CN107884578A - For echinococcosis antibody test card in Quantitative detection serum - Google Patents
For echinococcosis antibody test card in Quantitative detection serum Download PDFInfo
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Abstract
The invention discloses one kind to be used for echinococcosis antibody test card in Quantitative detection serum, including detection card shell and it is assemblied in test strips in detection card shell, the test strips include the plastic bottom board with pressure sensitive adhesive, paste sample pad successively on bottom plate, labeling pad, nitrocellulose filter and blotting paper, the labeling pad is made up of carrier substrate and label, label is to spray lanthanide fluoro in carrier substrate to detect the tunic that microballoon and lanthanide fluoro Quality Control microballoon are formed, it is detection line that echinococcosis recombinant antigen is coated with the nitrocellulose filter, it is nature controlling line to be coated with rabbit-anti chicken lgY antibody, label is the fluorescence Quality Control microballoon for being marked with echinococcosis recombinant antigen fluoroscopic examination microballoon and marking chicken lgY antibody.The achievable echinococcosis antibody scene Quantitative detection of the present invention, has higher practical value and application value.
Description
Technical field
The present invention relates to one kind detection card and use, be used for echinococcosis in Quantitative detection serum more particularly to one kind
Antibody test card.
Background technology
Echinococcosis (Hydatidosis) is also known as hydatidosis（Echinococcosis）, it is that a kind of serious harm people and animals are good for
The parasitic zoonoses of health, the Ministry of Agriculture are classified as two class animal epidemics, and the Ministry of Public Health is classified as Class C infectious disease.At me
State has confirmed that 25 provinces, municipalities and autonomous regions have cases of infection, wherein Gansu, Xinjiang, Ningxia, the Inner Mongol, Qinghai, Sichuan, Shan
The northwestward province such as west, Tibet is particularly acute.With the development of modern society, floating population's increase, and pet and economic animal
Cultivate quantity increase, the circulation of animal and population causes echinococcosis also progressively upcountry and the diffusion of coastal provinces and cities.China has 11 kinds
Domestic animal produces echinococcosis as the intermediate host of Echinococcus granulosus, and wherein sheep, ox, pig infection is particularly acute, in district occurred frequently,
The infection rate of sheep is more than 60%.Harm of the echinococcosis to domestic animal is mainly shown as impaired development, and the livestock products underproduction and internal organs give up
Abandon.
Echinococcosis is caused by the middle silk ribbon phase larva of Echinococcus tapeworm, Echinococcus granulosus（Echinococcus
granulosus, Eg）, Echinococcus multilocularis (E.multilocularis, Em) be China's Major Epidemic echinococcosis.China
It is one of echinococcosis incidence of disease highest country, echinococcosis has a strong impact on the economic development in China western part pastoral area and the body of the people
Health, while be one of the main reason for western China masses of farmers and herdsmen drive into poverty by medical crises and backed into poverty by medical crises, the parasitic disease
It has been listed in《Long-term animal epidemic control program in country》（2012-2020）Preferential preventing and treating and the animal epidemic of guard key.
China has implemented the pilot work of general prevention and treatment measure in hydatidosis district occurred frequently since the 1980s, but due to
Influenceed by factors such as pilot regional economy and culture, effect and unobvious.Therefore controlled by immunoprophylaxis approach and eliminate bag
Parasitosis is effective method.Therefore just there are the recombinant vaccine Eg95 and EgA31 for Eg, for Em recombinant vaccine
Em18 and Em14 etc..Therefore the diagnostic method of detection echinococcosis antibody is to determine resistance of the animal to echinococcosis, to daily prison
Survey vaccine and produce antibody, selection vaccine of the plant to colony, assess immune programme for children reasonability, grasp ruminant health status
Important means.
There is echinococcosis antibody colloidal gold rapid detection card on the market, pass through the detection to sample, you can judge animal roughly
The antibody content of internal echinococcosis, animal doctor judge whether to need to inject Echinococcus hydatid cyst disease vaccine according to testing result.Same in the market makes
With detection echinococcosis enzyme-labeled antibody kit, ELISA kit needs ELIASA, incubation reaction condition, board-washing condition, operates
Working environment and professional and technical personnel, the pre-treatment that sample also needs to complexity is detected in addition, such as gather blood, separation serum etc.；
And quickly collaurum is although easy to detect, professional and technical personnel and working environment are not required to, but sensitivity of its detection is low, and can only
It is qualitative, detect linear narrow range.And the purpose of patent of the present invention is exactly to overcome the respective shortcoming of the class kit of the above two, pass through letter
Single operation, realize quick, accurate, quantitative with sensitivity at the scene of cowboying and sheep（Sxemiquantitative）Detection.
The content of the invention
It is an object of the invention to provide one kind to be used for echinococcosis antibody test card in Quantitative detection serum, overcomes glue
The respective shortcoming of body gold rapid detection card and Kit, by shirtsleeve operation, is realized quick, accurate, high at ox, sheep scene
Delicately quantitatively detect, so as to be fully solved in place of above-mentioned the deficiencies in the prior art.
The purpose of the present invention is realized by following technical proposals：
One kind is for echinococcosis antibody test card in Quantitative detection serum, including detects card shell and be assemblied in outside detection card
Test strips in shell, the test strips include the plastic bottom board with pressure sensitive adhesive, paste sample pad, label successively on bottom plate
Pad, nitrocellulose filter and blotting paper, the labeling pad are made up of carrier substrate and label, and label is in carrier substrate
Spray the tunic that lanthanide fluoro detection microballoon and lanthanide fluoro Quality Control microballoon are formed, it is characterised in that：The nitrocellulose
It is detection line that echinococcosis recombinant antigen is coated with film, and coating rabbit-anti chicken lgY antibody is nature controlling line, and label is to be marked with echinococcosis
The fluoroscopic examination microballoon of recombinant antigen and the fluorescence Quality Control microballoon of mark chicken lgY antibody.
Further, the echinococcosis structural proteins are the recombinant protein of epitope expression, one-component or more component recombinant antigens
Combination, recombinant antigen refers to Eg95, EgA31, Em18 and Em14 recombinant protein.
Further, the rabbit-anti chicken lgY antibody that nature controlling line uses on the nitrocellulose filter for rabbit-anti chicken lgY lgG.One
The method that kind prepares above-mentioned echinococcosis, echinococcosis different genotype is compared using bioinformatics technique, is designed
Synthesize echinococcosis（Scrotiform and alveolitoid）Specific Eg95, EgA31, Em18 and Em14 antigen gene, and it is cloned into protokaryon table
Up in plasmid pET-30a, echinococcosis antigen Eg95, EgA31, Em18 and Em14 antigen gene recombinant expression plasmid pET- is built
30a- (Eg95), pET-30a- (EgA31), pET-30a- (Em18) and pET-30a- (Em14), are then converted large intestine bar
Bacterium competence cell BL21（DE3）PLysS screening recombination expression bacterium.Recombinantly express bacterium through IPTG induced expressions Eg95, EgA31,
Em18 and Em14 recombinant antigens, antigen is by being after purification detection antigen.
A kind of method for preparing above-mentioned echinococcosis, using bioinformatics technique principle, appliance computer to echinococcosis not
The antigen gene of homogenic type is compared, design synthesis Eg95, EgA31, Em18 and Em14 echinococcosis epitope antigen base
Cause, and prokaryotic expression plasmid structure echinococcosis epitope antigen DNA recombinant expression plasmid is cloned into, then converted big
Enterobacteria competent cell BL21（DE3）PLysS screening recombination expression bacterium, recombination expression bacterium is through IPTG induced expression echinococcosis tables
Position recombinant antigen, antigen are another detection antigen after affinitive layer purification.
A kind of application method of above-mentioned detection card, the detection card are quantitative chromatography detection card, will contain echinococcosis antibody
Prepare liquid instill detection card well in, chromatograph 15 minutes, then using chromatogram scanner to detection card be scanned, layer
Analysis scanner will scan the data obtained and radio to client, and the upper Quality Control of detection card is calculated according to scan data in client
The fluorescence signal intensity value of line and detection line, while client obtains the standard curve of detected echinococcosis antibody from cloud platform,
Client is calculated in prepare liquid according to the contrast relationship of standard curve and nature controlling line and the fluorescence signal intensity value of detection line
The content of echinococcosis antibody.
Preferably, the standard curve is to import cloud platform in the following way, a collection of detection card is prepared first, and
Echinococcosis antibody standard substance corresponding to preparation, block chromatography standard items with detection and detected with chromatogram scanner, chromatogram scanner will
Detect obtained data processing and pass to client, the fluorescence of detection line corresponding to standard items and nature controlling line is calculated in client
Signal strength values, and being returned according to this data so as to obtain the standard curve of echinococcosis antibody, client is by standard curve
Transmit to cloud platform and store.
Preferably, the standard curve that the client is calculated forms a file, and corresponding generation bar code, visitor
Family end can extract the standard curve by scanning bar code from cloud platform.
Explanation of nouns：
Rabbit-anti chicken lgY lgG：Rabbit-anti chicken lgY immunoglobulin G
Chicken lgY antibody：Chicken yolk antibody
Competent escherichia coli cell BL21（DE3）：The most frequently used host strain of escherichia expression system,
IPTG：Isopropyl-β-D-thiogalactoside.
Compared with prior art, the beneficial effects of the present invention are：High sensitivity, batch internal difference and difference between batch of detection are small,
There is preferable repeatability, stable performance, both can detect whole blood sample, serum and plasma sample can also be detected, with FMDV（Mouth hoof
Epidemic disease poison）、）PPRV (PPR virus), CPV（Capripox virus）There is no cross reaction Deng antibody.Achievable echinococcosis resists
Body field quick detection, there is higher practical value and promotional value, the recombinant protein expressed using multi-epitope can prevent
Missing inspection, detection is improved in susceptibility.
Brief description of the drawings
Fig. 1 is the structural representation of the present invention；
Fig. 2 is the structural representation of test strips in Fig. 1；
Fig. 3 is Fig. 2 top view；
Fig. 4 is echinococcosis antibody test standard curve；
Fig. 5 is another structural representation of present invention detection card.
Embodiment
With reference to specific embodiments and the drawings, the present invention is further illustrated.
As shown in Figures 1 to 4, it is a kind of to be used for echinococcosis antibody test card in Quantitative detection serum, including be assemblied in
Test strips in shell 10, the test strips include the plastic bottom board 1 with pressure sensitive adhesive, on plastic bottom board 1 from left to right successively
Sample pad 2, labeling pad 3, nitrocellulose filter 4 and blotting paper 5 are pasted, and the right-hand member of sample pad 2 rides over a left side for labeling pad 3
On end, the right-hand member of labeling pad 3 is ridden on the left end of nitrocellulose filter 4, and the left end of blotting paper 5 rides over nitrocellulose filter 4
On right-hand member.The recombinant antigen that echinococcosis is coated with the nitrocellulose filter 4 is detection line 6, and coating rabbit-anti chicken lgY antibody is matter
Line 7 is controlled, counter sample pad 2 opens up well 11 on shell 10, corresponds to detection line 6 and nature controlling line 7 on nitrocellulose filter 4
Open up form hole 12.
The echinococcosis recombinant antigen is the recombinant protein of epitope expression, or the combination of several recombinant antigens, recombinant antigen
Refer to Eg95, EgA31, Em18 and Em14 recombinant protein.
The rabbit-anti chicken lgY antibody that nature controlling line uses on the nitrocellulose filter 4 for rabbit-anti chicken lgY lgG.
The labeling pad 3 is made up of carrier substrate and label, and label is the recombinant antigen for being marked with echinococcosis
The fluorescence Quality Control microballoon of fluoroscopic examination microballoon and mark chicken lgY antibody.
In the blood sample Echinococcus hydatid cyst is coated with the label and nitrocellulose filter of echinococcosis antibody, echinococcosis recombinant antigen
Sick recombinant antigen is to detect echinococcosis antibody in sheep blood sample by dual-antigen sandwich method.
As a kind of optimal technical scheme, it is described detection card the surface of shell 10 correspond to blotting paper 5 position offer it is more
Individual through hole 13, be advantageous to solvent and volatilize shell 10, it is ensured that testing result is accurate, and the detection card shell for solving prior art is adopted
The technical problem that can not be volatilized away with enclosed construction, solvent, referring to Fig. 5.
In order to ensure volatilization effect, equidistantly uniformly opened in the region that the surface of shell 10 matches with the size of blotting paper 5
If through hole 13.
As another optimal technical scheme, the detection card body, which tilts, to be fixed in shell 10, and sample pad 2 is in
Low side, blotting paper 5 are in high-end.This structure causes the testing sample reduced velocity flow instilled from well 11, slowly toward upper strata
Analysis, ensures the enough chromatography time, can effectively prevent that liquid sample flow is too fast and influences testing result, avoid the too fast stream of sample
Entering nitrocellulose filter 4 causes testing result to fail.Because if testing sample flow velocity is too fast, easily cause to chromatograph it is insufficient,
And then influence testing result.The detection inclined angle of card body is specifically designed according to the difference of testing sample.
As another preferred structure of chromatography detection card, holding tank is provided with the shell 10 of the chromatography detection card, and
2 holding tanks are arranged with the both sides of detection card body, sheet drier is installed in holding tank.This design causes dry
Drying prescription keeps together with detection card body so that detection card body humidity is effectively controlled, so as to improve accuracy of detection.
As another preferred structure of chromatography detection card, can also be set on the surface of shell 10 of chromatography detection card with cover
Sample accumulator tank, sample can be sealed up for safekeeping.The hd top face of the sample accumulator tank flushes with the surface of shell 10, covers provided with recessed
Hole, convenient use person's finger force is turned-out to uncap.
Embodiment one, echinococcosis Eg95 albumen pronucleus expressions
Walk poly- one, echinococcosis antigen gene recombinant expression plasmid pET-30a- (Eg95) structure
According to NCBI websites echinococcosis Eg95 genes, the aim sequence of Eg95 genes is optimized for Escherichia coli preference codon sequence
Row, and using chemical synthesis method be respectively synthesized echinococcosis Eg95 express objective gene sequence.PET30a carriers are entered
Row BamH I and Xho I double digestions, digestion system are 40 μ L：The μ each 1 μ L of l, Ned I and Xho I of 10 × enzyme cutting buffering liquid 4, carrier
24 μ L, the μ L of deionized water 8, digestion products glue reclaim kit after 1% agarose electrophoresis reclaim.Then respectively by digestion
Eg95 coding gene sequences after good carrier and amplification are attached, and build 10 μ L linked system.Wherein, Eg95 encodes base
Because of the μ L of 5.5 μ L, pET30a carrier of fragment, 1.5 μ L, T4 DNA ligase, 1 μ L, T4 DNA connections buffer solution 2.Tube wall is rapped, up and down
It is reverse mix after wink from putting in 22 DEG C of connection instrument and connect 4 h.Recombinant plasmid is subjected to double digestion identification and purpose fragment sequence
Sequencing analysis, sequencing identification is named as pET30a- (Eg95) for positive plasmid.
Step 2: the structure of recombinant escherichia coli pET-30a- (Eg95)-BL21 (DE3) strain and Eg95 recombinant proteins
Induced expression
PET30a- (Eg95) connection product is transformed into BL21 (DE3) cell respectively.Under aseptic condition, appropriate BL21 bacterium are taken
Liquid is added to LB（Kan+）On solid medium flat board, bacterium solution is coated with uniformly, after bacterium solution fully absorbs, carries out mark, be inverted
In 37 DEG C of constant incubators, the h of quiescent culture 16.Picking monoclonal bacterium colony carries out bacterium solution identification, digestion identification, bacterium solution respectively
Sequencing, determines that Eg95 fragments have been transformed into pET30a carriers respectively.Take 1ml recombinant escherichia colis pET-30a- (
Eg95)-BL21 (DE3) strain is inoculated in 100ml Kan+LB culture mediums, while with the pET-30a-BL21 containing empty plasmid
（DE3）Strain is put 37 DEG C, cultivated about 150 minutes with 220r/min as control group, to OD600nm values up to 0.4~0.6.Respectively to
The IPTG of final concentration of 0.75mmol/ml amounts is added in test group and control group, 16 DEG C is put, is cultivated 13 hours with 220r/min.
Step 3: recombinant protein Eg95 purifying
Bacterium solution 60ml after inducing is taken, is centrifuged 10 minutes with 8000r/min, abandons supernatant, bacterial sediment is collected, uses PBS（0.01mol/
L, pH value 7.4）Wash bacterial sediment three times, centrifuged 10 minutes with 8000r/min, with 6ml PBS（0.01mol/L, pH value 7.4）Weight
Outstanding bacterial sediment, ice bath cracks in the presence of the ultrasonic wave of 150W power, and effective time is 10 minutes, and the working time is 5 seconds,
Interval 5 seconds.Ultrasonic degradation thing is centrifuged 15 minutes at 2 ~ 8 DEG C, with 12000r/min, retains supernatant.Use Ni SepharoseTM
6 Fast Flow resins purify to recombinant antigen, carry out washing impurity with the PBS containing 20mM imidazoles, then
It is purified by flash with the PBS containing 500mM imidazoles, and the destination protein sample of ni-sepharose purification is given birth to by GE companies
The Superdex200 gels column molecular sieve of production carries out further polishing purification, collects the collection liquid after molecular sieve polishing purification and enters
Row sds gel electrophoresis is analyzed and purity analysis, and its Eg95 antigen purity is up to more than 95%.Western blot（Western blotting
Experiment）As a result show, with echinococcosis positive serum specific immune response can occur for recombinant protein Eg95 respectively.Recombinated with this anti-
Original work are detection antigen.
EgA31, Em18, Em14 recombinant antigen are prepared with same method.
The Eg95 protein epitope antigen presentations of embodiment two, echinococcosis
A kind of method for preparing above-mentioned echinococcosis epitope antigen, echinococcosis difference pedigree Eg95 is resisted using bioinformatics technique
Protogene is compared, design synthesis echinococcosis Eg95 Primary epitope antigen genes, in order to ensure epitope antigen gene just
Really insertion expression vector, I/xho of specific cleavage site EcoR I, the precious biological work of gene commission are introduced at the both ends of gene
Journey（Dalian）Co., Ltd synthesizes, and is named as AE.With the enzymes of I/xho of EcoR I respectively to the Gene A E and recombinant expression plasmid of synthesis
PGEM-6P-1 carries out digestion, with DNA fragmentation QIAquick Gel Extraction Kit（Takara, Dalian）To genetic fragment and pGEM-6P-1
DNA fragmentation is reclaimed, then with two fragment connection construction recombination plasmid pGEM-AE of T4 ligases.Connection product is converted
BL21（DE3）PLysS competent cells, through containing ampicillin（Amp+）LAB screening positive restructuring bacterium.Select positive weight
Group clone bacterium adds 100ml and contained in Amp+ LB nutrient solutions, 37 DEG C of 220rpm shaken cultivations, works as OD600During=0.4-0.6, add
After the IPTG for entering final concentration of 0.6Mm, continue to cultivate 4-6 hours, culture is collected by centrifugation, after precipitation is resuspended with appropriate PBS, warp
Ultrasonication is handled, and 10000g centrifugation 30min, supernatant is precipitated and carries out SDS-PAGE electrophoresis（Polyacrylamide gel electricity
Swimming）, as a result show, recombinant protein is expressed with soluble form, purifies expression antigen by affinity column, its purity is up to 95%
More than.Western blot（Western blot test）As a result show, recombinant protein can occur special with echinococcosis Positive Sera
Specific immunological reacts, with this epitope recombinant antigen alternatively detection antigen.
EgA31, Em18, Em14 albumen of echinococcosis are prepared with same method.
Embodiment three, prepare echinococcosis antibody test card
Prepare echinococcosis antibody test standard curve：Configure the calibration solution containing echinococcosis antibody（Antibody standard substance containing echinococcosis）
6 parts, concentration is respectively 0,1/1024,1/256,1/64,1/16,1/4（Echinococcosis antibody standard substance doubling dilution）.By it is above-mentioned not
Calibration solution with concentration is separately added into the well of the detection card assembled, and chromatography is entered after 15 minutes by chromatogram scanner
Row detection, for the testing result that 6 times are obtained by client process, client calculates detection line corresponding to standard items and nature controlling line
Fluorescence signal intensity value, and carry out linear regression according to this data and make the standard curve of echinococcosis antibody, client calculates
The standard curve that draws forms a file, and it is corresponding generate bar code, client is by the file using bar code as filename（Bag
Containing standard curve）Transmit to cloud platform and store, referring to Fig. 4.
The standard curve that the client is calculated forms a file, and corresponding generation bar code, client pass through
Scanning bar code can extract the standard curve from cloud platform.
The application method of the detection card：By measuring samples（By taking serum as an example）1 is pressed with sample diluting liquid:50 dilution proportions,
The sample 80ul diluted is added in well 11, in the presence of blotting paper 5, sample is from sample pad 2 to the direction of blotting paper 5
It is mobile.15min is chromatographed in the case where avoiding strong illumination, then detection card is detected with chromatogram scanner, chromatography is swept
The fluorescence signal that instrument obtains detection line 6 and nature controlling line 7 is retouched, detection data are passed to client by chromatogram scanner by bluetooth, visitor
Family end calculates the fluorescence signal intensity value of detection line and the fluorescence signal intensity value of nature controlling line according to detection data, and client is led to
The bar code for over-scanning this batch of detection card obtains the standard curve of detected echinococcosis antibody from cloud platform, and client is according to standard
Containing for echinococcosis antibody in prepare liquid is calculated in the contrast relationship of curve and nature controlling line and the fluorescence signal intensity value of detection line
Amount, referring to Fig. 4 standard curve.Client can be mobile phone or tablet personal computer.
The preparation method of above-mentioned detection card is as follows：
Step 1, nitrocellulose filter is pasted onto on PVC bottom plates, using a film metal spraying special purpose machinery on nitrocellulose filter
The lgG that spraying has been diluted to 0.5mg/ml goat-anti chicken lgY forms nature controlling line and has been diluted to the weight of 0.5mg/ml echinococcosis
Histone forms detection line, and discharge rate is 1 μ l/cm, is then baked 8 hours at a temperature of 37 DEG C；
Step 2, the lanthanide fluoro for preparing the lanthanide fluoro microballoon for being marked with chicken lgY and the recombinant antigen for being marked with echinococcosis are micro-
Ball, 1mL lanthanide fluoro microballoon is added to 50mg MES（2-（N- morpholines）Ethyl sulfonic acid）Buffer solution（0.1M, pH7.0）
In, add 10mg carbodiimides（EDC）With 10mg n-hydroxysuccinimide sulfonate sodium stirring and dissolvings, room temperature reaction 30
Centrifugally operated is carried out after minute, by centrifugal sediment 50mM borate buffers（pH8.2）Redissolve, add the chicken that 2mg dialysed
LgY, stirring reaction 24 hours at ambient temperature, after being then centrifuged for, closing, then preserved in dilution（Conservation environment temperature
For 2~8 DEG C）, produce the lanthanide fluoro microballoon for being marked with chicken lgY；Restructuring using above-mentioned same method mark echinococcosis resists
Former lanthanide fluoro microballoon；
Step 3, be marked with chicken lgY and be marked with echinococcosis recombinant antigen lanthanide fluoro microballoon and be diluted to respectively dense
0.1 μ g/ml and 3 μ g/ml are spent, is sprayed on using a film metal spraying machine in carrier substrate and forms label pad, discharge rate is 2.5 μ
L/cm, then baked 8 hours at a temperature of 37 DEG C；
Step 4, sample pad, label pad, blotting paper are bonded on PVC bottom plates successively, are assembled into kilocalorie, then cut with cutter
The test strips wide into 5mm, it is assemblied in detection card shell, that is, obtains this echinococcosis antibody test card.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should be included in the scope of the protection.
Claims (8)
1. one kind is used for echinococcosis antibody test card in Quantitative detection serum, including detects card shell and be assemblied in detection card
Test strips in shell, the test strips include the plastic bottom board with pressure sensitive adhesive, paste sample pad, label successively on bottom plate
Pad, nitrocellulose filter and blotting paper, the labeling pad are made up of carrier substrate and label, and label is in carrier substrate
Spray the tunic that lanthanide fluoro detection microballoon and lanthanide fluoro Quality Control microballoon are formed, it is characterised in that：The nitrocellulose
It is detection line that echinococcosis recombinant antigen is coated with film, and coating rabbit-anti chicken lgY antibody is nature controlling line, and label is to be marked with echinococcosis
Recombinant antigen fluoroscopic examination microballoon and mark chicken lgY antibody fluorescence Quality Control microballoon.
2. according to claim 1 be used for echinococcosis antibody test card in Quantitative detection serum, it is characterised in that：Institute
State echinococcosis antigen refer mainly to echinococcosis epitope expression recombinant protein refer to Eg95, EgA31, Em18 and Em14 recombinant protein,
The combination of one-component or more component recombinant antigens.
3. according to claim 1 be used for echinococcosis antibody test card in Quantitative detection serum, it is characterised in that：Institute
State the lgG of rabbit-anti chicken lgY antibody that nature controlling line on nitrocellulose filter uses for rabbit-anti chicken lgY.
4. a kind of prepare the method that echinococcosis is prepared described in claim 2, it is characterised in that：Prokaryotic expression is believed using biology
Echinococcosis different genotype is compared breath technology, design synthesis echinococcosis（Scrotiform and alveolitoid）Specific Eg95,
EgA31, Em18 and Em14 antigen gene, and be cloned into prokaryotic expression plasmid Pet-30a, build echinococcosis antigen
Eg95, EgA31, Em18 and Em14 antigen gene recombinant expression plasmid pET-30a- (Eg95), pET-30a- (EgA31), pET-
30a- (Em18) and pET-30a- (Em14), is then converted competent escherichia coli cell BL21（DE3）PLysS screening weights
Group expression bacterium, recombination expression bacterium is through IPTG induced expression Eg95, EgA31, Em18 and Em14 recombinant antigens, and antigen is by after purification
To detect antigen.
5. a kind of prepare the method that echinococcosis is prepared described in claim 2, it is characterised in that：It is former using bioinformatics technique
The antigen gene of echinococcosis different genotype is compared for reason, appliance computer, design synthesis Eg95, EgA31, Em18
With Em14 echinococcosis epitope antigen genes, and be cloned into prokaryotic expression plasmid structure echinococcosis epitope antigen genetic recombination table
Up to plasmid, competent escherichia coli cell BL21 is then converted（DE3）PLysS screening recombination expression bacterium, recombinantly express bacterium
Through IPTG induced expression echinococcosis epitope recombinant antigens, antigen is another detection antigen after affinitive layer purification.
A kind of 6. application method of the detection card of claim 1 or 2 or 3 or 4, it is characterised in that：The detection card is quantitative
Chromatography detection card, the prepare liquid containing echinococcosis antibody is instilled in the well of detection card, chromatographed 15 minutes, then using layer
Analysis scanner is scanned to detection card, and chromatogram scanner will scan the data obtained and radio to client, client according to
The fluorescence signal intensity value of the upper nature controlling line of detection card and detection line is calculated in scan data, while client obtains from cloud platform
Echinococcosis antibody standard curve is detected, client is according to standard curve and nature controlling line and the fluorescence signal intensity value pair of detection line
Echinococcosis antibody content in prepare liquid is calculated according to relation.
7. application method according to claim 6, it is characterised in that：The standard curve is to import cloud in the following way
Platform, a collection of detection card, and echinococcosis antibody standard substance corresponding to preparation are prepared first, are blocked chromatography standard items with detection and are used in combination
Chromatogram scanner is detected, and the data processing that detection obtains is passed to client by chromatogram scanner, and standard is calculated in client
The fluorescence signal intensity value of detection line corresponding to product and nature controlling line, and returned according to this data so as to obtain echinococcosis antibody
Standard curve is transmitted to cloud platform and stored by standard curve, client.
8. application method according to claim 7, it is characterised in that：The standard curve that the client is calculated is formed
One file, and corresponding generation bar code, client can extract the standard curve by scanning bar code from cloud platform.
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Application publication date: 20180406 |