CN101955956A - Eimeria tenella calcium-dependent protein kinase gene and application thereof - Google Patents

Abstract

The invention discloses an eimeria tenella gene. The nucleotide sequence of the gene coded calcium-dependent protein kinase comprises a DNA sequence for coding an SEQ ID NO.1 amino acid sequence or a DNA sequence for coding an amino acid sequence with calcium-dependent protein kinase activity obtained by deleting, adding, inserting or replacing one or a plurality of amino acids in the SEQ ID NO.1 amino acid sequence. The invention also discloses a protein coded by the gene. The eimeria tenella gene is a new gene highly expressed in a sporozoite phase of the eimeria tenella, and is related to the invasion of a to a host cell. The gene has high application value in developing new vaccines or new medicines for inhibiting the invasion of the sporozoite to the host cell and blocking the life cycle of the eimeria tenella.

Description

Eimeria tenella calcium dependent kinases gene and application

Technical field

The present invention relates to technical field of bioengineering, relate in particular to a kind of Eimeria tenella calcium dependent kinases gene and application.

Background technology

Coccidiosis of chicken is that the coccidia by Amy otology, Eimeria (Eimeria) parasitizes caused a kind of protozoal disease in the chicken intestinal epithelial cells.And wherein with the pathogenic and hazardness maximum of Eimeria tenella (Eimeria tenella), its main parasitic is in caecum and near zone thereof, enteritis can cause bleeding, maximum to chick harm, can cause higher mortality ratio when serious, cause enormous economic loss for global aquaculture.Control coccidiosis mainly still relies on the use of anticoccidial drug at present, but because the life-time service of anticoccidial drug, the chemical sproof generation of chicken coccidia is inevitable, makes medical treatment coccidiosis face significant challenge.

In view of the harm and the drug-fast generation of coccidiosis of chicken, how it is prevented and treats effectively to become the problem that receives much concern.And the chicken coccidia has the life history of a complexity, both comprised the extracellular invasion stage, also comprise the intracellular breeding stage, in growth course, all experience exophytic growth (sporogony) and endogenous growth (scissiparity and syngenesis), scissiparity and syngenesis are carried out in the chicken body, and sporogony carries out external.Coccidian oocyst is discharged in the external environment with ight soil, and the egg capsule of this moment is sporeization not, does not have the ability of infected chicken, under optimal temperature, humidity and sufficient oxygen condition, growing through a couple of days is the spore egg capsule, after being eaten by chicken, finishes its life history in the chicken body again.The chicken coccidia can survive in the chicken enteron aisle and breed, and (sporozoite and merozoite) removes identification, combination and invasion intestinal epithelial cell to need the invasion stage.Sporozoite is first important stage of invasion host cell, should have many stage specific expression genes, particularly with the associated molecule of invading the host cell needs, therefore strengthen research to sporozoite invasion host cell associated molecule and mechanism thereof, find and the relevant target molecule of coccidia invasion, significant to the control of coccidiosis.

Summary of the invention

The present invention will solve at medical treatment coccidiosis and very easily produce the technical problem that chemical sproof problem is badly in need of development of new vaccine and new drug, a kind of Eimeria tenella calcium dependent kinases (EtCDPK) gene is provided, it is special that this EtCDPK plays a part during in Eimeria tenella sporozoite identification with in conjunction with the invasion site of chicken intestinal epithelial cells, and the novel vaccine or the new drug that exploitation are suppressed sporozoite invasion chicken intestinal epithelial cells, blocking-up coccidia life history have very high using value.

In addition, also need to provide a kind of application of Eimeria tenella calcium dependent kinases gene.

In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:

In one aspect of the invention, provide a kind of Eimeria tenella gene, described genes encoding calcium dependent kinases, its nucleotide sequence comprises one of following dna sequence dna:

(1) dna sequence dna of coding SEQ ID NO.1 aminoacid sequence;

(2) lack, add, insert or replace the resulting dna sequence dna of one or more amino acid in the aminoacid sequence shown in the coding SEQ ID NO.1 with the active aminoacid sequence of calcium dependent kinases.

Preferably, described gene has the dna sequence dna shown in the SEQ ID NO.2.

In another aspect of this invention, provide a kind of eimeria tenella protein, described protein has calcium dependent protein kinase enzymic activity, and this protein is selected from:

(1) has the protein of SEQ ID NO.1 aminoacid sequence;

(2) has the active fragments or the conservative property variant protein matter of partial amino-acid series among the SEQ ID NO.1;

(3) lack, add, insert or replace the resulting protein of one or more amino acid shown in the SEQ ID NO.1 in the aminoacid sequence with the active aminoacid sequence of calcium dependent kinases.

Preferably, described protein has aminoacid sequence shown in the SEQ ID NO.1.

In the present invention, because the modification etc. of protein itself or owing to the polymorphism and the variation of encoding egg white gene, amino acid whose disappearance, interpolation, insertion, replacement or other variations can appear in naturally occurring protein in the body or during the purifying.In fact, exist the albumen that is equal to no variant protein matter on some physiology and the biological activity substantially.These structures are different from corresponding proteins matter but do not have the polypeptide of tangible function difference or albumen to be called the functional equivalent varient with this protein.

The functional equivalent varient is equally applicable to import the polypeptide that this class variation is made by artificial means in a kind of proteinic aminoacid sequence.Even now can obtain how multi-form varient, but the varient of gained is the activity that its physiologically active is equal to original no variant protein matter substantially as the prerequisite of functional equivalent varient.

For example, replace the activity [Science, 224,1431 (1984)] that the specific resulting polypeptide of cysteine residues is still keeping IL-2 with Serine in human interleukin 2's (IL-2) the aminoacid sequence.

In addition, when producing protein with gene engineering method, often desired protein is expressed as fused protein, for example, be added to the N-end of desired protein to improve the expression of desired protein coming from the terminal peptide chain of other proteinic N-, perhaps a suitable peptide chain is added to the N-or the C-end of desirable proteins, express this albumen and use a kind of to adding the carrier of peptide chain with avidity the purifying of desirable proteins is become be more prone to.

With regard to the codon (combination of triplet base) of specific amino acids on determining gene, there is 1 to 6 codon in every seed amino acid.Although therefore depend on aminoacid sequence, still have a kind of amino acid whose gene of many codings.At occurring in nature, gene is also unstable, and the nucleic acid variation often takes place.The variation of gene may not influence coded aminoacid sequence (silent variant), in this case, can produce the different genes of coding same acid sequence.Therefore, even isolated the gene of coding specific amino acids sequence,, still can produce the different genes of coding same acid sequence inevitably along with containing going down to posterity of this gene biological body.

The range gene that manually produces the coding same acid sequence with the range gene engineering is not difficult.For example, when the coding desired protein natural gene codon be used for genetic engineering technique produce proteinic host's utilizability lower, when the expressed proteins amount is not enough, can manually codon be transformed into another codon that utilizability is high in this host and not change coded aminoacid sequence, can strengthen the expression of desired protein.Therefore, the artificial different polynucleotide of producing of this class are also included within the scope of the present invention the aminoacid sequence disclosed by the invention as long as it can be encoded out.

In addition, by the polypeptide of at least a change (as disappearance, interpolation, insertion or the replacement of one or more amino-acid residues are arranged in the proteinic aminoacid sequence) gained or albumen one have be equal on the function as described in activity of proteins, encode this class polypeptide or proteic gene is also included within the scope of the present invention, still is artificial production no matter it separates from natural origin.

One, the gene that encoding function is equal to varient is a homologous.Therefore, can have the active proteinic nucleic acid molecule of Eimeria tenella calcium dependent kinases with gene recombination of the present invention and coding is also included within the scope of the present invention.

In another aspect of this invention, also provide a kind of recombinant vectors that comprises all or part of sequence of above-mentioned Eimeria tenella gene DNA sequence.

Described recombinant vectors comprises recombinant cloning vector or recombinant expression vector, and recombinant expression vector comprises recombinant prokaryotic expression vector, recombinant eukaryon expression vector.

In another aspect of this invention, also provide a kind of host cell, comprised above-mentioned recombinant vectors, or transformed or transfection with above-mentioned gene order.

In another aspect of this invention, also provide a kind of application of above-mentioned Eimeria tenella gene, be used to prepare the vaccine or the medicine of prevention or treatment coccidiosis.

Described vaccine or medicine are blocked coccidia by inhibition Eimeria tenella sporozoite invasion host cell and are played a role the life history.

In another aspect of this invention, also provide a kind of application of above-mentioned eimeria tenella protein, be used to prepare the vaccine or the medicine of prevention or treatment coccidiosis.

Described vaccine or medicine are blocked coccidia by inhibition Eimeria tenella sporozoite invasion host cell and are played a role the life history.

In another aspect of this invention, also provide a kind of application of above-mentioned Eimeria tenella gene, be used to screen the medicine of prevention or treatment coccidiosis.

Eimeria tenella calcium dependent kinases gene of the present invention (EtCDPK) is a new gene of Eimeria tenella (Eimeria tenella) sporozoite stage high expression level.Prokaryotic expression product and the eukaryotic expression product of Western-blot analysis revealed EtCDPK of the present invention all have good immunogenicity; The immunofluorescence positioning experiment shows that with the body outer suppressioning experiment result EtCDPK is relevant with the invasion of sporozoite, has brought into play vital role when coccidia sporozoite invasion host cell.Therefore, EtCDPK of the present invention, the novel vaccine or the new drug that suppress sporozoite invasion, blocking-up coccidia life history for exploitation provide new approaches.

Description of drawings

The present invention is further detailed explanation below in conjunction with the drawings and specific embodiments.

Fig. 1 is the full-length cDNA product electrophoresis evaluation figure of the embodiment of the invention 1 Eimeria tenella sporozoite EtCDPK gene RT-PCR amplification;

Fig. 2 is that the embodiment of the invention 2 real-time quantitative PCRs detect the expression figure of EtCDPK gene in the Eimeria tenella different developmental phases;

Fig. 3 is the double digestion evaluation figure of the embodiment of the invention 3 reorganization prokaryotic expression plasmid pET28a-EtCDPK;

Fig. 4 is the not SDS-PAGE electrophorograms of phase expressing protein simultaneously of the embodiment of the invention 3 recombinant expression plasmid pET28a-EtCDPK;

Fig. 5 is the EtCDPK protein expression form analysis figure of the embodiment of the invention 3;

Fig. 6 is the EtCDPK recombinant protein analysis chart of the embodiment of the invention 3 purifying;

Fig. 7 is the His-EtCDPK recombinant protein Western-blot analysis chart of the embodiment of the invention 3;

Fig. 8 is the embodiment of the invention 4 amplification EtCDPK gene electrophoresis detection figure;

Fig. 9 is the double digestion analysis chart of the embodiment of the invention 4 recombinant plasmid pPIC9k-EtCDPK and empty carrier pPIC9k;

Figure 10 is the proteic SDS-PAGE analysis charts of the embodiment of the invention 4 Pichia anomala expressions;

Figure 11 is the EtCDPK yeast expression albumen Western-blot analysis chart of the embodiment of the invention 4;

Figure 12 is that the embodiment of the invention 5 immunofluorescences detect the distribution plan of EtCDPK in sporozoite invasion host cell process;

Figure 13 is the flow cytometer detection figure of the DF-1 cell that do not infect of the embodiment of the invention 5;

Figure 14 is the flow cytometer detection figure of the DF-1 cell of the embodiment of the invention 5 sporozoites infection;

Figure 15 is the flow cytometer detection figure of the DF-1 cell that infects of sporozoite that the anti-recombinant protein His-EtCDPK of the embodiment of the invention 5 usefulness rabbits polyclonal antibody is handled.

Embodiment

In the following example, the experimental technique of unreceipted actual conditions, condition routinely usually is as " molecular cloning experiment guide " (J. Sa nurse Brooker, D.W. the Russell is outstanding, Huang Peitang, Wang Jiaxi, Zhu Houchu, Deng translating. the 3rd edition, Beijing: Science Press, 2002) described in method carry out.

The present invention utilizes suppression subtractive hybridization technique and cDNA microarray technology to screen Eimeria tenella sporozoite and spore egg capsule difference expression gene on a large scale, obtained on the basis of a large amount of difference expression gene ESTs, selected 1 ESTs (clone number for ZB1-H07), found highly homology of ZB1-H07 and plasmodium falciparum calcium dependent kinases (CDPK) through Blast homology compare of analysis at sporozoite stage cance high-expression gene.Utilize RACE technology amplification to obtain the full-length cDNA of this gene, ZB1-H07 full length gene 1336bp contains the ORF of a 1302bp, 433 amino acid of encode, and the proteic molecular weight of prediction expression is 49.3kDa.To signal peptide, the hydrophobicity of this gene coded protein, stride membrane structure, physico-chemical property antigen site and conservative functional domain etc. and carry out the searching analysis prediction, the result shows: ZB1-H07 encoded protein and toxoplasma gondii calcium dependent kinases (CDPK) height homology, therefore be speculated as Eimeria tenella calcium dependent kinases, called after EtCDPK.

The EtCDPK gene is connected to prokaryotic expression carrier pET28a (+), makes up recombinant expression plasmid pET (28a)-EtCDPK, transformed into escherichia coli BL21 (DE3) carries out abduction delivering again, and the fusion rotein of expression occurs with the inclusion body form.Utilize nickel affinity chromatography column purification recombinant protein His-EtCDPK, analyze, show that recombinant protein His-EtCDPK has good immunogenicity through Western-blot.In order to obtain soluble proteins, further analyze its function, the EtCDPK gene is connected among the yeast expression vector pPIC-9K, after transforming GS115 yeast competent cell, express through methanol induction, obtained soluble proteins, Western-blot analyzes and shows also have good immunogenicity.

For understanding the distribution situation of EtCDPK in sporozoite invasion host cell process, carry out the immunofluorescence positioning experiment, the result shows that EtCDPK is relevant with growth with the invasion of sporozoite.Body outer suppressioning test has verified that further EtCDPK is relevant with the sporozoite invasion, and plays a significant role when coccidia sporozoite invasion cell.These experimental results show, Eimeria tenella calcium dependent kinases EtCDPK of the present invention provides new approaches for novel vaccine or the new drug that exploitation suppresses sporozoite invasion, blocking-up coccidia life history.

Clone and the analysis of embodiment 1 Eimeria tenella EtCDPK full length gene cDNA

In earlier stage utilize suppression subtractive hybridization technique and cDNA microarray technology to screen sporozoite and spore egg capsule difference expression gene on a large scale in the laboratory, obtained on the basis of a large amount of difference expression gene ESTs, selected 1 ESTs (gene clone number: ZB1-H07) at sporozoite stage cance high-expression gene, find ZB1-H07 and plasmodium falciparum calcium dependent kinases (CDPK) height homology through Blast homology compare of analysis, be speculated as Eimeria tenella calcium dependent kinases, called after EtCDPK.

Utilize the amplification of RACE technology to obtain the full-length cDNA of EtCDPK gene, concrete steps are as follows:

1.RACE design of primers

With the ESTs sequence of ZB1-H07 earlier with E.tenella gene order-checking website ( Http:// www.Sanger. Ac.uk. / projects/E_tenella/OmniBLAST) carry out the similarity comparison, determine 3 ' end and the 5 ' end of ESTs, again according to sequences Design 3 ' end and 5 ' the RACE amplimer of holding (seeing the following form 1).

Table 1RACE primer

2. the collection of Eimeria tenella sporozoite and purifying

The method that the collection of sporozoite is set up with reference to (1998) such as Song Cuiping is carried out the preparation of sporozoite, and slightly modified.

1) with the spore egg capsule of purifying, adds HBBS damping fluid (a kind of PBS damping fluid), discharge with the broken egg capsule microscopy of glass homogenizer sporocyst.

2) sporocyst is packed in the 50mL centrifuge tube, with HBBS washing 2～3 times, the centrifugal 10min of 2500r/min, collecting precipitation.

3) precipitation is packed in the 50mL Erlenmeyer flask, add the trypsinase of 2.5g/L and 2%～5% Fel Gallus domesticus digestion, 41 ℃ of water-baths digestion 1h (microscopy sporozoite release conditions at any time).

4) with HBBS washing 2～3 times, 3000r/min, centrifugal 5min collecting precipitation.

5) with G3 sand core funnel negative pressure filtration, microscopy filtrate, sporozoite is banana-shaped.

6) filtrate is packed in the 10mL centrifuge tube, 2500r/min, centrifugal 10min collecting precipitation.

Wash 2～3 times with HBBS again, in the cell pipe of packing into, high speed freezing centrifuge 12000r/min, centrifugal 2min, it is standby that precipitation is put into liquid nitrogen.

3. the extraction of Eimeria tenella sporozoite Total RNA

To be used to extract glassware and the metallic weapon of total RNA, and clean the oven dry back and wrap 180 ℃ of roasting 6h with masking foil.Plastics steep 2h with 0.1% DEPC water logging, and autoclave sterilization is removed RNase.

1) go bail for and be stored in E.tenella sporozoite in the liquid nitrogen, sample adds Trizol reagent and abundant mixing by 50～100mg/mL Trizol, and room temperature leaves standstill 6min.

2) in step 1), add chloroform (adding 200 μ L chloroforms), cover tight centrifuge tube lid, vibration 15s by 1mL Trizol.After treating solution fully emulsified (no noted phase separation phenomena), room temperature leaves standstill 15min again.

3) 4 ℃ of centrifugal 15min of 12000r/min.

4) the careful centrifuge tube that takes out from whizzer, this moment, homogenate was divided into three layers, that is: colourless supernatant liquor, intermediary white protein layer and have lower floor's organic phase of color.Drawing supernatant liquor is transferred in another new centrifuge tube.

5) in supernatant, add isopyknic Virahol, put upside down the abundant mixing of centrifuge tube after, under 15～30 ℃, leave standstill 10mim.

6) 4 ℃, the centrifugal 10min of 12000r/min, RNA are sunken to the pipe end.

7) carefully abandon supernatant, add 75% ethanol 1mL (preparation of DEPC water), the vibration precipitation.

8) 4 ℃, discard ethanol behind the centrifugal 5min of 12000r/min, the precipitation room temperature is dried to no ethanol smell, with a small amount of DEPC water dissolution precipitation.Electrophoretic analysis, and measure its concentration and analyze its purity.

4. according to GeneRacer ^TMKit (American I nvitrogen company) specification sheets operation steps will be extracted synthetic cDNA first chain of Eimeria tenella sporozoite RNA reverse transcription.Utilize the RACE primer amplification 3 ' and 5 ' the cDNA end of above-mentioned design.After 3 ' RACE and the recovery of 5 ' RACE pcr amplification product, be connected with the pGEM-T-easy carrier, transformed into escherichia coli TOP10 competent cell is selected white colony and is carried out the PCR evaluation, if amplified fragments is consistent with expection, then gets 500 μ L bacterium liquid and send company's order-checking.For the positive colony sequencing result, search the lap of 3 ' RACE and 5 ' RACE amplified production sequence and former est sequence with DNAstar software, the splicing full length cDNA sequence.

According to the full length cDNA sequence design two ends primers (seeing the following form 2) of splicing, reverse transcription synthetic cDNA first chain is a template during with 3 ' RACE and 5 ' RACE amplification, carries out pcr amplification.

Table 2 full-length cDNA amplimer

Carry out 1% agarose gel electrophoresis after reaction finishes and analyze (see figure 1).In Fig. 1, the M:DNA molecular weight standard; The full-length cDNA product of 1:EtCDPK gene amplification.The PCR product that reclaims is connected the transformed into escherichia coli competent cell with the pGEM-T-easy carrier.Select white colony and carry out the PCR evaluation.Send company's order-checking with the positive bacterium colony of identifying.

Utilize conventional information biology software that the full length cDNA sequence and the proteins encoded structure thereof that obtain are carried out forecast analysis.

5. result

According to the est sequence of ZB1-H07, its cDNA sequence that amplification obtains, total length 1336bp (SEQ ID NO.2), analysis revealed has the complete ORF of a 1302bp, 433 amino acid (SEQ ID NO.1) of encoding between 30～1331.The supposition theoretical molecular is 49265.5Da, and iso-electric point is 6.00, and the instability index in solution is 39.48, total wetting ability-0.309.The homology search finds that albumen and toxoplasma gondii, plasmodium falciparum, the people Cryptosporidium calcium dependent kinases of this genes encoding have very high homology, and the albumen of inferring this genes encoding is Eimeria tenella calcium dependent kinases, called after EtCDPK.

The albumen of striding this genes encoding of membrane structure analysis revealed contains one and strides membrane structure.The signal peptide analysis shows that the albumen of this genes encoding does not have signal peptide, shows that this albumen is not secreted protein.Hydrophobicity analysis shows that maximum value is 2.967, and minimum value is-3.233.This proteic antigen site is analyzed, and the result has 20 possible antigen sites.The structure function domain analysis finds that this albumen has than complex construction, comprising some more special structures, as two-pack nuclear localization signal, tetradecyl acylations site, pseudopeptidoglycan binding site etc.

The functional structure domain analysis finds that the albumen of EtCDPK genes encoding has the typical structure of CDPKs, and tangible CDPKs feature is arranged, and holds the C end to have 4 structural domains from N, is followed successively by variable region, catalytic domain, joining region and control region, wherein 4 and Ca ²⁺Bonded EF chiral structure, under the situation that lacks calmodulin and phosphatide, can be by calcium ion activated CDPKs activity, thus make CDPKs participation Ca ²⁺Rely on the signal conduction of signal pathway with phosphorylation, and in genetic expression, metabolism, signal pathway formation, ion transport and cytoskeleton form, play regulating effect.

The structure function domain analysis finds that the albumen of EtCDPK genes encoding contains protein kinase C phosphorylation site and casein kinase i I phosphorylation site.

Functional domain analysis simultaneously finds that also the albumen of EtCDPK genes encoding has the two-pack nuclear localization signal.Nuclear localization signal is a kind ofly to be present in the cell in the numerous protein and aminoacid sequence that mediating protein is transported in nucleus by tenuigenin.

Embodiment 2EtCDPK gene is in the differential expression analysis of Eimeria tenella different developmental phases

Extract total RNA of 4 etap of Eimeria tenella polypide (spore egg capsule, not spore egg capsule, sporozoite and s-generation merozoite) respectively, with Eimeria tenella not cDNA first chain of spore egg capsule, spore egg capsule, sporozoite, s-generation merozoite be template, utilize real-time fluorescence quantitative PCR, select 18s rRNA as confidential reference items, the expression of checking EtCDPK gene in Eimeria tenella different developmental phases polypide.Table 3 is a real-time fluorescence quantitative PCR amplimer sequence.The result shows that the EtCDPK gene is expressed (see figure 2) in the sporocyst level interval.

Table 3 real-time quantitative PCR amplimer sequence

Embodiment 3EtCDPK Prokaryotic Expression and immunogenicity analysis

1. the structure of prokaryotic expression plasmid

The EtCDPK gene is connected to prokaryotic expression carrier pET28a (+), make up recombinant expression plasmid pET (28a)-EtCDPK, after this pET (28a)-EtCDPK recombinant plasmid transformed gone into DH5 α competent cell, positive colony checks order and BamHI, Hind III double digestion are identified.The enzyme that Figure 3 shows that reorganization prokaryotic expression plasmid pET (28a)-EtCDPK is cut evaluation figure, and in Fig. 3, " 1 " represents BamHI, the HindIII double digestion product of recombinant expression plasmid pET (28a)-EtCDPK.

Through identifying correct recombinant expression plasmid (pET28a-EtCDPK) transformed into escherichia coli BL21 (DE3), through 1mmol/L IPTG abduction delivering.The EtCDPK predicted molecular weight is 49.3KDa, adopts His gene fusion expression system, and the fusion rotein of expression has the His label that molecular weight is 3.5kDa, and recombinant protein (His-EtCDPK) molecular weight that recombinant plasmid pET28a-EtCDPK expresses is approximately 53kDa.SDS-PAGE result shows that the fusion rotein of recombinant plasmid pET28a-EtCDPK expression is consistent substantially with the size of predictive analysis.For the optimization expression condition, carried out different time the expression analysis (0h, 2h, 4h, 6h, 8h, 10h), the result shows that transformed bacteria is 2h after 1mmol/L IPTG induces, Expression of Fusion Protein level basicly stable (Fig. 4).Therefore, the best abduction delivering time all is selected in 2h.In Fig. 4, M: protein standard molecular weight (being 94.0KDa, 66.2kDa, 45.0kDa, 35.0kDa, 26.0kDa, 20.0kDa, 14.4kDa from top to bottom); 1～6: recombinant plasmid is induced the expression product of back 0h-10h at IPTG.

2.His-EtCDPK expression of recombinant proteins form analysis and purifying

(1) His-EtCDPK protein expression form analysis

Behind ultrasonic degradation, centrifugal through IPTG abduction delivering bacterium liquid, cleer and peaceful precipitation in the SDS-PAGE electrophoretic analysis.Concrete steps are as follows:

1) get 20mL and induce the BL21 bacterium liquid that contains recombinant plasmid that spends the night through IPTG, the centrifugal 5min of 12000r/min abandons supernatant.

2) add the resuspended thalline of 4mL sterilization PBS in the precipitation in the 10mL centrifuge tube.

3) ultrasonication 2～5min on ice.

4) 12000r/min, centrifugal 5min.

5) supernatant sucks in the 1.5mL sterile tube, with the resuspended precipitation of aqua sterilisa of 2mL.

6) precipitation and supernatant respectively get 40 μ L respectively with the abundant mixing of 10 μ L, 2 * SDS-PAGE sample-loading buffer, sample is boiled 5min in water-bath, precipitate the centrifugal 5min of 4000r/min, carry out electrophoretic analysis respectively.

The result: fusion rotein His-EtCDPK is with the inclusion body formal representation.(Fig. 5).Among Fig. 5, M: protein standard molecular weight (big or small the same Fig. 4); 1: supernatant after the supersound process; 2: the supersound process postprecipitation.

(2) purifying of His-EtCDPK recombinant expression protein

Because the His-EtCDPK expressing protein is the inclusion body form, need to use the urea soluble protein earlier, use His Resin affinity column chromatography column purification of recombinant proteins again, the recombinant protein purity of SDS-PAGE analysis revealed purifying higher (Fig. 6).Among Fig. 6, M: protein standard molecular weight (big or small the same Fig. 4); 1: the EtCDPK recombinant protein of purifying.

3.His-EtCDPK the preparation of protein polyclone antibody

Because His-EtCDPK protein expression form is the inclusion body form, use several different methods behind the purifying and dialyse, but precipitation appears in albumen, so adopt the method for cutting the glue immunity to prepare polyclonal antibody, concrete steps are as follows:

(1) the bacterium liquid of centrifugal collection abduction delivering is removed supernatant.

(2) add 4mL PBS suspension bacterium, smudge cells (ultrasonic 2s stops 2s for 50W, 15min) on ice.

(3) 5000r/min, 4 ℃ of centrifugal 15min abandon supernatant, and precipitation adds 6mL ddH ₂O suspends, and adds 1mL2 * SDS-PAGE sample-loading buffer again, abundant mixing, and 100 ℃ are boiled sample 5min, the centrifugal 5min of 4000r/min.

(4) get supernatant and carry out SDS-PAGE.

(5) finish the back and dye and decolour, determine the target protein position from one of the middle cutting-out of glue.

(6) downcut the target protein of monoblock glue, be put into and smash the back in the mortar to pieces and add liquid nitrogen and fully grind, make gel become fine powder, add an amount of PBS (PH 8.0) afterwards, add freund's adjuvant while grinding, make its thorough mixing and emulsification, suck disposable syringe (5mL).

(7) give multi-point injection in the rabbit skin, carry out exempting from, two exempt from after two weeks, and adjuvant is a Freund's incomplete adjuvant, intradermal injection; Two exempt from then to carry out 3 two weeks exempts from, and adjuvant is a Freund's incomplete adjuvant.Three take rabbit serum to be the anti-His-EtCDPK polyclonal antibody of rabbit after exempting from.

4. the sero-fast preparation of Eimeria tenella

The Eimeria tenella spore egg capsule that the multigelation purifying is good under liquid nitrogen and the room temperature condition, ultrasonic on ice (ultrasonic 2s stops 2s for 50W, 30min), the centrifugal 30min of 12000r/min collects supernatant.To go up cleer and peaceful Freund's complete adjuvant by 1: 1 mixed emulsification, multi-point injection under rabbit skin carries out exempting from, and albumen dosage is 0.5mL altogether; Two week backs two exempt from (two exempt from and later on adjuvant be Freund's incomplete adjuvant), dosage is 1mL altogether; Exempted from direct injection supernatant 2mL in 14 days three.Blood sampling after 7 days, it is ℃ frozen to collect serum-20.

5. recombinant protein Western-blot analyzes

The recombinant protein His-EtCDPK of purifying is carried out SDS-PAGE, transfer on the pvdf membrane, recombinant protein His-EtCDPK respectively with the anti-His antibody of antiserum(antisera), rabbit of Eimeria tenella soluble proteins immune rabbit preparation and rabbit negative serum as an anti-Western-blot that is.Two anti-goat anti-rabbit iggs with the HRP mark carry out Western-blot and analyze.The recombinant protein His-EtCDPK band that responds as a result, molecular weight and expection size consistent (Fig. 7) show that this recombinant protein has certain immunogenicity.Among Fig. 7, M: protein standard molecular weight (being 170kDa, 130kDa, 95kDa, 72kDa, 55kDa, 43kDa, 34kDa, 26kDa, 17kDa, 11kDa from top to bottom); 1: the antiserum(antisera) of Eimeria tenella egg capsule whole protein immune rabbit; 2: the rabbit negative serum; 3: anti-His serum.

The eukaryotic expression of embodiment 4EtCDPK gene and immunogenicity analysis

In order to obtain soluble proteins, further analyze the EtCDPK function, the EtCDPK gene is connected among the yeast expression vector pPIC-9K, behind the conversion GS115 yeast competent cell, express through methanol induction, obtained soluble proteins, concrete steps are as follows:

(1) amplification EtCDPK gene

With pET28a-EtCDPK bacterium liquid is template, and redesign upstream and downstream primer carries out pcr amplification, and primer sequence is:

Upstream primer: 5 '-GCAGAATTCATGCAGCGAGCAGTCAAGACC-3 ' (SEQ ID NO.13);

Downstream primer: 5 '-GCAGCGGCCGCTCATTTGCTGCTCTTCTTTTTC-3 ' (SEQ ID NO.14);

The primer two ends are introduced EcoRI, Not I restriction enzyme site respectively, get 5 μ L after the PCR reaction finishes and carry out 1% agarose gel electrophoresis analysis.The purpose fragment that the result obtains and the (see figure 8) that conforms to of expectation.Among Fig. 8, the M:DNA molecular weight standard; The 1:EtCDPK goal gene.The EtCDPK goal gene is connected with the pGEM-T-easy carrier, the picking hickie extracts plasmid again.

(2) pPIC9k-EtCDPK construction of recombinant plasmid

With being connected with the empty carrier pPIC9k of same double digestion behind purpose fragment Not I, the EcoRI double digestion, made up recombinant plasmid pPIC9k-EtCDPK.This recombinant plasmid pPIC9k-EtCDPK transforms the TOP10 competent cell, and the positive colony that PCR identifies extracts plasmid.Recombinant plasmid and empty carrier are identified with Not I, EcoRI double digestion respectively qualification result shows, it is consistent with PCR product size that enzyme is cut the product fragment, all is about 1400bp.A macromolecule band (see figure 9) after cutting, the empty carrier enzyme is only arranged.Among Fig. 9, the M:DNA molecular weight standard; The 1:pPIC9k-EtCDPK recombinant plasmid is analyzed with Not I, EcoRI double digestion; The pPIC9k empty carrier is analyzed with Not I, EcoRI double digestion.

(3) conversion of pPIC9k-EtCDPK recombinant plasmid and screening

Behind recombinant plasmid vector pPIC9k-EtCDPK and empty carrier pPIC9k usefulness Sac I linearization for enzyme restriction, transform GS115 yeast competent cell.After transforming 2d, on the MD flat board, as seen grow white colony, after the amplification of third round YPD substratum, wherein 30 colony inoculations to YPD (containing G418 2.0mg/mL) flat board, are grown 4 bacterium colonies behind the 2d.

(4) abduction delivering of Yeast protein

1) gets two test tubes that contain the fresh YPD substratum of 4mL, inoculate mono-clonal bacterium colony that grows on YPD (the containing G418 2.0mg/mL) flat board and the empty carrier that the conversion back grows respectively and compare on the YPD flat board, place 30 ℃ of incubator overnight incubation.

2) respectively get 2mL and be inoculated in the fresh BMGY substratum of 50mL, 30 ℃ of shaking culture are to OD ₆₀₀Reach more than 10.

3) the centrifugal 5min of room temperature abandons supernatant, adds the fresh BMMY substratum of 50mL, 30 ℃ of shaking culture 24h.

4) in substratum, add 5mL 10 * methyl alcohol, 30 ℃ of shaking culture 24h.

5) the centrifugal 5min of room temperature, 4 ℃ of supernatant preservations is standby.

Collect methanol induction and express the supernatant liquid of 4d, supernatant is packed in the dialysis tubing, concentrate with polyoxyethylene glycol, salt is removed in dialysis in distilled water again, is SDS-PAGE and analyzes.The result shows, As time goes on, expressing quantity reaches maximum at 2d, and the 3rd～4d is basicly stable, the expressed proteins molecular weight with expect size conform to (Figure 10).Cell conditioned medium is a solubility through detecting albumen after concentrating.Among Figure 10, M: protein standard molecular weight (being 94.0KDa, 66.2kDa, 45.0kDa, 35.0kDa, 26.0kDa, 20.0kDa, 14.4kDa from top to bottom); 1～4: be respectively the expression product of inducing 1～4d; 5: the empty carrier negative control.

(5) recombinant protein Western-blot analyzes

The process of getting yeast expression concentrates albumen dialyse and is Western-blot and analyzes, and does blank with the pPIC9k empty carrier, the anti-Eimeria tenella immunize rabbit serum of using, and two resist and are the goat-anti rabbit anteserum of HRP mark, and the DAB substrate develops the color.The result shows that this albumen can be illustrated that the yeast expression product also has certain immunogenicity (Figure 11) by the identification of Eimeria tenella immunize rabbit serological specificity.Among Figure 11, M: protein standard molecular weight (being 170kDa, 130kDa, 95kDa, 72kDa, 55kDa, 43kDa, 34kDa, 26kDa, 17kDa, 11kDa from top to bottom); 1:pPIC9k empty carrier blank; 2: yeast expression albumen.

Embodiment 5EtCDPK functional analysis

1. immunofluorescence detects the distribution of EtCDPK in sporozoite invasion host cell process

Utilize the anti-EtCDPK polyclonal antibody of rabbit of preparation and the goat-anti rabbit two of green fluorescence mark to resist, checked respectively that PBS hatches the EtCDPK distribution situation after the sporozoite of 1h and sporozoite insert 2h, 12h behind the chick embryo fibroblast DF-1,24h, 36h, 48h, observed growth course and EtCDPK albumen distribution in sporozoite (Figure 12 A～F) of sporozoite in the DF-1 cell.Figure 12 A is that the fluorescence position observation mainly is positioned at sporozoite one end to EtCDPK albumen sporozoite is hatched 1h in PBS after, 2h mainly concentrates and is distributed in sporozoite front end (Figure 12 B) after sporozoite adds DF-1, in 48h subsequently, along with polypide further grows, expression amount increases, and in band worm cavity, find in a large number, infer that EtCDPK may be relevant with the sporozoite invasion (Figure 12 C～F).

2. whether body outer suppressioning test analysis EtCDPK is relevant with sporozoite invasion host cell

For further whether checking EtCDPK is relevant with the sporozoite invasion, carry out body outer suppressioning test.

The fresh sporozoite that extracts is hatched 1h with the mark sporozoite with fluorescence dye CFDA SE cell proliferation and spike detection reagent 37 ℃ of water-baths earlier, and then adding anti-EtCDPK polyclonal antibody of rabbit and sporozoite effect 2h, after low-speed centrifugal is collected the good sporozoite of mark, the sporozoite of mark is joined in the well-developed individual layer DF-1 cell of cultivation, cultivate and grow 20h, centrifugal going utilizes flow cytometry to detect the influence of EtCDPK polyclonal antibody to the sporozoite invasion in cell behind the supernatant, simultaneously not to be blank (Figure 13～Figure 15) with the sporozoite of polyclonal antibody effect and blank DF-1 cell.Figure 13 is that the DF-1 cell that does not infect is counted as blank through the Flow Cytometry instrument; Figure 14 is that the DF-1 cell that the good sporozoite of mark infects is counted in contrast through the Flow Cytometry instrument, and wherein A represents all cells of counting, and the DF-1 cell of sporozoite is not infected in the B1 representative, the DF-1 cell of B2 representative infection sporozoite; Figure 15 is that mark DF-1 cell good and handle the sporozoite infection with anti-EtCDPK polyclonal antibody is through Flow Cytometry instrument counting, wherein A represents all cells of counting, the DF-1 cell of sporozoite is not infected in the B1 representative, and the DF-1 cell of sporozoite is infected in the B2 representative.The result shows that the mark sporozoite of EtCDPK polyclonal antibody useless effect has 7.7% invasion to enter into the DF-1 cell, and has only 2.4% invasion to enter into the DF-1 cell through the sporozoite of anti-EtCDPK polyclonal antibody effect, and inhibiting rate reaches 68.8%.This result shows that EtCDPK is relevant with the invasion of sporozoite, may bring into play vital role when coccidia sporozoite invasion cell.

The above embodiment has only expressed embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.

Claims (10)

1. an Eimeria tenella gene is characterized in that, described genes encoding calcium dependent kinases, and its nucleotide sequence comprises one of following dna sequence dna:

(1) dna sequence dna of coding SEQ ID NO.1 aminoacid sequence;

(2) lack, add, insert or replace the resulting dna sequence dna of one or more amino acid in the aminoacid sequence shown in the coding SEQ ID NO.1 with the active aminoacid sequence of calcium dependent kinases.

2. Eimeria tenella gene according to claim 1 is characterized in that, described gene has the dna sequence dna shown in the SEQ ID NO.2.

3. an eimeria tenella protein is characterized in that, described protein has calcium dependent protein kinase enzymic activity, and this protein is selected from:

(1) has the protein of SEQ ID NO.1 aminoacid sequence;

(2) has the active fragments or the conservative property variant protein matter of partial amino-acid series among the SEQ ID NO.1;

(3) lack, add, insert or replace the resulting protein of one or more amino acid shown in the SEQ ID NO.1 in the aminoacid sequence with the active aminoacid sequence of calcium dependent kinases.

4. eimeria tenella protein according to claim 3 is characterized in that described protein has aminoacid sequence shown in the SEQ IDNO.1.

5. a recombinant vectors is characterized in that, comprises all or part of sequence of the described Eimeria tenella gene DNA sequence of claim 1.

6. a host cell is characterized in that, comprises the described recombinant vectors of claim 5, or transforms or transfection with the described gene order of claim 1.

7. an energy and the described eimeria tenella protein specificity of claim 3 bonded antibody or antiserum(antisera).

8. the application of the described Eimeria tenella gene of claim 1 is characterized in that, is used to prepare the vaccine or the medicine of prevention or treatment coccidiosis.

9. the application of the described eimeria tenella protein of claim 3 is characterized in that, is used to prepare the vaccine or the medicine of prevention or treatment coccidiosis.

10. the application of the described Eimeria tenella gene of claim 1 is characterized in that, is used to screen the medicine of prevention or treatment coccidiosis.

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