CN110330556A - Highly expressed gene and its coding albumen and application in Schistosomula Japonicum - Google Patents
Highly expressed gene and its coding albumen and application in Schistosomula Japonicum Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
- C07K14/43559—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from trematodes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/10—Anthelmintics
- A61P33/12—Schistosomicides
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
- G01N2333/43526—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms
- G01N2333/43547—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms from trematodes
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present invention provides highly expressed gene and its coding albumen and application in Schistosomula Japonicum.The present invention screens a series of genes highly expressed in Schistosomula Japonicum using Schistosoma japonicum full-length genome chip of expression spectrum, the wherein antigen protein of gene SjScP27, SjScP80, SjScP84, SjScP88 coding, can be by snail fever patients serum's specific recognition, and stronger positive reaction is presented.ELISA detection shows that these antigen proteins detection blood fluke has hypersensitivity and specificity, can be used for the exploitation of Japanese schistosomiasis diagnostic reagent.
Description
Technical field
The invention belongs to field of biotechnology, specifically, be related in Schistosomula Japonicum highly expressed gene and
It encodes albumen and application.
Background technique
Snail fever is a kind of infectiousness parasitic disease for seriously threatening human health, and Major Epidemic is in Asian, African and Latin American 76
A countries and regions, the whole world have more than 200,000,000 people to be infected, and separately have nearly 800,000,000 people to be threatened by its infection.China's prevalence is Japanese blood
Fluke disease, by the unremitting effort of decades, China's schistosomiasis control is made remarkable achievements, snail fever
It is under control in Prevalent district, but realizes that the final target for eliminating snail fever still shoulders heavy responsibilities.
Diagnosis is the key link in prevention and cure of snail fever field.Accurate diagnostic techniques is not only to the morning of Patients with Schistosomiasis Japonica
It was found that early treatment has important clinical significance, judgment criteria can be also provided for Endemic Areas of Schistosomiasis Japonica grade, be to assess popular state
Gesture and examination control effect provide essential information and scientific basis.Lacking efficiently accurate diagnostic techniques is snail fever
The major reason that can not thoroughly eliminate.With the increasing of China's blood fluke preventing and controlling dynamics, snail fever becomes in China
In a kind of fashion trend of low infection.If the Patients with Schistosomiasis Japonica of these low-grade infections cannot be made a definite diagnosis and be controlled in time
It treats, worm's ovum contained in excreta will cause the continued popularity of snail fever to be propagated.Therefore, researching and developing new has high sensitivity
It is imperative with the schistosomiasis diagnosis method of specificity.
Currently, the diagnosis of snail fever depends on parasite morphologic detection method, such as the World Health Organization (WHO) more
The Kato- Katz technique (Kato-Katz) of recommendation.This method mainly passes through the japonice ovum checked in Feces of Patients or urine
It diagnoses the illness, it is not only time-consuming and laborious and lower to the sensibility of slight infection by Schistosoma diagnosis, be not suitable for extensive
Snail fever field monitoring.
Compare traditional Morphologic Diagnosis method, enzyme-linked immunosorbent assay (Enzyme-linked immune
Sorbent assay, ELISA) diagnostic method has sensitivity simple and efficient to handle and higher, and can be used for extensive scene
Monitoring.Currently, the most common antigen for snail fever immunodiagnosis is the Adult Antigens component for extracting blood fluke polypide
(Adult worm antigen, AWA) and egg antigen component (Soluble egg antigen, SEA), since both are thick
The complicated component (being made of thousands of kinds of blood fluke albumen) of antigen is mentioned, and there is more serious with other parasitic infection serum
The specificity of cross reaction, schistosomiasis diagnosis is not high, and the reagent of production should not standardize.
Summary of the invention
The object of the present invention is to provide gene highly expressed in Schistosomula Japonicum and its coding albumen and applications.
Present inventive concept is as follows: being screened using Schistosoma japonicum full-length genome chip of expression spectrum a series of in the suction of Japanese blood
Highly expressed gene in worm child worm, virgin worm are the polypides of relatively early contact host's peripheral circulation, can cause host body fluids earlier
Immune response, generates corresponding antibody.The present invention will wherein 4 Schistosomula Japonicum protein gene SjScP27, SjScP80,
SjScP84, SjScP88 are recombinantly expressed in Escherichia coli by the hydrophilic section of PCR amplification gene, and by it, ELISA test
Show that diagnosis of the gained recombinant protein for Japanese schistosomiasis has hypersensitivity and specificity, is that potential diagnostic antigen is waited
Select target.
In order to achieve the object of the present invention, in a first aspect, the present invention provides the highly expressed gene in Schistosomula Japonicum,
The gene is selected from least one of SjScP27, SjScP80, SjScP84, SjScP88, their nucleotide sequence difference
It is as follows:
Gene SjScP27:
I) nucleotide sequence shown in SEQ ID NO:1；
Ii) nucleotide sequence shown in SEQ ID NO:1 be substituted, lack and/or increase one or more nucleotide and
Express the nucleotide sequence of identical function protein；
Iii) hybridize under strict conditions with sequence shown in SEQ ID NO:1 and express the nucleotide of identical function protein
Sequence, the stringent condition are in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS, at 65 DEG C
Lower hybridization, and film is washed with the solution；Or
Iv) and i), ii) or nucleotide sequence iii) there is 90% or more homology and express identical function protein
Nucleotide sequence；
Gene SjScP80:
I) nucleotide sequence shown in SEQ ID NO:2；
Ii) nucleotide sequence shown in SEQ ID NO:2 be substituted, lack and/or increase one or more nucleotide and
Express the nucleotide sequence of identical function protein；
Iii) hybridize under strict conditions with sequence shown in SEQ ID NO:2 and express the nucleotide of identical function protein
Sequence, the stringent condition are in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS, at 65 DEG C
Lower hybridization, and film is washed with the solution；Or
Iv) and i), ii) or nucleotide sequence iii) there is 90% or more homology and express identical function protein
Nucleotide sequence；
Gene SjScP84:
I) nucleotide sequence shown in SEQ ID NO:3；
Ii) nucleotide sequence shown in SEQ ID NO:3 be substituted, lack and/or increase one or more nucleotide and
Express the nucleotide sequence of identical function protein；
Iii) hybridize under strict conditions with sequence shown in SEQ ID NO:3 and express the nucleotide of identical function protein
Sequence, the stringent condition are in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS, at 65 DEG C
Lower hybridization, and film is washed with the solution；Or
Iv) and i), ii) or nucleotide sequence iii) there is 90% or more homology and express identical function protein
Nucleotide sequence；
Gene SjScP88:
I) nucleotide sequence shown in SEQ ID NO:4；
Ii) nucleotide sequence shown in SEQ ID NO:4 be substituted, lack and/or increase one or more nucleotide and
Express the nucleotide sequence of identical function protein；
Iii) hybridize under strict conditions with sequence shown in SEQ ID NO:4 and express the nucleotide of identical function protein
Sequence, the stringent condition are in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS, at 65 DEG C
Lower hybridization, and film is washed with the solution；Or
Iv) and i), ii) or nucleotide sequence iii) there is 90% or more homology and express identical function protein
Nucleotide sequence.
Second aspect, the present invention provide the antigen protein of gene coding, gene SjScP27, SjScP80,
The amino acid sequence of the antigen protein of SjScP84, SjScP88 coding is respectively as shown in SEQ ID NO:5-8.
The clipped form of the antigen protein of gene SjScP27, SjScP80, SjScP84, SjScP88 coding passes through modification
Protein derivatives or fusion protein, and have it is identical or approximate antigenic as antigen protein shown in SEQ ID NO:5-8
Protein variant belongs to protection category of the present invention.
The third aspect, the present invention provide following any application of the antigen protein:
1) Schistosoma japonicum detection reagent or kit are used to prepare；
2) it is used to prepare anti schistosoma vaccine；
3) it is used to prepare anti schistosoma drug；
4) it is used for the detection of non-diagnostic purpose Schistosoma japonicum；
5) it is used for diagnosing Japanese schistosomiasis.
Specifically, Schistosoma japonicum SjScP27, SjScP80, SjScP84, SjScP88 albumen of the invention is as special
Property schistosome antigen, the application in terms of serodiagnosis；Application as immunogene, in terms of preparing anti-schistosome vaccine；
Application as potential drug target, in terms of screening chemistry and other categories of drugs；As Schistosoma japonicum
The encoding gene of SjScP27, SjScP80, SjScP84, SjScP88 albumen, the application in terms of gene therapy.
Fourth aspect, the present invention provide a kind of Schistosoma japonicum detection reagent, in the detection reagent containing it is following 1.~
4. at least one of:
1. Japan schistosome antigen Protein S jScP27, or the DNA molecular of the coding antigen protein, or by containing described
The recombinant protein that the recombinant bacterium of DNA molecular generates；
2. Japan schistosome antigen Protein S jScP80, or the DNA molecular of the coding antigen protein, or by containing described
The recombinant protein that the recombinant bacterium of DNA molecular generates；
3. Japan schistosome antigen Protein S jScP84, or the DNA molecular of the coding antigen protein, or by containing described
The recombinant protein that the recombinant bacterium of DNA molecular generates；
4. Japan schistosome antigen Protein S jScP88, or the DNA molecular of the coding antigen protein, or by containing described
The recombinant protein that the recombinant bacterium of DNA molecular generates.
5th aspect, the present invention provide the kit for containing the Schistosoma japonicum detection reagent.
6th aspect, the present invention provide Japanese schistosomiasis ELISA immunodiagnosis kit, and the kit includes:
1) it is coated with the micro reaction plate of 1-5 μ g/mL antigen protein；With the carbonate-of the TWEEN20 containing 0-0.05%v/v
Bicarbonate buffer (being purchased from Sigma, article No. C3041) is as coating buffer；
2) washing buffer: PBST liquid, the i.e. PBS solution of the TWEEN20 containing 0.05%v/v, pH7.4；
3) Sample dilution: 5-10%BSA solution is prepared by solvent of PBS buffer solution；
4) enzyme labelled antibody: the goat anti-human immunoglobulin antibody of alkali phosphatase enzyme mark；
5) substrate developing solution: pNPP developing solution；
PNPP is purchased from Sigma, article No. N2640.The preparation method of pNPP developing solution is as follows: 15mg pNPP is dissolved in 15mL
(0.1M glycine, 1mM MgCl in the glycine buffer of 0.1M2, 1mM ZnCl2, it is dissolved in pure water, pH10.4) to get.
6) reaction terminating liquid: 120g/L NaOH aqueous solution；
7) positive control: with 1 μ g/mL human immunoglobulin(HIg) IgG wrapper sheet.
Meanwhile negative control is arranged: with corresponding antigen protein wrapper sheet, enzyme labelled antibody replaces with Sample dilution.
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
(1) antigen protein detection sensitivity provided by the invention is high, to the Schistosomiasis patients (EPG < 100) of low-grade infection
It can still detect, susceptibility is up to 96%.
(2) detection kit provided by the invention has high specific, and Japanese schistosomiasis specificity is up to
100%.
(3) kit can be used for the early diagnosis of infection by Schistosoma, can detect after experimental animal infection 3 weeks
Anti- SjScP27 in serum, SjScP80, SjScP84, the presence of SjScP88 protein antibodies.
(4) easy to operate, as a result stable, repeatability is high.
Detailed description of the invention
Fig. 1 is Schistosoma japonicum SjScP27, SjScP80, SjScP84, SjScP88 gene order in the embodiment of the present invention 1
PCR amplification result schematic diagram.Wherein, M:DNA molecular weight standard, 1,2,3,4 be respectively SjScP27, SjScP80,
The pcr amplification product of SjScP84, SjScP88 gene.
Fig. 2 is SjScP27, SjScP80, SjScP84 in the embodiment of the present invention 2, the SDS-PAGE of SjScP88 recombinant protein
Analyze result schematic diagram.Wherein, M: Protein Marker, 1:SjScP27,2:SjScP80,3:SjScP84,4:SjScP88.
Fig. 3-Fig. 6 is SjScP27 (Fig. 3), SjScP80 (Fig. 4), SjScP84 (Fig. 5) in the embodiment of the present invention 3,
The Western of SjScP88 (Fig. 6) recombinant protein antigen and snail fever human serum and different infection by Schistosoma animal blood serums
Blot analyzes result schematic diagram.Wherein, M: Protein Marker, 1: Schistosoma japonicum patients serum, 2: infecting Japanese blood and inhale
42 days mice serums of worm, 3: infection 42 days rabbit anteserums of Schistosoma japonicum, 4: the anti-His tag antibody positive control of mouse, 5:
Normal Mouse Serum negative control.
Fig. 7 is rSP13-ELISA kit and SjScP27-ELISA, SjScP80-ELISA in the embodiment of the present invention 5,
SjScP84-ELISA, SjScP88-ELISA kit are used for Japanese schistosomiasis diagnostic evaluation comparison result schematic diagram.Its
In, A is SjScP27-ELISA kit results, and B is SjScP80-ELISA kit results, and C is SjScP84-ELISA reagent
For box as a result, D is SjScP88-ELISA kit results, E is the result of rSP13-ELISA kit.Sj is Japanese schistosomiasis
People's group, Sj-3M are three months groups of Schistosoma japonicum patient chemotherapy, and Cs is clonorchis sinensis patient group, and Healthy is normal healthy controls
Group.Horizontal straight line is 2.1 times of Healthy class mean of cutoff value in figure, is considered as the positive on the line, is considered as feminine gender under line.
Fig. 8 is Schistosoma japonicum SjScP27, SjScP80, SjScP84, SjScP88 gene in the embodiment of the present invention 6 in worm
Fluorescence real-time quantitative PCR in ovum (E), cercaria (C), liver phase child worm (S), male insect (M) and female insect (F) analyzes result
Schematic diagram.
Fig. 9 is that anti-SjScP27 and SjScP84 protein antibodies are infecting Babl/c mouse and Xin Xi in the embodiment of the present invention 7
Dynamics comparative analysis result schematic diagram in blue White Rabbit serum.Wherein, A, B are respectively anti-SjScP27 and SjScP84 albumen
Dynamic analysis of the antibody in infection Babl/c mice serum is as a result, C, D are respectively that anti-SjScP27 and SjScP84 albumen is anti-
Dynamic analysis result of the body in infection new zealand white rabbit serum.
Figure 10 is that anti-SjScP80 and SjScP88 protein antibodies are infecting Babl/c mouse and Xin Xi in the embodiment of the present invention 7
Dynamics comparative analysis result schematic diagram in blue White Rabbit serum.Wherein, A, B are anti-SjScP80 and SjScP88 egg
Dynamic analysis of the Bai Kangti in infection Babl/c mice serum is as a result, C, D are respectively anti-SjScP80 and SjScP88 albumen
Dynamic analysis result of the antibody in infection new zealand white rabbit serum.
Specific embodiment
General technical process of the invention is as follows:
Verified first with result of the method for qPCR to chip screening, confirmation SjScP27, SjScP80, SjScP84,
Expression of the SjScP88 gene in virgin worm is significantly higher than the expression of other period polypides really.Then SignalP- is utilized
4.1Server (http://www.cbs.dtu.dk/services/SignalP/) and TMHMM server are v.2.0
(http://www.cbs.dtu.dk/services/TMHMM-2.0/) will correspond to signal peptide and hydrophobic region in corresponding nucleic sequence
Sequence removal.Utilize NCBI Primer designing tool (https: //www.ncbi.nlm.nih.gov/tools/
Primer-blast/ design of primers and primer specificity analysis) are carried out.Then Invitrogen-Gateway is utilized
II kit of Technology with clonase carries out clone's building.Specific step is as follows, is required to specifications by primer
One end increases the site attB, then carries out PCR expansion to Schistosoma japonicum SjScP27, SjScP80, SjScP84, SjScP88 gene
Increase, by amplified production after purification using on BP Clonase II enzyme clone to pDONR221 carrier provided in kit, passes through
Transformation and selection recycles II enzyme of LR Clonase provided in kit to shift gene order after plasmid order-checking identification confirmation
Into expression vector pDEST17, converted after plasmid order-checking identification confirmation into colibacillus host cell by transformation and selection
Carry out recombinant protein inducing expression；Inclusion body protein is purified through denaturation and affinity chromatography, finally obtains the recombination egg of purifying
White SjScP27, SjScP80, SjScP84, SjScP88 complete SjScP27, SjScP80, SjScP84, SjScP88 gene
Body outer clone expression and purifying.Then, SjScP27, the SjScP80 purified using Western blot technical identification,
SjScP84, SjScP88 recombinant protein can be infected experimental animal serum and the identification of snail fever human serum of Schistosoma japonicum.
In turn, with the SjScP27 of purifying, SjScP80, SjScP84, SjScP88 recombinant protein is that diagnostic antigen progress snail fever is examined
The preparation of disconnected reagent and kit, passes through its application value in snail fever immunodiagnosis of elisa technique assay
And anti-SjScP27, SjScP80, SjScP84 are analyzed, changing rule of the SjScP88 antibody in infection animal serum.Finally,
SjScP27 is analyzed using fluorescence real-time quantitative PCR technology, SjScP80, SjScP84, SjScP88 gene is in Schistosoma japonicum
The gene expression rule of different developmental phases.
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW,
Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.
The clone of 1 Schistosoma japonicum SjScP27, SjScP80, SjScP84, SjScP88 gene of embodiment
According to the SjScP27 announced in GenBank, SjScP80, SjScP84, SjScP88 gene (the putative
Transcriptome data of S.japonicum BU802382, FN315317.1, FN357371.1, FN357552.1)
Sequence, separately design primer and introduce restriction enzyme site, primer sequence is as follows:
SjScP27:
PF:5 '-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAATCTCATAGTATCAGATACAAC-3 ',
PR:5 '-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTATCACTTACCATCAAGATGAAAT-3’
SjScP80:
PF:5 '-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAAAGCCTTTCGTCGGTGATGC-3 ',
PR:5 '-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGTGAAACCCAACTGGACATA-3’
SjScP84:
PF:5 '-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTTCCAGGAAGTTAAATTATGCCGT-3 ',
PR:5 '-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTATCAACTCACCTCACCAACATAA-3’
SjScP88:
PF:5 '-GGGGACAAGTTTGTACAAAAAAGCAGGCTTGGTAATGGAAAACGAAATTTCAAC-3 ',
PR:5 '-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAACACTCCAAACAGGAACAGG-3’
Underscore part is according to II reagent of Invitrogen Gateway Technology with Clonase in PF
The site attB1 (SEQ ID NO:9 or 10) of box specification design, for being connected to shuttle vector pDONR221；Under in PR
Dashed part is designed according to II kit specification of Invitrogen Gateway Technology with Clonase
The site attB2 (SEQ ID NO:11), for being connected to shuttle vector pDONR221；Specific primer is raw by Suzhou gold only intelligence
The synthesis of object Science and Technology Ltd..
Using 14 days child worm cDNA of Schistosoma japonicum (Schistosoma japonicum) as template, PCR reaction is carried out, is expanded
Increase SjScP80 gene ORF segment, reaction system is as follows: 12.5 μ l, cDNA template of high-fidelity DNA polymerase Mix, 1 μ l, upstream
0.5 μ l of primer, downstream primer 0.5 μ l, ddH210.5 μ l of O, 25 μ l of total volume.PCR amplification program: 95 DEG C of 5min, 95 DEG C of denaturation
30sec, 55 DEG C of annealing 30sec, 72 DEG C of extension 1min, totally 35 recycle；Last 72 DEG C of extensions 10min.
With 1.2% agarose gel electrophoresis detect PCR product, see whether that there are purpose bands, as a result as shown in Figure 1,
M:DNA molecular weight standard, 1:SjScP27,2:SjScP80,3:SjScP84,4:SjScP88.Agarose gel electrophoresis results are aobvious
Show, SjScP27, SjScP80, SjScP84, SjScP88 have at 198bp, 811bp, 120bp, 720bp respectively one clearly
Band is consistent with the size of expected purpose segment, shows successfully to amplify SjScP27, SjScP80, SjScP84 from cDNA,
The ORF segment of SjScP88 gene.
Use AxyPrep DNA gel QIAquick Gel Extraction Kit (AXYGEN company) purification and recovery PCR product.
II kit of Invitrogen Gateway Technology with Clonase constructs shuttle vector, reactant
Be as follows: PCR recycling DNA product (is dissolved in ddH2O) 150ng, pDONR221 carrier (are dissolved in ddH2O) 150ng, BP clonase
II enzyme, 2 μ l, adds ddH2O to 10 μ l of total volume.25 DEG C of incubation 1h after mixing.Above-mentioned connection product is converted to bacillus coli DH 5 alpha
Competent cell after conversion is applied to LB culture medium flat plate by competent cell (Beijing Quanshijin Biotechnology Co., Ltd)
(containing 50 μ g/ml kanamycins), 37 DEG C of overnight incubations.Picking single colonie carries out PCR identification, and PCR is accredited as to positive clone
Further use II kit construction of expression vector of Invitrogen Gateway Technology with Clonase.Reactant
Be as follows: the positive shuttle vector of PCR identification (is dissolved in ddH2O) 150ng, pDEST17 carrier (are dissolved in ddH2O) 150ng adds TE
Then II enzyme of LR clonase, 2 μ l is added in Buffer (pH8.0) to 8 μ l of total volume.25 DEG C of incubation 1h after mixing.It adds
Protein K (2 μ g/ μ l) 1 μ l, 37 DEG C of incubation 10min remove isolating protein.Above-mentioned connection product is converted to Escherichia coli
Competent cell after conversion is applied to LB culture medium by B21DE3 competent cell (Beijing Quanshijin Biotechnology Co., Ltd)
Plate (contains 50 μ g/ml ampicillins), 37 DEG C of overnight incubations.Picking single colonie carries out PCR identification, and PCR is accredited as the positive
Clone send Suzhou Jin Weizhi Biotechnology Co., Ltd carry out DNA sequencing confirmation sequence it is whether correct.Sequencing analysis result table
The exogenous genetic fragment sequence of bright insertion is correct, recombinant plasmid pDEST17-SjScP27, pDEST17-SjScP80, pDEST17-
SjScP84, pDEST17-SjScP88 are constructed successfully.The nucleotides sequence of gene SjScP27, SjScP80, SjScP84, SjScP88
Column are respectively as shown in SEQ ID NO:1-4, and the amino acid sequence for the antigen protein that they are encoded is respectively such as SEQ ID NO:5-8 institute
Show.
The expression and purifying of 2 Schistosoma japonicum SjScP27, SjScP80, SjScP84, SjScP88 recombinant protein of embodiment
Above-mentioned PCR is accredited as positive clone and is inoculated into LB liquid medium (containing 50 μ g/ml ampicillins) 15mL,
37 DEG C, 200rpm overnight incubation takes 10mL culture medium to transfer into LB culture medium (containing 50 μ g/ml ampicillins) 1L for second day,
Continue culture to OD600nmValue is 0.8, and the IPTG for adding final concentration of 1mM is induced, and 18 DEG C of 140rpm are expressed 16 hours,
Thalline were collected by centrifugation, -80 DEG C freeze it is spare.
It is resuspended in PBS buffer solution before taking a small amount of induction with the thallus after induction, SDS-PAGE sample-loading buffer is added, mixes
Boiling 5min after even in boiling water bath makes albuminous degeneration.
It is separately added into each 10 μ l of sample before induction and after induction in each loading hole, carries out SDS-PAGE analysis (concentration
Glue is 5%, separation gel 12%).
PDEST17-SjScP27, pDEST17-SjScP80, pDEST17-SjScP84, pDEST17-SjScP88 are recombinated
Plasmid converts expression competent cell respectively, and obvious band of expression occurs in the thallus before relatively inducing after IPTG inducing expression.
Thallus after induction is resuspended in 40mL bacterial lysate, ultrasonication, 4 DEG C, 12,000rpm centrifugation 30min,
Inclusion body precipitating and supernatant are collected respectively.
Inclusion body precipitating and supernatant are subjected to SDS-PAGE analysis, identify the dissolubility of recombinant protein.The results show that weight
Histone is primarily present in inclusion body precipitating.
Inclusion body is resuspended in the PBS solution supersound washing 5min containing 1%Triton-X100,12,000rpm centrifugations
15min collects inclusion body precipitating；
Inclusion body is resuspended in 8M urea, 4 DEG C of rotation mixing overnights, sufficiently dissolution inclusion body, 12,000rpm centrifugations
30min collects supernatant；
Supernatant, which is crossed nickel ion chelating rubber column gel column (QIAGEN company), makes the recombination SjScP80 with 6 histidine tags
Protein binding in rubber column gel column, with 50mM imidazoles rinse foreign protein, then use 250mM imidazoles wash-out recombinant protein, collection eluent；
Purifying gained recombinant protein is through SDS-PAGE analysis detection purity of protein.Electrophoresis result as shown in Fig. 2, SjScP27,
SjScP80, SjScP84, SjScP88 molecular weight of albumen respectively may be about 10.2kDa, 33.0kDa, 7.1kDa, 29.5kDa, point
Son amount size is consistent with the theoretical Mr of purpose recombinant protein, shows to obtain after purification through nickel ion chelating rubber column gel column pure
Spend higher recombinant protein.
Use the dense of BCA quantification of protein kit (Thermo Fisher Scientific company) measurement recombinant protein
Degree, is operated to specifications.Purifying resulting recombinant protein concentration after measured is 1.0mg/mL.
The detection of antigenicity of 3 Schistosoma japonicum SjScP27, SjScP80, SjScP84, SjScP88 recombinant protein of embodiment
SDS-PAGE electrophoresis: recombinant protein 100ng loading, deposition condition are taken are as follows: 100V 20min, 120V 1h.
Transferring film: using wet robin by the protein delivery in PAGE glue to pvdf membrane, electricity turns condition are as follows: ice bath, 100V1h.
Closing: 5% skimmed milk power room temperature of pvdf membrane is closed into 2h, TBST buffer washs 3 times.
Add primary antibody to be incubated for: being separately added into infection 42 days BALB/c mouse serum of Schistosoma japonicum, infection Schistosoma japonicum 42
Its new zealand white rabbit serum and Schistosoma japonicum patients serum, with the anti-His-tag antibody of mouse, (Ai Bimate biological medicine has
Limit company) it is positive control, using healthy mice serum as negative control (with confining liquid 1:500 dilution), 4 DEG C of overnight incubations,
TBST buffer washs 3 times.
Add secondary antibody to be incubated for: being separately added into the anti-mouse IgG antibody, anti-rabbit IgG antibody and anti-human IgG antibodies of fluorescent marker
(using confining liquid 1:10,000 dilution), 37 DEG C are protected from light incubation 1h, and TBST buffer washs 3 times.
It sweeps film: using Odyssey infrared laser imaging system scanning imagery.
As a result as shown in figures 3 to 6, using the anti-His-tag antibody of mouse as positive control, SjScP27, SjScP80,
SjScP84, SjScP88 have an apparent identification band at 10.2kDa, 33.0kDa, 7.1kDa, 29.5kDa respectively, with strong
Health mice serum is negative control, without obvious band.Wherein SjScP27, SjScP80, SjScP88 albumen can be infected Japan
Bilharzial BALB/c mouse serum, new zealand white rabbit serum and Schistosoma japonicum patients serum identification, SjScP84 can be by people
Serum identification, also there is weaker identification band in rabbit anteserum.Show recombinant protein SjScP27, SjScP80, SjScP84,
SjScP88 has antigenicity well.
Embodiment 4 prepares Japanese schistosomiasis SjScP27, SjScP80, SjScP84, SjScP88-ELISA immunodiagnosis
Kit
1, the main component of kit includes:
The solid phase carrier of envelope antigen: with the carbonate-bicarbonate buffer solution (purchase of the TWEEN20 containing 0-0.05%v/v
From Sigma, article No. C3041) as coating buffer, dilution destination protein to 1 μ g/mL is coated in polystyrene reactant hole,
100 holes μ L/.
Washing buffer: PBST liquid, the i.e. PBS solution of the TWEEN20 containing 0.05%v/v, pH7.4.
Sample dilution: 10%BSA solution is prepared by solvent of PBS buffer solution.
Enzyme labelled antibody: goat anti-human immunoglobulin antibody (α, γ and the μ-chain of alkali phosphatase enzyme mark
Specific) antibody (Sigma company).
Substrate developing solution: pNPP developing solution.
PNPP is purchased from Sigma, article No. N2640.The preparation method of pNPP developing solution is as follows: 15mg pNPP is dissolved in 15mL
(0.1M glycine, 1mM MgCl in the glycine buffer of 0.1M2, 1mM ZnCl2, it is dissolved in pure water, pH10.4) to get.
Reaction terminating liquid: 120g/L NaOH aqueous solution.
Positive control: with human immunoglobulin(HIg) IgG (1 μ g/mL) wrapper sheet.
Negative control: with corresponding antigen protein wrapper sheet, enzyme labelled antibody replaces with Sample dilution.
2, the operation sequence and detection method of kit:
Closing: liquid in drying hole adds 200 hole μ L/ of dilution, 37 DEG C, closes 2h, washed three times with PBST, 200 μ L/
Hole 2min/ times, pats dry for the last time.
Add sample to be tested: measuring samples serum and dilution set positive, negative and blank by 1:100 dilution proportion
It compares (only plus Sample dilution), adds each sample by 100 holes μ L/, 3 multiple holes of each sample Parallel testing, are incubated for 1h by 37 DEG C.
Washing: liquid in drying hole is washed four times, 200 holes μ L/ 2min/ times, pat dry for the last time with PBST buffer.
Enzyme labeling antibody: goat anti-human immunoglobulin (α, γ and the μ-chain of alkali phosphatase enzyme mark is added
Specific) antibody, 100 holes μ L/, are incubated for 1h, as above wash, pat dry by 37 DEG C.
Colour developing: being added chromogenic enzyme substrate liquid, and 100 holes μ L/, 37 DEG C are protected from light incubation 30min, and terminate liquid, 25 holes μ L/ are added.
Reading data and processing: OD is read with microplate reader405nmNumerical value, with 2.1 times of works of the OD mean value of negative control sample
For the critical value (cut-off) for judging yin and yang attribute.
Embodiment 5 SjScP27, SjScP80, SjScP84, SjScP88-ELISA kit are immune to Japanese schistosomiasis
Diagnostic Value
1, kit sensibility
Hunan Province's excrement, which is collected, using Kato- Katz technique (Kato-Katz) examines worm's ovum positive Schistosoma japonicum patients serum 35
Part, the sensibility of this kit is evaluated, and (preparation method of rSP13-ELISA kit is the same as real with rSP13-ELISA kit
Example 4 is applied, the amino acid sequence of rSP13 antigen protein is shown in SEQ ID NO:12) it is compared, the same embodiment of ELISA experimental implementation
3。
As a result as shown in table 1 and Fig. 7, in 35 parts of Schistosoma japonicum patients serums, 32 parts are examined by rSP13-ELISA kit
Break as the positive, the susceptibility of rSP13-ELISA kit diagnosing Japanese schistosomiasis be 91.4% (95% confidence interval,
75.8%-97.7%), this result is close to 90.4% (95% confidence area of rSP13-ELISA kit susceptibility reported in the literature
Between, 75.0%-95.0%)；34 parts are diagnosed as the positive by SjScP27-ELISA kit, SjScP27-ELISA kit it is quick
Sensitivity is 97.1% (95% confidence interval, 83.4%-99.9%)；35 parts by SjScP80-ELISA, SjScP84-ELISA,
SjScP88-ELISA kit is diagnosed as the positive, the susceptibility of these three kits be 100% (95% confidence interval,
87.7%-100%).
The result shows that the sensibility of SjScP27, SjScP80, SjScP84, SjScP88-ELISA kit is significantly higher than
RSP13-ELISA kit, p < 0.05.
Table 1 rSP13-ELISA and SjScP27-ELISA, SjScP80-ELISA, SjScP84-ELISA, SjScP88-
The comparative analysis of ELISA kit sensibility
*: the sensibility of SjSAPLLP5-ELISA kit is significantly higher than rSP13-ELISA kit, p < 0.05.
2, kit specificity
35 parts of Heilongjiang Province's normal human serum sample is collected, 18 parts of clonorchiasis in Guangdong Province, China patients serum, evaluation is originally
False positive rate of the kit in normal population, and the cross reaction situation with other parasitic disease human serums, and with
RSP13-ELISA kit is compared, and ELISA experimental implementation is the same as embodiment 3.
As a result as shown in table 2 and Fig. 7,35 portions of normal human serums by rSP13-ELISA and SjScP27-ELISA,
SjScP80-ELISA, SjScP84-ELISA, SjScP88-ELISA kit are diagnosed as feminine gender, the specificity of five kinds of kits
It is 100% (95% confidence interval, 87.7%-100%)；18 parts of clonorchis sinensis patients serums are diagnosed by five kinds of kits
For feminine gender, no cross reaction.
Table 2 rSP13-ELISA and SjScP27-ELISA, SjScP80-ELISA, SjScP84-ELISA, SjScP88-
The comparative analysis of ELISA kit specificity
3, value of the kit in terms of assessing Japanese schistosomiasis development situation
Above-mentioned 35 Schistosoma japonicum patients, acquire serum after chemotherapy 3 months again, evaluate this kit in the date of the valuation
Value in terms of schistosomiasis resistant development situation, and be compared with rSP13-ELISA kit, ELISA experimental implementation is the same as real
Apply example 3.As a result as shown in Table 3 and Fig. 7, SjScP80-ELISA, SjScP88-ELISA, after rSP13-ELISA kit chemotherapy
Patient OD value is remarkably decreased compared with the Current Infection stage.SjScP27-ELISA and SjScP84-ELISA kit in Current Infection and
The stage does not have notable difference after chemotherapy.
Table 3 rSP13-ELISA and SjScP27-ELISA, SjScP80-ELISA, SjScP84-ELISA, SjScP88-
The comparative analysis that ELISA kit is worth in terms of assessing Japanese schistosomiasis development situation
6 Schistosoma japonicum SjScP27, SjScP80, SjScP84, SjScP88 gene expression law-analysing of embodiment
It is female that schistosoma japonice ovum, cercaria, liver phase child worm and 42 days are extracted using RNeasy kit (QIAGEN company)
The total serum IgE of male imago, after dnase digestion removes DNA, with SuperScriptTM III reverse transcription reagent box (Invitrogen
Company) synthesis cDNA, using 7300Real-Time PCR (Applied Biosystems) detection SjScP27, SjScP80,
Transcriptional level of SjScP84, SjScP88 gene in Schistosoma japonicum different developmental phases.
SjScP27, SjScP80, SjScP84, SjScP88 gene primer are respectively as follows:
SjScP27 upstream primer: 5 '-AATCTCATAGTATCAGATACAAC-3 ',
Downstream primer: 5 '-TCACTTACCATCAAGATGAAAT-3 '；
SjScP80 upstream primer: 5 '-CGTCCTACAGTGGGCATTCT-3 ',
Downstream primer: 5 '-AAGAACGCCGAACCAGAACC-3 '；
SjScP84 upstream primer: 5 '-TTCCAGGAAGTTAAATTATGCCGT-3 ',
Downstream primer: 5 '-TCAACTCACCTCACCAACATAA-3 '；
SjScP88 upstream primer: 5 '-GGTAATGGAAAACGAAATTTCAACA-3 ',
Downstream primer: 5 '-TTCCAACCAGCTGTTGTCCA-3 '；
Subunit gene PSMD4 is adjusted as internal reference, upstream primer 5 '-using proteasome
CCTCACCAACAATTTCCACATCT-3 ', downstream primer 5 '-GATCACTTATAGCCTTGCGAACAT-3 '.
PCR reaction system: 12.5 μ l, cDNA template of SYBR Green Mix (Agilent company), 1 μ l, upstream primer 0.5
μ l, downstream primer 0.5 μ l, ddH210.5 μ l of O, 25 μ l of total volume.
PCR amplification program: 95 DEG C, 10min；95 DEG C, 30sec, 60 DEG C, 1min (circulation 40 times).
Data analysis is carried out using 1.4 software of SDS (Applied Biosystems).
As a result as shown in figure 8, Schistosoma japonicum SjScP27, SjScP80, SjScP84, SjScP88 gene are mainly in virgin worm
The expression of stage height, it is relatively low in the expression of other stages (worm's ovum, cercaria and adult stage), it is cercarial Development of Schistosoma Japonicum early stage
Key gene is potential antischistosomal drug target spot and vaccine candidate antigen.
Embodiment 7 anti-SjScP27, SjScP80, SjScP84, SjScP88 protein antibodies are dynamic in infection animal serum
Mechanical analysis
Before acquiring 6 Babl/c mouse and 5 New Zealand White Rabbit schistosoma japonicum infections, the 1-8 weeks serum after infection,
ELISA experimental implementation is the same as embodiment 3.
As a result as shown in Fig. 9-Figure 10,6 Babl/c mouse and 5 New Zealand White Rabbit are after infection Schistosoma japonicum 2 weeks
It can detect that corresponding albumen is anti-in serum by SjScP27, SjScP80, SjScP84, SjScP88-ELISA kit
Body, the test result show that kit has the value of potential Diagnosis of Schistosomiasis early infection.Wherein SjScP80,
The corresponding protein antibodies of serum are begun to decline at SjScP88-ELISA kit 8 weeks, blood at SjScP27-ELISA kit 7 weeks
Clear antibody level is remarkably decreased.These three kits are prompted to have the potential value for distinguishing acute infection and chronic infection.
Embodiment 8 SjScP27, SjScP80, the application of SjScP84 and SjScP88-ELISA joint-detection
The antigen protein used in the application is virgin worm phase cance high-expression gene coding for antigens, in the expression water of virgin worm phase
It is flat higher, until polypide development is expression reduction after adult, the decline of corresponding antigens level.Animal blood serum antibody power in embodiment 7
Learn analysis shows that, this four protein antibodies can be detected in serum at zoogenetic infection two weeks by corresponding ELISA kit
Corresponding antibodies.Thus this four kits have potential diagnostic value in Schistosoma japonicum early infection.
In existing blood fluke diagnosis, SjScP80 and SjScP88 the OD value when three months patients of chemotherapy organize ELISA detection
It is remarkably decreased, and SjScP27 and SjScP84 do not decline.
SjScP80 and SjScP88 OD value decline explanation polypide death after praziquantel treatment after chemotherapy is degraded, thus phase
Antigen degradation is answered, so that patient antibodies' level is gradually reduced.Facilitate therapeutic evaluation.Although there is the potency decline of antibody after chemotherapy
Help therapeutic evaluation, but will be unable to detect when antibody titer is lower than certain level.
OD value does not decline after SjScP27 and SjScP84 chemotherapy, facilitates the evaluation of previous infection.Therefore, by SjScP80
Cooperate SjScP27 and SjScP84 with antigen proteins such as SjScP88, it will help distinguish Current Infection and previous infection.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Institute of Pathogen Biology, Chinese Academy of Medical Sciences
<120>highly expressed gene and its coding albumen and application in Schistosomula Japonicum
<130> KHP191112980.2
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 201
<212> DNA
<213>Schistosoma japonicum (Schistosoma japonicum)
<400> 1
ttcaatctca tagtatcaga tacaacaaca aaatcatatg aatataactt aataaatgat 60
aatgatgatg atgatgtaat aaaagatgtg acaactgaat taagagattt aaattggcca 120
aatttattac caaaatcatc taatcaatca gaaaataaaa cttatcatat tcaaacagta 180
tttcatcttg atggtaagtg a 201
<210> 2
<211> 834
<212> DNA
<213>Schistosoma japonicum (Schistosoma japonicum)
<400> 2
ttaaagcctt tcgtcggtga tgctgtatat ggatatggga ttactaactg tggtattggt 60
ttatgcgaaa agaagcaatg caaaaataat gctgcttgtt tgcaaacatc cggctcgact 120
gtcatctgtc agtgtccttt gggaacttat ggacaattat gtgaaaaaag aggtgagcta 180
attataccgt cctacagtgg gcattcttac acagagtata ttggattaag tgggacatca 240
tcatcattta ctaccttgga actccaatac agaccaataa aacccgatgg gctgatttta 300
tatgaaggtt atagtcatga cagtcgtggt gattttttgg ctattatttt ggttaatgga 360
tacgttgtag tggcattcga cttaggttct ggttcagcgt tcttaaagag tgaggatcaa 420
atgaaattaa atgagtggca tttaatacgt ttctggcgaa ttggtcgtgt gggttatcta 480
caagttgact taaatgagca tattatgaca aactactcat ttggtttaca agttcaacta 540
acattaactt actttttatt tcttggcgga catccaaatt taaatattgt gtcaacacat 600
ataaaagaat atattggatt cccaaataat ctaagtattg gattccaagg atgcataagt 660
aaaattgaaa caaacggaat actgattgat cctatcaaga atgcgatagg aggtgcaaat 720
gtttataatt gcgcaggaca agaatgtggt ttttcaaatc ctgtatgttt aaacaacgga 780
aaatgtgtac aatactcaaa tggctataag tgtatatgtc cagttgggtt tcac 834
<210> 3
<211> 123
<212> DNA
<213>Schistosoma japonicum (Schistosoma japonicum)
<400> 3
ttcttccagg aagttaaatt atgccgtaag acatcaaatg aaaaattcgg tttaacactt 60
tgttatcgac aaggtgatac atgcgataca agttgtaatg tttatgttgg tgaggtgagt 120
tga 123
<210> 4
<211> 720
<212> DNA
<213>Schistosoma japonicum (Schistosoma japonicum)
<400> 4
ttggtaatgg aaaacgaaat ttcaacaaag aaaagttcat acaaaactaa tataccagtg 60
aattatgatg gtgcagtagt aggtttagct agacgatcaa cattatttcc atttacaaca 120
cctttaccag gtatttcaac agctgcttta caattggaac atttaatagg taaaaataaa 180
aaatggacaa cagctggttg gaaattaagt tgtgaatcag gattagtatt tcgtactcaa 240
ttacaagata aagaatatgt ttacttgaat gcagctgaat ttggtgaatt agaagttgtt 300
cgagatttat tagatgatac aaaacttgat gtgaattgtg tagattatat gggacgtaat 360
gctttactac tggcaatgaa aaatgaaaat attgatttag tagaactatt agtgaataga 420
ttagattttt atgctgttga agatgcctta ttaaatgcaa ttagtcaaca aaaaaatcat 480
ttagttaaat taattgttga tcatccacaa tatattagaa tggaaaaaca atgcaaaagt 540
aaagtgtctg gaccatcaag tggaacaaat aatcgtggaa aacgatcaca attttcttca 600
gacatttcac ctttaatgct tgcagcacat attaacaatc atgaaattat tcagttatta 660
ttagatagag ggttaaaact agaaatgcca catgatcgtg cctgttcctg tttggagtgt 720
<210> 5
<211> 66
<212> PRT
<213>Schistosoma japonicum (Schistosoma japonicum)
<400> 5
Phe Asn Leu Ile Val Ser Asp Thr Thr Thr Lys Ser Tyr Glu Tyr Asn
1 5 10 15
Leu Ile Asn Asp Asn Asp Asp Asp Asp Val Ile Lys Asp Val Thr Thr
20 25 30
Glu Leu Arg Asp Leu Asn Trp Pro Asn Leu Leu Pro Lys Ser Ser Asn
35 40 45
Gln Ser Glu Asn Lys Thr Tyr His Ile Gln Thr Val Phe His Leu Asp
50 55 60
Gly Lys
65
<210> 6
<211> 278
<212> PRT
<213>Schistosoma japonicum (Schistosoma japonicum)
<400> 6
Leu Lys Pro Phe Val Gly Asp Ala Val Tyr Gly Tyr Gly Ile Thr Asn
1 5 10 15
Cys Gly Ile Gly Leu Cys Glu Lys Lys Gln Cys Lys Asn Asn Ala Ala
20 25 30
Cys Leu Gln Thr Ser Gly Ser Thr Val Ile Cys Gln Cys Pro Leu Gly
35 40 45
Thr Tyr Gly Gln Leu Cys Glu Lys Arg Gly Glu Leu Ile Ile Pro Ser
50 55 60
Tyr Ser Gly His Ser Tyr Thr Glu Tyr Ile Gly Leu Ser Gly Thr Ser
65 70 75 80
Ser Ser Phe Thr Thr Leu Glu Leu Gln Tyr Arg Pro Ile Lys Pro Asp
85 90 95
Gly Leu Ile Leu Tyr Glu Gly Tyr Ser His Asp Ser Arg Gly Asp Phe
100 105 110
Leu Ala Ile Ile Leu Val Asn Gly Tyr Val Val Val Ala Phe Asp Leu
115 120 125
Gly Ser Gly Ser Ala Phe Leu Lys Ser Glu Asp Gln Met Lys Leu Asn
130 135 140
Glu Trp His Leu Ile Arg Phe Trp Arg Ile Gly Arg Val Gly Tyr Leu
145 150 155 160
Gln Val Asp Leu Asn Glu His Ile Met Thr Asn Tyr Ser Phe Gly Leu
165 170 175
Gln Val Gln Leu Thr Leu Thr Tyr Phe Leu Phe Leu Gly Gly His Pro
180 185 190
Asn Leu Asn Ile Val Ser Thr His Ile Lys Glu Tyr Ile Gly Phe Pro
195 200 205
Asn Asn Leu Ser Ile Gly Phe Gln Gly Cys Ile Ser Lys Ile Glu Thr
210 215 220
Asn Gly Ile Leu Ile Asp Pro Ile Lys Asn Ala Ile Gly Gly Ala Asn
225 230 235 240
Val Tyr Asn Cys Ala Gly Gln Glu Cys Gly Phe Ser Asn Pro Val Cys
245 250 255
Leu Asn Asn Gly Lys Cys Val Gln Tyr Ser Asn Gly Tyr Lys Cys Ile
260 265 270
Cys Pro Val Gly Phe His
275
<210> 7
<211> 40
<212> PRT
<213>Schistosoma japonicum (Schistosoma japonicum)
<400> 7
Phe Phe Gln Glu Val Lys Leu Cys Arg Lys Thr Ser Asn Glu Lys Phe
1 5 10 15
Gly Leu Thr Leu Cys Tyr Arg Gln Gly Asp Thr Cys Asp Thr Ser Cys
20 25 30
Asn Val Tyr Val Gly Glu Val Ser
35 40
<210> 8
<211> 240
<212> PRT
<213>Schistosoma japonicum (Schistosoma japonicum)
<400> 8
Leu Val Met Glu Asn Glu Ile Ser Thr Lys Lys Ser Ser Tyr Lys Thr
1 5 10 15
Asn Ile Pro Val Asn Tyr Asp Gly Ala Val Val Gly Leu Ala Arg Arg
20 25 30
Ser Thr Leu Phe Pro Phe Thr Thr Pro Leu Pro Gly Ile Ser Thr Ala
35 40 45
Ala Leu Gln Leu Glu His Leu Ile Gly Lys Asn Lys Lys Trp Thr Thr
50 55 60
Ala Gly Trp Lys Leu Ser Cys Glu Ser Gly Leu Val Phe Arg Thr Gln
65 70 75 80
Leu Gln Asp Lys Glu Tyr Val Tyr Leu Asn Ala Ala Glu Phe Gly Glu
85 90 95
Leu Glu Val Val Arg Asp Leu Leu Asp Asp Thr Lys Leu Asp Val Asn
100 105 110
Cys Val Asp Tyr Met Gly Arg Asn Ala Leu Leu Leu Ala Met Lys Asn
115 120 125
Glu Asn Ile Asp Leu Val Glu Leu Leu Val Asn Arg Leu Asp Phe Tyr
130 135 140
Ala Val Glu Asp Ala Leu Leu Asn Ala Ile Ser Gln Gln Lys Asn His
145 150 155 160
Leu Val Lys Leu Ile Val Asp His Pro Gln Tyr Ile Arg Met Glu Lys
165 170 175
Gln Cys Lys Ser Lys Val Ser Gly Pro Ser Ser Gly Thr Asn Asn Arg
180 185 190
Gly Lys Arg Ser Gln Phe Ser Ser Asp Ile Ser Pro Leu Met Leu Ala
195 200 205
Ala His Ile Asn Asn His Glu Ile Ile Gln Leu Leu Leu Asp Arg Gly
210 215 220
Leu Lys Leu Glu Met Pro His Asp Arg Ala Cys Ser Cys Leu Glu Cys
225 230 235 240
<210> 9
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
ggggacaagt ttgtacaaaa aagcaggctt c 31
<210> 10
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
ggggacaagt ttgtacaaaa aagcaggctt 30
<210> 11
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
ggggaccact ttgtacaaga aagctgggtc cta 33
<210> 12
<211> 177
<212> PRT
<213>Schistosoma japonicum (Schistosoma japonicum)
<400> 12
Met Leu Lys Arg Leu Phe Ile Leu Ile Val Ile Leu Gly Val Asn Glu
1 5 10 15
Val Thr Leu Gly Leu Glu Asn Ser Val Ser Pro Leu Lys Gln Pro Asn
20 25 30
Cys Arg Leu Leu Cys Gly Thr Cys Leu Phe Met Gly Arg Met Thr Lys
35 40 45
Val Phe Leu Glu Ser Glu Pro Phe Ile Pro Ile Met Ala Arg Ile Ile
50 55 60
Ser Pro Leu Cys His Leu Ile Pro Asn Glu Glu Cys Lys His Asn Cys
65 70 75 80
Leu Asn Val Thr His Glu Leu Pro Arg Glu Ile Lys Thr Trp Ala Lys
85 90 95
His Met Asn Val Ser His Asp Cys Ser Lys Leu Gly Leu Cys His Lys
100 105 110
Asn His Ser Met Val Ser Ser Phe Glu Phe Thr Ser Phe Leu Lys Glu
115 120 125
His Met Asn Tyr Trp Leu Ser Leu Asp Gln Asn Gly Lys Tyr Lys Asn
130 135 140
Thr Phe Ile Lys Asn Leu Cys Lys His His Ala Ala Asp Thr Asp Lys
145 150 155 160
Cys Ile Glu Thr Leu Glu Thr Ile Val Lys Phe Leu Val Gln Phe Thr
165 170 175
Ile
Claims (6)
1. the highly expressed gene in Schistosomula Japonicum, which is characterized in that the gene be SjScP27 and/or
SjScP84, their nucleotide sequence difference are as follows:
Gene SjScP27:
I) nucleotide sequence shown in SEQ ID NO:1；
Ii) nucleotide sequence shown in SEQ ID NO:1 is substituted, lacks and/or increases one or more nucleotide and expression
The nucleotide sequence of identical function protein；
Iii) hybridize under strict conditions with sequence shown in SEQ ID NO:1 and express the nucleotides sequence of identical function protein
Column, the stringent condition are in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS, at 65 DEG C
Hybridization, and film is washed with the solution；Or
Iv) and i), ii) or nucleotide sequence iii) there is 90% or more homology and express the nucleosides of identical function protein
Acid sequence；
Gene SjScP84:
I) nucleotide sequence shown in SEQ ID NO:3；
Ii) nucleotide sequence shown in SEQ ID NO:3 is substituted, lacks and/or increases one or more nucleotide and expression
The nucleotide sequence of identical function protein；
Iii) hybridize under strict conditions with sequence shown in SEQ ID NO:3 and express the nucleotides sequence of identical function protein
Column, the stringent condition are in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS, at 65 DEG C
Hybridization, and film is washed with the solution；Or
Iv) and i), ii) or nucleotide sequence iii) there is 90% or more homology and express the nucleosides of identical function protein
Acid sequence.
2. the antigen protein of the coding of gene described in claim 1, which is characterized in that gene SjScP27, SjScP84 coding resist
The amino acid sequence of former albumen is respectively as shown in SEQ ID NO:5 and 7.
3. following any application of antigen protein described in claim 2:
1) Schistosoma japonicum detection reagent or kit are used to prepare；
2) it is used to prepare anti schistosoma vaccine；
3) it is used to prepare anti schistosoma drug；
4) it is used for the detection of non-diagnostic purpose Schistosoma japonicum.
4. Schistosoma japonicum detection reagent, which is characterized in that in the detection reagent containing it is following 1.~at least one of 2.:
1. Japan schistosome antigen Protein S jScP27, or the DNA molecular of the coding antigen protein, or by containing the DNA points
The recombinant protein that the recombinant bacterium of son generates；
2. Japan schistosome antigen Protein S jScP84, or the DNA molecular of the coding antigen protein, or by containing the DNA points
The recombinant protein that the recombinant bacterium of son generates；
Wherein, the amino acid sequence of antigen protein SjScP27, SjScP84 is respectively as shown in SEQ ID NO:5 and 7.
5. the kit containing detection reagent described in claim 4.
6. Japanese schistosomiasis ELISA immunodiagnosis kit, which is characterized in that the kit includes:
1) it is coated with the micro reaction plate of antigen protein described in 1-5 μ g/mL claim 2；With TWEEN20 containing 0-0.05%v/v
Carbonate-bicarbonate buffer solution as coating buffer；
2) washing buffer: PBST liquid, the i.e. PBS solution of the TWEEN20 containing 0.05%v/v, pH7.4；
3) Sample dilution: 5-10%BSA solution is prepared by solvent of PBS buffer solution；
4) enzyme labelled antibody: the goat anti-human immunoglobulin antibody of alkali phosphatase enzyme mark；
5) substrate developing solution: pNPP developing solution；
6) reaction terminating liquid: 120g/L NaOH aqueous solution；
7) positive control: with 1 μ g/mL human immunoglobulin(HIg) IgG wrapper sheet；
8) negative control: with corresponding antigen protein wrapper sheet, enzyme labelled antibody replaces with Sample dilution.
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