CN102676554A - Eimeria tenella protein tyrosine phosphatase gene and expression method as well as application thereof - Google Patents
Eimeria tenella protein tyrosine phosphatase gene and expression method as well as application thereof Download PDFInfo
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Abstract
The invention relates to an eimeria tenella (E. tenella) protein tyrosine phosphatase gene and an expression method as well as application thereof. The eimeria tenella protein tyrosine phosphatase gene has an amino acid sequence with a code SEQ ID NO. 1. By utilizing a RACE (Rapid Amplification of cDNA Ends) technology, a serine/threonine protein phosphatase (EtPP) gene is firstly amplified to E. tenella; the total length of the gene is 2,562 bp; the gene contains complete 1,647 bp ORF (Open Reading Frame); 549 amino acids are coded; the theoretical molecular weight is 60,822.4 Da; the molecular formula is C2705H4235N739O803S27; the isoelectric point is 5.89; the total number (Asp+Glu) of amino acids with negative charges is 71; the total number (Arg+Lys) of amino acids with positive charges is 62; and the gene is generally acidic. Antigenic site analysis discovers 19 possible antigenic sites. According to the invention, the gene is connected to a prokaryotic expression vector pET-28a, so that prokaryotic expression recombinant plasmid pET-28a-EtPP is constructed, and expression in an escherichia coli BL21 (DE3) system is realized, expressed fusion protein exists in the form of an inclusion body, recombinant fusion protein EtPP is obtained by using a His.Bind resin purification method, a theoretical foundation is laid for subsequently developed screening and confirmation of new drug target sites, an objective problem of drug resistance of coccidium is solved, and the gene has broad market prospect and economic benefit.
Description
Technical field
(Eimeria tenella, E.tenella) protein phosphatase gene and expression method thereof and application belong to biological technical field to the present invention relates to a kind of Eimeria tenella.
Background technology
Coccidiosis of chicken is that protozoon parasitizes in the Eimeria cell by the multiple device door in top, the most serious a kind of protozoal disease of the most common in aviculture, the most harmful, the most fatal, economic damage.This disease often is acute breaking out, and cause the chicken diarrhea, have blood in stool, anaemia, so that large quantities of death; Chronic case then causes chicken crowd appetite stimulator, grow be obstructed, anaemia, influence weightening finish and reduce feed conversion rate.This disease with 3 monthly ages with the susceptible of interior chicken, the M & M of chick is all higher.The chick growth of recovering is obstructed, and weightening finish slowly usually causes conditionality pathogenic bacterium such as intestinal bacteria easily and fall ill, and makes the chicken crowd occur that egg laying performance reduces, intractable is had loose bowels, immunosuppression, symptoms such as disease resistance reduction.Mostly adult chicken is the carrier, although there is not manifest symptom, growth performance and egg laying performance has all been produced very big influence, has brought enormous economic loss to poultry husbandry.In addition, characteristics such as coccidia is short with its life cycle, and environmental compatibility is strong, and transmission capacity is big are the most obstinate pathogenic agent that endangers aviculture at present, and are hard to guard against, the enormous economic loss of bringing to aviculture.Especially in the s-generation scissiparity stage, a large amount of merozoite of generation is invaded the intestinal epithelial cell that does not infect again, causes intestines mucosa flat spline structure to occur; The intestinal villus atrophy, large-area necrosis and erosion are subsided, and have hindered the absorption of nutritive substance; Make chicken infected discharge bloody stool, digestion and absorptive function descend, and feed conversion rate reduces; The chicken weightening finish descends, and brings out other pathogen infections and death, has seriously influenced the development of poultry husbandry.
Medical treatment remains the main means of preventing and treating coccidiosis at present; And to chemical sproof generation; Press for the research and development of anticoccidial new drug; And the research and development of new drug need be sought important action target, and are general main through screening the agent that is inhibited, and perhaps go to survey the characteristic of target protein through the design with suppressed molecule.Yet the screening of drug target is a very long process.
Summary of the invention
The object of the present invention is to provide a kind of Eimeria tenella protein phosphatase gene.
The present invention also aims to provide of the application of a kind of Eimeria tenella protein phosphatase gene at the anti-coccidiosis of chicken medicine thing of preparation.
To achieve these goals, technical scheme of the present invention has adopted a kind of Eimeria tenella protein phosphatase gene, has the aminoacid sequence of coding SEQ ID NO.1.
Described full length gene 2562bp contains complete 1647bp ORF, 549 amino acid of encoding, and theoretical molecular is 60822.4Da, molecular formula is C
2705H
4235N
739O
803S
27, iso-electric point is 5.89, and electronegative amino acid sum (Asp+Glu) is 71, and positively charged amino acid sum (Arg+Lys) is 62, overall slant acidity, the antigen site analysis finds to have 19 antigen sites.
Technical scheme of the present invention has also adopted a kind of expression method of Eimeria tenella protein phosphatase gene; May further comprise the steps: be the strain of experiment worm with E.tenella; Set up medicine anticoccidial action model; With E.tenella s-generation merozoite is research object; Made up diclazuril effect E.tenella s-generation merozoite differential expression cDNA subtractive library, filtered out the key molecule of a kind of diclazuril anticoccidial effect, be indicated as E.tenella serine/threonine protein Phosphoric acid esterase (EtPP) est sequence through the BlastP compare of analysis by the cDNA microarray technology.
Described expression method specifically may further comprise the steps:
(1) preparation of E.tenella s-generation merozoite
(1) foundation of trial model
The public young bird of the yellow plumage egg of high-quality is raised in University Of Science and Technology Of He'nan experimental animal room available from Luoyang City's brilliance kind chicken house, and feeding environment keeps hygienically clean, and does not have any pathogen contamination.During experimental animal feeding to 12 age in days, be equally divided into two groups, test 14 ages in days; All inoculate coccidia for two kinds, be divided into and infect control group (inoculation coccidia, 0mg/kg diclazuril feed) and drug-treated group (inoculation coccidia; 1mg/kg diclazuril feed is from testing 96h to experiment 120h).Set up diclazuril anticoccidial experimental animal model;
(2) preparation of E.tenella s-generation merozoite
120h slaughters test chicken after infection, cuts off the caecum wall, removes cecal content, with containing remaining content on two anti-ice-cold PBS flush away caecums; The caecum of cleaning is cut into about 1cm in petridish
3The fragment of size is transferred in the beaker, adds the enzymic digestion liquid of 10 times of volumes, digests 45min in 37 ℃ of water-baths, and stirring frequently, makes merozoite fully dissociate out; And, remove big tissue block and impurity through four layers of sterile gauzes filtration; Filtrating is through the centrifugal 10min of 3000r/min; With the resuspended deposition of PBS, washing, the centrifugal 10min of 3000r/min; Adopt the resuspended deposition of erythrocyte cracked liquid, 4 ℃ of cracking 10min, and stirring frequently, the centrifugal 10min of 2500r/min then; With the PBS washing precipitation once, the centrifugal 10min of 3000r/min;
To precipitate dilution with PBS, add the Percoll parting liquid, be configured to the 30%Percoll suspension; The merozoite suspension that will contain 30%Percoll slowly adds to above 50% the Percoll PBS solution, notes not destroying liquid level; Behind the centrifugal 15min of 3200r/min, carefully draw 50% Percoll solution layer in another clean centrifuge tube; Contain merozoite sucking-off liquid with the PBS cleaning, behind the centrifugal 5min of 3500r/min, collecting precipitation; The PBS washing, the centrifugal 5min of 3500r/min, deposition is the s-generation merozoite of acquisition;
(2) clone of E.tenella s-generation merozoite EtPP gene
(1) extraction of the total RNA of E.tenella s-generation merozoite
The extraction of total RNA is carried out according to the Trizol reagent extraction operation instructions of Invitrogen company.
(2) E.tenella s-generation merozoite EtPP gene 5 ' and 3 ' amplification
Sequence with after the screening order-checking in the subtractive library is EST, with according to the SMARTer of clonetech company
TMRACE cDNA Amplification Kit specification sheets carries out the amplification of gene 3 ' and 5 ',
5 ' RACE, 3 ' primer: be 5 '-GACAGTTTGCGTGCGTCCGTTGAAG-3 ',
3 ' RACE, 5 ' primer is 5 '-CCGTCTGCTACCCCTGGCTTTTGTG-3 ', and amplified production carries out 1% agarose gel electrophoresis analysis;
(3) recovery of E.tenella s-generation merozoite EtPP gene 5 ' and 3 ' pcr amplification product and and order-checking
Adopt the QIA quick Gel Extraction kit of QIAGEN company to reclaim the amplified production of gene 5 ' and 3 '; And be connected with pMD19-T Vector, conversion DH5 α competent cell carries out blue hickie screening; After selecting hickie bacterium colony incubated overnight, carry out PCR and identify; Through the positive bacterium colony of identifying that the molecular weight size is consistent with expection, serve extra large handsome Bioisystech Co., Ltd and check order;
(4) splicing of E.tenella s-generation merozoite EtPP full length gene sequence
With 3 ' RACE and 5 ' the RACE PCR product sequence that order-checking obtains, utilize DNAstar software to search the sequence of amplified production and the lap of former est sequence, be full length cDNA sequence with three sections sequence assemblies;
(5) amplification and the order-checking of E.tenella s-generation merozoite EtPP gene
Be the basis with the splicing full-length gene order; Utilize Primer 5.0 software design upstream and downstream primers; With cDNA is template; The total length amplimer is: Sense primer:5 '-TGCATGCGACAGGATATTTG-3 ' and Antisense primer:5 '-GCGAAGTTTTCAATGCCAAG-3 ', carry out RT-PCR amplification total length; Reaction finishes the back and adopts the QIA quick Gel Extraction kit of QIAGEN company that the PCR product is connected with pMD 19-T Vector, transforms DH5 α competent cell, carries out blue hickie and screens, select hickie bacterium colony incubated overnight after, carry out the PCR evaluation; Through the positive bacterium colony of identifying that the molecular weight size is consistent with expection, to serve extra large handsome Bioisystech Co., Ltd and check order, sequence is identical with former sequence;
(3) the proteic expression of E.tenella s-generation merozoite EtPP
(1) structure of EtPP expression plasmid
With cDNA is template, obtains sequence (Fig. 3) and expression vector pET-28a MCS according to order-checking, selects EcoR I and Not I restriction enzyme site at aim sequence ORF (Fig. 4) two ends,
Primer sequence is: Sense primer:5 '-CTAGAATTCATGGCGGAAGGCAGCGAC-3 ' and Antisense primer:5 '-GCAGCGGCCGCTTAAATGAAGCTTAGC-3 '; Utilize pcr amplification EtPP gene; PCR recovery product and pET-28a plasmid are carried out double digestion with EcoR I and Not I respectively, and after the purifying and recovering, the T4DNA ligase enzyme connects; And transformed into escherichia coli DH5 α cell; The picking mono-clonal, the downstream that PCR and order-checking proof EtPP gene correctly insert the HIS gene, called after pET-28a-EtPP;
(2) expression of EtPP expression plasmid in intestinal bacteria
With identifying that correct recombinant expression plasmid pET-28a-EtPP changes e. coli bl21 (DE3) host cell, 37 ℃ of incubated overnight, picking list bacterium colony over to; Be inoculated in enlarged culturing in the LB liquid nutrient medium that contains Kan, to be cultured to OD260 at 37 ℃ about 0.4～0.6 the time when bacterium liquid, add IPTG to final concentration be 0.8mmol/L; 37 ℃ of abduction deliverings are collected thalline in the different culture time, after the ultrasonication; Carry out the SDS-PAGE electrophoretic analysis, expression product mainly exists with the inclusion body form;
(3) the proteic purifying of EtPP
The EtPP albumen that BL21 (DE3) expresses mainly exists with the form of inclusion body.Get above-mentioned thalline, 12000r/min gets deposition behind 4 ℃ of centrifugal 15min, PBS washing and resuspended thalline, and after the ice-bath ultrasonic, 12000r/min, 4 ℃ of centrifugal back collecting precipitations promptly get purified inclusion body.With reference to HisBind resin purification method purifying inclusion body protein, the SDS-PAGE technology for detection is analyzed then.
That the present invention adopts is novel at present, low toxicity, wide spectrum, anticoccidial drug diclazuril efficiently; With E.tenella is the strain of experiment worm; Having set up medicine anticoccidial action model, is research object with E.tenella s-generation merozoite, has made up diclazuril effect E.tenella s-generation merozoite differential expression cDNA subtractive library; Filter out the key molecule of a kind of diclazuril anticoccidial effect by the cDNA microarray technology; Be indicated as E.tenella serine/threonine protein Phosphoric acid esterase (EtPP) est sequence through the BlastP compare of analysis, and pass through the full length sequence of RACE technology amplification acquisition EtPP, and carried out the expression of full-length gene and used discussion.
The present invention utilizes the RACE technology, serine/threonine protein Phosphoric acid esterase (EtPP) gene of the E.tenella that increases first, and this full length gene 2562bp contains complete 1647bp ORF, 549 amino acid of encoding, theoretical molecular is 60822.4Da, molecular formula is C
2705H
4235N
739O
803S
27, iso-electric point is 5.89, and electronegative amino acid sum (Asp+Glu) is 71, and positively charged amino acid sum (Arg+Lys) is 62, overall slant acidity.The antigen site analysis finds to have 19 possible antigen sites.The present invention is connected to this gene among the prokaryotic expression carrier pET-28a; Made up prokaryotic expression recombinant plasmid pET-28a-EtPP, and realized the expression in e. coli bl21 (DE3) system, the fusion rotein of expression exists with the inclusion body form; And adopt HisBind resin purification method to obtain recombination fusion protein EtPP; Establish theoretical basis for follow-up screening and the affirmation of carrying out the new drug target position, solved the chemical sproof objective difficult problem of coccidia, had vast market prospect and economic benefit.
Description of drawings
Fig. 1 is EtPP gene 5 ' pcr amplification product; Wherein, M, dna molecular amount standard, 1 is 5 ' PCR product;
Fig. 2 is EtPP gene 3 ' pcr amplification product; Wherein, M, dna molecular amount standard, 1 is 5 ' PCR product;
Fig. 3 is an EtPP full length gene sequence;
Fig. 4 is an EtPP gene ORF pcr amplification product;
Fig. 5 detects the expression of recombinant plasmid pET-28a-EtPP in E.coli BL21 (DE3) cell for SDS-PAGE; Wherein, M, LMWP molecular weight standard; NC, negative control (not carrying out abduction delivering); (1h, 2h, 4h, 6h, 8h, 9h), different time inductive expression product; Su, supernatant; Pe, deposition;
Fig. 6 detects EtPP fusion rotein purified product for SDS-PAGE; M, the LMWP molecular weight standard; 1 and 2, the protein purification product.
Embodiment
Employed in the present invention term only if other explanation is arranged, generally has the implication of those of ordinary skills' common sense.
The expression method of Eimeria tenella protein phosphatase gene of the present invention is following:
One, the preparation of E.tenella s-generation merozoite
(1) foundation of trial model
The public young bird of the yellow plumage egg of high-quality is raised in University Of Science and Technology Of He'nan experimental animal room available from Luoyang City's brilliance kind chicken house, and feeding environment keeps hygienically clean, and does not have any pathogen contamination.During experimental animal feeding to 12 age in days, be equally divided into two groups, test 14 ages in days; All inoculate coccidia for two kinds, be divided into and infect control group (inoculation coccidia, 0mg/kg diclazuril feed) and drug-treated group (inoculation coccidia; 1mg/kg diclazuril feed is from testing 96h to experiment 120h).Set up diclazuril anticoccidial experimental animal model.
(2) preparation of E.tenella s-generation merozoite
120h slaughters test chicken after infection, cuts off the caecum wall, removes cecal content, with containing remaining content on two anti-ice-cold PBS flush away caecums; The caecum of cleaning is cut into about 1cm in petridish
3The fragment of size is transferred in the beaker, adds the enzymic digestion liquid of 10 times of volumes, digests 45min in 37 ℃ of water-baths, and stirring frequently, makes merozoite fully dissociate out; And, remove big tissue block and impurity through four layers of sterile gauzes filtration; Filtrating is through the centrifugal 10min of 3000r/min; With the resuspended deposition of PBS, washing, the centrifugal 10min of 3000r/min; Adopt the resuspended deposition of erythrocyte cracked liquid, 4 ℃ of cracking 10min, and stirring frequently, the centrifugal 10min of 2500r/min then; With the PBS washing precipitation once, the centrifugal 10min of 3000r/min;
To precipitate dilution with PBS, add the Percoll parting liquid, be configured to the 30%Percoll suspension; The merozoite suspension that will contain 30%Percoll slowly adds to above 50% the Percoll PBS solution, notes not destroying liquid level; Behind the centrifugal 15min of 3200r/min, carefully draw 50% Percoll solution layer in another clean centrifuge tube; Contain merozoite sucking-off liquid with the PBS cleaning, behind the centrifugal 5min of 3500r/min, collecting precipitation; The PBS washing, the centrifugal 5min of 3500r/min, deposition is the s-generation merozoite of acquisition.
Two, the clone of E.tenella s-generation merozoite EtPP gene
(1) extraction of the total RNA of E.tenella s-generation merozoite
The extraction of total RNA is carried out according to the Trizol reagent extraction operation instructions of Invitrogen company.
(2) E.tenella s-generation merozoite EtPP gene 5 ' and 3 ' amplification
Sequence with after the screening order-checking in the subtractive library is EST; To carry out the amplification of gene 3 ' and 5 ' according to clonetech company SMARTerTM RACE cDNA Amplification Kit specification sheets; 5 ' RACE, 3 ' primer: be 5 '-GACAGTTTGCGTGCGTCCGTTGAAG-3 '; 3 ' RACE, 5 ' primer is 5 '-CCGTCTGCTACCCCTGGCTTTTGTG-3 ', and 1% agarose gel electrophoresis analysis is carried out in amplification.
(3) recovery of E.tenella s-generation merozoite EtPP gene 5 ' and 3 ' pcr amplification product and and order-checking
Adopt the amplified production of the QIA quick Gel Extraction kit recovery gene 5 ' (Fig. 1) and 3 ' (Fig. 2) of QIAGEN company; And be connected with pMD19-T Vector, conversion DH5 α competent cell carries out blue hickie screening; After selecting hickie bacterium colony incubated overnight, carry out PCR and identify.Through the positive bacterium colony of identifying that the molecular weight size is consistent with expection, serve extra large handsome Bioisystech Co., Ltd and check order.
(4) splicing of E.tenella s-generation merozoite EtPP full length gene sequence
With 3 ' RACE and 5 ' the RACE PCR product sequence that order-checking obtains, utilize DNAstar software to search the sequence of amplified production and the lap of former est sequence, be full length cDNA sequence (Fig. 3) with three sections sequence assemblies.
(5) amplification and the order-checking of E.tenella s-generation merozoite EtPP gene
Be the basis with the splicing full-length gene order; Utilize Primer 5.0 software design upstream and downstream primers; With cDNA is template; The total length amplimer is: Sense primer:5 '-TGCATGCGACAGGATATTTG-3 ' and Antisense primer:5 '-GCGAAGTTTTCAATGCCAAG-3 ', carry out RT-PCR amplification total length.Reaction finishes the back and adopts the QIA quick Gel Extraction kit of QIAGEN company that the PCR product is connected with pMD 19-T Vector, transforms DH5 α competent cell, carries out blue hickie and screens, select hickie bacterium colony incubated overnight after, carry out the PCR evaluation.Through the positive bacterium colony of identifying that the molecular weight size is consistent with expection, to serve extra large handsome Bioisystech Co., Ltd and check order, sequence is identical with former sequence.
Three, the proteic expression of E.tenella s-generation merozoite EtPP
(1) structure of EtPP expression plasmid
With cDNA is template; Obtain sequence (Fig. 3) and expression vector pET-28a MCS according to order-checking, select EcoR I and Not I restriction enzyme site at aim sequence ORF (Fig. 4) two ends, primer sequence is: Sense primer:5 '-CTAGAATTCATGGCGGAAGGCAGCGAC-3 ' and Antisense primer:5 '-GCAGCGGCCGCTTAAATGAAGCTTAGC-3 '; Utilize pcr amplification EtPP gene; PCR recovery product and pET-28a plasmid are carried out double digestion with EcoR I and Not I respectively, and after the purifying and recovering, the T4DNA ligase enzyme connects; And transformed into escherichia coli DH5 α cell; The picking mono-clonal, the downstream that PCR and order-checking proof EtPP gene correctly insert the HIS gene, called after pET-28a-EtPP.
(2) expression of EtPP expression plasmid in intestinal bacteria
With identifying that correct recombinant expression plasmid pET-28a-EtPP changes e. coli bl21 (DE3) host cell, 37 ℃ of incubated overnight, picking list bacterium colony over to; Be inoculated in enlarged culturing in the LB liquid nutrient medium that contains Kan, to be cultured to OD260 at 37 ℃ about 0.4～0.6 the time when bacterium liquid, add IPTG to final concentration be 0.8mmol/L; 37 ℃ of abduction deliverings are collected thalline in the different culture time, after the ultrasonication; Carry out the SDS-PAGE electrophoretic analysis, expression product mainly exists with the inclusion body form, like Fig. 5.
(3) the proteic purifying of EtPP
The EtPP albumen that BL21 (DE3) expresses mainly exists with the form of inclusion body.Get above-mentioned thalline, 12000r/min gets deposition behind 4 ℃ of centrifugal 15min, PBS washing and resuspended thalline, and after the ice-bath ultrasonic, 12000r/min, 4 ℃ of centrifugal back collecting precipitations promptly get purified inclusion body.Then with reference to HisBind resin purification method purifying inclusion body protein, SDS-PAGE check and analysis such as Fig. 6.
The aminoacid sequence of the expression method of the Eimeria tenella protein phosphatase gene of present embodiment is shown in sequence table SEQ ID NO.1
Sequence table
SEQUENCE?LISTING
< 110>University Of Science and Technology Of He'nan
< 120>a kind of Eimeria tenella protein phosphatase gene and expression method and application
<170>PatentIn?version?3.5
<210>1
<211>558
< 213>Eimeria tenella protein phosphatase gene
<400>1
MAEGSDSAAISCNSAESLKREEGQIIKDSAMAVDGGGSCAAKEAGNVVPCSEKDVS 57
ASAAVLERAEALKNEGNVLFKQHQYAEAVAKYSAAIDLVDEAPVDPRETQLHVFLC?114
NRAFAHLRMENFGSAIIDAERALKLNPKFSKGYYRRGTGYFCLGNLKAAQRDFERV?171
CQLRGGKDADAQSRLNECKKQLRAQAFAAAIRTKQTIPASKQVLAEGLEKLFPVPA?228
DYAGPVYKKGKVDEEFLQALRTWLQNPENRLHPQYAYAMAVDFIEILEPLPSLVDL?285
EIPADKEITICGDVHGQFYDLLNIFTINGMPSEENPYLFNGDIVDRGSFSVEVLLM?342
MVACKLAYPNHMHITRGNHETSEMNAMYGFKGEMLNKYDERLYQIFSEAFRLLPLA?399
FVVNKKAFVVHGGLSRQENVTLDEIRKIDRNREPGSDVLITDLLWSDPSPLPGLTP?456
SKRGVACQFGPDITEKFLKTNNLSFVIRSHEMKEAGYEVEHGGKLITVFSAPNYCD?513
EMGNKGAFIRLKGSEMVPKFHQFTAVPHPPGPAMQYANPMLSFI 558
Claims (5)
1. an Eimeria tenella protein phosphatase gene is characterized in that: the aminoacid sequence with coding SEQ ID NO.1.
2. Eimeria tenella protein phosphatase gene according to claim 1 is characterized in that: described full length gene 2562bp, contain complete 1647bp ORF, and 549 amino acid of encoding, theoretical molecular is 60822.4Da, molecular formula is C
2705H
4235N
739O
803S
27, iso-electric point is 5.89, and electronegative amino acid sum (Asp+Glu) is 71, and positively charged amino acid sum (Arg+Lys) is 62, overall slant acidity.The antigen site analysis finds to have 19 possible antigen sites.
3. the expression method of an Eimeria tenella protein phosphatase gene; It is characterized in that: may further comprise the steps: be the strain of experiment worm with E.tenella; Set up medicine anticoccidial action model; With E.tenella s-generation merozoite is research object; Made up diclazuril effect E.tenella s-generation merozoite differential expression cDNA subtractive library, filtered out the key molecule of a kind of diclazuril anticoccidial effect, be indicated as E.tenella serine/threonine protein Phosphoric acid esterase (EtPP) est sequence through the BlastP compare of analysis by the cDNA microarray technology.
4. the expression method of Eimeria tenella protein phosphatase gene according to claim 1 is characterized in that: described expression method specifically may further comprise the steps:
(1) preparation of E.tenella s-generation merozoite
(1) foundation of trial model
The yellow plumage egg of high-quality is public young, during experimental animal feeding to 12 age in days, is equally divided into two groups; Test 14 ages in days, all inoculate coccidia for two kinds, be divided into and infect control group (inoculation coccidia; 0mg/kg diclazuril feed) and the drug-treated group (inoculation coccidia; 1mg/kg diclazuril feed is from testing 96h to experiment 120h), set up diclazuril anticoccidial experimental animal model;
(2) preparation of E.tenella s-generation merozoite
120h slaughters test chicken after infection, cuts off the caecum wall, removes cecal content, with containing remaining content on two anti-ice-cold PBS flush away caecums; The caecum of cleaning is cut into about 1cm in petridish
3The fragment of size is transferred in the beaker, adds the enzymic digestion liquid of 10 times of volumes, digests 45min in 37 ℃ of water-baths, and stirring frequently, makes merozoite fully dissociate out; And, remove big tissue block and impurity through four layers of sterile gauzes filtration; Filtrating is through the centrifugal 10min of 3000r/min; With the resuspended deposition of PBS, washing, the centrifugal 10min of 3000r/min; Adopt the resuspended deposition of erythrocyte cracked liquid, 4 ℃ of cracking 10min, and stirring frequently, the centrifugal 10min of 2500r/min then; With the PBS washing precipitation once, the centrifugal 10min of 3000r/min;
To precipitate dilution with PBS, add the Percoll parting liquid, be configured to the 30%Percoll suspension; The merozoite suspension that will contain 30%Percoll slowly adds to above 50% the Percoll PBS solution, notes not destroying liquid level; Behind the centrifugal 15min of 3200r/min, carefully draw 50% Percoll solution layer in another clean centrifuge tube; Contain merozoite sucking-off liquid with the PBS cleaning, behind the centrifugal 5min of 3500r/min, collecting precipitation; The PBS washing, the centrifugal 5min of 3500r/min, deposition is the s-generation merozoite of acquisition;
(2) clone of E.tenella s-generation merozoite EtPP gene
(1) extraction of the total RNA of E.tenella s-generation merozoite
The extraction of total RNA is carried out according to the Trizol reagent extraction operation instructions of Invitrogen company;
(2) E.tenella s-generation merozoite EtPP gene 5 ' and 3 ' amplification
Sequence with after the screening order-checking in the subtractive library is EST, with according to the SMARTer of clonetech company
TMRACE cDNAAmplification Kit specification sheets carries out the amplification of gene 3 ' and 5 ',
5 ' RACE, 3 ' primer: be 5 '-GACAGTTTGCGTGCGTCCGTTGAAG-3 ',
3 ' RACE, 5 ' primer is 5 '-CCGTCTGCTACCCCTGGCTTTTGTG-3 ', and amplified production carries out 1% agarose gel electrophoresis analysis;
(3) recovery of E.tenella s-generation merozoite EtPP gene 5 ' and 3 ' pcr amplification product and and order-checking
Adopt the QIA quick Gel Extraction kit of QIAGEN company to reclaim the amplified production of gene 5 ' and 3 '; And be connected with pMD19-T Vector, conversion DH5 α competent cell carries out blue hickie screening; After selecting hickie bacterium colony incubated overnight, carry out PCR and identify;
(4) splicing of E.tenella s-generation merozoite EtPP full length gene sequence
With 3 ' RACE and 5 ' the RACE PCR product sequence that order-checking obtains, utilize DNAstar software to search the sequence of amplified production and the lap of former est sequence, be full length cDNA sequence with three sections sequence assemblies;
(5) amplification and the order-checking of E.tenella s-generation merozoite EtPP gene
Be the basis with the splicing full-length gene order; Utilize Primer 5.0 software design upstream and downstream primers; With cDNA is template; The total length amplimer is: Sense primer:5 '-TGCATGCGACAGGATATTTG-3 ' and Antisense primer:5 '-GCGAAGTTTTCAATGCCAAG-3 ', carry out RT-PCR amplification total length; Reaction finishes the back and adopts the QIA quick Gel Extraction kit of QIAGEN company that the PCR product is connected with pMD19-T Vector, transforms DH5 α competent cell, carries out blue hickie and screens, select hickie bacterium colony incubated overnight after, carry out the PCR evaluation;
(3) the proteic expression of E.tenella s-generation merozoite EtPP
(1) structure of EtPP expression plasmid
With cDNA is template, obtains sequence and expression vector pET-28a MCS according to order-checking, selects EcoR I and Not I restriction enzyme site at aim sequence ORF two ends,
Primer sequence is: Sense primer:5 '-CTAGAATTCATGGCGGAAGGCAGCGAC-3 ' and Antisense primer:5 '-GCAGCGGCCGCTTAAATGAAGCTTAGC-3 '; Utilize pcr amplification EtPP gene; PCR recovery product and pET-28a plasmid are carried out double digestion with EcoR I and Not I respectively, and after the purifying and recovering, the T4DNA ligase enzyme connects; And transformed into escherichia coli DH5 α cell; The picking mono-clonal, the downstream that PCR and order-checking proof EtPP gene correctly insert the HIS gene, called after pET-28a-EtPP;
(2) expression of EtPP expression plasmid in intestinal bacteria
With identifying that correct recombinant expression plasmid pET-28a-EtPP changes e. coli bl21 (DE3) host cell, 37 ℃ of incubated overnight, picking list bacterium colony over to; Be inoculated in enlarged culturing in the LB liquid nutrient medium that contains Kan, to be cultured to OD260 at 37 ℃ about 0.4～0.6 the time when bacterium liquid, add IPTG to final concentration be 0.8mmol/L; 37 ℃ of abduction deliverings are collected thalline in the different culture time, after the ultrasonication; Carry out the SDS-PAGE electrophoretic analysis, expression product mainly exists with the inclusion body form;
(3) the proteic purifying of EtPP
The EtPP albumen that BL21 (DE3) expresses mainly exists with the form of inclusion body.Get above-mentioned thalline, 12000r/min gets deposition behind 4 ℃ of centrifugal 15min, PBS washing and resuspended thalline, and after the ice-bath ultrasonic, 12000r/min, 4 ℃ of centrifugal back collecting precipitations promptly get purified inclusion body; With reference to HisBind resin purification method purifying inclusion body protein, SDS-PAGE carries out check and analysis then.
5. application that utilizes the Eimeria tenella protein phosphatase gene to prepare anti-coccidiosis of chicken medicine.
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