CN110420322A - The DC vaccine for the trichina ES co-induction agent induction for preventing, treating and diagnosing for pigs trichina disease - Google Patents
The DC vaccine for the trichina ES co-induction agent induction for preventing, treating and diagnosing for pigs trichina disease Download PDFInfo
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- CN110420322A CN110420322A CN201910742898.2A CN201910742898A CN110420322A CN 110420322 A CN110420322 A CN 110420322A CN 201910742898 A CN201910742898 A CN 201910742898A CN 110420322 A CN110420322 A CN 110420322A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/002—Protozoa antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
Abstract
The DC vaccine for the trichina ES co-induction agent induction that the present invention relates to a kind of to prevent, treat and diagnose for pigs trichina disease, belongs to field of biotechnology.It is prepared including trichina ES, is separately added into camptothecine, Hypericum Chinense 0.61 μ g, 0.25 μ g, Trichinella sui DC vaccine construction, the separation of pig peripheral blood monocyte, the acquisition of attached cell, the acquisition of DC vaccine.Advantage, it is mature using trichina ES compound formulation induction DC using complete medium using pig peripheral blood monocyte, immunization therapy is carried out to the carnivorous pig of infection trichina.The trichina ES co-induction agent of use is camptothecine and hypericin, trichina secretion can be enhanced to the inducing action of DC cell, the present invention can be used for preventing, treating, diagnose pigs trichina disease, it promotes pork and imports and exports quality, it ensures food safety, pigs trichina disease economic loss caused by pig breeding industry is reduced, avoids being negatively affected by pigs trichina disease to food safety and pork international trade bring.
Description
Technical field
The invention belongs to field of biotechnology, the DC vaccine for preventing, treating and diagnosing for pigs trichina disease is referred in particular to.
Background technique
Pigs trichina disease not only influences people's health, also causes huge economic loss to pig breeding industry, and directly bring state
The negative prestige of border trade, current prevention and control situation propose new challenge to trichinous prevention and treatment.
With the development of international meat trade and tourist industry, and afforestation, the improvement for the ecological environments such as concede the land to forestry,
In addition living standards of the people improve, under the consumer factors Co stituation such as food diversification and excessive pursuit ecosystem product,
Trichinosis generally shows new feature in recent years: 1, the regional disease incidence for eating raw meat custom reduces, and eats the ground of cold cuts
Area's disease incidence increases；2, based on Local primitive exponent, infection sources diversification.Current prevention and control situation is proposed to trichinous prevention and treatment
New challenge.Albendazole, which is used as pig feed additive, has good prevention effect to pigs trichina disease, but acetysalicylic acid phenobarbital is rattled away
Medicament residue problem and its side effect of the azoles in pork are unclear, therefore wouldn't advocate that (such as acetysalicylic acid phenobarbital is rattled away using anthelmintic drug
Azoles) expelling parasite raising is carried out to pig, and international trichina committee member (ICT) can not agree with the way yet.
Antigen presenting cell (Antigen.presenting cells, APC) intake, processing and present antigen are that starting is suitable
Answering property immune response key link, Dendritic Cells (dendritic cell, DC) as now generally acknowledge in vivo functionality most
Strong professional antigen presenting cell, on the one hand can be by antigen submission and secrete cytokines starting, regulating cell are immunized and T is thin
The specific humoral immunity reaction that born of the same parents rely on, on the other hand also can induce regulatory T cells or remove corresponding T cell clone and
Have the function that immunosupress or inducing immune tolerance, it is now recognized that DC is the key that the connection innate immunity and adaptive immunity ring
Section also plays a crucial role in maintaining immunologic balance.In recent years, with the development of DC vaccine application study, DC
Vaccine has become one of the focus being concerned now.The type of DC vaccine mainly has the DC vaccine of antigen sensibilization, resists at present
DC vaccine and the DC vaccine of gene modification of former polypeptide sensitization etc..Compared to preceding two classes DC vaccine, the DC vaccine of gene modification has
More advantage, thus cause the concern of more and more scholars.Although there is more than ten of DC vaccine to grind in third stage in recent years
Study carefully the stage, greatly motivates the further exploitation of DC vaccine, also indicate that DC vaccine has broad application prospects.It there is no benefit at present
The pig DC vaccine induced with trichina ES compound formulation.
Summary of the invention
The present invention provide it is a kind of prevent, treat and diagnose for pigs trichina disease trichina ES co-induction agent induction
DC vaccine.
The technical solution adopted by the present invention is that: include the following steps:
(1), prepared by the agent of trichina ES co-induction
(1) prepared by trichina ES
The Wistar rat cervical dislocation of trichina 10 of oral infection 300 is put to death, and takes small intestine, reject adipose tissue and
Mesenterium, longitudinally splits small intestine, and the tap water undershoot intestinal lavage content of flowing takes 37 DEG C of 300 grams of small intestines added with 200U/ml mould
Small intestine is cleaned in the 2000ml physiological saline of element and 200U/ml streptomycin sulphate, artificial digestion method is collected Adult Antigens of Trichinella and counted
Number；Liquid 50ml containing 50,000 Adult Antigens of Trichinella is poured into the clean glass dish of a diameter 15cm, is stood
15min inhales and abandons supernatant liquid, cleans the Adult Antigens of Trichinella being collected into and is transferred in 15ml centrifuge tube, with containing dual anti-
After RPMI-1640 culture solution 1000rpm/min, 2min centrifuge washing 2 times, the trichina of centrifugation bottom of the tube will be deposited in after cleaning
Adult is transferred to 25cm3Tissue Culture Flask, 15ml culture medium, 37 DEG C, 5%CO2Incubator culture 12h, 12h after be centrifuged,
1000rpm/min, 2min collect centrifuged supernatant；It is centrifuged repeatedly supernatant, 9 DEG C, 6000rpm/min, 30min are concentrated into ES
Liquid becomes 8-10ml colourless liquid, as trichina ES, and colourless liquid is transferred in the centrifuge tube of 1.5mL, sealing, and -20
DEG C save；
(2) prepared by the agent of trichina ES co-induction
It is spare that every milliliter of ES is separately added into camptothecine, 0.61 μ g, 0.25 μ g of Hypericum Chinense；
Drug is configured to 200mg/mL according to storage concentration by camptothecine, hypericin configuration, and when use is diluted to
0.1ug/ml；
(2), Trichinella sui DC vaccine construction
(1) separation of pig peripheral blood monocyte:
The outer venous blood 10ml of pig ante-chamber is taken under aseptic condition, absorption upper serum is spare, and isometric PBS dilution is added
Blood will dilute peripheral blood 14ml with suction pipe and the 1.077A of NycoPrepTM containing 7ml separation of lymphocytes is added slowly along tube wall
In the centrifuge tube of liquid, 1800rpm is centrifuged 20min, and the Interphase cells being gently sucked out in centrifuge tube separation liquid layer with suction pipe are i.e. single
Nucleus is transferred in another sterile centrifugation tube, and PBS is gently blown and beaten uniformly, makes sufficiently to dilute, and 1500rpm is centrifuged 8min, washing
Twice, collecting monocytic cell；
(2) acquisition of attached cell
The RPMll640 liquid containing 10% fetal calf serum is used to suspend the monocyte obtained, piping and druming uniformly, takes 10ul cell
Suspension adds 90ul trypan blue, mixes well, and then adds in counting scale, carries out viable count, and Trypan Blue proves cell
Vigor > 95%,
Calculation formula: the big lattice total viable cell/4 × 104 × extension rate of cell number/L=4
Adjusting monocyte concentration is 2 × 106A/ml is added in 6 porocyte culture plates, every hole 2ml, in 5%CO237℃
Under the conditions of be incubated for, wash off non-adherent cell after 90min, the RPMI1640 without FCS be added and continues to be incubated for 30min, after 30min again
Secondary to wash off non-adherent cell, remaining cell is adherent monocyte；
(3) acquisition of DC vaccine
Complete medium (recombination colony stimulating factor (rmGSF) 160ng/ml+rmIL-4 50ng/ml+ is added in every hole
10% fetal calf serum) 2ml, culture plate is put into aseptic plastic bag, wraps up sack, cell is in 5%CO2It is cultivated under the conditions of 37 DEG C,
Culture hole is sufficiently washed with suction pipe after 7 days, cell suspension is drawn and is added in centrifuge tube, 1500rpm is centrifuged 8min, and it is heavy to collect centrifugation
It forms sediment；The centrifugation that complete medium culture obtains, changes culture medium on the 3rd day, and put english caterpillar ES co-induction agent 5ml on the 6th day,
7th day plus TNF-α 100u/ml, induced maturation, piping and druming mixed, and all suspension cells being collected into are Trichinella sui DC, hanged
Floating cell count, makes suspension cell concentration 2 × 106A/ml, as Trichinella sui DC vaccine；The every 100Kg weight usage amount of pig
For 1ml.
Advantages of the present invention utilizes trichina ES compound formulation using complete medium using pig peripheral blood monocyte
It induces DC mature, immunization therapy is carried out to the carnivorous pig of infection trichina.The trichina ES co-induction agent of use be by from
Icariin, podophyllotoxin, Kaempferol, chlorogenic acid, oleanolic acid, rheum emodin, Quercetin, Forsythoside, camptothecine, rheum officinale
It screens and obtains in 15 kinds of effective components of Chinese medicinal such as phenol, indigo red, indigo, hypericin, procyanidine, catnip oil, and confirm to like
Tree alkali and hypericin can enhance trichina secretion (ES) to the inducing action of DC cell, the present invention can be used for preventing, treating,
Pigs trichina disease is diagnosed, pork is promoted and imports and exports quality, ensure food safety, pigs trichina disease is reduced and is passed through caused by pig breeding industry
Ji loss avoids being negatively affected by pigs trichina disease to food safety and pork international trade bring.
Detailed description of the invention
Fig. 1 is the DC of ES co-induction agent induction and the DC figure without ES co-induction agent induction；
Fig. 2 is the mature DC streaming qualification figure of ES co-induction agent induction；
Fig. 3 is regulation figure of the trichina ES co-induction agent induction DC to pig blood cell factor；
Fig. 4 is hypericin concentration screening figure of the present invention；
Fig. 5 is camptothecine concentration screening figure of the present invention.
Specific embodiment
Include the following steps:
(1), prepared by the agent of trichina ES co-induction
(1) prepared by trichina ES
The Wistar rat cervical dislocation of trichina 10 of oral infection 300 is put to death, and takes small intestine, reject adipose tissue and
Mesenterium, longitudinally splits small intestine, and the tap water undershoot intestinal lavage content of flowing takes 37 DEG C of 300 grams of small intestines added with 200U/ml mould
Small intestine is cleaned in the 2000ml physiological saline of element and 200U/ml streptomycin sulphate, artificial digestion method is collected Adult Antigens of Trichinella and counted
Number；Liquid 50ml containing 50,000 Adult Antigens of Trichinella is poured into the clean glass dish of a diameter 15cm, is stood
15min inhales and abandons supernatant liquid, cleans the Adult Antigens of Trichinella being collected into and is transferred in 15ml centrifuge tube, with containing dual anti-
After RPMI-1640 culture solution 1000rpm/min, 2min centrifuge washing 2 times, the trichina of centrifugation bottom of the tube will be deposited in after cleaning
Adult is transferred to 25cm3Tissue Culture Flask, 15ml culture medium, 37 DEG C, 5%CO2Incubator culture 12h, 12h after be centrifuged,
1000rpm/min, 2min collect centrifuged supernatant；It is centrifuged repeatedly supernatant, 9 DEG C, 6000rpm/min, 30min are concentrated into ES
Liquid becomes 8-10ml colourless liquid, as trichina ES, and colourless liquid is transferred in the centrifuge tube of 1.5mL, sealing, and -20
DEG C save；
(2) prepared by the agent of trichina ES co-induction
It is spare that every milliliter of ES is separately added into camptothecine, 0.61 μ g, 0.25 μ g of Hypericum Chinense；
Drug is configured to 200mg/mL according to storage concentration by camptothecine, hypericin configuration, and when use is diluted to
0.1ug/ml；
(2), Trichinella sui DC vaccine construction
(1) separation of pig peripheral blood monocyte:
The outer venous blood 10ml of pig ante-chamber is taken under aseptic condition, absorption upper serum is spare, and isometric PBS dilution is added
Blood will dilute peripheral blood 14ml with suction pipe and the 1.077A of NycoPrepTM containing 7ml separation of lymphocytes is added slowly along tube wall
In the centrifuge tube of liquid, 1800rpm is centrifuged 20min, and the Interphase cells being gently sucked out in centrifuge tube separation liquid layer with suction pipe are i.e. single
Nucleus is transferred in another sterile centrifugation tube, and PBS is gently blown and beaten uniformly, makes sufficiently to dilute, and 1500rpm is centrifuged 8min, washing
Twice, collecting monocytic cell；
(2) acquisition of attached cell
The RPMll640 liquid containing 10% fetal calf serum is used to suspend the monocyte obtained, piping and druming uniformly, takes 10ul cell
Suspension adds 90ul trypan blue, mixes well, and then adds in counting scale, carries out viable count, and Trypan Blue proves cell
Vigor > 95%,
Calculation formula: the big lattice total viable cell/4 × 104 × extension rate of cell number/L=4
Adjusting monocyte concentration is 2 × 106A/ml is added in 6 porocyte culture plates, every hole 2ml, in 5%CO237℃
Under the conditions of be incubated for, wash off non-adherent cell after 90min, the RPMI1640 without FCS be added and continues to be incubated for 30min, after 30min again
Secondary to wash off non-adherent cell, remaining cell is adherent monocyte；
(3) acquisition of DC vaccine
Complete medium (recombination colony stimulating factor (rmGSF) 160ng/ml+rmIL-4 50ng/ml+ is added in every hole
L0% fetal calf serum) 2ml, culture plate is put into aseptic plastic bag, wraps up sack, cell is in 5%CO2It is cultivated under the conditions of 37 DEG C,
Culture hole is sufficiently washed with suction pipe after 7 days, cell suspension is drawn and is added in centrifuge tube, 1500rpm is centrifuged 8min, and it is heavy to collect centrifugation
It forms sediment；The centrifugation that complete medium culture obtains, changes culture medium on the 3rd day, and put english caterpillar ES co-induction agent 5ml on the 6th day,
7th day plus TNF-α 100u/ml, induced maturation, piping and druming mixed, all suspension cells being collected into, and suspension cell counts, and made to hang
Floating cell concentration is 2 × 106A/ml, as Trichinella sui DC vaccine；The every 100Kg weight usage amount of pig is 1ml.
Experimental example 1, Morphological Identification
1, difference inverted microscope identification: observation cell growth state and metamorphosis daily, shooting cell shines within the 7th day
Piece.
2, it scans electric border identification: the suspension cell of collection being centrifuged, removes 2/3 supernatant, again suspension cell；Draw cell
Suspension, it is careful to be added dropwise on the slide for having film again, it lies against and stands 30min in culture dish, be then added in culture dish
2.5% glutaraldehyde is immersed in slide wherein, and fixed 30min, 0.1M buffer sufficiently rinses, and the fixed 30min of 1% osmic acid delays
Fliud flushing rinsing, 30% alcohol start serial dehydration, the own pentyl ester transition 5min of acetic acid, and critical point drying takes out slide with conductive
Glue is on object sland, ion sputtering film coating；Electric border observation is scanned to take pictures.
Transmit electric border identification: cell is centrifuged to obtain cell mass, and 4% glutaraldehyde is fixed 2h (preceding fixation), and 0.1M phosphoric acid is slow
Fliud flushing rinsing.2h, the rinsing of 0.1M phosphate buffer are fixed after 1% osmic acid, 30% ethyl alcohol starts serial dehydration；Acetone, resin are set
It changes and is impregnated with, embed and polymerize, repair block slice, acetic acid uranium, lead citrate dyeing；Electric border observation is transmitted to take pictures.Such as Fig. 1 (B, D institute
Showing) surface the DC dendritic processes of ES co-induction agent induced maturation are long and more, and there are a large amount of folds, shows thickness, size, length
The different dendroid such as short, some protrusions are more straight, and some comparison bendings are covered with entire cell peripheral, and cell surface is rough,
It is rough and uneven in surface.Meet the morphological feature of typical maturation DC.
If Fig. 1 (shown in A, C) is the immature DC cell induced without inducer.
3, DC vaccine streaming is identified
With the expression (Fig. 2 E, B) of Flow cytometry maturation DC and immature DC cell CD86, CD11c is marrow source property
The characteristic surface molecule of DC, CD11c+ shows that extracted cell is marrow source property, rather than derives from lymphatic system.Not at
The all high expression CD11c (Fig. 2A, D) of ripe and mature DC, and mature DC than immature expression MHC-II (Fig. 2 F, C) and
The amount of CD86 is higher.
2. pigs trichina disease immunization therapy of experimental example
1.ES answers inducer induction DC vaccine worm reduction rate
Health pig 40 are infected respectively by 2000 trichinas (trichina international numbering ISS534)/head, respectively with physiology
Salt water, DC, ES and ES answer inducer induction DC vaccine therapy and infect trichina pig each 10；The every 100Kg weight usage amount of pig is
1ml；
Immunization strategy: after infection, the 0th day after infection, the 2nd day single times of immunizing dose DC vaccine immunity is all controls
Treat pig, the 7th day doubling dosage immunization therapy pig, the 13rd day, the 17th day doubling dosage immunization therapy pig；
It is cutd open after 18 days and kills each group pig, each group pig muscle and enteron aisle trichina are collected with digestion method and counted, and by following
Formula, which calculates worm reduction rate, the results are shown in Table 1.
Worm reduction rate=(saline therapy group trichina average-treatment group's trichina average)/saline therapy
Group trichina average
1 ES of table answers inducer induction DC vaccine worm reduction rate
| Group | Physiological saline | DC | ES | ES answers inducer induction DC |
| Worm reduction rate (%) | 28.53 | 35.71 | 44.82 | 48.41 |
2. trichina ES co-induction agent induces regulation of the DC to pig blood cell factor
Health pig 40 are infected respectively by 2000 trichinas (trichina international numbering ISS534)/head, respectively with physiology
Salt water, DC, ES and ES answer inducer induction DC vaccine therapy and infect trichina pig each 10, and the every 100Kg weight usage amount of pig is
1ml；It is right respectively at vena cava anterior blood sampling in 3,6,9,12,15,18,21,24,27 days according to eBioscience kit specification
The situation of change of cell factor in serum carries out ELISA detection.
Efficacy evaluation:
The situation of change of various cell factors is as shown in Figure 4.Represent IL-6 (Fig. 3 B), the IL-4 of trichina immune response
The expression quantity of (Fig. 3 C) and IL-10 (Fig. 3 D) are lowered, IFN-γ (Fig. 3 A) up-regulation.
3. hypericin of experimental example and camptothecine concentration screening
By hypericin and camptothecine according to 8 μ g/mL, 1.6 μ g/mL, 0.32 μ g/mL, 0.064 μ g/mL, six concentration groups
Wind not and porcine fetus fibroblasts (G418) effect, each concentration be arranged for 24 hours, tri- time groups of 48h, 72h, and solvent is set
Control wells and blank well, every group sets 3 parallel holes.By the drug concentration for having 5% toxicity to cell, (i.e. cell survival rate is 95%
When drug concentration) as the drug to the basic non-toxic concn of cell, with for 24 hours when this concentration carry out follow-up test.Cell
Survival rate=(OD value experimental group-OD is worth control group)/(OD value control group-OD is worth blank group) × 100%.As a result see Fig. 4 and figure
5。
Claims (4)
1. a kind of DC vaccine for the trichina ES co-induction agent induction for preventing, treating and diagnosing for pigs trichina disease, special
Sign is: including the following steps:
(1), prepared by the agent of trichina ES co-induction
(1) prepared by trichina ES
The Wistar rat cervical dislocation of trichina 10 of oral infection 300 is put to death, and small intestine is taken, and rejects adipose tissue and intestines system
Film, longitudinally splits small intestine, the tap water undershoot intestinal lavage content of flowing, take 37 DEG C of 300 grams of small intestines added with 200U/ml penicillin and
Small intestine is cleaned in the 2000ml physiological saline of 200 U/ml streptomycin sulphates, artificial digestion method is collected Adult Antigens of Trichinella and counted；
Liquid 50ml containing 50,000 Adult Antigens of Trichinella is poured into the clean glass dish of a diameter 15cm, stands 15min,
It inhales and abandons supernatant liquid, clean the Adult Antigens of Trichinella that is collected into and be simultaneously transferred in 15ml centrifuge tube, with containing dual anti-RPMI-1640
After culture solution 1000rpm/min, 2min centrifuge washing 2 times, the Adult Antigens of Trichinella that centrifugation bottom of the tube is deposited in after cleaning is shifted
To 25cm3Tissue Culture Flask, 15ml culture medium, 37 DEG C, 5%CO2Incubator culture 12h, 12h after be centrifuged, 1000rpm/min,
2min collects centrifuged supernatant；It is centrifuged repeatedly supernatant, 9 DEG C, 6000rpm/min, 30min, being concentrated into ES liquid becomes 8-
10ml colourless liquid, as trichina ES, colourless liquid is transferred in the centrifuge tube of 1.5mL, sealing, -20 DEG C of preservations；
(2) prepared by the agent of trichina ES co-induction
It is spare that every milliliter of ES is separately added into camptothecine, 0.61 μ g, 0.25 μ g of Hypericum Chinense；
(2), Trichinella sui DC vaccine construction
(1) separation of pig peripheral blood monocyte:
The outer venous blood 10ml of pig ante-chamber is taken under aseptic condition, absorption upper serum is spare, and isometric PBS diluted blood is added
Liquid will dilute peripheral blood 14ml with suction pipe and the 1.077A of NycoPrepTM containing 7ml lymphocyte separation medium is added slowly along tube wall
Centrifuge tube in, 1800rpm be centrifuged 20min, be gently sucked out with suction pipe centrifuge tube separation liquid layer in the i.e. single core of Interphase cells
Cell is transferred in another sterile centrifugation tube, and PBS is gently blown and beaten uniformly, makes sufficiently to dilute, and 1500rpm is centrifuged 8min, washing two
It is secondary, collecting monocytic cell；
(2) acquisition of attached cell
The RPMll640 liquid containing 10% fetal calf serum is used to suspend the monocyte obtained, piping and druming uniformly, takes 10ul cell suspension
Add 90ul trypan blue, mix well, then adds in counting scale, progress viable count, Trypan Blue proof cell viability >
95%,
Calculation formula: the big lattice total viable cell/4 × 104 × extension rate of cell number/L=4
Adjusting monocyte concentration is 2 × 106A/ml is added in 6 porocyte culture plates, every hole 2ml, in 5%CO237 DEG C of conditions
Non-adherent cell is washed off in lower incubation after 90min, and the RPMI1640 without FCS is added and continues to be incubated for 30min, washes again after 30min
Fall non-adherent cell, remaining cell is adherent monocyte；
(3) acquisition of DC vaccine
Complete medium 2ml is added in every hole, and culture plate is put into aseptic plastic bag, wraps up sack, cell is in 5%CO237 DEG C of conditions
Lower culture sufficiently washed culture hole with suction pipe after 7 days, draws cell suspension and is added in centrifuge tube, and 1500rpm is centrifuged 8min, receives
Collect centrifugation；The centrifugation that complete medium culture obtains, changes culture medium on the 3rd day, putting english within the 6th day, caterpillar ES is compound to be lured
Agent 5ml is led, the 7th day plus TNF-α 100u/ml, induced maturation, piping and druming mixed, and all suspension cells being collected into are pig rotation hair
Worm DC, suspension cell count, and make suspension cell concentration 2 × 106A/ml, as Trichinella sui DC vaccine.
2. a kind of trichina ES co-induction for preventing, treating and diagnosing for pigs trichina disease according to claim 1
The DC vaccine of agent induction, it is characterised in that: (2) trichina ES prepared by step (1), trichina ES co-induction agent is compound to be lured
It leads in agent preparation, camptothecine, hypericin configuration, drug is configured to 200mg/mL according to storage concentration, when use is diluted to
0.1ug/ml。
3. a kind of trichina ES co-induction for preventing, treating and diagnosing for pigs trichina disease according to claim 1
Agent induction DC vaccine, it is characterised in that: step (2), Trichinella sui DC vaccine construction (3) DC vaccine acquisition in, every hole
The complete medium of addition is: recombination colony stimulating factor (rmGSF) 160ng/ml+rmIL-4 50ng/ml+10% tire ox blood
Clearly.
4. a kind of trichina ES co-induction for preventing, treating and diagnosing for pigs trichina disease according to claim 1
The DC vaccine of agent induction, it is characterised in that: the every 100Kg weight usage amount of pig is 1ml.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113186160A (en) * | 2021-04-25 | 2021-07-30 | 大连大学 | Continuous cell line of porcine DC cells and establishment method and application thereof |
| CN117018018A (en) * | 2023-10-09 | 2023-11-10 | 吉林大学 | Application of yeast-derived beta-glucan in preparation of medicines for preventing, treating or assisting in treating trichinosis |
| CN117050980A (en) * | 2023-10-12 | 2023-11-14 | 吉林大学 | Application of Ts-TTPA antigen in preparation of early diagnosis reagent for trichinosis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5541075A (en) * | 1993-02-05 | 1996-07-30 | Heska Corporation | Carbohydrate-based vaccine and diagnostic reagent for trichinosis |
| US20210393681A1 (en) * | 2020-06-22 | 2021-12-23 | Therapeutic Solutions International, Inc. | Treatment of SARS-CoV-2 with Dendritic Cells for Innate and/or Adaptive Immunity |
-
2019
- 2019-08-12 CN CN201910742898.2A patent/CN110420322B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5541075A (en) * | 1993-02-05 | 1996-07-30 | Heska Corporation | Carbohydrate-based vaccine and diagnostic reagent for trichinosis |
| US20210393681A1 (en) * | 2020-06-22 | 2021-12-23 | Therapeutic Solutions International, Inc. | Treatment of SARS-CoV-2 with Dendritic Cells for Innate and/or Adaptive Immunity |
Non-Patent Citations (5)
| Title |
|---|
| JING DING ET AL.: "Murine hepatoma treatment with mature dendritic cells stimulated by Trichinella spiralis excretory/secretory products", 《PARASITE》 * |
| ROBINSON K ET AL.: "Vaccination against the nematode Trichinella spiralis in high- and low-responder mice. Effects of different adjuvants upon protective immunity and immune responsiveness", 《IMMUNOLOGY.》 * |
| 刘菁: "旋毛虫ES抗原诱导DC对肝细胞癌预防性作用的实验研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 周玲等主编: "《感染性疾病的处置策略与防治》", 30 June 2017, 吉林科学技术出版社 * |
| 路丽群: "旋毛虫病ELISA和PCR诊断方法的建立", 《万方数据知识服务平台》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113186160A (en) * | 2021-04-25 | 2021-07-30 | 大连大学 | Continuous cell line of porcine DC cells and establishment method and application thereof |
| CN117018018A (en) * | 2023-10-09 | 2023-11-10 | 吉林大学 | Application of yeast-derived beta-glucan in preparation of medicines for preventing, treating or assisting in treating trichinosis |
| CN117050980A (en) * | 2023-10-12 | 2023-11-14 | 吉林大学 | Application of Ts-TTPA antigen in preparation of early diagnosis reagent for trichinosis |
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