CN106237322A - DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine and preparation method thereof - Google Patents

DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine and preparation method thereof Download PDF

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CN106237322A
CN106237322A CN201610584707.0A CN201610584707A CN106237322A CN 106237322 A CN106237322 A CN 106237322A CN 201610584707 A CN201610584707 A CN 201610584707A CN 106237322 A CN106237322 A CN 106237322A
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plga
mannose
inactivated vaccine
nanometer
ear disease
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王钦富
刘欢
胡亚萍
王鹏
张正
张正一
王美辰
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Dalian University
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Dalian University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N2770/10011—Arteriviridae
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Abstract

The present invention relates to biovaccine technical field, specially DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine and preparation method thereof, in terms of 10 part vaccines, including following material: PRRSV TCID50=5 × 105‑5×107, mannose 1 20wt%, PLGA 0.01 5wt%, Poly (I:C) 0.01 5wt%.The preparation method of this vaccine mainly includes that two steps: S1 prepares PLGA nanometer adjuvant inactivated vaccine；The PLGA nanometer Adjuvanted vaccines of S2 synthesis mannose-modified.Mannose residue is utilized to modify PLGA, it is intended to build the induction system of the targeting vaccine with the dendritic cell (DC) of mannose receptor, macrophage；Add Poly (I:C) to be to strengthen immunity pig immunity, strengthen cellullar immunologic response；Using non-toxic degradable material PLGA is that carrier prepares nano vaccine, it is therefore intended that the release time delaying virus, the toxic reducing medicine, the half-life of prolongation medicine.

Description

DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine and preparation method thereof
Technical field
The present invention relates to a kind of vaccine and preparation method thereof, specially DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivation epidemic disease Seedling and preparation method thereof, belongs to biovaccine technical field.
Background technology
Pig blue-ear disease, also known as Porcine reproductive and respiratory syndrome (PRRS), is to be caused by PRRS virus (PRRSV), numerous with sow Grow obstacle and high degree in contact that piglet respiratory system disease is principal character, high fatality rate deadly infectious disease.From the U.S. 1987 Since year is reported, oneself is through each main pig-raising countries that spreads all over the world, the animal A circulated a notice of by World Organization for Animal Health (OIE) in recent years One of class deadly infectious disease, for high incidence and the serious infectious diseases of high mortality.Rising since in June, 2006, one is referred to as The epidemic disease of " hyperpyrexia disease " or " innominate high fever disease " is that calamity is broken out on some pig farms of south China with varying number and scale, with After almost sweep over the country, within 2006, enter Winter Solstice 2007, " porcine hyperthermia " by acute transfer to chronic sustained infect, mainly encroach on Sow and piglet.So far this disease the most constantly occurs, and is the most scabrous great epidemic disease in current pig farm.The generation of pig blue-ear disease Causing the biggest economic loss to China's aquaculture, there is presently no effective medicine can be to its thorough healing, to sow and son It is to control the most cost-effective method of pig blue-ear disease that pig carries out immunoprophylaxis, but existing pig blue-ear disease inactivated vaccine and attenuation Live vaccine immunne response is slower and effect is undesirable.
Summary of the invention
For making up the deficiencies in the prior art, the present invention provides a kind of novel DC targeting pig blue-ear disease PLGA nanometer adjuvant to go out Live vaccine and preparation method thereof, can effective prevention and control PRRS virus, solve the economic loss that reproductive and respiratory syndrome comes to cultivation industrial belt. Technical scheme is as follows:
A kind of DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine (10 parts), including following material: PRRSV TCID50=5 × 105-5×107, mannose 1-20wt%, PLGA0.01-5wt%, Poly (I:C) 0.01-5wt%, PLGA are Poly(D,L-lactide-co-glycolide well known in the art.
The present invention is claimed the preparation method of described DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine simultaneously, The method comprises the steps:
S1. PLGA nanometer adjuvant inactivated vaccine is prepared；
S2. the PLGA nanometer Adjuvanted vaccines of mannose-modified is synthesized.
Further, described step S1 is:
A. prepare W2 phase: PVA is added to the water, all dissolve；
B. prepare O phase: PLGA and join in dichloromethane, all dissolve；
C. prepare W1 phase: Poly (I:C) to mix with PRRSV, dissolve fully；
D. W1 is added in O phase, carries out ultrasonic Treatment and obtain mixture I；
E. mixture I is joined in W2 phase, remove dichloromethane after carrying out ultrasonic emulsification and obtain mixture II；
F. mixture II is centrifuged off supernatant, retains precipitation and be PLGA nanometer Adjuvanted vaccines.Further, described Step S2 be:
A. be separately added in the PLGA nanometer Adjuvanted vaccines that step S1 obtains 1-10wt%N-N-Hydroxysuccinimide and 1-10wt% dicyclohexylcarbodiimide solution, mix homogeneously；
B. the liquid of step a mix homogeneously is placed in bag filter dialysis 6-8 hour, removes unnecessary N-hydroxysuccinimidyl acyl Imines, dicyclohexylcarbodiimide；
C. in the liquid after dialysis, add ethylenediamine, regulate pH to 5.0 with concentrated hydrochloric acid, form ammonium PLGA；
D. 1-20wt% mannose is dissolved in 0.1mol/L sodium acetate solution, mannose-sodium acetate solution is added In the ammonium PLGA that step c obtains, process 2 days 37 DEG C of concussions；
E. the liquid after step d being processed is centrifuged, and is precipitated, i.e. obtains mannose by brine, resuspended precipitation The PLGA nanometer Adjuvanted vaccines modified.
Further, in described W2 phase, the mass fraction of PVA is 0.25-1.25%.
Further, the condition of described ultrasonic emulsification is: time 110s, 0.6s/ time, 55%.
PLGA is nontoxic biodegradable polymer, the U.S. by FDA approval listing, product for pharmaceutic adjuvant, Bio-medical material.Including medicinal slow release agent, controlled release preparation carrier (including coronary artery bracket medicine carrying material), such as amycin slow release Agent, many peptides biodegradation sustained-release micro-spheres etc., it is also used for multiple vaccine carrier, such as HIV, hepatitis B, foot and mouth disease etc..Poly(I:C) Chinese name is polyinosinic acid, confirms for the copolymer of poly, pharmacological research and clinical position, and polyinosini is timely Efficient endogenous interferon derivant, is again immunomodulator, has broad-spectrum antiviral, antitumor action, stimulation phagocytosis work With with enhancing human body immunity function.It addition, experimental results demonstrate that mannose can combine the manna of dendritic cell with targeting Saccharide acceptor.Therefore this problem is intended using mannose-modified PLGA nanometer adjuvant, add Poly (I:C) and assist a ruler in governing a country PRRSV, accelerate and strengthen Cellullar immunologic response to PRRSV, to effective prevention and control pig blue-ear disease poison, solves the economic damage that reproductive and respiratory syndrome comes to cultivation industrial belt Lose.
Beneficial effects of the present invention is as follows: the present invention utilizes mannose residue to modify PLGA, is aided with Poly (I:C) for stimulating Agent, prepares DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine.Mannose residue is utilized to modify PLGA, it is intended to build and have The dendritic cell (DC) of mannose receptor, the induction system of targeting vaccine of macrophage；Add Poly (I:C) to be to increase Strong immunity pig immunity, strengthens cellullar immunologic response；Using non-toxic degradable material PLGA is that carrier prepares nano vaccine, mesh Be delay virus release time, reduce medicine toxic, extend medicine half-life.
Accompanying drawing explanation
Fig. 1 is the nanometer Adjuvanted vaccines laser particle size testing result figure of the present invention；
Fig. 2 is the viral releasing curve diagram of the nanometer Adjuvanted vaccines of the present invention；
Fig. 3 is the nanometer Adjuvanted vaccines infrared spectrum detection figure of the present invention；
Fig. 4 is that Phenol-sulphate acid method of the present invention surveys polyoses content canonical plotting；
Fig. 5 is the nanometer Adjuvanted vaccines lymphopoiesis result figure of the present invention；
Fig. 6 is the nanometer Adjuvanted vaccines ELISPOT testing result figure of the present invention.
Wherein: 1, mannose-modified PLGA nanometer Adjuvanted vaccines, 2, PLGA nanometer Adjuvanted vaccines.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention will be further described, if no special instructions, raw materials used the most commercially available can , as preferably, edible sodium bicarbonate is purchased from Shandong Haitian Biochemical Co., Ltd..DC targeting is prepared according to such as table 1 below formula Pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine:
Table 1
Embodiment 1
Preparation PLGA nanometer Adjuvanted vaccines
(1) W2 phase: with PVA (polyvinyl alcohol) solution of 30mL distilled water preparation 0.25wt%, put into rotor, use sealed membrane Sealing, is placed in constant-temperature heating magnetic stirring apparatus, and constant-temperature heating magnetic stirring apparatus added water no iron ring, regulated suitable rotating speed, Regulation voltage is maximum, and after water seethes with excitement, regulation voltage is gradually reduced, until PVA takes out after all dissolving.
(2) O phase: take 5mL centrifuge tube, adds 2mL dichloromethane, weighs 0.01wt%PLGA (polylactic-co-glycolic acid) and adds Enter in dichloromethane, turbula shaker shakes, until PLGA all dissolves.
(3) W1 phase: weigh 0.01wt%Poly (I:C) (polyinosinic acid-polycytidylicacid) and put in 1mL centrifuge tube, with TCID50=5 × 105PRRSV (high-pathogenicity porcine reproductive and respiration syndrome inactivation of viruses) mixes, mixed system is joined from In heart pipe, concussion is dissolved.
(4) material in W1 phase being joined in O phase, put in ice chest, W1/O is in new sesame Ultrasound Instrument 1min, amplitude 80%, 2s.
(5) material in W1/O phase is joined in W2 phase, put in ice chest, W1/O/W2 ultrasonic emulsification 110s (UP400 Type Ultrasound Instrument, amplitude 55%, 0.6s).
(6) liquid of aspiration step (5) ultrasonic emulsification is on microscope slide, observes under inverted microscope, and W1/O/W2 phase is put In constant-temperature heating magnetic stirring apparatus, 3h is stirred at room temperature to remove dichloromethane.
(7) W1/O/W2 phase is incorporated in centrifuge tube, and 10000rpm is centrifuged 15min, abandons supernatant, with distilled water wash, by upper State step cyclic washing three times.
The PLGA nanometer Adjuvanted vaccines of synthesis mannose-modified
(1) weigh 1wt%N-N-Hydroxysuccinimide and 1wt% dicyclohexylcarbodiimide dissolve with distilled water respectively, Add in PLGA nanometer Adjuvanted vaccines and mix.
(2) mixing is placed in bag filter dialysis 6h, removes unnecessary N-hydroxy-succinamide, dicyclohexyl carbon two sub- Amine.
(3) taking out bag filter, poured into by liquid in centrifuge tube, add 96 μ L ethylenediamines, now PH is 9.0, uses concentrated hydrochloric acid Regulate its PH to 5.0, form ammonium PLGA.
(4) 1wt% mannose is dissolved in the 0.1mol/L sodium acetate solution of 1mL, adds in above-mentioned solution, 37 DEG C of shakes Swing 2 days.
(5) above-mentioned solution is taken out centrifugal 8000rpm, 10min, abandon supernatant, with brine, repeated washing two Secondary.Finally will precipitate resuspended with normal saline, for the PLGA nanometer Adjuvanted vaccines of mannose-modified.
Embodiment 2
Preparation PLGA nanometer Adjuvanted vaccines
(1) W2 phase: with PVA (polyvinyl alcohol) solution of 30mL distilled water preparation 0.75wt%, put into rotor, use sealed membrane Sealing, is placed in constant-temperature heating magnetic stirring apparatus, and constant-temperature heating magnetic stirring apparatus added water no iron ring, regulated suitable rotating speed, Regulation voltage is maximum, and after water seethes with excitement, regulation voltage is gradually reduced, until PVA takes out after all dissolving.
(2) O phase: take 5mL centrifuge tube, adds 2mL dichloromethane, weighs 0.5wt%PLGA (polylactic-co-glycolic acid) and adds Enter in dichloromethane, turbula shaker shakes, until PLGA all dissolves.
(3) W1 phase: weigh 0.1wt%Poly (I:C) (polyinosinic acid-polycytidylicacid) and put in 1mL centrifuge tube, with TCID50=5 × 106PRRSV (high-pathogenicity porcine reproductive and respiration syndrome inactivation of viruses) mixes, mixed system is joined from In heart pipe, concussion is dissolved.
(4) material in W1 phase being joined in O phase, put in ice chest, the W1/O new sesame ultrasonic 1min of Ultrasound Instrument, 80% shakes Width, 2s.
(5) material in W1/O phase is joined in W2 phase, put in ice chest, W1/O/W2 ultrasonic emulsification 110s (UP400 Type Ultrasound Instrument 55% amplitude, 0.6s).
(6) liquid of aspiration step (5) ultrasonic emulsification is on microscope slide, observes under inverted microscope, and W1/O/W2 phase is put In constant-temperature heating magnetic stirring apparatus, 3h is stirred at room temperature to remove dichloromethane.
(7) W1/O/W2 phase is incorporated in centrifuge tube, and 10000rpm is centrifuged 15min, abandons supernatant, with distilled water wash, by upper State step cyclic washing three times.
The PLGA nanometer Adjuvanted vaccines of synthesis mannose-modified
(8) weigh 3.5wt%N-N-Hydroxysuccinimide and 4.5wt% dicyclohexylcarbodiimide is water-soluble with distillation respectively Solve, add in PLGA nanometer Adjuvanted vaccines and mix.
(9) mixing is placed in bag filter dialysis 6h, removes unnecessary N-hydroxy-succinamide, dicyclohexyl carbon two sub- Amine.
(10) taking out bag filter, poured into by liquid in centrifuge tube, add 96 μ L ethylenediamines, now PH is 9.0, uses concentrated hydrochloric acid Regulate its pH to 5.0, form ammonium PLGA.
(11) 5wt% mannose is dissolved in the 0.1mol/L sodium acetate solution of 1mL, adds in above-mentioned solution, 37 DEG C Shake 2 days.
(12) above-mentioned solution is taken out centrifugal 8000rpm, 10min, abandon supernatant, with brine, repeated washing two Secondary.Finally will precipitate resuspended with normal saline, for the PLGA nanometer Adjuvanted vaccines of mannose-modified.
Embodiment 3
Preparation PLGA nanometer Adjuvanted vaccines
(1) W2 phase: with PVA (polyvinyl alcohol) solution of 30mL distilled water preparation 1.25wt%, put into rotor, use sealed membrane Sealing, is placed in constant-temperature heating magnetic stirring apparatus, and constant-temperature heating magnetic stirring apparatus added water no iron ring, regulated suitable rotating speed, Regulation voltage is maximum, and after water seethes with excitement, regulation voltage is gradually reduced, until PVA takes out after all dissolving.
(2) O phase: take 5mL centrifuge tube, adds 2mL dichloromethane, weighs 5wt%PLGA (polylactic-co-glycolic acid) and adds In dichloromethane, turbula shaker shakes, until PLGA all dissolves.
(3) W1 phase: weigh 5wt%Poly (I:C) (polyinosinic acid-polycytidylicacid) and put in 1mL centrifuge tube, with TCID50 =5 × 107PRRSV (high-pathogenicity porcine reproductive and respiration syndrome inactivation of viruses) mixes, and mixed system is joined centrifuge tube In, concussion is dissolved.
(4) material in W1 phase being joined in O phase, put in ice chest, the W1/O new sesame ultrasonic 1min of Ultrasound Instrument, 80% shakes Width, 2s.
(5) material in W1/O phase is joined in W2 phase, put in ice chest, W1/O/W2 ultrasonic emulsification 110s (UP400 Type Ultrasound Instrument 55% amplitude, 0.6s).
(6) liquid of aspiration step (5) ultrasonic emulsification is on microscope slide, observes under inverted microscope, and W1/O/W2 phase is put In constant-temperature heating magnetic stirring apparatus, 3h is stirred at room temperature to remove dichloromethane.
(7) W1/O/W2 phase is incorporated in centrifuge tube, and 10000rpm is centrifuged 15min, abandons supernatant, with distilled water wash, by upper State step cyclic washing three times.
The PLGA nanometer Adjuvanted vaccines of synthesis mannose-modified
(8) weigh 6wt%N-N-Hydroxysuccinimide and 8wt% dicyclohexylcarbodiimide dissolve with distilled water respectively, Add in PLGA nanometer Adjuvanted vaccines and mix.
(9) mixing is placed in bag filter dialysis 6h, removes unnecessary N-hydroxy-succinamide, dicyclohexyl carbon two sub- Amine.
(10) taking out bag filter, poured into by liquid in centrifuge tube, add 96 μ L ethylenediamines, now pH is 9.0, uses concentrated hydrochloric acid Regulate its pH to 5.0, form ammonium PLGA.
(11) 20wt% mannose is dissolved in the 0.1mol/L sodium acetate solution of 1mL, adds in above-mentioned solution, 37 DEG C Shake 2 days.
(12) above-mentioned solution is taken out centrifugal 8000rpm, 10min, abandon supernatant, with brine, repeated washing two Secondary.Finally will precipitate resuspended with normal saline, for the PLGA nanometer Adjuvanted vaccines of mannose-modified.
The detection of embodiment 4-nanometer particle size
Take the PLGA nanometer Adjuvanted vaccines of above-mentioned mannose-modified.Measure particle diameter and distribution with laser particle size analyzer, see Fig. 1.Laser particle analyzer detects, and DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine granularity is 434.8nm.
Embodiment 5-envelop rate, drug loading measure
Envelop rate (EE%)=WBag/WMedicine× 100%
Drug loading (DL%)=WBag/WAlways× 100%
W in formulaMedicine: total dose (mg) of input
WAlways: the gross weight (mg) of nanoparticle
WBag: the medication amount (mg) of parcel in nanoparticle
Result: envelop rate is about 46.9%, and drug loading is about 6.36%.
The safety of embodiment 6-detects
Giving 50 above-mentioned samples of mice by intraperitoneal injection, normal raising one month respectively, observe its upgrowth situation, result is not Performance body temperature, search for food, the abnormal response of aspect such as spirit.
Embodiment 7-bicinchoninic acid method (BCA method) protein determination
(1) it is heavy that DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine embodiment 1 obtained obtains after being centrifuged 3 times Shallow lake adds distilled water, is settled to 10ml.
(2) take 3 1.5ml centrifuge tubes, each addition step (1) gained solution 0.5ml, be centrifuged 10000rpm, 10min, Remove supernatant, each addition 0.1mol/L NaOH 1ml, dispel precipitation, be placed in 37 DEG C of shaking table 1h, BCA method measures.
(3) take 3 1.5mL centrifuge tubes, numbering 1,2,3, each addition step (2) gained solution 1.5ml, be placed in 37 DEG C and shake Bed, takes 80 microlitres every day, centrifugal 10000rpm, and 10min, BCA method measures supernatant protein content, observes virus release profiles, knot Fruit sees Fig. 2.
The infrared spectrum analysis of embodiment 8-
(1) DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine lyophilization embodiment 1 obtained processes.
(2) potassium bromide and DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine lyophilized powder are ground until powdery.
(3) powder compressing machine aerofluxus: first handwheel is regulated pressure leading screw very good during use, and by fuel outlet valve handwheel up time Pin is twisted, the tension do not twisted, and swings up and down hands handle, stops pressurization after gauge hand moves upward, and pressure is the most too high, Aerofluxus sound can be heard, then tightened by air bleeding valve that the most open fuel tap handwheel unclamps pressure leading screw after unclamping air bleeding valve.
(4) mould is placed on tablet machine work space middle position, swings up and down hands handle.Observe Pressure gauge indicating value to read simultaneously Number, stops pressurization after reaching desirable pressure, keeps 1min, take out the sample suppressed.
(5) record data, and finishing analysis, are shown in Fig. 3.
Embodiment 9-Phenol-sulphate acid method surveys polyoses content
Precision measures D-MANNOSE reference substance solution 50 μ g/ml 0ml, 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, Put respectively in tool plug test tube, respectively add water to 1ml, then add 3% phenol solution 1ml, shake up, be rapidly added sulphuric acid 4.5ml, shake up, Placing to room temperature, with 0 pipe as blank, scan between 200-650nm wavelength, D-MANNOSE derivant has maximum at 490nm Absorb, therefore select 490nm for detection wavelength.Absorbance, with reference substance concentration (x) as abscissa, absorbance is measured at 490nm Y () is vertical coordinate, carry out linear regression, and obtaining regression equation is y=16.284x-0.0685 (r=0.9991).Result shows D- Mannose derivative concentration is good linear relation with absorbance in the range of 10.2～51 μ g/ml, meets Lambert Beer Law, illustrates that it is reliable for calculating D-MANNOSE content in test sample according to absorbance.
(1) foundation of standard curve, as shown in Figure 4.
(2) in vaccine, the measurement result of mannose is shown in Table 2.
Table 2 sample total sugar content measurement result
The targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine immunity clinical observation of embodiment 10-DC
Take 5 sow nest farrowing pigs, 8, every nest, totally 40 17-22 age in days piglets in Large-scale pig farm, divide three groups, point Zhu She DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine 2ml (containing 1 part), commercially available pig blue-ear disease attenuated live vaccine 2ml (containing 1 part) and normal saline 2ml, carries out clinical observation, result: immune nano adjuvant inactivated vaccine and commercially available attenuated live Vaccine compares by pig with matched group, at body temperature, searches for food, is not clearly distinguished from terms of the clinical manifestation such as speed of production.Illustrate that immunity is received Rice adjuvant inactivated vaccine and commercially available attenuated live vaccine do not show side effect.
Embodiment 11-lymphocyte proliferation assay
Took a blood sample after above-mentioned immune one month, separate single core lymphocyte, detect T cell by lymphocyte proliferation assay Cause of disease proliferated specifically and nonspecific proliferation.
1) separation of pig peripheral blood mononuclearcell.By the pig peripheric venous blood of fresh collection, use anticoagulant heparin.With 1.5 The RPMI-1640 culture medium dilution of times volume, is added slowly to lymphocyte separation medium liquid level by it along tube wall in l:l ratio afterwards On, 2000r/min is centrifuged 20 minutes.Cell is divided into four layers afterwards, and ground floor is blood plasma or tissue homogenate liquid layer；The second layer is Ring-type milky lymphocyte enriched layer；Third layer is transparent separation liquid layer: the 4th layer is red blood cell layer.Sucking-off more intermediate annular breast White PBMC enriched layer, adds equal-volume RPMI-1640 culture medium and rinses, and 2000r/min is centrifuged 10 minutes, rinses 2 times Rear acquisition PBMC.Use 1ml RPMI-1640 culture medium resuspended the PBMC of fresh separated.
2) dyeing of pig peripheral blood mononuclearcell and counting.The trypan blue solution of cell suspension and 0.08wt% is according to 1: The ratio of 1 fully mixes (final concentration of 0.04wt%), be placed under inverted microscope observation counting.
3) lymphocyte proliferation assay
A. animal peripheral blood lymphocyte separation medium is utilized to extract lymphocyte.
B.1000rpm 10min it is centrifuged, supernatant discarded, resuspended with erythrocyte cracked liquid.
C. washing, 1000rpm is centrifuged 6min, is repeated twice.
D., after carrying out cell counting, it is separately added in 96 orifice plates, 5 × 105Individual/hole.
E. stimulus object, respectively TJM-F92 (MOI=0.01), PRRSV (MOI=0.01), positive control PHA (20 are added μ g/mL), negative control LPS (10ng/mL), blank, in 37 DEG C of incubators cultivate 70h.
F. add MTT solution 10 μ L/ hole, put in 37 DEG C of incubators and hatch 4h.
G. terminate hatching, carefully suck liquid in hole, add dimethyl sulfoxide 100 μ L/ hole, after concussion is fully mixing gently Absorbance is detected at 490nm and 630nm.Result is shown in Fig. 5, DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine immune swine After, its lymphocyte is for nonspecific stimulation thing LPS and PHA and specific antigen high-pathogenicity blue ear disease vaccine strain (TJM-F92) and the cell immune response of street strain's (TJ-F10 strain) be all significantly better than commercial available vaccines and matched group (* p < 0.05, * * p < 0.01).
Embodiment 12-ELISPOT detects
Above-mentioned test swinery was taken a blood sample after immune one month, separated single core lymphocyte, with ELISPOT experiment detection IFN γ secretion level.
1) design of reproductive and respiratory syndrome virus polypeptide pond and synthesis
NetMHC4 server polypeptide MHCI quasi-molecule is selected to combine artificial neural network (ANN) prediction.Peptide sequence from The virus protein aminoacid sequence searched in NCBI, utilizes computer software to be designed, and selects nm minimum, and rank Value minimum, it is the effective polypeptide epitope of most probable.
2) ELISpot detection method
A. take out the commercially available pvdf membrane plate being coated IFN γ antibody, wash plate 5 times with aseptic PBS/HBSS, 200 μ l/ holes.
B. close, add the RPMI-1640 culture medium containing 10wt% hyclone, incubated at room 30min.
C. cell is adjusted to 2 × 105Individual/hole, 100 μ l/ holes, detection hole is separately added into the disease of 10 μ l variable concentrations dilutions Poison stimulus object, positive control adds PHA, concentration 10 μ g/ml；The every hole of negative control adds the Cell sap of 110 μ l, the every hole of blank Add the RPMI-1640 culture medium of 110 μ l；It is all provided with multiple hole.
D. culture plate is put into 37 DEG C, 5%CO2In incubator, cultivate 24h.
E. discard culture fluid, wash plate 5 times with aseptic PBS/HBSS, 200 μ l/ holes.
F. with the PBS/HBSS dilution anti-IFN γ of biotin labeling containing 0.5% hyclone to 1 μ g/ml, 100 μ l/ holes, Room temperature 2h.
G. discarding culture fluid, wash plate 5 times with aseptic PBS/HBSS, 200 μ l/ holes, with the PBS/ containing 0.5% hyclone HBSS dilution alkali phosphatase enzyme mark Streptavidin to 1g/ml, 100 μ l/ holes, room temperature 2h.
H. discard culture fluid, wash plate 5 times with aseptic PBS/HBSS, 200 μ l/ holes, add substrate colour developing 15min, until speckle Point occurs.Result is shown in Fig. 6, after showing DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine immune swine, and its lymphocyte pair Reproductive and respiratory syndrome GP5 protein polypeptide pond produces stronger reaction, shows generation IFN γ showed increased (* p < 0.001).

Claims (6)

1.DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine, it is characterised in that in terms of 10 part vaccines, including following thing Matter: PRRSV TCID50=5 × 105-5×107, mannose 1-20wt%, PLGA 0.01-5wt%, Poly (I:C) 0.01- 5wt%.
2. the method preparing DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine described in claim 1, its feature Being, the method comprises the steps:
S1. PLGA nanometer adjuvant inactivated vaccine is prepared；
S2. the PLGA nanometer Adjuvanted vaccines of mannose-modified is synthesized.
3. the method preparing DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine as claimed in claim 2, its feature exists In, described step S1 is:
A. prepare W2 phase: PVA is added to the water, all dissolve；
B. prepare O phase: PLGA and join in dichloromethane, all dissolve；
C. prepare W1 phase: Poly (I:C) to mix with PRRSV, dissolve fully；
D. W1 is added in O phase, carries out ultrasonic Treatment and obtain mixture I；
E. mixture I is joined in W2 phase, remove dichloromethane after carrying out ultrasonic emulsification and obtain mixture II；
F. mixture II is centrifuged off supernatant, retains precipitation and be PLGA nanometer Adjuvanted vaccines.
4. the method preparing DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine as claimed in claim 2, its feature exists In, described step S2 is:
A. in the PLGA nanometer Adjuvanted vaccines that step S1 obtains, it is separately added into 1-6wt%N-N-Hydroxysuccinimide and 1- 8wt% dicyclohexylcarbodiimide solution, mix homogeneously；
B. the liquid of step a mix homogeneously is placed in bag filter dialysis 6-8 hour, removes unnecessary N-hydroxysuccinimidyl acyl sub- Amine, dicyclohexylcarbodiimide；
C. in the liquid after dialysis, add ethylenediamine, regulate pH to 5.0 with concentrated hydrochloric acid, form ammonium PLGA；
D. 1-20wt% mannose is dissolved in 0.1mol/L sodium acetate solution, mannose-sodium acetate solution is joined step In the ammonium PLGA that rapid c obtains, process 2 days 37 DEG C of concussions；
E. the liquid after step d being processed is centrifuged, and is precipitated, i.e. obtains mannose-modified by brine, resuspended precipitation PLGA nanometer Adjuvanted vaccines.
5. the method preparing DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine as claimed in claim 3, its feature exists In, in described W2 phase, the mass fraction of PVA is 0.25-1.25wt%.
6. the method preparing DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine as claimed in claim 3, its feature exists In, the condition of described ultrasonic emulsification is: time 110s, 0.6s/ time, amplitude 55%.
CN201610584707.0A 2016-07-23 2016-07-23 DC targeting pig blue-ear disease PLGA nanometer adjuvant inactivated vaccine and preparation method thereof Pending CN106237322A (en)

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