CN103033626B - Colloidal gold strip for TGEV antibody and PEDV antibody - Google Patents
Colloidal gold strip for TGEV antibody and PEDV antibody Download PDFInfo
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- CN103033626B CN103033626B CN201210577099.2A CN201210577099A CN103033626B CN 103033626 B CN103033626 B CN 103033626B CN 201210577099 A CN201210577099 A CN 201210577099A CN 103033626 B CN103033626 B CN 103033626B
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Abstract
The invention discloses a test strip for quickly detecting a PEDV (Porcine Epidemic Diarrhea Virus) serum antibody and a TGEV (Transmissible Gastroenteritis Virus) serum antibody and a preparation method of the test strip. The test strip comprises a support layer, a sample loading layer, a gold labeling protein release pad, a detection layer and an absorption layer, wherein a gold labeling protein and a detection line protein of the test strip are an S1 protein of a PEDV and a recombination protein of a TGEV, which are obtained by an efficient prokaryotic expression system; the recombination protein comprises an S protein AD site; and two quality control line proteins are monoclonal antibodies for the two proteins. Compared with the traditional test strip for detecting the PEDV antibody and the TGEV antibody, the test strip is high in specificity and safety and simple to operate, and judges results quickly.
Description
Technical field
The invention belongs to technical field of veterinary biology, it is related to a kind of quick detection antibody technique, more particularly, to pig transmissible
Marcy agent (tgev) and the colloidal gold colloidal gold detection test paper strip of Porcine epidemic diarrhea virus (pedv) two kinds of antibody, also relate to
The preparation method and application method of this test strips.
Background technology
Transmissible gastroenteritis of swine (transmissible gastroenteritis, tge) and porcine epizootic diarrhea
(porcine transmissible gastroenteritis, ped) is to be infected by the pig of coronaviridae, coronavirus genuses
Property marcy agent (transmissible gastroenteritis virus tgev) and Porcine epidemic diarrhea virus
The high degree in contact intestinal disease of the pig that (porcine transmissible gastroenteritis virus, pedv) causes
Disease, with beginning in autumn, Winter-Spring aggravation and the main epidemic characteristic of Xia Shaowei, various age pigs all can fall ill, and suckling pig case fatality rate is very high,
Within 10 ages in days, piglet case fatality rate is up to 100%.
In recent years, diarrhea of pigs disease brings massive losses to China's pig industry.In order to preferably control both diarrhea of pigs diseases
Disease, is necessary for understanding and grasp the infection conditions of pig farm virus in time, and therefore, exploitation is set up one kind and swinery can be carried out simultaneously
The quick effective ways of pedv and tgev Serum Antibody Detection are very necessary.The method of existing detection serum antibody at present
Mainly there are enzyme linked immunosorbent assay (elisa method) and neutralization test method, there are some in these traditional detection methods more difficult
The shortcoming overcoming, such as: neutralization test step is relatively complicated, detection required time is longer, and result judgement need certain special
Industry technology；Need in elisa detection method to coordinate microplate reader to be used, the professional of detection is also required to through professional training,
Simultaneously elisa detection method also exist specificity not high stability difference the features such as.Therefore exigence is set up one kind and is saved time province
The detection method that power can be popularized rapidly again.
Immune colloidal gold technique is widely used to clinical diagnosises at present, in invention " one or more pig virus of detection
Just detected with reference to immune colloidal gold technique in the test strips of diarrhea disease antibody ".Its gold mark albumen is spa albumen or to be checked
Antibody, the fixing tgev of detection line and pedv totiviruss, the igg albumen that nature controlling line is sheep or the anti-spa of rabbit produces or sheep or rabbit resist
The igg albumen of tgev and pedv virus.Have the characteristics that sensitivity is high, specificity is not strong using totiviruss and spa, if made
Its sensitivity and specific problem so can be solved with strong antigen albumen in virus and its monoclonal antibody long.
Content of the invention
It is an object of the present invention to provide the test strips of a kind of quick detection pedv and tgev serum antibody.
Further object is that providing the preparation method of this test strips.
Test strips of the present invention include supporting layer, and the sample-adding pad setting gradually on described supporting layer, gold mark albumen are released
Put pad, detection layers and absorbed layer, wherein, described gold mark protein delivery pad is embedded with two kinds of expressing proteins of colloid gold label, point
It is not Porcine epidemic diarrhea virus s1Albumen and the recombinant expression protein piece containing transmissible gastro-enteritis viruss s albumen ad site
Section；Two detection lines and two nature controlling lines are provided with described detection layers, described two detection lines fixing described s successively1Egg
The recombinant expression protein fragment in the s albumen ad site described in Pseudobulbus Bletillae (Rhizoma Bletillae), described two nature controlling lines are fixed with successively for two albumen
The monoclonal antibody of fragment.
Wherein, supporting layer preferably selects polyethylene fibre plate；Middling speed qualitative filter paper preferably selected by sample-adding pad；Release pad is preferred
From glass fibre；Detection layers preferably select nitrocellulose filter；Absorbed layer preferably selects glass fibre.
The preparation method of the described test strip that the present invention provides comprises the following steps:
1) Porcine epidemic diarrhea virus s is fixed on the diverse location of detection layers respectively1Albumen and transmissible gastroenteritis of swine
The ad site recombinant expression protein fragment of viral s albumen and its corresponding monoclonal antibody, form two detection lines and matter respectively
Control line；
2) release pad containing two kinds of gold mark albumen for the preparation；
3) assembling supporting layer, sample-adding pad, gold mark protein delivery pad, detection layers and absorbed layer.
1) Porcine epidemic diarrhea virus are fixed on the diverse location of detection layers successively
Wherein, preparing gold mark protein delivery pad, comprise the steps: will be to be marked with the labelling ratio of 1:100 ~ 3000
Porcine epidemic diarrhea virus s1The recombinant expression protein fragment in albumen or transmissible gastro-enteritis viruss s albumen ad site is added to
In the colloidal gold solution of ph7.0 ~ 10.0, delayed with the 0.001 ~ 0.1mol/l phosphate containing 0.1 ~ 5%bsa in the ratio of 1:10 ~ 500
Rush the s that liquid dilutes colloid gold label1Albumen or the recombinant expression protein in s albumen ad site, by the colloid gold label egg after dilution
White liquor presses 60 μ l/cm2Speed be sprayed on glass fibre element film in, 4 DEG C of low-temperature vacuum dryings.
Wherein, will be coated concentration is 0.1 ~ 2mg/ml, the Porcine epidemic diarrhea virus s of preferably 0.2 ~ 0.8mg/ml1Egg
It is sprayed on as detection line 1 in detection layers by the speed of 1 μ l/cm in vain, will be coated concentration is 0.1 ~ 2mg/ml, preferably 0.2 ~
The recombinant expression protein in the transmissible gastro-enteritis viruss s albumen ad site of 0.8mg/ml is sprayed on detection layers by the speed of 1 μ l/cm
On as detection line 2, will be coated concentration be 0.1 ~ 5mg/ml, the Porcine epidemic diarrhea virus s of preferably 0.2 ~ 0.8mg/ml1Egg
White monoclonal antibody is sprayed on as nature controlling line 1 in the detection layers below detection line by the speed of 1 μ l/cm, and will be coated concentration is 0.1
The recombinant expression protein monoclonal in the transmissible gastro-enteritis viruss s albumen ad site of ~ 5mg/ml, preferably 0.2 ~ 0.8mg/ml
Antibody is sprayed in the detection layers of nature controlling line 1 lower section by the speed of 1 μ l/cm and 30min is dried as at 2,37 DEG C of nature controlling line, thus making
Standby detection layers.
Used in the present invention, gold mark albumen and detection linear protein are Porcine epidemic diarrhea virus s1Albumen with contain pig
The recombinant expression protein in Transmissible gastroenteritis virus s albumen ad site, Quality Control linear protein is then the respective monoclonal of two kinds of albumen
Antibody, solves sensitivity and specificity issues it is established that detection method that is time saving and energy saving and can popularizing rapidly.
With traditional pedv compared with tgev antibody detection method, the present invention has an obvious superiority:
1) high specificity, using the pedv s of prokaryotic expression1Albumen and the recombinant expression protein in tgev s albumen ad site
With its corresponding monoclonal antibody, greatly reduce the false-positive probability of appearance it is ensured that test strips specificity and sensitivity,
Meanwhile, two nature controlling lines also ensure that the accuracy of testing result；
2) safe, it is to avoid the operation of virus in detection process；
3) easy and simple to handle, the present invention does not need technical professional to carry out operating the auxiliary detection with special instrument；
4) result of determination is quick, and with traditional neutralization test and compared with the time used by elisa test, this testing result is directly perceived
Easily judge and detection time only needs 10min extremely rapid.
Brief description
Fig. 1 show test strips side structure schematic diagram of the present invention.In figure: 1 is sample-adding pad, and 2 is release pad, and 3 is detection
Layer, 4 is absorbed layer, and 5 is supporting layer, and 6 is detection line -1, and 7 is detection line -2, and 8 is nature controlling line -1, and 9 is nature controlling line -2.
It is gold test paper strip positive structure schematic of the present invention shown in Fig. 2.In figure:
A detection line -1,2 and nature controlling line -1,2 all manifest, be pedv and tgev antibody positive sample detection result；
B detection line -1 is all developed the color with two nature controlling lines, and it is pedv antibody positive sample detection result；
C detection line -2 is all developed the color with two nature controlling lines, and it is tgev antibody positive sample detection result；
Only one nature controlling line colour developing of d, is invalid detection；
E nature controlling line and detection line all do not manifest, and are invalid detection.
Specific embodiment
Following examples are used for the present invention is described, but are not limited to the scope of the present invention.
Embodiment 1pedv s1The expression and purification of albumen and its preparation of monoclonal antibody
1、pedv s1The expression and purification of albumen
1. the extraction of genes of interest and amplification
The gold mark albumen that this colloidal gold strip selects is preferable fragment s of reactionogenicity in pedv s albumen1Albumen, pin
To domestic epidemic strain, according to its corresponding s1Protein gene designs following primer
Forward primer: 5 '-acggatccgatggcaccaggtgatc-3 '
Downstream primer: 5 '-ctgaattcatgactgtccatgactc-3 '
Extract the rna of Porcine epidemic diarrhea virus using test kit (roche test kit).Concrete operation step: in 1.5ml
Ep pipe in add the binding buffer that 200 μ l virus liquids and 400 μ l contain poly a, be transferred to after mixing standing 1min
In high-efficiency filtering pipe, 8000 × g centrifugation 15s discards liquid；Add 500 μ l inhibitor removal in filter tube
Buffer8000 × g is centrifuged 1min, discards liquid；Add 450 μ l wash buffer8000 in filter tube × g centrifugation 1min,
Discard liquid, be centrifuged 15s after being repeated twice again and dry to liquid；Finally add 50 μ l elution in filter tube
Buffer8000 × g is centrifuged the rna that 1min is just purified.
The concrete operation step of reverse transcription:
Carry out reverse transcription using full formula gold reverse transcription reagent box, 20 μ l systems consist of: the rna5 μ l, random of extraction
Primer (0.1 μ g/ μ l) 1 μ l, 2 × ts reactiom mix10 μ l, transscript rt/ri enzyme mix1 μ l,
nase-free water3μl.Reverse transcription reaction condition is 25 DEG C of incubation 10min, 42 DEG C of incubation 30min.85 DEG C of heating 5min.
The 50 μ l reaction systems of conventional pcr consist of: take pcr template 1 μ l, (concentration is each 1 μ l of upstream and downstream primer
50mmol/l), 10 × pcrbuffer is (containing mg2+) 5 μ l, 2.5mmdntps4 μ l (concentration is 2.5mmol/l), taq dna gather
Synthase 1 μ l, dd h2o38μl.The reaction condition of pcr is 94 DEG C of denaturations 5min, 94 DEG C of degeneration 40s, 55 DEG C of annealing 40s, 72 DEG C
Extend 45s, 35 circulations, then 72 DEG C of extension 7min.
Reaction takes 8 μ lpcr products to check the size of product in 1% agarose gel electrophoresiies after terminating.Result shows pedv's
s1Gene size, about near 1500bp, is consistent with actual 1445bp.
After electrophoresis terminates, cut purpose band, by dna gel reclaims kit (Shanghai Hua Shun biological engineering company limited)
Operating procedure carry out, the dna obtaining purification is put -20 DEG C and saves backup.
2. the connection of pcr product, conversion and identification
pedv s1The identification of gene: by the s of purification1- dna is connected to pmd19-t carrier, transformed competence colibacillus escherichia coli
Tg1, carries out blue white macula screening to transformed bacteria, selects white macula, and lb culture medium culturing simultaneously extracts plasmid, with pcr method Rapid identification
Positive colony.Positive colony plasmid is sent Beijing Bo Maide biotech firm to be sequenced, sequencing result display positive recombinant plasmid is correct.
Plasmid pet-32a-s1Preparation: connect pedv s1Gene takes the pedv of pet-32a vector plasmid and recovery
s1The each 20 μ l of pcr product, add bamh i and each 2.5 μ l of ecor i enzyme, 10 × buffer5 μ l, water 15 μ l, anti-in 37 DEG C of water-baths
Digestion products are reclaimed after answering 3h.Carrier 1 μ l, pcr product 4 μ l, 10 after addition bamh i and the recovery of ecor i enzyme action ×
Buffer1 μ l, t4Dna ligase 1 μ l, water 3 μ l, 16 DEG C overnight connect.
Prepare jm109 competent cell according to a conventional method: take Resulting plasmid pet-32a-s after connection15 μ l, add impression
State cell, 42 DEG C of heat shocks, place 2min on ice, add non-resistant culture medium, concussion and cultivate 50min, take 100 μ l culture coatings
Containing the lb flat board blocking that resistance, 37 DEG C of incubated overnight.The bacterium colony of picking overnight growth, using alkaline lysis method of extracting plasmid, uses
Bamh i, ecor i double digestion identification recombiant plasmid pet-32a-s1.Further pcr mirror is carried out to positive two recombiant plasmid
Fixed.Result is the positive, determines and converts successfully.
3. the abduction delivering of positive restructuring bacterium
Picking be accredited as the positive recombinant bacterium be inoculated in lb culture medium, overnight proceed to afterwards triangular flask carry out 37 DEG C cultivate to
od600During ≈ 0.6, add the final concentration of 1.5mmol/l of isopropylthiogalactoside (iptg) to carry out abduction delivering, proceed to 25
DEG C culture 6h.
Take out culture, the supernatant after recombinant bacterium ultrasonic degradation is carried out sds-page electrophoretic analysiss respectively with precipitation, cloudy
Property compares the jm109 competent cell for a conversion carrier, coomassie brilliant blue staining.Electrophoresis result shows the lysate of recombinant bacterium
All there is the band that molecular weight is about 80ku size in precipitation and supernatant, in negative control, have no above-mentioned band.
4. the purification of destination protein
Fusion protein after induction 6h is harvested bacterium solution, 5000r/min is centrifuged 10min, add 10ml combination buffer, abandon
Go the combination buffer resuspended bacterium solution with 10ml after supernatant, by sample be placed in ultrasonic treatment 5s in ice bath, interval 5s, ultrasonic extremely
Liquid-transparent.Then 10000r/min centrifugation 15min, removes cell debriss, supernatant is moved in new pipe.Supernatant is taken to utilize
The his-tag selectivity label application nitrogen company description purifying protein that pet-32a carrier carries.
2、pedv s1The preparation of protein monoclonal antibody
1. animal immune program
The good albumen of purification and Freund's complete adjuvant equal-volume are fully mixed emulsifying, mouse peritoneal injection, dosage of inoculation
75 μ g/ are only；Mouse peritoneal is injected with the emulsion of the good albumen of same purification and incomplete Freund's adjuvant after 2 weeks；After surrounding, then
The secondary emulsion with same purifying protein and incomplete Freund's adjuvant injects mouse peritoneal；It is not added with the purification of adjuvant for the last time
Albumen booster immunization.
2. the foundation of screening technique
Docking blood sampling before gathering 21d, 35d of mice after immunity and taking spleen, obtains positive serum after centrifugation.With
The expressing protein of purification as envelope antigen, is diluted with being coated liquid and being l:200, l:400,1:600,1:800,1:1000 times.Will
Diluent is pressed 100 μ l/ holes and is added ELISA Plate, and oscillating flat plate is so that Antigen distribution is uniform.Coated ELISA Plate plastic foil is sealed
Good, 4 DEG C overnight.Next day discards liquid in elisa plate, adds cleaning mixture (pbst solution 1l: sodium chloride 8g, potassium chloride 0.2g, phosphorus
Sour disodium hydrogen 2.7g, potassium dihydrogen phosphate 0.2g.Ph:7.4,500 μ l tween 20s) 250 μ l/ holes, wash 3min under room temperature, with clean
Absorbent paper gently pats dry on the table, cyclic washing three times.Diluted as confining liquid using pbst with 5% defatted milk powder, 100 μ l/
Hole.Coated ELISA Plate plastic foil is sealed, 37 DEG C of placement 2h.Discard liquid in elisa plate, (pbst is molten to add cleaning mixture
Liquid 250 μ l/ hole), wash 3 each 3min under room temperature.Positive serum is one to resist, and makees l:50,1:80 respectively, 1:100,1:150,1:
200 dilutions, add in ELISA Plate, and 100 μ l/ holes set blank simultaneously, and 37 DEG C of effect l h wash three times, pat dry.Add and use
The hrp- rabbit anti-Mus igg of 5% defatted milk powder 1:8000 dilution, 100ul/ hole, 37 DEG C of effect 1h, pat dry after washing three times.Plus bottom
Thing solution (tmb.h2o2), 100 μ l/ holes, room temperature acts on 15min, adds terminate liquid (2m h2so4Solution) 50 μ l/ holes, in enzyme mark
Od value is read on instrument.With the od value of positive blood borehole cleaning close to 1.0, p/n > 2.0 antigen serum greatest dilution as antigen and
The most suitable working concentration of control serum, it is respectively 1:600 and 1:100.
3. cell fusion
Separating Morr. cell: take the balb/c mice of three days after booster immunization, eyeball excise blood-letting, separate serum for positive anti-
Body is used；Mice cervical dislocation is put to death, after 75% alcohol wipe sterilization, moves in superclean bench, be fixed on dissection plate,
Cut off skin and peritoneum, aseptic taking-up spleen respectively with sterilizing shears, tweezers, put into oneself and fill the sterilized petri dishes of basic culture solution
In clear Xian, peel off the connective tissue on envelope.Spleen is moved in another plate with copper mesh filling 10ml basic culture solution,
It is lightly ground with dismembyator afterwards, splenocyte suspension in plate is transferred in 50ml centrifuge tube, 1000r/min is centrifuged 10min, carefully
Born of the same parents are precipitated with basic culture solution with method centrifuge washing once, then with culture fluid by resuspended for cell to 10～20ml, mix, platform is expected
Blue stain living cells are standby after counting.
Preparation myeloma cell: merge the last fortnight recovery myeloma sp2/0 cell, and by myeloma sp2/0 cell through 8-
Ag selects culture.After cell recovery, the dmem complete culture solution of 8-ag (20ug/ml) is added to select more than culture three generations, to protect
Hold hgprt deficiency.Merge the same day, take the sp2/0 cell that growth conditions are good, with pbs buffer solution three times, will just locate
In the cell of exponential phase is collected to centrifuge tube, 900r/min is centrifuged 10min, discards nutritional solution, is washed with basic culture solution
Wash once afterwards by cell Eddy diffusion, platform expects that blue staining cell counts, and cell is diluted to 106Individual/ml, places standby in centrifuge tube
With.
Cell fusion: merge according to a conventional method, take immune mouse spleen cell and myeloma cell sp2/0 by cell quantity 5:1 ~
Mix between 10:1, add in the disposable sterilized centrifuge tube of 50ml, 1000r/min is centrifuged 10min, abandons supernatant, uses aseptic suction
Paper is sufficiently absorbed through residual liquid, with finger attack ttom of pipe, so that two kinds of cells is fully mixed, until becoming pasty state；Centrifuge tube is placed in
In the beaker of 37 DEG C of water, 50% Polyethylene Glycol (peg) the solution l ml drawing 37 DEG C of preheatings is slowly dropped into, used in water in 1min
Centrifuge tube l min is shaken gently on bath, slowly instill in lmin 37 DEG C preheating basic culture solution l ml, rotate when dripping from
Heart pipe, is subsequently slowly dropped into the basic culture solution 4ml of 37 DEG C of preheatings in 1min, adds and add basis training in subsequent 4min
Nutrient solution is gently agitated for centrifuge tube to 30ml makes the thorough dilution of peg lose fusion.1000r/min is centrifuged 10min, abandons
Clearly, cell precipitation is gently suspended from pre-temperature hat containing 20% hyclone and selects in culture fluid 40ml, mix homogeneously, it is added to
In 96 porocyte culture plates added with feeder cells, every hole 100 μ l, culture plate is moved to 37 DEG C, 5%co2Cultivate in incubator.
4. positive hole cloning amplification culture
Positive hybridoma cell carries out the detection of specific antibody with indirect elisa method, using limiting dilution assay to
The positive hybridoma cell of identification carries out cloning.
Observe after cell 3d after fusion, and change culture fluid once every 7d, using partly changing liquid mode.Treat that hybridoma is thin
When born of the same parents cover with bottom hole 1/3～1/2, can use cell culture supernatant 100ul after changing liquid 1～2d, do not dilute, with merging egg
Coated indirect elisa method carries out the detection of specific antibody in vain, uses sp2/0 cell culture supernatant to make negative right simultaneously
According to positive Mus serum is positive control.
It has been detected as hybridoma colonies in the particular bore secrete the positive and blown afloat mixing, cell Trypan Blue meter
Number, takes above-mentioned cell suspension, is diluted to l ml with the complete culture solution containing 20% hyclone and contains 10 cells, by diluted
Cell suspension 100 μ l is added to and cultivates in 96 porocyte culture plates added with feeder cells in advance.Change liquid, screening and clone in good time
Change, be enlarged cultivating by cloning remaining cell frozen simultaneously every time.Through the operation of 2-3 time cloningization, until all clones
When changing cell hole Positive rate for 100%, you can determine the hybridoma cell strain having obtained secrete monoclonal antibody.
5. slightly the carrying of animal inoculation and odd contradictive hydroperitoneum
1 week before the hybridoma of inoculation secrete monoclonal antibody, first give 8～12 week old female balb/c mices every
Lumbar injection 0.5ml liquid paraffin.The hybridoma of culture is rinsed from Tissue Culture Flask, 1000r/min is centrifuged
5min, then wash 2 times with pbs buffer (ph7.2) suspended centrifugal, Trypan Blue does cell density number after viable count
It is adjusted to l ~ 2 × 106Individual/ml, every mouse peritoneal injection 0.5ml cell suspension, containing about 5 ~ 10 × 105Individual cell.Note after inoculation
Observe mice state, treat that mouse web portion substantially expands, when handicapped, after iodine tincture cotton balls sterilization hypogastric region skin, can use
20ml syringe needle pierces abdominal cavity it is seen that there being flaxen ascites to ooze, and is collected with sterile centrifugation tube, 4 DEG C stand overnight,
3000r/min is centrifuged 5min, removes precipitation of grease, supernatant is ascites, after subpackage labelling, -80 DEG C of preservations；Interval 3d, treats abdomen
After water reuse (treatment accumulation, take again with method, a mice extracts 2～3 times.
Slightly carrying of monoclonal antibody is carried out using ammonium sulfate precipitation method.Take 5ml mouse ascites, 4 DEG C of 10000r/min, be centrifuged 15min,
Remove cell debriss and other impurities；Supernatant is moved in small beaker, press pump is slowly added dropwise saturated ammonium sulfate (ph7.8)
2.5ml, stirring while adding；After stirring 30min, 10000r/min is centrifuged 15min, abandons supernatant；Precipitate is with 1/3 saturation sulphuric acid
Ammonium suspends, stirring action 30min, with method centrifugation.Precipitate is dissolved in the pbs of lml, loads in dialysis band, 4 DEG C of dialysed overnight,
Change liquid 4 times therebetween, until can't detect nh4+And so4 2-.Removal protein solution, 10000r/min is centrifuged 15min, collects supernatant
It is the monoclonal antibody slightly carrying.Ultraviolet spectrophotometer surveys protein content, and subpackage is labeled as s1Albumen, -80 DEG C save backup.
The expression and purification of embodiment 2tgev s recombiant protein fragment and its preparation of monoclonal antibody
1st, the expression and purification of the recombinant expression protein in s albumen ad site
1. the extraction of genes of interest and amplification
Select reactionogenicity preferable a, d site in tgev s albumen to be expressed, for domestic epidemic strain, pass through
The following primer of recombinant expression protein gene design in genbank comparison design s albumen ad site:
The recombinant expression protein in tgev s albumen ad site:
Forward primer: acgtcgactgctacgatgatttca
Downstream primer: gtaagcttatagccccaactatgg
Extract the routine operation that rna is roche test kit.Extract pig transmissible stomach using test kit (roche test kit)
The rna of enteritis virus: add the binding that 200 μ l virus liquids and 400 μ l contain poly a in the ep pipe of 1.5ml
Buffer, is transferred in high-efficiency filtering pipe after mixing standing 1min, and 8000 × g centrifugation 15s discards liquid；Add in filter tube
500 μ l inhibitor removal buffer8000 × g centrifugation 1min, discard liquid；Add 450 μ lwash in filter tube
Buffer8000 × g is centrifuged 1min, discards liquid, is centrifuged 15s again and dries to liquid after being repeated twice；Finally add in filter tube
Enter 50 μ l elution buffer8000 × g and be centrifuged the rna that 1min is just purified.
The concrete operation step of reverse transcription:
Carry out reverse transcription using full formula gold reverse transcription reagent box, 20 μ l systems consist of: rna5 μ l, random primer
(0.1 μ g/ μ l) 1 μ l, 2 × ts reactiom mix10 μ l, transscript rt/ri enzyme mix 1 μ l, nase-
free water3μl.Reverse transcription reaction condition is 25 DEG C of incubation 10min, 42 DEG C of incubation 30min, 85 DEG C of heating 5min.
The 50 μ l reaction systems of conventional pcr consist of: take pcr template 1 μ l, (concentration is each 1 μ l of upstream and downstream primer
50mmol/l), 10 × pcr buffer(contains mg2+) 5 μ l, 2.5mm dntps4 μ l (concentration is 2.5mmol/l), taq dna
Polymerase 1 μ l, dd h2o38μl.The reaction condition of pcr is popular response condition: 94 DEG C of denaturations 5min, 94 DEG C of degeneration 40s,
55 DEG C of annealing 40s, 72 DEG C of extension 45s, 35 circulations, then 72 DEG C of extension 7min.
Reaction takes 8 μ lpcr products to check the size of product in 1% agarose gel electrophoresiies after terminating.Result display detection base
It is consistent with actual 1202bp because size is about 1200bp.
After electrophoresis terminates, cut purpose band, by dna gel reclaims kit (Shanghai Hua Shun biological engineering company limited)
Operating procedure carry out, the dna obtaining purification is labeled as st- dna, puts -20 DEG C and saves backup.
2. the connection of pcr product, conversion and identification
stThe identification of-dna: by the s of purificationt- dna is connected to pmd19-t carrier, and transformed competence colibacillus escherichia coli, to conversion
Bacterium carries out blue white macula screening, selects white macula, and lb culture medium culturing simultaneously extracts plasmid, with pcr method Rapid identification positive colony.Will
Positive colony plasmid send Beijing Bo Maide biotech firm to be sequenced, and sequencing result display positive recombinant plasmid is correct.
Plasmid pet-32a-stPreparation: connect tgev stGene takes the tgev of pet-32a vector plasmid and recovery
stThe each 20 μ l of pcr product, add bamh i and each 2.5 μ l of hind iii enzyme, 10 × buffer5 μ l, water 15 μ l, in 37 DEG C of water-baths
Digestion products are reclaimed after reaction 3h.Carrier 1 μ l, pcr product 4 μ l, 10 after addition bamh i and the recovery of hind iii enzyme action ×
Buffer1 μ l, t4Dna ligase 1 μ l, water 3 μ l, 16 DEG C overnight connect.
Prepare jm109 competent cell according to a conventional method: take Resulting plasmid pet-32a-s after connectiont5 μ l, add impression
State cell, 42 DEG C of heat shocks, place 2min on ice, add non-resistant culture medium, concussion and cultivate 50min, take 100 μ l culture coatings
Containing the lb flat board blocking that resistance, 37 DEG C of incubated overnight.
The bacterium colony of picking overnight growth, using alkaline lysis method of extracting plasmid, with bamh i, hind iii double digestion identification weight
Group plasmid pet-32a-st.Positive recombinant plasmid is carried out with further pcr identification.Result is the positive, determines and converts successfully.
3. the abduction delivering of positive restructuring bacterium
Picking be accredited as the positive recombinant bacterium be inoculated in lb culture medium, overnight proceed to afterwards triangular flask carry out 37 DEG C cultivate to
od600During ≈ 0.5, add the final concentration of 1.6mmol/l of isopropylthiogalactoside (iptg) to carry out abduction delivering, proceed to 25
DEG C culture 6h.
Take out culture, the supernatant after recombinant bacterium ultrasonic degradation is carried out sds-page electrophoretic analysiss respectively with precipitation, cloudy
Property compares the jm109 competent cell for a conversion carrier, coomassie brilliant blue staining.Electrophoresis result shows the lysate of recombinant bacterium
All there is the band that molecular weight is about 65ku size in precipitation and supernatant, be consistent with theoretical 66ku, have no in negative control
State band.
4. the purification of destination protein
Fusion protein after induction 6h is harvested bacterium solution, 5000r/min is centrifuged 10min, add 10ml combination buffer, abandon
Go the combination buffer resuspended bacterium solution with 10ml after supernatant, by sample be placed in ultrasonic treatment 5s in ice bath, interval 5s, ultrasonic extremely
Liquid-transparent.Then 10000r/min centrifugation 15min, removes cell debriss, supernatant is moved in new pipe.Supernatant is taken to utilize
The his-tag selectivity label application nitrogen company description purifying protein that pet-32a carrier carries.
2nd, the preparation of monoclonal antibody
1. animal immune program
The good albumen of purification and Freund's complete adjuvant equal-volume are fully mixed emulsifying, mouse peritoneal injection, dosage of inoculation
50 μ g/ are only；Mouse peritoneal is injected with the emulsion of the good albumen of same purification and incomplete Freund's adjuvant after 2 weeks；After surrounding, then
The secondary emulsion with same purifying protein and incomplete Freund's adjuvant injects mouse peritoneal；It is not added with the purification of adjuvant for the last time
Albumen booster immunization.
2. the foundation of screening technique
Docking blood sampling before gathering 21d, 35d of mice after immunity and taking spleen, obtains positive serum after centrifugation.With
The expressing protein of purification as envelope antigen, is diluted with being coated liquid and being l:200, l:400,1:600,1:800,1:1000 times.Will
Diluent is pressed 100 μ l/ holes and is added ELISA Plate, and oscillating flat plate is so that Antigen distribution is uniform.Coated ELISA Plate plastic foil is sealed
Good, 4 DEG C overnight.Next day discards liquid in elisa plate, adds cleaning mixture (pbst solution) 250 μ l/ hole, washes 3min under room temperature, uses
Clean absorbent paper gently pats dry on the table, cyclic washing three times.Diluted as confining liquid using pbst with 5% defatted milk powder, 100
μ l/ hole.Coated ELISA Plate plastic foil is sealed, 37 DEG C of placement 2h.Discard liquid in elisa plate, add cleaning mixture (pbst
Solution) the every hole of 250 μ l, wash 3 each 3min under room temperature.Positive serum is one to resist, and makees l:50,1:80 respectively, 1:100,1:
150,1:200 dilutions, add in ELISA Plate, and 100 μ l/ holes set blank simultaneously, and 37 DEG C of effect l h wash three times, pat dry.
Add the hrp- rabbit anti-Mus igg with 5% defatted milk powder 1:8000 dilution, the every hole of 100ul, 37 DEG C of effect 1h, wash three times, clap
Dry.Plus substrate solution (tmb.h2o2), 100 μ l/ holes, room temperature acts on 15min, adds terminate liquid (2m h2so4Solution) 50 μ l/
Hole, reads od value in microplate reader.Made close to 1.0, p/n > 2.0 antigen serum greatest dilution with the od value of positive blood borehole cleaning
For the most suitable working concentration of antigen and control serum, it is respectively 1:600 and 1:150.
3. cell fusion
Separating Morr. cell: take the balb/c mice of three days after booster immunization, eyeball excise blood-letting, separate serum for positive anti-
Body is used；Mice cervical dislocation is put to death, after 75% alcohol wipe sterilization, moves in superclean bench, be fixed on dissection plate,
Cut off skin and peritoneum, aseptic taking-up spleen respectively with sterilizing shears, tweezers, put into oneself and fill the sterilized petri dishes of basic culture solution
In clear Xian, peel off the connective tissue on envelope.Spleen is moved in another plate with copper mesh filling 10ml basic culture solution,
It is lightly ground with dismembyator afterwards, splenocyte suspension in plate is transferred in 50ml centrifuge tube, 1000r/min is centrifuged 10min, carefully
Born of the same parents are precipitated with basic culture solution with method centrifuge washing once, then with culture fluid by resuspended for cell to 10～20ml, mix, platform is expected
Blue stain living cells are standby after counting.
Preparation myeloma cell: merge the last fortnight recovery myeloma sp2/0 cell, and by myeloma sp2/0 cell through 8-
Ag selects culture.After cell recovery, the dmem complete culture solution of 8-ag (20ug/ml) is added to select more than culture three generations, to protect
Hold hgprt deficiency.Merge the same day, take the sp2/0 cell that growth conditions are good, with pbs buffer solution three times, will just locate
In the cell of exponential phase is collected to centrifuge tube, 900r/min is centrifuged 10min, discards nutritional solution, is washed with basic culture solution
Wash once afterwards by cell Eddy diffusion, platform expects that blue staining cell counts, and cell is diluted to 106Individual/ml, places standby in centrifuge tube
With.
Cell fusion: merge according to a conventional method, take immune mouse spleen cell and myeloma cell sp2/0 by cell quantity 5:1 ~
Mix between 10:1, add in the disposable sterilized centrifuge tube of 50ml, 1000r/min is centrifuged 10min, abandons supernatant, uses aseptic suction
Paper is sufficiently absorbed through residual liquid, with finger attack ttom of pipe, so that two kinds of cells is fully mixed, until becoming pasty state；Centrifuge tube is placed in
In the beaker of 37 DEG C of water, 50% Polyethylene Glycol (peg) the solution l ml drawing 37 DEG C of preheatings is slowly dropped into, used in water in 1min
Centrifuge tube l min is shaken gently on bath, slowly instills the basic culture solution l ml of 37 DEG C of preheatings in l min, rotate when dripping
Centrifuge tube, is subsequently slowly dropped into the basic culture solution 4ml of 37 DEG C of preheatings in 1min, adds and add basis in subsequent 4min
Culture fluid is gently agitated for centrifuge tube to 30ml makes the thorough dilution of peg lose fusion.1000r/min is centrifuged 10min, abandons
Clearly, cell precipitation is gently suspended from pre-temperature hat containing 20% hyclone and selects in culture fluid 40ml, mix homogeneously, it is added to
In 96 porocyte culture plates added with feeder cells, every hole 100 μ l, culture plate is moved to 37 DEG C, 5%co2Cultivate in incubator.
4. positive hole cloning amplification culture
Positive hybridoma cell carries out the detection of specific antibody with indirect elisa method, using limiting dilution assay to
The positive hybridoma cell of identification carries out cloning.
Observe after cell 3d after fusion, and change culture fluid once every 7d, using partly changing liquid mode.Treat that hybridoma is thin
When born of the same parents cover with bottom hole 1/3～1/2, can use cell culture supernatant 100ul after changing liquid 1～2d, do not dilute, with merging egg
Coated indirect elisa method carries out the detection of specific antibody in vain, uses sp2/0 cell culture supernatant to make negative right simultaneously
According to positive Mus serum is positive control.
It has been detected as hybridoma colonies in the particular bore secrete the positive and blown afloat mixing, cell Trypan Blue meter
Number, takes above-mentioned cell suspension, is diluted to l ml with the complete culture solution containing 20% hyclone and contains 10 cells, by diluted
Cell suspension 100 μ l is added to and cultivates in 96 porocyte culture plates added with feeder cells in advance.Change liquid, screening and clone in good time
Change, be enlarged cultivating by cloning remaining cell frozen simultaneously every time.Through the operation of 2-3 time cloningization, until all clones
When changing cell hole Positive rate for 100%, you can determine the hybridoma cell strain having obtained secrete monoclonal antibody.
5. slightly the carrying of animal inoculation and odd contradictive hydroperitoneum
1 week before the hybridoma of inoculation secrete monoclonal antibody, first give 8～12 week old female balb/c mices every
Lumbar injection 0.5ml liquid paraffin.The hybridoma of culture is rinsed from Tissue Culture Flask, 1000r/min is centrifuged
5min, then wash 2 times with pbs buffer (ph7.2) suspended centrifugal, Trypan Blue does cell density number after viable count
It is adjusted to l ~ 2 × 106Individual/ml, every mouse peritoneal injection 0.5ml cell suspension, containing about 5 ~ 10 × 105Individual cell.Note after inoculation
Observe mice state, treat that mouse web portion substantially expands, when handicapped, after iodine tincture cotton balls sterilization hypogastric region skin, can use
20ml syringe needle pierces abdominal cavity it is seen that there being flaxen ascites to ooze, and is collected with sterile centrifugation tube, 4 DEG C stand overnight,
3000r/min is centrifuged 5min, removes precipitation of grease, supernatant is ascites, after subpackage labelling, -80 DEG C of preservations；Interval 3d, treats abdomen
After water reuse (treatment accumulation, take again with method, a mice can extract 2～3 times.
Slightly carrying of monoclonal antibody is carried out using ammonium sulfate precipitation method.Take 5ml mouse ascites, 4 DEG C of 10000r/min, be centrifuged 15min,
Remove cell debriss and other impurities；Supernatant is moved in small beaker, press pump is slowly added dropwise saturated ammonium sulfate (ph7.8)
2.5ml, stirring while adding；After stirring 30min, 10000r/min is centrifuged 15min, abandons supernatant；Precipitate is with 1/3 saturation sulphuric acid
Ammonium suspends, stirring action 30min, with method centrifugation.Precipitate is dissolved in the pbs of lml, loads in dialysis band, 4 DEG C of dialysed overnight,
Change liquid 4 times therebetween, until can't detect nh4+And so4 2-.Removal protein solution, 10000r/min is centrifuged 15min, collects supernatant
It is the monoclonal antibody slightly carrying.Ultraviolet spectrophotometer surveys protein content, subpackage, and -80 DEG C save backup.
The assembling of embodiment 3 colloidal gold strip
1st, colloid gold label antigen protein
Gold solution is prepared with reduction of sodium citrate method: add 4ml's in 0.02% aqueous solution of chloraurate of 50ml boiling
1.2% citric acid three sodium solution, continues agitating heating until solution is in stable and bright redness.Glue is observed under transmission electron microscope
Body gold solution, finds that its size of colloid gold particle is homogeneous, no oval or other are irregularly shaped, illustrate to obtain stably
Gold colloidal.
K with 0.1mol/l2co3Adjust gold colloidal ph value 7.5, with the labelling ratio of 1:1000 by albumen (s to be marked1Or
st) be added in the colloidal gold solution regulating ph value, after standing 10min, add 20% Polyethylene Glycol (peg) 10000 to final
Concentration be 0.05%, at 4 DEG C 2000r/min centrifugation 20min, remove unconjugated colloid gold particle, at 4 DEG C 15000r/min from
After heart 40min, supernatant discarded obtains the good albumen of colloid gold label, with 4 DEG C after propylene glucosan s-400 chromatography column separating purification
Lower short-term preservation.
2nd, test strips layers of material prepares
The test strips basic structure of invention design is supporting layer, sample-adding layer, detection layers and absorbed layer.Each layer is pressed following suitable
Sequence is pasted: detection layers are pasted onto on supporting layer；Filter pad is bonded at composition sample-adding layer in release pad, is connected to by release pad and leans on
Detection line one end of nearly detection layers, edge is attached in detection layers, is defined as top；Absorbed layer is pasted onto in detection layers near matter
The side of control line, is defined as end.In the present invention, sample-adding pad is chosen as middling speed qualitative filter paper (Shanghai Chu Bai laboratory equlpment is limited
Company), nc film be ff85(U.S. whatman), rapid27(U.S. whatman) as release pad material, absorbed layer select
Absorption layer glass fibre cf4(U.S. whatman) material.
3rd, the selection of expressing protein and MAb concentration
The concentration of detection albumen and Quality Control albumen selects: 0.01mol/l pb(is contained 1%bsa) gold of 1:200 times of dilution marks
What albumen carried out detecting albumen and Quality Control albumen is coated concentration.Selected s1Three, albumen is coated concentration and is respectively 0.5,1.0 and
1.5mg/ml, three of selected monoclonal antibody are coated concentration is 0.5,1 and 2mg/ml, and by cross matching, result shows: s1
Albumen be coated concentration be 1mg/ml, its monoclonal antibody be coated concentration be 1mg/ml when, Red Sigil on nc film ff85
The most obvious；The recombinant expression protein three in selected s albumen ad site is coated concentration and is respectively 0.5,1.0 and 1.5mg/ml, selectes
It is 0.5,1 and 2mg/ml that three of monoclonal antibody are coated concentration, and by cross matching, result shows: the weight in s albumen ad site
Group expressing protein be coated concentration be 0.5mg/ml, its monoclonal antibody be coated concentration be 1mg/ml when, on nc film ff85
Red Sigil is the most obvious.
The selection of gold mark protein concentration: contain 1%bsa with 0.01mol/l pb() dilution 1:100,1:200,1:300 and 1:
400 times of two kinds of gold mark albumen press 60 μ l/cm respectively2Volume be uniformly sprayed in gold standard pad, at 37 DEG C dry 1h after, be pasted onto
Each on the nc film of the good detection line of labelling and nature controlling line, Deca 100 μ l normal saline compares respective nature controlling line in release pad
The colour developing depth, result shows:
①s1Albumen
Note :+colour developing is very shallow；++ colour developing is shallow；+++ colour developing is deep
Therefore, select gold mark s1The dilution factor of albumen is 1:300.
2. the recombinant expression protein in s albumen ad site
Note :+colour developing is very shallow；++ colour developing is shallow；+++ colour developing is deep
Therefore, the dilution factor selecting the recombinant expression protein in gold mark s albumen ad site is 1:300.
The checking test of 4 colloidal gold strips of embodiment
1st, the specific detection of pedv and tgev antibody colloidal gold test paper bar
Using pedv with tgev antibody colloidal gold test paper bar to pig circular ring virus 1 type (pcv-1), porcine circovirus 2 type
(pcv-2), pig colyliform disease virus (porv), porcine pseudorabies virus (prv), Porcine epidemic diarrhea virus (pedv) and pig are infected
Property marcy agent (tgev) positive serum and Porcine epidemic diarrhea virus (pedv) and transmissible gastro-enteritis viruss
(tgev) mixing positive serum carries out double sample detection.Result shows pcv-1, pcv-2, porv, prv and pcv-1 positive blood
It is clearly negative (only showing two nature controlling lines), the testing result of pedv and tgev positive serum is respective positive and noiseless
(pedv positive serum result display detection line 1 and nature controlling line 1, tgev positive serum result display detection line 2 and nature controlling line 2),
(two nature controlling lines of test strips and two detection lines are all aobvious for the positive entirely for the testing result of pedv and tgev mixing positive serum
Existing), thus prove this Test paper specificity good and with other diarrhea of pigs disease virus-positive serum no cross reaction.
2nd, the sensitivity technique of pedv and tgev antibody colloidal gold test paper bar
Pedv the and tgev positive hyper-immune serum obtaining during this laboratory animal is tested dilutes 8 gradients (2 respectively3、24、
25、26、27、28、29With 210), two viruses are separately neutralized testing inspection and colloidal gold strip detection.Result shows
The limit of identification of the detection of pedv neutralization test and colloidal gold strip detection is all in same range (28~29) in；In tgev and examination
The limit of identification testing detection with colloidal gold strip detection is all in same range (29~210) in.The testing result of two kinds of antibody
Coincidence rate all reaches more than 98% it was demonstrated that this test strips has higher sensitivity.
3rd, the Detection of Stability of pedv and tgev antibody colloidal gold test paper bar
Take pedv positive serum, tgev positive serum, both mixing positive serums and each 5 parts of normal serum, make respectively
Detected that with this test strips every part of sample duplicate detection three times continuously repeats three days, calculate batch in and interassay coefficient of variation.
Result of the test shows, average variation within batch coefficient is 3.9%, and average interassay coefficient of variation is 4.5% it was demonstrated that this reagent paper
Bar has good stability.
The clinical practice of 5 colloidal gold strips of embodiment
Carry out the Blight control detection on Virus monitory and pig farm mainly for morbidity pig farm, whether detect in pig body to be checked
Containing pedv antibody or tgev antibody.
Clinical sample gathers clinical 50 parts of porcine blood serum sample for this laboratory, using pedv and tgev antibody colloidal gold test paper
The method of bar and neutralization test detects sample to be checked respectively.Testing result shows:
Pedv detects the result of serum sample with tgev antibody colloidal gold test paper bar: pedv antibody positive rate (comprises full sun
Result) it is 84% for 80%, tgev antibody positive rate (comprise full constipation caused by accumulation of heat fruit), full positive findingses are 80%.Test inspection with neutralizing antibody
The identical rate surveying result is all higher than 97%.
Test example 1
Carry out installation according to the method described in cn200810141163.6 and detected pedv and two kinds of diseases of tgev simultaneously
Colloidal gold colloidal gold detection test paper strip x: the supporting layer of malicious antibody selects plastic cement slip, test lead fibrous layer glass cotton, layers of absorbent material
From absorbent paper, cellulose film layer nitrocellulose filter；Gold mark albumen is spa albumen, and detection albumen is the good pedv of purification
And tgev, Quality Control albumen is rabbit anti-spa polyclonal antibody.Using parallel lines imprinting mark.
Carry out the Blight control detection on Virus monitory and pig farm mainly for morbidity pig farm, whether detect in pig body to be checked
Containing pedv antibody or tgev antibody.
Clinical sample gathers clinical 50 parts of porcine blood serum sample for this laboratory, using antibody colloidal gold test paper in the present invention
The method of bar, similar test strips x and neutralization test detects sample to be checked respectively.Testing result shows:
The pedv and tgev antibody colloidal gold test paper bar of the present invention detects the result of serum sample: pedv antibody positive rate
(comprising full constipation caused by accumulation of heat fruit) is 84% for 80%, tgev antibody positive rate (comprising full constipation caused by accumulation of heat fruit), and full positive findingses are 80%.With neutralization
The identical rate of antibody test testing result is all higher than 97%.
Similar colloidal gold strip x detects the result of serum sample: pedv antibody positive rate (comprising full constipation caused by accumulation of heat fruit) is
72%, tgev antibody positive rate (comprising full constipation caused by accumulation of heat fruit) is 76%, and full positive findingses are 70%.With neutralizing antibody testing inspection result
Identical rate be less than 75%.
The above is only the preferred embodiment of the present invention it is noted that ordinary skill people for the art
For member, on the premise of without departing from the technology of the present invention principle, some improvements and modifications can also be made, these improvements and modifications
Also should be regarded as protection scope of the present invention.
Claims (5)
1. a kind of Porcine epidemic diarrhea virus antibody and transmissible gastro-enteritis viruss antibody colloidal gold Rapid detection test strip, bag
Supporting layer, and sample-adding pad, gold mark protein delivery pad, detection layers and the absorbed layer setting gradually are included on described supporting layer, its
It is characterised by, described gold mark protein delivery pad is embedded with two kinds of recombinant expression proteins of colloid gold label, be that pig is popular respectively
Diarrhea viruses s1Albumen and the ad site recombinant expression protein of transmissible gastro-enteritis viruss s albumen；Arrange in described detection layers
There are two detection lines and two nature controlling lines, described two detection lines fixing described s successively1Albumen and the ad of described s albumen
Site recombinant expression protein fragment, described two nature controlling lines are fixed with the monoclonal antibody for two protein fragments successively,
Wherein, the Porcine epidemic diarrhea virus s that concentration is 1mg/ml will be coated1Albumen is sprayed in detection layers by the speed of 1 μ l/cm
As detection line 1, the ad site recombinant expression protein of the transmissible gastro-enteritis viruss s albumen that concentration is 0.5mg/ml will be coated
It is sprayed on as detection line 2 in detection layers by the speed of 1 μ l/cm, the Porcine epidemic diarrhea virus s that concentration is 1mg/ml will be coated1
Protein monoclonal antibody is sprayed on as nature controlling line 1 in the detection layers below detection line by the speed of 1 μ l/cm, will be coated concentration and be
The ad site recombinant expression protein monoclonal antibody of the transmissible gastro-enteritis viruss s albumen of 0.5mg/ml presses the speed of 1 μ l/cm
It is sprayed on and 30min is dried as at 2,37 DEG C of nature controlling line in the detection layers of nature controlling line 1 lower section, thus preparing detection layers.
2. test strips as claimed in claim 1 are it is characterised in that supporting layer selects polyethylene fibre plate；During sample-adding pad is selected
Fast qualitative filter paper；Release pad selects glass fibre；Detection layers select nitrocellulose filter；Absorbed layer selects glass fibre.
3. the preparation method of the test strips described in claim 1 or 2, it comprises the steps:
1) Porcine epidemic diarrhea virus s is fixed on the diverse location of detection layers respectively1Albumen and transmissible gastro-enteritis viruss s
The ad site recombinant expression protein fragment of albumen and its corresponding monoclonal antibody, form two detection lines and nature controlling line respectively；
2) release pad containing two kinds of gold mark albumen for the preparation；
3) assembling supporting layer, sample-adding pad, gold mark protein delivery pad, detection layers and absorbed layer.
4. method as claimed in claim 3 is it is characterised in that preparation gold mark protein delivery pad comprises the steps: with 1:100
~ 3000 labelling ratio is by Porcine epidemic diarrhea virus s to be marked1Albumen or the ad of transmissible gastro-enteritis viruss s albumen
Site recombinant expression protein fragment is added in ph 7.0 ~ 10.0 colloidal gold solution, in 1:10 ~ 500 ratio with containing 0.1 ~ 5%
0.001 ~ 0.1mol/l phosphate buffer of bsa dilutes the s of colloid gold label1The ad site of albumen or s albumen is recombinant expressed
Albumen, the colloid gold label protein liquid after dilution is pressed 60 μ l/cm2Speed be sprayed on glass fibre element film in, 4 DEG C of cryogenic vacuums
It is dried.
5. method as claimed in claim 3 will be it is characterised in that the Porcine epidemic diarrhea virus s that concentration is 1mg/ml will be coated1
Albumen is sprayed on as detection line 1 in detection layers by the speed of 1 μ l/cm, will be coated the pig transmissible gastrointestinal that concentration is 0.5mg/ml
The ad site recombinant expression protein of scorching virus s albumen is sprayed on as detection line 2 in detection layers by the speed of 1 μ l/cm, will be coated dense
Spend the Porcine epidemic diarrhea virus s for 1mg/ml1Protein monoclonal antibody is sprayed on the inspection below detection line by the speed of 1 μ l/cm
Survey on layer as nature controlling line 1, the ad site restructuring table of the transmissible gastro-enteritis viruss s albumen that concentration is 0.5mg/ml will be coated
Reach protein monoclonal antibody and be sprayed in the detection layers of nature controlling line 1 lower section by the speed of 1 μ l/cm and be dried as at 2,37 DEG C of nature controlling line
30min, thus prepare detection layers.
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| CN103454419B (en) * | 2013-07-22 | 2015-03-25 | 东北农业大学 | Immune colloidal gold test strip for detecting porcine epidemic diarrhea virus as well as preparation method and application thereof |
| CN105527437B (en) * | 2015-12-17 | 2018-04-20 | 洛阳普莱柯万泰生物技术有限公司 | A kind of detection kit and its application |
| CN109232736B (en) * | 2017-07-10 | 2021-11-09 | 洛阳普泰生物技术有限公司 | Monoclonal antibody specifically binding to porcine transmissible gastroenteritis virus, pharmaceutical composition, kit and application thereof |
| CN108761076A (en) * | 2018-05-24 | 2018-11-06 | 深圳出入境检验检疫局动植物检验检疫技术中心 | PEDV immune detections chromatograph test strip and its preparation method and application in milk |
| CN109609695A (en) * | 2018-12-29 | 2019-04-12 | 华南农业大学 | Detect the RPA-LED visualizing agent box of Porcine epidemic diarrhea virus |
| CN109975544A (en) * | 2019-04-26 | 2019-07-05 | 安徽荣达生物技术有限公司 | A kind of PEDV IgY antibody colloidal gold test strip and its preparation method and application |
| CN111596064B (en) * | 2020-04-28 | 2023-03-31 | 江苏省农业科学院 | Rapid test card for simultaneously detecting PEDV and TGEV and preparation and use methods thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101339192A (en) * | 2008-08-28 | 2009-01-07 | 河南省农业科学院 | Test paper for one-step detection for pig virus diarrhoea disease pathogen |
| CN101358972A (en) * | 2008-08-28 | 2009-02-04 | 河南省农业科学院 | Test paper strip for detecting one or more porcine virus diarrhea disease antibody |
| CN102735680A (en) * | 2012-07-05 | 2012-10-17 | 北京大北农科技集团股份有限公司 | Test strip for quickly detecting porcine circovirus 2 (PCV2) antibody by adopting colloidal gold |
-
2012
- 2012-12-27 CN CN201210577099.2A patent/CN103033626B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101339192A (en) * | 2008-08-28 | 2009-01-07 | 河南省农业科学院 | Test paper for one-step detection for pig virus diarrhoea disease pathogen |
| CN101358972A (en) * | 2008-08-28 | 2009-02-04 | 河南省农业科学院 | Test paper strip for detecting one or more porcine virus diarrhea disease antibody |
| CN102735680A (en) * | 2012-07-05 | 2012-10-17 | 北京大北农科技集团股份有限公司 | Test strip for quickly detecting porcine circovirus 2 (PCV2) antibody by adopting colloidal gold |
Non-Patent Citations (5)
| Title |
|---|
| Spike protein region (aa636-789) of procine epidemic diarrhea virus essential for induction of neutralizing antibodies.;D.B.Sun等;《Acta Viruologica》;20071231;第51卷;第149-156页 * |
| 猪传染性胃肠炎病毒S蛋白在昆虫细胞中的表达及间接ELISA抗体诊断方法的建立;李菁;《中国优秀硕士学位论文全文数据库》;20080515(第2008/05期);第二章摘要、第46页第3段 * |
| 猪传染性胃肠炎病毒胶体金免疫层析试纸条的研制;畅丹;《中国优秀硕士学位论文全文数据库》;20090315(第2009/03期);第21-24页 * |
| 猪流行性腹泻病毒S蛋白基因在昆虫细胞中的表达及间接ELISA抗体检测方法的初步建立;熊静;《中国优秀硕士学位论文全文数据库》;20090815(第2009/08期);第二章摘要 * |
| 猪流行性腹泻胶体金抗体检测技术的建立及其应用;张利勃 等;《中国农学通报》;20111231;第27卷;第374-377页 * |
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