CN102827260A - Recombinant antigen protein for detecting yellow fever virus antibody, kit and application of recombinant antigen protein - Google Patents
Recombinant antigen protein for detecting yellow fever virus antibody, kit and application of recombinant antigen protein Download PDFInfo
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Abstract
The invention discloses a recombinant antigen protein for detecting a yellow fever virus antibody. The sequence of the recombinant protein is shown as SEQ ID NO.1; the sequence of an encoding gene of the recombinant antigen protein is shown as SEQ ID NO.2, and comprises an extension segment for encoding a flexible arm of the recombinant antigen protein; and the flexible arm can be used for eliminating masking of a peptide epitope by a fusion protein of an expression carrier, so that the expressed recombinant antigen protein is folded correctly. The invention further discloses a preparation method of the recombinant antigen protein. Moreover, a yellow fever virus kit disclosed by the invention comprises an antigen detection plate coated with the recombinant antigen protein and an ELISA (Enzyme-Linked Immunosorbent Assay) reaction liquid. The recombinant antigen protein provided by the invention has the advantages of high specificity and high affinity, does not undergo any serological cross reaction with other congenial arthropod-borne viruses, has ultrahigh affinity with the yellow fever virus antibody, and can be used for detecting the yellow fever virus antibody rapidly and accurately for diagnosing the infection condition of a yellow fever virus.
Description
Technical field
The present invention relates to gene engineering technology field, relate in particular to a kind of recombinant antigen protein of diagnosing yellow fever virus antibody and preparation method thereof, and with the yellow fever virus detection kit of this antigen protein as envelope antigen.
Background technology
(Yellow Fever Virus YFV) belongs to the Flavivirus of flaviviridae to yellow fever virus, and viral genome is the sub-thread positive chain RNA of non-segmented negative, and several different genotype are arranged, but a serotype is only arranged, and antigenicity is conservative.Yellow jack is the acute infectious disease that is caused by yellow fever virus, propagates through Aedes aegypti.Clinical symptoms has heating, severe headache, jaundice, hemorrhage and proteinuria etc., and the main fluidity of this disease is in torrid areas such as South America, Central America and Africa.Estimate global annual number of the infected 20 examples, dead 30,000 examples according to WHO.Up to now; China does not still have the report of the popular or confirmed cases of yellow jack, but my each provinces and regions, south are located in the torrid zone, subtropics, and weather is hot moist; The media mosquito specie is various; The host and the communication media that extensively have yellow fever virus, there is the possibility of importing once more, breaking out in frequent day by day along with personnel in the global range and economic interaction.Yellow fever virus is mainly propagated in the crowd through mosquito bite, and velocity of propagation is fast, and harm is big, causes the common people panic easily.Therefore, carry out its Study on rapid detection technique and products-supply in advance, guarantee has crucial meaning to public health security.
Be used for the virological immunology detection method at present; EUSA (ELISA) method speed is fast, and is not high to requirement of experiment, and can robotization, a large amount of samples of high-throughout detection; Also has simultaneously advantage highly sensitive, good reliability; And in different pathogen detection, be used widely, it is operated by vast basic health epidemic prevention worker on top of, has higher using value.Immune colloid gold quick diagnosis technology then is based on the new technique of EUSA, latex agglutination test, immune colloidal gold technique; The antibody that was used to detect anti-human immunodeficiency virus in 1989 first, its operation is more easy fast, only needs several minutes; The positive reaction naked eyes are visible; Do not need specific apparatus, not influenced by ambient temperature, stable reagent is prone to prolonged preservation, specificity and susceptibility all than advantages such as height; Be specially adapted to the place use that field and experiment condition do not possess, and in the detection of multiple virus, be applied.In grass-roots unit, more than two kinds of methods have the additive method can't displaced advantage, and its key all is high specificity, the high antigenic preparation of avidity, to improve specificity and recall rate.
In the prior art; About the research aspect of yellow fever virus amynologic diagnostic method, domesticly also met many pieces of reports, but owing to have serious serological cross reaction between the different arbovirusess; There is multiple arboviruses popular zone at those; Because there is cross-reacting antibody in polyinfection in patient's body, make immunology diagnosis and differential diagnosis very difficult.Simultaneously, because IgG antibody can exist for a long time in host, what have surpasses 10 months, even carries throughout one's life, and feasible discriminating past, still present infection recently be the ten minutes difficulty also, and antigenic quality is also had higher requirement.
Summary of the invention
In view of this; The technical problem that solves of the present invention is to provide a kind of recombinant antigen protein that detects yellow fever virus antibody and preparation method thereof; This recombinant antigen protein has high specificity, advantage that avidity is high; The arthropod-borne virus serum-free close with other learned cross reaction, and has high avidity with yellow fever virus antibody.
The technical problem that solves of the present invention is to provide a kind of detection kit of yellow fever virus, utilizes prepared recombinant antigen protein, sets up the ELISA Fast Detection Technique of yellow fever virus antibody.
In order to solve the problems of the technologies described above, on the one hand, the present invention provides a kind of recombinant antigen protein that detects yellow fever virus, and said recombinant antigen protein has aminoacid sequence shown in SEQ ID NO.1.
Preferably, the coding gene sequence of said recombinant antigen protein has the nucleotide sequence shown in SEQ ID NO.2.This recombinant antigen protein is as target gene fragment through the encoding sox selecting to present viral high specific peptide section in the yellow fever virus albumen; And this target gene fragment imported to show to answer in the host cell through prokaryotic expression carrier prepare; This target gene fragment has the nucleotide sequence shown in SEQ ID NO.2; The extension of section that comprises coding recombinant antigen protein flexible arm in this target gene fragment; The fusion rotein that said flexible arm can be eliminated expression vector is covered polypeptide epitope, makes the recombinant antigen protein of expression correctly folding.
On the other hand, a kind of preparation method who detects the recombinant antigen protein of yellow fever virus antibody is provided also in the specific embodiment of the invention, has comprised the steps:
One, the encoding sox that presents viral high specific peptide section in the selection yellow fever virus albumen is as target gene fragment; This target gene fragment has the nucleotide sequence shown in SEQ ID NO.2; The extension of section that comprises coding recombinant antigen protein flexible arm in this target gene fragment; The fusion rotein that said flexible arm can be eliminated expression vector is covered polypeptide epitope, makes the recombinant antigen protein of expression correctly folding;
Two, design pcr amplification primer, and through the said target gene fragment of pcr amplification;
Three, pass through restriction enzyme digestion and be connected, the target gene fragment body is connected in the prokaryotic expression carrier, make up the recombinant expression plasmid that contains said target gene fragment;
Four, said recombinant expression plasmid is converted in the competent expression host cell; Cultivating said expression host cell makes it produce recombinant antigen protein; And obtaining recombinant antigen protein through purifying, said recombinant antigen protein has aminoacid sequence shown in SEQ ID NO.1.
Preferably, the pcr amplification primer sequence that adopts in the said step 2 is following:
Forward primer: CGGGATCCTGGAAGATGAAAACTGGACGC < SEQ ID NO.3 >
Reverse primer: ACGCAAGCTTTTATAACTTTGCGGTCCCCCTGGA < SEQ ID NO.4 >.
Preferably, select pMAL c2x prokaryotic expression carrier for use at prokaryotic expression carrier described in the specific embodiment of the present invention, said expression host cell is intestinal bacteria.
For recombinant antigen protein of the present invention, it has high specificity, advantage that avidity is high, and the arthropod-borne virus serum-free close with other learned cross reaction, and has high avidity with yellow fever virus antibody.In an embodiment of the present invention; Through expressing the antigen protein that obtains after SDS-PAGE separates; Carry out Western-blot with viral antiserum(antisera); Verified that recombinant antigen protein of the present invention has high specificity; And obtained the engineering strain that can efficiently express yellow fever virus specificity recombinant antigen, the yellow fever virus detection kit that combines simultaneously to have adopted this recombinant antigen protein is in its sensitivity test, specificity test-results, and the highly sensitive that recombinant antigen protein of the present invention shows when being used to detect yellow fever virus, the advantage of high specific further have been described.
For recombinant antigen protein preparation method of the present invention; Because its encoding sox of having selected to present viral high specific peptide section in the yellow fever virus albumen is as target gene fragment; The fusion rotein that the extension of section that comprises coding recombinant antigen protein flexible arm in this target gene fragment, said flexible arm can be eliminated expression vector is covered polypeptide epitope, makes the recombinant antigen protein of expression correctly folding; Combine the primer special that relates in the such scheme simultaneously; Obtained this goal gene effectively, combined the prokaryotic expression carrier and the expressive host bacterium of adopting in this method simultaneously, made recombinant antigen protein possess expression effect efficiently.In addition; In an embodiment of the present invention; Through expressing the antigen protein that obtains after SDS-PAGE separates; Carry out Western-blot with viral antiserum(antisera); The yellow fever virus detection kit that combines simultaneously to have adopted this recombinant antigen protein is in its sensitivity test, specificity test-results, and the preparation method that recombinant antigen protein of the present invention further has been described is in the advantage that shows aspect the highly sensitive that efficiently expresses recombinant antigen protein and assurance recombinant antigen protein of the present invention, the high specific.
In addition; The present invention also provides a kind of detection kit of yellow fever virus; Comprise antibody test plate and ELISA reaction solution, be coated with the recombinant antigen protein that detects yellow fever virus on the said antibody test plate, said recombinant antigen protein has aminoacid sequence shown in SEQ ID NO.1.
Wherein, the ELISA reaction solution comprises in the test kit: enzyme conjugates working fluid, positive control, negative control; Concentrated cleaning solution, colour developing liquid A, colour developing liquid B and stop buffer; Enzyme conjugates working fluid, positive control are the yellow fever virus antibody standard substance, and negative control is a yellow fever virus negative antibody serum.
Preferably, be the goat anti-human igg of horseradish peroxidase-labeled at the working fluid of enzyme conjugates described in the specific embodiment of the invention.
Preferably, in a specific embodiment of the present invention, the preparation process of said antibody test plate comprises the steps:
One, the preparation of recombinant antigen protein comprises the steps:
(1), the encoding sox that presents viral high specific peptide section in the selection yellow fever virus albumen is as target gene fragment; This target gene fragment has the nucleotide sequence shown in SEQ ID NO.2; The extension of section that comprises coding recombinant antigen protein flexible arm in this target gene fragment; The fusion rotein that said flexible arm can be eliminated expression vector is covered polypeptide epitope, makes the recombinant antigen protein of expression correctly folding;
(2), design pcr amplification primer, and through the said target gene fragment of pcr amplification;
(3), through restriction enzyme digestion and being connected, the target gene fragment body is connected in the prokaryotic expression carrier, make up the recombinant expression plasmid that contains said target gene fragment;
(4), said recombinant expression plasmid is converted in the competent expression host cell; Cultivating said expression host cell makes it produce recombinant antigen protein; And obtaining recombinant antigen protein through purifying, said recombinant antigen protein has aminoacid sequence shown in SEQ ID NO.1;
Two, the recombinant antigen protein behind the purifying is fixed in elisa plate; This process comprises that the recombinant antigen protein that purifying in the step 1 is obtained encapsulates elisa plate with step concentration respectively; Every hole 50-200ul, 4 ℃ encapsulate and spend the night, then with phosphoric acid salt tween damping fluid washing 1-2 minute; After it clap is done, sealed 1-3 hour in 37 ℃, promptly obtain said antibody test plate after drying with 5% bovine serum albumin solution.
Moreover; The embodiment that is used for the detection kit of yellow fever virus in conjunction with recombinant antigen protein of the present invention; Prove absolutely the utilization of recombinant antigen protein of the present invention in preparation diagnosis yellow fever virus test kit, can guarantee sensitivity and specificity in the yellow fever virus diagnostic procedure.
Need to prove that also research and development unit of the present invention is the entire PLA and Guangdong Province's arboviruses specialized laboratory, collected domestic comparatively comprehensively arboviruses strain, and prepared its standard serum, for good basis has been established in the specificity checking of recombinant antigen protein.Can find out in conjunction with the experiment content that discloses among the embodiment; Guaranteeing on the basis of reactionogenicity in the experiment; Shortened antigen fragment length as far as possible; Guarantee that the prokaryotic expression antigen that is screened possesses good specificity, with the Syndebis of being tested, Xi Menlike, Ma Yaluo, Luo Sihe, aigret mountain, Gai Ta, chikungunya, step on the leather 1～4 type, epidemic encephalitis type B and the equal no cross reaction of west Nile virus, S/N ratio is more than 12.Simultaneously; The preparation method who is set up and the detection kit of yellow fever virus all possess higher sensitivity and detect scope; The experiment of carrying out with yellow fever virus mouse immune serum shows, in serum 1:100～1:6400 times dilution range, all can obtain positive findings; And do not have non-specific result, detect the OD value more than 0.16.
Description of drawings
Fig. 1 is yellow fever virus in the embodiment of the invention 1, dengue 1-type virus, dengue 2-type virus, dengue 3 virus, 4-type dengue virus, encephalitis b virus and west nile virus polyclonal antibody and the antigenic western blot of prokaryotic expression results of hybridization; Wherein, a:SDS PAGE electrophorogram; B: yellow fever virus polyclonal antibody Wester blot result; C: dengue 1-type virus polyclonal antibody Wester blot result; D: dengue 2-type virus polyclonal antibody Wester blot result; E: dengue 3 virus polyclonal antibody Wester blot result; F: 4-type dengue virus polyclonal antibody Wester blot result; G: encephalitis b virus polyclonal antibody Wester blot result; H: west nile virus polyclonal antibody Wester blot result.1.YFV22+pMAL c2x; 2.pMAL c2x; M. molecular weight of albumen Marker.
Fig. 2 is an antigen coated concentration experimental result picture in the embodiment of the invention 2.
Fig. 3 is ELISA test kit and application thereof in the embodiment of the invention 2; Wherein, the experiment of b:ELISA specificity, the A1 hole adds dengue 1-type virus antibody, and B1 is a dengue 2-type virus antibody; C1 is a dengue 3 virus antibody, and D1 is a 4-type dengue virus antibody, and E1 is a B encephalitis virus antibody, and F1 is a yellow fever virus antibody; G1 is the Ma Yaluo antiviral antibody, and H1 is a west Nile virus antibody, and the A2 hole adds ross river virus antibody, and B2 is a sagiyama virus antibody; C2 is a getah virus antibody, and D2 is a CHIK antibody, and E2 is a sindbis alphavirus antibody; F2 is that Xi Menli restrains viral malicious antibody, and the negative serum of G2, H2 are blank; The experiment of c:ELISA susceptibility; The Orthogonal Method method is diluted mouse-anti yellow fever virus polyclonal antibody, measures ELISA method susceptibility, and wherein A1 is that the how anti-1:200 of mouse-anti yellow fever virus doubly dilutes; And do serial doubling dilution by A1-H1, do serial doubling dilution by first row to the 4th row subsequently.
Embodiment
For making the present invention be more prone to understand,, further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.
The experimental technique of unreceipted actual conditions among the following embodiment, usually according to normal condition, for example the Sambrook equimolecular is cloned the condition described in the enforcement manual.
In the following example of the present invention, the yellow fever virus of selecting for use: 17D strain, toxic strain sample in the applicant laboratory; And the restriction endonuclease, ligase enzyme, T carrier used in the experiment are available from Dalian TaKaRa company.Gel-purified recovery and plasmid rapid extraction test kit are available from OMEGA company.Trizol and reverse transcription reagent are available from Invitrogen company.The pMAL-C2x expression vector is available from New England Biolabs company, and Amylose Resin affinity chromatography reagent is available from NEB company, and expressive host bacterium Rosetta (DE3) is by the preservation of going down to posterity of this laboratory.
The preparation of the recombinant antigen protein of embodiment 1, detection yellow fever virus
Detect the recombinant antigen protein of yellow fever virus according to the following steps preparation:
The encoding sox that presents viral high specific peptide section in step 1, the selection yellow fever virus albumen is as target gene fragment; This target gene fragment has the nucleotide sequence shown in SEQ ID NO.2; The extension of section that comprises coding recombinant antigen protein flexible arm in this target gene fragment; The fusion rotein that said flexible arm can be eliminated expression vector is covered polypeptide epitope, makes the recombinant antigen protein of expression correctly folding;
Step 3, through restriction enzyme digestion and being connected, the target gene fragment body is connected in the prokaryotic expression carrier, make up the recombinant expression plasmid that contains said target gene fragment;
Step 4, said recombinant expression plasmid is converted in the competent expression host cell; Cultivating said expression host cell makes it produce recombinant antigen protein; And obtaining recombinant antigen protein through purifying, said recombinant antigen protein has aminoacid sequence shown in SEQ ID NO.1.
During concrete the realization; In the above-mentioned steps one; With reference to yellow fever virus 17D pnca gene group sequence, utilize DNAstar and ANTHEPROT software that its proteins encoded hydrophobicity, antigenicity, accessibility and plasticity-are carried out detailed analysis, and with reference to the sequence information among the Genbank; Epitope to prediction is compared, 33 sections of the possible antigenicity fragments of preliminary screening.
In the step 2; According to a kind of selected antigenicity fragment of step; And the extension of section that needs in the combination polypeptide structure, and through the optimization design primer corresponding, through pcr amplification corresponding target gene fragment with each section; Thereby make the fusion rotein that to eliminate expression vector by extension of section coding flexible arm to the covering of polypeptide epitope, make the recombinant antigen protein of expression correctly folding.For this reason, in the preferred process of this step, 33 pairs of design pcr amplification primers, shown in table one:
Table one antigen fragment clone the primer sequence
Wherein, the design of primers process is according to 33 pairs of the primers described in the antigenicity analysis consequence devised table one, and respectively at primer 5 ' terminal and 3 ' terminal BamH I and the Hind III restriction enzyme site of importing.
As the preliminary step that obtains goal gene sheet degree; Also need the genome of reverse transcription yellow fever virus; This transcriptive process,reversed is: yellow fever virus 17D strain is through the cultivation of going down to posterity of C6/36 cell cultures; Adopt the Trizol reagent of Invitrogen to extract viral RNA, adopt ordinary method to carry out reverse transcription and obtain its cDNA.
Then carry out PCR by following condition:
Get cDNA2 μ l, 10 * PCR damping fluid, 5 μ l, forward primer, each 2 μ l of reverse primer, dNTP 8 μ l, Taq 2U, moisturizing to TV 50 μ l carry out pcr amplification.Reaction parameter is: 95 ℃ of 5min, 94 ℃ of 30s then, 55 ℃ of 40s, 72 ℃ of 60s, totally 32 circulations circulate for the last time back 72 ℃ and extend 7min, amplified fragments is purified, reclaim after ,-20 ℃ of preservations are subsequent use.
In the step 3, through restriction enzyme digestion and being connected, the target gene fragment body is connected in the prokaryotic expression carrier, makes up the recombinant expression plasmid that contains target gene fragment.In embodiments of the present invention, said prokaryotic expression carrier is a pMAL c2x prokaryotic expression carrier, and said expression host cell is intestinal bacteria.This step is specific as follows:
Enzyme is cut: respectively gets the purified amplified production of 10ul, adds Buffer K 3ul respectively, and BamH I and each 1ul of Hind III (10U), sterilization deionized water 15ul, 37 ℃ of enzymes are cut and are spent the night.Other gets pMAL c2x expression vector plasmid 2ug, adds Buffer K 3ul, BamH I and each 1ul of Hind III (10U), and the sterilization deionized water is mended to 30ul, and 37 ℃ of enzymes are cut and are spent the night.Enzyme is cut product and is reclaimed the test kit purifying and recovering with TaKaRa company gel respectively.
Connect: get 2ul through the pMAL of above processing c2x carrier (about 130ng), the 5ul enzyme is cut purpose fragment (about 40ng), 45 ℃ of heating 5min, so that the annealed cohesive end unwinds, ice bath 10sec adds T immediately
4Dna ligase 0.5U, ligase enzyme Buffer 1ul heats up in a steamer water with sterilization four and mends to 10ul, and 16 ℃ of connections are spent the night.Get recombinant plasmid 5ul transformed competence colibacillus bacterium DH5a, add the LB liquid nutrient medium 300ul of antibiotic-free, behind 37 ℃ of recovery 45min, get 100ul and be coated with penbritin (100ug/ml) plate, cultivate 18h for 37 ℃.
Recon is identified: the picking white colony carries out bacterium colony PCR and identifies the PCR reactant: 10 * PCR damping fluid, 2 μ l, and forward primer, each 1 μ l of reverse primer, dNTP 0.4 μ l, Taq 2U, moisturizing to TV 20 μ l carry out pcr amplification.The same step 2 of PCR reaction conditions, positive colony send the order-checking of biotechnology (Shanghai) Co., Ltd. to confirm.Confirm to obtain 25 groups of correct recombinant expression plasmids through order-checking, and extract plasmid, and-20 ℃ of preservations are subsequent use.
In the step 4, recombinant expression plasmid is converted in the competent expression host cell, the culture expression host cell, and detect its expression of recombinant proteins situation; This step is specially in the present embodiment: the expressive host bacterium list bacterium colony of choosing through transforming recombinant expression vector is inoculated in respectively in the LB liquid nutrient medium, and 4h are cultivated in 37 ℃ of concussions, when the OD value reaches 0.6; Draw and not induce contrast bacterium 1ml, and in the residue bacterium, adding inductor IPTG, to make its final concentration be 0.5mmol/L, in 37 ℃ of shaking culture 4h; Behind the ice bath 5min, 4 ℃, the centrifugal 2min receipts of 12000g bacterium, and carry out SDS-PAGE and detect; With reference to the molecular cloning experiment guide, prepare reaction liquid, spacer gel concentration is 4%; Resolving gel concentration is 12%, spacer gel constant current 10mA, separation gel constant current 25mA electrophoresis; After electrophoresis finishes, coomassie brilliant blue staining, checking expression of recombinant proteins situation.Through the experiment of this step, 25 constructed recombinant expression vectors all obtain and efficiently express.
Then, the recombinant expressed antigen of obtain being carried out western blot analyzes; To verify the specificity of its immunoreation originality and immunological response; This step is specially in the present embodiment: the same step 4 of SDS-PAGE electrophoresis; After electrophoresis finished, electricity changeed nitrocellulose filter, yellow fever virus, dengue 1-type virus, dengue 2-type virus, dengue 3 virus, 4-type dengue virus, encephalitis b virus and west nile virus polyclonal antibody 1:5000 dilution; Carry out western blotting (western-blot), antigenic atopic is expressed in checking.Acquisition can efficiently express engineering strain one strain of yellow fever virus specificity recombinant antigen: E.coli-YFV22.Therefore; In embodiments of the present invention; The target gene fragment that final optimization pass obtains has the nucleotide sequence shown in SEQ ID NO.2, and recombinant antigen protein then has aminoacid sequence shown in SEQ ID NO.1, corresponding with it; In step 2, the pcr amplification primer sequence of selected employing is following:
Forward primer: CGGGATCCTGGAAGATGAAAACTGGACGC < SEQ ID NO.3 >
Reverse primer: ACGCAAGCTTTTATAACTTTGCGGTCCCCCTGGA < SEQ ID NO.4 >
Be YFAg22-1 and YFAg22-2 in the correspondence table one.
In addition, carry out the expression and purification experiment of above-mentioned recombinant antigen protein, obtain the recombinant antigen protein that is suitable for the ELISA experiment through this efficient expression strain; This step is specially in the present embodiment: E.coli-YFV22 bacterium overnight culture 10ml inoculates 1L LB substratum (containing 100 μ g/ml penbritins), and 37 ℃ of shaking culture 2～3 hours treat that the OD value reaches at 0.6 o'clock; Add IPTG to final concentration 0.5mM; Continue 37 ℃ of shaking culture after 4 hours, centrifugal results bacterium, and use the PBS washed twice; 50mlPBS is resuspended; Behind the ultrasonic disruption bacterium, adopt the NEB Amylose Resin of company affinity chromatography column purification purification of recombinant proteins, its concentration of nucleic acid-protein analysis-e/or determining is 1200ug/ml.
As shown in Figure 1, carry out SDS-PAGE experiment and western blotting, with reference to the molecular cloning experiment guide; Prepare reaction liquid, spacer gel concentration is 4%, and resolving gel concentration is 12%; Spacer gel constant current 10mA, separation gel constant current 25mA electrophoresis is after electrophoresis finishes; Electricity changes nitrocellulose filter, and western blotting (western-blot) is carried out in chikungunya, Syndebis, ross river virus polyclonal antibody 1:5000 dilution; Antigenic atopic is expressed in checking, and obtains to efficiently express the engineering strain of yellow fever virus specificity recombinant antigen.Then carry out the expression and purification experiment of above-mentioned recombinant antigen protein, obtain the recombinant antigen protein that is suitable for the ELISA experiment through this efficient expression strain.
In the present embodiment need to recombinant antigen protein of the present invention encapsulate condition and concentration is optimized; Also need earlier ELISA reaction solution composition in the test kit to be described in order to reach this purpose:
Enzyme conjugates working fluid: the goat anti-human igg of horseradish peroxidase-labeled;
Positive control: yellow fever virus antibody positive mouse serum.
Negative control: yellow fever virus negative antibody human serum.
Concentrated cleaning solution: contain 1% foetal calf serum in the phosphate buffered saline buffer of 0.1mol/L pH7.4,0.5%Tween-20 and 20mg/L qingfengmeisu qiong;
Colour developing liquid A:0.02%H2O2; With 0.1M Hydrocerol A-0.2M Sodium phosphate, dibasic, PH4.5～5.0 dilutions.
Colour developing liquid B:0.4 ‰ TMB-HCl: use the 50mM Trisodium Citrate, dense HCl accent pH value is 2.8 solution dissolving.
Stop buffer: 2M H
2SO
4
With the purification of Recombinant antigen protein antigen that obtains in the foregoing description 1 respectively with 10,5,2.5,1.25,0.63,0.32,0.16,0.08,0.04,0.02ug/ml concentration encapsulates elisa plate, every hole consumption 100ul, 4 ℃ encapsulate and spend the night PBST washing 3 minutes; Clap and do, 37 ℃ of sealings of 5%BSA 2 hours are got 100ul and are added reacting hole after mouse-anti yellow fever virus polyclonal antibody 1:5000 doubly dilutes, and 37 ℃ were reacted 1 hour; PBST washing 4 times each 1 minute, is clapped and is done, after the anti-goat anti-mouse igg 1:10000 of HRP mark doubly dilutes; Every hole adds 100ul, and 37 ℃ were reacted 40 minutes, PBST washing 4 times, each 1 minute; Clap and do, every hole adds tmb substrate solution 100ul, and 37 ℃ were reacted 15 minutes; Add 50ul 2M H2SO4 termination reaction, under the 450nm wavelength, measure the OD value with ELIASA, as shown in Figure 2; The result is presented at and encapsulates in concentration 2.5～10ug/ml scope, the no significant difference of experiment OD value, and finally selecting antigen coated concentration is 5ug/ml.
Embodiment 3, ELISA reaction condition optimization
Antigen coated condition and ELISA reaction conditions are carried out system optimization, and confirm that finally best ELISA reaction conditions is: recombinant antigen 100ul (concentration is 10ug/ml) encapsulates for 4 ℃ and spends the night, and PBST washing 3 minutes is clapped and done; 37 ℃ of sealings of 5%BSA 2 hours are got 100ul and are added reacting hole after serum 1:100 to be checked doubly dilutes, 37 ℃ were reacted 1 hour, PBST washing 4 times; Each 1 minute, clap and do, HRP mark IgG, every hole adds 100ul; 37 ℃ were reacted 40 minutes, and PBST washing 4 times each 1 minute, is clapped and done; Every hole adds tmb substrate solution 100ul, and 37 ℃ were reacted 15 minutes, adds 50ul 2M H
2SO
4Termination reaction is measured the OD value with ELIASA under the 450nm wavelength.
The specificity experiment of embodiment 4, test kit
Adopt the optimal conditions among the embodiment 3 to carry out the ELISA experiment; Selection contains Syndebis, Xi Menlike, Ma Yaluo, Luo Sihe, aigret mountain, Gai Ta, steps on leather 1～4 type, epidemic encephalitis type B, yellow heat and west Nile virus polyclonal antibody sample as specificity laboratory reference sample; Shown in table two; The detection kit of yellow fever virus of the present invention and the recombinant antigen protein of employing thereof have good specificity, with the equal no cross reaction of above-mentioned reference sample.
The experiment of table two yellow fever virus ELISA detection method specificity
The sensitivity experiment of embodiment 5, test kit
The purification of Recombinant antigen protein that obtains among the embodiment 1 is encapsulated elisa plate with 10ug/ml; Through the Orthogonal Method method mouse-anti yellow fever virus polyclonal antibody is diluted; The optimal conditions of adopt implementing in 3 is carried out the ELISA experiment, and shown in table three, measuring it, to detect scope be 1: 100～1:12800.
The experiment of table three yellow fever virus ELISA detection method susceptibility
Should be noted that at last; Above embodiment is only in order to technical scheme of the present invention to be described but not to the restriction of protection domain of the present invention; Although the present invention has been done detailed description with reference to preferred embodiment; Those of ordinary skill in the art should be appreciated that and can make amendment or be equal to replacement technical scheme of the present invention, and do not break away from the essence and the scope of technical scheme of the present invention.
Claims (10)
1. recombinant antigen protein that detects yellow fever virus antibody, it is characterized in that: said recombinant antigen protein has aminoacid sequence shown in SEQ ID NO.1.
2. the recombinant antigen protein of detection yellow fever virus antibody according to claim 1 is characterized in that: the coding gene sequence of said recombinant antigen protein has the nucleotide sequence shown in SEQ ID NO.2.
3. a preparation method who detects the recombinant antigen protein of yellow fever virus antibody is characterized in that, comprises the steps:
One, the encoding sox that presents viral high specific peptide section in the selection yellow fever virus albumen is as target gene fragment; This target gene fragment has the nucleotide sequence shown in SEQ ID NO.2; The extension of section that comprises coding recombinant antigen protein flexible arm in this target gene fragment; The fusion rotein that said flexible arm can be eliminated expression vector is covered polypeptide epitope, makes the recombinant antigen protein of expression correctly folding;
Two, design pcr amplification primer, and through the said target gene fragment of pcr amplification;
Three, pass through restriction enzyme digestion and be connected, the target gene fragment body is connected in the prokaryotic expression carrier, make up the recombinant expression plasmid that contains said target gene fragment;
Four, said recombinant expression plasmid is converted in the competent expression host cell; Cultivating said expression host cell makes it produce recombinant antigen protein; And obtaining recombinant antigen protein through purifying, said recombinant antigen protein has aminoacid sequence shown in SEQ ID NO.1.
4. preparation method according to claim 3 is characterized in that, the pcr amplification primer sequence that adopts in the said step 2 is following:
Forward primer: CGGGATCCTGGAAGATGAAAACTGGACGC < SEQ ID NO.3 >
Reverse primer: ACGCAAGCTTTTATAACTTTGCGGTCCCCCTGGA < SEQ ID NO.4 >.
5. preparation method according to claim 3 is characterized in that: said prokaryotic expression carrier is a pMAL c2x prokaryotic expression carrier, and said expression host cell is intestinal bacteria.
6. the detection kit of a yellow fever virus; Comprise antibody test plate and ELISA reaction solution; It is characterized in that: be coated with the recombinant antigen protein that detects yellow fever virus antibody on the said antibody test plate, said recombinant antigen protein has aminoacid sequence shown in SEQ ID NO.1.
7. detection kit according to claim 6 is characterized in that, the ELISA reaction solution comprises in the test kit: the enzyme conjugates working fluid; Positive control, negative control, concentrated cleaning solution; Colour developing liquid A, colour developing liquid B and stop buffer, enzyme conjugates working fluid; Positive control is the yellow fever virus antibody standard substance, and negative control is a yellow fever virus negative antibody serum.
8. detection kit according to claim 7 is characterized in that: said enzyme conjugates working fluid is the goat anti-human igg of horseradish peroxidase-labeled.
9. detection kit according to claim 6 is characterized in that, the preparation process of said antibody test plate comprises the steps:
One, the preparation of recombinant antigen protein comprises the steps:
(1), the encoding sox that presents viral high specific peptide section in the selection yellow fever virus albumen is as target gene fragment; This target gene fragment has the nucleotide sequence shown in SEQ ID NO.2; The extension of section that comprises coding recombinant antigen protein flexible arm in this target gene fragment; The fusion rotein that said flexible arm can be eliminated expression vector is covered polypeptide epitope, makes the recombinant antigen protein of expression correctly folding;
(2), design pcr amplification primer, and through the said target gene fragment of pcr amplification;
(3), through restriction enzyme digestion and being connected, the target gene fragment body is connected in the prokaryotic expression carrier, make up the recombinant expression plasmid that contains said target gene fragment;
(4), said recombinant expression plasmid is converted in the competent expression host cell; Cultivating said expression host cell makes it produce recombinant antigen protein; And obtaining recombinant antigen protein through purifying, said recombinant antigen protein has aminoacid sequence shown in SEQ ID NO.1;
Two, the recombinant antigen protein behind the purifying is fixed in elisa plate; This process comprises that the recombinant antigen protein that purifying in the step 1 is obtained encapsulates elisa plate with step concentration respectively; Every hole 50-200ul, 4 ℃ encapsulate and spend the night, then with phosphoric acid salt tween damping fluid washing 1-2 minute; After it clap is done, sealed 1-3 hour in 37 ℃, promptly obtain said antibody test plate after drying with 5% bovine serum albumin solution.
10. according to claim 1 or claim 2 the utilization of recombinant antigen protein in preparation diagnosis yellow fever virus test kit.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103235131A (en) * | 2013-03-22 | 2013-08-07 | 深圳国际旅行卫生保健中心 | Lateral flow immunochromatographic determination product for detecting yellow fever viruses and preparation method of lateral flow immunochromatographic determination product |
| CN105732777A (en) * | 2016-05-05 | 2016-07-06 | 中国人民解放军军事医学科学院微生物流行病研究所 | Yellow fever virus specific detection antigen and application thereof |
| CN108226507A (en) * | 2016-12-15 | 2018-06-29 | 江苏维赛科技生物发展有限公司 | A kind of yellow fever antigen near-infrared fluorescent detection kit and application thereof |
| CN108546706A (en) * | 2018-03-29 | 2018-09-18 | 杭州泰熙生物技术有限公司 | A kind of preparation of yellow fever virus recombinant antigen and its kit production method |
| CN112813089A (en) * | 2021-03-22 | 2021-05-18 | 苏州诺威百奥生物科技有限公司 | Preparation method of multi-site recombinant antigen for flaviviridae detection |
| CN113429479A (en) * | 2018-04-04 | 2021-09-24 | 中国科学院微生物研究所 | High-sensitivity yellow fever virus humanized monoclonal antibody and application thereof |
| CN113621058A (en) * | 2021-08-19 | 2021-11-09 | 中国科学院微生物研究所 | Yellow fever virus antibody and application thereof |
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-
2012
- 2012-08-31 CN CN201210322510.1A patent/CN102827260B/en active Active
Non-Patent Citations (4)
| Title |
|---|
| 任瑞文等: "主要黄病毒E蛋白抗原表位分析与初步鉴定", 《新发和再发传染病防治热点研讨会》 * |
| 无: "polyprotein [Yellow fever virus]", 《GENBANK》 * |
| 无: "Yellow fever virus clone HONG9 polyprotein gene, complete", 《GENBANK》 * |
| 胡文龙等: "黄热病毒特异性抗原片段的筛选与鉴定", 《中国预防医学杂志》 * |
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| CN103235131B (en) * | 2013-03-22 | 2015-07-01 | 深圳国际旅行卫生保健中心 | Lateral flow immunochromatographic determination product for detecting yellow fever viruses and preparation method of lateral flow immunochromatographic determination product |
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| CN113429479A (en) * | 2018-04-04 | 2021-09-24 | 中国科学院微生物研究所 | High-sensitivity yellow fever virus humanized monoclonal antibody and application thereof |
| CN113651886B (en) * | 2018-04-04 | 2022-04-15 | 中国科学院微生物研究所 | High-sensitivity yellow fever virus humanized monoclonal antibody and application thereof |
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