CN106546753B - A kind of application of angiostrongylus cantonensis protein Gal-9 ectin 1 - Google Patents

A kind of application of angiostrongylus cantonensis protein Gal-9 ectin 1 Download PDF

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CN106546753B
CN106546753B CN201611055886.5A CN201611055886A CN106546753B CN 106546753 B CN106546753 B CN 106546753B CN 201611055886 A CN201611055886 A CN 201611055886A CN 106546753 B CN106546753 B CN 106546753B
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黄慧聪
闫宝龙
闫兰竹
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Wenzhou Medical University
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract

The present invention relates to the application of angiostrongylus cantonensis (Angiostrongylus Cantonensis) protein Gal-9 ectin 1 a kind of, the application is the application of the immunosuppressive action of angiostrongylus cantonensis protein Gal-9 ectin 1.The present invention is the application of angiostrongylus cantonensis protein Gal-9 ectin 1, the application for the immunosuppressive action that the application is angiostrongylus cantonensis protein Gal-9 ectin 1, applications of the particularly angiostrongylus cantonensis protein Gal-9 ectin 1 in the medicine with immune suppression function is prepared, theoretical foundation is provided for the treatment of angiostrongyliasis cantonensis, latent effect target spot is provided for the development of medicine；Lay the foundation at the same time for the research and development of angiostrongyliasis cantonensis medicine, provide target for the personalized treatment of angiostrongylus cantonensis, have suitable market prospects and application.

Description

A kind of application of angiostrongylus cantonensis protein Gal-9 ectin-1
Technical field
The invention belongs to technical field of life science, especially a kind of angiostrongylus cantonensis protein Gal-9 ectin-1's should With.
Background technology
Angiostrongylus cantonensis (Angiostrongylus Cantonensis) is the important pathogen for causing eosinophilic meningoencephalitis Body, can cause central lesion, and severe patient causes death.Galectins is a large family, in terms of immunological regulation Play a significant role, but there is no angiostrongylus cantonensis Galectin-1 to the research and application in terms of host immune adjusting.
It is as follows by retrieval, the existing research similar to present patent application：
(1) Galectins can be used as medical diagnosis on disease site.
(2) Galectin-1 is a kind of important immune modulatory molecules, contributes to the immunologic escape of tumour to react.
By contrast, there is the difference of essence with above-mentioned relevant open source literature in present patent application.
The content of the invention
It is an object of the invention in place of overcome the deficiencies in the prior art, supplement and angiostrongylus cantonensis immunological regulation is studied Deficiency, there is provided a kind of application of angiostrongylus cantonensis protein Gal-9 ectin-1, this application be angiostrongylus cantonensis albumen The application of the immunosuppressive action of Galectin-1, particularly angiostrongylus cantonensis protein Gal-9 ectin-1 are being prepared with immune Suppress the application in the medicine of function, theoretical foundation is provided for the treatment of angiostrongyliasis cantonensis, carried for the development of medicine For latent effect target spot.
The technical proposal for solving the technical problem of the invention is：
A kind of application of angiostrongylus cantonensis (Angiostrongylus Cantonensis) protein Gal-9 ectin-1, institute State the application using the immunosuppressive action for angiostrongylus cantonensis protein Gal-9 ectin-1.
Moreover, the application is preparing the medicine with immune suppression function for angiostrongylus cantonensis protein Gal-9 ectin-1 In application.
Moreover, the application is controlled preparing the immune of angiostrongyliasis cantonensis for angiostrongylus cantonensis protein Gal-9 ectin-1 Treat the application in medicine.
Moreover, the angiostrongylus cantonensis protein Gal-9 ectin-1 has immunosuppressive action.
Moreover, the angiostrongylus cantonensis protein Gal-9 ectin-1 has the verification method of immunosuppressive action, step is such as Under：
(1) mouse is immunized
By angiostrongylus cantonensis Galectin-1 albumen respectively with isometric Freund's complete adjuvant, incomplete Freund's adjuvant It is sufficiently mixed, blows and beats emulsification repeatedly with syringe until there is Water-In-Oil state, immune C57BL/6 mouse is subcutaneously injected；Specifically Immunization method is：
Initial immunity：Every 100 μ g albumen of mouse, mixes with Freund's complete adjuvant；
Booster immunization：Every 50 μ g albumen of mouse, mixes with incomplete Freund's adjuvant；
Each immunization interval one week, is immunized three times altogether；
(2) III phase of angiostrongylus cantonensis larva infecting mouse
1. the acquisition of III phase larva
Positive SD rats fresh excreta is added in the Pomacea canaliculata living environment manually established, consecutive infection 3-4 times；2 weeks Pomacea canaliculata is shelled afterwards, takes out internal organ and musculature, 10mL artificial digestion liquid is added according to 1g spiral shells meat after mincing, in 37 DEG C of guarantors 2-3h is digested in incubator；Then through strainer filtering, staticly settle, washed 2-3 times with physiology salt, be deposited in microscopy under anatomical lens；
2. III phase larva infects
Choose III phase larva under microscope, and according to every mouse with 30 ± 2 larva infecting mouses；
(3) humoral immunity is adjusted
Before 0 week i.e. infection after III phase larva infects, 1 week, 2 weeks, 3 weeks eyeball blood sampling collection mice serums, mouse blood is obtained Final proof product, Total IgG in different times mice serum, IgG1, IgG2a and IgE antibody level are detected using ELISA method, Assess Humoral Immunological Regulation Roles；
Using Total IgG and IgE as index, compared with control group, reduced if experimental group is horizontal, illustrate Galectin-1 Albumen has immunosuppressive action；
Antibody level of serum is detected using ELISA detection kit；
Concretely comprise the following steps：
1. it is coated with：Antibody will be captured respectively with coating buffer according to capture antibody：The volume ratio of coating buffer is 1:250 ratio After dilution, 100 μ L/HoleAdd in elisa plate, 4 DEG C of coatings are overnight；
Wherein, the coating buffer is PBS, captures antibody as corresponding anti-mouse monoclonal antibody, i.e., anti-mouse IgG, anti-mouse IgG1, anti-mouse IgG2a, anti-mouse IgE monoclonal antibodies；
2. board-washing：Next day discards liquid in hole, 300 μ L/HoleLavation buffer solution board-washing 2 times, by orifice plate after last time board-washing Tip upside down on blotting paper and all discard liquid in hole；
3. close：250μL/HoleConfining liquid, room temperature closing 2h；
4. board-washing：Discard liquid in hole, 300 μ L/HoleOrifice plate, is buckled to after last time board-washing by lavation buffer solution board-washing 2 times Liquid in hole is all discarded on blotting paper；
5. prepare standard items and sample：By standard items mouse Total IgG antibodies, mouse IgG1 antibody, mouse IgG2a antibody, mouse The concentration that IgE antibody is diluted to mouse Total IgG antibodies with deionized water respectively is 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL、6.25ng/mL、3.13ng/mL、1.56ng/mL；The concentration of mouse IgG1 antibody for 200ng/mL, 100ng/mL, 50ng/mL、25ng/mL、12.5ng/mL、6.25ng/mL、3.13ng/mL；The concentration of mouse IgG2a antibody for 250ng/mL, 125ng/mL、62.5ng/mL、31.25ng/mL、15.63ng/mL、7.81ng/mL、3.90ng/mL；The concentration of mouse IgE antibody For 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.63ng/mL, 7.81ng/mL, 3.90ng/mL；100μ L/HoleAdd in standard sample wells, if 3 multiple holes；
Meanwhile dilute mice serum sample with confining liquid, diluted volume ratio is mouse IgG- serum：Confining liquid=1: 100000；Mouse IgG1- serum：Confining liquid=1:10；Mouse IgG2a- serum：Confining liquid=1:10000；Mouse IgE- serum：Closing Liquid=1:50；
6. incubate primary antibody：100μL/HoleDiluted blood serum sample is added in sample well, and every group sets 3 multiple holes, while set blank pair According to hole；
7. incubate secondary antibody：50μL/HoleDiluted detection antibody is added in elisa plate, is incubated at room temperature 3h；
Wherein, the diluted detection antibody is the HRP anti-mouse IgG monoclonal antibodies marked or biotin-avidin mark Remember anti-mouse IgE monoclonal antibodies, diluted using confining liquid, dilution volume ratio is confining liquid：Antibody=250:1；
8. board-washing：Discard liquid in hole, 300 μ L/HoleOrifice plate, is buckled to after last time board-washing by lavation buffer solution board-washing 4 times Liquid in hole is all discarded on blotting paper；
9. develop the color：100μL/HoleTMB nitrite ions, room temperature lucifuge are incubated 15min；
10. terminate：100μL/Hole2M H2SO4Terminate liquid terminates reaction；
Finally elisa plate is placed in microplate reader, reads the absorbance value at 450nm wavelength, draws standard curve, according to Standard curve calculating antibody concentration；
(4) Culture in vitro
In order to observe adjustment effect of the destination protein to cellular immunity, mouse spleen lymphocyte cell factor IFN- is carried out γ, IL-4, IL-5 and IL-10 are detected；
Angiostrongylus cantonensis infection is acute infection, and acute stage based on Th1 immune responses, refers to IFN-γ as confirmation Mark, compared with control group, if experimental group IFN-γ reduces, then it is assumed that Galectin-1 albumen has immunosuppressive action；
Concretely comprise the following steps：
1. mouse spleen lymphocyte separates
Take spleen：Cervical dislocation is put to death after eyeball of mouse is taken blood, is put into equipped with the beaker that percentage by volume is 75% ethanol In, solution takes mouse spleen under aseptic condition；
Grinding：Mouse spleen is put into 1mL DMEM cell culture fluids and is cleaned, is fully ground, and constantly dripped to tissue Add DMEM nutrient solutions, until more than 95% cell is separated in tissue, cell suspension is carefully moved in sterile centrifuge tube；
Centrifugation：1400rpm, room temperature centrifugation 5min；
Separation of lymphocytes：Supernatant is abandoned, adds 500 μ L Ack erythrocyte cracked liquids, 1min is stood, adds DMEM nutrient solutions to determine Hold to 2mL；
Centrifugation：1400rpm, room temperature centrifugation 5min；
Filtering：Abandon supernatant, add 500 μ L DMEM that cell is resuspended, through 200 mesh filter screens filter to another it is clean it is sterile from In heart pipe, cleaning solution is moved in new centrifuge tube together after being cleaned with DMEM；
Dyeing：98 μ L trypan blues and 2 μ L cell suspensions fill pond after fully mixing；
Cell count：The cell quantity in 4 block plaids is counted under microscope, according to C=N/4 × 10 × 103× 50 meters Calculate cell concentration；
2. Secreted by Mouse Splenic cytokines measurement
Splenic lymphocytes suspension is diluted to 1 × 10 with containing the DMEM that percentage by volume is 10%FBS6A cell/mL, adds Into 96 orifice plates, 100 μ L/Hole, then add angiostrongylus cantonensis Galectin-1 protein 25s ng/mL, 100 μ L/HoleStimulate spleen thin Born of the same parents；Set canavalin ConA25ng/mL, 100 μ L/ at the same timeHolePositive controls, 100 μ L/ of sterilizing PBSHoleNegative control group；5% CO2Cell conditioned medium is collected after 37 DEG C of culture 72h of incubator, cell conditioned medium sample is obtained, is preserved for cytokines measurement or -80 DEG C It is spare；
Horizontal, the concrete operations step using ELISA detection kit detection cell factor IFN-γ, IL-4, IL-5 and IL-10 Suddenly it is：
Coating：The anti-mouse IFN-γ of antibody, anti-mouse IL-4, anti-mIL5, anti-mouse IL-10 will be captured respectively with coating buffer Monoclonal antibody is according to capture antibody：The volume ratio of coating buffer is 1:After 250 dilution proportion, it is added to according to every 100 μ L of hole In elisa plate, 4 DEG C of coatings are overnight；Wherein, the anti-mouse IFN-γ, anti-mouse IL-4, the bag of anti-mIL5 monoclonal antibody It is 7.13g NaHCO by liquid3+1.59gNa2CO3Add the mixed liquor after 1L distilled water；The bag of anti-mouse IL-10 monoclonal antibodies It is 12.49g Na by liquid2HPO4+15.47g NaH2PO4Add the mixed liquor after 1L distilled water；
Board-washing：Next day, liquid in hole is discarded, with 300 μ L/HoleLavation buffer solution board-washing 3 times, for the last time falls orifice plate It is buckled on blotting paper and pats dry liquid in hole；
Closing：Elisa plate is closed containing the PBS that percentage by volume is 10%FBS per 200 μ L of hole, is incubated at room temperature 2h；
Board-washing：Liquid in hole is discarded, with 300 μ L/HoleLavation buffer solution board-washing 3 times, tips upside down on suction by orifice plate for the last time Liquid in hole is patted dry on water paper；
Prepare standard items and sample：By standard items recombinant murine IFN-γ, recombinant murine IL-4, recombinant murine IL-5, recombinant murine IL- 10 are diluted to 7 concentration gradients, i.e. IFN-γ by multiple proportions with the PBS for being 10%FBS containing percentage by volume respectively:2000pg/mL、 1000pg/mL、500pg/mL、250pg/mL、125pg/mL、62.5pg/mL、31.3pg/mL；IL-4:500pg/mL、250pg/ mL、125pg/mL、62.5pg/mL、31.3pg/mL、15.6pg/mL、7.8pg/mL；IL-5:1000pg/mL、500pg/mL、 250pg/mL、125pg/mL、62.5pg/mL、31.3pg/mL、15.6pg/mL；IL-10:2000pg/mL、1000pg/mL、 500pg/mL、250pg/mL、125pg/mL、62.5pg/mL、31.3pg/mL；The cells and supernatant sample of collection is used and contains body The PBS that product percentage is 10%FBS carries out 1:20 dilutions, 3 multiple holes of every group of setting, while set blank control wells；
Incubate primary antibody：Standard item group, sample sets and blank control group are added in elisa plate per 100 μ L of hole, are incubated at room temperature 1h；
Board-washing：Liquid in hole is discarded, with 300 μ L/HoleLavation buffer solution board-washing 5 times, tips upside down on suction by orifice plate for the last time Liquid in hole is patted dry on water paper；
Incubate secondary antibody：Add the enzyme marking reagent of the diluted detection antibody of 100uL and SAv-HRP marks per hole, be incubated at room temperature 1h；
Wherein, it is described detection antibody be respectively Biotinylate mark anti-mouse IFN-γ monoclonal antibody, Biotinylate mark anti-mouse IL-4 monoclonal antibodies, Biotinylate mark anti-mIL5 monoclonal antibody, The anti-mouse IL-10 monoclonal antibodies of Biotinylate marks；Using dilute containing the PBS that percentage by volume is 10%FBS during dilution Release, PBS：Detect antibody：The volume ratio of SAv-HRP enzyme marking reagents is 500:1:2；
Board-washing：Liquid in hole is discarded, with 300 μ L/HoleLavation buffer solution board-washing 7 times, each board-washing is by lavation buffer solution It is placed in elisa plate and stops 1min；
Colour developing：Add 100 μ L TMB nitrite ions per hole, lucifuge is incubated 15min at room temperature；
Terminate reaction：Add 50 μ L2M H per hole2SO4Terminate liquid；
Read plate：Elisa plate is placed in microplate reader, reads the absorbance value at 450nm wavelength；
Calculate concentration：Standard curve is drawn, cytokine concentrations are calculated according to standard curve；
(5) mouse spleen lymphocyte differentiation group detection
Using flow cytomery CD4+T cell and CD4+CD25+PoxP3+Treg cellular changes；Compared with control group, Experimental group CD4+T cell reduces and/or CD4+CD25+PoxP3+Treg cells raise, then illustrate that Galectin-1 albumen tool is immune Inhibitory action；
Isolated splenic lymphocytes suspension is adjusted to 1.5 × 10 with DMEM7A cell/mL, is added to 96 orifice plates 100 μL/HoleIn, operated in accordance with the following steps using detection kit：
1. adding 200 μ L Facs buffer solutions per hole, 1400rpm, room temperature centrifugation 5min, abandons supernatant；
2. 25 μ L Facs buffer solutions are added per hole closes 1min on ice；
3. the 25 diluted mixtures of antibodies of μ L are added per hole is incubated 30min on ice, wherein, dilution volume ratio delays for Facs Fliud flushing：CD4-FITC：CD8-PerCP-Cy5.5：CD25-APC=200：1：1：1；
4. adding 200 μ L Facs buffer solutions per hole, 1400rpm, room temperature centrifugation 5min, abandons supernatant；
5. adding the rupture of membranes of 100 μ L Fresh to fix buffer solution per hole, 30min is incubated on ice；
6. directly into hole plus 200 μ L cleaning solutions, 1400rpm, room temperature centrifuge 5min, abandon supernatant；
7. repeat step is 6.；
8. adding 50 μ L cleaning solutions per hole, FoxP3-PE antibody presses cleaning solution：The volume ratio of FoxP3-PE is 100:1 directly adds Enter in hole, 4 DEG C of lucifuges are incubated 30min；
9. repeat step is 6., clean 2 times；
10. 100 μ L cleaning solutions are added to add upper machine testing in streaming pipe after cell is resuspended per hole；
(6) brain tissue HE is dyed
Mice brain tissues are taken to carry out HE dyeing, the micro- sem observation cerebral tissue pathology injury order of severity and inflammatory cell become Change, further verify the immunoregulation effect of Galectin-1 albumen；If in the meningitis predilection site inflammation of experimental mice Cellular infiltration significantly reduces, and illustrates that Galectin-1 albumen has immunosuppressive action；
Step is as follows：
1. materials are with fixing
It is in 4% paraformaldehyde fixer to take the mass percent that fresh Mice brain tissues input prepares in advance, makes group Knit, the solidification of the protein denaturation of cell, to prevent cell postmortem autolysis or bacterial decomposition, so as to keep the original form knot of cell Structure；
2. it is dehydrated transparent
Make dehydrating agent with by low concentration to alcohol in high concentration, gradually slough tissue moisture in the block, then tissue block is placed in Not only alcohol is dissolved in, but also is dissolved in transparent in the clarifier dimethylbenzene of paraffin, tissue alcohol in the block is replaced out with dimethylbenzene；
3. waxdip embeds
Transparent tissue block is placed in the paraffin dissolved, is put into the insulation of dewaxing case；
Embedded after paraffin is completely immersed in tissue block：Container is prepared, pours into the paraffin dissolved, rapid gripping is The tissue block for being impregnated with paraffin is put into wherein, and cooled and solidified is blocking；
Treat that embedded tissue block is hardened, very thin section could be cut on slicer；
4. section and patch
Embedded wax stone is fixed on slicer, is thinly sliced, generally 5-8 μ m-thicks；The thin slice cut often wrinkles Pleat, will be put into the water of heating and plate, then be attached on glass slide, put in 45 DEG C of insulating boxs and dry；
5. dewaxing dyeing
The sample of drying is subjected to HE dyeing, to increase the heterochromia of tissue cellularity each several part, beneficial to observation；Soviet Union Another name for coloring agent is a kind of basic-dyeable fibre, nucleus and intracellular ribosomes can be dyed bluish violet；Yihong is a kind of acid dye Cytoplasm, can be dyed red or pale red by material.Before dyeing, the paraffin in section is sloughed with dimethylbenzene, then arrive via high concentration Low-concentration ethanol, finally enters distilled water, you can dyeing；
HE dyeing courses are：
The section after distilled water 1-2s will have been entered it has been put into hematoxylin coloring agent to dye 3min, tap water rinses 5-10s；
It is 70% ethanol and concentrated hydrochloric acid 100-200 by volume to be put into percentage by volume:Break up 5- in the mixed liquor of 1 composition 10s；
Flowing water rinses 1-2s；
It is put into concentrated ammonia liquor and distilled water by volume 1:5-10s in 99 mixed liquors prepared；
Flowing water rinses 15-30s；
Enter eosin stains liquid dyeing 8-10s；
6. it is dehydrated transparent
Section after dyeing is dehydrated through absolute alcohol, then makes section transparent through dimethylbenzene；
7. sealing
By the transparent upper canada balsam of section drop, covered sealing, after natural gum is slightly dry, sticks mark writing paper, cuts Piece sample can be used, and carry out microscope observation.
Moreover, (2) middle mode of infection infects the step for gavage.
Moreover, the step is (3) middle using eBioscience ELISA detection kits detection antibody level of serum；Or Person, the ox blood that the step Tween-20 that (3) it is 1% containing percentage by volume that middle confining liquid, which is, percentage by volume are 10% are pure The PBS of albumen.
Moreover, the step is (4) 2. middle using BD Biosciences ELISA detection kits detection cell factor IFN- γ, IL-4, IL-5 and IL-10 are horizontal.
Moreover, the step PBS that (5) it is 2%FBS containing percentage by volume that middle Facs buffer solutions, which are,；The step (5) middle inspection Test agent box is BD Biosciences；(5) middle rupture of membranes fixes the article No. of buffer solution to the step：562574, batch 51- 9008100 and 51-9008101；The article No. of the step (5) middle cleaning solution：562574, batch 51-9008102.
Moreover, (6) middle inflammatory cell is neutrophil leucocyte, eosinophil to the step；Alternatively, the step (6) in Meningitis predilection site is the ventricles of the brain, cavum subarachnoidale；Alternatively, (6) 2. the volume of middle low concentration to alcohol in high concentration is dense for the step Degree is respectively：70% alcohol 30min；90% alcohol 30min；95% alcohol 30min；100% alcohol 1h；100% alcohol 1h；Or Person, (6) 5. the volumetric concentration of middle and high concentration to low-concentration ethanol is respectively the step：100% alcohol 2min；95% alcohol 1min；80% alcohol 1min；75% alcohol 1min.
The advantages of present invention obtains and good effect are：
1st, the present invention is the application of angiostrongylus cantonensis protein Gal-9 ectin-1, which is angiostrongylus cantonensis albumen The application of the immunosuppressive action of Galectin-1, particularly angiostrongylus cantonensis protein Gal-9 ectin-1 are being prepared with immune Suppress the application in the medicine of function, theoretical foundation is provided for the treatment of angiostrongyliasis cantonensis, carried for the development of medicine For latent effect target spot；Lay the foundation at the same time for the research and development of angiostrongyliasis cantonensis medicine, be of angiostrongylus cantonensis Propertyization treatment provides target, has suitable market prospects and application.
2nd, verification methods of the angiostrongylus cantonensis protein Gal-9 ectin-1 with immunosuppressive action is simple in the present invention, grasps Facilitate, analyze that host body fluids are immunized in angiostrongylus cantonensis Galectin-1 albumen, cellular immunity and modulability are immunized Effect, and is further verified by histopathology, and the never immunological regulation of Tongfang surface analysis angiostrongylus cantonensis albumen is made With to more fully understand that the mechanism of causing a disease of angiostrongylus cantonensis provides foundation, being established for the immunization therapy of angiostrongyliasis cantonensis Theoretical foundation.
Brief description of the drawings
Fig. 1 is mouse different times Serum Antibody Detection level view after III phase larva infection in the present invention；Wherein, Fig. 1-A For Total IgG level views；Fig. 1-B are IgG1 level views；Fig. 1-C are IgG2a level views；Fig. 1-D are IgE level views；
Fig. 2 is mouse different times cytokines measurement level view after III phase larva infection in the present invention；Wherein, Fig. 2-A For IFN-γ level view；Fig. 2-B are IL-4 level views；Fig. 2-C are IL-5 level views；Fig. 2-D are IL-10 level views；
Fig. 3 is mouse different times flow cytometer detection result figure after III phase larva infection in the present invention；Wherein, Fig. 3-A are III 2 weeks CD4 after the infection of phase larva+T cell streaming figure；Fig. 3-B are 2 weeks CD4 after the infection of III phase larva+CD25+FoxP3+T cell stream Formula figure；Fig. 3-C are CD4+T cell change level figure；Fig. 3-D are CD4+CD25+FoxP3+Treg cellular change level views；
Fig. 4 is different times Mice brain tissues pathological change figure after III phase larva infection in the present invention；Wherein, Fig. 4-A are 1 All control group histopathology figures；Fig. 4-B are 2 weeks control group histopathology figures；Fig. 4-C are 3 weeks control group histopathologies Figure；Fig. 4-D are 1 week experimental group histopathology figure；Fig. 4-E are 2 weeks experimental group histopathology figures；Fig. 4-F are 3 weeks experimental groups Histopathology figure.
Embodiment
For that can further appreciate that present disclosure, feature and effect, the following examples are hereby given, and coordinates attached drawing detailed It is described as follows.It should be noted that the present embodiment is descriptive, it is not limited, it is impossible to thus limit the guarantor of the present invention Protect scope.
Reagent used in the present invention, is the conventional reagent of this area unless otherwise required；Used in the present invention Method, be the conventional method of this area unless otherwise required.
A kind of application of angiostrongylus cantonensis (Angiostrongylus Cantonensis) protein Gal-9 ectin-1, institute State the application using the immunosuppressive action for angiostrongylus cantonensis protein Gal-9 ectin-1.
Further, the application is being prepared with immune suppression function for angiostrongylus cantonensis protein Gal-9 ectin-1 Application in medicine.
Further, the application is preparing exempting from for angiostrongyliasis cantonensis for angiostrongylus cantonensis protein Gal-9 ectin-1 Application in epidemic disease medicine.
Further, the angiostrongylus cantonensis protein Gal-9 ectin-1 has immunosuppressive action.
Above-mentioned angiostrongylus cantonensis protein Gal-9 ectin-1 has the verification method of immunosuppressive action, and step is as follows：
(1) mouse is immunized
By angiostrongylus cantonensis Galectin-1 albumen respectively with isometric Freund's complete adjuvant, incomplete Freund's adjuvant It is sufficiently mixed, blows and beats emulsification repeatedly with syringe until there is Water-In-Oil state, immune C57BL/6 mouse is subcutaneously injected；Specifically Immunization method is：
Initial immunity：Every 100 μ g albumen of mouse, mixes with Freund's complete adjuvant；
Booster immunization：Every 50 μ g albumen of mouse, mixes with incomplete Freund's adjuvant；
Each immunization interval one week, is immunized three times altogether；
(2) III phase of angiostrongylus cantonensis larva infecting mouse
1. the acquisition of III phase larva
Positive SD rats fresh excreta is added in the Pomacea canaliculata living environment manually established, consecutive infection 3-4 times；2 weeks Pomacea canaliculata is shelled afterwards, takes out internal organ and musculature, 10mL artificial digestion liquid is added according to 1g spiral shells meat after mincing, in 37 DEG C of guarantors 2-3h is digested in incubator；Then through strainer filtering, staticly settle, washed 2-3 times with physiology salt, be deposited in microscopy under anatomical lens；
2. III phase larva infects
Choose III phase larva under microscope, and according to every mouse with 30 ± 2 larva infecting mouses；
(3) humoral immunity is adjusted
Before 0 week i.e. infection after III phase larva infects, 1 week, 2 weeks, 3 weeks eyeball blood sampling collection mice serums, mouse blood is obtained Final proof product, Total IgG in different times mice serum, IgG1, IgG2a and IgE antibody level are detected using ELISA method, Assess Humoral Immunological Regulation Roles；
Using Total IgG and IgE as index, compared with control group, reduced if experimental group is horizontal, illustrate Galectin-1 Albumen has immunosuppressive action；
Antibody level of serum is detected using ELISA detection kit；
Concretely comprise the following steps：
1. it is coated with：Antibody will be captured respectively with coating buffer according to capture antibody：The volume ratio of coating buffer is 1:250 ratio After dilution, 100 μ L/HoleAdd in elisa plate, 4 DEG C of coatings are overnight；
Wherein, the coating buffer is PBS, captures antibody as corresponding anti-mouse monoclonal antibody, i.e., anti-mouse IgG, anti-mouse IgG1, anti-mouse IgG2a, anti-mouse IgE monoclonal antibodies；
2. board-washing：Next day discards liquid in hole, 300 μ L/HoleLavation buffer solution board-washing 2 times, by orifice plate after last time board-washing Tip upside down on blotting paper and all discard liquid in hole；
3. close：250μL/HoleConfining liquid, room temperature closing 2h；
4. board-washing：Discard liquid in hole, 300 μ L/HoleOrifice plate, is buckled to after last time board-washing by lavation buffer solution board-washing 2 times Liquid in hole is all discarded on blotting paper；
5. prepare standard items and sample：By standard items mouse Total IgG antibodies, mouse IgG1 antibody, mouse IgG2a antibody, mouse The concentration that IgE antibody is diluted to mouse Total IgG antibodies with deionized water respectively is 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL、6.25ng/mL、3.13ng/mL、1.56ng/mL；The concentration of mouse IgG1 antibody for 200ng/mL, 100ng/mL, 50ng/mL、25ng/mL、12.5ng/mL、6.25ng/mL、3.13ng/mL；The concentration of mouse IgG2a antibody for 250ng/mL, 125ng/mL、62.5ng/mL、31.25ng/mL、15.63ng/mL、7.81ng/mL、3.90ng/mL；The concentration of mouse IgE antibody For 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.63ng/mL, 7.81ng/mL, 3.90ng/mL；100μ L/HoleAdd in standard sample wells, if 3 multiple holes；
Meanwhile dilute mice serum sample with confining liquid, diluted volume ratio is mouse IgG- serum：Confining liquid=1: 100000；Mouse IgG1- serum：Confining liquid=1：10；Mouse IgG2a- serum：Confining liquid=1:10000；Mouse IgE- serum：Closing Liquid=1:50；
6. incubate primary antibody：100μL/HoleDiluted blood serum sample is added in sample well, and every group sets 3 multiple holes, while set blank pair According to hole；
7. incubate secondary antibody：50μL/HoleDiluted detection antibody is added in elisa plate, is incubated at room temperature 3h；
Wherein, the diluted detection antibody is the HRP anti-mouse IgG monoclonal antibodies marked or biotin-avidin mark Remember anti-mouse IgE monoclonal antibodies, diluted using confining liquid, dilution volume ratio is confining liquid：Antibody=250:1；
8. board-washing：Discard liquid in hole, 300 μ L/HoleOrifice plate, is buckled to after last time board-washing by lavation buffer solution board-washing 4 times Liquid in hole is all discarded on blotting paper；
9. develop the color：100μL/HoleTMB nitrite ions, room temperature lucifuge are incubated 15min；
10. terminate：100μL/Hole2M H2SO4Terminate liquid terminates reaction；
Finally elisa plate is placed in microplate reader, reads the absorbance value at 450nm wavelength, draws standard curve, according to Standard curve calculating antibody concentration；
(4) Culture in vitro
In order to observe adjustment effect of the destination protein to cellular immunity, mouse spleen lymphocyte cell factor IFN- is carried out γ, IL-4, IL-5 and IL-10 are detected；
Angiostrongylus cantonensis infection is acute infection, and acute stage based on Th1 immune responses, refers to IFN-γ as confirmation Mark, compared with control group, if experimental group IFN-γ reduces, then it is assumed that Galectin-1 albumen has immunosuppressive action；
Concretely comprise the following steps：
1. mouse spleen lymphocyte separates
Take spleen：Cervical dislocation is put to death after eyeball of mouse is taken blood, is put into equipped with the beaker that percentage by volume is 75% ethanol In, solution takes mouse spleen under aseptic condition；
Grinding：Mouse spleen is put into 1mL DMEM cell culture fluids and is cleaned, is fully ground, and constantly dripped to tissue Add DMEM nutrient solutions, until more than 95% cell is separated in tissue, cell suspension is carefully moved in sterile centrifuge tube；
Centrifugation：1400rpm, room temperature centrifugation 5min；
Separation of lymphocytes：Supernatant is abandoned, adds 500 μ L Ack erythrocyte cracked liquids, 1min is stood, adds DMEM nutrient solutions to determine Hold to 2mL；
Centrifugation：1400rpm, room temperature centrifugation 5min；
Filtering：Abandon supernatant, add 500 μ L DMEM that cell is resuspended, through 200 mesh filter screens filter to another it is clean it is sterile from In heart pipe, cleaning solution is moved in new centrifuge tube together after being cleaned with DMEM；
Dyeing：98 μ L trypan blues and 2 μ L cell suspensions fill pond after fully mixing；
Cell count：The cell quantity in 4 block plaids is counted under microscope, according to C=N/4 × 10 × 103× 50 meters Calculate cell concentration；
2. Secreted by Mouse Splenic cytokines measurement
Splenic lymphocytes suspension is diluted to 1 × 10 with containing the DMEM that percentage by volume is 10%FBS6A cell/mL, adds Into 96 orifice plates, 100 μ L/Hole, then add angiostrongylus cantonensis Galectin-1 protein 25s ng/mL, 100 μ L/HoleStimulate spleen thin Born of the same parents；Set canavalin ConA25ng/mL, 100 μ L/ at the same timeHolePositive controls, 100 μ L/ of sterilizing PBSHoleNegative control group；5% CO2Cell conditioned medium is collected after 37 DEG C of culture 72h of incubator, cell conditioned medium sample is obtained, is preserved for cytokines measurement or -80 DEG C It is spare；
Horizontal, the concrete operations step using ELISA detection kit detection cell factor IFN-γ, IL-4, IL-5 and IL-10 Suddenly it is：
Coating：The anti-mouse IFN-γ of antibody, anti-mouse IL-4, anti-mIL5, anti-mouse IL-10 will be captured respectively with coating buffer Monoclonal antibody is according to capture antibody：The volume ratio of coating buffer is 1:After 250 dilution proportion, it is added to according to every 100 μ L of hole In elisa plate, 4 DEG C of coatings are overnight；Wherein, the anti-mouse IFN-γ, anti-mouse IL-4, the bag of anti-mIL5 monoclonal antibody It is 7.13g NaHCO by liquid3+1.59gNa2CO3Add the mixed liquor after 1L distilled water；The bag of anti-mouse IL-10 monoclonal antibodies It is 12.49gNa by liquid2HPO4+15.47g NaH2PO4Add the mixed liquor after 1L distilled water；
Board-washing：Next day, liquid in hole is discarded, with 300 μ L/HoleLavation buffer solution board-washing 3 times, for the last time falls orifice plate It is buckled on blotting paper and pats dry liquid in hole；
Closing：Elisa plate is closed containing the PBS that percentage by volume is 10%FBS per 200 μ L of hole, is incubated at room temperature 2h；
Board-washing：Liquid in hole is discarded, with 300 μ L/HoleLavation buffer solution board-washing 3 times, tips upside down on suction by orifice plate for the last time Liquid in hole is patted dry on water paper；
Prepare standard items and sample：By standard items recombinant murine IFN-γ, recombinant murine IL-4, recombinant murine IL-5, recombinant murine IL- 10 are diluted to 7 concentration gradients, i.e. IFN-γ by multiple proportions with the PBS for being 10%FBS containing percentage by volume respectively:2000pg/mL、 1000pg/mL、500pg/mL、250pg/mL、125pg/mL、62.5pg/mL、31.3pg/mL；IL-4:500pg/mL、250pg/ mL、125pg/mL、62.5pg/mL、31.3pg/mL、15.6pg/mL、7.8pg/mL；IL-5:1000pg/mL、500pg/mL、 250pg/mL、125pg/mL、62.5pg/mL、31.3pg/mL、15.6pg/mL；IL-10:2000pg/mL、1000pg/mL、 500pg/mL、250pg/mL、125pg/mL、62.5pg/mL、31.3pg/mL；The cells and supernatant sample of collection is used and contains body The PBS that product percentage is 10%FBS carries out 1:20 dilutions, 3 multiple holes of every group of setting, while set blank control wells；
Incubate primary antibody：Standard item group, sample sets and blank control group are added in elisa plate per 100 μ L of hole, are incubated at room temperature 1h；
Board-washing：Liquid in hole is discarded, with 300 μ L/HoleLavation buffer solution board-washing 5 times, tips upside down on suction by orifice plate for the last time Liquid in hole is patted dry on water paper；
Incubate secondary antibody：Add the enzyme marking reagent of the diluted detection antibody of 100uL and SAv-HRP marks per hole, be incubated at room temperature 1h；
Wherein, it is described detection antibody be respectively Biotinylate mark anti-mouse IFN-γ monoclonal antibody, Biotinylate mark anti-mouse IL-4 monoclonal antibodies, Biotinylate mark anti-mIL5 monoclonal antibody, The anti-mouse IL-10 monoclonal antibodies of Biotinylate marks；Using dilute containing the PBS that percentage by volume is 10%FBS during dilution Release, PBS：Detect antibody：The volume ratio of SAv-HRP enzyme marking reagents is 500:1:2；
Board-washing：Liquid in hole is discarded, with 300 μ L/HoleLavation buffer solution board-washing 7 times, each board-washing is by lavation buffer solution It is placed in elisa plate and stops 1min；
Colour developing：Add 100 μ L TMB nitrite ions per hole, lucifuge is incubated 15min at room temperature；
Terminate reaction：Add 50 μ L2M H per hole2SO4Terminate liquid；
Read plate：Elisa plate is placed in microplate reader, reads the absorbance value at 450nm wavelength；
Calculate concentration：Standard curve is drawn, cytokine concentrations are calculated according to standard curve；
(5) mouse spleen lymphocyte differentiation group detection
Using flow cytomery CD4+T cell and CD4+CD25+PoxP3+Treg cellular changes；Compared with control group, Experimental group CD4+T cell reduces and/or CD4+CD25+PoxP3+Treg cells raise, then illustrate that Galectin-1 albumen tool is immune Inhibitory action；
Isolated splenic lymphocytes suspension is adjusted to 1.5 × 10 with DMEM7A cell/mL, is added to 96 orifice plates 100 μL/HoleIn, operated in accordance with the following steps using detection kit：
1. adding 200 μ L Facs buffer solutions per hole, 1400rpm, room temperature centrifugation 5min, abandons supernatant；
2. 25 μ L Facs buffer solutions are added per hole closes 1min on ice；
3. the 25 diluted mixtures of antibodies of μ L are added per hole is incubated 30min on ice, wherein, dilution volume ratio delays for Facs Fliud flushing：CD4-FITC：CD8-PerCP-Cy5.5：CD25-APC=200：1：1：1；
4. adding 200 μ L Facs buffer solutions per hole, 1400rpm, room temperature centrifugation 5min, abandons supernatant；
5. adding the rupture of membranes of 100 μ L Fresh to fix buffer solution per hole, 30min is incubated on ice；
6. directly into hole plus 200 μ L cleaning solutions, 1400rpm, room temperature centrifuge 5min, abandon supernatant；
7. repeat step is 6.；
8. adding 50 μ L cleaning solutions per hole, FoxP3-PE antibody presses cleaning solution：The volume ratio of FoxP3-PE is 100:1 directly adds Enter in hole, 4 DEG C of lucifuges are incubated 30min；
9. repeat step is 6., clean 2 times；
10. 100 μ L cleaning solutions are added to add upper machine testing in streaming pipe after cell is resuspended per hole；
(6) brain tissue HE is dyed
Mice brain tissues are taken to carry out HE dyeing, the micro- sem observation cerebral tissue pathology injury order of severity and inflammatory cell become Change, further verify the immunoregulation effect of Galectin-1 albumen；If in the meningitis predilection site inflammation of experimental mice Cellular infiltration significantly reduces, and illustrates that Galectin-1 albumen has immunosuppressive action；
Step is as follows：
1. materials are with fixing
It is in 4% paraformaldehyde fixer to take the mass percent that fresh Mice brain tissues input prepares in advance, makes group Knit, the solidification of the protein denaturation of cell, to prevent cell postmortem autolysis or bacterial decomposition, so as to keep the original form knot of cell Structure；
2. it is dehydrated transparent
Make dehydrating agent with by low concentration to alcohol in high concentration, gradually slough tissue moisture in the block, then tissue block is placed in Not only alcohol is dissolved in, but also is dissolved in transparent in the clarifier dimethylbenzene of paraffin, tissue alcohol in the block is replaced out with dimethylbenzene；
3. waxdip embeds
Transparent tissue block is placed in the paraffin dissolved, is put into the insulation of dewaxing case；
Embedded after paraffin is completely immersed in tissue block：Container is prepared, pours into the paraffin dissolved, rapid gripping is The tissue block for being impregnated with paraffin is put into wherein, and cooled and solidified is blocking；
Treat that embedded tissue block is hardened, very thin section could be cut on slicer；
4. section and patch
Embedded wax stone is fixed on slicer, is thinly sliced, generally 5-8 μ m-thicks；The thin slice cut often wrinkles Pleat, will be put into the water of heating and plate, then be attached on glass slide, put in 45 DEG C of insulating boxs and dry；
5. dewaxing dyeing
The sample of drying is subjected to HE dyeing, to increase the heterochromia of tissue cellularity each several part, beneficial to observation；Soviet Union Another name for coloring agent is a kind of basic-dyeable fibre, nucleus and intracellular ribosomes can be dyed bluish violet；Yihong is a kind of acid dye Cytoplasm, can be dyed red or pale red by material.Before dyeing, the paraffin in section is sloughed with dimethylbenzene, then arrive via high concentration Low-concentration ethanol, finally enters distilled water, you can dyeing；
HE dyeing courses are：
The section after distilled water 1-2s will have been entered it has been put into hematoxylin coloring agent to dye 3min, tap water rinses 5-10s；
It is 70% ethanol and concentrated hydrochloric acid 100-200 by volume to be put into percentage by volume:Break up 5- in the mixed liquor of 1 composition 10s；
Flowing water rinses 1-2s；
It is put into concentrated ammonia liquor and distilled water by volume 1:5-10s in 99 mixed liquors prepared；
Flowing water rinses 15-30s；
Enter eosin stains liquid dyeing 8-10s；
6. it is dehydrated transparent
Section after dyeing is dehydrated through absolute alcohol, then makes section transparent through dimethylbenzene；
7. sealing
By the transparent upper canada balsam of section drop, covered sealing, after natural gum is slightly dry, sticks mark writing paper, cuts Piece sample can be used, and carry out microscope observation.
Further, (2) middle mode of infection infects the step for gavage.
Further, the step is (3) middle using eBioscience ELISA detection kits detection antibody level of serum； Alternatively, the cow's serum that the step Tween-20 that (3) it is 1% containing percentage by volume that middle confining liquid, which is, percentage by volume are 10% The PBS of albumin.
Further, the step is (4) 2. middle using BD Biosciences ELISA detection kits detection cell factor IFN-γ, IL-4, IL-5 and IL-10 are horizontal.
Further, the step PBS that (5) it is 2%FBS containing percentage by volume that middle Facs buffer solutions, which are,；The step is (5) Middle detection kit is BD Biosciences；(5) middle rupture of membranes fixes the article No. of buffer solution to the step：562574, batch 51- 9008100 and 51-9008101；The article No. of the step (5) middle cleaning solution：562574, batch 51-9008102.
Further, (6) middle inflammatory cell is neutrophil leucocyte, eosinophil to the step；Alternatively, the step (6) middle meningitis predilection site is the ventricles of the brain, cavum subarachnoidale；Alternatively, the step (6) 2. body of the middle low concentration to alcohol in high concentration Accumulating concentration is respectively：70% alcohol 30min；90% alcohol 30min；95% alcohol 30min；100% alcohol 1h；100% alcohol 1h；Alternatively, (6) 5. the volumetric concentration of middle and high concentration to low-concentration ethanol is respectively the step：100% alcohol 2min；95% wine Smart 1min；80% alcohol 1min；75% alcohol 1min.
Angiostrongylus cantonensis protein Gal-9 ectin-1 has the principle of the verification method of immunosuppressive action such as in the present invention Under：
Total IgG in different times mice serum, IgG1, IgG2a and IgE antibody level are detected by ELISA method, Assess Humoral Immunological Regulation Roles.
Mouse spleen lymphocyte cell factor IFN-γ, IL-4, IL-5, IL-10 detection, assessment Culture in vitro are made With.
Flow cytomery CD4+T cell and CD4+CD25+PoxP3+Treg cells, research purpose protein immunization are adjusted Mechanism.
Mice brain tissues are taken to carry out HE dyeing, the observation cerebral tissue pathology injury order of severity, further verifies Galectin- The immunoregulation effect of 1 albumen.
The related test results of the method for the present invention and discussion：
Antibody test result shows that Galectin-1 albumen can suppress Total IgG, IgG2a and IgE antibody is horizontal, Promote IgG1 synthesis；
Cytokines measurement is the results show that Galectin-1 albumen suppresses IFN-γ, IL-5 secretions, promotion IL-10, IL-4 Synthesis；
Flow cytometer detection the result shows that, the inhibitory action of Galectin-1 albumen mainly passes through CD4+CD25+FoxP3+Treg What cell was adjusted；
Brain tissue HE dyeing proves, infects different times after III phase larva, control group mice brain tissue impairment degree ratio Galectin-1 experimental mices are serious.
The present invention have studied immunoregulation effect of the angiostrongylus cantonensis Galectin-1 albumen to host from different aspect. Tentative confirmation Galectin-1 albumen has certain immunosuppressive action, and is mainly to suppress humoral immunity and cellular immunity Th1 approach.
Discuss：
The present invention illustrates the immunosuppressive action of angiostrongylus cantonensis Galectin-1 albumen in different aspect.Guangzhou Guan Yuan Nematodiasis is a kind of acute infectious diseases, although having self limiting, being in the infection infringement caused by body in early days cannot It is ignored.Galectin-1 albumen can suppress immune response to a certain extent, reduce the damage to host, can conduct Treat the potential drug action target spot of angiostrongyliasis cantonensis.

Claims (8)

1. a kind of application of angiostrongylus cantonensis (Angiostrongylus Cantonensis) protein Gal-9 ectin-1, it is special Sign is：The application answering in medicine of the preparation with immune suppression function for angiostrongylus cantonensis protein Gal-9 ectin-1 With.
2. the application of angiostrongylus cantonensis protein Gal-9 ectin-1 according to claim 1, it is characterised in that：The Guangzhou Pipe strongylid protein Gal-9 ectin-1 has immunosuppressive action.
3. the application of angiostrongylus cantonensis protein Gal-9 ectin-1 according to claim 2, it is characterised in that：The Guangzhou Pipe strongylid protein Gal-9 ectin-1 has the verification method of immunosuppressive action, and step is as follows：
(1) mouse is immunized
Angiostrongylus cantonensis Galectin-1 albumen is filled with isometric Freund's complete adjuvant or incomplete Freund's adjuvant respectively Divide mixing, blow and beat emulsification repeatedly with syringe until there is Water-In-Oil state, be subcutaneously injected and C57BL/6 mouse are immunized；Specifically exempt from Epidemic disease method is：
Initial immunity：Every 100 μ g albumen of mouse, mixes with Freund's complete adjuvant；
Booster immunization：Every 50 μ g albumen of mouse, mixes with incomplete Freund's adjuvant；
Each immunization interval one week, is immunized three times altogether；
(2) III phase of angiostrongylus cantonensis larva infecting mouse
1. the acquisition of III phase larva
Positive SD rats fresh excreta is added in the Pomacea canaliculata living environment manually established, consecutive infection 3-4 times；Will after 2 weeks Pomacea canaliculata shells, and takes out internal organ and musculature, 10mL artificial digestion liquid is added according to 1g spiral shells meat after mincing, in 37 DEG C of incubators Middle digestion 2-3h；Then through strainer filtering, staticly settle, washed 2-3 times with physiology salt, be deposited in microscopy under anatomical lens；
2. III phase larva infects
Choose III phase larva under microscope, and according to every mouse with 30 ± 2 larva infecting mouses；
(3) humoral immunity is adjusted
0 week after III phase larva infects, 1 week, 2 weeks, 3 weeks eyeball blood sampling collection mice serums, mice serum sample is obtained, is used Total IgG, IgG1, IgG2a and IgE antibody are horizontal in ELISA method detection different times mice serum, assess humoral immunity Adjustment effect；
Using Total IgG and IgE as index, compared with control group, reduced if experimental group is horizontal, illustrate Galectin-1 albumen With immunosuppressive action；
Antibody level of serum is detected using ELISA detection kit；
Concretely comprise the following steps：
1. it is coated with：Antibody will be captured respectively with coating buffer according to capture antibody：The volume ratio of coating buffer is 1:250 dilution proportion Afterwards, 100 μ L/HoleAdd in elisa plate, 4 DEG C of coatings are overnight；
Wherein, the coating buffer is PBS, captures antibody as corresponding anti-mouse monoclonal antibody, i.e., anti-mouse IgG, anti-mouse IgG1, anti-mouse IgG2a, anti-mouse IgE monoclonal antibodies；
2. board-washing：Next day discards liquid in hole, 300 μ L/HoleOrifice plate, is buckled to after last time board-washing by lavation buffer solution board-washing 2 times Liquid in hole is all discarded on blotting paper；
3. close：250μL/HoleConfining liquid, room temperature closing 2h；
4. board-washing：Discard liquid in hole, 300 μ L/HoleLavation buffer solution board-washing 2 times, tips upside down on suction after last time board-washing by orifice plate Liquid in hole is all discarded on water paper；
5. prepare standard items and sample：Standard items mouse Total IgG antibodies, mouse IgG1 antibody, mouse IgG2a antibody, mouse IgE are resisted The concentration that body is diluted to mouse Total IgG antibodies with deionized water respectively is 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/ mL、6.25ng/mL、3.13ng/mL、1.56ng/mL；The concentration of mouse IgG1 antibody for 200ng/mL, 100ng/mL, 50ng/mL, 25ng/mL、12.5ng/mL、6.25ng/mL、3.13ng/mL；The concentration of mouse IgG2a antibody for 250ng/mL, 125ng/mL, 62.5ng/mL、31.25ng/mL、15.63ng/mL、7.81ng/mL、3.90ng/mL；The concentration of mouse IgE antibody is 250ng/ mL、125ng/mL、62.5ng/mL、31.25ng/mL、15.63ng/mL、7.81ng/mL、3.90ng/mL；100μL/HoleAdd to mark In quasi- sample wells, if 3 multiple holes；
Meanwhile dilute mice serum sample with confining liquid, diluted volume ratio is mouse IgG- serum：Confining liquid=1: 100000；Mouse IgG1- serum：Confining liquid=1:10；Mouse IgG2a- serum：Confining liquid=1:10000；Mouse IgE- serum：Closing Liquid=1:50；
6. incubate primary antibody：100μL/HoleDiluted blood serum sample is added in sample well, and every group sets 3 multiple holes, while set blank control Hole；
7. incubate secondary antibody：50μL/HoleDiluted detection antibody is added in elisa plate, is incubated at room temperature 3h；
Wherein, the diluted detection antibody is the anti-mouse IgG monoclonal antibody of HRP marks, is diluted using confining liquid, dilutes body Product ratio is confining liquid：Antibody=250:1；
8. board-washing：Discard liquid in hole, 300 μ L/HoleLavation buffer solution board-washing 4 times, tips upside down on suction after last time board-washing by orifice plate Liquid in hole is all discarded on water paper；
9. develop the color：100μL/HoleTMB nitrite ions, room temperature lucifuge are incubated 15min；
10. terminate：100μL/Hole2M H2SO4Terminate liquid terminates reaction；
Finally elisa plate is placed in microplate reader, reads the absorbance value at 450nm wavelength, standard curve is drawn, according to standard Curve calculating antibody concentration；
(4) Culture in vitro
In order to observe adjustment effect of the destination protein to cellular immunity, mouse spleen lymphocyte cell factor IFN-γ, IL- are carried out 4th, IL-5 and IL-10 detections；
Angiostrongylus cantonensis infection is acute infection, acute stage based on Th1 immune responses, using IFN-γ as confirming index, Compared with control group, if experimental group IFN-γ reduces, then it is assumed that Galectin-1 albumen has immunosuppressive action；
Concretely comprise the following steps：
1. mouse spleen lymphocyte separates
Take spleen：Take cervical dislocation after blood to put to death eyeball of mouse, be put into equipped with percentage by volume as in the beaker of 75% ethanol, nothing Solution takes mouse spleen under the conditions of bacterium；
Grinding：Mouse spleen is put into 1mLDMEM cell culture fluids and is cleaned, is fully ground, and is constantly added dropwise to tissue DMEM nutrient solutions, until more than 95% cell is separated in tissue, cell suspension is carefully moved in sterile centrifuge tube；
Centrifugation：1400rpm, room temperature centrifugation 5min；
Separation of lymphocytes：Supernatant is abandoned, adds 500 μ LAck erythrocyte cracked liquids, 1min is stood, adds DMEM nutrient solutions to be settled to 2mL；
Centrifugation：1400rpm, room temperature centrifugation 5min；
Filtering：Supernatant is abandoned, adds 500 μ L DMEM that cell is resuspended, is filtered through 200 mesh filter screens to another clean sterile centrifuge tube In, cleaning solution is moved in new centrifuge tube together after being cleaned with DMEM；
Dyeing：98 μ L trypan blues and 2 μ L cell suspensions fill pond after fully mixing；
Cell count：The cell quantity in 4 block plaids is counted under microscope, according to C=N/4 × 10 × 103× 50 calculate cell Concentration；
2. Secreted by Mouse Splenic cytokines measurement
Splenic lymphocytes suspension is diluted to 1 × 10 with containing the DMEM that percentage by volume is 10%FBS6A cell/mL, is added to 96 In orifice plate, 100 μ L/Hole, then add angiostrongylus cantonensis Galectin-1 protein 25s ng/mL, 100 μ L/HoleStimulate splenocyte；Together When set canavalin ConA25ng/mL, 100 μ L/HolePositive controls, 100 μ L/ of sterilizing PBSHoleNegative control group；In 5%CO2Training Cell conditioned medium is collected after supporting 37 DEG C of culture 72h of case, obtains cell conditioned medium sample, -80 DEG C save backup, for cytokines measurement；
Horizontal, the concrete operation step using ELISA detection kit detection cell factor IFN-γ, IL-4, IL-5 and IL-10 For：
Coating：The anti-mouse IFN-γ of antibody, anti-mouse IL-4, anti-mIL5, anti-mouse IL-10 Dan Ke will be captured respectively with coating buffer Grand antibody is according to capture antibody：The volume ratio of coating buffer is 1:After 250 dilution proportion, elisa plate is added to according to every 100 μ L of hole In, 4 DEG C of coatings are overnight；Wherein, the anti-mouse IFN-γ, anti-mouse IL-4, the coating buffer of anti-mIL5 monoclonal antibody are 7.13g NaHCO3+1.59gNa2CO3Add the mixed liquor after 1L distilled water；The coating buffer of anti-mouse IL-10 monoclonal antibodies is 12.49gNa2HPO4+15.47g NaH2PO4Add the mixed liquor after 1L distilled water；
Board-washing：Next day, liquid in hole is discarded, with 300 μ L/HoleLavation buffer solution board-washing 3 times, for the last time tips upside down on orifice plate Liquid in hole is patted dry on blotting paper；
Closing：Elisa plate is closed containing the PBS that percentage by volume is 10%FBS per 200 μ L of hole, is incubated at room temperature 2h；
Board-washing：Liquid in hole is discarded, with 300 μ L/HoleLavation buffer solution board-washing 3 times, tips upside down on blotting paper by orifice plate for the last time On liquid in hole is patted dry；
Prepare standard items and sample：By standard items recombinant murine IFN-γ, recombinant murine IL-4, recombinant murine IL-5, IL-10 points of recombinant murine 7 concentration gradients, i.e. IFN-γ are not diluted to the PBS for being 10%FBS containing percentage by volume by multiple proportions:2000pg/mL、 1000pg/mL、500pg/mL、250pg/mL、125pg/mL、62.5pg/mL、31.3pg/mL；IL-4:500pg/mL、250pg/ mL、125pg/mL、62.5pg/mL、31.3pg/mL、15.6pg/mL、7.8pg/mL；IL-5:1000pg/mL、500pg/mL、 250pg/mL、125pg/mL、62.5pg/mL、31.3pg/mL、15.6pg/mL；IL-10:2000pg/mL、1000pg/mL、 500pg/mL、250pg/mL、125pg/mL、62.5pg/mL、31.3pg/mL；The cells and supernatant sample of collection is used and contains body The PBS that product percentage is 10%FBS carries out 1:20 dilutions, 3 multiple holes of every group of setting, while set blank control wells；
Incubate primary antibody：Standard item group, sample sets and blank control group are added in elisa plate per 100 μ L of hole, are incubated at room temperature 1h；
Board-washing：Liquid in hole is discarded, with 300 μ L/HoleLavation buffer solution board-washing 5 times, tips upside down on blotting paper by orifice plate for the last time On liquid in hole is patted dry；
Incubate secondary antibody：Add the enzyme marking reagent of the diluted detection antibody of 100uL and SAv-HRP marks per hole, be incubated at room temperature 1h；
Wherein, the detection antibody is respectively the anti-mouse IFN-γ monoclonal antibody of Biotinylate marks, Biotinylate The anti-mouse IL-4 monoclonal antibodies of mark, the anti-mIL5 monoclonal antibody of Biotinylate marks, Biotinylate marks Anti- mouse IL-10 monoclonal antibodies；Diluted during dilution using containing the PBS that percentage by volume is 10%FBS, PBS：Detect antibody： The volume ratio of SAv-HRP enzyme marking reagents is 500:1:2；
Board-washing：Liquid in hole is discarded, with 300 μ L/HoleLavation buffer solution board-washing 7 times, lavation buffer solution is placed in by each board-washing 1min is stopped in elisa plate；
Colour developing：Add 100 μ LTMB nitrite ions per hole, lucifuge is incubated 15min at room temperature；
Terminate reaction：Add 50 μ L2M H per hole2SO4Terminate liquid；
Read plate：Elisa plate is placed in microplate reader, reads the absorbance value at 450nm wavelength；
Calculate concentration：Standard curve is drawn, cytokine concentrations are calculated according to standard curve；
(5) mouse spleen lymphocyte differentiation group detection
Using flow cytomery CD4+T cell and CD4+CD25+PoxP3+Treg cellular changes；Compared with control group, experiment Group CD4+T cell reduces and/or CD4+CD25+PoxP3+Treg cells raise, then illustrate that Galectin-1 albumen has immunosupress Effect；
Isolated splenic lymphocytes suspension is adjusted to 1.5 × 10 with DMEM7A cell/mL, is added to 96 orifice plate, 100 μ L/Hole In, operated in accordance with the following steps using detection kit：
1. adding 200 μ LFacs buffer solutions per hole, 1400rpm, room temperature centrifugation 5min, abandons supernatant；
2. 25 μ LFacs buffer solutions are added per hole closes 1min on ice；
3. the 25 diluted mixtures of antibodies of μ L are added per hole is incubated 30min on ice, wherein, dilution volume ratio buffers for Facs Liquid：CD4-FITC：CD8-PerCP-Cy5.5：CD25-APC=200：1：1：1；
4. adding 200 μ LFacs buffer solutions per hole, 1400rpm, room temperature centrifugation 5min, abandons supernatant；
5. adding the rupture of membranes of 100 μ L Fresh to fix buffer solution per hole, 30min is incubated on ice；
6. directly into hole plus 200 μ L cleaning solutions, 1400rpm, room temperature centrifuge 5min, abandon supernatant；
7. repeat step is 6.；
8. adding 50 μ L cleaning solutions per hole, FoxP3-PE antibody presses cleaning solution：The volume ratio of FoxP3-PE is 100:1 is directly added into hole Interior, 4 DEG C of lucifuges are incubated 30min；
9. repeat step is 6., clean 2 times；
10. 100 μ L cleaning solutions are added to add upper machine testing in streaming pipe after cell is resuspended per hole；
(6) brain tissue HE is dyed
Mice brain tissues are taken to carry out HE dyeing, the micro- sem observation cerebral tissue pathology injury order of severity and inflammatory cell change, into One step demonstrate,proves the immunoregulation effect of Galectin-1 albumen；If soaked in the meningitis predilection site inflammatory cell of experimental mice Profit significantly reduces, and illustrates that Galectin-1 albumen has immunosuppressive action；
Step is as follows：
1. materials are with fixing
It is to make tissue, thin in 4% paraformaldehyde fixer to take the mass percent that fresh Mice brain tissues input prepares in advance The protein denaturation solidification of born of the same parents, to prevent cell postmortem autolysis or bacterial decomposition, so as to keep the original morphosis of cell；
2. it is dehydrated transparent
Make dehydrating agent with by low concentration to alcohol in high concentration, gradually slough tissue moisture in the block, then tissue block is placed in both molten In alcohol, and it is dissolved in transparent in the clarifier dimethylbenzene of paraffin, tissue alcohol in the block is replaced out with dimethylbenzene；
3. waxdip embeds
Transparent tissue block is placed in the paraffin dissolved, is put into the insulation of dewaxing case；
Embedded after paraffin is completely immersed in tissue block：Container is prepared, pours into the paraffin dissolved, rapid gripping has been impregnated with The tissue block of paraffin is put into wherein, and cooled and solidified is blocking；
Treat that embedded tissue block is hardened, very thin section could be cut on slicer；
4. section and patch
Embedded wax stone is fixed on slicer, is thinly sliced, is 5-8 μ m-thicks；The thin slice gauffer cut, will be put into heating Water in plate, then be attached on glass slide, put in 45 DEG C of insulating boxs and dry；
5. dewaxing dyeing
The sample of drying is subjected to HE dyeing, to increase the heterochromia of tissue cellularity each several part, beneficial to observation；Hematoxylin Coloring agent is a kind of basic-dyeable fibre, nucleus and intracellular ribosomes can be dyed bluish violet；Yihong is a kind of acid dyes, energy Cytoplasm is dyed into red or pale red.Before dyeing, the paraffin in section is sloughed with dimethylbenzene, then via high concentration to low concentration Alcohol, finally enters distilled water, you can dyeing；
HE dyeing courses are：
The section after distilled water 1-2s will have been entered it has been put into hematoxylin coloring agent to dye 3min, tap water rinses 5-10s；
It is 70% ethanol and concentrated hydrochloric acid 100-200 by volume to be put into percentage by volume:Break up 5-10s in the mixed liquor of 1 composition；
Flowing water rinses 1-2s；
It is put into concentrated ammonia liquor and distilled water by volume 1:5-10s in 99 mixed liquors prepared；
Flowing water rinses 15-30s；
Enter eosin stains liquid dyeing 8-10s；
6. it is dehydrated transparent
Section after dyeing is dehydrated through absolute alcohol, then makes section transparent through dimethylbenzene；
7. sealing
By the transparent upper canada balsam of section drop, covered sealing, after natural gum is slightly dry, sticks mark writing paper, section mark This can be used, and carry out microscope observation.
4. the application of angiostrongylus cantonensis protein Gal-9 ectin-1 according to claim 3, it is characterised in that：The step (2) middle mode of infection infects for gavage.
5. the application of angiostrongylus cantonensis protein Gal-9 ectin-1 according to claim 3, it is characterised in that：The step (3) it is middle using eBioscience ELISA detection kits detection antibody level of serum；(3) middle confining liquid is containing body to the step Product percentage be 1% Tween-20, percentage by volume be 10% bovine serum albumin(BSA) PBS.
6. the application of angiostrongylus cantonensis protein Gal-9 ectin-1 according to claim 3, it is characterised in that：The step (4) it is 2. middle using BD Biosciences ELISA detection kits detection cell factor IFN-γ, IL-4, IL-5 and IL-10 water It is flat.
7. the application of angiostrongylus cantonensis protein Gal-9 ectin-1 according to claim 3, it is characterised in that：The step (5) the PBS that it is 2%FBS containing percentage by volume that middle Facs buffer solutions, which are,；(5) middle detection kit is the step BDBiosciences；(5) middle rupture of membranes fixes the article No. of buffer solution to the step：562574, batch 51-9008100 and 51- 9008101；The article No. of the step (5) middle cleaning solution：562574, batch 51-9008102.
8. the application of angiostrongylus cantonensis protein Gal-9 ectin-1 according to claim 5, it is characterised in that：The step (6) middle inflammatory cell is neutrophil leucocyte, eosinophil；(6) middle meningitis predilection site is the ventricles of the brain, arachnoid to the step Cavity of resorption；(6) 2. the volumetric concentration of middle low concentration to alcohol in high concentration is respectively the step：70% alcohol 30min；90% alcohol 30min；95% alcohol 30min；100% alcohol 1h；100% alcohol 1h；The step (6) 5. middle and high concentration to low-concentration ethanol Volumetric concentration be respectively：100% alcohol 2min；95% alcohol 1min；80% alcohol 1min；75% alcohol 1min.
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