CN106841601A - The preparation of zika virus multiple clips fusion protein and IgG/IgM antibody assay kits - Google Patents
The preparation of zika virus multiple clips fusion protein and IgG/IgM antibody assay kits Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
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- G—PHYSICS
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Abstract
It is an object of the invention to provide a kind of simple and rapid zika virus detection kit.The preferred amalgamation and expression albumen of the kit is used as diagnostic antigen, anti-human igg monoclonal antibody A374, anti-human IgM monoclonal antibody A371 and biotin BSA conjugate are coated on nitrocellulose filter as detection line and nature controlling line respectively, it is equipped with the amalgamation and expression albumen of colloid gold label and the Streptavidin of colloid gold label and other reagents, using the specific IgM antibody of zika virus, IgG antibody in immunochromatography prize law principle qualitative detection human serum, so as to realize quick, the specific diagnostic of zika virus infection.
Description
Technical field
Data Bio technical field of the present invention, is related to a kind of using the quick expression technology preparation of genetic engineering multiple clips fusion
Zika virus diagnostic antigen, and zika virus IgG/IgM antibody fast detecting kits (colloidal gold method) preparation and its use.
Background technology
2015, zika virus quickly spread in the whole world, and epidemic situation report, world health occur in existing 24 countries and regions
Declaration on 2 1st, 2016 is organized in, zika virus and microcephaly are classified as global emergency public health event.
Zika virus disease (Zika virus disease) belongs to the Flavivirus of yellow fever virus section, is by zika virus
(Zika virus), a kind of self limiting acute infectious disease for causing.Zika virus passes through the Aedes mosquito bite being infected
Travel to people, it is also possible to fetus or baby are broadcast in pregnancy or production process by the mother being pregnant, it is also possible to which generation property is passed
Broadcast, or Transfusion Transmission.After infection, there is symptom, including heating, skin disease, conjunctivitis, muscle and joint in only 20% patient
Bitterly, malaise and headache etc., these symptoms are typically lasted for 2~7 days.Zika virus is likely to result in nerve and autoimmunity
The complication of system, such as actue infectious polyradiculoneuritis (Guillian-Barr é syndrome), infection of pregnant women zika virus may
Cause baby's microcephalus.
At present, the whole world is had found more than 4000 neonate's microcephalus cases.China has found Imported cases totally 8
Example.Zika virus is abnormal severe in Global prevalence situation.Accordingly, it would be desirable to a kind of quick, easy, special detection kit is come
Reply.And zika virus and the dengue fever virus of same kind have a homology very high, and two kinds of virus be through killing propagation, because
How this, differentiate zika virus and dengue virus infection is just particularly important.
In the recent period, also some producer's zika virus diagnostic antigens both domestic and external come out, but often due to the gene of antigen upstream
The corresponding epitope of sequence does not do any modification, causes the cross reaction inevitably with dengue fever virus.
Therefore, the present invention carries out epitope analysis using bioinformatic analysis technology to zika virus and dengue fever virus,
Difference epitope is chosen, amalgamation and expression then is carried out to special epitope using one-step method amalgamation and expression technology, be respectively compared
Its diagnosis effect, and finally select that sensitivity is high, high specificity (with dengue fever virus no cross reaction) fusion protein conduct
Diagnostic antigen.
Immune colloidal gold technique is very rapid as a kind of class development of new techniques that is immunized in recent years, in biomedical each research
Field is particularly and increasingly extensive application has been obtained in medical test, it has, and simple to operate, detection is quick, specificity is good,
Sensitivity advantage high.
On this basis, the preparation of kit is carried out using fast and convenient colloidal gold technique, the diagnosis of zika virus is allowed
It is sensitiveer, special, convenient, so that preferably for public health provides Fast Detection Technique.
The present invention is the advantage using genetic engineering amalgamation and expression technology and collaurum methodology, and succeeding in developing one kind can
Distinguish the quick diagnosis reagent kit of the zika virus of dengue virus infection.This kit has sensitivity high, high specificity, steady
The qualitative remarkable advantage such as good, easy to operate and with low cost.
The content of the invention
It is an object of the invention to provide a kind of simple and rapid zika virus detection kit (colloidal gold method).The reagent
Box is using the preferred amalgamation and expression albumen of independent research as diagnostic antigen, and anti-human igg monoclonal antibody A374, anti-human IgM are mono-
Clonal antibody A371 and biotin-BSA conjugate are coated on nitrocellulose filter as detection line and nature controlling line respectively, are equipped with
The amalgamation and expression albumen of colloid gold label and the Streptavidin of colloid gold label and other reagents, using immunochromatography prize law
The specific IgM antibody of zika virus in principle qualitative detection human serum, IgG antibody, so as to realize zika virus infection
Quickly, specific diagnostic.
The invention reside in application bioinformatics technique com-parison and analysis zika virus and dengue fever virus gene order, choose
The distinctive linear epitope of zika virus carries out multi-epitope amalgamation and expression purifying, and preferably goes out recombinant protein ZIKV-Ag2 as diagnosis
Antigen, zika virus in sample is detected with colloid gold label.
Kit of the invention include 1) detection reagent card, 2) sample treatment liquid and 3) separate and concentrate packaging these reagents
The packing box of bottle or pipe.Wherein, detection reagent card by 1) sample pad, 2) gold conjugation pad, 3) chromatographic film, 4) absorbent material and
5) liner plate composition, and it is above-mentioned 1)~4) overlap from beginning to end and be attached on 5)；And the gold conjugation pad is to be coated with gold
The glass fibre membrane of mark fused antigen ZIKV-Ag2 and golden labelled streptavidin；Chromatographic film is to be coated with anti-human igg list
Clonal antibody A371, anti-human IgM monoclonal antibody A372, the nitrocellulose filter of biotin-BSA；Liner plate is PVC materials；Sample
Product treatment fluid is by the heterophile antibody blocking agent of 1~50mg/mL and 0.02%~0.06%NaN3Preservative is constituted.
In order to realize the present invention, inventor employs following technical scheme：
(1) bioinformatics is compared：Choose zika virus carries out bioinformatics with dengue fever virus full length gene sequence
Compare analysis, the special linear epitope of analysis zika virus；
(2) combination of gene-specific fragments：Filter out the corresponding genetic fragment of the special linear epitope of zika virus
Sequence 1,2,3；And fragment 1,2,3 is carried out alone or in combination, respectively respectively：
1. fragment 1；
2. fragment 2；
3. fragment 3；
4. fragment 1+ fragments 2 are merged；
5. fragment 1+ fragments 3 are merged；
6. fragment 2+ fragments 3 are merged；
7. fragment 1+ fragments 2+ fragments 3 are merged；
(4) clonal expression of genetic fragment and purifying：7 kinds of genetic fragment combinations are each carried out into PCR primer design, is expanded
Increasing, construction recombination plasmid and induced expression, extracted recombinant plasmid simultaneously carry out a large amount of induced expressions fusions after digestion, sequence verification
Albumen；
(5) cross match experiment：4) the fusion protein mark gold that will be obtained in respectively with anti-human igg monoclonal antibody, anti-
People's IgM monoclonal antibody carries out cross match experiment, it is determined that being merged to sensitivity, specificity highest with what monoclonal antibody was matched somebody with somebody
Albumen, as the antigen of gold-labelled diagnosis；
(6) colloid gold reagent is prepared：Zika virus IgG/IgM antibody colloidal golds are prepared using the antigen filtered out in 5) to examine
Test agent.
Zika virus specific gene fragment is respectively following 3 fragment gene sequences in the present invention,
Preferably, in 7 kinds in above-mentioned technical proposal restructuring 4., 5. with 6. three kinds combine and enter respectively performing PCR amplification, weigh
Group, expression, finally obtain positive recombinant plasmid；Three kinds of corresponding amplimers of combination are as follows successively：
Preferably, above-mentioned 4., 5. with 6. three kinds of amplified productions for combining are carried out into weight with the expression vector of linearisation respectively
Group reaction construction recombination plasmid ZIKV-P1, ZIKV-P2 and ZIKV-P3.
Preferably, induced expression is carried out to recombinant plasmid, by three kinds of recombinant plasmids ZIKV-P1, ZIKV-P2 and ZIKV-P3
The Amplification Culture to Escherichia coli is converted respectively, and extracted recombinant plasmid simultaneously carries out a large amount of induced expressions after digestion, sequence verification
Fusion protein ZIKV-Ag1, ZIKV-Ag2 and ZIKV-Ag3 are simultaneously purified.
Preferably, will obtain after purification ZIKV-Ag1, ZIKV-Ag2, ZIKV-Ag3 mark gold respectively with anti-human igg list
Clonal antibody, anti-human IgM monoclonal antibody carry out cross match experiment, to determine that sensitivity, specificity highest and antibody are matched somebody with somebody
To antigen (fusion protein)；It is final to determine ZIKV-Ag2 as the antigen for colloidal gold method detection zika virus.
Brief description of the drawings
Fig. 1 multiple clips clonal expression schematic diagrames.
Fig. 2 multiple clips clone-fragment is chosen.
Fig. 3 multiple clips clone-PCR primer (note：M, DL2000marker；1, pcr-product 1；2, pcr-product
2；3, pcr-product 3；NC, negative control without template.).
Fig. 4 multiple clips clone-protein expression and purifications product (note：M, albumen marker；1, recombination fusion protein ZIKV-
Ag1；2, recombination fusion protein ZIKV-Ag2；3, recombination fusion protein ZIKV-Ag3.).
Fig. 5 collaurum principle schematics.
Fig. 6 kit results interpretation schematic diagrames.
Specific embodiment
The expression of the fusion protein of embodiment 1, purifying
1st, the preparation of expression vector is linearized：(such as Fig. 1) chooses prokaryotic expression carrier pET28a, using restriction enzyme site Bam
H I and Xhol I are linearized to prokaryotic expression carrier double digestion to it, and carrier is standby after the double digestion of recovery purifying linearisation.
Double digestion system is as follows：
2nd, the preparation (principle is see accompanying drawing 2) of multiple clips PCR primer：Design corresponding amplimer, Insert Fragment 1,2,3
PCR amplifications respectively.
PCR reaction systems are as follows：
PCR reaction conditions：
Pcr amplification product is as shown in Figure 3, it is seen that amplify required purpose band.
3rd, the preparation of recombinant plasmid：Linearisation expression vector and Insert Fragment amplified production prepared by above-mentioned steps 1,2
Recombining reaction system is prepared, 37 DEG C of reaction 30min can be rapidly completed recombining reaction, realize the external cyclisation of two linear DNAs, i.e.,
Obtain recombinant plasmid.
4th, product conversion, coated plate：Recombinant products are directly converted, picking positive monoclonal, are put into LB culture mediums, 37
DEG C, 200~250rpm, 4~5h of oscillation incubation culture.
5th, the plasmid checking of row double digestion, rear sequence verification again are extracted.
6th, recombinant plasmid ZIKV-P1, ZIKV-P2, ZIKV-P3 are converted to e. coli bl21/DE3 respectively, IPTG is lured
Lead expressed fusion protein, purified fusion protein ZIKV-Ag1, ZIKV-Ag2, ZIKV-Ag3, as a result as shown in figure 4, institute can be obtained
The destination protein for needing.
The fused antigen of embodiment 2 matches screening experiment
1. fusion protein ZIKV-Ag1, ZIKV-Ag2, ZIKV-Ag3 after purification is marked into gold respectively, it is anti-human with outsourcing
IgG monoclonal antibody A371, anti-human IgM monoclonal antibody A372 carry out pairing experiment respectively, with Checkerboard titration method cross match,
Sensitivity, specificity highest pairing antigen-antibody.The results are shown in Table -4.
The antigen-antibody of table -4 matches screening experiment result
Result shows：When ZIKV-Ag2 is used as labelled antigen, when A371, A372 are as labelled antibody, sensitivity, specificity
Equal highest (up to 100%), so selection ZIKV-Ag2 and A371, A372 are used for the pairing antigen-antibody of this detection reagent.
The composition of the kit of embodiment 3
1st, detection reagent card：1. sample pad, 2. gold conjugation pad (glass fibre membrane)：Dry gold mark is adsorbed on film
Antigen and gold mark Streptavidin (fluxion strap), 3. chromatographic film (nitrocellulose filter), and antigen or antibody band are coated with film
With Quality Control thing band (detection band) that can be directly reacted with label, 4. absorbent material (blotting paper), 5. liner plate (PVC glue
Plate).Above each component head and the tail overlap and are attached on PVC offset plates (as shown in Figure 5).
2nd, sample treatment liquid：1 bottle (10 person-portions are filled：1.0mL；25 person-portions are filled：2.5mL；50 person-portions are filled：5.0mL), mainly into
It is divided into heterophile antibody blocking agent (1~50mg/mL of concentration range).Preservative：0.02%~0.06%NaN3。
The application method of the kit of embodiment 4
1. reagent is taken out, reagent card packaging is torn, drop 2 drips sample treatment liquid to well.
2. drop 1 drips test serum to well, judged result in 5~20min, and sentence read result is invalid after 20 minutes.
3. result interpretation：
1. it is positive (+)：There are three aubergine bands to occur in observation window, respectively positioned at detection zone (M, G) and quality control region (C)
It is interior, prompt for containing zika virus specific IgM, IgG antibody in sample.
2. it is positive (+)：There are two aubergine bands to occur in observation window, respectively positioned at detection zone (M) and quality control region (C)
It is interior, prompt for containing zika virus specific IgM antibodies in sample.
3. it is positive (+)：There are two aubergine bands to occur in observation window, respectively positioned at detection zone (G) and quality control region (C)
It is interior, prompt for containing zika virus specific IgG antibodies in sample.
4. it is negative (-)：Only there is an aubergine band in quality control region (C), is taken out of without aubergine bar in detection zone (M, G)
It is existing, point out to be free of zika virus specific IgM, IgG antibody in sample.
5. it is invalid：There is not aubergine band in quality control region (C), shows that incorrect operating process or reagent have gone bad damage
It is bad.In this case, should retest.
Direct schematic diagram, is shown in accompanying drawing 6.
The foundation of the reaction system of embodiment 5 and optimization
1. the pretreatment of protein：After obtaining preferred recombination fusion protein ZIKV-Ag2, pretreatment side need to be carried out to it
Can be used for golden mark.1. protein first to the water dialysis of LIS should remove salt composition.Salt composition can influence colloid
Absorption of the gold to protein, and collaurum coagulation can be made, the presence of phosphate anion and borate ion should be avoided.2. micropore is used
Filter membrane or ultracentrifugation remove the fine particles in protein solution.3. absorption of the collaurum to albumen depends primarily on pH value,
Under conditions of the isoelectric point close to protein or slightly biased alkali, the two easily forms firm conjugate.If the pH of collaurum
When value is less than the isoelectric point of protein, then can assemble and lose binding ability.
2. protein optimum dose is determined：After protein (ZIKV-Ag2) storing liquid to be marked is serially diluted, point
0.1ml is not taken to be added in 1ml colloidal gold solutions, the control tube that a pipe is not added with protein is separately set, and 0.1ml 10% is added after 5 minutes
NaCl solution, stands 2 hours, shown in table -6 specific as follows after mixing.
The protein of table -6 optimum dose is determined
Result judges：If control tube adds protein content not enough, there is coacervation from red to blue in collaurum；Add albumen
Quality meets or exceeds stable quantity, then collaurum keeps red constant.
Unstable aurosol will occur coagulation, control tube (not plus protein) and add the amount of protein to be not enough to stabilization
Each pipe of collaurum, shows coagulation phenomenon from red to blue；And add protein content to meet or exceed each of minimum stable quantity
Pipe still keeps red constant.To stablize the red constant minimum enzyme protein dosage of 1ml colloidal gold solutions, the as labelled protein
Minimum amount, in real work, can suitably increase by 10%, in the case absorption of the protein molecule on gold grain surface
Amount is maximum.
After collaurum and protein molecule, stabilizer is added, to avoid producing aggegation.It is general that from PEG, (molecular weight is
20000) with bovine serum albumin(BSA) used as stabilizers.The amount of addition：3%BSA makes solution final concentration of 1%；10% polyethylene glycol adds
To the 1/10 of total solution.
Concrete operations are as follows：
1. 0.1mol/L K are used2CO3Or 0.1mol/L HCl adjust aurosol to required pH.
2. the protein solution of optimum mark amount is added to stir in 100ml aurosols 2~3 minutes.
3. 5ml1%PEG20000 solution is added.
4. carefully supernatant (never toppling over) is sucked within 30~60 minutes in 10000~100000g centrifugations.
5. will precipitate and be suspended in buffer solution of the certain volume containing 0.2~0.5mg/ml PEG20000, after centrifugation,
Recovered with same buffer solution again, concentration is advisable with A540nm=1.5 or so, and anti-corrosion puts 4 DEG C of preservations.
6. the aurosol after being coated with can also concentrate and carry out gel chromatography after Sephadex G-200 posts and isolate and purify, to contain
The cushioning liquid wash-out of 0.1%BSA.It is 8.2 generally with the coated aurosol eluent pH of IgG.
It should be noted that not answering impure particulate in all solution in operating above, can in advance be located with high speed centrifugation or miillpore filter
Reason.
3. the preparation (by taking 100ml colloidal gold solutions as an example) of collaurum：
3.1 prepare colloidal gold solution (100ml)：
3.1.1 preparation：Measure 100ml purified waters to pour into round-bottomed flask, addition stirrer is on magnetic stirring apparatus
Boiling is heated with stirring to, water is outwelled, cleaned with purified water twice.
3.1.2 measure 100ml purified waters to pour into the round-bottomed flask for having cleaned, after addition stirrer under the conditions of 180 DEG C
Magnetic stirring apparatus on heat；When being heated to 80-90 DEG C, 3%HAuCl is added4Solution 2ml；Continue heating stirring to seething with excitement, turn
Velocity modulation is to 900rpm；After fully boiling 1-2min, the existing 5.8% trisodium citrate aqueous solution 3ml for matching somebody with somebody is added；Deng colour stable
Afterwards, 300rpm, continues to close magnetic stirring apparatus after heating 10min, and the colloidal gold solution that will be prepared is cooled to room temperature, with 0.22
It is stand-by that μm filter is filled into clean vial, 2-8 DEG C of preservation.
The mark of 3.2 collaurums, spray film, drying：
3.2.1 metal spraying pipeline is cleaned according to metal spraying pen machine flushing procedure.
3.2.2 the preparation and purification (as a example by 100ml colloidal gold solutions) of zika virus recombinant antigen-colloidal gold conjugate
3.2.2.1 the preparation (as a example by 100ml colloidal gold solutions) of zika virus recombinant antigen-colloidal gold conjugate
(1) to addition 12ml 0.8M K in 100ml colloidal gold solutions2CO3And stir.
(2) under magnetic stirring, 50 μ g zika virus recombinant antigens are added by every milliliter of colloidal gold solution, is stirred at room temperature
40min。
(3) under magnetic stirring, 10ml 1%BSA solution is added, 20min is stirred.
3.2.2.2 the purifying (as a example by 100ml colloidal gold solutions) of zika virus recombinant antigen-colloidal gold conjugate
(1) by gold mark zika virus recombinant antigen 4~10 DEG C, 8000rpm in refrigerated centrifuge obtained by 3.2.2.1
Centrifugation 5min, collection is precipitated in clean centrifuge tube, by supernatant after centrifugation be transferred in another centrifuge tube in 4~10 DEG C,
It is centrifuged 20min under the conditions of 6000rpm, collects precipitation, supernatant is transferred in another centrifuge tube in 4~10 after this is centrifuged
DEG C, 20min be centrifuged under the conditions of 6000rpm collect precipitation, it is so repeated multiple times to collect precipitation, untill supernatant is colourless.
(2) during the precipitation that will be collected into combines in a centrifuge tube, mixed after adding 20ml purified waters, 4 in refrigerated centrifuge
~10 DEG C, 6000rpm centrifugation 5min in collecting precipitation as net centrifuge tube, supernatant after centrifugation are transferred in another centrifuge tube
It is centrifuged 5min under the conditions of 4~10 DEG C, 6000rpm, collects precipitation, during supernatant transfers to another centrifuge tube after this is centrifuged
20min is centrifuged under the conditions of 4~10 DEG C, 6000rpm and collects precipitation, it is so repeated multiple times to collect precipitation, until supernatant is colourless being
Only.
(3) precipitation physiological saline solution, per 100ml, original colloidal gold solution precipitation first plus after 150 μ l physiological saline is mixed,
Be taken out 10 μ l solution physiological saline do 1: 20 dilution after, measured with spectrophotometric as blank using physiological saline
Absorption value under 535nm wavelength, so as to calculate redissolve after gold solution OD values [for example：If solution absorbs after 1: 200 dilution
It is X to be worth, and the absorption value of blank is Y；OD values=(X-Y) * 200 of gold solution after then redissolving], according to the initial OD values for determining
Situation add appropriate physiological saline [for example：The OD values for calculating are X, and product is V1, then the physiological saline volume to be added
V=(X*V1/90)-V1)] so that OD values are saved backup for 20,2-8 DEG C.
3.2.3 the preparation and purification (as a example by 100ml colloidal gold solutions) of Streptavidin-colloidal gold conjugate
3.2.3.1 the preparation of Streptavidin-colloidal gold conjugate
(1) to adding 500 μ l0.1M Na in 100ml colloidal gold solutions2CO3。
(2) under magnetic stirring, 5 μ g Streptavidins are added by every milliliter of colloidal gold solution, 40min is stirred at room temperature.
(3) under magnetic stirring, 2ml 1%BSA solution is added, 20min is stirred.
3.2.3.2 the purifying of Streptavidin-colloidal gold conjugate
(1) by the golden labelled streptavidin obtained by 3.2.3.1 4~10 DEG C in refrigerated centrifuge, 8000rpm centrifugations
20min, in collecting precipitation as net centrifuge tube, supernatant after centrifugation is transferred in another centrifuge tube in 4~10 DEG C, 10000rpm
Under the conditions of be centrifuged 20min, collect precipitation, after this is centrifuged supernatant transfer in another centrifuge tube in 4~10 DEG C,
20min is centrifuged under the conditions of 12000rpm and collects precipitation, it is so repeated multiple times to collect precipitation, untill supernatant is colourless.
(2) during the precipitation that will be collected into combines in a centrifuge tube, mixed after adding 30ml PB cleaning fluids, in refrigerated centrifuge
In 4~10 DEG C, 8000rpm centrifugation 20min, collection be precipitated in clean centrifuge tube, supernatant after centrifugation is transferred to another centrifugation
Be centrifuged 20min in pipe under the conditions of 4~10 DEG C, 10000rpm, collect precipitation, after this is centrifuged supernatant transfer to it is another from
20min is centrifuged under the conditions of 4~10 DEG C, 12000rpm in heart pipe and collects precipitation, it is so repeated multiple times to collect precipitation, until supernatant
Untill colourless.
(3) precipitation physiological saline solution, per 100ml, original colloidal gold solution precipitation first plus after 150 μ l physiological saline is mixed,
Be taken out 10 μ l solution physiological saline do 1: 200 dilution after, measured with spectrophotometric as blank using physiological saline
Absorption value under 535nm wavelength, so as to calculate redissolve after gold solution OD values, according to initial measure OD values situation again
Add appropriate physiological saline so that OD values are saved backup for 20,2-8 DEG C.
3.2.4 by OD535=20 gold mark Streptavidin and OD535=90 gold mark zika virus recombinant antigen is isometric
Mix and be colloid gold label thing.
3.2.5 the colloid gold label thing obtained by 3.2.4 is loaded in metal spraying pen machine, according to the μ l/mm of metal spraying amount 2, spray
Golden speed 20mm/s is sprayed onto on processed golden pad glass fibre, after the completion of put it into 20~37 DEG C, humidity≤40%
16~18 hours in hothouse, drying dries sealing room temperature preservation, as gold conjugation pad.
4 line：
4.1 prepare C lines, G lines and M line solution：
4.1.1 it is coated with buffer solution with 50mM PBS (pH7.0 ± 0.1) and dilutes biotin-BSA conjugate, diluted concentration is extremely
2mg/ml is the line solution of C lines.
4.1.2 it is coated with buffer solution with 100mM PBS (pH7.0 ± 0.1) and dilutes anti-human igg monoclonal antibody A371, dilution
Concentration to 0.2mg/ml for G lines line solution.
4.1.3 anti-human IgM monoclonal antibody A372 is diluted with 100mM PB (pH7.0 ± 0.1) solution, diluted concentration is extremely
0.2mg/ml is the line solution of M lines.
4.2 diaphragms for opening NC films location for paste in PVC board, NC films (305 ± 1) mm is uniformly advanced from left to right, is made
It is firmly pasted onto in PVC board.
4.3 line：
4.3.1 according to metal spraying pen machine flushing procedure cleaning spray membrane tube road.
4.3.2 rule on the NC films being affixed in PVC board, the mark of C lines, G lines and M lines, C are carried out in one end pen of film
The distance of line and G lines is 4mm, and G lines are 4mm with the distance of M lines, and M lines are 6mm with the distance of NC film lower edges, and line amount is 1 μ
L/mm, speed 10mm/s, first draw G, M line, and C lines are drawn afterwards, after the completion of sheet material is put into 20~37 DEG C, in humidity≤40% hothouse
16~18 hours, drying dried sealing room temperature preservation.
5 assemblings：
5.1 get the raw materials ready：Blotting paper is cut into 19mm × 300mm and is absorption pad；Through processing and cut standby sample
Pad and gold conjugation pad；In 22~30 DEG C of temperature, assembled in the environment of relative humidity≤40%.
5.2 are laid on work top PVC board.
5.3 diaphragms for opening PVC board top absorption pad location for paste, absorption pad is adhered thereto, it is uniform, slight to roll
Formula is advanced, to strengthen bonding force, absorption pad covering NC films 2-3mm.
5.4 diaphragms for gently opening NC film lower edge gold conjugation pads location for paste, it is adhered to by gold conjugation pad
On, the same absorption pad of method.Gold conjugation pad should cover NC films 2-3mm.
5.5 diaphragms for opening PVC board bottom, sample pad is adhered on gold conjugation pad, bottom alignment, method
Same absorption pad.Sample pad covering gold conjugation pad 3-4mm.
5.6 PVC boards that will be posted on automatic strip-cutting machine are cut into (3.5 ± 0.1) mm reagent strips wide.
5.7 load in plastic clip reagent strip, and reagent is placed in single part aluminum foil bag, and add 1g drier 1 to wrap,
Heat sealing, it is labelled, as reagent semi-finished product, room temperature preservation.
The stabilization of kit of embodiment 6 is tested
Kit is put in 37 DEG C, 4 DEG C, performance evaluation is done after 21 days, it is desirable to：1. negative reference product coincidence rate：10 parts of the moon
Property reference material testing result should be all negative, false positive must not occur, coincidence rate is 100% (10/10).2. positive reference product are accorded with
Conjunction rate：10 parts of positive reference product testing results should be all positive, false negative must not occur, and coincidence rate is 100% (10/10).③
Accuracy：With the person-portion reagent of accuracy reference material parallel determination 10, reaction result should be consistent, and colour developing degree should be homogeneous.
Experimental result shows：Compared with 4 DEG C, 37 DEG C of indices after 21 days are remained to meet and required.Therefore, initial setting should
Preserved at the shady and cool lucifuge drying of 2~30 DEG C of kit, from packing, the term of validity 18 months.
The performance obtained in laboratory of the kit of embodiment 7
Kit to embodiment 3 carries out performance obtained in laboratory analysis, as a result as follows：
1) negative reference product coincidence rate：10 parts of negative reference product testing results should be all negative, false positive must not occur, accord with
Conjunction rate is 100% (10/10).
2) positive reference product coincidence rate：10 parts of positive reference product testing results should be all positive, false negative must not occur, accord with
Conjunction rate is 100% (10/10).
3) accuracy：With the person-portion reagent of accuracy reference material parallel determination 10, reaction result should be consistent, and colour developing degree should be equal
One.
4) cross reaction：20 dengue fever virus positive serums of detection, have no cross reaction.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of without departing substantially from spirit or essential attributes of the invention, the present invention can be in other specific forms realized.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit requires to be limited rather than described above, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.People in claim and reference should not be considered as the claim involved by limitation.
Claims (7)
1. a kind of preparation of zika virus multiple clips fusion protein and IgG/IgM antibody assay kits, it is characterised in that application
Bioinformatics technique com-parison and analysis zika virus and dengue fever virus gene order, choose the distinctive linear epitope of zika virus
Multi-epitope amalgamation and expression purifying is carried out, and preferably goes out recombinant protein ZIKV-Ag2 as diagnostic antigen, examined with colloid gold label
Zika virus in test sample sheet；And kit include 1) detection reagent card, 2) sample treatment liquid and 3) separate and concentrate packaging these
The packing box of reagent bottle or pipe.
2. a kind of preparation of zika virus multiple clips fusion protein and IgG/IgM antibody assay kits, it is characterised in that stockaded village's card
The preparation process of viral multiple clips fusion protein is comprised the following steps：
1) bioinformatics is compared：Choose zika virus and dengue fever virus full length gene sequence and carry out bioinformatics and compare point
Analysis, the special linear epitope of analysis zika virus；
2) combination of gene-specific fragments：Filter out the corresponding gene fragment order 1 of the special linear epitope of zika virus,
2、3；And fragment 1,2,3 is carried out respectively alone or in combination, respectively 1. fragment 1；2. fragment 2；3. fragment 3；4. fragment 1+ pieces
Section 2 is merged；5. fragment 1+ fragments 3 are merged；6. fragment 2+ fragments 3 are merged；7. fragment 1+ fragments 2+ fragments 3 are merged；
4) clonal expression of genetic fragment and purifying：7 kinds of genetic fragment combinations are each carried out into PCR primer design, amplification, is built
Recombinant plasmid and induced expression, extracted recombinant plasmid simultaneously carry out a large amount of induced expression fusion proteins after digestion, sequence verification；
5) cross match experiment：4) the fusion protein mark gold that will be obtained in respectively with anti-human igg monoclonal antibody, anti-human IgM
Monoclonal antibody carries out cross match experiment, it is determined that with monoclonal antibody match somebody with somebody to sensitivity, specificity highest fusion protein,
As it is used for the antigen of gold-labelled diagnosis；
6) colloid gold reagent is prepared：The detection examination of zika virus IgG/IgM antibody colloidal golds is prepared using the antigen filtered out in 5)
Agent.
3. kit according to claim 1, it is characterised in that detection reagent card in the kit by 1) sample pad, 2)
Gold conjugation pad, 3) chromatographic film, 4) absorbent material and 5) liner plate composition, and it is above-mentioned 1)~4) overlap from beginning to end and be attached to
5) on；And the gold conjugation pad is the glass for being coated with gold mark fused antigen ZIKV-Ag2 and golden labelled streptavidin
Glass tunica fibrosa；Chromatographic film is to be coated with anti-human igg monoclonal antibody A371, anti-human IgM monoclonal antibody A372, biotin-BSA
Nitrocellulose filter；Liner plate is PVC materials.
4. kit according to claim 1, it is characterised in that the sample treatment liquid in the kit is by 1~50mg/mL
Heterophile antibody blocking agent and 0.02%~0.06%NaN3Preservative is constituted.
5. preparation method according to claim 2, is further characterized in that in step 2) described in specific gene be following 3
Fragment gene sequence,
1) fragment 1：
ATGGAAATAATAAAGAAGTTCAAGAAAGATCTGGCTGCCATGCTGAGAATAATCAATGCTAGGAAGGAGAAGA
AGAGACGAGGCGCAGATACTAATGTCGGAATTGTTGGCCTCCTGCTGACCACAGCTATGGCAGCGGAGGTCACTAGA
CGTGGGAGTGCATACTATATGTACTTGGACAGA；
2) fragment 2：
GAATGCCCTATGCTGGATGAGGGGGTGGAACCAGATGACGTCGATTGTTGGTGCAACACGACGTCAACTTGGG
TTGTGTACGGAACCTGCCATCACAAAAAAGGTGAAGCACGGAGATCTAGAAGAGCTGTGACGCTCCCCTCCCATTCC
ACTAGGAAGCTGCAAACGCGGTCGCAAACTTGGTTGGAATCAAGAGAATACACAAAGCACTTGATTAGAGTCGAAAA
TTGGATATTCAGGAACCCTGGCTTCGCGTTAGCAGCAGCTGCCATCGCTTGGCTTTTGGGAAGCTCAACGAGCCAAA
AAGTCATATACTTGGTCATGATACTGCTGATTGCCCCGGCATACAGCATCAGGTGCATAGGAGTCAGCAATAGG；
2) fragment 3：
ATGGCTTCGGACAGCCGCTGCCCAACACAAGGTGAAGCCTACCTTGACAAGCAATCAGACACTCAATATGTCT
GCAAAAGAACGTTAGTGGACAGAGGCTGGGGAAATGGATGTGGACTTTTTGGCAAAGGGAGCCTGGTGACATGCGCT
AAGTTTGCATGCTCCAAGAAAATGACCGGGAAGAGCATCCAGCCAGAGAATCTGGAGTACCGGATAATGCTGTCAGT
TCATGGCTCCCAGCACAGTGGGATGATCGTTAATGACACAGGACATGAAACTGATGAGAATAGAGCGAAGGTTGAGA
TAACGCCCAATTCACCAAGAGCCGAAGCCACCCTGGGGGGTTTTGGAAGCCTAGGACTTGATTGTGAACCGAGGACA
GGCCTTGACTTTTCAGATTTGTATTACTTGACTATGAATAACAAGCACTGGTTGGTTCACAAGGAG。
6. preparation method according to claim 2, is further characterized in that the fragment combination that kit is finally screened is：5. piece
Section 1+ fragments 3 are merged, and corresponding pcr amplification primer thing sequence is respectively：
1)Primer3F：5′-CAGCAAATGGGTCGCGGATCCATGG-3′
Primer3R：5′-TAGGGCATTCTCTGTCCAAGTACATA-3′；
2)Primer4F：5′-ACTTGGACAGAATGGCTTCGGACAGCCGC-3′
Primer4R：5′-GTGGTGGTGGTGGTGCTCGAGCTCCTTGT-3′.
7. preparation method according to claim 5, is further characterized in that step 5) described in antigen be ZIKV-Ag2.
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| CN112114139A (en) * | 2020-09-11 | 2020-12-22 | 博奥赛斯（天津）生物科技有限公司 | Novel coronavirus IgM-IgA-IgG antibody colloidal gold detection kit |
| CN112194709A (en) * | 2020-12-07 | 2021-01-08 | 北京纳百生物科技有限公司 | Immunochromatographic test strip for detecting feline panleukopenia syndrome virus antibody, and preparation method and application thereof |
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