CN1803195A - Immunity regulating type Eimeria tenella DNA vaccine for preventing and treating chicken coccidiosis - Google Patents
Immunity regulating type Eimeria tenella DNA vaccine for preventing and treating chicken coccidiosis Download PDFInfo
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Abstract
The invention provides an immunoregulation type DNA vaccine with actions of preventing and treating chicken coccidiosis, which comprises calcium regulation bonding domain protein kinase genes pE+K2 of Eimeria tenella and chicken interleukin-2 genes, and a section of proteinase hydrolytic sequence is introduced to the 5' end of the chIL-2 gene.
Description
Technical field
The present invention relates to field of biological veterinary, is a kind of dna vaccination with immunity regulating type of prevention chicken coccidiosis effect.This vaccine contains Eimeria tenella (Eimeria tenella) calcium transfers binding domain protein kinase gene pEtK2 and chicken interleukin-2 gene (chIL-2).And introduced one section protease specificity hydrolysis sequence at 5 ' end of chIL-2 gene.This vaccine includes a plurality of T cell immune response elements.
Background technology
Chicken coccidiosis is the important parasite disease of harm aviculture, and the economic loss that the whole world causes because of coccidiosis every year is about 2,000,000,000 pounds.This disease causes by 7 kinds of coccidiosiss of Eimeria, wherein the harm maximum that causes with Eimeria tenella (E.tenella).The medical treatment chicken coccidiosis has brought a series of problem at present.As chemical sproof generation, harm that drug residue causes environment or the like.These problems have caused people's growing interest.People urgently wish to find a kind of effective way to prevent and treat chicken coccidiosis.
Use strong malicious Seedling and cause the weak coccidiosis Seedling a kind of means of can yet be regarded as that inoculate against, but strong malicious Seedling is only applicable to kind of a chicken, not exclusively be applicable to laying hen and broiler, though the available weak malicious coccidiosis Seedling of broiler, but there is poor stability, inoculum concentration is restive, and problems such as expense height make these coccidiosis Seedlings can not obtain good development and utilization.Along with molecular biology and immunologic fast development, people urgently wish to develop the novel gene engineered vaccine by genetic engineering means, for the control of chicken coccidiosis provides new means.
Chicken is the comprehensive defence performance of body to pathogen to the immunoreation of coccidiosis, and existing nonspecific immunity has specificity acquired immunity-humoral immunization and cellular immunization again.Humoral immunoresponse(HI) realizes by the bone-marrow-derived lymphocyte that relies on the bursa of Fabricius that mainly cellullar immunologic response is mainly realized by the T cell that relies on thymus.The immunity that has been found that chicken coccidiosis can be suppressed because of extracing thymus, extracts the bursa of Fabricius and does not then have influence.The effect that antibody in the protective immunity of coccidiosis is described is less.But the result by adoptive immunity lymphocyte shift experiment is verified, and t cell immune response is in core status in immunity to coccidiosis is replied.
Nucleic acid vaccine is a kind of new generation vaccine that grew up in recent years, easy and simple to handle because of it, the safety that had both had recombinant subunit vaccine has the advantage that attenuated live vaccine is induced efficient, the persistence of comprehensive immunne response again, becomes one of focus of current vaccine research.In recent years, aspect anti-parasitic-infectious, nucleic acid vaccine research has launched positive exploration.
The pEtK2 gene is Dunn had proliferation function to lymphocyte by use in 1996 the prepared antiserum of antigen, it is C α Em70/l antiserum, from E.tenella zygoblast cDNA library, screen, this gene size is 1464bp, the calcium that contains of coding 55KD is transferred protein kinase (EtCDPK, the registration number AY389513 of Genebank) in conjunction with the territory.This albumen is present in zygoblast and the merozoite apical organ, can stimulate the T lymphopoiesis of infected chicken, can provide the better protect effect for infected chicken.
Cytokine is one group of immunoregulatory factor with extensive biologic activity that immunocyte or non-immunocyte produce in the body, can activate and regulate immunologically competent cell in vivo, and the generation and the adjusting of immunne response had important function.The main effect of IL-2 is to promote CD8
+T cell and CD4
+T cell differentiation propagation is safeguarded the growth of T cell, promotes natural killer cell (NK) and B cell function.So be T cell growth factor (TCGF) again.Now confirmed the effect of IL-2 in anti-E.tenella and anti-E.acerv μ lina infection.(Caarter LL.et al. such as Gaarter, Regulation of T cell subsets fronmnative to memory.J Immunol.1998,21 (3): 181-187) be reported in and infect the 3rd day and the 5th day and can make egg capsule output reduction to chicken injection IL-2.Li Xiangrui etc. (Li Xiangrui, golden red, Lu Jingliang. the clone of chicken interleukin-2 cDNA. Agricultural University Of Nanjing's journal, 1999,22 (2): 80-84) cloned and expressed the IL-2 gene of chicken.
Summary of the invention
The invention provides a kind of dna vaccination with immunity regulating type of prevention or the effect of treatment chicken coccidiosis.
The invention provides the method that a kind of preparation has the immunity regulating type DNA vaccine of prevention or the effect of treatment chicken coccidiosis.
In addition, the present invention also provides the purposes of described dna vaccination.
The present invention partly is based upon on the inventor's the following discovery: contain Eimeria tenella (E.tenella) calcium and transfer the eucaryon plasmid carrier of binding domain protein kinase gene pEtK2 to provide effective immunoprotection at Eimeria tenella for chicken; Contain chIL-2 and can improve inducing of Eimeria (E.tenella) infected chicken cecal tonsil and spleen pouring commentaries on classics level and chIL-2, and can reduce the lesion score of coccidium infection and the egg sac number in the feces; During dna vaccination, chicken interleukin-2-2 gene is introduced eucaryon plasmid with coccidiosis protective gene (calcium accent binding domain protein kinase gene pEtK2) in preparation, can further strengthen described coccidiosis protecting group because of immune protective effect.
An aspect, the invention provides a kind of dna vaccination with immunity regulating type of prevention or the effect of treatment chicken coccidiosis, this vaccine comprises the eucaryon plasmid carrier that has wherein inserted Eimeria tenella (E.tenella) calcium accent binding domain protein kinase gene pEtK2.
Dna vaccination of the present invention uses the eucaryon plasmid expression vector as structural framing, and described eucaryon plasmid carrier removes has the expression vector primary element, and the place has inserted genes of interest at restriction enzyme site.Any eucaryon plasmid carrier known in the art all can be used for the present invention, for example, includes but not limited to pcDNA3.0, pcDNA3.1, pVAX1.0 and pcDNA4/His Max.In a concrete embodiment preferred of the present invention, described eucaryon plasmid carrier is pcDNA4/His Max (available from an American I nvitrogen company).
The present invention uses has immunogenic pEtK2 gene; this gene is a kind of coccidiosis protective antigen gene; size is 1464bp; be to use C α Em70/1 antiserum (using a kind of antiserum that lymphocyte is had the antigen preparation of proliferation function), from E.tenella zygoblast cDNA library, screen.The protein of this gene code is present in zygoblast and the merozoite apical organ, can stimulate the T lymphopoiesis of infected chicken, can provide good protective effect for infected chicken.
In a preferred embodiment of the invention, described vaccine further also comprises to link together with described gene pEtK2 and inserts the chicken interleukin-2 gene (chIL-2) of described eucaryon plasmid carrier, to strengthen the immunogenicity of dna vaccination.
The inventor successfully clones and has expressed the IL-2 gene (chIL-2) of chicken, and its nucleotide sequence is shown in 1483-1905 position among the SEQ ID NO:1.The inventor has further studied the influence of chicken IL-2 to the sick cellular immune level of Eimeria Tenella.In brief, test is established matched group, various dose oocyst infection group and various dose oocyst infection and is added interleukin II (IL-2) group, on the basis of infecting first, attack worm, the cecal tonsil of different time after infecting the back first and attacking worm and the pouring commentaries on classics level of splenocyte are detected, and the while keeps the score in conjunction with caecum lesion and the numeration of feces egg capsule is analyzed the result.The result shows that after infecting first, simple infection group cecal tonsil lymphocyte drenches the commentaries on classics level to be continued to be suppressed; Each oocyst infection group splenocyte level of conversion all descended at the 4th day.Injection IL-2 made 1,2,50,000 oocyst infection group cecal tonsils pouring commentaries on classics level be higher than the simple infection group respectively at the 4th, 6,9 day when infecting; 1,20,000 oocyst infection group splenocyte level of conversion obviously raise.Attacking behind the worm different infected group cecal tonsils and spleen drenches the commentaries on classics level and downward trend all occurs.Can improve cecal tonsil lymphocyte transformation level after adding IL-2, but the diversity of show dose not; The splenocyte level of conversion is higher than the simple infection group, is higher than matched group on the 9th day.This result of the test shows: 1) add reorganization chicken IL-2 and can make to infect first and attack in the lesion score of 10,000,20,000 oocyst infection groups behind the worm and the feces egg capsule output and obviously reduce; 2) cause behind the coccidium infection chickling that spleen and cecal tonsil lymphocyte immunity suppress, with the increasing of infectious agent amount, the immunosuppressant heighten degree; 3) recombinant il-2 can improve and infects cecal tonsil and splenocyte level of conversion first, infects spleen and the cecal tonsil immunosuppressant that causes first thereby improve coccidiosis; 4). recombinant il-2 can improve attacks cecal tonsil and splenocyte level of conversion behind the worm, and the worm of attacking against each other has immanoprotection action.
Based on above-mentioned discovery; therefore in one embodiment of the invention; introducing links together with described gene pEtK2 and inserts the chicken interleukin-2 gene (chIL-2) of described eucaryon plasmid carrier in dna vaccination of the present invention, found that the immanoprotection action of this dna vaccination further strengthens.
In a particularly preferred embodiment of the present invention, described vaccine also is included in and inserts one section protease Stuart factor between described gene pEtK2 and the gene chIL-2
aSpecificity hydrolysis sequential coding nucleotide sequence CCT ACC CTC GAT.Between coccidiosis protective gene and chIL-2, insert protease specificity hydrolysis sequential coding nucleotide sequence, so just can make and contain protease specificity site in the fusion rotein of coccidiosis protective antigen that dna vaccination of the present invention is expressed and chIL-2 in muscle cell or antigen-presenting cell.Thereby, can guarantee that protease can obtain coccidiosis antigen and chIL-2 albumen at this fusion rotein of protease specificity site hydrolysis, thereby makes these two kinds of albumen can form higher structure alone and bring into play function corresponding.
In a preferred specific embodiments of the present invention,, introduce the protease Stuart factor at 5 ' end of chIL-2 gene by the method for design of primers
aSpecificity hydrolysis sequential coding nucleotide sequence GAATTCCCTACCCTCGAT (shown in 1465-1482 position nucleotide among the SEQ ID NO:1), thus prepare a kind of fusion gene pEtK2-X that inserts in the eucaryon plasmid carrier
a-chIL-2 (direction by from 5 ' to 3 '), this fusion gene has the nucleotide sequence shown in the SEQ ID NO:1.
Aspect second of the present invention, provide a kind of method of production Eimeria tenella (Eimeriatenella) immunity regulating type DNA vaccine.In brief, its technology path is as follows:
1.PCR amplification preparation coccidiosis protective antigen gene pEtK2 and chicken interleukin-2 2 genes (chIL-2)
By the pcr amplification means, the clone obtains gene pEtK2 and chIL-2.Wherein in a concrete preferred embodiment of the present invention,, introduced one section protease factor Xa specificity hydrolysis sequential coding nucleotide sequence GAA TTC CCTACC CTC GAT at 5 ' end of chIL-2 gene by the design of primers means;
2.DNA the structure of vaccine reorganization eucaryon plasmid and corresponding gene engineering bacteria
Utilize the recombination method of molecular biology routine, prepare the reorganization eucaryon plasmid that contains pEtK2 or contain pEtK2-(Xa)-chIL-2, transform the host bacterium with it then, in a technical scheme of the present invention, described host bacterium is an e. coli jm109, and the screening back obtains the recombination engineering bacteria;
3. the extensive separation and purification of the fermentation of genetic engineering bacterium and dna vaccination reorganization eucaryon plasmid.
Aspect the 3rd of the present invention is the purposes of Eimeria tenella of the present invention (Eimeriatenella) immunity regulating type DNA vaccine.With after the containing pEtK2 or contain the recombinant DNA vaccine direct immunization animal of pEtK2-(Xa)-chIL-2 of purification; by every gram feces index, egg capsule slip, caecum lesion is kept the score and Multitest index such as anticoccidial index is found, the two all has good immune protective effect (wherein the latter protect effect more remarkable).Therefore, dna vaccination of the present invention can be used for preparing the pharmaceutical preparation that can prevent or treat chicken coccidiosis.
Description of drawings
Fig. 1 is the structural representation of immunity regulating type Eimeria tenella DNA vaccine for preventing and treating chicken coccidiosis of the present invention.
Fig. 2 is the structural representation of the recombiant plasmid pBV220-chIL-2 of the coding chicken interleukin-2 2 of this laboratory structure.
Fig. 3 is the structure flow chart of recombiant plasmid pMD18-T-pEtK2.
Fig. 4 is the structure flow chart of recombiant plasmid pET-chIL-2.
Fig. 5 is the structure flow chart of recombiant plasmid pcDNA4/His Max-pEtK2.
Fig. 6 is the structure flow chart of recombiant plasmid pcDNA4/His Max-pEtK2-Xa-chIL-2.
Fig. 7 for RT-PCR detect dna vaccination pcDNA4/His Max-pEtK2, pVAX1.0-pEtK2,
PcDNA4/His Max-pEtK2-chIL-2 and pVAX 1.0-pEtK2-chIL-2 transcribe situation in the chicken muscle cell, RT-PCR product agarose gel electrophoresis figure.
1 is DL2000 Marker; 2 and 3 is the RT-PCR amplified production (using the pEtK2 primer amplification) of pcDNA4/His Max-pEtK2 and pVAX 1.0-pEtK2 recombiant plasmid injection site muscle (chest muscle); 4 and 5 is the RT-PCR amplified production (using the pEtK2 primer amplification) of pcDNA4/His Max-pEtK2-chIL-2 and pVAX1.0-pEtK2-chIL-2 recombiant plasmid injection site muscle (chest muscle); 6 and the 7 RT-PCR amplified productions (using the IL-2 primer amplification) 8,9,10 and 11 for pcDNA4/His Max-pEtK2-Xa-chIL-2 and pVAX1.0-pEtK2-Xa-chIL-2 recombiant plasmid injection site muscle (chest muscle) are respectively pcDNA4/His Max-pEtK2, pVAX1.0-pEtK2, the RT-PCR amplified production (using the pEtK2 primer amplification) of pcDNA4/His Max-pEtK2-Xa-chIL-2 and the non-injection site of pVAX1.0-pEtK2-Xa-chIL-2 recombiant plasmid muscle (shank); 12 and 13 are respectively the RT-PCR amplified production (using the chIL-2 primer amplification) of pcDNA4/His Max-pEtK2-chIL-2 and the non-injection site of pVAX1.0-pEtK2-chIL-2 recombiant plasmid muscle (shank).
Fig. 8 detects the expression of results figure of dna vaccination in muscle that contains pEtK2 or pEtK2-(Xa)-chIL-2 for the Western-trace.
1 and 2 protein expression results for Western-trace detection pcDNA4/His Max-pEtK2 and the non-injection site of pVAX1.0-pEtK2 recombiant plasmid muscle (shank); 3 and 4 protein expression results for Western-trace detection pcDNA4/His Max-pEtK2-chIL-2 and the non-injection site of pVAX1.0-pEtK2-chIL-2 recombiant plasmid muscle (shank); 5 and 6 protein expression results for Western-trace detection pcDNA4/His Max-pEtK2-Xa-chIL-2 and pVAX1.0-pEtK2-Xa-chIL-2 recombiant plasmid injection site muscle (chest muscle); 7 and 8 protein expression results for Western-trace detection pcDNA4/His Max-pEtK2 and pVAX1.0-pEtK2 recombiant plasmid injection site muscle (chest muscle).
Fig. 9 is a pVAX1.0-pEtK2-Xa-chIL-2 vaccine immunity group, attacks night blindness enteropathy change figure behind the worm.
Figure 10 is the PBS immune group, attacks night blindness enteropathy change figure behind the worm.
Embodiment
Basic material: the recombiant plasmid pBV220-chIL-2 (this laboratory makes up, and structure is seen accompanying drawing 2) that contains chicken interleukin-2 gene (chIL-2); Carrier for expression of eukaryon pcDNA4/His Max and pVAX1.0 (American I nvitrogen company product); PMD18-T (precious biological (TaKaRa) Engineering Co., Ltd in Dalian) pET-28 (+) (U.S. Novagen company product).
The preparation of embodiment 1.pEtK2 gene:
1. synthetic primer P1, P2, sequence is respectively:
Disclosed nucleotide sequence (Genebank registration number AY389513) according to pEtK2 designs following primer:
P1：5’-AA
GGATCC CCAGCAGCGTCCAA-3′
P2：5’-GAA
GAATTCCGCTGCGGTATCTCCG-3’
Wherein underscore partly is respectively the restriction enzyme site BamHI and the EcoRI of introducing; Square frame partly is a start codon.
2. the extraction of the total RNA of coccidiosis:
Get the purebred spore egg capsule of E.tenella 0.1g (about 107) and put in the glass homogenizer, add 1ml Trizol reagent, grinds and the egg capsule wall was broken in 5 minutes, the total RNA of one-step method extraction zygoblast.Concrete steps are as follows: solution after the homogenate is put in the 1.5ml eppendorf pipe, add chloroform/iso pentane alcohol mixture of 24: 1 of 0.2mL, firmly shake sample 15s, put 5min on ice, leave heart 15min for 4 ℃ 12000, the water (upper strata) that carefully will contain RNA forwards in another eppendorf pipe and puts on ice, add the 0.5ml isopropyl alcohol, mixing sample and put room temperature 10min gently, 4 ℃ 12000 leaves heart 15min, RNA forms the yellow-white precipitation at the bottom of test tube, carefully remove isopropyl alcohol, add 1ml 70% washing with alcohol RNA precipitation, the RNA precipitation is suspended again by rocking or inhale to beat, leave heart 8min for 4 ℃ 12000, remove ethanol, drying at room temperature is surveyed A260/A280 ratio, add 20 μ l DEPC treated waters, put-20 ℃ of preservations.
3.cDNA synthetic
Add synthetic cDNA first chain of reagent in following ratio:
Total RNA 10 μ l
500μg/ml Oligo(dT) 1.0μl
Placed 5 minutes for 70 ℃ behind the mentioned reagent mixing, put cooled on ice immediately, add following ingredients again:
2.5mmol/L dNTP 3.5μl
RNASin (TaKaRa company) 1.0 μ l
5×RT buffer 5.0μl
200U/μl M-MLV 1.5μl
25mmol/L MgCL
2 23.0μl
No RNase water is mended to 25.0 μ l mixings, and low speed is instantaneous centrifugal, gets rid of down the drop on the tube wall, 37 ℃ of reaction 1h.90 ℃ of 5min deactivation reverse transcription.Reverse transcription product carries out PCR immediately or-20 ℃ of preservations are standby.
4.pEtK2 the pcr amplification of gene
In thin-walled PCR pipe, add following ingredients and carry out pcr amplification:
cDNA 2.5μl
2.5mmol/L dNTP 4.0μl
P1(50pmol) 1.0μl
P2(50pmol) 1.0μl
10×PCR buffer 5.0μl
25mmol/L MgCL
2 23.0μl
Taq archaeal dna polymerase (5U/ μ l) 0.5 μ l
ddH
2
O 33.0μl
Amount to 50.0 μ l
Behind the centrifugal mixing of mentioned component, 94 ℃ of degeneration 5min on the PCR instrument; 94 ℃ of 45sec, 60 ℃ of 60sec, 72 ℃ of 75sec, totally 30 circulations; 72 ℃ are extended 10min then.
Show that through order-checking the pEtK2 gene that amplification obtains has the nucleotide sequence shown in 1-1464 position among the SEQ ID NO:1.
The preparation of embodiment 2.chIL-2 gene:
1. synthetic primer P3, P4, sequence is respectively:
P3：5’-CTA
GAATTCCCTACCCTCGAT
TGCAAAGTACTGATCT-3’
P4：5’-TTA
GTCGACTTGCAGATATCTCACAAAGTT-3’
Wherein underscore partly is restriction enzyme site EcoRI and the SalI that introduces; Square frame partly is a start codon; Tilted letter partly is a Stuart factor
aSpecificity hydrolysis sequential coding nucleotide sequence (nucleotide sequence shown in 1465-1482 position among the SEQID NO:1).
2.chIL-2 the pcr amplification of gene
In thin-walled PCR pipe, add following ingredients and carry out pcr amplification:
Plasmid pBV220-chIL-2 (50ng/ μ l) 1.0 μ l
2.5mmol/L dNTP 4.0μl
P3(50pmol) 1.0μl
P4(50pmol) 1.0μl
10 * PCR buffer, 5.0 μ l
25mmol/L MgCL
2 23.0μl
Taq archaeal dna polymerase (5U/ μ l) 0.5 μ l
ddH
2
O 34.5μl
Amount to 50.0 μ l
Behind the centrifugal mixing of mentioned component, 94 ℃ of degeneration are 5 minutes on the PCR instrument; 94 ℃ 30 seconds, 58 ℃ 45 seconds, 72 ℃ 45 seconds, totally 30 circulations; 72 ℃ were extended 10 minutes then.
Owing to use P3 and P4 as primer, 5 ' end of the chIL-2 gene that obtains contains factor Xa specificity hydrolysis sequential coding nucleotide sequence (that is, Xa-chIL-2)
Show that through order-checking the chIL-2 gene that amplification obtains has the nucleotide sequence shown in 1483-1905 position among the SEQ ID NO:1.
The preparation of embodiment 3.DNA vaccine
1. the structure (see figure 3) of recombiant plasmid pMD18-T-pEtK2
Get the PCR product 50 μ l that obtain among the embodiment 1, electrophoresis on 1% agarose gel downcuts purpose band place agarose gel under uviol lamp, uses the glue recovery test kit of the precious biotech firm in Dalian to reclaim purification purpose fragment, and method is with reference to description.The PCR product of getting purification be connected with linearizing pMD18-T carrier behind the EcoRI enzyme action through BamHI, reaction system is as follows:
PCR reclaims product 4.0 μ l
PMD18-T carrier 1.0 μ l
Ligation solution I 5.0μl
Cumulative volume 10.0 μ l
With mentioned component in thin-walled eppendorf pipe behind the mix homogeneously 4 ℃ of connections spend the night.Connect product transformed competence colibacillus e. coli jm109, extract plasmid,, be defined as pMD18-T-pEtK2 with BamHI and evaluations such as EcoRI double digestion and order-checking.
2. the structure (see figure 4) of recombiant plasmid pET-Xa-chIL-2
Get the PCR product 50 μ l that obtain among the embodiment 2, electrophoresis on 1% agarose gel downcuts purpose band place agarose gel under uviol lamp, uses the glue recovery test kit of the precious biotech firm in Dalian to reclaim purification purpose fragment, and method is with reference to description.Get the PCR product and the plasmid pET-28 (+) of purification, use EcoRI and Sal I double digestion respectively, product carries out purification and reclaims.Then two kinds of purified products are connected with the T4DNA ligase.The coupled reaction system is as follows:
PCR product/EcoR I+Sal I (20ng/ μ l) 5.0 μ l
pET-28(+)/EcoRI+Sal I(25ng/μl) 2.0μl
5 * T4 connects buffer 2.0 μ l
T4DNA ligase (2U/ μ l) 1.0 μ l
Cumulative volume 10.0 μ l
With mentioned component in thin-walled eppendorf pipe behind the mix homogeneously 4 ℃ of connections spend the night.Connect product transformed competence colibacillus e. coli bl21, extract plasmid,, be defined as pET-Xa-chIL-2 with EcoRI and evaluations such as SalI double digestion and order-checking.
3. the structure (see figure 5) of recombiant plasmid pVAX1.0-pEtK2 (or pcDNA4/HisMax-pEtK2)
Use BamHI and EcoRI double digestion cloned plasmids carrier pMD18-T-pEtK2 and pVAX1.0 (or pcDNA4/His Max) respectively, reclaim the big fragment of pEtK2 genes of interest and pVAX1.0 (or pcDNA4/HisMax), connect according to proper ratio, connect product transformed competence colibacillus e. coli jm109, extract plasmid, identify with BamHI and EcoRI double digestion, be defined as pVAX1.0-pEtK2 (or pcDNA4/His Max-pEtK2) dna vaccination.
4. the structure (see figure 6) of recombiant plasmid pcDNA-pEtK2-Xa-chIL-2
Use EcoRI+NotI double digestion cloned plasmids carrier pET-Xa-chIL-2 and pVAX1.0-pEtK2 (or pcDNA4/His Max-pEtK2) respectively, reclaim the big fragment of Xa-chIL-2 genes of interest and pVAX1.0-pEtK2 (or pcDNA4/His Max-pEtK2) then, connect according to proper ratio, connect product transformed competence colibacillus e. coli jm109, extract plasmid, identify with EcoRI and NotI double digestion, be defined as pVAX1.0-pEtK2-Xa-chIL-2 (pcDNA4/His Max-pEtK2-Xa-chIL-2) dna vaccination.
A large amount of respectively pVAX1.0-pEtK2 (or pcDNA4/His Max-pEtK2) and pVAX1.0-pEtK2-Xa-chIL-2 (pcDNA4/His Max-pEtK2-Xa-chIL-2) recombiant plasmid of extracting, only inject 14 Japanese instar chicklings, 100 μ g/ through chest muscle, get injection site (chest muscle) and non-injection site muscle (shank) after 7 days respectively
1, RT-PCR detects transcribing of dna vaccination
One-step method is extracted the total RNA of muscle, with RT-PCR method increase respectively pEtK2 gene and chIL-2 gene.The result shows that dna vaccination has obtained to transcribe (see figure 7) in the chicken muscle cell.
2, the Western-trace detects the translation of dna vaccination
Get injection site (chest muscle) and non-injection site muscle (shank) sample preparation respectively, transfer printing nitrocellulose filter behind the SDS-PAGE electrophoresis, Ponceaux dyeing, the washing back added the irrelevant protein binding site of 5% defatted milk powder sealing 1 hour, add anti-pEtK2 recombiant protein immunize rabbit serum (one is anti-) effect 1 hour then, the goat anti-rabbit antibody (two is anti-) that lavation buffer solution washing back adds the HRP labelling acts on 1 hour, the colour developing of last DAB colour developing liquid.Found that dna vaccination has obtained good expression (see figure 8) in the chicken muscle cell
The zoopery result of embodiment 5. dna vaccinations of the present invention
120 plumages, 1 Japanese instar chickling is divided into 4 groups at random, every group 30 plumage.Difference intramuscular injection 100 μ l PBS (first group), 100 μ l PBS (second group), 100 μ g pVAX 1.0-pEtK2 (the 3rd group), 100 μ g pVAX 1.0-pEtK2-Xa-chIL-2 (the 4th group) when 14 ages in days and 21 ages in days.Peroral infection E.tenella spore egg capsule 0 (first group), 10 respectively during 28 ages in days
5Individual (second group), 10
5Individual (the 3rd group), 10
5Individual (the 4th group).Immune protective effect is carried out statistical analysis.
1, gaining effect
Every group of chicken be by only weighing before the injection dna vaccination, then before attacking worm, attack behind the worm the 7th day chicken to correspondence again by only weighing, observe before attacking worm, attack the body weight change situation of chicken behind the worm, calculate average weight gain and the relative weight gain then.It is heavy when weight-head exempted from when average weight gain 1=attacked worm; It is heavy when average weight gain 2=weigh-attacks worm when slaughtering at last; The relative weight gain=(the non-immune group average weight gain of each test group average weight gain/non-infection) * 100%.Weightening finish result (seeing Table 1) shows that the injection of vaccine does not have influence to the weightening finish of chicken, and promptly the toxic and side effects of vaccine is suitable with PB S.And the weightening finish of pVAX 1.0-pEtK2-Xa-chIL-2DNA vaccine immunity group is not remarkable with the non-immune group difference of non-infection after attacking worm, illustrates that pVAX 1.0-pEtK2-Xa-chIL-2DNA vaccine has significant protective effect.
Table 1: the gaining effect of dna vaccination of the present invention
Represent that with containing same letter in the string difference is not remarkable, do not contain same letter and represent significant difference that different lower cases are represented significant difference (p＜0.05).
| Group | | The relative weight gain 1 (%) | | The relative weight gain 2 (%) |
| The non-immune group of non-infection (the one Zu) infects non-immune group (the 2nd Zu) pVAX1.0-pEtK2 (the 3rd Zu) pVAX1.0-pEtK2-Xa-chIL-2 (the 4th Zu) | 66.30±12.58 a 56.92±8.28 a 62.52±12.66 a 66.75±9.36 a | 100.00 85.85 94.30 100.83 | 220.15±19.59 a 99.23±25.66 c 187.90±15.87 b 215.30±21.11 a | 100 45.0 85.35 97.80 |
2, every gram feces egg sac number
Attack behind the worm and to check feces every day, collected respectively in the 7th day and respectively to organize feces, behind the mixing, get 10g for every group, add the 90mL distilled water and make 10 times of diluents, through stirring after in microscopically count the worm's ovum number in every gram feces of plate that counts with erythrocyte.
The every gram feces of table 2. egg sac number
| Group | Every gram feces egg sac number * 10 6 |
| The non-immune group of non-infection (first group) infects non-immune group (second group) pVAX1.0-pEtK2 recombiant plasmid (the 3rd group) pVAX1.0-pEtK2-Xa-chIL-2 recombiant plasmid (the 4th group) | 0 4.68 0.82 0.710 |
3, egg capsule slip
The egg capsule slip is an another one index of judging vaccine protection effect, and its computing formula is as follows: egg capsule slip=[the non-immune group feces egg capsule the number of discharge of (infecting non-immune group feces egg capsule the number of discharge-test group feces egg capsule the number of discharge)/infection] * 100%.
Table 3: egg capsule slip
| Group | The egg capsule slip |
| The non-immune group of non-infection (first group) infects non-immune group (second group) pVAX1.0-pEtK2 recombiant plasmid (the 3rd group) pVAX1.0-pEtK2-Xa-chIL-2 recombiant plasmid (the 4th group) | 100％ 0％ 82.47％ 84.83％ |
4, caecum lesion is kept the score
Attacked behind the worm the 7th day, and got 10 chickens for every group and cut open and kill, observe caecum lesion.Carrying out caecum lesion by the Johnson method keeps the score.Just caecum lesion is divided into 0～+ 4 five grade.0 minute: expression was normal, no naked eyes pathological changes; + 1 minute: the petechiae that the caecum wall has amount seldom to be dispersed in, intestinal wall does not thicken, and content is normal; + 2 minutes: pathological changes quantity was more, and cecal content obviously is with blood, and the caecum wall thickens slightly, and content is normal; + 3 minutes: volume blood or caecum core (the caseous Fructus Musae type of blood clot or canescence block) are arranged in the caecum, and the caecum wall was obviously plump, and feces content is few in the caecum; + 4 minutes: because of being full of a large amount of blood or the enlargement of intestinal core caecum, containing the excrement slag in the intestinal core or do not contain, dead chicken also remembers+4 minutes.When the both sides caecum lesion is inconsistent, be as the criterion with a serious side.Relative lesion score=(the non-immune group lesion score of experimental group lesion score/infection) * 100% (pathological changes is seen Fig. 9,10).
Table 4. caecum lesion is kept the score
| Group | Caecum lesion is kept the score |
| The non-immune group of non-infection (first group) infects non-immune group (second group) pVAX1.0-pEtK2 recombiant plasmid (the 3rd group) pVAX1.0-pEtK2-Xa-chIL-2 recombiant plasmid (the 4th group) | 0±0 a 3.60±0.46 c 1.30±0.53 ab 0.40±0.31 ab |
Annotate: represent that with containing same letter in the string difference is not remarkable, do not contain same letter and represent significant difference,
Different lower cases are represented significant difference (p＜0.05).
5, anticoccidial index (ACI)
ACI comprises multiple parameters such as survival rate, weightening finish, pathological changes, egg capsule output, as the index of judging coccidiosis drug resistance or pharmaceutical efficacy, also can be used for the index of coccidial vaccine protection effect detection after the Comprehensive Assessment.ACI judges formula: ACI=(survival rate+the relative weight gain rate)-(pathological changes value+egg capsule value).(ACI 〉=180 are obvious for the protection effect, and ACI=160～179 are general for the protection effect, and ACI＜160 are the unprotect effect.)
Result such as table 5, pVAX1.0-pEtK2-Xa-chIL-2 dna vaccination ACI of the present invention reaches 198.20, has good immune protective effect.
Table 5: anticoccidial index (ACI)
| Group | Survival rate | The relative weight gain rate | The pathological changes value | The egg capsule value | ACI |
| The non-immunity of non-infection, (first group) infects non-immunity, (second group) pVAX1.0-pEtK2, (the 3rd group) pVAX1.0-pEtK2-Xa-chIL-2, (the 4th group) | 100 100 100 100 | 100 45.07 85.35 97.8 | 0 36 13 4 | 0 1 1 1 | 200.00 108.07 161.35 192.80 |
Correspondingly replace pVAX1.0-pEtK2 and pVAX1.0-pEtK2-Xa-chIL-2 to carry out above-mentioned test (other contrasts, step and parameter are all identical) respectively with pcDNA4/His Max-pEtK2 and pcDNA4/His Max-pEtK2-Xa-chIL-2, also obtained gratifying approximation (data do not provide).
Sequence table
＜110〉Agricultural University Of Nanjing
＜120〉a kind of immunity regulating type Eimeria tenella DNA vaccine for preventing and treating chicken coccidiosis
<210>1
<211>1905
<212>DNA
＜213〉artificial sequence
<220>
＜223〉fusion gene
<220>
＜221〉CDS (calcium is transferred binding domain protein kinase gene pEtK2)
<222>(1)..(1464)
<220>
＜221〉protease Stuart factor
aThe coded sequence of specificity hydrolysis sequence
<222>(1465)..(1482)
<220>
＜221〉CDS (chicken interleukin-2-2 gene)
<222>(1483)..(1905)
<400>1
atg cca gca gcg tcc aag tca gac aaa ctg gct gcc acg ccg ggc atg 48
Met Pro Ala Ala Ser Lys Ser Asp Lys Leu Ala Ala Thr Pro Gly Met
1 5 10 15
ttt gtg cag cac tct aca gca gcc ttc agc gat agg tac aag ggc cag 96
Phe Val Gln His Ser Thr Ala Ala Phe Ser Asp Arg Tyr Lys Gly Gln
20 25 30
cgc gtg ttg ggc aag ggc agt ttc ggt gaa gtt att ttg tgc aaa gat 144
Arg Val Leu Gly Lys Gly Ser Phe Gly Glu Val Ile Leu Cys Lys Asp
35 40 45
aaa gta act gga cag gaa tat gcc gtg aag gtg att tca aaa cgg caa 192
Lys Val Thr Gly Gln Glu Tyr Ala Val Lys Val Ile Ser Lys Arg Gln
50 55 60
gtg aag cag aag acg gac aaa gag ctg ctg ctc aag gag gtg gag ctc 240
Val Lys Gln Lys Thr Asp Lys Glu Leu Leu Leu Lys Glu Val Glu Leu
65 70 75 80
ctt aag aag ctg gtc cac ccc aac atc atg aag ctc tac gag ttc ttc 288
Leu Lys Lys Leu Val His Pro Asn Ile Met Lys Leu Tyr Glu Phe Phe
85 90 95
gag gac aag ggc tac ttc tac tta gtc aca gaa gtc tat acc ggc gga 336
Glu Asp Lys Gly Tyr Phe Tyr Leu Val Thr Glu Val Tyr Thr Gly Gly
100 105 110
gag ctc ttc ggc gag atc atc agc aga aag agg ttc agc gag gtt gac 384
Glu Leu Phe Gly Glu Ile Ile Ser Arg Lys Arg Phe Ser Glu Val Asp
115 120 125
gct gcc cgc atc att cga caa gtg ctt tcg ggc att acg tat atg cac 432
Ala Ala Arg Ile Ile Arg Gln Val Leu Ser Gly Ile Thr Tyr Met His
130 135 140
aag aac aaa atc gtt cac aga gac ctc aag cct gaa aat ctg ctg ctg 480
Lys Asn Lys Ile Val His Arg Asp Leu Lys Pro Glu Asn Leu Leu Leu
145 150 155 160
gaa aac aaa aga aaa gat gct aac att cgc atc att gac ttc ggc ctt 528
Glu Asn Lys Arg Lys Asp Ala Asn Ile Arg Ile Ile Asp Phe Gly Leu
165 170 175
tcc act cac ttt gag tcc acc aag gaa atg aag gac aag atc gga acg 576
Ser Thr His Phe Glu Ser Thr Lys Glu Met Lys Asp Lys Ile Gly Thr
180 185 190
gcc tat tac att gcc cca gag gtc ttg cat ggg gcc tac gac gag aag 624
Ala Tyr Tyr Ile Ala Pro Glu Val Leu His Gly Ala Tyr Asp Glu Lys
195 200 205
tgc gac gtg tgg tcc aca ggg gtc att ttg tat atc ctt ctt tct gga 672
Cys Asp Val Trp Ser Thr Gly Val Ile Leu Tyr Ile Leu Leu Ser Gly
210 215 220
tgc ccc cca ttt aac ggg gca aac gaa ttt gac att ttg aag aaa gtc 720
Cys Pro Pro Phe Asn Gly Ala Asn Glu Phe Asp Ile Leu Lys Lys Val
225 230 235 240
gaa aaa gga aag ttc acc ttg gat ctg ccg caa tgg aag aag gtg agc 768
Glu Lys Gly Lys Phe Thr Leu Asp Leu Pro Gln Trp Lys Lys Val Ser
245 250 255
gag tca gcc aaa gac ttg att cgt aag atg ctg gcg tac gtg ccc aca 816
Glu Ser Ala Lys Asp Leu Ile Arg Lys Met Leu Ala Tyr Val Pro Thr
260 265 270
atg cgg ata tct gca cga gac gcc ctc gag cac gag tgg ctc aag act 864
Met Arg Ile Ser Ala Arg Asp Ala Leu Glu His Glu Trp Leu Lys Thr
275 280 285
aca gac gca gcc act gac agt att gat gtg cct tcg ctt gag agc acc 912
Thr Asp AlaAla Thr Asp Ser Ile Asp Val Pro Ser Leu Glu Ser Thr
290 295 300
ata ctt aac atc aga cag ttc cag ggg acg caa aag ctt gcc gcg gct 960
Ile Leu Asn Ile Arg Gln Phe Gln Gly Thr Gln Lys Leu Ala Ala Ala
305 310 315 320
gcg ctg ctc tat atg ggc agc aag ctg acc acc aac gaa gag aca gtt 1008
Ala Leu Leu Tyr Met Gly Ser Lys Leu Thr Thr Asn Glu Glu Thr Val
325 330 335
gag ctc aac aag att ttc caa agg atg gac aaa aac gga gat ggc cag 1056
Glu Leu Asn Lys Ile Phe Gln Arg Met Asp Lys Asn Gly Asp Gly Gln
340 345 350
ctt gat aag cag gag ctc atg gaa ggc tac gtg gag ctg atg aag ctg 1104
Leu Asp Lys Gln Glu Leu Met Glu Gly Tyr Val Glu Leu Met Lys Leu
355 360 365
aaa ggc gaa gac gtt tcg gcg ctg gac cag agc gcc atc gag ttc gag 1152
Lys Gly Glu Asp Val Ser Ala Leu Asp Gln Ser Ala Ile Glu Phe Glu
370 375 380
gtg gag cag gtg ctc gac gct gtg gac ttt ggc aag aac ggc ttc ata 1200
Val Glu Gln Val Leu Asp Ala Val Asp Phe Gly Lys Asn Gly Phe Ile
385 390 395 400
gag tac tcg gag ttc gtc aca gtg gca atg gac cgc aag aca ctc ttg 1248
Glu Tyr Ser Glu Phe Val Thr Val Ala Met Asp Arg Lys Thr Leu Leu
405 410 415
tct cgc cag cga ctg gaa aga gcc ttt gga atg ttc gac gct gac ggc 1296
Ser Arg Gln Arg Leu Glu Arg Ala Phe Gly Met Phe Asp Ala Asp Gly
420 425 430
tcg ggc aag att tcc tct tca gag ttg gcc acc ata ttc ggg gtg agc 1344
Ser Gly Lys Ile Ser Ser Ser Glu Leu Ala Thr Ile Phe Gly Val Ser
435 440 445
gag gtc gac tcc gaa acc tgg cgc cgc atc ctc gcg gag gtg gac aga 1392
Glu Val Asp Ser Glu Thr Trp Arg Arg Ile Leu Ala Glu Val Asp Arg
450 455 460
aac aac gac ggg gaa gtg gac ttt gag gag ttc cgg cag atg ctc ctg 1440
Asn Asn Asp Gly Glu Val Asp Phe Glu Glu Phe Arg Gln Met Leu Leu
465 470 475 480
aag ttg tgc gga gat acc gcg gcg gaattcccta ccctcgat atg tgc aaa 1491
Lys Leu Cys Gly Asp Thr Ala Ala Met Cys Lys
485 490
gta ctg atc ttt ggc tgt att tcg gta gca acg cta atg act aca gct 1539
Val Leu Ile Phe Gly Cys Ile Ser Val Ala Thr Leu Met Thr Thr Ala
495 500 505
tat gga gca tct cta tca tca gca aaa agg aaa cct ctt caa aca tta 1587
Tyr Gly Ala Ser Leu Ser Ser Ala Lys Arg Lys Pro Leu Gln Thr Leu
510 515 520
ata aag gat tta gaa ata ttg gaa aat atc aag aac aag att cat ctc 1635
Ile Lys Asp Leu Glu Ile Leu Glu Asn Ile Lys Asn Lys Ile His Leu
525 530 535
gag ctc tac aca cca act gag acc cag gag tgc acc cag caa act ctg 1683
Glu Leu Tyr Thr Pro Thr Glu Thr Gln Glu Cys Thr Gln Gln Thr Leu
540 545 550 555
cag tgt tac ctg gga gaa gtg gtt act ctg aag aaa gaa act gaa gat 1731
Gln Cys Tyr Leu Gly Glu Val Val Thr Leu Lys Lys Glu Thr Glu Asp
560 565 570
gac act gaa att aaa gaa gaa ttt gta act gct att caa aat atc gaa 1779
Asp Thr Glu Ile Lys Glu Glu Phe Val Thr Ala Ile Gln Asn Ile Glu
575 580 585
aag aac ctc aag agt ctt acg ggt cta aat cac acc gga agt gaa tgc 1827
Lys Asn Leu Lys Ser Leu Thr Gly Leu Asn His Thr Gly Ser Glu Cys
590 595 600
aag atc tgt gaa gct aac aac aag aaa aaa ttt cct gat ttt ctc cat 1875
Lys Ile Cys Glu Ala Ash Asn Lys Lys Lys Phe Pro Asp Phe Leu His
605 610 615
gaa ctg acc aac ttt gtg aga tat ctg caa 1905
Glu Leu Thr Asn Phe Val Arg Tyr Leu Gln
620 625
Claims (7)
1. an Eimeria tenella (Eimeria tenella) immunity regulating type DNA vaccine, this vaccine comprise and have wherein inserted the eucaryon plasmid carrier that Eimeria tenella calcium is transferred binding domain protein kinase gene pEtK2.
2. by the described dna vaccination of claim 1, wherein further also comprising links together with described gene pEtK2 inserts the chicken interleukin-2 gene of described eucaryon plasmid carrier.
3. by the described dna vaccination of claim 2, wherein further also be included in and insert one section protease Stuart factor between described gene pEtK2 and the gene chIL-2
aSpecificity hydrolysis sequential coding nucleotide sequence GAA TTC CCT ACCCTC GAT forms a fusion gene pEtK2-X
a-chIL-2.
4. the described dna vaccination of claim 3, wherein said fusion gene has the nucleotide sequence shown in SEQ ID NO:1.
5. each described dna vaccination among the claim 1-4, wherein said eucaryon plasmid carrier is pcDNA3.0, pcDNA3.1, pVAX1.0 or pcDNA4/His Max.
6. each described dna vaccination in the claim claim 5, wherein said eucaryon plasmid carrier is pVAX1.0.
7. the purposes of each described dna vaccination in the pharmaceutical preparation of preparation prevention or treatment chicken coccidiosis among the claim 1-6.
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|---|---|---|---|
| CN 200510124049 CN1803195A (en) | 2005-11-23 | 2005-11-23 | Immunity regulating type Eimeria tenella DNA vaccine for preventing and treating chicken coccidiosis |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510124049 CN1803195A (en) | 2005-11-23 | 2005-11-23 | Immunity regulating type Eimeria tenella DNA vaccine for preventing and treating chicken coccidiosis |
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| Publication Number | Publication Date |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102018687A (en) * | 2010-11-24 | 2011-04-20 | 广东省农业科学院兽医研究所 | Application of hexachlorophene in resistance to Eimeria tenella |
| CN102028680A (en) * | 2010-11-24 | 2011-04-27 | 广东省农业科学院兽医研究所 | Application of fisetin for resisting against Eimeria tenella |
| CN102028677A (en) * | 2010-11-24 | 2011-04-27 | 广东省农业科学院兽医研究所 | Application of juglone nucin to resistance to eimeria tenella |
| CN107904247A (en) * | 2017-12-26 | 2018-04-13 | 吉林大学 | One kind restructuring Eimeria species Alphavirus particle vaccines and preparation method |
| CN110559431A (en) * | 2019-10-11 | 2019-12-13 | 南京农业大学 | Eimeria maxima nano subunit vaccine and preparation method and application thereof |
| CN110559432A (en) * | 2019-10-11 | 2019-12-13 | 南京农业大学 | Eimeria acervulina nano subunit vaccine and preparation method and application thereof |
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2005
- 2005-11-23 CN CN 200510124049 patent/CN1803195A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102018687A (en) * | 2010-11-24 | 2011-04-20 | 广东省农业科学院兽医研究所 | Application of hexachlorophene in resistance to Eimeria tenella |
| CN102028680A (en) * | 2010-11-24 | 2011-04-27 | 广东省农业科学院兽医研究所 | Application of fisetin for resisting against Eimeria tenella |
| CN102028677A (en) * | 2010-11-24 | 2011-04-27 | 广东省农业科学院兽医研究所 | Application of juglone nucin to resistance to eimeria tenella |
| CN102018687B (en) * | 2010-11-24 | 2012-05-09 | 广东省农业科学院兽医研究所 | Application of hexachlorophene in preparing medicine for resistance to Eimeria tenella |
| CN102028680B (en) * | 2010-11-24 | 2012-05-09 | 广东省农业科学院兽医研究所 | Application of fisetin in preparing medicine for resisting against Eimeria tenella |
| CN102028677B (en) * | 2010-11-24 | 2012-05-30 | 广东省农业科学院兽医研究所 | Application of juglone nucin in preparing medicine for resistance to eimeria tenella |
| CN107904247A (en) * | 2017-12-26 | 2018-04-13 | 吉林大学 | One kind restructuring Eimeria species Alphavirus particle vaccines and preparation method |
| CN110559431A (en) * | 2019-10-11 | 2019-12-13 | 南京农业大学 | Eimeria maxima nano subunit vaccine and preparation method and application thereof |
| CN110559432A (en) * | 2019-10-11 | 2019-12-13 | 南京农业大学 | Eimeria acervulina nano subunit vaccine and preparation method and application thereof |
| CN110559432B (en) * | 2019-10-11 | 2023-06-13 | 南京农业大学 | Eimeria acervulina nano subunit vaccine and preparation method and application thereof |
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