyoxyethylene glycol, the centrifugal 5min of cell mixture 1000 × g room temperature, abandon supernatant, add HAT nutrient solution (xanthoglobulin (H), aminopterin-induced syndrome (A) and thymidine (T) (HAT, Sigma)) re-suspended cell gently, cell is divided in 96 orifice plates, every hole 200 μ l.Cultivate after three days, observation of cell merges situation, changes half HAT nutrient solution, the continuous a few days, until there is Clone formation, merges and within latter seven days, change HT nutrient solution (xanthoglobulin (H) and thymidine (T) (HT, Sigma)) cultivation.
5) hybridoma of the anti-TgVP1 monoclonal antibody of screening secretion
Indirect elisa method screening cells and supernatant, the positive colony hybridoma of tiring higher is selected to carry out subcloning, and with limiting dilution assay continuous cloning 2-3 time, until to 100% cell positive rate, finally obtain the anti-TgVP1 cell strain of monoclonal antibody of stably excreting, be labeled as 3A6.Cell amplification positive rate after cloning being reached 100% cultivates rear liquid nitrogen cryopreservation.
6) preparation of ascites and purifying
By 3A6 hybridoma cell strain with 1 × 10
6the BALB/c female mice abdominal cavity in/amount injection liquid paraffin body pretreated 8-10 age in week only, extracts ascites when mouse web portion expands after breeding observing 10-14 days.Adopt affinity chromatography Protein G Sepharose Fast Flow monoclonal antibody purification, measure the purity of monoclonal antibody with SDS-PAGE, purity reaches more than 90%.
2, the CHARACTERISTICS IDENTIFICATION of monoclonal antibody of the present invention
1) mensuration of antibody concentration: obtain TgVP1 monoclonal antibody 3A6 after the ascites prepared through hybridoma CCTCC NO:C2014231 is purified, the Smart Spec plus nucleic acid-protein determinator using BIO-RAD company to produce measures, and its concentration is 1.24mg/ml.
2) antibody subtype qualification: the hypotype adopting the mouse monoclonal antibody hypotype identification kit qualification hybridoma cell strain of Roche company, the hypotype of 3A6 secretory antibody is IgG1 type, and light chain is κ chain.
3) qualification of tiring of antibody purification: the 0.05M carbonate bag that 50 μ g synthetic peptide TgVP1-P1 are dissolved in 10ml pH9.6 is buffered in liquid, adds 96 orifice plates, every hole 100 μ L, 4 DEG C are spent the night.PBS (containing 0.05% (V/V) Tween-20) washes plate three times, with 10mM PBS containing 1%BSA confining liquid 150 μ l/ hole, 37 DEG C of closed 2h, PBS (containing 0.05% (V/V) Tween-20) is used to wash plate three times, every hole adds 100 μ l antibody purifications, hatch 1h for 37 DEG C, PBS (containing 0.05% (V/V) Tween-20) washes plate three times, the sheep anti-mouse igg polyclonal antibody adding horseradish peroxidase-labeled is two to resist, hatch 30min for 37 DEG C, PBS (containing 0.05% (V/V) Tween-20) washes plate three times, every hole adds 100 μ l, TMB develops the color, 37 DEG C hatch 15min after, add 2MH
2sO
4solution termination reaction, microplate reader detects at absorbance 450nm place.
4) the Western blot of antibody identifies: 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, Bio-Rad electrotransfer device is used by protein delivery on pvdf membrane after 10%SDS-PAGE, 1h closed by 5% skim-milk, the Tris-HCl damping fluid (containing 0.1% (V/V) Tween-20) of pH7.4 washes film 3 times, each 5min, 1:1000 adds anti-TgVP1 monoclonal antibody 3A6 prepared by purified hybridoma CCTCC NO:C2014231, 4 DEG C of overnight incubation, the Tris-HCl damping fluid (containing 0.1% (V/V) Tween-20) of pH7.4 washes film 3 times, each 5min, add 1:10000 dilution sheep anti-mouse igg polyclonal antibody (Sigma) be two resist, incubated at room 2h, TBST washes film 3 times, unnecessary solution is sucked with filter paper, be laid on clean tin foil, add 1.4ml Pierce-Thermo Scientific ECL series Western chemical luminous substrate reaction solution (A:B=1:1), make film complete wetting in reaction solution, rapid taking-up, surplus liquid is sucked with filter paper, be laid on another tin foil, with tin foil, film is wrapped, put into X-ray magazine, develop in dark place.There is single specific band to toxoplasma gondii crack protein in anti-TgVP1 monoclonal antibody 3A6 prepared by hybridoma CCTCC NO:C2014231, result as shown in Figure 1.
5) identified by immunofluorescence of antibody: 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 monoclonal antibody 3A6 (PBST 1:500 dilutes) the incubated at room 1.5h of the anti-TgVP1 in the present invention.PBS washs 3 times, sheep anti-mouse 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 mouse serum simultaneously.
Result: all observe red fluorescence in the toxoplasma gondii tenuigenin that the anti-TgVP1 monoclonal antibody 3A6 that in the present invention prepared by hybridoma CCTCC NO:C2014231 dyes, proves the TgVP1 albumen that anti-TgVP1 antibody identifiable design in the present invention is natural.
Claims (3)
1. a toxoplasma protein TgVP1 monoclonal antibody hybridoma 3A6, is characterized in that being preserved in China typical culture collection center, and preservation day is on December 16th, 2014, and preserving number is CCTCCNO:C2014231.
2. the preparation method of toxoplasma protein TgVP1 monoclonal antibody hybridoma 3A6 described in claim 1, it is characterized in that the blue carrier protein KLH of keyhole blood comprising the steps: that TgVP1 antigenic peptide and maleinamide activate, as immunogen and adjuvant mixed immunity BALB/c mouse after desalting and purifying, period carries out more than twice immunity, gets serum titer and is greater than 1:10
5bALB/c mouse splenocyte and SP2/0 myeloma cell merge with 50%PEG-4000; With the HAT RPMI-1640 Screening of Media fused cell containing 20% calf serum, with TgVP1 antigenic peptide bag by elisa plate, carry out ELISA screening; Through repeatedly limiting dilution, finally obtain stably excreting anti-TgVP1 monoclonal antibody hybridoma 3A6.
3. the anti-TgVP1 monoclonal antibody that toxoplasma protein TgVP1 monoclonal antibody hybridoma 3A6 described in claim 1 secretes is identifying the application in TgVP1 albumen.
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| CN105738618A (en) * | 2016-02-02 | 2016-07-06 | 浙江大学 | Toxoplasma detection kit |
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