CN105384803A - Schistosoma japonicum katsurada recombinant protein SjSAPLP4 as well as encoding gene and application thereof - Google Patents
Schistosoma japonicum katsurada recombinant protein SjSAPLP4 as well as encoding gene and application thereof Download PDFInfo
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Abstract
The invention provides a schistosoma japonicum katsurada recombinant protein SjSAPLP4 as well as encoding gene and application thereof. The protein has an amino acid sequence shown as SEQ ID NO.2 or an amino acid sequence having a same function, formed by replacing, omitting and/or adding one or more amino acid residues for the amino acid sequence shown as SEQ ID No.2. The invention also provides a gene sequence for encoding the protein. The recombinant protein SjSAPLP4 is good in immunogenicity, can be used as an excellent diagnostic antigen, can be used for preparing a schistosoma japonicum katsurada diagnosis kit having high sensitivity and high specificity, also can be used for preparing an anti-schistosome vaccine and can be used as a potential drug acting target spot to screen the drug for treating the schistosoma japonicum katsurada.
Description
Technical field
The present invention relates to biological technical field, specifically, relate to a kind of Schistosoma japonicum recombinant protein SjSAPLP4 and encoding gene thereof and application.
Background technology
Schistosomicide is a kind of infectivity parasitic disease of serious threat human health, and Major Epidemic is in 76 countries and regions of Asia, Africa and Latin America, and the whole world has people more than 200,000,000 to be infected, and separately has nearly 800,000,000 people to infect by it and threatens.What China was popular is schistosomiasis japanica, through the unremitting effort of decades, China's schistosomiasis control achieves remarkable achievement, and schistosomicide is under control in Prevalent district, but the target realizing final elimination schistosomicide still shoulders heavy responsibilities.
Diagnosis is the key link in prevention and cure of snail fever field.To the morning of Patients with Schistosomiasis Japonica, accurate diagnostic techniques not only finds that early treatment has important clinical significance, also can be Endemic Areas of Schistosomiasis Japonica grade and provide judging criterion, for assessing epidemic status and examining control effect to provide requisite information and scientific basis.Lacking efficient accurate diagnostic techniques is the major reason that schistosomicide cannot thoroughly be eliminated.Along with the increasing of China's schistosomicide preventing and controlling dynamics, schistosomicide is tending towards a kind of fashion trend of minuent infection in China.If the Patients with Schistosomiasis Japonica of these low-grade infections can not be made a definite diagnosis in time and treat, worm's ovum contained in its movement is propagated causing the continued popularity of schistosomicide.Therefore, research and develop and new there is high sensitivity and specific schistosomiasis diagnosis method is imperative.
At present, the diagnosis of schistosomicide depends on parasite morphologic detection method more, the Kato-Katz technique (Kato-Katz) that the such as World Health Organization (WHO) is recommended.This method, mainly through checking that the japonice ovum in Feces of Patients or urine diagnoses the illness, not only wastes time and energy, and the susceptibility diagnosed slight schistosomicide is lower, is not suitable for large-scale schistosomicide field monitoring.Compared to traditional Morphologic Diagnosis method, enzyme linked immunosorbent assay (Enzyme-linkedimmunesorbentassay, ELISA) diagnostic method has simple and efficient to handle and higher susceptibility, and can be used for extensive field monitoring.At present, the most frequently used antigen for schistosomicide immunodiagnosis is the Adult Antigens component (Adultwormantigen extracting schistosomicide polypide, and egg antigen component (Solubleeggantigen AWA), SEA), because these two kinds are slightly carried the complicated component (being made up of thousands of kinds of schistosomicide albumen) of antigen, and there is more serious cross reaction with other parasitic infection serum, the specificity of schistosomiasis diagnosis is not high, the unsuitable stdn of reagent of production.
The schistosomicide recombinant antigen molecule relying on genetic engineering technique to produce has that composition is simple and clear, cross reaction is few, can the advantage such as large-scale mass production, be considered to the target antigen molecule of desirable schistosomiasis diagnosis and control.2014, China scientist reports Immunodiagnosis of Schistosomiasis Japonica mark molecule (SjSP-13), and developed as rSP13-ELISA test kit, its diagnostic sensitivity improves 6 times than traditional form method, this achievement in research in the world top academic journal " lancet transmissible disease " magazine deliver online with treatise form.But the susceptibility (90%) of this diagnostic kit remains the space promoted further.
The present invention utilizes bioinformatics method to find to there is a Saposin sample albumen (SchistosomajaponicumSaposin-likeprotein in its genome to the forecast analysis of Schistosoma japonicum full-length genome encoding gene, SjSAPLP) family, this gene family has 15SjSAPLP genomic constitution, and SjSP-13 is wherein a member.By sequence alignment analysis, the present invention finds that the sequence homology between SjSAPLP family member is extremely low, the amino acid sequence homology wherein between SjSAPLP4 and the SjSP-13 reported is lower than 30%.Before making the present invention, also do not occur relating to Schistosoma japonicum SjSAPLP4 recombinant antigen protein of the present invention and the open report for schistosomiasis japanica diagnosis thereof.The present invention is recombinant expressed in intestinal bacteria through gene clone by SjSAPLP4 and SjSP-13, ELISA tests the diagnosis that comparative analysis two recombinant proteins are used for schistosomiasis japanica and has hypersensitivity and specificity, and to finding, susceptibility is higher, specificity better schistosomicide immunodiagnostic antigen.
Summary of the invention
One object of the present invention is to provide the Schistosoma japonicum recombinant protein that a Species sensitivity is high, specificity is good.
Another object of the present invention is to provide the application of this recombinant protein in Schistosoma japonicum serodiagnosis, vaccine development and medicine screening.
Technical scheme of the present invention is: first utilize Protocols in Molecular Biology carry out pcr amplification to Schistosoma japonicum SjSAPLP4 gene and be cloned into recombinant plasmid pET-28a (+)-SjSAPLP4 that expression plasmid carrier pET-28a (+) is built into Schistosoma japonicum SjSAPLP4 gene prokaryotic, pass through transformation and selection, after plasmid order-checking qualification confirms, be converted in colibacillus host cell and carry out recombinant protein abduction delivering; Inclusion body protein, through sex change and affinity chromatography purifying, finally obtains the recombinant protein SjSAPLP4 of purifying, completes the body outer clone Expression and purification of SjSAPLP4 gene.Utilize the SjSAPLP4 recombinant protein of Westernblotting technical identification purifying can the laboratory animal serum of infected Schistosoma japonicum and the identification of schistosomicide human serum.And then with the SjSAPLP4 recombinant protein of purifying for diagnostic antigen carries out the preparation of schistosomiasis diagnosis reagent and test kit, its using value in schistosomicide immunodiagnosis by elisa technique assay also analyzes the Changing Pattern of anti-SjSAPLP4 antibody in infection animal serum.Finally utilize fluorescence real-time quantitative PCR technical Analysis SjSAPLP4 gene in the genetic expression rule of Schistosoma japonicum different developmental phases.
Thus, the invention provides a kind of Schistosoma japonicum recombinant protein SjSAPLP4, its aminoacid sequence is:
A) aminoacid sequence shown in SEQIDNo.2; Or
B) aminoacid sequence with same function that the aminoacid sequence shown in SEQIDNo.2 is formed through replacing, lacking and/or add one or several amino-acid residue.
Should be appreciated that those skilled in the art according to the aminoacid sequence of Protein S jSAPLP4 disclosed by the invention (SEQIDNo.2), can replace, lack and/or increase one or several amino acid, obtain the mutant nucleotide sequence of described albumen.Therefore, Schistosoma japonicum recombinant protein SjSAPLP4 of the present invention also comprises aminoacid sequence shown in SEQIDNo.2 and is substituted, lacks or increases one or several amino acid, has to derive by SjSAPLP4 the protein obtained with recombinant protein SjSAPLP4 with isoreactivity.
Further, the invention provides the gene of the above-mentioned Schistosoma japonicum recombinant protein SjSAPLP4 that encodes, be following a) or b):
A) its nucleotide sequence is as shown in SEQ ID No .1; Or
B) be substituted one or several Nucleotide by nucleotide sequence shown in SEQIDNo.1, obtain the nucleotide sequence of coding SjSAPLP4.
Further, the invention provides a kind of recombinant expression vector, it is containing the expression cassette of said gene or clone or recombinant bacterium.
Present invention also offers the Auele Specific Primer pair for the above-mentioned coding Schistosoma japonicum recombinant protein SjSAPLP4 gene that increases, its nucleotide sequence is respectively as shown in SEQIDNO.3,4.
In one embodiment of the invention, provide the amplification method of Schistosoma japonicum SjSAPLP4 gene ORF, comprising: with Schistosoma japonicum 42 days female male imago cDNA for template, carry out PCR reaction, amplification SjSAPLP4 gene ORF fragment;
The primer sequence that described PCR reaction adopts is:
PF:5 '-
gGATCCaACCACACTGAGTTGGACAT-3 ' (as shown in SEQIDNO.3, the protectiveness base of what italicized item represented is upstream primer, underscore part is the BamHI restriction enzyme site of upstream primer);
PR:5 '-
cTCGAGtGGGCATAACTGTATTGTCT-3 ' (as shown in SEQIDNO.4, the protectiveness base of what italicized item represented is downstream primer, underscore part is the XhoI restriction enzyme site of downstream primer);
System and the PCR response procedures of described PCR reaction are respectively:
Reaction system: high-fidelity DNA polymerase Mix12.5 μ l, cDNA template 1 μ l, upstream primer 0.5 μ l, downstream primer 0.5 μ l, ddH
2o10.5 μ l, cumulative volume 25 μ l.
Pcr amplification program: 98 DEG C of 1min, 98 DEG C of sex change 10sec, 55 DEG C of annealing 30sec, 72 DEG C extend 1min, totally 35 circulations; Last 72 DEG C extend 10min.
Present invention also offers a kind of method preparing Schistosoma japonicum recombinant protein SjSAPLP4, comprise the following steps:
(1) recombinant expression vector containing Schistosoma japonicum recombinant protein SjSAPLP4 encoding gene is transformed into competent cell, obtained Schistosoma japonicum recombinant protein SjSAPLP4 expresses engineering bacteria;
(2) Schistosoma japonicum recombinant protein SjSAPLP4 is expressed engineering bacteria through IPTG abduction delivering; Collection inclusion body precipitates, and fully dissolves inclusion body, centrifugal, collects supernatant liquor, and after the correct expression product of qualification is crossed nickel ion chelating glue column purification, wash-out obtains the Schistosoma japonicum recombinant protein SjSAPLP4 of purifying.
Detection kit containing Schistosoma japonicum recombinant protein SjSAPLP4 of the present invention or diagnostic reagent belong to protection scope of the present invention.
Biological products containing Schistosoma japonicum recombinant protein SjSAPLP4 of the present invention belong to protection scope of the present invention.
Preferably, described biological products are anti schistosoma vaccine.
The present invention is studied Schistosoma japonicum SjSAPLP4 genetic expression rule, find not express at worm's ovum stage SjSAPLP4 gene, from the cercaria stage to entering in host the stage (virgin worm and adult stage) in up-regulated expression, illustrating that growing of SjSAPLP4 gene function and polypide is closely related, is potential antischistosomal drug target spot and vaccine candidate antigen.With Schistosoma japonicum recombinant protein SjSAPLP4 of the present invention for the medicine of latent effect target spot also belongs to protection scope of the present invention.
The invention provides above-mentioned Schistosoma japonicum recombinant protein SjSAPLP4 or its encoding gene or the recombinant expression vector containing this gene and prepare the application in Schistosoma japonicum serological diagnostic kit or diagnostic reagent.
The invention provides above-mentioned Schistosoma japonicum recombinant protein SjSAPLP4 or its encoding gene or the recombinant expression vector containing this gene and prepare the application in anti schistosoma vaccine.
The invention provides above-mentioned Schistosoma japonicum recombinant protein SjSAPLP4 or its encoding gene or the recombinant expression vector containing this gene application in screening treatment Schistosoma japonicum medicine.
The Schistosoma japonicum recombinant protein SjSAPLP4 that the present invention obtains has good immunogenicity and antigenicity, and can identify the BALB/c mouse serum of infection Schistosoma japonicum, new zealand white rabbit serum and Schistosoma japonicum patients serum, specificity is 100%; With SjSAPLP4 Protein Detection Schistosoma japonicum patients serum, still can detect the Schistosomiasis patients (EPG < 100) of low-grade infection, detection sensitivity reaches 98%; The dynamic variation of anti-SjSAPLP4 protein antibodies in infection animal serum is detected by ELISA method, just the anti-SjSAPLP4 protein antibodies in serum detected by SjSAPLP4-ELISA test kit after 3 weeks, can be used for the diagnosis of schistosomiasis japanica early infection, the present invention finds that growing of SjSAPLP4 gene function and polypide is closely related, for genetic method treatment schistosomiasis japanica provides foundation from now on.
Accompanying drawing explanation
Fig. 1 is the pcr amplification result schematic diagram of Schistosoma japonicum SjSAPLP4 gene order in embodiment 1, and wherein M is DNA molecular amount standard, and 1 is the pcr amplification product of SjSAPLP4 gene.
Fig. 2 is the SDS-PAGE analytical results schematic diagram of SjSAPLP4 recombinant protein in embodiment 2.Swimming lane M is Protein Marker, and swimming lane 1 is not for induce full bacterium to contrast, and swimming lane 2 is induction latter 4 hours full bacterium, and swimming lane 3 is supernatant after cellular lysate after induction, and 4 is the rear cellular lysate postprecipitation of induction, and 5 is the SjSAPLP4 recombinant protein of purifying.
Fig. 3 is the Westernblotting analytical results schematic diagram of SjSAPLP4 recombinant protein antigen and different schistosomicide animal serum and schistosomicide human serum in embodiment 3.In figure, M is Protein Marker, 1, and be little mouse-anti His tag antibody positive control, 2 to be Normal Mouse Serum negative control, 3 be that to infect the Schistosoma japonicum mice serum of 42 days, 4 be that to infect the Schistosoma japonicum rabbit anteserum of 42 days, 5 be Schistosoma japonicum patients serum.
Fig. 4 is the result schematic diagram that in embodiment 5, rSP13-ELISA test kit compares for schistosomiasis japanica diagnostic evaluation with SjSAPLP4-ELISA test kit.A figure is the result of rSP13-ELISA test kit, and B figure is SjSAPLP4-ELISA kit results.
Fig. 5 is that in embodiment 6, anti-SjSAPLP4 protein antibodies and anti-SjSP-13 protein antibodies are infecting the kinetics comparative analysis result schematic diagram in new zealand white rabbit serum.A figure is that anti-SjSP-13 protein antibodies is infecting the dynamic analysis result in new zealand white rabbit serum, and B figure is that anti-SjSAPLP5 protein antibodies is infecting the dynamic analysis result in new zealand white rabbit serum.
Fig. 6 be in embodiment 7 Schistosoma japonicum SjSAPLP4 gene worm's ovum, cercaria, the liver phase virgin worm and female male imago in fluorescence real-time quantitative PCR analytical results schematic diagram.E is worm's ovum, C is cercaria, S is virgin worm, M is male insect, F is female insect.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.If do not specialize, reagent used in embodiment is commercially available.
The clone of embodiment 1 Schistosoma japonicum SjSAPLP4 gene
Sequence (as shown in SEQIDNO.1 sequence) according to SjSAPLP4 gene (GenBankFN315317.1) designs primer and introduces restriction enzyme site, as follows:
PF:5 '-
gGATCCaACCACACTGAGTTGGACAT-3 ' (as shown in SEQIDNO.3, the protectiveness base of what italicized item represented is upstream primer, underscore part is the BamHI restriction enzyme site of upstream primer);
PR:5 '-
cTCGAGtGGGCATAACTGTATTGTCT-3 ' (as shown in SEQIDNO.4, the protectiveness base of what italicized item represented is downstream primer, underscore part is the XhoI restriction enzyme site of downstream primer);
Auele Specific Primer is synthesized by Jin Weizhi bio tech ltd, Suzhou.
With Schistosoma japonicum 42 days female male imago cDNA for template, carry out PCR reaction, amplification SjSAPLP4 gene ORF fragment, reaction system is as follows: high-fidelity DNA polymerase Mix12.5 μ l, cDNA template 1 μ l, upstream primer 0.5 μ l, downstream primer 0.5 μ l, ddH
2o10.5 μ l, cumulative volume 25 μ l.Pcr amplification program: 98 DEG C of 1min, 98 DEG C of sex change 10sec, 55 DEG C of annealing 30sec, 72 DEG C extend 1min, totally 35 circulations; Last 72 DEG C extend 10min.
With 1.2% agarose gel electrophoresis detect PCR primer, observe whether there is object band, result as shown in Figure 1, M:DNA molecular weight standard, 1:SjSAPLP4.Agarose gel electrophoresis result shows, and has a band clearly about 531bp place, and the size after removing signal peptide sequence with expection object fragment conforms to, and shows the ORF fragment successfully amplifying SjSAPLP4 gene from cDNA.
Use AxyPrepDNA gel to reclaim test kit (AXYGEN company) purifying and reclaim PCR primer.The PCR object fragment of recovery and pET28a empty carrier plasmid are spent the night respectively at 37 DEG C of water-bath double digestions, endonuclease reaction system is as follows: DNA fragmentation or pET28a expression vector (are dissolved in ddH
2o) 40 μ l, BamHI restriction endonuclease 2.5 μ l, XhoI restriction endonuclease 2.5 μ l, enzyme cuts buffer5 μ l, cumulative volume 50 μ l.
PCR primer after being cut by enzyme and pET28a expression vector carry out 1.2% agarose gel electrophoresis qualification and glue reclaims purifying.Use T4DNA ligase enzyme to be connected the expression vector fragment after recovery and external source goal gene fragment according to the ratio of 1:7 (mol ratio), reaction conditions is 16 DEG C, connects 6h.Product conversion will be connected to intestinal bacteria Trans1-T1 competent cell (Beijing Quanshijin Biotechnology Co., Ltd), competent cell after transforming is applied to LB culture medium flat plate (containing 50 μ g/ml kantlex), 37 DEG C of overnight incubation.Picking list bacterium colony carries out PCR qualification, PCR is accredited as positive clone and send Jin Weizhi bio tech ltd, Suzhou to carry out DNA sequencing to confirm that whether sequence is correct.Sequencing analysis result shows that the exogenous genetic fragment sequence inserted is correct, and recombinant plasmid pET28a (+)-SjSAPLP4 successfully constructs.
The expression and purification of embodiment 2 Schistosoma japonicum SjSAPLP4 recombinant protein
By pET28a (+)-SjSAPLP4 recombinant plasmid transformed correct for order-checking to expressing competent cell Transetta (DE3) (Beijing Quanshijin Biotechnology Co., Ltd); LB liquid nutrient medium (containing 50 μ g/ml kantlex) 15mL is inoculated into by being accredited as positive clone through PCR, 37 DEG C of overnight incubation, within second day, get 10mL substratum to transfer into LB substratum (containing 50 μ g/ml kantlex) 1L, continue to be cultured to OD
600nmvalue is 0.8, then to add final concentration be that the IPTG of 1mM carries out abduction delivering 4 hours, collected by centrifugation thalline, and-80 DEG C frozen for subsequent use.
Front and after inducing the thalline of the induction that takes a morsel is resuspended in PBS damping fluid, adds SDS-PAGE sample-loading buffer, boils 5min and make protein denaturation after mixing in boiling water bath.The each 10 μ l of sample before adding induction in each loading hole respectively and after induction, carry out SDS-PAGE analysis (concentrated glue is 5%, and separation gel is 12%).As shown in Figure 2, pET28a (+)-SjSAPLP4 recombinant plasmid transformed is expressed competent cell, thalline before comparatively inducing after IPTG abduction delivering has occurred that molecular weight is about the obvious band of expression of 23kDa, and its molecular size range conforms to the theoretical Mr of object recombinant protein.
Be resuspended in 40mL bacterial lysate by the thalline after induction, ultrasonication, 4 DEG C, the centrifugal 30min of 12,000rpm, collects inclusion body precipitation and supernatant liquor respectively.Inclusion body precipitation and supernatant liquor are carried out SDS-PAGE analysis, the solvability of qualification recombinant protein.Result shows, and recombinant protein is mainly present in inclusion body precipitation.Inclusion body is resuspended in the PBS solution supersound washing 5min containing 1%Triton-X100, the centrifugal 15min of 12,000rpm collects inclusion body precipitation; Inclusion body is resuspended in 8M urea, 4 DEG C rotate mixing overnight, fully dissolve inclusion body, and the centrifugal 30min of 12,000rpm collects supernatant liquor; Supernatant liquor is crossed nickel ion chelating glue post (QIAGEN company) to make, with 6 histidine-tagged restructuring SjSAPLP4 protein binding on glue post, to rinse foreign protein with 50mM imidazoles, then use 250mM imidazoles wash-out recombinant protein, collect elutriant; Purifying gained recombinant protein is through SDS-PAGE analyzing and testing purity of protein.Electrophoresis result as shown in Figure 2, shows after nickel ion chelating glue column purification, obtain the higher restructuring SjSAPLP4 albumen of purity.Use BCA quantification of protein test kit (ThermoFisherScientific company) to measure the concentration of recombinant protein, operate to specifications.The recombinant protein concentration of purifying gained is 2.1mg/mL after measured.
The detection of antigenicity of embodiment 3 Schistosoma japonicum SjSAPLP4 recombinant protein
SDS-PAGE electrophoresis: the SjSAPLP4 recombinant protein 100ng loading that Example 2 obtains, deposition condition is: 100V20min, 120V1h.
Transferring film: adopt wet robin by the protein delivery in PAGE glue to pvdf membrane, electricity turns condition and is: ice bath, 100V1h.
Close: pvdf membrane 5% skim-milk room temperature is closed 2h, TBST buffer solution 3 times.
Add primary antibodie to hatch: add respectively and infect Schistosoma japonicum 42 days BALB/c mouse serum, infection Schistosoma japonicum 42 days new zealand white rabbit serum and Schistosoma japonicum patients serums, with little mouse-anti His-Tag antibody (Ai Bimate biological medicine company limited) be positive control, with healthy mice serum for negative control (with confining liquid 1:500 dilution), 4 DEG C of overnight incubation, TBST buffer solution 3 times.Add two anti-to hatch: add fluorescently-labeled anti-mouse IgG antibody, anti-rabbit IgG antibody and anti-human IgG antibodies's (use confining liquid 1:10,000 dilutes) respectively, 37 DEG C of lucifuges hatch 1h, TBST buffer solution 3 times.
Sweep film: use the imaging of Odyssey infrared laser imaging system scan.
Result as shown in Figure 3, with little mouse-anti His-tag antibody for positive control, one is had significantly to identify band at about 23kDa place, with healthy mice serum for negative control, 23kDa place does not have obvious band, infect the BALB/c mouse serum of Schistosoma japonicum, new zealand white rabbit serum and the equal identifiable design target protein of Schistosoma japonicum patients serum, show that recombinant protein SjSAPLP4 has good immunogenicity and antigenicity.
Embodiment 4 prepares schistosomiasis japanica SjSAPLP4-ELISA immunodiagnosis kit
(1) main ingredient of test kit comprises:
The solid phase carrier of envelope antigen: be buffered with carbonate bag SjSAPLP4 recombinant protein to the 1 μ g/mL that liquid (pH9.6) dilutes embodiment 2 acquisition, be coated in polystyrene reactant hole, 100 μ L/ holes.
Enzyme labelled antibody: goat anti-human immunoglobulin (alpha, gamma and the μ-chainspecific) antibody (sigma company) of alkali phosphatase enzyme mark
Substrate: pNPP (sigma company)
Lavation buffer solution: PBST
Diluent: 5g skim-milk is dissolved in PBST damping fluid 100mL.
Substrate buffer solution: diethanolamine 0.1mol/L (9.7ml), MgCl
21mmol/L (10mg), concentrated hydrochloric acid adjusts pH to 9.8, and distilled water is settled to 100mL.
Reaction terminating liquid: 12gNaOH is dissolved in distilled water, is finally settled to 100mL.
(2) schedule of operation of test kit and detection method:
Close: dry liquid in hole, add diluent 200 μ L/ hole, 37 DEG C, close 2h, wash three times with PBST, 200 μ L/ holes, 2min/ time, pat dry for the last time.
Add measuring samples: measuring samples serum and diluent, by 1:100 dilution proportion, establish the positive, feminine gender and blank (only adding Sample dilution) simultaneously, add each sample by 100 μ L/ holes, the multiple hole of each sample Parallel testing 3, hatches 1h by 37 DEG C.
Washing: dry liquid in hole, wash four times with PBST, 200 μ L/ holes, 2min/ time, pat dry for the last time.
Add enzyme labelled antibody: goat anti-human immunoglobulin (alpha, gamma and the μ-chainspecific) antibody adding alkali phosphatase enzyme mark, 100 μ L/ holes, 37 DEG C, hatch 1h, as above wash, pat dry.
Colour developing: add chromogenic enzyme substrate liquid, 100 μ L/ holes, 37 DEG C of lucifuges hatch 30min, add stop buffer, 25 μ L/ holes.
Digital independent and process: read OD by microplate reader
405nmnumerical value, using 2.1 times of the OD average of negative control sample as the threshold value (cut-off) judging yin and yang attribute.
Embodiment 5SjSAPLP4-ELISA test kit is to Immunodiagnosis of Schistosomiasis Japonica evaluating characteristics
(1) test kit susceptibility
Kato-Katz technique (Kato-Katz) is utilized to collect the positive Schistosoma japonicum patients serum 50 parts of Hunan Province's excrement inspection worm's ovum, wherein 46 parts, low-grade infection person's sample (EPG<100), 4 parts, grade and moderate infection person's sample (400>EPG >=100), the susceptibility of the test kit of Evaluation operation example 4, and with rSP13-ELISA test kit, namely document (XuX is adopted, ZhangY, LinD, etal.SerodiagnosisofSchistosomajaponicuminfection:genome-wideidentificationofaproteinmarker, andassessmentofitsdiagnosticvalidityinafieldstudyinChina .LancetInfectDis, 2014, S1473-3099 (14) 70067-2) the obtained test kit of disclosed SjSP-13 albumen compares, ELISA experimental implementation is with embodiment 3.
Result is as shown in table 1 and Fig. 4, in 50 parts of Schistosoma japonicum patients serums, 44 parts are diagnosed as the positive by rSP13-ELISA test kit, the susceptibility of rSP13-ELISA test kit diagnosing Japanese schistosomiasis is 88% (95% fiducial interval, 75.0%-95.0%), this result is close to document (LancetInfectDis, 2014, S1473-3099 (14) 70067-2.) the rSP13-ELISA test kit susceptibility 90.4% (95% fiducial interval, 86.5%-93.5%) reported.
In 50 parts of Schistosoma japonicum patients serums, 49 parts of SjSAPLP4-ELISA test kits being implemented example 4 obtained are diagnosed as the positive, and the susceptibility of SjSAPLP4-ELISA test kit is 98.0% (95% fiducial interval, 88.0%-99.9%); The susceptibility of SjSAPLP4-ELISA test kit is significantly higher than rSP13-ELISA test kit, p<0.05.
The comparative analysis of table 1rSP13-ELISA and SjSAPLP4-ELISA test kit susceptibility
* the susceptibility of SjSAPLP4-ELISA test kit is significantly higher than rSP13-ELISA test kit, p<0.05.
(2) test kit specificity
Collect 50 parts, Heilongjiang Province's normal human serum sample, Xinjiang region hepatic echinococcosis human serum 20 parts, Trichinosis In Yunnan Province human serum 18 parts, evaluate the false positive rate of test kit in normal population of the embodiment of the present invention 4, and with the cross reaction situation of other parasitosis human serum, and compare with rSP13-ELISA test kit, ELISA experimental implementation is with embodiment 4.
Result is as shown in table 2 and Fig. 4,50 portions of normal human serums are all diagnosed as feminine gender by rSP13-ELISA and SjSAPLP4-ELISA test kit, the specific degree of two kinds of test kits is 100% (95% fiducial interval, 91.1%-100%), this result is close to document (LancetInfectDis, 2014, S1473-3099 (14) 70067-2.) the rSP13-ELISA test kit specific degree 98.9% (95% fiducial interval, 95.9%-99.9%) reported; 20 parts of hepatic echinococcosis human serums and 18 parts of trichonematosis human serums are all diagnosed as feminine gender, no cross reaction by two kinds of test kits.
The specific comparative analysis of table 2rSP13-ELISA and SjSAPLP4-ELISA test kit
The dynamic analysis of the anti-SjSAPLP4 protein antibodies of embodiment 6 in infection animal serum
Before utilizing ear vein blood-collecting method to collect 5 New Zealand white rabbit schistosoma japonicum infections respectively, (mode of infection is identical in infection, infective dose is that every rabbit infects 1000 articles of cercarias) serum in 1-6 week afterwards, the dynamic variation of anti-SjSAPLP4 protein antibodies in infection animal serum is detected by ELISA method, and compare with the dynamic variation of anti-SjSP-13 protein antibodies in infection animal serum, ELISA experimental implementation is with embodiment 4.
Result as shown in Figure 5,5 New Zealand white rabbit just detect anti-SjSAPLP4 protein antibodies in serum infecting Schistosoma japonicum by SjSAPLP4-ELISA test kit of the present invention after 3 weeks, and rSP13-ELISA test kit infects Schistosoma japonicum in New Zealand white rabbit and anti-SjSP-13 protein antibodies in serum just can be detected after 4 weeks.This test-results shows that SjSAPLP4-ELISA test kit has the value of potential Diagnosis of Schistosomiasis early infection.Meanwhile, because SjSAPLP4 proteantigen just can be produced antibody by the immune system recognition of host at the commitment of schistosomicide, therefore SjSAPLP4 albumen is potential anti-schistosomiasis vaccine candidate antigen.
Embodiment 7 Schistosoma japonicum SjSAPLP4 genetic expression law-analysing
Utilize RNeasy test kit (QIAGEN company) extract schistosoma japonice ovum, cercaria, the liver phase virgin worm and the total serum IgE of 42 days female male imagos, remove after DNA through dnase digestion, synthesize cDNA with SuperScriptTMIII Reverse Transcription box (Invitrogen company), utilize 7300Real-TimePCR (AppliedBiosystems) to detect the transcriptional level of SjSAPLP4 gene in Schistosoma japonicum different developmental phases.SjSAPLP4 upstream region of gene primer is 5 '-CGATAAAGTGTGCTCACTAACTGG-3 ' (SEQIDNO.5), and downstream primer is 5 '-GCTGAATTTGATGACATTTGGGT-3 ' (SEQIDNO.6); Proteasome is adopted to regulate subunit gene PSMD4 as internal reference, its upstream primer is 5 '-CCTCACCAACAATTTCCACATCT-3 ' (SEQIDNO.7), and downstream primer is 5 '-GATCACTTATAGCCTTGCGAACAT-3 ' (SEQIDNO.8).
Reaction system is as follows: SYBRGreenMix (Agilent company) 12.5 μ l, cDNA template 1 μ l, upstream primer 0.5 μ l, downstream primer 0.5 μ l, ddH
2o10.5 μ l, cumulative volume 25 μ l.
Pcr amplification program: 95 DEG C, 10min; 95 DEG C, 30sec, 60 DEG C, 1min (circulating 40 times).
SDS1.4 software (AppliedBiosystems) is utilized to carry out data analysis.Result as shown in Figure 6, Schistosoma japonicum SjSAPLP4 gene was not expressed in the worm's ovum stage, from the cercaria stage to entering in host the stage (virgin worm and adult stage) in up-regulated expression, illustrate that growing of SjSAPLP4 gene function and polypide is closely related, disturb the expression of this gene in polypide or suppress its biologic activity may affect growing of polypide and even kill and wound polypide.Therefore, this gene is potential antischistosomal drug target spot.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a Schistosoma japonicum recombinant protein SjSAPLP4, its aminoacid sequence is:
A) aminoacid sequence shown in SEQIDNo.2; Or
B) aminoacid sequence with same function that the aminoacid sequence shown in SEQIDNo.2 is formed through replacing, lacking and/or add one or several amino-acid residue.
2. to encode the gene of Schistosoma japonicum recombinant protein SjSAPLP4 described in claim 1, be following a) or b):
A) its nucleotide sequence is as shown in SEQ ID No .1; Or
B) be substituted one or several Nucleotide by nucleotide sequence shown in SEQIDNo.1, obtain the nucleotide sequence of coding SjSAPLP4.
3. a recombinant expression vector, it is the expression cassette containing gene described in claim 2 or clone or recombinant bacterium.
4. for the Auele Specific Primer pair of the gene described in claim 2 that increases, it is characterized in that, its nucleotide sequence is respectively as shown in SEQIDNO.3,4.
5. the detection kit containing Schistosoma japonicum recombinant protein SjSAPLP4 described in claim 1 or diagnostic reagent.
6. the biological products containing Schistosoma japonicum recombinant protein SjSAPLP4 described in claim 1.
7. with the medicine that Schistosoma japonicum recombinant protein SjSAPLP4 described in claim 1 is latent effect target spot.
8. the application of recombinant expression vector in preparation Schistosoma japonicum serological diagnostic kit or diagnostic reagent described in Schistosoma japonicum recombinant protein SjSAPLP4 described in claim 1 or gene according to claim 2 or claim 3.
9. recombinant expression vector described in Schistosoma japonicum recombinant protein SjSAPLP4 described in claim 1 or gene according to claim 2 or claim 3 is preparing the application in anti schistosoma vaccine.
10. the application of recombinant expression vector in screening treatment Schistosoma japonicum medicine described in Schistosoma japonicum recombinant protein SjSAPLP4 described in claim 1 or gene according to claim 2 or claim 3.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107778363A (en) * | 2016-08-25 | 2018-03-09 | 中国农业科学院上海兽医研究所 | Schistosoma japonicum katsurada rSjMRP1 and its application |
| CN110305871A (en) * | 2019-06-24 | 2019-10-08 | 中国医学科学院病原生物学研究所 | Highly expressed gene and its coding albumen and application in Schistosomula Japonicum |
| CN110330556A (en) * | 2019-06-24 | 2019-10-15 | 中国医学科学院病原生物学研究所 | Highly expressed gene and its coding albumen and application in Schistosomula Japonicum |
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| CN115043922A (en) * | 2022-03-30 | 2022-09-13 | 中国医学科学院病原生物学研究所 | Schistosoma japonicum antigen protein rSjScP57 and application thereof |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1425918A (en) * | 2003-01-14 | 2003-06-25 | 江苏省血吸虫病防治研究所 | Quick test paper strip diagnostic reagent kits for bilharziasis and its preparing method and use |
| 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 |
| CN1618965A (en) * | 2003-11-18 | 2005-05-25 | 陈晓光 | New type Japanese blood fluxes dominant pesition antigen molecule |
| CN1629188A (en) * | 2003-12-15 | 2005-06-22 | 国家人类基因组南方研究中心 | Specific antigen of Japanese blood fluke and its use |
-
2015
- 2015-11-23 CN CN201510818639.5A patent/CN105384803B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1425918A (en) * | 2003-01-14 | 2003-06-25 | 江苏省血吸虫病防治研究所 | Quick test paper strip diagnostic reagent kits for bilharziasis and its preparing method and use |
| 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 |
| CN1618965A (en) * | 2003-11-18 | 2005-05-25 | 陈晓光 | New type Japanese blood fluxes dominant pesition antigen molecule |
| CN1629188A (en) * | 2003-12-15 | 2005-06-22 | 国家人类基因组南方研究中心 | Specific antigen of Japanese blood fluke and its use |
Non-Patent Citations (2)
| Title |
|---|
| LIU,F.等: "Accession No.: FN315317.1，Schistosoma japonicum isolate Anhui full length mRNA clone SJFCA3648.003|FSA001-P00015-I16, complete sequence", 《GENBANK》 * |
| THE SCHISTOSOMA JAPONICUM GENOME SEQUENCING AND FUNCTIONAL ANALY: "The Schistosoma japonicum genome reveals features of host-parasite interplay", 《NATURE》 * |
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