CN105254732B - A kind of Schistosoma japonicum recombinant protein SjSAPLP5 and its encoding gene and application - Google Patents
A kind of Schistosoma japonicum recombinant protein SjSAPLP5 and its encoding gene and application Download PDFInfo
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- CN105254732B CN105254732B CN201510818349.0A CN201510818349A CN105254732B CN 105254732 B CN105254732 B CN 105254732B CN 201510818349 A CN201510818349 A CN 201510818349A CN 105254732 B CN105254732 B CN 105254732B
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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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
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- G—PHYSICS
- 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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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 a kind of Schistosoma japonicum recombinant protein SjSAPLP5 and its encoding gene and applications.The albumen of the present invention has the amino acid sequence with same function that amino acid sequence or the sequence as shown in SEQ ID No.2 are formed through replacing, lacking and oring add one or several amino acid residues.The present invention also provides the gene orders for encoding above-mentioned albumen.The recombinant protein SjSAPLP5 immunogenicities of the present invention are good, it can be used as excellent diagnostic antigen, it is used to prepare highly sensitive, high specific Schistosoma japonicum diagnostic kit, it can also be used to prepare anti-schistosome vaccine and as potential drug target, the drug of screening treatment Japanese schistosomiasis.
Description
Technical field
The present invention relates to biotechnologies, specifically, be related to a kind of Schistosoma japonicum recombinant protein SjSAPLP5 and
Its encoding gene and application.
Background technology
Snail fever is a kind of infectiousness parasitic disease seriously threatening human health, and Major Epidemic is in Asian, African and Latin American 76
A countries and regions, the whole world have people more than 200,000,000 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 makes 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, criterion can be also provided for Endemic Areas of Schistosomiasis Japonica grade, be the popular state of assessment
Gesture and examination control effect provide essential information and scientific basis.It is snail fever to lack efficiently accurate diagnostic techniques
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 will cause the continued popularity of snail fever to be propagated contained in excreta.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 is mainly by checking the japonice ovum in Feces of Patients or urine
It diagnoses the illness, it is not only time-consuming and laborious and relatively low to the sensibility of slight infection by Schistosoma diagnosis, be not suitable for extensive
Snail fever field monitoring.Compared to 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 big
Scale on-site monitors.The most common antigen for being presently used for snail fever immunodiagnosis is that the adult of extraction blood fluke polypide is anti-
Stock blend (Adult worm antigen, AWA) and egg antigen component (Soluble egg antigen, SEA), due to this
Two kinds of thick complicated components (being made of thousands of kinds of blood fluke albumen) for carrying antigen, and with other parasitic infection serum there is compared with
The specificity of serious cross reaction, schistosomiasis diagnosis is not high, and the reagent of production should not standardize.
By the blood fluke recombinant antigen molecule of technique for gene engineering production is concise with ingredient, cross reaction is few, can be big
The advantages that scale is produced in batches, it is considered to be the target antigen molecule of ideal schistosomiasis diagnosis and prevention.2014, China
Scientist reports an Immunodiagnosis of Schistosomiasis Japonica mark molecule (SjSP-13), and it is rSP13-ELISA to be developed
Kit, diagnostic sensitivity improve 6 times than traditional form method, and the achievement in research is in international top academic journal《Willow
Leaf knife infectious disease》Magazine is delivered online in the form of treatise.However, the sensibility (90%) of the diagnostic kit remains further
The space of promotion.
The present invention has found its base using bioinformatics method to Schistosoma japonicum full-length genome encoding gene forecast analysis
Because in group there are a Saposin sample albumen (Schistosoma japonicum Saposin-like protein,
SjSAPLP) family, the gene family share 15SjSAPLP genes composition, and SjSP-13 is wherein a member.The present invention passes through
Sequence alignment analysis finds that the sequence homology between SjSAPLP family members is extremely low, wherein SjSAPLP5 and has reported
Amino acid sequence homology between SjSP-13 is less than 30%.The present invention by SjSAPLP5 and SjSP-13 through gene cloning and
It is recombinantly expressed in Escherichia coli, ELISA, which tests diagnosis of two recombinant proteins of comparative analysis for Japanese schistosomiasis, to be had
Hypersensitivity and specificity, to find susceptibility higher, specificity preferably snail fever immunodiagnostic antigen.
Invention content
It is an object of the present invention to provide the good Schistosoma japonicum recombinant proteins of Species sensitivity height, specificity.
Another object of the present invention is to provide the recombinant proteins in Schistosoma japonicum serodiagnosis, vaccine development and to control
Treat the application in drug screening.
The technical scheme is that：Schistosoma japonicum full-length genome is encoded first with bioinformatics method
SjSAPLP genes are predicted, using Protocols in Molecular Biology to Schistosoma japonicum SjSAPLP5 genes carry out PCR amplification and gram
The grand recombinant plasmid pET- that Schistosoma japonicum SjSAPLP5 gene prokaryotics are built into expression plasmid carrier pET-28a (+)
28a (+)-SjSAPLP5 after plasmid order-checking identification confirms, is carried out by transformation and selection in conversion to colibacillus host cell
Recombinant protein induced expression；Inclusion body protein is purified through denaturation and affinity chromatography, finally obtains the recombinant protein of purifying
SjSAPLP5 completes the body outer clone expression and purifying of SjSAPLP5 genes.Utilize Western blotting technical identifications
The SjSAPLP5 recombinant proteins of purifying can be infected experimental animal serum and the identification of snail fever human serum of Schistosoma japonicum.
And then the preparation of schistosomiasis diagnosis reagent and kit is carried out using the SjSAPLP5 recombinant proteins of purifying as diagnostic antigen, pass through
Its application value in snail fever immunodiagnosis of elisa technique assay simultaneously analyzes anti-SjSAPLP5 antibody and is feeling
Contaminate the changing rule in animal blood serum.Finally SjSAPLP5 genes are analyzed in Japanese blood using fluorescence real-time quantitative PCR technology
The gene expression rule of fluke different developmental phases.
The present invention provides a kind of Schistosoma japonicum recombinant protein SjSAPLP5 as a result, and amino acid sequence is：
A) amino acid sequence shown in SEQ ID No.2；Or
B) amino acid sequence shown in SEQ ID No.2 is through replacing, lacking and oring add one or several amino acid residues
The amino acid sequence with same function formed.
It should be appreciated that those skilled in the art can be according to the amino acid sequence (SEQ of Protein S jSAPLP5 disclosed by the invention
ID No.2), Schistosoma japonicum recombinant protein SjSAPLP5 of the invention further includes the warp of amino acid sequence shown in SEQ ID No.2
Replace, lack or increase one or several amino acid, has and recombinant protein SjSAPLP5 is same active is spread out by SjSAPLP5
Raw obtained protein.
Further, it is as follows the present invention provides the gene for encoding above-mentioned Schistosoma japonicum recombinant protein SjSAPLP5
A) or b)：
A) in its nucleotide sequence such as sequence table shown in SEQ ID No.1；Or
B) nucleotide sequence shown in SEQ ID No.1 is substituted one or several nucleotide, obtains coding SjSAPLP5
Nucleotide sequence.
Further, the present invention provides a kind of recombinant expression carrier, is expression cassette or cell containing said gene
System or recombinant bacterium.
The present invention also provides the specificity for expanding above-mentioned coding Schistosoma japonicum recombinant protein SjSAPLP5 genes
Primer pair, nucleotide sequence is respectively as shown in SEQ ID NO.3,4.
In one embodiment of the invention, the amplification method of Schistosoma japonicum SjSAPLP5 genes ORF is provided, is wrapped
It includes：Using 42 days female male imago cDNA of Schistosoma japonicum as template, PCR reactions are carried out, expand SjSAPLP5 gene ORF segments；
The primer sequence that PCR reaction uses for：
PF：5’-CGCGGATCCTCAACATTGAACAGTAATAAT-3 ' (as shown in SEQ ID NO.3, italicized item table
What is shown is the protectiveness base of sense primer, and underscore part is the BamH I restriction enzyme sites of sense primer)；
PR：5’-CTGCTCGAG(as shown in SEQ ID NO.4, italicized item indicates TGCAAAAGGATATTTCAAA-3 '
Be downstream primer protectiveness base, underscore part is the Xho I restriction enzyme sites of downstream primer)；
The system and PCR response procedures of PCR reaction be respectively：
Reaction system：12.5 μ l, cDNA templates of high-fidelity DNA polymerase Mix 1 μ l, 0.5 μ l of sense primer, downstream primer
0.5 μ l, ddH210.5 μ l of O, 25 μ l of total volume.
PCR amplification program：98 DEG C of 1min, 98 DEG C denaturation 10sec, 55 DEG C annealing 30sec, 72 DEG C extension 1min, totally 35
Cycle；Last 72 DEG C of extensions 10min.
The present invention also provides a kind of methods preparing Schistosoma japonicum recombinant protein SjSAPLP5, include the following steps：
(1) recombinant expression carrier containing Schistosoma japonicum recombinant protein SjSAPLP5 encoding genes is transformed into competence
Cell is made Schistosoma japonicum recombinant protein SjSAPLP5 and expresses engineering bacteria；
(2) Schistosoma japonicum recombinant protein SjSAPLP5 is expressed into engineering bacteria through IPTG induced expressions；It is heavy to collect inclusion body
It forms sediment, and fully dissolves inclusion body, centrifuge, collect supernatant, will identify that correct expression product crosses nickel ion chelating rubber column gel column purifying
Afterwards, the Schistosoma japonicum recombinant protein SjSAPLP5 of purifying is afforded.
Detection kit or diagnostic reagent containing Schistosoma japonicum recombinant protein SjSAPLP5 of the present invention belong to this
The protection domain of invention.
Biological products containing Schistosoma japonicum recombinant protein SjSAPLP5 of the present invention belong to the protection model of the present invention
It encloses.
Preferably, the biological products are anti schistosoma vaccine.
The present invention studies Schistosoma japonicum SjSAPLP5 gene expression rules, finds in the worm's ovum stage
SjSAPLP5 genes are not expressed, and are in up-regulated expression from the cercaria stage to the stage (virgin worm and adult stage) in host is entered, are said
The growth and development of bright SjSAPLP5 gene functions and polypide is closely related, is potential antischistosomal drug target spot and vaccine candidate
Antigen.Also belong to the present invention's by the drug of latent effect target spot of Schistosoma japonicum recombinant protein SjSAPLP5 of the present invention
Protection domain.
The present invention provides above-mentioned Schistosoma japonicum recombinant protein SjSAPLP5 or its encoding gene or contain the gene
Application of the recombinant expression carrier in preparing Schistosoma japonicum serological diagnostic kit or diagnostic reagent.
The present invention provides above-mentioned Schistosoma japonicum recombinant protein SjSAPLP5 or its encoding gene or contain the gene
Application of the recombinant expression carrier in preparing anti schistosoma vaccine.
The present invention provides above-mentioned Schistosoma japonicum recombinant protein SjSAPLP5 or its encoding gene or contain the gene
Application of the recombinant expression carrier in Schistosoma japonicum medicine is treated in screening.
The Schistosoma japonicum recombinant protein SjSAPLP5 that the present invention obtains has good immunogenicity and antigenicity, can
BALB/c mouse serum, new zealand white rabbit serum and the Schistosoma japonicum patients serum of identification infection Schistosoma japonicum, specifically
Property is 100%；With SjSAPLP5 Protein Detection Schistosoma japonicum patients serums, to Schistosomiasis patients (the EPG ＜ of low-grade infection
100) it can still detect, detection sensitivity reaches 96%；Anti- SjSAPLP5 protein antibodies are detected by ELISA method infecting
Dynamic variation in animal blood serum can be detected anti-in serum after 3 weeks by SjSAPLP5-ELISA kits
SjSAPLP5 protein antibodies can be used for the diagnosis of Japanese schistosomiasis early infection, present invention discover that SjSAPLP5 gene functions
It is closely related with the growth and development of polypide, it treats Japanese schistosomiasis for genetic method from now on and foundation is provided.
Description of the drawings
Fig. 1 is the PCR amplification result schematic diagram of Schistosoma japonicum SjSAPLP5 gene orders in embodiment 1, and wherein M is
DNA molecular amount standard, 1 is the pcr amplification product of SjSAPLP5 genes.
Fig. 2 is the SDS-PAGE analysis result schematic diagrames of SjSAPLP5 recombinant proteins in embodiment 2.Swimming lane M is albumen point
Sub- amount standard, swimming lane 1 are that full bacterium is not induced to compare, and swimming lane 2 is 4 hours full bacterium after induction, after swimming lane 3 is cellular lysate after induction
Supernatant, 4 is precipitate after cellular lysate after induction, 5 be the SjSAPLP5 recombinant proteins of purifying.
Fig. 3 is SjSAPLP5 recombinant protein antigens and different infection by Schistosoma animal blood serums and snail fever in embodiment 3
The Western blotting analysis result schematic diagrames of human serum.M is Protein Marker, 1 is the anti-His labels of mouse in figure
Antibody positive control, 2 be Normal Mouse Serum negative control, 3 be infect Schistosoma japonicum 42 days mice serums, 4 be infection
42 days rabbit anteserums of Schistosoma japonicum, 5 are Schistosoma japonicum patients serum.
Fig. 4 is that rSP13-ELISA kits are used for Japanese schistosomiasis with SjSAPLP5-ELISA kits in embodiment 5
Diagnostic evaluation result of the comparison schematic diagram.A figures are rSP13-ELISA kits as a result, B figures are SjSAPLP5-ELISA reagents
Box result.
Fig. 5 is that 6 moderate resistance SjSAPLP5 protein antibodies of embodiment are infecting new zealand white rabbit with anti-SjSP-13 protein antibodies
Dynamics comparative analysis result schematic diagram in serum.A figures are anti-SjSP-13 protein antibodies in infection new zealand white rabbit serum
In dynamic analysis as a result, B figures are power credit of the anti-SjSAPLP5 protein antibodies in infecting new zealand white rabbit serum
Analyse result.
Fig. 6 be in embodiment 7 Schistosoma japonicum SjSAPLP5 genes in worm's ovum, cercaria, liver phase child worm and female male imago
Fluorescence real-time quantitative PCR analysis result schematic diagram.E is worm's ovum, C is cercaria, S is virgin worm, M is male insect, F be female at
Worm.
Specific implementation mode
The content that following embodiment further illustrates the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case of spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to
Range.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.
Unless otherwise specified, reagent used in embodiment is commercially available.
The clone of 1 Schistosoma japonicum SjSAPLP5 genes of embodiment
It is designed according to the sequence (sequence as shown in SEQ ID NO.1) of SjSAPLP5 genes (GenBank FN315317.1)
Primer simultaneously introduces restriction enzyme site, as follows：
PF：5’-CGCGGATCCTCAACATTGAACAGTAATAAT-3 ' (as shown in SEQ ID NO.3, italicized item table
What is shown is the protectiveness base of sense primer, and underscore part is the BamH I restriction enzyme sites of sense primer)；
PR：5’-CTGCTCGAG(as shown in SEQ ID NO.4, italicized item indicates TGCAAAAGGATATTTCAAA-3 '
Be downstream primer protectiveness base, underscore part is the Xho I restriction enzyme sites of downstream primer)；
Specific primer is synthesized by the Suzhou bio tech ltd Jin Weizhi.
Using 42 days female male imago cDNA of Schistosoma japonicum as template, PCR reactions are carried out, expand SjSAPLP5 gene ORF pieces
Section, reaction system are as follows：12.5 μ l, cDNA templates of high-fidelity DNA polymerase Mix 1 μ l, 0.5 μ l of sense primer, downstream primer
0.5 μ l, ddH210.5 μ l of O, 25 μ l of total volume.PCR amplification program：98 DEG C of 1min, 98 DEG C of denaturation 10sec, 55 DEG C of annealing
30sec, 72 DEG C of extension 1min, totally 35 recycle；Last 72 DEG C of extensions 10min.
PCR product is detected with 1.2% agarose gel electrophoresis, sees whether that, there are purpose band, the results are shown in Figure 1,
M：DNA molecular amount standard, 1：SjSAPLP5.Agarose gel electrophoresis at 588bp the results show that about there is a clearly item
Band, the size after removing signal peptide sequence with expected purpose segment are consistent, show successfully to amplify SjSAPLP5 bases from cDNA
The ORF segments of cause.
Recycling PCR product is purified using AxyPrep DNA gel QIAquick Gel Extraction Kits (AXYGEN companies).By the PCR of recycling
Target fragment and pET28a empty carriers plasmid are stayed overnight respectively at 37 DEG C of water-bath double digestions, and endonuclease reaction system is as follows：DNA fragmentation or
PET28a expression vectors (are dissolved in ddH2O) 2.5 μ l, digestion buffer 5 of 40 μ l, BamH I restriction endonucleases, 2.5 μ l, Xho I restriction endonucleases
μ l, 50 μ l of total volume.
By after digestion PCR product and pET28a expression vectors carry out 1.2% agarose gel electrophoresis identification and glue recycling
Purifying.According to 1：The ratio of 7 (molar ratios) by after recycling expression vector segment and external source target gene fragment connected using T4DNA
It connects enzyme to be attached, reaction condition is 16 DEG C, connects 6h.Connection product is converted thin to Escherichia coli Trans1-T1 competence
Competent cell after conversion is applied to LB culture medium flat plates and (contains 50 μ g/ml by born of the same parents (Beijing Quanshijin Biotechnology Co., Ltd)
Kanamycins), 37 DEG C of overnight incubations.Picking single bacterium colony carries out PCR identifications, and PCR, which is accredited as positive clone, send Suzhou gold only
Intelligence bio tech ltd carries out DNA sequencing and confirms whether sequence is correct.Sequencing analysis is the result shows that the foreign gene being inserted into
Fragment sequence is correct, and recombinant plasmid pET28a (+)-SjSAPLP5 is built successfully.
The expression and purifying of 2 Schistosoma japonicum SjSAPLP5 recombinant proteins of embodiment
Correct pET28a (+)-SjSAPLP5 recombinant plasmid transformeds will be sequenced to expression competent cell Transetta
(DE3) (Beijing Quanshijin Biotechnology Co., Ltd)；It is inoculated into LB liquid medium by positive clone is accredited as through PCR
(containing 50 μ g/ml kanamycins) 15mL, 37 DEG C of overnight incubations take 10mL culture mediums to transfer for second day and (contain 50 μ g/ into LB culture mediums
Ml kanamycins) 1L, continues culture to OD600nmValue is 0.8, and the IPTG progress induced expression 4 for adding final concentration of 1mM is small
When, thalline were collected by centrifugation, -80 DEG C freeze it is spare.
It is resuspended in PBS buffer solution with the thalline after induction before taking a small amount of induction, SDS-PAGE sample-loading buffers is added, mix
Boiling 5min in boiling water bath after even makes albuminous degeneration.Each 10 μ of sample before induction and after induction is separately added into each loading hole
L carries out SDS-PAGE analyses (concentration glue is 5%, separation gel 12%).As shown in Fig. 2, by pET28a (+)-SjSAPLP5 weights
Group plasmid conversion expression competent cell, it is about 25kDa that molecular weight, which occurs, in the thalline before relatively being induced after IPTG induced expressions
Apparent band of expression, molecular size range is consistent with the theoretical Mr of purpose recombinant protein.
Thalline after induction is resuspended in 40mL bacterial lysates, ultrasonication, 4 DEG C, 12,000rpm centrifugation 30min,
Inclusion body precipitation and supernatant are collected respectively.Inclusion body precipitation and supernatant are subjected to SDS-PAGE analyses, identify recombinant protein
Dissolubility.The results show that recombinant protein is primarily present in inclusion body precipitation.Inclusion body is resuspended in containing 1%Triton-
PBS solution the supersound washing 5min, 12,000rpm centrifugation 15min of X100 collects inclusion body precipitation；Inclusion body is resuspended in 8M urine
Element, 4 DEG C of rotation mixing overnights fully dissolve inclusion body, and 12,000rpm centrifugation 30min collect supernatant；By supernatant cross nickel from
Son chelating rubber column gel column (QIAGEN companies) makes the recombination SjSAPLP5 protein bindings with 6 histidine tags in rubber column gel column, uses
50mM imidazoles rinse foreign protein, then use 250mM imidazoles wash-out recombinant proteins, collection eluent；Purifying gained recombinant protein warp
SDS-PAGE analysis detection purity of protein.Electrophoresis result is as shown in Fig. 2, show to obtain after purification through nickel ion chelating rubber column gel column pure
Spend higher recombination SjSAPLP5 albumen.Using BCA quantification of protein kit, (Thermo Fisher Scientific are public
Department) measure recombinant protein concentration, operated to specifications.The recombinant protein of purifying gained is a concentration of after measured
2.2mg/mL。
The detection of antigenicity of 3 Schistosoma japonicum SjSAPLP5 recombinant proteins of embodiment
SDS-PAGE electrophoresis：The SjSAPLP5 recombinant protein 100ng loadings that Example 2 obtains, deposition condition are：100V
20min, 120V 1h.
Transferring film：Using wet robin by the protein delivery in PAGE glue to pvdf membrane, electricity turns condition and is：Ice bath, 100V 1h.
Closing：5% skimmed milk power room temperature of pvdf membrane is closed into 2h, TBST buffer solutions wash 3 times.
Primary antibody is added to be incubated：It is separately added into 42 days BALB/c mouse serum of infection 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 medicines have
Limit company) it is positive control, (uses confining liquid 1 by negative control of healthy mice serum:500 dilutions), 4 DEG C of overnight incubations,
TBST buffer solutions wash 3 times.Secondary antibody is added to be incubated：Be separately added into the anti-mouse IgG antibody of fluorescent marker, anti-rabbit IgG antibody and
Anti-human IgG antibodies (use confining liquid 1:10,000 dilutions), 37 DEG C are protected from light incubation 1h, and TBST buffer solutions wash 3 times.
Sweep film：Use Odyssey infrared laser imaging system scanning imageries.
The results are shown in Figure 3, using the anti-His-tag antibody of mouse as positive control, there is an apparent identification at about 25kDa
Band, using healthy mice serum as negative control, no apparent band, infects the BALB/c mouse of Schistosoma japonicum at 25kDa
The recognizable destination protein of serum, new zealand white rabbit serum and Schistosoma japonicum patients serum, shows recombinant protein
SjSAPLP5 has good immunogenicity and antigenicity.
Embodiment 4 prepares Japanese schistosomiasis SjSAPLP5-ELISA immunodiagnosis kits
(1) key component of kit includes：
The solid phase carrier of envelope antigen：Dilute what embodiment 2 obtained with carbonate coating buffer solution (pH 9.6)
SjSAPLP5 recombinant proteins are coated in 1 μ g/mL in polystyrene reactant hole, 100 holes μ L/.
Enzyme labelled antibody：Goat anti-human immunoglobulin (α, γ and μ-chain specific) antibody of alkali phosphatase enzyme mark
(sigma companies)
Substrate：PNPP (sigma companies)
Washing buffer：PBST
Dilution：5g skimmed milk powers are dissolved in PBST buffer solutions 100mL.
Substrate buffer solution：Diethanol amine 0.1mol/L (9.7ml), MgCl21mmol/L (10mg), concentrated hydrochloric acid tune pH is extremely
9.8, distilled water is settled to 100mL.
Reaction terminating liquid：12g NaOH are dissolved in distilled water, are finally settled to 100mL.
(2) operation sequence and detection method of kit：
Closing：Liquid in hole is dried, adds 200 holes 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 measuring samples：Measuring samples serum and dilution press 1:100 dilution proportions, while setting positive, negative and blank
It compares (only plus Sample dilution), adds each sample, 3 multiple holes of each sample Parallel testing 37 DEG C, to be incubated 1h by 100 holes μ L/.
Washing：Liquid in hole is dried, is washed four times with PBST, 200 holes μ L/ 2min/ times, pat dry for the last time.
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 1h, as above wash, pat dry by 37 DEG C.
Colour developing：Chromogenic enzyme substrate liquid is added, 100 holes μ L/, 37 DEG C are protected from light incubation 30min, and terminate liquid, 25 holes μ L/ are added.
Digital independent and processing：OD is read with microplate reader405nmNumerical value, with 2.1 times of works of the OD mean values of negative control sample
To judge the critical value (cut-off) of yin and yang attribute.
5 SjSAPLP5-ELISA kits of embodiment are to Immunodiagnosis of Schistosomiasis Japonica evaluating characteristics
(1) kit sensibility
Hunan Province's excrement inspection worm's ovum positive Schistosoma japonicum patients serum 50 is collected using Kato- Katz technique (Kato-Katz)
Part, wherein 46 parts of (EPG of low-grade infection person's sample<100), 4 part (400 of grade and moderate infection person's sample>EPG >=100), evaluate embodiment
The sensibility of 4 kit, and with rSP13-ELISA kits, that is, use document (Xu X, Zhang Y, Lin D, et
al.Serodiagnosis of Schistosoma japonicum infection:genome-wide
identification of a protein marker,and assessment of its diagnostic validity
In a field study in China.Lancet Infect Dis, 2014, S1473-3099 (14) 70067-2) it is disclosed
Kit is compared made from SjSP-13 albumen, and ELISA experimental implementations are the same as embodiment 3.
As a result as shown in table 1 and Fig. 4, in 50 parts of Schistosoma japonicum patients serums, 44 parts are examined by rSP13-ELISA kits
Break as the positive, the susceptibility of rSP13-ELISA kit diagnosing Japanese schistosomiasis is 88% (95% confidence interval, 75.0%-
95.0%), which reports close to document (Lancet Infect Dis, 2014, S1473-3099 (14) 70067-2.)
RSP13-ELISA kits susceptibility 90.4% (95% confidence interval, 86.5%-93.5%).
In 50 parts of Schistosoma japonicum patients serums, 49 parts are carried out SjSAPLP5-ELISA kits made from example 4 and diagnose
Susceptibility for the positive, SjSAPLP5-ELISA kits is 96.0% (95% confidence interval, 85.1.0%-99.3%)；
The sensibility of SjSAPLP5-ELISA kits is significantly higher than rSP13-ELISA kits, p<0.05.
The comparative analysis of 1 rSP13-ELISA of table and SjSAPLP5-ELISA kit sensibility
* the sensibility of SjSAPLP5-ELISA kits is significantly higher than rSP13-ELISA kits, p<0.05.
(2) kit specificity
50 parts of Heilongjiang Province's normal human serum sample, 20 parts of Xinjiang region hepatic echinococcosis human serum are collected, hair is revolved in Yunnan Province
18 parts of parasitosis human serum, evaluates false positive rate of the kit in normal population of the embodiment of the present invention 4, and with other parasitisms
The cross reaction situation of parasitosis human serum, and be compared with rSP13-ELISA kits, the same embodiment of ELISA experimental implementations
4。
As a result as shown in table 2 and Fig. 4,50 portions of normal human serums are by rSP13-ELISA and SjSAPLP4-ELISA reagents
Box is diagnosed as feminine gender, and the specificity of two kinds of kits is 100% (95% confidence interval, 91.1%-100%), and the result is close
The rSP13-ELISA kits of document (Lancet Infect Dis, 2014, S1473-3099 (14) 70067-2.) report are special
Different degree 98.9% (95% confidence interval, 95.9%-99.9%)；20 parts of hepatic echinococcosis human serums and 18 parts of trichinosis human serums
Feminine gender, no cross reaction are diagnosed as by two kinds of kits.
The comparative analysis of 2 rSP13-ELISA of table and SjSAPLP4-ELISA kit specificity
Dynamic analysis of the 6 anti-SjSAPLP5 protein antibodies of embodiment in infection animal serum
Before collecting 5 New Zealand White Rabbit schistosoma japonicum infections respectively using ear vein blood collection method, infection (mode of infection phase
Together, infective dose is that every rabbit infects 1000 articles of cercarias) the 1-6 weeks serum afterwards, anti-SjSAPLP5 is detected by ELISA method
Dynamic variation of the protein antibodies in infection animal serum, and with anti-SjSP-13 protein antibodies in infection animal serum
Dynamic variation is compared, and ELISA experimental implementations are the same as embodiment 4.
The results are shown in Figure 5, and 5 New Zealand White Rabbit can be through the invention after infecting Schistosoma japonicum 3 weeks
SjSAPLP5-ELISA kits detect the anti-SjSAPLP5 protein antibodies in serum, and rSP13-ELISA kits are new
Western orchid white rabbit infection Schistosoma japonicum just can detect the anti-SjSP-13 protein antibodies in serum after 4 weeks.The test result shows
SjSAPLP5-ELISA kits have the value of potential Diagnosis of Schistosomiasis early infection.Simultaneously as SjSAPLP5 eggs
Bai Kangyuan can be identified and be generated antibody, therefore SjSAPLP4 eggs by the immune system of host in the early stage of infection by Schistosoma
It is potential anti-schistosomiasis vaccine candidate antigen in vain.
7 Schistosoma japonicum SjSAPLP5 gene expression law-analysings of embodiment
It is female using RNeasy kits (QIAGEN companies) extraction schistosoma japonice ovum, cercaria, liver phase child worm and 42 days
The total serum IgE of male imago, after dnase digestion removes DNA, with SuperScriptTM III reverse transcription reagent box (Invitrogen
Company) synthesis cDNA, SjSAPLP5 genes are detected in Japan using 7300Real-Time PCR (Applied Biosystems)
The transcriptional level of blood fluke different developmental phases.SjSAPLP5 upstream region of gene primers are 5 '-
GATATTCTGTTTACTTTTCGCTGAG-3 ' (SEQ ID NO.5), downstream primer 5 '-
TCAAAAGGTTAATGAAAGTGCATAG-3’(SEQ ID NO.6)；It is adjusted in subunit gene PSMD4 conducts using proteasome
Ginseng, sense primer 5 '-CCTCACCAACAATTTCCACATCT-3 ' (SEQ ID NO.7), downstream primer 5 '-
GATCACTTATAGCCTTGCGAACAT-3’(SEQ ID NO.8)。
Reaction system is as follows：12.5 μ l, cDNA templates of SYBR Green Mix (Agilent companies), 1 μ l, sense 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 of 30sec, 60 DEG C of 1min are recycled 40 times altogether.
Data analysis is carried out using 1.4 softwares of SDS (Applied Biosystems).The results are shown in Figure 6, Japanese blood
Fluke SjSAPLP5 genes are not expressed in the worm's ovum stage, the stage (virgin worm and adult stage) in from the cercaria stage to entrance host
In up-regulated expression, illustrate that the growth and development of SjSAPLP5 gene functions and polypide is closely related, is potential antischistosomal drug
Target spot and vaccine candidate antigen.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, several improvements and modifications can also be made, these improvements and modifications
Also it should be regarded as protection scope of the present invention.
Claims (3)
1. Schistosoma japonicum recombinant protein SjSAPLP5 is preparing Japanese schistosomiasis immunological diagnostic reagent box or diagnostic reagent
In application, the amino acid sequence of the Schistosoma japonicum recombinant protein SjSAPLP5 is as shown in SEQ ID NO.2.
2. Schistosoma japonicum recombinant protein SjSAPLP5 is being prepared using it as the Schistosomicides of latent effect target spot
In application, the amino acid sequence of the Schistosoma japonicum recombinant protein SjSAPLP5 is as shown in SEQ ID NO.2.
3. applications of the Schistosoma japonicum recombinant protein SjSAPLP5 in preparing anti schistosoma vaccine, the Schistosoma japonicum
The amino acid sequence of recombinant protein SjSAPLP5 is as shown in SEQ ID NO.2.
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| CN1477202A (en) * | 2003-03-11 | 2004-02-25 | 中国农业科学院上海家畜寄生虫病研究 | Schistosomiasis japonica (Chinese Continental strain) MFS4 gene clone, expression amd DNA vacine |
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