CN102827261B - Recombinant antigenic protein for detecting west nile virus antibody, kit and application thereof - Google Patents
Recombinant antigenic protein for detecting west nile virus antibody, kit and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant antigenic protein for detecting a west nile virus antibody. The sequence of the recombinant antigenic protein is shown as SEQ ID NO.1. The encoding gene sequence of the recombinant antigenic protein is shown as SEQ ID NO.2. The encoding gene sequence comprises an extended section that encodes the flexible arm of the recombinant antigenic protein, wherein the flexible arm can eliminate cover of a fusion protein of an expression carrier to peptide epitope so that the expressed recombinant antigenic protein is folded correctly. The invention further discloses a preparation method of the recombinant antigenic protein. In addition, a detection kit of west nile virus provided by the invention comprises an antibody detection plate coated with the recombinant antigenic protein and an ELISA (Enzyme-Linked Immuno Sorbent Assay) reaction liquid. The recombinant antigenic protein provided by the invention has the advantages of strong specificity and high affinity, and is free from cross reaction with other similar arthropod-borne viruses in serology but has extremely high affinity with the west nile virus antibody. The west nile virus can be quickly and accurately detected, so that infection of the west nile virus can be diagnosed.
Description
Technical field
The present invention relates to gene engineering technology field, relate in particular to a kind of recombinant antigen protein of diagnosing west nile virus antibody and preparation method thereof, and west nile virus detection kit using this antigen protein as envelope antigen.
Background technology
West nile virus (West Nile virus, WNV) belong to flaviviridae Flavivirus, for single strand plus RNA virus, belong to japanese encephalitis virus (Japanese encephalitis virus together with Saint Louis' encephalitis virus (Saint-Louis encephalitis virus) and Murray paddy fever virus (Murray Valley fever virus), JEV) serogroups, can cause humans and animals generation west Nile fever and west nile virus meningoencephalitis, culicine mosquito is its main communication media, birds are these viral intermediate hosts, and people and Ma Ze are terminal hosts.WNV disease is distributed widely in the some areas in Africa, the Middle East, Europe, and the torrid areas in USSR (Union of Soviet Socialist Republics), India, Indonesia and Asia etc.Summer in 1999, WNV logs in the U.S. first, and the inherent North America of coming years and Central America bamboo telegraph, cause the maximum of the national arbovirus infections in history such as America & Canada popular.Although China does not have WNV epidemic situation to occur so far, but neighboring countries and regions are own through breaking out this epidemic disease, the possibility that has at any time Introduced cases to propagate, and as a kind of potential biological warfare agent, existence is used to the possibility that bio-terrorism attacks, strengthen the tachnical storage of association area, ensure significant for public health and the Biosafety of China.
At present for virological immunology detection method, enzyme linked immunosorbent assay (ELISA) method speed is fast, not high to requirement of experiment, and can automatization, a large amount of samples of high-throughout detection, also there is the advantage highly sensitive, reliability is strong simultaneously, and in different pathogen detection, be used widely, its operation is skillfully grasped by vast basic health epidemic prevention worker, has higher using value.Immune colloid gold quick diagnosis technology is the new technique based on enzyme linked immunosorbent assay, latex agglutination test, immune colloidal gold technique, the antibody for detection of anti-human immunodeficiency virus first in 1989, its operation is more easy fast, only need several minutes, positive reaction naked eyes are visible, do not need specific apparatus, not influenced by ambient temperature, the easily long-term preservation of stable reagent, specificity and susceptibility are all compared with advantages of higher, 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, above two kinds of advantages that method has additive method to replace, and its key is all the preparation of high specificity, the high antigen of avidity, to improve specificity and recall rate.
In prior art, about the research aspect of west nile virus amynologic diagnostic method, domesticly many pieces of reports were also met, but owing to there being serious serological cross reaction between different arbovirusess, there is at those region that multiple arboviruses is popular, due to polyinfection, in patient body, there is cross-reacting antibody, make immunology diagnosis and differential diagnosis very difficult.Meanwhile, because IgG antibody can exist for a long time in host, what have exceedes 10 months, carries even throughout one's life, makes discriminating infection in the past, recently or now also very difficult, and the quality of antigen is also had higher requirement.
Summary of the invention
In view of this, of the present invention solved technical problem is to provide a kind of recombinant antigen protein that detects west nile virus antibody and preparation method thereof, this recombinant antigen protein has advantages of that high specificity, avidity are high, the arthropod-borne virus serum-free close with other learned cross reaction, and has high avidity with west nile virus antibody.
Of the present invention solved technical problem is to provide the detection kit of a kind of west nile virus, utilizes prepared recombinant antigen protein, sets up the ELISA Fast Detection Technique of west nile virus antibody.
In order to solve the problems of the technologies described above, on the one hand, the invention provides a kind of recombinant antigen protein that detects west nile virus, described recombinant antigen protein has aminoacid sequence as shown in SEQ ID NO.1.
Preferably, the coding gene sequence of described recombinant antigen protein has the nucleotide sequence as shown in SEQ ID NO.2.This recombinant antigen protein is by selecting the encoding gene that presents viral high specific peptide section in west nile virus albumen as goal gene fragment, and this goal gene fragment is imported to show to answer in host cell by prokaryotic expression carrier prepare, this goal gene fragment has the nucleotide sequence as shown in SEQ ID NO.2, this goal gene fragment comprises the extension of section of coding recombinant antigen protein flexible arm, the fusion rotein that described flexible arm can be eliminated expression vector is covered polypeptide epitope, makes the recombinant antigen protein of expressing correctly folding.
On the other hand, in the specific embodiment of the invention, also provide a kind of preparation method of the recombinant antigen protein that detects west nile virus antibody, comprised the steps:
One, the encoding gene that presents viral high specific peptide section in selection west nile virus albumen is as goal gene fragment, this goal gene fragment has the nucleotide sequence as shown in SEQ ID NO.2, this goal gene fragment comprises the extension of section of coding recombinant antigen protein flexible arm, the fusion rotein that described flexible arm can be eliminated expression vector is covered polypeptide epitope, makes the recombinant antigen protein of expressing correctly folding;
Two, design pcr amplification primer, and by goal gene fragment described in pcr amplification;
Three, by restriction enzyme digestion and connection, goal gene sheet segment body is connected in prokaryotic expression carrier, build the recombinant expression plasmid that contains described goal gene fragment;
Four, described recombinant expression plasmid is converted in competent expression host cell, cultivating described expression host cell makes it produce recombinant antigen protein, and obtaining recombinant antigen protein by purifying, described recombinant antigen protein has aminoacid sequence as shown in SEQ ID NO.1.
Preferably, the pcr amplification primer sequence adopting in described step 2 is as follows:
Forward primer: CGGGATCCCCACCAGGCACTTCAGATCCA<SEQ ID NO.3>
Reverse primer: ACGCAAGCTTTTAGGCAATCTCATTCCCCATCTT<SEQ ID NO.4>.
Preferably, select pMAL c2x prokaryotic expression carrier at prokaryotic expression carrier described in a specific embodiment of the present invention, described expression host cell is intestinal bacteria.
For recombinant antigen protein of the present invention, it has advantages of high specificity, the arthropod-borne virus serum-free cross reaction that avidity is high, close with other, and has high avidity with west nile virus antibody.In an embodiment of the present invention, by expressing the antigen protein obtaining after SDS-PAGE separates, carry out Western-blot with viral antiserum(antisera), verify that recombinant antigen protein of the present invention has high specificity, and obtained can high efficient expression west nile virus specificity recombinant antigen engineering strain, simultaneously in conjunction with having adopted the west nile virus detection kit of this recombinant antigen protein in its sensitivity test, specific test result, furthermore understand the highly sensitive that recombinant antigen protein of the present invention shows when for detection of west nile virus, the advantage of high specific.
For recombinant antigen protein preparation method of the present invention, because it has selected the encoding gene that presents viral high specific peptide section in west nile virus albumen as goal gene fragment, this goal gene fragment comprises the extension of section of coding recombinant antigen protein flexible arm, described flexible arm can be eliminated fusion rotein the covering polypeptide epitope of expression vector, the recombinant antigen protein of expressing is correctly folded, simultaneously in conjunction with the primer special relating in such scheme, effectively obtain this goal gene, simultaneously in conjunction with the prokaryotic expression carrier adopting in the method and expressive host bacterium, make recombinant antigen protein possess efficient expression effect.In addition, in an embodiment of the present invention, by expressing the antigen protein obtaining after SDS-PAGE separates, carry out Western-blot with viral antiserum(antisera), in conjunction with having adopted the west nile virus detection kit of this recombinant antigen protein in its sensitivity test, specific test result, furthermore understand that the preparation method of recombinant antigen protein of the present invention is in the advantage showing aspect the highly sensitive of high efficient expression recombinant antigen protein and assurance recombinant antigen protein of the present invention, high specific simultaneously.
In addition, the present invention also provides the detection kit of a kind of west nile virus, comprise antibody test plate and ELISA reaction solution, be coated with the recombinant antigen protein that detects west nile virus on described antibody test plate, described recombinant antigen protein has aminoacid sequence as shown in SEQ ID NO.1.
Wherein, in test kit, ELISA reaction solution comprises: enzyme conjugates working fluid, positive control, negative control, sample diluting liquid, concentrated cleaning solution, nitrite ion A, nitrite ion B and stop buffer, enzyme conjugates working fluid, positive control is west nile virus antibody standard substance, and negative control is west nile virus negative antibody serum.
Preferably, the anti-human IgG that is 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 described antibody test plate comprises the steps:
One, the preparation of recombinant antigen protein, comprises the steps:
(1) encoding gene that, presents viral high specific peptide section in selection west nile virus albumen is as goal gene fragment, this goal gene fragment has the nucleotide sequence as shown in SEQ ID NO.2, this goal gene fragment comprises the extension of section of coding recombinant antigen protein flexible arm, the fusion rotein that described flexible arm can be eliminated expression vector is covered polypeptide epitope, makes the recombinant antigen protein of expressing correctly folding;
(2), design pcr amplification primer, and by goal gene fragment described in pcr amplification;
(3), by restriction enzyme digestion and connection, goal gene sheet segment body is connected in prokaryotic expression carrier, build contain described goal gene fragment recombinant expression plasmid;
(4), described recombinant expression plasmid is converted in competent expression host cell, cultivating described expression host cell makes it produce recombinant antigen protein, and obtaining recombinant antigen protein by purifying, described recombinant antigen protein has aminoacid sequence as shown in SEQ ID NO.1;
Two, the recombinant antigen protein after purifying is fixed on to elisa plate, this process comprises that the recombinant antigen protein that purifying in step 1 is obtained is respectively with the coated elisa plate of step concentration, every hole 50-200ul, 4 ℃ of coated spending the night, then with phosphoric acid salt tween damping fluid washing 1-2 minute; After being patted dry, in 37 ℃ of sealing 1-3 hour, after drying, obtain described antibody test plate with 5% bovine serum albumin solution.
Moreover, embodiment in conjunction with recombinant antigen protein of the present invention for the detection kit of west nile virus, absolutely prove the utilization of recombinant antigen protein of the present invention in preparation diagnosis west nile virus test kit, can guarantee sensitivity and specificity in west nile virus diagnostic procedure.
Also it should be noted that, research and development unit of the present invention is the entire PLA and arboviruses specialized laboratory of Guangdong Province, has collected domestic comparatively comprehensively arboviruses strain, and has prepared its standard serum, for good basis has been established in the specificity checking of recombinant antigen protein.The experiment content disclosing in conjunction with the embodiments can be found out, in experiment, guaranteeing on the basis of reactionogenicity, shortened antigen fragment length as far as possible, guarantee that screened prokaryotic expression antigen possesses good specificity, with tested Syndebis, Xi Menlike, Ma Yaluo, Luo Sihe, aigret mountain, Gai Ta, chikungunya, Dengue 1 ~ 4 type, epidemic encephalitis type B and the equal no cross reaction of yellow fever virus, S/N ratio is more than 25.Simultaneously, the preparation method who sets up and the detection kit of west nile virus all possess higher sensitivity and inspection range, the experiment of carrying out with west nile virus mouse immune serum shows, in serum 1:100 ~ 1:25600 times dilution range, all can obtain positive findings, detect OD value more than 0.19, and without non-specific result.
Accompanying drawing explanation
Fig. 1 is the western blot results of hybridization of west nile virus in the embodiment of the present invention 1, encephalitis b virus, dengue 1-type virus polyclonal antibody and prokaryotic expression antigen; Wherein, a:SDS PAGE electrophorogram; B: west nile virus virus polyclonal antibody Wester blot result; C: encephalitis b virus polyclonal antibody Wester blot result; D: dengue 1-type virus polyclonal antibody Wester blot result.1. not induction contrast; M. molecular weight of albumen Marker; 2.WnV 16+pMAL c2x; 3.pMAL c2x.
Antigen coated concentration experimental result picture in Fig. 2 embodiment of the present invention 2.
Fig. 3 is ELISA test kit and application thereof in the embodiment of the present invention 2; Wherein, the experiment of b:ELISA specificity, A1 hole adds dengue 1-type virus antibody, B1 is dengue 2-type virus antibody, and C1 is dengue 3 virus antibody, and D1 is 4-type dengue virus antibody, E1 is B encephalitis virus antibody, F1 is yellow fever virus antibody, and G1 is west nile virus antibody, and H1 is Ma Yaluo antiviral antibody, A2 hole adds ross river virus antibody, B2 is sagiyama virus antibody, and C2 is getah virus antibody, and D2 is Chikungunya virus antibody, E2 is sindbis alphavirus antibody, F2 is the Xi Menli gram of malicious antibody of virus, the negative serum of G2, and H2 is blank; C:ELISA sensitivity experiments, Orthogonal Method method is diluted mouse-anti west nile virus polyclonal antibody, measure ELISA method susceptibility, wherein A1 is that the how anti-1:200 of mouse-anti west nile 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 easier to understand, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.
The experimental technique of unreceipted actual conditions in embodiment below, conventionally according to normal condition, for example condition described in Sambrook equimolecular clone enforcement manual.
In the following example of the present invention, the west nile virus international standard strain of selecting is introduced by Nat'l Pharmaceutical & Biological Products Control Institute, toxic strain sample in applicant laboratory; And the restriction endonuclease, ligase enzyme, T carrier used in experiment are purchased from Dalian TaKaRa company.Gel-purified recovery and plasmid rapid extraction test kit are purchased from OMEGA company.Trizol and reverse transcription reagent are purchased from Invitrogen company.PET32a expression vector is purchased from Novagen company, pMAL-C2x expression vector is purchased from New England Biolabs company, Amylose Resin affinity chromatography reagent is purchased 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 west nile virus
Preparation detects the recombinant antigen protein of west nile virus in accordance with the following steps:
The encoding gene that presents viral high specific peptide section in step 1, selection west nile virus albumen is as goal gene fragment, this goal gene fragment has the nucleotide sequence as shown in SEQ ID NO.2, this goal gene fragment comprises the extension of section of coding recombinant antigen protein flexible arm, the fusion rotein that described flexible arm can be eliminated expression vector is covered polypeptide epitope, makes the recombinant antigen protein of expressing correctly folding;
When specific implementation realizes, in above-mentioned steps one, with reference to wnv cdna group sequence, utilize DNAstar and ANTHEPROT software to carry out detailed analysis to its proteins encoded hydrophobicity, antigenicity, accessibility and plasticity-, and with reference to the sequence information in Genbank, epitope to prediction is compared, 30 sections of the possible antigenicity fragments of preliminary screening.
In step 2, according to a kind of selected antigenicity fragment of step, and the extension of section needing in Binding peptide structure, and by the optimization design primer corresponding with each section, by the corresponding goal gene fragment of pcr amplification, thereby the fusion rotein that makes can to eliminate expression vector by extension of section coding flexible arm is covered polypeptide epitope, make the recombinant antigen protein of expressing correctly folding.For this reason, in the preferred process of this step, design pcr amplification primer 30 is right, as shown in Table 1:
Table one antigen fragment clone the primer sequence
Wherein, design of primers process is right according to the primer 30 described in antigenicity analysis consequence devised table one, and imports BamH I and Hind III restriction enzyme site respectively at primer 5 ' end and 3 ' end.
As the preliminary step that obtains goal gene sheet degree, also need the genome of reverse transcription west nile virus, this transcriptive process,reversed is: west nile virus 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, the each 2 μ l of reverse primer, dNTP 8 μ l, Taq 2U, moisturizing is carried out pcr amplification to cumulative volume 50 μ l.Reaction parameter is: 95 ℃ of 5min, then 94 ℃ of 30s, 55 ℃ of 40s, 72 ℃ of 60s, totally 32 circulations, circulate for the last time latter 72 ℃ and extend 7min, amplified fragments is purified, reclaim after ,-20 ℃ save backup.
In step 3, by restriction enzyme digestion and connection, goal gene sheet segment body is connected in prokaryotic expression carrier, builds the recombinant expression plasmid that contains goal gene fragment.In embodiments of the present invention, described prokaryotic expression carrier is pMAL c2x prokaryotic expression carrier, and described expression host cell is intestinal bacteria.This step is specific as follows:
Enzyme is cut: respectively get the purified amplified production of 10ul, add respectively Buffer K 3ul, BamH I and the each 1ul(10U of Hind III), sterilizing deionized water 15ul, 37 ℃ of enzymes are cut and are spent the night.Separately get pMAL c2x expression vector plasmid 2ug, add Buffer K 3ul, BamH I and the each 1ul(10U of Hind III), sterilizing deionized water is mended to 30ul, and 37 ℃ of enzymes are cut and are spent the night.Enzyme is cut product and is reclaimed test kit with TaKaRa company gel respectively and purify recovery.
Connect: get 2ul through the above pMAL c2x carrier (about 130ng) of processing, 5ul enzyme is cut object fragment (about 40ng), 45 ℃ of heating 5min, so that the cohesive end of annealing unwinds, ice bath 10sec immediately, adds T4DNA ligase enzyme 0.5U, ligase enzyme Buffer 1ul, heat up in a steamer water with sterilizing four and mend to 10ul, 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, after 37 ℃ of recovery 45min, get 100ul and be coated with penbritin (100ug/ml) plate, cultivate 18h for 37 ℃.
Recon is identified: picking white colony carries out bacterium colony PCR evaluation, PCR reactant: 10 × PCR damping fluid, 2 μ l, and forward primer, the each 1 μ l of reverse primer, dNTP 0.4 μ l, Taq 2U, moisturizing is carried out pcr amplification to cumulative volume 20 μ l.The same step 2 of PCR reaction conditions, positive colony send the order-checking of biotechnology (Shanghai) Co., Ltd. to confirm.Confirm to obtain 17 groups of correct recombinant expression plasmids through order-checking, and extract plasmid, and-20 ℃ save backup.
In step 4, recombinant expression plasmid is converted in competent expression host cell, culture expression host cell, and detect its expression of recombinant proteins situation, this step is specially in the present embodiment: the single bacterium colony of expressive host bacterium of choosing through transforming recombinant expression vector is inoculated in respectively in LB liquid nutrient medium, 4h is cultivated in 37 ℃ of concussions, in the time that OD value reaches 0.6, draw not induction contrast bacterium 1ml, and to add inductor IPTG to make its final concentration to residue in bacterium be 0.5mmol/L, in 37 ℃ of shaking culture 4h, after ice bath 5min, 4 ℃, the centrifugal 2min of 12000g receives bacterium, and carry out SDS-PAGE detection, with reference to 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, coomassie brilliant blue staining, checking expression of recombinant proteins situation.Through the experiment of this step, in 17 constructed recombinant expression vectors, 11 are obtained high efficient expression.
Then, obtained recombinant expressed antigen is carried out to western blot analysis, to verify the specificity of its immune response originality and immunological response, this step is specially in the present embodiment: the same step 4 of SDS-PAGE electrophoresis, after electrophoresis, electricity turns nitrocellulose filter, chikungunya, Syndebis, ross river virus polyclonal antibody 1:5000 dilution, carry out western blotting (western-blot), the atopic of checking antigen expressed.Obtain engineering strain one strain of the high efficient expression west nile virus specificity recombinant antigen of energy: E.coli-WnV16.Therefore, in embodiments of the present invention, the goal gene fragment that final optimization pass obtains has the nucleotide sequence as shown in SEQ ID NO.2, recombinant antigen protein has aminoacid sequence as shown in SEQ ID NO.1, corresponding with it, in step 2, the pcr amplification primer sequence of selected employing is as follows:
Forward primer: CGGGATCCCCACCAGGCACTTCAGATCCA<SEQ ID NO.3>
Reverse primer: ACGCAAGCTTTTAGGCAATCTCATTCCCCATCTT<SEQ ID NO.4>.
Be WnAg16-1 and WnAg16-2 in corresponding table one.
In addition, carry out the expression and purification experiment of above-mentioned recombinant antigen protein by this efficient expression strain, obtain the recombinant antigen protein that is suitable for ELISA experiment, in the present embodiment, this step is specially: E.coli-WnV16 bacterium overnight culture 10ml inoculation 1L LB substratum (containing 100 μ g/ml penbritins), 37 ℃ of shaking culture 2 ~ 3 hours, reach at 0.6 o'clock until OD value, add IPTG to final concentration 0.5mM, continue 37 ℃ of shaking culture after 4 hours, centrifugal results bacterium, and use PBS washed twice, 50mlPBS is resuspended, after ultrasonic disruption bacterium, adopt the Amylose Resin of NEB company affinity chromatography column purification purification of recombinant proteins, its concentration of nucleic acid-protein analysis-e/or determining is 2316ug/ml.
As shown in Figure 1, carry out SDS-PAGE experiment and western blotting, with reference to 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, electricity turns nitrocellulose filter, chikungunya, Syndebis, ross river virus polyclonal antibody 1:5000 dilution, carry out western blotting (western-blot), the atopic of checking antigen expressed, and the engineering strain of the high efficient expression west nile virus specificity recombinant antigen of acquisition energy.Then carry out the expression and purification experiment of above-mentioned recombinant antigen protein by this efficient expression strain, obtain the recombinant antigen protein that is suitable for ELISA experiment.
The coated experiment of embodiment 2, recombinant antigen protein
Need in the present embodiment coated condition and the concentration of recombinant antigen protein of the present invention to be optimized; Also need first ELISA reaction solution composition in test kit to be described in order to reach this object:
Enzyme conjugates working fluid: the anti-human IgG polyclonal antibody of horseradish peroxidase-labeled;
Positive control: west nile virus Positive Sera.
Negative control: west nile 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 gentamicin;
Nitrite ion A:0.02%H2O2; With 0.1M citric acid-0.2M Sodium phosphate dibasic, PH4.5～5.0 dilution.
Nitrite ion B:0.4 ‰ TMB-HCl: use 50mM Trisodium Citrate, dense HCl adjusts the solution that pH value is 2.8 to dissolve.
Stop buffer: 2M H
2sO
4
By the purification of Recombinant antigen protein antigen obtaining in above-described embodiment 1 respectively with 10, 5, 2.5, 1.25, 0.63, 0.32, 0.16, 0.08, 0.04, 0.02ug/ml concentration is coated with elisa plate, every hole consumption 100ul, 4 ℃ of coated spending the night, PBST washing 3 minutes, pat dry, 37 ℃ of sealings of 5%BSA 2 hours, after mouse-anti west nile virus polyclonal antibody 1:5000 doubly dilutes, get 100ul and add reacting hole, 37 ℃ are reacted 1 hour, PBST washing 4 times, each 1 minute, pat dry, after the anti-1:10000 of the anti-mouse two of HRP mark doubly dilutes, every hole adds 100ul, 37 ℃ are reacted 40 minutes, PBST washing 4 times, each 1 minute, pat dry, every hole adds tmb substrate solution 100ul, 37 ℃ are reacted 15 minutes, add 50ul 2M H2SO4 termination reaction, under 450nm wavelength, measure OD value by microplate reader, as shown in Figure 2, result is presented within the scope of coated concentration 2.5 ~ 10ug/ml, experiment OD value is without significant difference, the antigen coated concentration of final selection is 5ug/ml.
Antigen coated condition and ELISA reaction conditions are carried out to system optimization, final determine that best ELISA reaction conditions is: recombinant antigen 100ul(concentration is 10ug/ml) 4 ℃ of coated spending the night, PBST washing 3 minutes, pat dry, 37 ℃ of sealings of 5%BSA 2 hours, after serum 1:100 to be checked doubly dilutes, get 100ul and add reacting hole, 37 ℃ are reacted 1 hour, PBST washing 4 times, each 1 minute, pat dry, after the anti-1:10000 of HRP mark two doubly dilutes, every hole adds 100ul, 37 ℃ are reacted 40 minutes, PBST washing 4 times, each 1 minute, pat dry, every hole adds tmb substrate solution 100ul, 37 ℃ are reacted 15 minutes, add 50ul 2M H
2sO
4termination reaction is measured OD value under 450nm wavelength by microplate reader.
The specificity experiment of embodiment 4, test kit
Adopt the optimal conditions in embodiment 3 to carry out ELISA experiment, the sample that selection contains Syndebis, Xi Menlike, Ma Yaluo, Luo Sihe, aigret mountain, Gai Ta, Dengue 1 ~ 4 type, epidemic encephalitis type B, yellow heat and west nile virus polyclonal antibody is as specificity laboratory reference sample, as shown in Table 2, the detection kit of west nile 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 west nile virus ELISA detection method specificity
The sensitivity experiment of embodiment 5, test kit
The purification of Recombinant antigen protein obtaining in embodiment 1 is coated with to elisa plate with 10ug/ml, by Orthogonal Method method, mouse-anti west nile virus polyclonal antibody is diluted, adopt the optimal conditions of implementing in 3 to carry out ELISA experiment, as shown in Table 3, measuring its inspection range is 1:100 ~ 1:12800.
Table three west nile virus ELISA detection method sensitivity experiments
Finally should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although the present invention is explained in detail with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can modify or be equal to replacement technical scheme of the present invention, and not depart from essence and the scope of technical solution of the present invention.
Claims (10)
1. a recombinant antigen protein that detects west nile virus antibody, is characterized in that: the aminoacid sequence of described recombinant antigen protein is as shown in SEQ ID NO.1.
2. the recombinant antigen protein of detection west nile virus antibody according to claim 1, is characterized in that: the nucleotide sequence of the encoding gene of described recombinant antigen protein is as shown in SEQ ID NO.2.
3. a preparation method who detects the recombinant antigen protein of west nile virus antibody, is characterized in that, comprises the steps:
One, the encoding gene that presents viral high specific peptide section in selection west nile virus albumen is as goal gene fragment, the nucleotide sequence of this goal gene fragment is as shown in SEQ ID NO.2, this goal gene fragment comprises the extension of section of coding recombinant antigen protein flexible arm, the fusion rotein that described flexible arm can be eliminated expression vector is covered polypeptide epitope, makes the recombinant antigen protein of expressing correctly folding;
Two, design pcr amplification primer, and by goal gene fragment described in pcr amplification;
Three, by restriction enzyme digestion and connection, goal gene sheet segment body is connected in prokaryotic expression carrier, build the recombinant expression plasmid that contains described goal gene fragment;
Four, described recombinant expression plasmid is converted in competent expression host cell, cultivating described expression host cell makes it produce recombinant antigen protein, and obtaining recombinant antigen protein by purifying, the aminoacid sequence of described recombinant antigen protein is as shown in SEQ ID NO.1.
4. preparation method according to claim 3, is characterized in that, the pcr amplification primer sequence adopting in described step 2 is as follows:
Forward primer: CGGGATCCCCACCAGGCACTTCAGATCCA
Reverse primer: ACGCAAGCTTTTAGGCAATCTCATTCCCCATCTT.
5. preparation method according to claim 3, is characterized in that: described prokaryotic expression carrier is pMAL c2x prokaryotic expression carrier, and described expression host cell is intestinal bacteria.
6. the detection kit of a west nile virus, comprise antibody test plate and ELISA reaction solution, it is characterized in that: on described antibody test plate, be coated with the recombinant antigen protein that detects west nile virus antibody, the aminoacid sequence of described recombinant antigen protein is as shown in SEQ ID NO.1.
7. detection kit according to claim 6, it is characterized in that, in test kit, ELISA reaction solution comprises: enzyme conjugates working fluid, positive control, negative control, concentrated cleaning solution, nitrite ion A, nitrite ion B and stop buffer, enzyme conjugates working fluid, positive control is west nile virus antibody standard substance, and negative control is west nile virus negative antibody serum.
8. detection kit according to claim 7, is characterized in that: the goat anti-mouse igg that described enzyme conjugates working fluid is horseradish peroxidase-labeled.
9. detection kit according to claim 6, is characterized in that, the preparation process of described antibody test plate comprises the steps:
One, the preparation of recombinant antigen protein, comprises the steps:
(1) encoding gene that, presents viral high specific peptide section in selection west nile virus albumen is as goal gene fragment, the nucleotide sequence of this goal gene fragment is as shown in SEQ ID NO.2, this goal gene fragment comprises the extension of section of coding recombinant antigen protein flexible arm, the fusion rotein that described flexible arm can be eliminated expression vector is covered polypeptide epitope, makes the recombinant antigen protein of expressing correctly folding;
(2), design pcr amplification primer, and by goal gene fragment described in pcr amplification;
(3), by restriction enzyme digestion and connection, goal gene sheet segment body is connected in prokaryotic expression carrier, build contain described goal gene fragment recombinant expression plasmid;
(4), described recombinant expression plasmid is converted in competent expression host cell, cultivating described expression host cell makes it produce recombinant antigen protein, and obtaining recombinant antigen protein by purifying, the aminoacid sequence of described recombinant antigen protein is as shown in SEQ ID NO.1;
Two, the recombinant antigen protein after purifying is fixed on to elisa plate, this process comprises that the recombinant antigen protein that purifying in step 1 is obtained is respectively with the coated elisa plate of step concentration, every hole 50-200ul, 4 ℃ of coated spending the night, then with phosphoric acid salt tween damping fluid washing 1-2 minute; After being patted dry, in 37 ℃ of sealing 1-3 hour, after drying, obtain described antibody test plate with 5% bovine serum albumin solution.
10. the utilization of recombinant antigen protein as claimed in claim 1 or 2 in preparation diagnosis west nile virus test kit.
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