CN107904247A - One kind restructuring Eimeria species Alphavirus particle vaccines and preparation method - Google Patents
One kind restructuring Eimeria species Alphavirus particle vaccines and preparation method Download PDFInfo
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Abstract
The present invention provides a kind of restructuring Eimeria species Alphavirus particle vaccines and preparation method; the Alphavirus vector pSMART2b based on SFV is selected to be connected with Eimeria tenella protective antigen gene Repair_PSII; construct recombinant plasmid pSMART2b Repair_PSII and the Alphavirus particle vaccines of the gene containing Repair_PSII are packaged into helper plasmid pSHCAR, there is the advantage that protecting effect is good, induction immune level is high, safe.
Description
Technical field
The present invention provides one kind to recombinate Eimeria species Alphavirus particle vaccines, which can be used for prevention chicken ball
Parasitosis, while restructuring Eimeria species Alphavirus preparation method of granules is also disclosed, belong to gene engineering technology field.
Background technology
Chicken coccidiasis be by sporozoa (Sporozoa), Amy otology (Eimeriidae), Eimeria (Eimeria)
Coccidia caused by it is a kind of colonize in chicken intestinal parasitic protozoa disease.The disease is in worldwide distribution, infection rate height, harmfulness
Greatly.The Chicks ' Growth that infects coccidia is slow, apathetic, feather is inverse vertical, causes chick dead when serious, the death rate can reach
To 20%-80%.Adult Chicken is mostly carrier, the illnesss such as inverse vertical, the laying rate decline of growth retardation, feather occurs.According to incomplete system
Meter, economic loss caused by being often only chicken coccidiasis one worldwide are just up to more than 3,000,000,000 dollars.
Prevention chicken coccidiasis relies primarily on medicine at present, but is just a makeshift arrangement for a long time by medical treatment.It is first
The problem of drug resistance, classical diclazuril, salinomycin, sulfanilamide (SN) quinoline dislike woods, sulfaclozine, sulfadimethoxine etc.
Has there is height drug resistance in medicine；Secondly medicament residue problem, it is anti-that the Ministry of Agriculture's long-term abuse of withdrawal time regulation is run counter in chicken farm
Coccidia medicine, caused result are exactly that meat egg product medicament residue is exceeded, and it is healthy and safe to seriously endanger human food；It is finally poultry
Factory researches and develops cost increase caused by new anticoccidial drug for detection drug resistance and largely using anticoccidial drug and pharmaceutical factory.By with
These upper factors influence, and existing more and more veterinary drug drugmakers remove from the field of prevention coccidia medicine both at home and abroad at present
Go out, it is existing at this stage to have no that new chemical synthesis anticoccidial drug comes out for many years.
Many drawbacks existing for medical treatment so that sight is turned to vaccines arts by researcher, to be exempted from by vaccine
The approach of epidemic disease come achieve the purpose that prevent chicken coccidiasis.
The more vaccine studied at this stage can be generally divided into three classes：Live seedling, subunit vaccine and DNA vaccination.Live seedling
Preparation process is complicated, transport is inconvenient, and returns the shortcomings of strong in the presence of scattered poison and virulence, under the conditions of unsuitable large-scale intensive culture
Multilayered cage culture pattern, therefore live seedling is huge to large-scale promotion difficulty；Subunit vaccine there is protected protein expression quantity it is low,
The defects of modification is difficult, immunogenicity is relatively low, can not provide strong anticoccidial protective effect；DNA vaccination is compared to tradition
Vaccine safety it is good, itself will not dissipate poison or produce toxin and trigger Other diseases, but there is also problems for DNA vaccination：
As conventional carrier for expression of eukaryon expression quantity is low, antigen immunogenicity is weak and foreign gene and host gene are integrated and cause to be mutated
Deng, therefore DNA vaccination also only exists in the laboratory research stage at present, not commercialization.And the appearance of alphavirus vectors, to solve
Problem above provides good thinking.
Alphavirus (Alphavirus) belongs to Togaviridae (Togavirus family), and inhereditary material is single-stranded positive
RNA, the size of its genome is about 12kb, there is 2 open reading frames, and 5,End be Alphavirus self-replication, transcription needed for enzyme
With the non-structural protein white area of signal, and the end has cap sequence and a CMV strong promoters, the area include nsP1, nsP2,
NsP3 and nsP4, the part have virulent packaging signal；3,The marismortui structural protein sequence for encoding alphavirus is held, RNA (-) is multiple
The RNA and subgenomic RNA of full-length genome are synthesized under the action of enzyme processed, is finally translated under the action of sub-genomic promoter
Capsid protein (Capsid), the structural proteins such as E1, E2,6K of virus, and the end has termination signal (A69) and the strong transcription whole
Stop signal SV40poly (A), termination signal make its normal transcription and polyA (A) stern construction strengthen its stability, make its completion table
Reach.Alphavirus vector can be replicated quickly at a high level after importing cell, and research confirms the duplication amount energy of SFV in the cell
Up to 2 × 105A/cell, remote unconventional carrier for expression of eukaryon.Alphavirus vector can also inducing cell apoptosis, outside
Source gene just occurs apoptosis and is cleared up by host cell without being incorporated on host cell after being expressed only in endochylema, greatly improve
The security of vaccine.
The content of the invention
The object of the present invention is to provide one kind restructuring Eimeria species Alphavirus particle vaccines and preparation method, is that one kind includes chicken
The recombinant alphavirus particle vaccine of coccidia Repair_PSII genes.
A kind of restructuring Eimeria species Alphavirus particle vaccines of the present invention and preparation method, its technical solution is such as
Under：
It is packaged into the recombinant alphavirus particle of the genes of Repair_PSII containing Eimeria species it may first have to which acquisition can transmit and express this
The recombinant plasmid pSMART2b-Repair_PSII of gene, and need to confirm that its structure is correct and can express.Then matter will be recombinated
Grain pSMART2b-Repair_PSII and helper plasmid pSHCAR is transfected into the host cell of Alphavirus（Such as 293 cells）, then
Collect virus stock solution used and infect BHK-21 cells, finally obtain the recombinant alphavirus particle of the genes of Repair_PSII containing Eimeria species
Vaccine.
1st, the structure of recombinant plasmid pSMART2b-Repair_PSII and expression
It is cDNA to extract the total serum IgE of Eimeria tenella and reverse transcription first, goes out Repair_PSII genes by PCR amplification；
Then connect, convert and clone into plasmid pMD18-T-Repair_PSII, and by restriction enzyme BamHI and ClaI with
And biological order-checking identification connection, sticky end Repair_PSII genes are connected to Alphavirus vector with method
On pSMART2b；Then recombinant plasmid pSMART2b-Repair_PSII is transfected into 293 cells, Western blot identifications
Repair_PSII expression conditions；
2nd, Eimeria species Alphavirus particle vaccines are recombinated to prepare
It will identify and express correct recombinant plasmid pSMART2b-Repair_PSII and helper plasmid pSHCAR liposome methods
293 cells are transfected into, 6 change liquid when small, 48 collect virus stock solution used when small.α-gruel albumen of 1/20 volume is added into virus stock solution used
Enzyme solutions（10 g/L）, p62 glycoprotein (is cracked into E2 and E3 albumen to activate disease by incubation at room temperature 40min to activate virus
Poison), then add the Aprotinin aprotinin of 1/15 volume（10 g/L）, it is incubated at room temperature 5 min；Take the disease after appropriate activation
Toxogen liquid inductance contaminates BHK-21 cells, 48 it is small when after collect restructuring Eimeria species Alphavirus particle be used to taking the photograph transmission electron microscope observing and
Western blot identify the expression of Repair_PSII genes；
3rd, Eimeria species Alphavirus particle vaccines Optimization of preparation is recombinated
With reference to the transfection reagent Lipofectamine of Invitrogen companiesTM3000 specifications, by recombinant plasmid pSMART2b-
Repair_PSII and helper plasmid pSHCAR is according to molar ratio 1:1.5 (common 2.5ug) cotransfection enters 293 cells, and 6 change when small
Liquid, 48 collect virus stock solution used when small；Meanwhile helper plasmid pSHCAR 2.5ug are mono- to be transfected into 293 cells using first taking, 6 it is small when
Change liquid, 24 hours whens, take recombinant plasmid pSMART2b-Repair_PSII 2.5ug are mono- to be transfected into 293 cells again, and 6 change liquid when small,
48 collect virus stock solution used when small；The virus stock solution used of optimization is finally infected into BHK-21 cells respectively, takes a small amount of restructuring Eimeria species first
Virion shoots transmission electron microscope.
The positive effect of the present invention is：Provide a kind of restructuring Alphavirus for including Eimeria species Repair_PSII genes
Particle vaccines, select Alphavirus vector pSMART2b and Eimeria tenella protective antigen gene based on SFV
Repair_PSII connections, construct recombinant plasmid pSMART2b-Repair_PSII and are packaged into helper plasmid pSHCAR and contain
The Alphavirus particle vaccines of Repair_PSII genes, have the advantage that protecting effect is good, induction immune level is high, safe.
Brief description of the drawings
Fig. 1 Repair_PSII gene electrophoretograms；
Fig. 2 restriction enzymes identify pMD18-T-Repair_PSII electrophoretograms；
Fig. 3 restriction enzymes identify recombinant plasmid pSMART2b-Repair_PSII electrophoretograms；
Fig. 4 Western blot methods are identified that recombinant plasmid is expressed in 293 cells and are schemed；
Fig. 5 Western blot methods identification restructuring Eimeria species Alphavirus particle figure；
Fig. 6 recombinates Eimeria species Alphavirus particle transmission electron microscope picture；
Fig. 7 recombinant alphavirus particles infect the X-gal analysis charts of BHK-21 cells；
Cell factor figure of changing in chicken serum after Fig. 8 double-antibody sandwich elisas measure is immune.
Embodiment
Further illustrated the description present invention by following embodiments, is not limit the invention in any way.Below with real
Example is applied to further illustrate the essentiality content of the present invention, but present disclosure is not limited thereto.
Embodiment 1
Structure, identification and the expression of recombinant plasmid pSMART2b-Repair_PSII
1st, 5.0 software Design primers PCR methods of Primer amplification Eimeria species Repair_PSII genes, sequence such as SEQ no.1 institutes
Show；
Upstream：5’-CGCGGATCCGATGGACTGGCTTTCGAGCGAG-3’(BamH I)
Downstream：5’-CCCATCGATACGATACTTGGCAATCTCG-3’ (ClaI)
2nd, using Eimeria species cDNA as template, PCR methods amplification Eimeria species Repair_PSII genes, do being connected to after purification process gram
Grand carrier pMD18-T, converts DH5 α competent cells and clones, and is cut with restriction enzyme BamHI and ClaI containing viscosity
The Repair_PSII genetic fragments of end, the pSMART2b carriers of the cohesive end notch containing BamHI and ClaI are obtained with method（Ginseng
See Fig. 1-4）；
3rd, according to the pSMART2b-Repair_PSII 2.5ug of plasmid containing endotoxin-free, transfection reagent
LipofectamineTMThe condition of 3000 7.5ul prepares transfection composite, and it is 293 thin to 60%-80% degree of converging to be transferred to culture
Born of the same parents, when transfection 6 is small after change liquid, using concentration for 2.0% hyclone and 1.0% dual anti-DMEM culture mediums continue to cultivate 48 it is small when.
293 cells after when collection culture 48 is small, cell lysis simultaneously obtains holoprotein, with rabbit-anti Eimeria species Repair_PSII albumen
Polyvalent antibody does Western blot identifications（Referring to Fig. 5）.
Embodiment 2
Eimeria species Alphavirus particle vaccines are recombinated to prepare
1st, it will identify and express correct recombinant plasmid pSMART2b-Repair_PSII, helper plasmid pSHCAR and transfection reagent
LipofectamineTM3000 according to 1ug:1.5ug:The ratio of 7.5ul is made transfection composite cotransfection and enters degree of converging 60-80%
293 cells, 6 changed into when small the dual anti-DMEM culture mediums containing 2.0% hyclone and 1.0% continue culture 48 it is small when, 48 it is small when
After collect virus stock solution used.
2nd, the Chymetin solution of 1/20 volume is added into virus stock solution used（10g/L）, be incubated at room temperature 40 min after (for
P62 glycoprotein is cracked into E2 and E3 albumen with activated virus) add 1/15 volume Aprotinin aprotinin（10g/L）, room
Temperature is incubated 5min.Then the virus stock solution used of activation is infected into BHK-21 cells, 2 it is small when after change into containing 2.0% hyclone and 1.0%
Dual anti-DMEM culture mediums continue culture 48 it is small when, 48 it is small when after collect restructuring Eimeria species Alphavirus particle.Finally take a small amount of first
Virion shoots transmission electron microscope and the identification Repair_PSII gene expressions of Western blot methods（Referring to Fig. 6）.
Embodiment 3
Infect the X-gal analyses of BHK-21 cells
Positive cell after X-gal native stainings is in large cortical cells, can rSFV-Repair_PSII particles titre according to a preliminary estimate, 400
Under × (40 × eyepiece+10 × object lens) multiple, 3 visuals field are looked for calculate the average of large cortical cells at random, it is then determined that titre.Each
Visual field large cortical cells number × platen area/mono- field area, the actually used viral suspension volumes (ml) of acquired results × lml/, i.e.,
Draw virus titer (IU/ml)（Referring to Fig. 7）.
Embodiment 4
Recombinate Eimeria species Alphavirus particle vaccines resisting Eimeria tenella protecting effect
The healthy chick of 160 13 ages in days is randomly divided into 4 groups, every group 40.Immune group is exempted from according to 14 ages in days one, 21 ages in days two
Exempt from, the mixed liquor of the 100 μ l recombinant alphavirus particles of every group of intramuscular injection of immune programme that 28 ages in days three are exempted from and 100 μ l adjuvants；Adjuvant pair
According to group 100 μ l adjuvants are only injected according to above-mentioned immune programme；Set up negative control group at the same time（White control）And positive controls（It is red
Control）, white control group is not immune not to attack worm, and red control group is not immune to attack worm.Before immune and attack before worm and randomly select 5 for every group
Chicken carries out Culling heart blood and spleen lymphocyte separation, measure Serum Antibody level, IL-2, IL-4, IL-10 and IFN-γ
The index such as expression, mtt assay measure chicken splenic lymphocytes proliferative conditions.The tender Chinese mugwort of oral vaccination after third time is 7 days immune
U.S. ear ball worm Sporulated Oocysts 1 × 104It is a/only to carry out attacking worm experiment, and carry out survival rate, body weight increase rate, caecum lesion meter
Point, the statistical survey of the index such as OPG, ACI.Finally to integrate anticoccidial index（ACI）Chicken is exempted to evaluate Alphavirus particle
Epidemic disease protecting effect（Referring to Fig. 8）.
The ACI overall merits of each group experimental chicken are shown in 1 table：
1. recombinant plasmid of table resistsE. tenellaAnticoccidial index
| Group | Survival rate (%) | Body weight increase rate (%) | Pathological changes value | Oocyst value | Anticoccidial index（ACI) |
| RSFV-Repair_PSII groups | 100 | 92.98 | 6 | 1 | 185.98 |
| Adjuvant group | 90 | 64.91 | 24 | 10 | 120.91 |
| Positive control | 90 | 56.14 | 27 | 10 | 109.14 |
| Negative control | 100 | 100 | 0 | 0 | 200 |
Conclusion：Ginseng is shown in Table 1, and the synthesis anticoccidial index that rSFV-Repair_PSII groups are immunized is 185.98, right with the positive
Compared according to the anticoccidial index 109.14 of group, hence it is evident that higher than positive controls, it is shown that good protecting effect.
Sequence table
<110>Jilin University
<120>One kind restructuring Eimeria species Alphavirus particle vaccines and preparation method
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 762
<212> DNA
<213>Eimeria tenella (Eimeriatenella)
<400> 1
atggactggc tttcgagcga gaaccaggcg ctgaagttgc cagtatggcc cactgcagtt 60
gagaaggtgc tgctttccga ggaacccact cctgatgatt tcaccgagga gctccaaggg 120
ggtgcaccca acatgaacat gttgcctggc ttcaccacac tggagaacta ccccaaccca 180
atggtcaact cgatggcctg cagaagatgg ggcatggaag tttccttcgt ctgcgaccca 240
gatcatgtgt tttctagcgg cacgttggac agaatagagg aacgcctttc caatatccga 300
cacaactcgt cccaccgctg cgtcgacgaa tacgtctcct acccctttgg agtggcgatt 360
gttcaagaac tgccatacgg tgttgatgcg aacacgtttg cgaccgagct catagatcgc 420
tggggacttg gacacagcaa gtgccacgat gggcttctgc tgttgtatgt tgcgtccgac 480
ggtgacgttt cgctcaagtg gaagaaagga gtcgagcctg agatcaactt caggacatat 540
agcgggctcc tgcgcgaatt cagaaagaat gggggaaaac tgtccgctgg aagggctctt 600
gaggccgatg tcattgcagt tggacagcat ctgacagggg aaattcttcc acctcgtcag 660
accccgcatc tcgccgtgtt gctcacaatc gccagtattg tcgcccttgc gtatcttgca 720
tgcgccgcca ctgtcatttc tgacgagatt gccaagtatc gt 762
Claims (2)
1. a kind of Repair_PSII genes, its sequence is as shown in SEQ no.1.
2. one kind restructuring Eimeria species Alphavirus particle vaccines preparation method, comprises the following steps：
1）The structure of recombinant plasmid pSMART2b-Repair_PSII and expression
The total serum IgE and reverse transcription for extracting Eimeria tenella are cDNA, go out Repair_PSII genes by PCR amplification；Even
Connect, convert and clone into plasmid pMD18-T-Repair_PSII, and pass through restriction enzyme BamHI and ClaI and biology
Sticky end Repair_PSII genes, are connected on Alphavirus vector pSMART2b by sequencing identification connection with method；
Then recombinant plasmid pSMART2b-Repair_PSII is transfected into 293 cells, Western blot identification Repair_PSII bases
Because of expression；
2）Eimeria species Alphavirus particle vaccines are recombinated to prepare
It will identify and express correct recombinant plasmid pSMART2b-Repair_PSII and helper plasmid pSHCAR liposome methods
293 cells are transfected into, 6 change liquid when small, 48 collect virus stock solution used when small；α-gruel albumen of 1/20 volume is added into virus stock solution used
Enzyme solutions（10 g/L）, it is incubated at room temperature 40min and p62 glycoprotein is cracked into E2 and E3 albumen with activated virus；Add 1/15 body
Long-pending Aprotinin aprotinin（10 g/L）, it is incubated at room temperature 5 min；Take the virus stock solution used infection BHK-21 after appropriate activation thin
Born of the same parents, 48 it is small when after collect restructuring Eimeria species Alphavirus particle and be used to take the photograph transmission electron microscope observing and Western blot identifications
The expression of Repair_PSII genes；
3）Recombinate Eimeria species Alphavirus particle vaccines Optimization of preparation
By recombinant plasmid pSMART2b-Repair_PSII and helper plasmid pSHCAR according to molar ratio 1:1.5 common 2.5ug is total to
293 cells are transfected into, 6 change liquid when small, 48 collect virus stock solution used when small；Contaminated using helper plasmid pSHCAR 2.5ug single-turns are first taken
Enter 293 cells, 6 change liquid when small, 24 hours whens take recombinant plasmid pSMART2b-Repair_PSII 2.5ug are mono- to be transfected into 293 again
Cell, 6 change liquid when small, 48 collect virus stock solution used when small；The virus stock solution used of optimization is infected into BHK-21 cells respectively, up to recombinating
Eimeria species Alphavirus particle.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113041344A (en) * | 2021-03-19 | 2021-06-29 | 吉林大学 | Eimeria tenella chitosan/nanoparticle and preparation method thereof |
| WO2022165789A1 (en) * | 2021-02-03 | 2022-08-11 | 郑州大学 | Cis replicon rna construct for efficiently expressing target protein |
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| CN113041344B (en) * | 2021-03-19 | 2023-07-18 | 吉林大学 | Chitosan/nanoparticle for eimeria tenella and preparation method thereof |
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