CN104353069B - Preparation method of oral subunit vaccine for porcine epidemic diarrhea virus - Google Patents
Preparation method of oral subunit vaccine for porcine epidemic diarrhea virus Download PDFInfo
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Abstract
The invention belongs to the field of biological medicine engineering, and particularly discloses a preparation method of an oral subunit vaccine for a porcine epidemic diarrhea virus. The prepared subunit vaccine can be directly used as an oral vaccine to immune a mouse, and the mouse can be stimulated to generate stronger humoral immune response and cellular immune response. The vaccine is not degraded easily in the stomach and intestines, the immune effect is remarkable, the preparation process is simple, and the oral subunit vaccine plays an active role in relieving harm caused by the porcine epidemic diarrhea virus to the pig industry and has important practical significance in promoting healthy development of the pig industry.
Description
Technical field
The present invention relates to animal organism pharmaceutical engineering field, more particularly, to a kind of orally available pig epidemic diarrhea
The preparation method of subunit viral vaccine.
Background technology
Porcine epidemic diarrhea virus（Porcine epidemic diarrhea virus, PEDV）It is a kind of sub-thread, just
Strand rna virus, belong to the many viraleses of Buddhist nun (Nidovirales), coronaviridae (Coronviridae), coronavirus genus
(Coronavims).Infection PEDV is the one of the main reasons of pig virus diarrhoea, and main feature is serious enteritis, vomits
Tell, watery diarrhea and losing weight, the high of the piglet death rate make PEDV infection become Chinese pig industry loss
One big factor.
PEDV until today, is not only effectively controlled after being found, and has become impact whole world pig industry on the contrary
Great virus, causes huge economic loss.The outburst in Asia for the PED causes the high death rate, has research display Japan
During 1993～1994 years outburst PED, just more than 10,000, the death rate is 30%～60% for 5 pig farm piglet death toll；It is reported that from
2 months in November, 2011 in 2010, the piglet death toll that causes of PED is broken out more than 50,000 in China, and the death rate up to 90%～
100%.2013, the U.S.'s also reported first presence of PEDV, 2014 years persistently outburst with popular.
At present vaccine inoculation is mainly although the inactivated vaccine of domestic PEDV and attenuated live vaccines obtain for the prevention of PED
Extensive promotion and application, decrease the incidence of disease of PED to a certain extent, but still cannot control completely.Reason is
PEDV mainly passes through enteric infection pig, has apparent biting property of intestinal tissue.Mucosal sIgA antibody is to through alimentary canal, breathing
Road, the mucosal infections virus of reproduction invasion have neutralization.Therefore exploitation produces the vaccine of mucosal sIgA antibody to prevention
PEDV plays the role of important.
For this feature, the stimulation intestinal mucosa of domestic-developed, the main vaccine producing mucoantibody is Attenuate vaccine, gene
The live bacterial vaccines of restructuring and subunit vaccine.There is the anti-strong risk of weak poison it is possible to cause secondary hazards in Attenuate vaccine, restructuring
The difficult point of bacterium live vaccine is to be difficult the quantity of control viable bacteria, easily inactivates, and when subunit vaccine is administered orally, by intestines and stomach
When, easily it is subject to enzyme hydrolysis to lose effect.
Content of the invention
Cannot be administered orally for Porcine epidemic diarrhea virus subunit vaccine in prior art, and easily degrade in the gastrointestinal tract
Defect, it is an object of the invention to provide a kind of orally available Porcine epidemic diarrhea virus subunit vaccine preparation method.
The present invention is achieved by the following technical programs.
A kind of preparation method of orally available Porcine epidemic diarrhea virus subunit vaccine, comprises the following steps：
S1. the shitosan being 80 ~ 85% by deacetylation is dissolved in 0.5 ~ 0.6%（v / v）In acetic acid, prepare 0.2%（w/v）
Chitosan solution；
S2. take the chitosan solution of 4 ml step S1 preparations, being added dropwise over 1 ml concentration while stirring is 50 ~ 100 μ g/
ML Porcine epidemic diarrhea virus antigens c OE albumen, albumen is stirred for 15 ~ 20 min after adding；
S3. by 1 ~ 2 ml concentration be 0.2%（W/V）Sodium tripolyphosphate, be added in S2 solution, continue stirring 5 ~
10min obtains final product vaccine.
Preferably, the deacetylation of shitosan described in S1 is 85%.
Preferably, during chitosan solution configuration, the shitosan that deacetylation is 85% is dissolved in 0.5% described in S1（v/ v）
In acetic acid.
Compared with prior art, the invention has the advantages that
The pig fluidity diarrhea virus subunit vaccine that the present invention is obtained can be administered orally, and is difficult to degrade in stomach intestines, energy
Body is enough stimulated to produce stronger HI and cellullar immunologic response, immune effect is notable, preparation process is simple, preparation
Mild condition it is only necessary to carry out routine stirring and normal temperature condition can prepare, preparation cost is cheap, is conducive to pushing away on a large scale
Extensively.
In addition, prior art report utilizes 0.3 ~ 1%（v / v）Acetic acid solution dissolving shitosan, encapsulating protein or core
The envelop rate of acid all 80% about, but, it is a discovery of the invention that for Porcine epidemic diarrhea virus antigens c OE albumen it is necessary to use
Deacetylation is 80 ~ 85%, and concentration is 0.5 ~ 0.6（v / v）Acetic acid solution to dissolve shitosan, can be only achieved good bag
Envelope rate.Analysis reason may be relevant with the space structure of Porcine epidemic diarrhea virus antigens c OE albumen and albumen size.
Brief description
Fig. 1 is embodiment 5 irriate spleen cell cultures supernatant GM-CSF concentration
Fig. 2 is embodiment 5 irriate spleen cell cultures supernatant IFN γ concentration
Fig. 3 is embodiment 5 irriate spleen cell cultures supernatant IL2 concentration
Fig. 4 is embodiment 5 irriate spleen cell cultures supernatant IL4 concentration
Fig. 5 is embodiment 5 irriate spleen cell cultures supernatant IL5 concentration
Fig. 6 is embodiment 5 irriate spleen cell cultures supernatant IL6 concentration
Fig. 7 is embodiment 5 irriate spleen cell cultures supernatant IL10 concentration
Fig. 8 is embodiment 5 irriate spleen cell cultures supernatant TNF α concentration.
Specific embodiment
With reference to Figure of description and specific embodiment, the present invention is expanded on further.These embodiments are merely to illustrate
The present invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in lower example embodiment, generally according to
This area normal condition or the condition according to manufacturer's suggestion.Unless otherwise defined, all specialties used in literary composition and section
Learn term identical with meaning familiar to the person skilled in the art.
Embodiment 1
The preparation of shitosan coated pig PEDV subunit vaccine：
S1. the configuration of chitosan solution：The shitosan that deacetylation is 85% is dissolved in 0.5%（v / v）In acetic acid, preparation
0.2%（w/v）Chitosan solution；
S2. take the chitosan solution of 4 ml step S1 preparations, being added dropwise over 1 ml concentration while stirring is 60 μ g/mL pig streams
Row diarrhea virus antigens c OE albumen, albumen is stirred for 15 min after adding；
S3. by 2 ml concentration be 0.2%（W/V）Sodium tripolyphosphate, be added in S2 solution, continue stirring 5min obtain final product
Vaccine.
Wherein, 60 μ g/mL Porcine epidemic diarrhea virus antigens c OE albumen are the normal saline dilution 500 μ g/ with 0.9%
ML Porcine epidemic diarrhea virus antigens c OE albumen stoste obtains, and Porcine epidemic diarrhea virus cAg COE albumen stoste
It is that poultry research department of animal science institute of Agricultural University Of South China obtains according to the method for conventional eukaryotic expression.
Embodiment 2
The preparation of shitosan coated pig PEDV subunit vaccine：
S1. the configuration of chitosan solution：The shitosan that deacetylation is 80% is dissolved in 0.6%（v / v）In acetic acid, preparation
0.2%（w/v）Chitosan solution；
Other steps are with embodiment 1.
Embodiment 3
The preparation of shitosan coated pig PEDV subunit vaccine：
S1. the configuration of chitosan solution：The shitosan that deacetylation is 85% is dissolved in 0.3%（v / v）In acetic acid, preparation
0.2%（w/v）Chitosan solution；
Other steps are with embodiment 1.
Embodiment 4
The preparation of shitosan coated pig PEDV subunit vaccine：
S1. the configuration of chitosan solution：The shitosan that deacetylation is 85% is dissolved in 1%（v / v）In acetic acid, preparation
0.2%（w/v）Chitosan solution；
Other steps are with embodiment 1.
Measure the envelop rate of the shitosan coated pig PEDV subunit vaccine that embodiment 1 ~ 4 prepares using BCA method,
The step of BCA method is with reference to the conventional method of the art, measurement result：The envelop rate of vaccine described in embodiment 1 ~ 4
It is respectively（86.31±2.05）%、（84.56±3.23）%、（70.12±1.96）%、（69.62±3.17）%.By embodiment 1,
3rd, 4 result understands, the concentration of acetum is put and be significantly affects envelop rate.Report in prior art, using 0.3 ~ 1%（v /
v）Acetic acid solution dissolving shitosan, the envelop rate of encapsulating protein or nucleic acid all 80% about, but, it is a discovery of the invention that right
In Porcine epidemic diarrhea virus antigens c OE albumen it is necessary to be 80 ~ 85% with deacetylation, concentration is 0.5 ~ 0.6（v / v）Second
Acid solution, to dissolve shitosan, can be only achieved good envelop rate.Analysis reason may be with Porcine epidemic diarrhea virus antigens c OE
The space structure of albumen is relevant with albumen size.
Embodiment 5
Shitosan coated pig PEDV subunit vaccine prepared by embodiment 1 for immune Balb/c female small white mouse,
If 3 test group.It is respectively：1st, NS group（Physiological saline group）；2nd, COE group（Not coated PEDV subunit vaccine group）；3、
COE-CS group（Shitosan coated pig PEDV subunit vaccine）.Every group 30, carry out intragastric using No. 8 gastric perfusion needle, in
1st d carries out first immunisation, and the 14th d carries out booster immunization, each continuous immunity 2 d.Experimental period is 56 d, blood-sample withdrawal weekly,
3 mouse of every group of blood-sample withdrawal every time, collect serum ELISA and detect anti-COE protein antibodies production.And in 35d dislocation
Put to death mouse, the spleen cell extracting mouse carries out in vitro culture, after stimulating using COE albumen, in detection cells and supernatant
The secretion concentration of cell factor.
The collection of blood sample is that the 7th, 14,21,28,35,42,49,56 d receive after first immunisation
Collection peripheral blood serum.Concrete operations are as follows：Fixing mouse, eye socket takes blood to be placed in standing in the centrifuge tube of 1.5 mL.All samples
Blood is placed in 37 DEG C of insulating boxs after terminating and stands 1 h by collection, and then at 4 DEG C, 5 000 rpm are centrifuged 15 min, takes after serum again
It is centrifuged again by back condition, be then stored in -80 DEG C, for ELISA detection antibody level after packing serum.
The collection of culture supernatant after splenocyte stimulation
After first immunisation, the 35th d obtains the spleen cell of mouse.Alcohol leaching after death, will be placed at mouse orbit blood sampling
Bubble, aseptic dissection in super-clean bench, take every group of 3 mouse spleen to merge and put in cell sieve, then be placed in containing appropriate RPMI-
1640 culture mediums（Containing dual anti-, similarly hereinafter）6 porocyte culture plates in, after spleen stamp being opened with syringe needle, slow using filling in syringe
Slow grind spleen, to there is no massive texture.Discard cell sieve, the cell suspension of in the hole is transferred to 15 mL centrifuge tubes.Use 2 mL
RPMI-1640 culture medium cleans 6 orifice plates, is transferred in the lump in 15 mL centrifuge tubes, complements to 10 mL, 20 DEG C, 200 g are centrifuged 10
Min, abandons supernatant, and the RPMI-1640 culture medium with 3 mL is resuspended, adds the ACK erythrocyte cracked liquid of 9 mL, and room temperature places 5
Min, period gently overturns and mixes.By back pelleted by centrifugation, discard centrifugation supernatant, cultivate basic weight with 10 mL RPMI-1640
Outstanding cell washs 2 times, and 22 DEG C every time, 200 g are centrifuged 10 min, finally with 3 mL RPMI-1640 complete mediums（Containing 10 %
FBS）Re-suspended cell.Refiltered once to 6 orifice plates with screen pack, be transferred to 15 new mL centrifuge tubes, with 2 mL complete mediums
Clean 6 orifice plates once, go to 15 mL centrifuge tubes in the lump, mended to 5 mL with RPMI-1640 complete medium.Using trypan blue meter
Number.Finally with RPMI-1640 complete medium dilution splenocyte to 5 × 106Individual cell/mL.
Take 96 orifice plates, 3 sample wells of every group of setting, it is 5 × 10 that every hole adds 200 μ L concentration6The spleen of individual cell/mL is thin
Born of the same parents' suspension, adds the COE albumen that 4.8 μ L purify to be stimulated（Final concentration of 5 μ g/mL）, to add the hole of equivalent culture medium
As comparison.Aseptic PBS is added to prevent in the hole cell liquid from evaporating around 96 orifice plates.After stimulating 80 h, draw cell liquid, 4
DEG C, 8 000 rpm are centrifuged 10 min.Supernatant is taken to be stored in -80 DEG C of mensure for cell factor.
Humoral is analyzed
By enzyme-linked immunosorbent assay（ELISA）Measure the shitosan coated pig PEDV subunit of embodiment 1 preparation
The antibody horizontal that vaccine is induced in animal body.Concrete mode is as follows：
1. with being coated buffer solution by COE albumen (222 μ g/mL) by 1:500 dilution proportion, is added by the every hole of 100 L
In ELISA Plate, it is placed in 4 DEG C overnight.
2. take out and be coated ELISA Plate overnight, abandon and be coated liquid, wash plate 3 times with every hole cleaning solution of 250 μ L, soak every time
Steep 1 min, be subsequently adding the confining liquid in 100 L/ holes, 37 DEG C of reaction 2h.
3. take out and wash plate（With 2）, serum is pressed 1 with serum dilution:After 20 dilutions, add ELISA Plate by 100 L/ holes,
37 DEG C of reaction 60 min.
4. take out and wash plate（With 2）, add 1 by the every hole of 100 L:1 000 ELIAS secondary antibody, 37 DEG C of reaction 60 min.
5. take out and wash plate（With 2）, add substrate buffer solution by the every hole of 100 L, room temperature reaction 5～15 min, by 100 L
Every hole adds terminate liquid, mixes, and surveys OD450 light absorption value on ELIASA.
Result of the test is as shown in table 1：As can be seen from the table, from the beginning of 14d, that is, there is the anti-of significantly anti-COE albumen
Body produces, wherein 14d, 21d, 42d and 49d, the specific antibody water that shitosan coated pig PEDV subunit vaccine produces
Flat is higher than naked PEDV subunit vaccine group.The effect that is coated that shitosan is described is significantly effective.
IgG antibody level in table 1 ELISA detection mice serum（OD450）
Annotation：1st, data coding method is mean value ± standard error；
2nd, data is according to being longitudinally compared, and data end does not mark letter or is labeled with same letter and represents difference not
Significantly（P ＞ 0.05,）, mark different letter explanation significant difference between any two（P ＜ 0.05）.
Cell factor tells on analysis
Stimulate the mouse boosting cell of in vitro culture using specific antigen COE albumen, and adopt Mouse Cytokine/
Chemokine Magnetic Bead Panel detection stimulates the secretion level of the cell factor in supernatant, can react mouse
The water skin of cell factor secreted when by antigenic stimulus of immunocyte.Specific implementation method is as follows：
The preparation of related reagent：
1) preparation of antibody labeling microballoon
This test uses the microballoon bottle of independent packing, processes 30 s, oscillator vibration 1 first on ultrasonoscope
Minute.Then plus Assay Buffer is to final volume from each antibody microballoon bottle, each liquid drawing 90 μ L adds hybrid bottle,
For 3 mL, vibration mixing, unused portion can preserve at most January at 2 ~ 8 DEG C.
2) preparation of quality control group
Before the test, respectively using deionized water dissolving quality control group 1 and the quality control group 2 of 250 μ L.Repeatedly turn over
Spin-the-bottle being sufficiently mixed, stands 5 ~ 10 min, and control group is transferred in the EP pipe of suitable mark, unused portion-
January is at most preserved under the conditions of 20 DEG C.
3) preparation of cleaning buffer solution (WashBuffer)
Take l0 × Wash Buffer to shake up after recovering room temperature, make all salt dissolvings, diluted with 540 mL deionized waters
60 mL 10 × Wash Buffer, unused portion can preserve at most January at 2 ~ 8 DEG C.
4) preparation of cytokine standards product
Using front, with 250 μ L deionized water dissolving cytokine standards product, now standard concentration reaches 10 000
Pg/mL, repeatedly to be sufficiently mixed, oscillator vibrates 10 s to upset bottle, stands 5-l0 min, standard items have been transferred to suitably
In the EP pipe of mark.This manages as 10 000 pg/mL standard QCs, and unused portion at most preserves January under the conditions of -20 DEG C.Point
Not 5 EP pipes of mark 2 000,400,80,16 and 3.2 pg/mL, is often separately added into 200 μ L Assay Buffer in pipe.
Serial dilution, draws 50 μ L from the primary standard QC of 10 000 pg/mL and adds in 2 000 pg/mL pipes, be sufficiently mixed,
Draw 50 μ L to add to 400 pg/mL pipes from 2 000 pg/mL pipes, be sufficiently mixed, middle absorption from 400 pg/mL pipes
50 μ L add in 80 pg/mL pipes, are sufficiently mixed, draw 50 μ L and add to 3.2 pg/mL pipes, fill from 80 pg/mL pipes
Divide mixing.Assay Buffer used by 0 pg/mL standard items (background).
Detecting step is carried out with reference to specification, and key step is as follows：
1）All reagent are recovered to room temperature（20-25℃）.
2）Take 96 orifice plates, every hole adds 200 μ L Wash Buffer to be prewetted, using 96 hole shaking tables in room temperature after sealing
Shake 10 min.
3）Reverse plank, after gently outwelling Wash Buffer, siphon away the liquid of residual using blotting paper.
4）Standard items or quality controling product are added in suitable hole, Assay Buffer is as the mark of 0 pg/mL
Quasi- product (simultaneously as background hole).
5）Plus 25 μ L Assay Buffer in sample well.
6）Plus 25 μ L suitable matrix liquid in background hole, standard sample wells or quality control hole, when detection sample be
During serum, Serum Matrix that kit provide is used as matrix liquid, when the sample of detection is cells and supernatant
Wait, use cell culture medium as matrix liquid.
7）Plus 25 μ L sample in sample well（Serum needs to do 1 using Assay Buffer:1 dilution）.
8）Rock magnetic bead mixing bottle, and add the magnetic bead of 25 μ L and enter in each hole（Uninterrupted during sample-adding
Ground shaking bottle to avoid sedimentation）.
9）First sealed after plate using plate film, reuse aluminium foil and plank is wrapped up.It is placed on 96 hole shaking tables, shake in 2-8 DEG C
Overnight（16-18 h）.
10）After removing aluminium foil and sealed membrane, it is placed on magnetic frame and stands 60 s, gently outwell the liquid in plate, and inhaling
The liquid of residual is gently sopped up on water paper.Subsequently take off 96 orifice plates, add the Wash Buffer of 200 μ L, be placed in 96 orifice plates and shake
60 s, the operation of repeated washing is shaken on bed.Altogether wash plate 2 times.
11）Anti- (the attention of 25 μ L bis- is added in every hole：Two anti-should warm to room temperature before addition again).
12）Sealed using sealed membrane, aluminium foil lucifuge, be incubated 1 h on (20-25 DEG C) shaking table at room temperature.
13）Streptavidin-Phycoerythrin (the streptomysin algae red egg of 25 μ L is added in the hole anti-containing two
In vain).
14）Sealed using sealed membrane, aluminium foil lucifuge, be incubated 1 h on (20-25 DEG C) shaking table at room temperature.
15）Repeat the（10）Wash plate process.
16）150 μ L sheath fluids are added, shaking table shakes 5 min making microballoon is in suspended state in all holes.
17）Plank is inserted in Luminex 200 and is detected.Entered with MILLIPLEX Analyst 5.1 software
Row data analysis.
Shown in result of the test such as Fig. 1 ~ 8 it can be seen that COE-CS group GM-CSF, IFN γ, IL2, IL4, IL5, IL6,
This 8 kinds of levels of cytokine secretion all pole of IL10, TNF α is significantly higher than NS group and COE group（P ＜ 0.01）, illustrate that shitosan is coated
Pig PEDV subunit vaccine can induce and produce stronger cell immune response.
The above be only the present invention be preferable to carry out method it is noted that ordinary skill people for the art
Member, under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (3)
1. a kind of preparation method of orally available Porcine epidemic diarrhea virus subunit vaccine is it is characterised in that include following walking
Suddenly：
S1. the shitosan being 80 ~ 85% by deacetylation is dissolved in the acetic acid of 0.5 ~ 0.6 percent by volume, prepares 0.2 mass body
The chitosan solution of long-pending percentage；
S2. take the chitosan solution of 4 mL step S1 preparations, being added dropwise over 1 mL concentration while stirring is 50 ~ 100 μ g/mL pigs
Epidemic diarrhea virus antigens c OE albumen, albumen is stirred for 15 ~ 20 min after adding；
S3. the sodium tripolyphosphate being 0.2 mass percent by volume by 1 ~ 2 mL concentration, is added in S2 solution, continues stirring 5
~ 10min obtains final product vaccine.
2. preparation method according to claim 1 is it is characterised in that the deacetylation of shitosan described in S1 is 85%.
3. preparation method according to claim 1 is it is characterised in that by deacetylation during chitosan solution configuration described in S1
Shitosan for 85% is dissolved in the acetic acid of 0.5 percent by volume.
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| CN109172817B (en) * | 2018-08-16 | 2021-10-26 | 华中农业大学 | Preparation method and application of chitosan microspheres of attenuated live vaccine of porcine epidemic diarrhea virus |
| CN110859955A (en) * | 2019-12-02 | 2020-03-06 | 黑龙江大学 | Preparation method of porcine epidemic diarrhea virus nano vaccine |
| CN111544583A (en) * | 2020-05-15 | 2020-08-18 | 西北农林科技大学 | Application of CS/HPMCP nano particles in oral vaccine delivery |
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| CN103074291A (en) * | 2012-12-20 | 2013-05-01 | 大连大学 | Recombinant porcine epidemic diarrhea virus (PEDV) lactococcus lactis and its construction method and use |
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