CN106729659A - A kind of application of angiostrongylus cantonensis albumen PAS 5 - Google Patents
A kind of application of angiostrongylus cantonensis albumen PAS 5 Download PDFInfo
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- CN106729659A CN106729659A CN201611056508.9A CN201611056508A CN106729659A CN 106729659 A CN106729659 A CN 106729659A CN 201611056508 A CN201611056508 A CN 201611056508A CN 106729659 A CN106729659 A CN 106729659A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/4806—Hydrolases (3) acting on peptide bonds (3.4) from animals other than mammals, e.g. snakes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6866—Interferon
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present invention relates to a kind of application of angiostrongylus cantonensis (Angiostrongylus Cantonensis) albumen PAS 5, the application is the application of the immunological enhancement of angiostrongylus cantonensis albumen PAS 5.The present invention is the application of angiostrongylus cantonensis albumen PAS 5, the application is the application of the immunological enhancement of angiostrongylus cantonensis albumen PAS 5, applications of the particularly angiostrongylus cantonensis albumen PAS 5 in the medicine with immune enhancing function is prepared, for the treatment of angiostrongyliasis cantonensis provides theoretical foundation, for the development of medicine provides latent effect target spot；Simultaneously for the research and development of angiostrongyliasis cantonensis medicine lay the foundation, for the personalized treatment of angiostrongylus cantonensis provides target, have suitable market prospects and application.
Description
Technical field
The invention belongs to technical field of life science, especially a kind of application of angiostrongylus cantonensis albumen PAS-5.
Background technology
Angiostrongylus cantonensis (Angiostrongylus Cantonensis) are the important pathogens for causing eosinophilic meningoencephalitis
Body, can cause central lesion, severe patient to cause death.PAS-5 is proteasome Alpha 5 subunit albumen, proteasome ginseng
With many pathological processes of body, host immune response is induced, but there is no angiostrongylus cantonensis PAS-5 albumen to host immune
Research and application in terms of regulation.
By retrieval, the existing research similar to present patent application is as follows：
(1) proteasome inhibitor can be used as the medicine of many diseases.
(2) blood fluke PAS-5 albumen can induce host immune to protect reaction.
By contrast, there is the difference of essence to above-mentioned related open source literature in present patent application.
The content of the invention
It is an object of the invention to overcome the deficiencies in the prior art part, supplement to study angiostrongylus cantonensis immunological regulation
Deficiency, there is provided a kind of application of angiostrongylus cantonensis albumen PAS-5, the application is exempted from for angiostrongylus cantonensis albumen PAS-5
The application of epidemic disease humidification, particularly angiostrongylus cantonensis albumen PAS-5 are in the medicine with immune enhancing function is prepared
Using for the treatment of angiostrongyliasis cantonensis provides theoretical foundation, for the development of medicine provides 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) albumen PAS-5, the application
It is the application of the immunological enhancement of angiostrongylus cantonensis albumen PAS-5.
And, the application is angiostrongylus cantonensis albumen PAS-5 in the medicine with immune enhancing function is prepared
Using.
And, the application is preparing the immunopotentiating drug of angiostrongyliasis cantonensis for angiostrongylus cantonensis albumen PAS-5
Application in thing.
And, the angiostrongylus cantonensis albumen PAS-5 has immunological enhancement.
And, the angiostrongylus cantonensis albumen PAS-5 has the verification method of immunological enhancement, and step is as follows：
(1) immune mouse
Angiostrongylus cantonensis PAS-5 albumen is abundant with isometric Freund's complete adjuvant, incomplete Freund's adjuvant respectively
Mixing, blows and beats emulsification until there is Water-In-Oil state repeatedly with syringe, the immune C57BL/6 mouse of hypodermic injection.It is specific immune
Method is：
Initial immunity：Every μ g albumen of mouse 100, mixes with Freund's complete adjuvant；
Booster immunization：Every μ g albumen of mouse 50, 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
In the Pomacea canaliculata living environment that the addition of positive SD rats fresh excreta is manually set up, 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 infection
Choose III phase larva under microscope, and according to every mouse with 30 ± 2 larva infecting mouses；
(3) humoral immunity regulation
Before 0 week after III phase larva infects infects, 1 week, 2 weeks, 3 weeks eyeball blood sampling collection mice serums, mouse blood is obtained
Final proof product, using Total IgG, IgG1, IgG2a and IgE antibody level in ELISA method detection different times mice serum,
Assessment Humoral Immunological Regulation Roles；
Using Total IgG and IgE as index, compared with control group, if experimental group level increases, PAS-5 albumen is illustrated
With immunological enhancement；
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, and capture antibody is 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 and all discard liquid in hole 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, by orifice plate back-off after last time board-washing
Liquid in hole is all discarded on blotting paper；
5. standard items and sample are prepared：By standard items mouse Total IgG antibodies, IgG1 antibody, IgG2a antibody, IgE antibody
It is 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/ to be diluted to the concentration of mouse Total IgG antibodies with deionized water respectively
mL、6.25ng/mL、3.13ng/mL、1.56ng/mL；The concentration of mouse IgG1 antibody be 200ng/mL, 100ng/mL, 50ng/mL,
25ng/mL、12.5ng/mL、6.25ng/mL、3.13ng/mL；The concentration of mouse IgG2a antibody be 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, 100 μ L/HoleAdd to mark
In quasi- sample wells, if 3 multiple holes；
Meanwhile, mice serum sample is diluted with confining liquid, the volume ratio of dilution is mouse Total IgG- serum：Confining liquid
=1:100000；Mouse IgG1- serum：Confining liquid=1:10；Mouse IgG2a- serum：Confining liquid=1:10000；Mouse IgE- serum：
Confining liquid=1:50；
6. primary antibody is incubated：100μL/HoleThe blood serum sample of dilution is added in sample well, and every group sets 3 multiple holes, while setting blank pair
According to hole；
7. secondary antibody is incubated：50μL/HoleThe detection antibody of dilution is added in elisa plate, is incubated at room temperature 3h；
Wherein, the detection antibody of the dilution is the anti-mouse IgG monoclonal antibody or biotin-avidin mark of HRP marks
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/HoleLavation buffer solution board-washing 4 times, by orifice plate back-off after last time board-washing
Liquid in hole is all discarded on blotting paper；
9. develop the color：100μL/HoleTMB nitrite ions, room temperature lucifuge is incubated 15min；
10. terminate：100μL/Hole2M H2SO4Terminate liquid terminating reaction；
Finally elisa plate is placed in ELIASA, the absorbance value at 450nm wavelength is read, 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- 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 is raised, then it is assumed that PAS-5 albumen has immunological enhancement；
Concretely comprise the following steps：
1. mouse spleen lymphocyte is separated
Take spleen：Cervical dislocation is put to death after eyeball of mouse is taken into 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 1mLDMEM cell culture fluids and is cleaned, be fully ground, and constantly to tissue drop
Plus DMEM nutrient solutions, until more than 95% cell is separated in tissue, during cell suspension carefully to move to aseptic centrifuge tube；
Centrifugation：1400rpm, room temperature centrifugation 5min；
Separation of lymphocytes：Supernatant is abandoned, 500 μ LAck erythrocyte cracked liquids are added, 1min is stood, plus DMEM nutrient solutions are determined
Hold to 2mL；
Centrifugation：1400rpm, room temperature centrifugation 5min；
Filtering：Abandon supernatant, plus 500 μ L DMEM re-suspended cells, through 200 mesh filter screens filter to another it is clean it is aseptic from
During cleaning fluid to move to new centrifuge tube together in heart pipe, after being cleaned with DMEM；
Dyeing：98 μ L trypan blues and 2 μ L cell suspensions fill pond after fully mixing；
Cell count：Cell quantity in 4 block plaids of counted under microscope, according to C=N/4 × 10 × 103× 50 meters
Calculate cell concentration；
2. Secreted by Mouse Splenic cytokines measurement
SPL suspension is diluted to 1 × 10 with containing the DMEM that percentage by volume is 10%FBS6Individual cell/mL, plus
To in 96 orifice plates, 100 μ L/Hole, it is subsequently adding angiostrongylus cantonensis PAS-5 protein 10s 0ng/mL, 100 μ L/HoleStimulate splenocyte；Together
When set canavalin ConA 100ng/mL, 100 μ L/HolePositive controls, sterilizing PBS 100 μ L/HoleNegative control group；In 5%CO2
Cell conditioned medium is collected after 37 DEG C of culture 72h of incubator, cell conditioned medium sample is obtained, it is standby for cytokines measurement or -80 DEG C of preservations
With；
Using ELISA detection kit detection cell factor IFN-γ, IL-4, IL-5 and IL-10 level, concrete operations step
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 the μ L of every hole 100
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：Per the μ L of hole 200 2h is incubated at room temperature containing percentage by volume for the PBS of 10%FBS closes elisa plate；
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 containing the PBS that percentage by volume is 10%FBS 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 that will be collected is with containing body
Product percentage carries out 1 for the PBS of 10%FBS:20 dilutions, 3 multiple holes of every group of setting, while setting blank control wells；
Incubate primary antibody：Standard item group, sample sets and blank control group are added in elisa plate per the μ L of hole 100, 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 detection antibody that 100uL dilutes and the enzyme marking reagent that SAv-HRP is marked per hole, be incubated at room temperature 1h；
Wherein, the 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：Detection 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 and 1min is stopped in elisa plate；
Colour developing：Add 100 μ LTMB nitrite ions per hole, lucifuge is incubated 15min at room temperature；
Terminating reaction：Add 50 μ L2M H per hole2SO4Terminate liquid；
Read plate：Elisa plate is placed in ELIASA, the absorbance value at 450nm wavelength is read；
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 is raised and/or CD4+CD25+PoxP3+The reduction of Treg cells, then illustrate PAS-5 albumen tool Immune-enhancing effect
Effect；
Isolated SPL suspension is adjusted to 1.5 × 10 with DMEM7Individual cell/mL, is added to 96 orifice plates 100
μL/HoleIn, operated in accordance with the following steps using detection kit：
1. 200 μ LFacs buffer solutions are added per hole, 1400rpm, room temperature centrifugation 5min abandon supernatant；
2. 25 μ LFacs buffer solutions are added to close 1min on ice per hole；
3. the mixtures of antibodies of 25 μ L dilutions is added to be incubated 30min on ice per hole, wherein, dilution volume ratio is slow for Facs
Fliud flushing：CD4-FITC：CD8-PerCP-Cy 5.5：CD25-APC=200：1：1：1；
4. 200 μ LFacs buffer solutions are added per hole, 1400rpm, room temperature centrifugation 5min abandon supernatant；
5. add the rupture of membranes of 100 μ L Fresh to fix buffer solution per hole, 30min is incubated on ice；
6. directly to 200 μ L cleaning fluids are added in hole, 1400rpm, room temperature centrifugation 5min abandon supernatant；
7. repeat step is 6.；
8. 50 μ L cleaning fluids, FoxP3-PE antibody are added to press cleaning fluid per hole：The volume ratio of FoxP3-PE is 100:1 directly adds
Enter in hole, 4 DEG C of lucifuges are incubated 30min；
9. 6. repeat step, cleans 2 times；
10. upper machine testing in streaming pipe is added after adding 100 μ L cleaning fluid re-suspended cells per hole；
(6) brain tissue HE dyeing
Taking Mice brain tissues carries out HE dyeing, and the micro- sem observation cerebral tissue pathology injury order of severity and inflammatory cell become
Change, further the immunoregulation effect of checking PAS-5 albumen；If in the meningitis predilection site inflammatory cell of experimental mice
Infiltration showed increased, illustrates that PAS-5 albumen has immunological enhancement；
Step is as follows：
1. draw materials and fix
Take during the mass percent that fresh Mice brain tissues input prepares in advance is 4% paraformaldehyde fixer, make group
Knit, the protein denaturation of cell solidifies, to prevent cell postmortem autolysis or bacterium from decomposing, 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 the moisture in tissue block, then tissue block is placed in
Both alcohol is dissolved in, is dissolved in again transparent in the clarifier dimethylbenzene of paraffin, the alcohol in tissue block is replaced out with dimethylbenzene；
3. waxdip embedding
Transparent tissue block is placed in the paraffin for having dissolved, the insulation of dewaxing case is put into；
Embedded after paraffin is completely immersed in tissue block：Container is prepared, the paraffin for having dissolved is poured into, 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. cut into slices and paster
Embedded wax stone is fixed on slicer, is thinly sliced, generally 5-8 μ m-thicks；The thin slice for cutting often wrinkles
Pleat, will be put into pressing in the water of heating, then be attached on slide, put drying in 45 DEG C of insulating boxs；
5. dewaxing dyeing
The sample of drying is carried out into HE dyeing, to increase the heterochromia of tissue cellularity each several part, beneficial to observation；Soviet Union
Another name for H is a kind of basic-dyeable fibre, nucleus and intracellular ribosomes can be dyed into bluish violet；Yihong E 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：
To enter during the section after distilled water 1-2s is put into brazilwood extract dyeing agent and dyeed 3min, running water has rinsed 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 makes section transparent through dehydration of alcohol, then through dimethylbenzene；
7. sealing
The transparent upper canada balsam of section drop, covered sealing after natural gum is slightly dry, are sticked mark writing paper, cut
Piece sample is that can be used, and carries out microscope observation.
And, (2) middle mode of infection is gavage infection to the step.
And, (3) middle use eBioscience ELISA detection kits detect antibody level of serum to the step；Or
Person, (3) middle confining liquid is pure containing the Tween-20 that percentage by volume is 1%, ox blood that percentage by volume is 10% to the step
The PBS of albumen.
And, (4) 2. middle use BD Biosciences ELISA detection kits detect cell factor IFN- to the step
γ, IL-4, IL-5 and IL-10 level.
And, (5) middle detection kit is BD Biosciences to the step；(5) middle rupture of membranes fixes buffering to the step
The article No. of liquid：562574, batch 51-9008100 and 51-9008101；The article No. of the step (5) middle cleaning fluid：562574, batch
Secondary 51-9008102.
And, (6) middle inflammatory cell is property granulocyte, eosinophil to the step；Or, the step (6) midbrain
Film inflammation predilection site is the ventricles of the brain, cavum subarachnoidale；Or, the step (6) 2. volumetric concentration of the middle low concentration to alcohol in high concentration
Respectively：70% alcohol 30min；90% alcohol 30min；95% alcohol 30min；100% alcohol 1h；100% alcohol 1h；Or
Person, (6) 5. middle and high concentration is respectively the step to the volumetric concentration of low-concentration ethanol：100% alcohol 2min；95% alcohol
1min；80% alcohol 1min；75% alcohol 1min.
The advantage for obtaining of the invention and good effect are：
1st, the present invention is the application of angiostrongylus cantonensis albumen PAS-5, and the application is angiostrongylus cantonensis albumen PAS-5's
The application of immunological enhancement, particularly angiostrongylus cantonensis albumen PAS-5 are in the medicine with immune enhancing function is prepared
Application, for the treatment of angiostrongyliasis cantonensis provides theoretical foundation, for the development of medicine provides latent effect target spot；Together
When lay the foundation for the research and development of angiostrongyliasis cantonensis medicine, for the personalized treatment of angiostrongylus cantonensis provides target,
Tool suitable market prospects and application.
2nd, angiostrongylus cantonensis albumen PAS-5 has that the verification method method of immunological enhancement is simple, behaviour in the present invention
Facilitate, analyze angiostrongylus cantonensis PAS-5 albumen immune to host body fluids, cellular immunity and the immune effect of modulability,
And further verify by histopathology that the never Tongfang surface analysis immunoregulation effect of angiostrongylus cantonensis albumen is
More fully understand that the mechanism of causing a disease of angiostrongylus cantonensis provides foundation, be that theoretical base is established in the immunization therapy of angiostrongyliasis cantonensis
Plinth.
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
It is TotalIgG 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
It is 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.
Specific embodiment
For present disclosure, feature and effect can be further appreciated that, following examples are hereby enumerated, and coordinate accompanying 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 guarantor of the invention
Shield scope.
Reagent used in the present invention, unless otherwise required, is the conventional reagent of this area；Used in the present invention
Method, unless otherwise required, be the conventional method of this area.
A kind of application of angiostrongylus cantonensis (Angiostrongylus Cantonensis) albumen PAS-5, the application
It is the application of the immunological enhancement of angiostrongylus cantonensis albumen PAS-5.
Further, the application is that angiostrongylus cantonensis albumen PAS-5 is preparing the medicine with immune enhancing function
In application.
Further, the application is preparing the immune increasing of angiostrongyliasis cantonensis for angiostrongylus cantonensis albumen PAS-5
Application in strong medicine.
Further, the angiostrongylus cantonensis albumen PAS-5 has immunological enhancement.
Further, the angiostrongylus cantonensis albumen PAS-5 has the verification method of immunological enhancement, and step is such as
Under：
(1) immune mouse
Angiostrongylus cantonensis PAS-5 albumen is abundant with isometric Freund's complete adjuvant, incomplete Freund's adjuvant respectively
Mixing, blows and beats emulsification until there is Water-In-Oil state repeatedly with syringe, the immune C57BL/6 mouse of hypodermic injection.It is specific immune
Method is：
Initial immunity：Every μ g albumen of mouse 100, mixes with Freund's complete adjuvant；
Booster immunization：Every μ g albumen of mouse 50, 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
In the Pomacea canaliculata living environment that the addition of positive SD rats fresh excreta is manually set up, 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 infection
Choose III phase larva under microscope, and according to every mouse with 30 ± 2 larva infecting mouses；
(3) humoral immunity regulation
Before 0 week after III phase larva infects infects, 1 week, 2 weeks, 3 weeks eyeball blood sampling collection mice serums, mouse blood is obtained
Final proof product, using Total IgG, IgG1, IgG2a and IgE antibody level in ELISA method detection different times mice serum,
Assessment Humoral Immunological Regulation Roles；
Using Total IgG and IgE as index, compared with control group, if experimental group level increases, PAS-5 albumen is illustrated
With immunological enhancement；
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, and capture antibody is 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 and all discard liquid in hole 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, by orifice plate back-off after last time board-washing
Liquid in hole is all discarded on blotting paper；
5. standard items and sample are prepared：By standard items mouse Total IgG antibodies, IgG1 antibody, IgG2a antibody, IgE antibody
It is 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/ to be diluted to the concentration of mouse Total IgG antibodies with deionized water respectively
mL、6.25ng/mL、3.13ng/mL、1.56ng/mL；The concentration of mouse IgG1 antibody be 200ng/mL, 100ng/mL, 50ng/mL,
25ng/mL、12.5ng/mL、6.25ng/mL、3.13ng/mL；The concentration of mouse IgG2a antibody be 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, 100 μ L/HoleAdd to mark
In quasi- sample wells, if 3 multiple holes；
Meanwhile, mice serum sample is diluted with confining liquid, the volume ratio of dilution is mouse Total IgG- serum：Confining liquid
=1:100000；Mouse IgG1- serum：Confining liquid=1:10；Mouse IgG2a- serum：Confining liquid=1:10000；Mouse IgE- serum：
Confining liquid=1:50；
6. primary antibody is incubated：100μL/HoleThe blood serum sample of dilution is added in sample well, and every group sets 3 multiple holes, while setting blank pair
According to hole；
7. secondary antibody is incubated：50μL/HoleThe detection antibody of dilution is added in elisa plate, is incubated at room temperature 3h；
Wherein, the detection antibody of the dilution is the anti-mouse IgG monoclonal antibody or biotin-avidin mark of HRP marks
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/HoleLavation buffer solution board-washing 4 times, by orifice plate back-off after last time board-washing
Liquid in hole is all discarded on blotting paper；
9. develop the color：100μL/HoleTMB nitrite ions, room temperature lucifuge is incubated 15min；
10. terminate：100μL/Hole2M H2SO4Terminate liquid terminating reaction；
Finally elisa plate is placed in ELIASA, the absorbance value at 450nm wavelength is read, 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- 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 is raised, then it is assumed that PAS-5 albumen has immunological enhancement；
Concretely comprise the following steps：
1. mouse spleen lymphocyte is separated
Take spleen：Cervical dislocation is put to death after eyeball of mouse is taken into 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 1mLDMEM cell culture fluids and is cleaned, be fully ground, and constantly to tissue drop
Plus DMEM nutrient solutions, until more than 95% cell is separated in tissue, during cell suspension carefully to move to aseptic centrifuge tube；
Centrifugation：1400rpm, room temperature centrifugation 5min；
Separation of lymphocytes：Supernatant is abandoned, 500 μ LAck erythrocyte cracked liquids are added, 1min is stood, plus DMEM nutrient solutions are determined
Hold to 2mL；
Centrifugation：1400rpm, room temperature centrifugation 5min；
Filtering：Abandon supernatant, plus 500 μ L DMEM re-suspended cells, through 200 mesh filter screens filter to another it is clean it is aseptic from
During cleaning fluid to move to new centrifuge tube together in heart pipe, after being cleaned with DMEM；
Dyeing：98 μ L trypan blues and 2 μ L cell suspensions fill pond after fully mixing；
Cell count：Cell quantity in 4 block plaids of counted under microscope, according to C=N/4 × 10 × 103× 50 meters
Calculate cell concentration；
2. Secreted by Mouse Splenic cytokines measurement
SPL suspension is diluted to 1 × 10 with containing the DMEM that percentage by volume is 10%FBS6Individual cell/mL, plus
To in 96 orifice plates, 100 μ L/Hole, it is subsequently adding angiostrongylus cantonensis PAS-5 protein 10s 0ng/mL, 100 μ L/HoleStimulate splenocyte；Together
When set canavalin ConA 100ng/mL, 100 μ L/HolePositive controls, sterilizing PBS 100 μ L/HoleNegative control group；In 5%CO2
Cell conditioned medium is collected after 37 DEG C of culture 72h of incubator, cell conditioned medium sample is obtained, it is standby for cytokines measurement or -80 DEG C of preservations
With；
Using ELISA detection kit detection cell factor IFN-γ, IL-4, IL-5 and IL-10 level, concrete operations step
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 the μ L of every hole 100
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：Per the μ L of hole 200 2h is incubated at room temperature containing percentage by volume for the PBS of 10%FBS closes elisa plate；
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 containing the PBS that percentage by volume is 10%FBS 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 that will be collected is with containing body
Product percentage carries out 1 for the PBS of 10%FBS:20 dilutions, 3 multiple holes of every group of setting, while setting blank control wells；
Incubate primary antibody：Standard item group, sample sets and blank control group are added in elisa plate per the μ L of hole 100, 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 detection antibody that 100uL dilutes and the enzyme marking reagent that SAv-HRP is marked per hole, be incubated at room temperature 1h；
Wherein, the 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：Detection 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 and 1min is stopped in elisa plate；
Colour developing：Add 100 μ LTMB nitrite ions per hole, lucifuge is incubated 15min at room temperature；
Terminating reaction：Add 50 μ L2M H per hole2SO4Terminate liquid；
Read plate：Elisa plate is placed in ELIASA, the absorbance value at 450nm wavelength is read；
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 is raised and/or CD4+CD25+PoxP3+The reduction of Treg cells, then illustrate PAS-5 albumen tool Immune-enhancing effect
Effect；
Isolated SPL suspension is adjusted to 1.5 × 10 with DMEM7Individual cell/mL, is added to 96 orifice plates 100
μL/HoleIn, operated in accordance with the following steps using detection kit：
1. 200 μ LFacs buffer solutions are added per hole, 1400rpm, room temperature centrifugation 5min abandon supernatant；
2. 25 μ LFacs buffer solutions are added to close 1min on ice per hole；
3. the mixtures of antibodies of 25 μ L dilutions is added to be incubated 30min on ice per hole, wherein, dilution volume ratio is slow for Facs
Fliud flushing：CD4-FITC：CD8-PerCP-Cy 5.5：CD25-APC=200：1：1：1；
4. 200 μ LFacs buffer solutions are added per hole, 1400rpm, room temperature centrifugation 5min abandon supernatant；
5. add the rupture of membranes of 100 μ L Fresh to fix buffer solution per hole, 30min is incubated on ice；
6. directly to 200 μ L cleaning fluids are added in hole, 1400rpm, room temperature centrifugation 5min abandon supernatant；
7. repeat step is 6.；
8. 50 μ L cleaning fluids, FoxP3-PE antibody are added to press cleaning fluid per hole：The volume ratio of FoxP3-PE is 100:1 directly adds
Enter in hole, 4 DEG C of lucifuges are incubated 30min；
9. 6. repeat step, cleans 2 times；
10. upper machine testing in streaming pipe is added after adding 100 μ L cleaning fluid re-suspended cells per hole；
(6) brain tissue HE dyeing
Taking Mice brain tissues carries out HE dyeing, and the micro- sem observation cerebral tissue pathology injury order of severity and inflammatory cell become
Change, further the immunoregulation effect of checking PAS-5 albumen；If in the meningitis predilection site inflammatory cell of experimental mice
Infiltration showed increased, illustrates that PAS-5 albumen has immunological enhancement；
Step is as follows：
1. draw materials and fix
Take during the mass percent that fresh Mice brain tissues input prepares in advance is 4% paraformaldehyde fixer, make group
Knit, the protein denaturation of cell solidifies, to prevent cell postmortem autolysis or bacterium from decomposing, 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 the moisture in tissue block, then tissue block is placed in
Both alcohol is dissolved in, is dissolved in again transparent in the clarifier dimethylbenzene of paraffin, the alcohol in tissue block is replaced out with dimethylbenzene；
3. waxdip embedding
Transparent tissue block is placed in the paraffin for having dissolved, the insulation of dewaxing case is put into；
Embedded after paraffin is completely immersed in tissue block：Container is prepared, the paraffin for having dissolved is poured into, 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. cut into slices and paster
Embedded wax stone is fixed on slicer, is thinly sliced, generally 5-8 μ m-thicks；The thin slice for cutting often wrinkles
Pleat, will be put into pressing in the water of heating, then be attached on slide, put drying in 45 DEG C of insulating boxs；
5. dewaxing dyeing
The sample of drying is carried out into HE dyeing, to increase the heterochromia of tissue cellularity each several part, beneficial to observation；Soviet Union
Another name for H is a kind of basic-dyeable fibre, nucleus and intracellular ribosomes can be dyed into bluish violet；Yihong E 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：
To enter during the section after distilled water 1-2s is put into brazilwood extract dyeing agent and dyeed 3min, running water has rinsed 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 makes section transparent through dehydration of alcohol, then through dimethylbenzene；
7. sealing
The transparent upper canada balsam of section drop, covered sealing after natural gum is slightly dry, are sticked mark writing paper, cut
Piece sample is that can be used, and carries out microscope observation.
Further, (2) middle mode of infection is gavage infection to the step.
Further, (3) middle use eBioscience ELISA detection kits detect antibody level of serum to the step；
Or, (3) middle confining liquid is containing the Tween-20 that percentage by volume is 1%, the cow's serum that percentage by volume is 10% to the step
The PBS of albumin.
Further, (4) 2. middle use BD Biosciences ELISA detection kits detect cell factor to the step
IFN-γ, IL-4, IL-5 and IL-10 level.
Further, (5) middle detection kit is BD Biosciences to the step；The step (5) fix by middle rupture of membranes
The article No. of buffer solution：562574, batch 51-9008100 and 51-9008101；The article No. of the step (5) middle cleaning fluid：
562574, batch 51-9008102.
Further, (6) middle inflammatory cell is property granulocyte, eosinophil to the step；Or, the step is (6)
Middle meningitis predilection site is the ventricles of the brain, cavum subarachnoidale；Or, the step (6) 2. volume of the middle low concentration to alcohol in high concentration
Concentration is respectively：70% alcohol 30min；90% alcohol 30min；95% alcohol 30min；100% alcohol 1h；100% alcohol 1h；
Or, (6) 5. middle and high concentration is respectively the step to the volumetric concentration of low-concentration ethanol：100% alcohol 2min；95% alcohol
1min；80% alcohol 1min；75% alcohol 1min.
Angiostrongylus cantonensis albumen PAS-5 has the principle of the verification method of immunological enhancement as follows in the present invention：
By Total IgG, IgG1, IgG2a, IgE antibody level in ELISA method detection different times mice serum,
Assessment 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, the regulation of research purpose protein immunization
Mechanism.
Taking Mice brain tissues carries out HE dyeing, and the observation cerebral tissue pathology injury order of severity further verifies PAS-5 albumen
Immunoregulation effect.
The related test results of the inventive method and discussion：
Antibody test result shows that PAS-5 albumen can promote Total IgG, IgG1, IgG2a and IgE antibody to synthesize；
Cytokines measurement result shows that PAS-5 albumen promotes IFN-γ, IL-4, IL-5 secretion, suppresses IL-10；
Flow cytometer detection result shows that the immunological enhancement of PAS-5 albumen is by promoting CD4+T cell, suppression CD4+
CD25+FoxP3+The common regulation of Treg cells secretion；
Brain tissue HE dyeing proves that different times after III phase larva of infection, experimental mice brain tissue impairment degree is compared
It is serious according to group mouse.
The present invention have studied immunoregulation effect of the angiostrongylus cantonensis PAS-5 albumen to host from different aspect.Tentatively
Confirm that PAS-5 albumen has certain immunological enhancement, and can simultaneously strengthen humoral immunity and cell immune response.
Discuss：
The present invention illustrates the immunological enhancement of angiostrongylus cantonensis PAS-5 albumen in different aspect.Angiostrongylus cantonensis
Disease is a kind of acute infectious diseases, although with self limiting, but is to be neglected to the infringement that body is caused in infection early stage
Slightly.PAS-5 albumen can to a certain extent strengthen immune response, aggravate the damage to host brain, can be as controlling
Treat the potential drug action target spot of angiostrongyliasis cantonensis.
Claims (10)
1. a kind of application of angiostrongylus cantonensis (Angiostrongylus Cantonensis) albumen PAS-5, its feature exists
In：The application is the application of the immunological enhancement of angiostrongylus cantonensis albumen PAS-5.
2. the application of angiostrongylus cantonensis albumen PAS-5 according to claim 1, it is characterised in that：The application is wide
Applications of the state pipe strongylid albumen PAS-5 in the medicine with immune enhancing function is prepared.
3. the application of angiostrongylus cantonensis albumen PAS-5 according to claim 1, it is characterised in that：The application is wide
Applications of the state pipe strongylid albumen PAS-5 in the medicament for immunity enhancement for preparing angiostrongyliasis cantonensis.
4. the application of the angiostrongylus cantonensis albumen PAS-5 according to any one of claims 1 to 3, it is characterised in that：It is described
Angiostrongylus cantonensis albumen PAS-5 has immunological enhancement.
5. the application of angiostrongylus cantonensis albumen PAS-5 according to claim 4, it is characterised in that：The Guangzhou Guan Yuan
Elegans proteins PAS-5 has the verification method of immunological enhancement, and step is as follows：
(1) immune mouse
Angiostrongylus cantonensis PAS-5 albumen is sufficiently mixed with isometric Freund's complete adjuvant, incomplete Freund's adjuvant respectively,
Emulsification is blown and beaten repeatedly with syringe until there is Water-In-Oil state, the immune C57BL/6 mouse of hypodermic injection.Specific immunization method
For：
Initial immunity：Every μ g albumen of mouse 100, mixes with Freund's complete adjuvant；
Booster immunization：Every μ g albumen of mouse 50, 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
In the Pomacea canaliculata living environment that the addition of positive SD rats fresh excreta is manually set up, 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 infection
Choose III phase larva under microscope, and according to every mouse with 30 ± 2 larva infecting mouses；
(3) humoral immunity regulation
Before 0 week after III phase larva infects infects, 1 week, 2 weeks, 3 weeks eyeball blood sampling collection mice serums, mice serum sample is obtained
Product, using Total IgG, IgG1, IgG2a and IgE antibody level, assessment in ELISA method detection different times mice serum
Humoral Immunological Regulation Roles；
Using Total IgG and IgE as index, compared with control group, if experimental group level increases, illustrate that PAS-5 albumen has
Immunological enhancement；
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, and capture antibody is 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 back-off after last time board-washing
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, suction is tipped upside down on after last time board-washing by orifice plate
Liquid in hole is all discarded on water paper；
5. standard items and sample are prepared：By standard items mouse Total IgG antibodies, IgG1 antibody, IgG2a antibody, IgE antibody difference
The concentration for being diluted to mouse Total IgG antibodies with deionized water be 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL,
6.25ng/mL、3.13ng/mL、1.56ng/mL；The concentration of mouse IgG1 antibody be 200ng/mL, 100ng/mL, 50ng/mL,
25ng/mL、12.5ng/mL、6.25ng/mL、3.13ng/mL；The concentration of mouse IgG2a antibody be 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, 100 μ L/HoleAdd to mark
In quasi- sample wells, if 3 multiple holes；
Meanwhile, mice serum sample is diluted with confining liquid, the volume ratio of dilution is mouse Total 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. primary antibody is incubated：100μL/HoleThe blood serum sample of dilution is added in sample well, and every group sets 3 multiple holes, while setting blank
Hole；
7. secondary antibody is incubated：50μL/HoleThe detection antibody of dilution is added in elisa plate, is incubated at room temperature 3h；
Wherein, the detection antibody of the dilution is anti-for the anti-mouse IgG monoclonal antibody or biotin-avidin of HRP marks are marked
Mouse IgE monoclonal antibodies, are diluted using confining liquid, and dilution volume ratio is confining liquid：Antibody=250:1；
8. board-washing：Discard liquid in hole, 300 μ L/HoleLavation buffer solution board-washing 4 times, suction is tipped upside down on 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 is incubated 15min；
10. terminate：100μL/Hole2M H2SO4Terminate liquid terminating reaction；
Finally elisa plate is placed in ELIASA, the absorbance value at 450nm wavelength is read, 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 is detected；
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 is raised, then it is assumed that PAS-5 albumen has immunological enhancement；
Concretely comprise the following steps：
1. mouse spleen lymphocyte is separated
Take spleen：Eyeball of mouse is taken into cervical dislocation after blood to put to death, is put into equipped with the beaker that percentage by volume is 75% ethanol, nothing
Solution takes mouse spleen under the conditions of bacterium；
Grinding：Mouse spleen is put into 1mL DMEM cell culture fluids and is cleaned, be fully ground, and constantly to tissue dropwise addition
DMEM nutrient solutions, until more than 95% cell is separated in tissue, during cell suspension carefully to move to aseptic centrifuge tube；
Centrifugation：1400rpm, room temperature centrifugation 5min；
Separation of lymphocytes：Supernatant is abandoned, 500 μ L Ack erythrocyte cracked liquids are added, 1min is stood, plus DMEM nutrient solutions are settled to
2mL；
Centrifugation：1400rpm, room temperature centrifugation 5min；
Filtering：Supernatant, plus 500 μ L DMEM re-suspended cells are abandoned, is filtered to another clean aseptic centrifuge tube through 200 mesh filter screens
In, during cleaning fluid to move to 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：Cell quantity in 4 block plaids of counted under microscope, according to C=N/4 × 10 × 103× 50 calculate cell
Concentration；
2. Secreted by Mouse Splenic cytokines measurement
SPL suspension is diluted to 1 × 10 with containing the DMEM that percentage by volume is 10%FBS6Individual cell/mL, is added to 96
In orifice plate, 100 μ L/Hole, it is subsequently adding angiostrongylus cantonensis PAS-5 protein 10s 0ng/mL, 100 μ L/HoleStimulate splenocyte；Set simultaneously
Canavalin ConA 100ng/mL, 100 μ L/HolePositive controls, sterilizing PBS 100 μ L/HoleNegative control group；In 5%CO2Culture
Cell conditioned medium is collected after 37 DEG C of culture 72h of case, cell conditioned medium sample is obtained, saved backup for cytokines measurement or -80 DEG C；
Using ELISA detection kit detection cell factor IFN-γ, IL-4, IL-5 and IL-10 level, concrete operation step
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 the μ L of every hole 100
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
12.49g Na2HPO4+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：Per the μ L of hole 200 2h is incubated at room temperature containing percentage by volume for the PBS of 10%FBS closes elisa plate；
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：Standard items recombinant murine IFN-γ, recombinant murine IL-4, recombinant murine IL-5, recombinant murine IL-10 are divided
7 concentration gradients, i.e. IFN-γ are not diluted to by multiple proportions with containing the PBS that percentage by volume is 10%FBS: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 that will be collected is with containing body
Product percentage carries out 1 for the PBS of 10%FBS:20 dilutions, 3 multiple holes of every group of setting, while setting blank control wells；
Incubate primary antibody：Standard item group, sample sets and blank control group are added in elisa plate per the μ L of hole 100,1h is incubated at room temperature；
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 detection antibody that 100uL dilutes and the enzyme marking reagent that SAv-HRP is marked per hole, be incubated at room temperature 1h；
Wherein, the detection antibody is respectively anti-mouse IFN-γ monoclonal antibody, the Biotinylate of Biotinylate marks
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 using containing the PBS that percentage by volume is 10%FBS during dilution, PBS：Detection 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, be placed in for lavation buffer solution by each board-washing
1min is stopped in elisa plate；
Colour developing：Add 100 μ L TMB nitrite ions per hole, lucifuge is incubated 15min at room temperature；
Terminating reaction：Add 50 μ L2M H per hole2SO4Terminate liquid；
Read plate：Elisa plate is placed in ELIASA, the absorbance value at 450nm wavelength is read；
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 is raised and/or CD4+CD25+PoxP3+The reduction of Treg cells, then illustrate PAS-5 albumen tool immunological enhancement；
Isolated SPL suspension is adjusted to 1.5 × 10 with DMEM7Individual cell/mL, is added to the μ L/ of 96 orifice plate 100Hole
In, operated in accordance with the following steps using detection kit：
1. 200 μ L Facs buffer solutions are added per hole, 1400rpm, room temperature centrifugation 5min abandon supernatant；
2. 25 μ L Facs buffer solutions are added to close 1min on ice per hole；
3. the mixtures of antibodies of 25 μ L dilutions is added to be incubated 30min on ice per hole, wherein, dilution volume ratio is buffered for Facs
Liquid：CD4-FITC：CD8-PerCP-Cy 5.5：CD25-APC=200：1：1：1；
4. 200 μ L Facs buffer solutions are added per hole, 1400rpm, room temperature centrifugation 5min abandon supernatant；
5. add the rupture of membranes of 100 μ L Fresh to fix buffer solution per hole, 30min is incubated on ice；
6. directly to 200 μ L cleaning fluids are added in hole, 1400rpm, room temperature centrifugation 5min abandon supernatant；
7. repeat step is 6.；
8. 50 μ L cleaning fluids, FoxP3-PE antibody are added to press cleaning fluid per hole：The volume ratio of FoxP3-PE is 100:1 is directly added into hole
Interior, 4 DEG C of lucifuges are incubated 30min；
9. 6. repeat step, cleans 2 times；
10. upper machine testing in streaming pipe is added after adding 100 μ L cleaning fluid re-suspended cells per hole；
(6) brain tissue HE dyeing
Taking Mice brain tissues carries out HE dyeing, and the micro- sem observation cerebral tissue pathology injury order of severity and inflammatory cell change, enter
One step demonstrate,proves the immunoregulation effect of PAS-5 albumen；If in the meningitis predilection site inflammatory cell infiltration of experimental mice
Showed increased, illustrates that PAS-5 albumen has immunological enhancement；
Step is as follows：
1. draw materials and fix
Take during the mass percent that fresh Mice brain tissues input prepares in advance is 4% paraformaldehyde fixer, make tissue, thin
The protein denaturation solidification of born of the same parents, to prevent cell postmortem autolysis or bacterium from decomposing, 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 the moisture in tissue block, then tissue block is placed in both molten
In alcohol, it is dissolved in again transparent in the clarifier dimethylbenzene of paraffin, the alcohol in tissue block is replaced out with dimethylbenzene；
3. waxdip embedding
Transparent tissue block is placed in the paraffin for having dissolved, the insulation of dewaxing case is put into；
Embedded after paraffin is completely immersed in tissue block：Container is prepared, the paraffin for having dissolved is poured into, 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. cut into slices and paster
Embedded wax stone is fixed on slicer, is thinly sliced, generally 5-8 μ m-thicks；The thin slice for cutting often gauffer,
Pressing in the water of heating is put into, then is attached on slide, put drying in 45 DEG C of insulating boxs；
5. dewaxing dyeing
The sample of drying is carried out into HE dyeing, to increase the heterochromia of tissue cellularity each several part, beneficial to observation；Haematine
H is a kind of basic-dyeable fibre, nucleus and intracellular ribosomes can be dyed into bluish violet；Yihong E is a kind of acid dyes, can carefully
Kytoplasm dyes red or pale red.Before dyeing, the paraffin in section is sloughed with dimethylbenzene, then via high concentration to low concentration wine
Essence, finally enters distilled water, you can dyeing；
HE dyeing courses are：
To enter during the section after distilled water 1-2s is put into brazilwood extract dyeing agent and dyeed 3min, running water has rinsed 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 makes section transparent through dehydration of alcohol, then through dimethylbenzene；
7. sealing
The transparent upper canada balsam of section drop, covered sealing after natural gum is slightly dry, are sticked into mark writing paper, section mark
This can be used, and carry out microscope observation.
6. the application of angiostrongylus cantonensis albumen PAS-5 according to claim 5, it is characterised in that：The step (2) in
Mode of infection infects for gavage.
7. the application of angiostrongylus cantonensis albumen PAS-5 according to claim 5, it is characterised in that：The step (3) in
Antibody level of serum is detected using eBioscience ELISA detection kits；Or, (3) middle confining liquid is containing body to the step
Product percentage is 1% Tween-20, the PBS of the bovine serum albumin(BSA) that percentage by volume is 10%.
8. the application of angiostrongylus cantonensis albumen PAS-5 according to claim 5, it is characterised in that：(4) 2. the step
Middle use BD Biosciences ELISA detection kits detection cell factor IFN-γ, IL-4, IL-5 and IL-10 level.
9. the application of angiostrongylus cantonensis albumen PAS-5 according to claim 5, it is characterised in that：The step (5) in
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 fluid：562574, batch 51-9008102.
10. the application of angiostrongylus cantonensis albumen PAS-5 according to claim 5, it is characterised in that：The step (6) in
Inflammatory cell is neutrophil leucocyte, eosinophil；Or, (6) middle meningitis predilection site is the ventricles of the brain, spider web to the step
Hypostegal cavity；Or, (6) 2. middle low concentration is respectively the step to the volumetric concentration of alcohol in high concentration：70% alcohol 30min；
90% alcohol 30min；95% alcohol 30min；100% alcohol 1h；100% alcohol 1h；Or, the step (6) 5. middle and high concentration
Volumetric concentration to low-concentration ethanol is respectively：100% alcohol 2min；95% alcohol 1min；80% alcohol 1min；75% alcohol
1min。
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| CN109306368A (en) * | 2018-08-03 | 2019-02-05 | 温州医科大学 | Application of the Thp-1 epicyte protein Annexin A2 in Ac-Gal-1 is apoptosis-induced |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1778848A2 (en) * | 2004-08-13 | 2007-05-02 | BASF Plant Science GmbH | Compositions and methods using rna interference for control of nematodes |
| CN104542505A (en) * | 2015-01-23 | 2015-04-29 | 温州医科大学 | In vitro culturing method for parasitic nematodes in parasitic stage |
| CN106546753A (en) * | 2016-11-25 | 2017-03-29 | 温州医科大学 | A kind of application of angiostrongylus cantonensis protein Gal-9 ectin 1 |
-
2016
- 2016-11-25 CN CN201611056508.9A patent/CN106729659A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1778848A2 (en) * | 2004-08-13 | 2007-05-02 | BASF Plant Science GmbH | Compositions and methods using rna interference for control of nematodes |
| CN104542505A (en) * | 2015-01-23 | 2015-04-29 | 温州医科大学 | In vitro culturing method for parasitic nematodes in parasitic stage |
| CN106546753A (en) * | 2016-11-25 | 2017-03-29 | 温州医科大学 | A kind of application of angiostrongylus cantonensis protein Gal-9 ectin 1 |
Non-Patent Citations (2)
| Title |
|---|
| 周燕蓉等: ""广州管圆线虫感染人体致脑损伤的机制分析"", 《川北医学院学报》 * |
| 闫兰竹等: ""广州管圆线虫蛋白酶体α5亚基基因免疫调节功能研究"", 《全国寄生虫病高峰论坛暨2016年浙江省热带病与寄生虫病学术年会论文汇编》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109306368A (en) * | 2018-08-03 | 2019-02-05 | 温州医科大学 | Application of the Thp-1 epicyte protein Annexin A2 in Ac-Gal-1 is apoptosis-induced |
| CN109306368B (en) * | 2018-08-03 | 2022-02-18 | 温州医科大学 | Application of Thp-1 cell membrane protein Annexin A2 in Ac-Gal-1 induced apoptosis |
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