CN104593385A - Eimeria tenella gametophyte antigen gam59 gene and application thereof - Google Patents
Eimeria tenella gametophyte antigen gam59 gene and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of gene engineering vaccines, and concretely relates to an eimeria tenella gametophyte antigen gene, expression and application of an expression product. The size of the open reading frame of the gene is 1617 bp, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1. A prokaryotic expression vector pET28a-Etgam59 is constructed based on the gene, and is transformed into BL21 host bacterium for inducible expression. The expression product can be recognized by chicken convalescent serum, which shows that recombinant protein possesses immunogenicity. The obtained serum of an immunized mouse of a recombinant eukaryotic expression plasmid pcDNA3.1-Etgam59 constructed by amplifying the gene is capable of specifically combined with the expression product of pET28a-Etgam59, which shows that the gene is applicable to development of a chicken coccidiosis gene engineering vaccine.
Description
Technical field
The present invention relates to the gametocyte antigen gene of Eimeria tenella (Eimeria tenella), by the polypeptide of described genes encoding, the acquisition methods of the carrier containing described gene and described gene and the vivoexpression method of polypeptide and Function Identification.Particularly, gene of the present invention comes from the gametophyte in Eimeria Tenella syngenesis stage.
Background technology
Coccidiosis of chicken is the protozoal disease being colonized in the serious harm poultry husbandry caused in chicken small intestine or caecum mucous membrane by one or several coccidias of Eimeria, estimate the annual whole world because of the loss of coccidiosis be 3,000,000,000 dollars.The year consumption just about 6 ~ 1,800,000,000 yuans of anticoccidial drug is often only by China, and the direct and indirect economic loss caused by coccidiosis of chicken is then difficult to estimate.For a long time, the control of coccidiosis of chicken mainly relies on medicine.But because coccidia generally creates resistance to medicine, cause drug effect to decline.And the long-term medicine that adds also causes drug residue meat egg in the fowl raising cycle, direct harm humans is healthy, and contaminate environment.For this reason, the prevention and controls of coccidiosis of chicken has turned to immunoprophylaxis by chemoprophylaxis.
At present, clinically for the vaccine of immunoprophylaxis coccidiosis of chicken by worm seedling and the large class of the subunit vaccine two that is made up of native protein of living.Although coccidiosis of chicken living vaccine has stronger immune effect, also exist as spread the risk of cause of disease, the strain of coccidia worm antigenic variation, cause that weak worm strain virulence is easily returned by force, vaccination doses is difficult to control, vaccine affect the price of deed, production cost is high, immune programme for children is loaded down with trivial details, operate and monitoring after immunity and the more high distinct issues of administrative skill requirement.It is formulated that commercial coccidiosis of chicken subunit vaccine is that origin comes from the gametophytic three kinds of purifying antigens of Eimeria maxima (molecular mass is respectively 230,82,56kDa), for disturbing the formation of egg capsule wall, namely block the syngenesis stage of Eimeria, thus reduce generation and the propagation of egg capsule.Because the antigen of such vaccine need through affinity column separation and purification from gametophyte albumen, gametophyte again need separation and purification from the intestinal epithelial cell of infected chicken body, therefore complex manufacturing, and cost is high, and difficulty or ease meet Production requirement.And utilize genetic engineering technique to produce such antigen in a large number, may be the optimal strategy addressed this problem.But Eimeria maxima gametocyte antigen Emgam82 and Emgam56 gene are subject to patent protection, and the immunity of Eimeria species has species specificity, the immunizing power namely produced after a kind of immunity to coccidiosis only has coccidium infection of the same race embraces protection.
Eimeria tenella is the main pathogen of coccidiosis of chicken, the chicken of main harm 15 ~ 50 age in days.From the functional antigen of Eimeria tenella isolation identification mainly from etap such as sporozoite, schizont, merozoites, wherein some protein localization is in the cytolemma of polypide, microneme, the structure such as rhoptry and refractive body, mainly comprises the gene such as SO7, MZ5-7, TA4 (A4), GX3262,3-1E, EtMic2 and LDH.Up to now, the rarely seen Etmgam22 gene of Eimeria tenella gametophyte full-length gene of bibliographical information, this gene contains the open reading frame of a 597bp, is a kind of multi-copy gene of not intron.Meanwhile, foreign literature also confirms the homologue that there are two kinds of Eimeria maxima gametophyte Emgam56 genes in Eimeria tenella, be respectively Etgam56 and Etgam59, but its nucleotide sequence there is no report.Eimeria tenella gametophyte Etgam22, Etgam56 are identical with Emgam59 gene with Eimeria maxima gametophyte Emgam56 with the function of Etgam59 gene, and the albumen of its coding is used for the formation of egg capsule wall.Therefore, Eimeria tenella gametophyte Etgam22, Etgam56 and Etgam59 gene may be used for building recombinant vaccine, the coccidiosis that control Eimeria tenella causes.
Summary of the invention
The present invention utilizes genetic engineering technique, clone Eimeria tenella gametocyte antigen gene Etgam59, and this gene is proceeded to engineering bacteria express, obtain a large amount of recombinant antigens, and the specificity, immunogenicity etc. of recombinant antigen are furtherd investigate, purport lays the foundation for developing Eimeria species restructuring gametocyte antigen vaccine.
The present invention relates to a kind of gametocyte antigen gene of Eimeria tenella, wherein, the open reading frame of described gene is 1617bp, and its nucleotide sequence is as shown in SEQ ID NO.1.
The aminoacid sequence of its coding is as shown in SEQ ID NO.2.
Remove the later sequence of signal peptide (front 22 amino acid) as shown in SEQ ID NO.3.
The present invention relates to a kind of expression vector pET28a, it contains described gametocyte antigen gene.Inserting restriction enzyme site by being separated the gametocyte antigen gene obtained, being connected in prokaryotic expression carrier pET28a, routine transformation e. coli bl21 (DE3), building genetic engineering bacterium, and abduction delivering, abstraction and purification is carried out to expression product.
In addition, the present invention relates to a kind of eukaryotic expression vector pcDNA3.1, it contains described gametocyte antigen gene.Insert restriction enzyme site by being separated the Etgam59 gene obtained, be connected in pcDNA3.1, be built into pcDNA3.1-Etgam59 eukaryon recombinant plasmid, immune mouse obtains polyclonal antibody; Resist for primary antibodie with this mouse more, with the Etgam59 recombinant protein of prokaryotic expression for antigen carries out Western-blot detection, the immunogenicity of the Etgam59 recombinant protein that qualification eucaryon plasmid is expressed.
As preferred scheme, concrete operations are:
1) from Eimeria tenella gametophytic development, Etgam59 gene is separated:
Ordinary method separation and purification Eimeria tenella gametophyte, extracts test kit with RNA and extracts total serum IgE.Design Auele Specific Primer:
Upstream primer: 5 '-CCCCGGGAACATGACTCGTCTCGCCGCC-3 ' (SEQ ID NO.4);
Downstream primer: 5 '-CGAATTCTTACTCAAATCCAAAAGAAGG-3 ' (SEQ ID NO.5).
The cDNA obtained with the reverse transcription of gametophyte total serum IgE is for template, and after the optimization repeatedly of PCR condition, Successful amplification goes out to conform to expected results and size is the specific fragment (Fig. 1) of about 1617bp, called after Etgam59.Containing object fragment PCR products after kits reclaims, will be cloned in pGEM-T-easy carrier, obtain pGEM-T-easy-Etgam59.
2) structure of prokaryotic expression carrier
According to ORF sequence and the plasmid pET-28a restriction enzyme site of Eimeria tenella Etgam59 gene, after removing the signal peptide of Etgam59 gene, design 1 pair of Auele Specific Primer:
Upstream primer F:5 '-CCGGAATTCATGCCCACGGCCATCCCC-3 ' (SEQ ID NO.6), containing EcoR I restriction enzyme site and 3 protectiveness bases,
Downstream primer R:5 '-CCGAAGCTTTTACTCAAATCCAAAAGA-3 ' (SEQ ID NO.7), containing Hind III restriction enzyme site and 3 protectiveness bases.
With the pGEM-T-easy-Etgam59 carrier built for template, Successful amplification goes out to conform to expected results and size is the specific fragment (Fig. 2) of about 1569bp, and this fragment is connected in prokaryotic expression carrier pET28a, obtain pET28a-Etgam59.
3) high expression of Eimeria tenella Etgam59 gene and purifying
The prokaryotic expression carrier pET28a-Etgam59 built is transformed BL21 Host Strains, after IPTG abduction delivering, through soluble analysis, finds that the albumen of this in-vitro recombination expression exists (Fig. 3) with the form of soluble proteins.During the great expression of recombinant protein, the soluble proteins of excusing from death cracking release, by containing after the Ni-NTA affinitive layer purification of HIS label, is the 8h that dialyses in the PBS of 8.0 in pH value, concentrate eventually through PEG8000, collect albumen, 12%SDS-PAGE analyzes (Fig. 4), 4 DEG C of preservations.
4) specificity of in-vitro recombination expression albumen and immunogenicity detect
Respectively with the rehabilitation serum (Fig. 7) of chicken after the polyclonal antibody (Fig. 6) of the monoclonal antibody of anti-HIS (Fig. 5), mouse-anti Etgam59 and E. pubescens Msxim three immunity for primary antibodie, detect the specificity of recombinant protein and immunogenicity by western-blot method.
The invention also discloses described Etgam59 gene and build the application in nucleic acid vaccine.
The structure of described nucleic acid vaccine is as follows:
According to the ORF sequence of Etgam59 gene and the characteristic of pcDNA3.1, design 1 pair of Auele Specific Primer: upstream primer F1:5 '-CCGAAGCTTATGCCCACGGCCATCCCC-3 ' (SEQ ID NO.8), containing Hind III restriction enzyme site and 3 protectiveness bases; Downstream primer R1:5 '-CCGGAATTCTTACTCAAATCCAAAAGA-3 ' (SEQ ID NO.9), containing EcoR I restriction enzyme site and 3 protectiveness bases.
With the pGEM-T-easy-Etgam59 carrier built for template, the Successful amplification size that obtains conforming to expected results is the specific fragment (Fig. 8) of about 1569bp, and this object fragment is connected in plasmid pcDNA3.1, obtain pcDNA3.1-Etgam59.
The recombinant plasmid pcDNA3.1-Etgam59 of structure is carried out EcoR I and Hind III double digestion qualification (Fig. 9), select positive clone strain order-checking, determine that reading frame is cloned for follow-up test after correct in a large number.
The Analysis of Immunogenicity of nucleic acid vaccine
The preparation of mouse-anti recombinant plasmid pcDNA3.1-Etgam59 polyclonal antibody: get the female mouse of BALB/c in 6 ~ 8 week age 6, intramuscular injection 0.75% lignocaine 50 μ l/ is auxiliary diffusion only, after 24h at same area injection recombinant plasmid pcDNA3.1-Etgam59 100 μ g/ only.After this once, operation is constant with dosage for the 2nd week the 3rd week each booster immunization.Within after 3 immunity the 10th day, pluck eyeball and get blood, 37 DEG C of standing 30min, 4 DEG C of standing 4h, the centrifugal 15min of 2500rpm, collect separation of serum, detect the Etgam59 recombinant protein antigen of prokaryotic expression for western-blot, the immunogenicity (Figure 10) of qualification nucleic acid vaccine.
Accompanying drawing explanation
Fig. 1 is the whole ORF amplification of Eimeria tenella Etgam59 gene.Wherein 1: standard molecular weight; 2 and 3 is the object fragments obtained that increase, and size is 1617bp.
Fig. 2 is that Eimeria tenella Etgam59 gene dispels signal peptide, adds the amplification after restriction enzyme site.Wherein 1: standard molecular weight; 2 and 3 is the object fragments obtained that increase, and size is 1569bp.
Fig. 3 is the outer recombinant expression protein soluble analysis result of Eimeria tenella Etgam59 genosome, wherein 1: standard protein molecular weight marker; 2:pET-28a/BL21 IPTG induced product; 3:pET-28a-Etgam59/BL21 IPTG induces the precipitation after cellular lysate; 4:pET-28a-Etgam59/BL21 IPTG induces the supernatant after cellular lysate.
Fig. 4 is the purification result analysis of Eimeria tenella Etgam59 genetic expression albumen, wherein 1: standard protein molecular weight marker; 2:pET-28a/BL21 TPTG induced product; Supernatant after the excusing from death of 3:pET-28a-Etgam59/BL21 recombinant bacterium; 4: under the effect of binding buffer liquid lysate be combined with Ni-NTA after effluent liquid; 5: the first time elutriant of lavation buffer solution; 6: the third time elutriant of lavation buffer solution; Target protein after 7:Ni-NTA affinity purification.
Fig. 5 is the specificity Western-blot qualification result of in-vitro recombination expression albumen, wherein 1: pre-dyed molecular weight protein marker; 2:pET-28a/BL21 IPTG induces; 3:pET-28a-Etgam59/BL21 IPTG induces.
Fig. 6 is the polyclonal antibody Western-blot detected result of mouse-anti Eimeria tenella Etgam59 in-vitro recombination expression albumen, wherein 1: pre-dyed molecular weight protein marker; 2:pET-28a/BL21IPTG induces; 3:pET-28a-Etgam59/BL21 IPTG induces.
Fig. 7 is the rehabilitation serum Western-blot detected result of Eimeria tenella gam59 in-vitro recombination expression albumen to Eimeria tenella, wherein 1: pre-dyed molecular weight protein marker; 2:pET-28a/BL21 IPTG induces; 3:pET-28a-Etgam59/BL21 IPTG induces.
Fig. 8 builds nucleic acid vaccine Etgam59 gene amplification result: wherein 1: standard molecular weight; 2,3 is the object fragments obtained that increase, and size is 1569bp.
Fig. 9 is recombinant plasmid pcDNA3.1-Etgam59 double digestion qualification result: wherein 1: standard molecular weight; 2,3,4 is double digestion results, and wherein plasmid fragments size is 5391bp, and object clip size is 1569bp.
Figure 10 be the outer recombinant expression protein of Eimeria tenella Etgam59 genosome to restructuring eucaryon plasmid polyclonal antibody Western-blot detected result, wherein 1: pre-dyed molecular weight protein marker; 2:BL21IPTG induces; 3:pET-28a/BL21 IPTG induces; 4:pET-28a-Etgam59/BL21 IPTG induces.
Embodiment
Below in conjunction with drawings and Examples, the present invention is further described.
Embodiment 1: be separated Etgam59 gene from Eimeria tenella gametophytic development
Ordinary method separation and purification Eimeria tenella gametophyte, extracts test kit with RNA and extracts total serum IgE.
RT-PCR fishes and gets Etgam59 gene:
With reference to (the TaKaRa RNA LA PCR of TaKaRa company
tMkit (AMV) Ver.1.1) method that provides: two-step approach RT-PCR kit amplification Etgam59 gene.
The fishing of Etgam59 gene is got and is adopted upstream primer (5 '-CCCCGGGAACATGACTCGTCTCGCCGCC-3 ') and downstream primer (5 '-CGAATTCTTACTCAAATCCAAAAGAAGG-3 ').
The first step: reverse transcription reaction is totally the total serum IgE (≤500ng total RNA) of 10 μ l:0.5 μ l, the MgCl of 25mM
22 μ l, 10 × RNA PCR Buffer 1 μ l, RNase Free dH
2o 4.25 μ l, concentration is the dNTP 1 μ l of 10mM, RNase Inhibitor 0.25 μ l, 5 μ/μ l AMV reversed transcriptive enzyme 0.5 μ l and Oligo dT adapter-primer 0.5 μ l.Reactions steps: 42 DEG C of reverse transcription reaction 30min; 99 DEG C of sex change 5min; 5 DEG C of cooling 5min.
Second step: PCR reacts, and cumulative volume is 25 μ l:dNTP (2.5Mm each) 4ul, 10 × LA PCR Buffer II (Mg
20+plus) 2.5ul, sterilizing ddH
2o 15.25 μ l, TaKaRa LA Taq 0.25 μ l, upstream primer (10 μMs) 1.0 μ l, downstream primer (10 μMs) 1.0 μ l, then join the product 1.0 μ l in the first step reverse transcription reaction in this system.PCR reactions steps: 95 DEG C of denaturation 3min; 95 DEG C of sex change 30s; 62 DEG C of annealing 1min; 72 DEG C extend 1.5min, totally 30 circulations.After reaction terminates, 1.2% agarose gel electrophoresis detects, and result is as Fig. 1.By the Etgam59 gene clone that obtains in pGEM-T-easy (purchased from Promega company) carrier, be sent to the order-checking of Hua Da genome company.Recombinant vectors called after: pGEM-T-Easy-Etgam59.
Embodiment 2: the amplification expressing fragment Etgam59
According to the ORF sequence of Eimeria tenella Etgam59 gene and the restriction enzyme site of plasmid pET28a (purchased from Invitrogen company), after dispelling signal peptide, design Auele Specific Primer, the gene fragment that amplification is expressed.
According to the restriction enzyme site that plasmid pET28a possesses; after sequential analysis is carried out to Etgam59 gene; dispel signal peptide; choose EcoR I and Hind III two restriction enzyme sites; designing the Auele Specific Primer obtained is: and upstream primer (5 '-CCGGAATTCATGCCCACGGCCATCCCC-3 ') containing EcoR I restriction enzyme site and 3 protectiveness bases, downstream primer (5 '-CCGAAGCTTTTACTCAAATCCAAAAGA-3 ') containing Hind III restriction enzyme site and 3 protectiveness bases.
50 μ l PCR reaction systems are as follows: the 10 × reaction buffer of 5 μ l; Concentration is each 2 μ l of the upstream and downstream primer of 10 μMs, 8 μ l dNTP (2.5mM); 30.5 μ l sterilizing ddH
2o; 0.5 μ l TaKaRa LA Taq enzyme, 2 μ l template pGEM-T-Easy-Etgam59.95 DEG C of sex change 3min; 95 DEG C of sex change 30s, 60 DEG C of annealing 30s, 72 DEG C extend 1.5min, totally 30 circulations; 72 DEG C, extend 10min.After reaction terminates, 1.2% agarose gel electrophoresis detects, and result is as Fig. 2.After expanding reaction system, reclaimed by the PCR primer purifying obtained ,-20 DEG C save backup.
Embodiment 3: the structure of prokaryotic expression carrier
Double digestion pET28a:
EcoR I and Hind III is Fermentas product.The 100 μ l enzyme systems of cutting are: 10 μ l 10 × FastDigest Green Buffer, each 5 μ l of 50 μ l carrier pET28a, FastDigest EcoR I and FastDigest Hind III, 30 μ l sterilizing ddH
2o.37 DEG C of reaction 12h, 1.2% agarose gel electrophoresis, glue reclaims product.
The PCR primer of purifying in double digestion embodiment 2:
EcoR I and Hind III is Fermentas product.100 μ l enzymes cut system: 10 μ l 10 × FastDigest Green Buffer; 20 μ l purifying PCR (Etgam59) products; The each 5 μ l of FastDigest EcoR I and FastDigest Hind III; 60 μ l sterilizing ddH
2o.37 DEG C of reaction 12h, 1.2% agarose gel electrophoresis, glue reclaims digestion products.
Ligation:
Solution I is TaKaRa product.9 μ l double digestion carrier pET28a, 1 μ l double digestion PCR primer, 10 μ l solution I, 16 DEG C connections are spent the night.
Get 10 μ l and connect product conversion competent cell DH5 α.The LB be applied to containing kalamycin resistance is dull and stereotyped, second day picking mono-clonal, after incubated overnight, extracts plasmid, adopt double digestion identification method to select positive colony (recombinant vectors called after: pET28a-Etgam59), and deliver the order-checking of Hua Da genome company.
The abduction delivering of embodiment 4:Etgam59 gene in intestinal bacteria
The Cloning Transformation competence BL21 (DE3) that picking checks order correct.The single colony inoculation of picking is in 3ml LB nutrient solution (containing 100 μ g/ml kantlex), 37 DEG C, 200rpm shaking culture is spent the night, 10ml is inoculated into containing in same concentrations microbiotic LB by 1: 100 turn, 37 DEG C of 200rpm shaking culture 4h, add the IPTG that final concentration is 0.5mM, 37 DEG C of 200rpm induce 3h, 12%SDS-PAGE to detect expression.Result, as Fig. 3, clearly shows that the molecular weight of albumen of expression is at about 57.5kDa, and is soluble proteins.
Embodiment 5: the purifying of expression product and renaturation
4 DEG C of centrifugal 5min of 12000rpm collect thalline, with 10ml Lysis Equilibrium Buffer (50mM NaH
2pO
4, 300mM NaCl, pH 8.0) and resuspended precipitation, then ice-bath ultrasonic (ultrasonic 2s, gap 3s, 15min).4 DEG C of centrifugal 15min of 12000rpm, collect supernatant and join in the Ni-NTA post using Lysis Equilibrium Buffer Balance Treatment in advance, and 4 DEG C of effect 30min, period will constantly rock.The operational manual purifying protein provided according to GenScript company.
Collect Elution Buffer (50mM NaH
2pO
4, 300mM NaCl, 500mM Imidazole, pH 8.0) and the albumen of wash-out, load dialysis tubing, dialysis 8h in PBS (pH8.0).PEG8000 protein concentrate, 12%SDS-PAGE detects, and protein-20 DEG C is preserved.The purification result of recombinant protein is as Fig. 4.
Embodiment 6: the specific detection of expression product
By the positive recombinant bacterium of induction and empty plasmid transformed bacteria protein 20 μ l, 12%SDS-PAGE electrophoresis, and transfer on nitrocellulose filter, after transfer printing terminates, NC film being put room temperature in containing the TBS damping fluid of 3%BSA and close 1h, is mouse-anti HIS tag monoclonal antibody (BBI, USA) (being diluted to 1 μ g/ml with confining liquid) by primary antibodie subsequently, 1.5h is acted at 37 DEG C, TBST washs 5min, repeats 3 times, then washs 5min with TBS; Then with HRP mark rabbit anti-mouse igg be two resist, with confining liquid 1: 2000 dilute, act on 1.5h under room temperature, TBST washs 5min, repeats 5 times, then washs 5min with TBS; Finally with DAB damping fluid colour developing 1min, drying at room temperature after rinsed with deionized water termination reaction, result is as Fig. 5.
Embodiment 7: the immunogenicity of expression product detects
By the positive recombinant bacterium of induction and empty plasmid transformed bacteria protein 20 μ l, 12%SDS-PAGE electrophoresis, and transfer on nitrocellulose filter, after transfer printing terminates, NC film is put room temperature in the TBS damping fluid containing 3%BSA and close 1h, be mouse-anti EtGAM59 polyclonal antibody prepared by this laboratory by primary antibodie subsequently, dilute with confining liquid 1: 200, at 37 DEG C, act on 1.5h; TBST washs 5min, repeats 3 times, then washs 5min with TBS; Then with HRP mark rabbit anti-mouse igg be two resist, with confining liquid 1: 1000 dilute, act on 1.5h under room temperature; TBST washs 5min, repeats 5 times, then washs 5min with TBS; Finally with DAB damping fluid colour developing 1min, drying at room temperature after rinsed with deionized water termination reaction, result is as Fig. 6.
By the positive recombinant bacterium of induction and empty plasmid transformed bacteria protein 20 μ l, 12%SDS-PAGE electrophoresis, and transfer on nitrocellulose filter, after transfer printing terminates, NC film is put room temperature in the TBS damping fluid containing 3%BSA and close 1.5h, primary antibodie is the rehabilitation serum that chicken after immune Eimeria tenella is prepared in this laboratory subsequently, dilute with confining liquid 1: 100, at 37 DEG C, act on 1h; TBST washs 5min, repeats 3 times, then washs 5min with TBS; Then with HRP mark the anti-chicken IgY (IgG) of rabbit be two resist, with confining liquid 1: 1000 dilute, act on 1.5h under room temperature; TBST washs 5min, repeats 5 times, then washs 5min with TBS; Finally with DAB damping fluid colour developing 1min, drying at room temperature after rinsed with deionized water termination reaction, result is as Fig. 7.Embodiment 8: build nucleic acid vaccine
Pcr amplification Etgam59 gene:
According to the ORF sequence of the characteristic sum Etgam59 gene of plasmid pcDNA3.1 (purchased from Invitrogen company), design Auele Specific Primer, amplification object fragment.
According to the characteristic of plasmid pcDNA3.1, choose EcoR I and Hind III two restriction enzyme sites, design Auele Specific Primer:
Upstream primer F1 (5 '-CCGAAGCTTATGCCCACGGCCATCCCC-3 '), containing Hind III restriction enzyme site and 3 protectiveness bases;
Downstream primer U1 (5 '-CCGGAATTCTTACTCAAATCCAAAAGA-3 '), containing EcoRI restriction enzyme site and 3 protectiveness bases.
50 μ l PCR reaction systems: 10 × reaction buffer 5 μ l; The each 2 μ l of upstream and downstream primer; The dNTP 8 μ l of 10mM; Autoclaving ddH
2o 30.5 μ l; The template pGEM-T-easy-Etgam59 plasmid 2 μ l of TaKaRa LA Taq enzyme 0.5 μ l, 100 μ g/mg.95 DEG C of sex change 3min; 95 DEG C of sex change 30s, 62 DEG C of annealing 1min, 68 DEG C extend 1.5min, 30 circulations; 68 DEG C, extend 10min.After reaction terminates, detect with 1.2% agarose gel electrophoresis, result is as Figure 10.Expand reaction system, the PCR primer PCR primer obtained is reclaimed kits and reclaims ,-20 DEG C save backup.
The PCR primer that double digestion purifying reclaims:
Hind III and EcoRI is purchased from Fermentas company.100 μ l enzymes cut system: 10 × FastDigest Green Buffer 10 μ l; PCR (Etgam59) the product 20 μ l of purifying; The each 5 μ l of FastDigest Hind III and FastDigest EcoRI; Autoclaving ddH
2o 60 μ l.37 DEG C of water-bath 2h, 1.2% agarose gel electrophoresis, glue reclaims test kit and reclaims digestion products.
Double digestion plasmid pcDNA3.1:
Hind III and EcoRI is purchased from Fermentas company.The 60 μ l enzyme systems of cutting are: 10 × FastDigest Green Buffer 6 μ l, carrier pcDNA3.1 30 μ l, FastDigest Hind III and FastDigest EcoRI each 3 μ l, autoclaving ddH
2o 18 μ l.37 DEG C of water-bath 2h, 1.2% agarose gel electrophoresis, glue reclaims test kit and reclaims digestion products.
Ligation:
Solution I is purchased from TaKaRa company.Double digestion carrier pcDNA3.11 μ l, double digestion PCR primer 3 μ l, solution I 10 μ l, autoclaving ddH
2o 6 μ l.16 DEG C of connections are spent the night.
Get 10 μ l and connect product conversion 200 μ l competent cell DH5 α.The LB be inoculated into containing ammonia benzyl resistance is dull and stereotyped, 37 DEG C of overnight incubation.Second day picking list bacterium colony, incubated overnight, extracts plasmid, and double digestion identification method is selected positive clone strain (recombinant plasmid called after: pcDNA3.1-Etgam59) 1.2% agarose gel electrophoresis and detected, result as Fig. 9, and delivers to the order-checking of Hua Da genome company.
Embodiment 9: the Analysis of Immunogenicity of nucleic acid vaccine
The preparation of polyclonal antibody:
Obtain serum: ordinary method heavy dose obtains recombinant plasmid, and measures plasmid concentration.Get BABL/c in 6 week age female mouse intramuscular injection plasmid solution (plasmid concentration 100 μ g).At after this 8th day, the 15th day each booster immunization once, injection site and immunizing dose the same.Before each immunity, 24h is at the lignocaine 50ml of immune injection location 0.75%, spreads the absorption of plasmid for auxiliary myocyte.3 exempt from after within the 10th day, pluck eyeball and get blood, separation of serum, detect the immunogenicity of eukaryon recombinant plasmid for Western-blot.
Western-blot detects: by the positive recombinant bacterium of induction, empty bacterium and empty plasmid transformed bacteria protein 20 μ l, 12%SDS-PAGE electrophoresis, and transfer on nitrocellulose filter, after transfer printing terminates, NC film is put containing 3%BSA TBS damping fluid in 4 DEG C close spend the night, the mouse-anti pcDNA3.1-Etgam59 polyclonal antibody prepared with above-mentioned test is subsequently as primary antibodie, dilute with confining liquid 1: 200, at 37 DEG C, act on 1.5h; TBST washs 5min, repeats 3 times, then washs 5min with TBS; Then with HRP mark rabbit anti-mouse igg be two resist, with confining liquid 1: 1000 dilute, act on 1.5h under room temperature; TBST washs 5min, repeats 5 times, then washs 5min with TBS; Finally with DAB damping fluid colour developing 1min, drying at room temperature after rinsed with deionized water termination reaction, result is as Figure 10, and the Etgam59 recombinant protein specific reaction of the antibody capable namely produced after recombinant plasmid pcDNA3.1-Etgam59 immune mouse and prokaryotic expression, show nucleic acid vaccine has immunogenicity.
Claims (4)
1. an Eimeria Tenella gametocyte antigen gene Et
gam59, its nucleotide sequence is as shown in SEQ ID NO.1.
2. an Eimeria Tenella gametocyte antigen, its aminoacid sequence is as shown in SEQ ID NO.2.
3. the preparation method of recombination chicken Eimeria tenella gametocyte antigen, is characterized in that comprising the following steps:
1) from Eimeria tenella gametophytic development, Et is separated
gam59gene:
Primer sequence is as shown in SEQ ID NO.4 and SEQ ID NO.5, and the cDNA obtained with the reverse transcription of gametophyte total serum IgE is for template, and amplifying size is 1617 bp specific fragments, called after Et
gam59; Containing object fragment PCR products after kits reclaims, will be cloned in pGEM-T-easy carrier, obtain pGEM-T-easy-Et
gam59;
2) structure of prokaryotic expression carrier
Specific primer sequence as SEQ ID NO.6 and SEQ ID NO.7, with the pGEM-T-easy-Et built
gam59carrier is template, the specific fragment of 1569 bp that increase, and this fragment is connected in prokaryotic expression carrier pET28a, obtains pET28a-Et
gam59;
3) Eimeria tenella Et
gam59the high expression of gene and purifying
By the prokaryotic expression carrier pET28a-Et built
gam59transform BL21 Host Strains, IPTG abduction delivering, after the soluble proteins that excusing from death cracking discharges passes through the Ni-NTA affinitive layer purification containing HIS label, be the 8h that dialyses in the PBS of 8.0 in pH value, concentrate eventually through PEG8000, collect albumen, 12% SDS-PAGE analyzes, 4 DEG C of preservations.
4. Et according to claim 1
gam59gene is building the application in control Eimeria Tenella nucleic acid vaccine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410745229.8A CN104593385A (en) | 2014-12-09 | 2014-12-09 | Eimeria tenella gametophyte antigen gam59 gene and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110204604A (en) * | 2019-05-13 | 2019-09-06 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | A kind of Eimeria tenella AN1 sample zinc finger protein and its application in inhibition coccidia invasion |
| CN113041344A (en) * | 2021-03-19 | 2021-06-29 | 吉林大学 | Eimeria tenella chitosan/nanoparticle and preparation method thereof |
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| CN103233017A (en) * | 2013-05-06 | 2013-08-07 | 扬州大学 | Gametophyte antigen gam22 gene of Eimeria necatrix and application of gene |
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| CN103233017A (en) * | 2013-05-06 | 2013-08-07 | 扬州大学 | Gametophyte antigen gam22 gene of Eimeria necatrix and application of gene |
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| JÜRGEN KRÜCKEN, ET AL.: "Excystation of Eimeria tenella Sporozoites Impaired by Antibody Recognizing Gametocyte/Oocyst Antigens GAM22 and GAM56", 《EUKARYOTIC CELL》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110204604A (en) * | 2019-05-13 | 2019-09-06 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | A kind of Eimeria tenella AN1 sample zinc finger protein and its application in inhibition coccidia invasion |
| CN110204604B (en) * | 2019-05-13 | 2022-02-08 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Eimeria tenella AN1-like zinc finger protein and application thereof in inhibition of coccidium invasion |
| CN113041344A (en) * | 2021-03-19 | 2021-06-29 | 吉林大学 | Eimeria tenella chitosan/nanoparticle and preparation method thereof |
| CN113041344B (en) * | 2021-03-19 | 2023-07-18 | 吉林大学 | Chitosan/nanoparticle for eimeria tenella and preparation method thereof |
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