CN102327623A - Preparation method of DNA (deoxyribonucleic acid) vaccine nanoparticles containing PLGA (Poly-lactide-co-glycolide) for ND (necastle disease) - Google Patents

Abstract

The invention provides a preparation method of DNA (deoxyribonucleic acid) vaccine nanoparticles containing PLGA (Poly-lactide-co-glycolide) for ND (necastle disease) and relates to a preparation method of a DNA vaccine nanoparticle for ND. The preparation method comprises the following steps: 1, dissolving PLGA in dichloromethane, adding F-gene DNA plasmids of a NDV (necastle disease virus) F48E9 strain, emulsifying, then adding polyvinyl alcohol, and re-emulsifying, thus obtaining a re-emulsified solution; 2, dropwise adding the re-emulsified solution into polyvinyl alcohol for curing nanoparticles, volatilizing redundant dichloromethane, and stirring, thus obtaining a cured solution; and 3, centrifuging the cured solution, collecting the nanoparticles, washing 3 times, and performing vacuum freeze drying, thus obtaining the DNA vaccine nanoparticles containing PLGA for ND. The DNA vaccine nanoparticle transfer system containing PLGA for ND provided by the invention has the characteristics of protecting DNA vaccines for ND, improving the cell transfer efficiency and the immunization effect, being injected in an intramuscular manner, dropped into eyes or taken orally, inducing mucosal immunization and the like, and can be used for immunization of ND; and the DNA vaccine nanoparticle obtained by the invention is easy to store, high in stability, high in entrapment rate, low in toxicity and high in bioavailability.

Description

The method for preparing of PLGA newcastle dna vaccination nanoparticle

Technical field

The present invention relates to the method for preparing of newcastle dna vaccination.

Background technology

Newcastle (Newcastle disease; ND) be by NDV (Newcastle disease virus; NDV) a kind of acute, height contact, the lethal infectious disease of chicken that causes and turkey; Often being septicemia, is one of most important infectious disease of harm aviculture, is decided to be I class infectious disease by World Organization for Animal Health.The most basic measure of control ND is to carry out effective vaccination, and vaccine commonly used at present is attenuated live vaccines and inactivated vaccine, but all there is certain limitation in the two in practical application.The ND oil emulsion inactivated vaccine can produce the blood circulation antibody of higher level after through injecting immune chicken body; But in the mucosa system of opposing respiratory tract and digestive tract pathogen infection, limitation is arranged; Be difficult to stop cause of disease to invade in the animal body, and the virus that gets into cell is difficult to play a role through mucous membrane tissue; Though the ND attenuated live vaccines can produce certain mucosa-immune and humoral immunization, influenced by immunization method and approach, the circulating antibody of generation is lower, and the phase of keeping is short.The drinking-water immunity is easy to cause vaccine to be degraded by digestive tract, causes the vaccine absorption efficiency low; The collunarium eye dripping also can only make vaccine have 15min at nasal cavity, causes vaccine can not effectively get into body and excites effective mucosa-immune; The mucosa-immune effect that aerosol immunization produces is better, but induces the chronic respiratory eqpidemic disease easily, and weak malicious Seedling all can not produce higher serum antibody etc.

Dna vaccination is with in the eukaryon expression plasmid direct inoculation body that contains the coding for antigens gene, and expressing corresponding antigens in vivo stimulates body to produce to this antigenic immunne response, produces protective immunity.At present, dna vaccination large animal experiment, human clinical's experimental result show: it is bigger that dna vaccination exists dosage, and bioavailability is low, and the immune effect individual variation is big, and dissatisfactory shortcoming has greatly hindered the progress of dna vaccination.Therefore, how to improve the immune effect of newcastle disease vaccine, develop and to produce stronger humoral immunization antibody and can produce the focus that the new generation vaccine of local mucosa-immune has efficiently become the ND vaccine research again.

In recent years along with new material is learned the development with the preparation new technique, press for a kind of new and the mortality rate of improved vaccine delivery method with the reduction infectious disease.At present, preparation is carried the carrier material that the vaccine antigen nanoparticle adopted and is mainly contained natural polymers chitosan, gelatin, synthetic polymer PLA, PLGA etc.At present, the research and development of external dna vaccination microsphere/nanoparticle mucosa-immune delivery system has obtained many phasic results, the clinical experiment of entering that has very rapidly.The research work of domestic dna vaccination microsphere/nanoparticle mucosa-immune delivery system is carried out less, especially the research of newcastle dna vaccination mucosa-immune delivery system also Shang Weijian report is arranged.

Summary of the invention

The invention provides the method for preparing of PLGA newcastle dna vaccination nanoparticle.

PLGA newcastle dna vaccination nanoparticle of the present invention prepares according to the following steps:

One, the polylactic acid/hydroxy acetate multipolymer (PLGA) that takes by weighing 40mg is dissolved in the dichloromethane of 1mL; Treat to dissolve fully the F gene DNA plasmid that the back adds 200 μ g NDV F48E9 strains; Ultrasonic emulsification 30s under the condition of ice bath, colostric fluid, add the 2mL mass concentration then and be 2% polyvinyl alcohol; Ultrasonic emulsification 60s under condition of ice bath gets double emulsion;

Two, double emulsion dropwise being joined the 10mL mass concentration is to carry out nanoparticle in 0.5% the polyvinyl alcohol to solidify and the unnecessary dichloromethane that volatilizees, and at the uniform velocity stirs 5h with 500r/min, consolidation liquid;

Three, get 4 ℃ of centrifugal 15min of 3000g consolidation liquid, collect nanoparticle, use ddH then ₂O washing 3 times promptly gets PLGA newcastle dna vaccination nanoparticle after vacuum lyophilization; Wherein the concentration of the F gene DNA plasmid of NDV F48E9 strain is 0.5% in the step 1; The power of ultrasonic emulsification is 50w in the step 1.

PLGA newcastle dna vaccination nanoparticle of the present invention has huge application potential; PLGA newcastle dna vaccination nanoparticle delivery system of the present invention has protective effect to the newcastle dna vaccination; Improve cell traffic efficient, improve immune effect, but intramuscular injection, eye dripping; Also orally-ingestible is induced characteristics such as mucosa-immune.Simultaneously; Under the pharmaceutically-active prerequisite of assurance, reduce dosage, alleviated or avoided toxicity, can prolong the immunoprotection phase, avoided repeatedly repetitively administered; And carrier material PLGA itself is nontoxic; Good biocompatibility, biodegradable, thereby be a kind of novel carriers vaccine with broad prospect of application.The present invention provides a good immune effect and the high delivery system of security performance for the extensive use clinically of newcastle dna vaccination, with the practical application that promotes the newcastle dna vaccination at poultry farming; Can this achievement in research be applied in more animal vaccine and the drug system on this basis; Be used to prevent, treat, diagnose animal and human's illness when the time comes; With rescue many ill domestic animals or patient's life or prolong its life-span, its economic benefit and social benefit are undoubtedly huge.

PDNA-PLGA-NPs complete form through transmission electron microscope observation the present invention preparation is spheroidal, smooth surface, and dispersibility is better, phenomenon such as do not have tangible adhesion, subside, particle diameter is (433.5 ± 7.5) nm, the particle size dispersion degree is 0.41.The Zeta potential that Zeta potential is analyzed the pDNA-PLGA-NPs of the present invention's preparation is+2.7mV that envelop rate is (91.8 ± 0.3) %.The pDNA-PLGA-NPs mucosa-immune of the present invention's preparation more can activate the mucosal immune response position actively than intramuscular injection; Produce high-caliber specific IgG and IgA; Explain that nanoparticle mucosa-immune delivery system not only in the local immunne response that produces of mucosa, also can cause the HI and the cellullar immunologic response of general; Overcome conventional vaccine simultaneously and in storage and transportation, be prone to shortcomings such as inactivation; And the NDV-CS-NPs with the prepared place of the inventive method is easy to preserve, and stability is high, envelop rate is high, toxicity is little, drug bioavailability is high, cost is low, be easy to suitability for industrialized production.

Use the dna vaccination nanoparticle of the inventive method preparation under the condition that guarantees dna vaccination stability, to have higher entrapment (envelop rate is 91.8 ± 0.3%); Can keep drug level best in the body; The extracorporeal releasing test result: two days plasmid rate of release of pro-are very fast, and the plasmid burst size accounts for 31.25% of total parcel amount; To between 10d, the burst size of the DNA every day among the pDNA-PLGA-NPs reaches 4.53% of parcel total amount at 2d; After 10d, be the later stage that DNA discharges, reach 93.14% to the release of 16d DNA.Therefore, the dna vaccination nanoparticle of the inventive method preparation can be realized the slow release of medicine.

The inventive method has that technology is easy, preparation cost is low, technology favorable reproducibility, sample requirement are few; The advantages such as activity that better kept dna vaccination; The method for preparing of the pDNA-PLGA-NPs of experiment sieving is also scalable to production application, thereby the application of industrialization is had directive significance.

PDNA-PLGA-NPs of the present invention can be used for the newcastle disease immunity, the oral administration administration, and dosage is 200 μ g DNAs.

Description of drawings

Fig. 1 is the transmission electron microscope observation figure of PLGA newcastle dna vaccination nanoparticle in the specific embodiment one;

Fig. 2 is the particle size distribution figure of PLGA newcastle dna vaccination nanoparticle in the specific embodiment one;

Fig. 3 is the Zeta potential figure of PLGA newcastle dna vaccination nanoparticle in the specific embodiment one;

Fig. 4 is the release in vitro curve chart of PLGA newcastle dna vaccination nanoparticle in the specific embodiment one;

Fig. 5 is the electrophoretogram of the anti-DNase I degraded of PLGA newcastle dna vaccination nanoparticle in the specific embodiment one, and 1 swimming lane is Markers, the naked DNA of 2 swimming lanes for not being wrapped, and the 3-6 swimming lane is the DNA that pDNA-PLGA-NPs wrapped up;

Fig. 6 is the electrophoretogram of the proteic expression of Western-blotting testing goal in the specific embodiment one; 1 swimming lane is Markers, and 2 swimming lanes are the pDNA-PLGA-NPs of transfection post processing, and 3 swimming lanes are plasmid pVAX1-F (o); 4 swimming lanes are blank nanoparticle, and 5 swimming lanes are the bhk cell of untransfected.

The specific embodiment

Technical scheme of the present invention is not limited to the following cited specific embodiment, also comprises the combination in any between each specific embodiment.

The specific embodiment one: this embodiment PLGA newcastle dna vaccination nanoparticle prepares according to the following steps:

One, the polylactic acid/hydroxy acetate multipolymer (PLGA) that takes by weighing 40mg is dissolved in the dichloromethane of 1mL; Treat to dissolve fully the F gene DNA plasmid that the back adds 200 μ g NDV F48E9 strains; Ultrasonic emulsification 30s under the condition of ice bath, colostric fluid, add the 2mL mass concentration then and be 2% polyvinyl alcohol; Ultrasonic emulsification 60s under condition of ice bath gets double emulsion;

Two, double emulsion dropwise being joined the 10mL mass concentration is to carry out nanoparticle in 0.5% the polyvinyl alcohol to solidify and the unnecessary dichloromethane that volatilizees, and at the uniform velocity stirs 5h with 500r/min, consolidation liquid;

Three, get 4 ℃ of centrifugal 15min of 3000g consolidation liquid, collect nanoparticle, use ddH then ₂O washing 3 times promptly gets PLGA newcastle dna vaccination nanoparticle after vacuum lyophilization; Wherein the concentration of the F gene DNA plasmid of NDV F48E9 strain is 0.5% in the step 1; The power of ultrasonic emulsification is 50w in the step 1.

The preparation and the purification of the F gene DNA plasmid of NDV F48E9 strain in this embodiment step 1 are to carry out as follows: with restricted enzyme EcoR V and Xba I the F gene eucaryon expression plasmid pVAX1-F (o) of NDV F48E9 strain is carried out double digestion; Positive plasmid is transformed among the E.coli. competent cell DH5 α; Behind the incubated overnight 12h; Picking list bacterium colony at random; Be inoculated in the LB fluid medium (containing the Kana resistance), 37 ℃ of air bath shaking tables are cultivated 16h, use in order to big upgrading grain.Adopt alkaline lysis large quantity extracting plasmid DNA, a large amount of extractions of plasmid and purification are referring to the SDS alkaline lysis and the Polyethylene Glycol (PEG of a large amount of preparations of " Molecular Cloning:A Laboratory Manual (3rd ed) " DNA ₈₀₀₀) purification process, and suitably improve.Eukaryon expression plasmid pVAX1-F (o) is made up and is preserved by Vet Biotechnology National Key Laboratory of Harbin Veterinary Medicine Inst., China Academy of Agriculture.Alkaline lysis large quantity extracting plasmid DNA concrete steps are following:

1) picking grows in single colony inoculation on the LB flat board in 5mL liquid LB culture medium (containing Kana, 100 μ g/mL), and 37 ℃ of shaken cultivation are spent the night to logarithm OD in late period ₆₀₀Value is 0.6;

2) in 1/1000 ratio bacterial cultures is transferred in the 1000mL LB fluid medium, 37 ℃, 300r/min concussion are cultivated 16h, stay 1mL bacterium liquid to be used for little upgrading grain and identify;

3) with 4 ℃ of bacterium liquid, the centrifugal 8min of 6000r/min, bacterial precipitation is resuspended with the STE vibration of 300mL pre-cooling, 4 ℃, 6000r/min recentrifuge 8min;

4) the bacterial precipitation thing that will wash is resuspended in the 100mL solution I, and thermal agitation adds 0.05g lysozyme powder, and mixing is put into 37 ℃ of water-bath temperature and bathed gently;

5) add the freshly prepared solution II of 100mL (existing), put upside down mixing repeatedly gently, room temperature effect 8min with join at present.The solution clarification of taking out this moment;

6) add the solution III of 100mL ice pre-cooling, gentle vibration is uniformly dispersed solution III for several times, can see that the adularescent deposition separates out this moment, leaves standstill 30min on ice, 4 ℃, the centrifugal 30min of 9000r/min;

7) with 4-6 layer gauze the supernatant after centrifugal is filtered in another centrifuge bottle, adds the isopropyl alcohol of 0.6 times of volume, mixing is abundant, and room temperature is placed 10min, 22 ℃, the centrifugal 30min of 9000r/min;

8) abandon supernatant, with 70% washing with alcohol deposition and tube wall, dissolve with 10mL TE (pH8.0) dry back; (can 4 ℃ spend the night)

The TE solution that 9) will be dissolved with thick DNA changes in another centrifuge tube, adds the LiCl of the 5mol/L of 10mL (with the TE equal-volume) ice pre-cooling, abundant mixing, 4 ℃, the centrifugal 15min of 10000r/min; (removing macromolecule RNA and foreign protein)

10) supernatant is transferred in another pipe, adds isopyknic isopropyl alcohol again, abundant mixing, room temperature leaves standstill 15min, 22 ℃, the centrifugal 15min of 10000r/min;

11) abandon supernatant,, wait to precipitate dry back and dissolve with 10mL TER (RNase concentration is 25mg/mL) with 70% washing with alcohol deposition and tube wall; (the little RNA that digestion is polluted, can 4 ℃ spend the night)

12) add equal-volume PEG-NaCl, mixing, 4 ℃, the centrifugal 15min of 10000r/min; (be settled out the macromole DNA, small-molecular weight DNA and RNA stayed in the supernatant)

13) abandon supernatant, dissolve DNA with 10mLTE;

14) phenol, phenol/chloroform/isoamyl alcohol (25/24/1), each extracting of chloroform that add equivalent once, 22 ℃, the centrifugal 15min of 10000r/min; (removing Deproteinization)

15) gentle aspiration upper strata aqueous phase solution adds the 3mol/L pH5.2 NaAC of 1/10 volume to another centrifuge tube, and it is above or spend the night to add the dehydrated alcohol deposit D NA 2h of ice pre-cooling of 2.5 times of volumes again;

16) 4 ℃, the centrifugal 30min of 10000r/min abandon supernatant, with 70% washing with alcohol deposition once, and the centrifugal supernatant of abandoning;

17) in super-clean bench, after the aeration-drying, precipitate with 1mL TE (pH8.0) dissolving DNA;

18) survey OD after being diluted to debita spissitudo with TE ₂₆₀And calculating plasmid DNA concentration; Put-20 ℃ in the good concentration of EP pipe marked with a day after date and preserve stock solution.

The PLGA newcastle dna vaccination nanoparticle of this embodiment method preparation (is called for short: pDNA-PLGA-NPs) under-20 ℃ of conditions, preserved 3 months; Transmission electron microscope observing (as shown in Figure 1) then, complete form is spheroidal, smooth surface; Dispersibility is better; Phenomenon such as do not have tangible adhesion, subside, particle diameter is (433.5 ± 7.5) nm (as shown in Figure 2), the particle size dispersion degree is 0.41.The Zeta potential that Zeta potential is analyzed the pDNA-PLGA-NPs of the present invention's preparation is+2.7mV (as shown in Figure 3).

The PLGA newcastle dna vaccination nanoparticle of this embodiment preparation; Entrapment efficiency determination before the preparation nanoparticle, carries out agarose gel electrophoresis with DNA; Warp is more accurately measured newcastle dna vaccination plasmid content with DNA standard molecule Marker, this total dose for dropping into.Draw the centrifuged supernatant after 50 μ L nanoparticles solidify, measure the dna content in the supernatant through agarose gel electrophoresis, definite not parcel advances the DNA content of nanoparticle.The computing formula of the envelop rate of PLGA newcastle dna vaccination nanoparticle is following:

Total dna content (μ g)=DNA concentration * volume

DNA concentration * volume in dna content (μ the g)=supernatant that does not wrap up

Envelop rate (%)=(total dna content-not the dna content of parcel)/total dna content * 100%

The envelop rate average out to 91.8 ± 0.3% of the pDNA-PLGA-NPs of this embodiment preparation.

The dna vaccination nanoparticle of this embodiment preparation has higher entrapment under the condition that guarantees dna vaccination stability; Can keep drug level best in the body; Extracorporeal releasing test result (as shown in Figure 4) shows; Two days plasmid rate of release of pro-are very fast, and the plasmid burst size accounts for 31.25% of total parcel amount; To between 10d, the burst size of the DNA every day among the pDNA-PLGA-NPs reaches 4.53% of parcel total amount at 2d; After 10d, be the later stage that DNA discharges, reach 93.14% to the release of 16d DNA.Therefore, the dna vaccination nanoparticle of this embodiment preparation can be realized the slow release of medicine.

This embodiment detects the anti-DNase I degraded of PLGA newcastle dna vaccination nanoparticle through 1% agarose gel electrophoresis; The result is that the naked DNA that is not wrapped is promptly degraded behind DNase I effect 30min fully; Even also can keep its integrity and pDNA-PLGA-NPs wrapped up DNA grows to 3h action time; Explain that the parcel of nanoparticle can resist the degraded of DNase I, played protective effect (as shown in Figure 5).

Detect the pDNA-PLGA-NPs vivoexpression through indirect immunofluorescence assay and Western-blotting:

(1) liposome transfection of pDNA-PLGA-NPs

After the monolayer bhk cell got off with trypsinization, be inoculated in the six porocyte culture plates 37 ℃, 5%CO with suitable density ₂Be cultured to the 70%-80% state.Inhale this moment and remove culture fluid,, wash 2 times with not containing antibiotic and fresh serum DMEM culture medium washed cell, for use.DNA in the nanoparticle is extracted, with DNA pVAX1-F (o) as positive plasmid respectively at liposome Lipofectamine ^TM2000 mediations are the transfection bhk cell down.The concrete operations step is carried out to specifications.Get 4.0 μ g plasmids and add the DMEM that 300 μ L do not contain serum and antibiotic optimization, this is an A liquid; 10 μ L liposomees are joined in another EP pipe that 300 μ L optimization DMEM is housed, and this is a B liquid; After A, B liquid leave standstill 2-3min respectively, A, B liquid are mixed gently, room temperature leaves standstill 30min (can not surpass 45min, otherwise liposome can be to the toxic effect of plasmid).In the ready cell culture hole, rock mixing all around gently, before mixed liquor joined in 37 ℃, 5%CO ₂After cultivating 5h, discard culture, change into and contain 1%GIBCO serum and an amount of antibiotic DMEM keeps culture medium, continue to cultivate.Simultaneously, with blank nanoparticle and blank cell as negative control.

Adopt the proteic expression of indirect immunofluorescence testing goal again.The concrete operations step is following: 1) cell culture fluid of cultivating 72h after the transfection is discarded, with PBS buffer (pH 7.4) washing 2 times, rock 5min gently at every turn; 2) with neutral formalin fixedly behind the 10min reuse PBS wash 3 times; 3) add the NDV positive serum that 1mL 1/100 dilutes, 37 ℃ of effect 1h, PBS washing 3 times, each 5min; 4) add two of FITC labelling and resist (1/5000 dilution) 37 ℃ of continuation effect 1h, reuse PBS washs three each 5min; 5) add an amount of glycerol-water (1/9), fluorescence microscope is observed the destination protein expression down.Result of the test is that the DNA and the positive plasmid pVAX1-optiF that from pDNA-PLGA-NPs, extract all can observe specificity fluorescent, and the blank nanoparticle of transfection and the cell of untransfected all do not detect fluorescence.

(2) the proteic expression of Western-blotting testing goal

72h after the bhk cell transfection, collecting cell with PBS washing 1 time, adds an amount of 2 * SDS-PAGE sample-loading buffer in cell precipitation, and boiling water bath boils 8min, carries out the SDS-PAGE protein isolate.Result of the test is the albumen that pDNA-PLGA-NPs and the plasmid pVAX1-F (o) of transfection post processing can both detect identical size, and consistent with expected results, the protein molecular size is about 58kDa; And the bhk cell of blank nanoparticle and untransfected does not all detect the expression (as shown in Figure 6) of destination protein.

Measure the safety evaluatio of the pDNA-PLGA-NPs delivery system of this embodiment preparation with CCK-8 test kit (available from Dojindo company): bhk cell is diluted to 2 * 10 with the DMEM culture medium that contains serum ⁶Individual/mL cell suspension, join in 96 well culture plates with the amount of every hole 100 μ L, cultivate 5h for 37 ℃; Afterwards, add the pDNA-PLGA-NPs that was diluted by the DMEM culture medium, 37 ℃, 5%CO to every hole ₂Continue overnight incubation, 2d adds 10 μ L CCK-8 reagent to every hole and cultivates 5h; The last light absorption value of under 450nm wavelength condition, measuring each hole calculates cell survival rate (%).

Cell survival rate (%)=[(As-Ab)/(Ac-Ab)] * 100%

As: test hole (culture medium, CCK-8, the nanoparticle suspension that contain cell)

Ab: control wells (culture medium, the CCK-8 that contain cell)

Ac: blank well (culture medium, the CCK-8 that do not contain cell and nanoparticle suspension)

The survival rate that experimental result records cell is (80.14 ± 8.27) %, shows that the cytotoxicity of pDNA-PLGA-NPs of preparation is less, and the newcastle dna vaccination PLGA nanoparticle mucosa-immune delivery system of structure has higher safety.

Through the inoculation test of SPF chicken immune, detect the immune effect of the pDNA-PLGA-NPs of this embodiment preparation.The detection step is following:

With 2 the week age SPF chicken be divided into 7 groups at random, 25 every group.The grouping situation is seen table 1.Wherein, naked DNA pVAX1-F (o) plasmid content is 200 μ g, and making the DNA content that carries the immunity of DNA nanoparticle group according to the conversion of envelop rate also is 200 μ g.The intramuscular injection group adopts the quadriceps femoris multi-point injection; One exempts from two weeks of back later on identical dosage booster immunization, and the 7th group of head exempts to adopt the collunarium immunization, carries out intramuscular injection with identical dosage during booster immunization.Exempt from back 13d and 27d respectively at one, get 3 chickens for every group and cut open and kill, get spleen and do lymphocyte proliferation assay.The investigation of whole immune effect continues to monitor one and exempts from back 49d.

Table 1 immunity is divided into groups

Group number	Group	Immunization ways
				1	PBS	Intramuscular injection (i.m.)
2	Blank nanoparticle	Intramuscular injection (i.m.)
			3	Blank nanoparticle	Collunarium immunity (i.n.)
4	pVAX1-F(o)	Intramuscular injection (i.m.)
			5	pDNA-PLGA-NPs	Intramuscular injection (i.m.)
6	pDNA-PLGA-NPs	Collunarium immunity (i.n.)
			7	pDNA-PLGA-NPs	Collunarium immunity/intramuscular injection (i.n./i.m.)

Immunity chicken spleen lymphproliferation response result of the test (seeing table 2) shows, compares with matched group PBS, intramuscular injection pDNA-PLGA-NPs and collunarium immunity pDNA-PLGA-NPs, and all the other each immune group all show significantly IR to Con A.Particularly exempt from back 28d one; Promptly two exempt from two weeks of back, the breeder reaction of pDNA-PLGA-NPs combined immunization group, pDNA-PLGA-NPs collunarium group and pDNA-PLGA-NPs intramuscular injection group immunity chicken spleen cell is extremely significantly in naked DNA pVAX1-F (o) intramuscular injection group (p＜0.01).One exempts from back 28d, and pDNA-PLGA-NPs collunarium group immunity chicken spleen cell proliferative response will significantly strengthen in intramuscular injection group (p＜0.05).

Splenic lymphocytes behind table 2 chicken immune

Annotate: the upper right corner does not have identical lower case person and shows group difference significantly (p＜0.05) in the same column data; Do not have identical capitalization person and represent group difference extremely significantly (p＜0.01)

Immunity back serum specific IgG antibodies changes: immunity back two all antibody titers still are in a lower level; In a week behind the booster immunization, the antibody titer ascensional range is very fast, pVAX1-F (o) intramuscular injection group, promptly positive controls one exempt from the back 28d reach top level, compared utmost point significant difference (p＜0.01) with negative control group with other immune group; And pDNA-PLGA-NPs collunarium group antibody horizontal is exempted from back 28d one and is begun to rise, and reaches summit at 35d, compares significant difference (p＜0.05) with positive control pVAX1-F (o); PDNA-PLGA-NPs head exempts from collunarium, and booster immunization is that the intramuscular injection group reaches summit at 35d, and can continue higher IgG antibody level to exempt from the back 49d, the utmost point is significantly higher than positive controls (p＜0.01).

IgA changes of contents in the serum of immunity back: each immune group IgA antibody content gap is little in the serum of being adopted weekly, but all the utmost point is significantly higher than negative control group (p＜0.01).PVAX1-F (o) intramuscular injection group and combined immunization group dynamic rule are similar, all exempt from back 28d one and reach summit, and can keep higher level to exempt from back 49d.And IgA antibody content growth rate is slower in the pDNA-PLGA-NPs collunarium group serum, exempts from back 28d to one and just significantly increases, and reaches the summit of IgA antibody content at 35d.

The immunity test result shows in the SPF chicken body; Newcastle dna vaccination PLGA nanoparticle mucosa-immune more can activate the mucosal immune response position actively than intramuscular injection; Produce high-caliber specific IgG and IgA; Explain that nanoparticle mucosa-immune delivery system not only in the local immunne response that produces of mucosa, also can cause the HI and the cellullar immunologic response of general.

Specific IgG antibodies level in the pDNA-PLGA-NPs immune serum of this embodiment of indirect elisa method mensuration preparation:

Step: at first, with 1/100 times of dilution of test serum, join in 96 orifice plates that encapsulated, set up the positive and negative control hole simultaneously, 37 ℃ of incubation 30min are with PBS (pH 7.4) washing 4 times; Every hole adds the goat-anti chicken ELIAS secondary antibody 100 μ L that test kit provides, and 37 ℃ are continued incubation 30min, reuse PBS washing 4 times; Then, add 100 μ L tmb substrate developers to every hole, the room temperature lucifuge is hatched 15min; At last, with 100 μ L stop buffer (2mol/L H ₂SO ₄) cessation reaction, under the 650nm wavelength, measure and record OD _650nmValue.Experimental result shows; Dna vaccination behind PLGA nanoparticle parcel; The binding antibody level that produces has had significant raising, and pDNA-PLGA-NPs group humoral immunization has played the effect of slow release apparently higher than naked newcastle dna vaccination group and antibody horizontal longer duration.

IgA antibody in the pDNA-PLGA-NPs immune serum of this embodiment of indirect elisa method detection preparation: method is following: supernatant is joined with 1/100 times of concentration dilution in 96 orifice plates that encapsulate, hatch 1h for 37 ℃, wash 5 times with PBST, each 5min; Every hole adds the good HRP labelling goat-anti chicken IgA of 100 μ L dilution, hatches 1h for 37 ℃, washes 5 times each 5min with PBST; Every hole adds 100 μ L TMB colour developing liquid, and after the room temperature lucifuge was hatched 15min, every hole added 100 μ L stop buffer (2mol/L H ₂SO ₄) the color development stopping reaction, the 450nm wavelength is measured OD down ₄₅₀Value.Simultaneously, add IgA normal gradients diluent drawing standard curve.Experimental result shows that pDNA-PLGA-NPs can stimulate specific b cells, and strengthens HI, thereby improves antibody titer.

The specific embodiment two: this embodiment is 40000～75000 with the molecular weight that is not both polylactic acid/hydroxy acetate multipolymer in the step 1 of the specific embodiment one, and LA/GA=50/50, specificity viscosity are 0.37dL/g.Other step and parameter are identical with the specific embodiment one.

Claims (2)

1.PLGA the method for preparing of newcastle dna vaccination nanoparticle is characterized in that PLGA newcastle dna vaccination nanoparticle prepares according to the following steps:

One, the PLGA that takes by weighing 40mg is dissolved in the dichloromethane of 1mL; Treat to dissolve fully the F gene DNA plasmid that the back adds 200 μ g NDV F48E9 strains; Ultrasonic emulsification 30s under the condition of ice bath, colostric fluid, add the 2mL mass concentration then and be 2% polyvinyl alcohol; Ultrasonic emulsification 60s under condition of ice bath gets double emulsion;

Two, double emulsion dropwise being joined the 10mL mass concentration is to carry out nanoparticle in 0.5% the polyvinyl alcohol to solidify and the unnecessary dichloromethane that volatilizees, and at the uniform velocity stirs 5h with 500r/min, consolidation liquid;

Three, get 4 ℃ of centrifugal 15min of 3000g consolidation liquid, collect nanoparticle, use ddH then ₂O washing 3 times promptly gets PLGA newcastle dna vaccination nanoparticle after vacuum lyophilization; Wherein the concentration of the F gene DNA plasmid of NDV F48E9 strain is 0.5% in the step 1; The power of ultrasonic emulsification is 50w in the step 1.

2. the method for preparing of PLGA newcastle dna vaccination nanoparticle according to claim 1 is characterized in that the PLGA molecular weight is 40000～75000 in the step 1, and LA/GA=50/50, specificity viscosity are 0.37dL/g.

Images