CN104730238A - ELISA detection kit for detecting porcine epidemic diarrhea virus antibody and use method and application of ELISA kit - Google Patents

Abstract

The invention discloses an ELISA detection kit for detecting a porcine epidemic diarrhea virus antibody. According to the ELISA detection kit, a recombined porcine epidemic diarrhea virus N protein represented by the amino acid sequence SEQ ID NO:2 is used as an antigen for detecting whether a sample to be detected contains the porcine epidemic diarrhea virus antibody. The invention also discloses a use method and application of the kit. The kit for detecting the porcine epidemic diarrhea virus antibody can be used for effectively detecting the porcine epidemic diarrhea virus antibody, has the characteristics of high specificity, high sensitivity, simplicity, quickness and the like, can be used for clinically detecting whether a pig carries the porcine epidemic diarrhea virus and is good in application prospect.

Description

A kind of detect porcine epidemic diarrhea resisting antiviral antibody ELISA detection kit and using method and purposes

Technical field

The present invention relates to a kind of detect porcine epidemic diarrhea resisting antiviral antibody ELISA detection kit and using method and purposes.

Background technology

Pig epidemic diarrhea (Porcine epidemic diarrhea, PED) be by Porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, what PEDV) cause is a kind of with the acute high degree in contact enteric infectious disease vomitted, suffer from diarrhoea, dewater for principal character, and suckling pig has high fatal rate and high infection rate after infecting.The equal easy infection of pig of all age group, this disease found in 1971 in Belgium and Britain at first, and after this, many countries report the popular of this disease successively, particularly serious piglet, this disease of China's reported first in 1976; Nineteen fifty-five, Porcine epidemic diarrhea virus is classified as coronavirus genus by ICTV (International Committeeon Taxonomyof Viruses, ICTV) in the 6th report.Although China's selected swine farms takes the vaccine immunity measure of PEDV, China is since 2010, and national most of hog area breaks out this disease once again, causes extremely serious loss.In recent years, it is popular also to there is this disease in Korea S, has also in succession occurred again, cause huge economic loss to this disease since 2013 in the U.S., Japan and other countries.Generally, this disease is throughout world hog area, causes serious economic loss to whole world pig industry.

At present, the diagnostic method such as clinical diagnosis, immune colloid gold, ELISA, RT-PCR is mainly contained to the diagnostic method of PEDV.Their accuracy, stability are valued by the people all very well gradually.ELISA and enzyme linked immunosorbent assay, it is that current Application comparison is many, and development is more a kind of antibody detection method than faster.The method gets final product detectable antigens, and can go out antigen in ight soil by direct-detection, also can detect antibody, can be used for detecting when there is the symptom of suffering from diarrhoea in the middle of pig farm, can accomplish early diagnosis, early treatment, is widely used in clinical detection.

The detection kit prepared in existing document, accuracy is not high, and the accuracy of commercially available kit is higher, e.g., the Porcine-PEDV kit of R & B company, but price is high, and use cost is high.

Summary of the invention

In order to solve the problem, the invention provides a kind of detection PEDV.

The present invention detects the ELISA detection kit of porcine epidemic diarrhea resisting antiviral antibody, it is with the Recombinant Swine epidemic diarrhea virus N protein of amino acid sequence as shown in SEQ ID NO:2 for antigen, to detect in measuring samples the antibody whether containing porcine epidemic diarrhea resisting virus.

Described ELISA detection kit is indirect ELISA testing kit, and it comprises following component:

(1) the Recombinant Swine epidemic diarrhea virus N protein of amino acid sequence as shown in SEQ ID NO:2;

(2) ELISA Plate;

(3) antigen coated liquid;

(4) cleansing solution;

(5) confining liquid;

(6) sample diluting liquid;

(7) ELIAS secondary antibody;

(8) substrate solution;

(9) stop buffer.

Wherein, described antigen coated liquid is carbonate solution.

Wherein, described cleansing solution is PBST solution.

PBST solution: pH7.4,0.2g KH2PO4,0.2g KCl, 2.9g Na2HPO4.12H2O, 8.0g NaCl, 0.5mL Tween-20, add deionized water and be settled to 1000mL.

Wherein, described confining liquid is the gelatin of the skim milk of 5%, the BSA of 1% or 3%.

Wherein, described sample diluting liquid is PBST solution.

Wherein, described ELIAS secondary antibody is the anti-pig IgG of horseradish peroxidase-labeled rabbit.

Wherein, the TMB solution of described substrate solution to be concentration be 0.208mmol/L.

Wherein, the H of described stop buffer to be concentration be 2mol/L ₂sO ₄solution.

Present invention also offers the using method of aforementioned detection kit, it comprises the steps:

(1) bag quilt: get the Recombinant Swine epidemic diarrhea virus N protein of amino acid sequence as shown in SEQ ID NO:2, being diluted to protein concentration with coating buffer is 0.313 ~ 10 μ g/mL, coated elisa plate, washing;

(2) close: add confining liquid and close, washing;

(3) add measuring samples: get measuring samples, by diluted 10 ~ 320 times, add in microwell plate, 37 DEG C of incubation 1h, washing;

(4) add ELIAS secondary antibody: get ELIAS secondary antibody, by diluted 2000 times, add in microwell plate, 37 DEG C of reaction 30min, washing;

(5) substrate is added: add substrate solution, room temperature lucifuge reaction 10 ~ 15min;

(6) cessation reaction: add stop buffer, cessation reaction, measures its OD450 value.

In step (1), it is 2.5 μ g/mL that described coating buffer is diluted to protein concentration.

In step (2), measuring samples, by diluted 20 times.

Present invention also offers the purposes of aforementioned detection kit in the detection kit of preparation Porcine epidemic diarrhea virus.

The ELISA detection kit that the present invention comprises recombinant N protein accurately can detect Porcine epidemic diarrhea virus, basically identical with the testing result of the Porcine-PEDV kit of commercially available R & B company, may be used for alternative commercially available kit to use, the simultaneously high specificity, highly sensitive, simple, quick of kit of the present invention, preparation method is simple, with low cost.

The embodiment of form by the following examples, is described in further detail foregoing of the present invention.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following embodiment.All technology realized based on foregoing of the present invention all belong to scope of the present invention.

Accompanying drawing explanation

The RT-PCR amplification of Fig. 1 PEDV N gene.M:DL2000; 1: negative control; 2-3:RT-PCR amplified production N gene; ;

The double digestion qualification of Fig. 2 recombinant plasmid P GEM-T-N.M:DNA Marker III; 1-2: plasmid PCR; 3: negative control; 4-5:BamH I and XhoI double digestion product;

Fig. 3 PEDV N gene nucleotide series tetraploid rice;

Fig. 4 PEDV N gene amino acid sequence tetraploid rice;

Fig. 5 PEDV N phylogenetic trees; The plasmid PCR of Fig. 6 recombinant plasmid.M:DNA Marker III; 1-3: plasmid PCR; 4: contrast;

The double digestion qualification of Fig. 7 recombinant plasmid pET-28a-N.M:DNA Marker III; 1-2:BamH I and XhoI double digestion product;

Fig. 8 recombinant protein different abduction delivering time.M: albumen Marker; Contrast before 1:pET-28a-N induction; 2: 1h after induction; 3: 2h after induction; 4: 3h after induction; 5: 4h after induction; 6: 5h after induction; 7: 6h after induction; 8: 7h after induction; Contrast after 9:pET-28a induction;

The different abduction delivering temperature of Fig. 9 recombinant protein.M: albumen Marker; 1:25 DEG C of induction; 2:30 DEG C of induction 3:37 DEG C of induction;

Figure 10 recombinant protein not isogeneous induction IPTG concentration.M: albumen Marker; Contrast after 1:pET-28a induction; Before 2:pET-28a-N induction; 3:0.2mM; 4:0.4mM; 5:0.6mM; 6:0.8; 7:1.0mM; 8:1.2mM;

Figure 11 recombinant protein soluble analysis.M: albumen Marker; Contrast 2 after 1:pET-28a induction: total bacterium liquid; 3: supernatant after broken bacterium; 4: solubilization of inclusion bodies liquid;

The purification result of Figure 12 recombinant protein.M: albumen Marker; 1-2: albumen after purifying;

Figure 13 two kinds of purification process contrasts.M: albumen Marker; 1: total bacterium liquid; 2: spread liquid; 3: supernatant 4:KCl purifying after broken bacterium; 5: affinitive layer purification;

Figure 14 recombinant N protein Western blotting divides.M: albumen Marker; 1:N albumen.

Embodiment

One, experiment material:

1.1 bacterial strains and plasmid

Bacillus coli DH 5 alpha and BL21 (DE3); PET-28a (+) prokaryotic expression carrier is preserved by this laboratory.

TGEV and PEDV Combined vaccine, RV, PRSSV, APP, HPS positive serum are all preserved by this laboratory.

1.2 reagent

The blue or green enzyme element of Reverse Transcription box, yeast extract, tryptone, ammonia benzyl, block that penicillin, total RNA extraction reagent box (centrifugal column type), 50mg/ml IPTG (isopropylthio-β-D-galactoside), DAB, TMB, 2xTaqPCR Mastermix, DNA Marker III, DL2000, pre-dyed Marker III purchased from Beijing Tian Gen biochemical technology company limited.Mini-scale plasmid extracts kit, glue reclaims kit purchased from OMEGA company; PrimeScript RT reagent Kit, BamHI, XhoI are all purchased from the precious bioengineering company limited in Dalian.PGEM-T carrier, SDS-PAGE gel reagent preparation box, PEDV antibody assay kit are purchased from Chengdu Fei Ke biotech company.

1.3 equipment

Sorvall ST 16R high speed freezing centrifuge, Thermo company of the U.S.; MyCyclerTMPCR instrument, Agarose horizontal electrophoresis tank, albumen Vertial electrophorestic tank, gel imaging system, nucleic acid-protein instrument, BIO-RAD company of the U.S..

The preparation of embodiment 1 Recombinant Swine epidemic diarrhea virus of the present invention N protein

One, experimental technique

The clone of 1.1 N genes and analysis of biological information

1.1.1 design of primers

With reference to the PEDV N gene order that GenBank has delivered, devise a pair Auele Specific Primer and carry out pcr amplification, upstream primer: 5'-GGATCCATGGCTTCTGTCAGCTTTCAG-3' downstream primer: 5 ' end of 5'-CTCGAGTTAATTTCCTGTATCGAAGATC-3' upstream and downstream primer introduces BamH I and Xho I restriction enzyme site respectively, underscore is restriction enzyme site, and primer is synthesized by Shanghai Ying Jun company.

1.1.2 viral RNA extracts

Method step according to total RNA extraction reagent box extracts total serum IgE.Specific as follows:

(1) extract the centrifuge tube that 250 μ L viral suspensions are placed in 1.5mL sterilizing, add 200 μ L chloroforms in centrifuge tube, thermal agitation 15s, room temperature leaves standstill 3min.4 DEG C, the centrifugal 10min of 12000r/min, is carefully drawn to the aqueous layer in three layers in another new 1.5mL centrifuge tube.

(2) absolute ethyl alcohol of 0.5 times of volume is slowly added, mixing.Solution is joined in adsorption column CR3 together with precipitation.4 DEG C, the centrifugal 30s of 12000r/min, discards the waste liquid in collection tube.

(3) add 500 μ L protein liquid removal RD (having added the ethanol of respective volume in advance) in adsorption column, 4 DEG C, the centrifugal 30s of 12000r/min, discards the waste liquid in collection tube as far as possible.

(4) in adsorption column CR3, room temperature leaves standstill 2min to add 700 μ L rinsing liquid RW (having added the ethanol of respective volume), and 4 DEG C, the centrifugal 30s of 12000r/min, throws away the waste liquid in collection tube.

(5) step (4) is repeated.Adsorption column is put into 1.5mL collection tube, 4 DEG C, 12000r/min is empty from 2min, outwells residual liquid.

(6) ventilate 5min in superclean bench, and ethanol is fully volatilized.

(7) proceeded to by adsorption column in the centrifuge tube of a new 1.5mL, add 30 μ L RNase-free ddH2O in adsorption column CR3 central authorities, room temperature leaves standstill 2min, 4 DEG C, and the centrifugal 2min of 12000r/min, discards adsorption column, puts-20 DEG C and saves backup.

1.1.3 the RT-PCR of N gene

The total serum IgE of extraction is carried out RT-PCR amplification, and system is as follows:

CDNA reaction system:

Response procedures: 37 DEG C, 15min; 85 DEG C, 5S; 12 DEG C of Forever.

PCR reaction system:

Response procedures: 95 DEG C of 5min, 95 DEG C of 30S, 55 DEG C of 30s, 72 DEG C of 1min, 30 circulations, last 72 DEG C extend 10min.PCR primer 1.0% agarose gel electrophoresis is analyzed.

1.1.4 the choning and sequencing of N gene

(i) PCR primer glue reclaims

The PCR primer of amplification is after 1% agarose gel electrophoresis, and use DNA gel to reclaim purification kit and reclaim object band, method is as follows:

(1) gel containing genes of interest is cut with scalpel blade.Add Binding Buffer (namely every 100mg gel adds the Binding Buffer of 100 μ l) according to the weight of gel, be placed in 60 DEG C of baking ovens and melt completely to gel;

(2), after dissolving, proceed in adsorption column by the liquid of above-mentioned dissolving, the centrifugal 1min of 10000r/min, discards filtrate;

(3) in adsorption column, add the Binding Buffer of 300 μ l, the centrifugal 1min of 10000r/min, discards filtrate,

(4) add in adsorption column by 700 μ l rinsing liquid SPW Wash Buffer, the centrifugal 1min of 10000r/min, discards filtrate, repeats the operation of step (4);

(5) 13000r/min is empty from 2min, and take out adsorption column and put into new centrifuge tube, the eluent Elution Buffer of 20 μ l is added the central authorities of adsorption column, room temperature leaves standstill 2min, the centrifugal 2min of 13000r/min;

(6) product getting 3 μ l glue recovery carries out the agarose gel electrophoresis (85V, 25min) of 1%, observes and detect organic efficiency under gel imaging system.

(ii) genes of interest is connected with carrier T

The genes of interest fragment of the genes of interest fragment reclaimed through glue or directly synthesis is connected with PGEM-TVector, and reaction system is:

Above-mentioned reaction product mixing is placed on 16 DEG C of connections and spends the night, and gets and connects product conversion DH5 α competent cell.

(iii) the preparation of DH5 α competent cell

(1) streak inoculation DH5 α bacterium liquid on LB flat board, is placed in incubator 37 DEG C of incubated overnight.The mono-bacterium colony of DH5 α that picking LB grow on plates is good, be inoculated in 5ml LB fluid nutrient medium, 37 DEG C of 220r/min are cultured to exponential phase;

(2) the activation bacterium liquid getting 500 μ l is placed in the LB fluid nutrient medium of 50ml with the ratio of 1:100, it is about 0.5 that 37 DEG C of 220r/min expansions are cultured to OD600;

(3) transfer in the centrifuge tube of 50ml ice precooling by expanding the bacterium liquid cultivated, the centrifugal 10min of ice bath 30min, 4000r/min collects thalline (subsequent step all carries out on ice);

(4) outwell supernatant, by the resuspended precipitation of 0.1mol/L CaCl2 solution of 25ml ice precooling, leave standstill 20min, 4 DEG C of centrifugal 10min of 4000r/min;

(5) outwell supernatant, by the resuspended precipitation of 0.1mol/L CaCl2 solution of 10ml ice precooling, leave standstill 20min, 4 DEG C of centrifugal 10min of 4000r/min;

(6) outwell supernatant, by the resuspended precipitation of CaCl2 of cold 15% glycerine of 2ml ice bath, place on ice and namely make competent cell suspension in a moment, packing, often pipe 100ul is placed in-70 DEG C and saves backup;

(iv) the conversion of product is connected

(1) competence is put into 20min on ice from-70 DEG C of taking-ups rapidly;

(2) the connection product of 10 μ l is joined in 100 μ l competent cells slowly, stir and evenly mix gently with rifle head;

(3) EP pipe is placed 30min on ice, put into 42 DEG C fast and make its heat shock 90sec, then ice bath 5min;

(4) in mixed liquor, add the LB fluid nutrient medium of 600 μ l, 37 DEG C of 180r/min continue shaken cultivation 1h, and thalline is recovered;

(5) draw 200 μ l bacterium liquid in EP pipe to coat on the LB solid medium flat board containing Amp, flat board is placed in 37 DEG C and is inverted and cultivates observations after 16h.

(v) the PGEM-T-N qualification of recombinant plasmid

Random choose puts down containing Amp LB the white colony pulled, and inoculation 5ml LB fluid nutrient medium (containing Amp 100 μ l/ml) overnight incubation, adopts the mini-scale plasmid of OMEGA company to extract kit and extract recombinant plasmid.

(1) get 2ml incubated overnight bacterium liquid in centrifuge tube, the centrifugal 2min of 13000r/min, outwells supernatant as far as possible;

(2) in centrifuge tube, add 250 μ l Solution I (adding RNase A), thermal agitation fully mixes, and forms suspension;

(3) in centrifuge tube, add 250 μ l Solution II, turning upside down of gentleness makes the abundant cracking of thalline for 6 ~ 8 times immediately, and solution becomes limpid;

(4) in centrifuge tube, add 350 μ l Solution III, gentleness turns upside down and fully mixes gently for 6 ~ 8 times immediately, until produce the floccus of a large amount of white;

(5) the centrifugal 10min of 13000r/min, outwells filtrate;

(6) in adsorption column, add the HB of 700 μ l, the centrifugal 1min of 12000r/min, outwells filtrate;

(7) this step is repeated once:

(8) add in adsorption column by 700 μ l rinsing liquid PW, the centrifugal 1min of 12000r/min, discards filtrate; Repeat this step once;

(9) 13000r/min is empty from 2min, and take out adsorption column CP3 and put into new centrifuge tube, room temperature leaves standstill 3 ~ 5min, treats that alcohol volatilizees completely;

(10) add the elution buffer EB 50 μ l of prior preheating again to adsorption column central authorities, after room temperature leaves standstill 2min, the centrifugal 2min of 13000r/min, in-20 DEG C of preservations;

As reaction template after the plasmid DNA of extracting is diluted through 50 ~ 100 times, carry out plasmid PCR qualification.Get the agarose gel electrophoresis analysis that 5 μ l PCR primer carry out 1% after reaction terminates, system is as follows:

Carry out the qualification of BamHI, XhoI double digestion to recombinant plasmid PGEM-T-N, carry out endonuclease reaction, reaction time 180min at 37 DEG C, get the agarose gel electrophoresis analysis that 5 μ l digestion products carry out 1% after reaction terminates, system is as follows:

The recombinant plasmid being accredited as the positive is delivered to Hua Da biotech firm and carries out sequencing.With software, the nucleotide sequence measured and coded amino acid are analysed and compared, check the correctness of reading frame.

1.1.5N the bioinformatic analysis of gene coded protein

By the EditSeq workspace of the complete reading frame typing DNAStar of the row N gene that checks order, select Translate DNA function to translate the amino acid sequence of N gene; The signal peptide of SingalP v3.0 (http://www.cbs.dtu.dk/services/SignalP/) software to its amino acid sequence is utilized to analyze; The cross-film district of TMHMM v2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) software to this sequence is utilized to analyze; ProtScale (http://www.expasy.ch/tools/protscale.html) is utilized to analyze the hydrophobicity of albumen; Http:// imed.med.ucm.es/Tools/antigenic.pl is utilized to carry out on-line analysis to the epitope of N protein; Do you utilize Prositescan (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl? page=/NPSA/npsa_proscan.html) structure of N protein is analyzed.

The prokaryotic expression of 1.2 N genes

1.2.1 the recovery of genes of interest and carrier

Also reclaimed with a small amount of gel purification kit with BamH I and XhoI double digestion respectively by positive recombinant plasmid PGEM-T-N and expression vector pET28a, concrete grammar is the same.

1.2.2 the connection of object fragment and expression vector

The object fragment of being cut by enzyme and pET28a carrier carry out spending the night being connected, and linked system is as follows:

1.2.3 the preparation of BL21 competent cell

Method is same as above

1.2.4 transform

Above-mentioned 10ul is connected product to be forwarded in the competent escherichia coli cell DH5 α of 100ul, be transformed in BL21 (DE3) competence further by being accredited as positive recombinant plasmid, bacterium liquid after transforming is coated on the LB agar plate containing Kan, 37 DEG C of incubated overnight.Select single bacterium colony next day to cultivate in the LB fluid nutrient medium of 5ml containing Kan.

1.2.5 the qualification of recombinant plasmid

Extract plasmid with extracting plasmid kit in a small amount, carry out plasmid PCR qualification and BamH I and XhoI double digestion and identify, system method is the same, send Hua Da biotech firm to carry out sequencing by being tentatively defined as positive plasmid simultaneously.

1.2.6 the abduction delivering of recombinant expression carrier and analysis

(i) the optimization of IPTG induction time

(1) 5ml is inoculated in respectively containing in the LB fluid nutrient medium of 50 μ g/ml Kan by being accredited as positive pET-28a-N and pET28 α bacterium liquid with the ratio of 1:100,220r/min 37 DEG C of shaken cultivation are about 0.4 ~ 0.6 to bacterium liquid OD600 value, get 1ml bacterium liquid in 4 DEG C of preservations, using this as pre-induction sample;

(2) added by IPTG in residue bacterium liquid, make its final concentration be 1.0mmol/L, after induction, 1h, 2h, 3h, 4h, 5h, 6h, 7h get 1ml pET-28a-N bacterium liquid in 4 DEG C of preservations, respectively as postinduction sample;

(3) with the centrifugal 2min of postinduction sample 12000r/min before inducing, supernatant will be abandoned;

(4) 10 μ l 5 × SDS LoadingBuffer are added after getting the resuspended sample of 40 μ l aqua sterilisa more wherein, denatured by boiling 10min after mixing, the centrifugal 2min of 12000r/min after cooling;

(5) get 10 μ l supernatants and carry out SDS-PAGE electrophoresis, deposition condition is: 60V, 30min; 100V, 90min;

(6) after electrophoresis terminates, take out gel, to spend the night dyeing to polyacrylamide gel with R-250 Coomassie brilliant blue, dyeing is placed on decolouring in destainer until the background colour of colloid observes electrophoresis result in gel imaging system after substantially taking off, the best induction time that analyzing proteins is expressed;

(ii) the optimization of IPTG induced concentration

(1) pET-28a-N and pET28 α bacterium liquid is inoculated in 7 pipe 5mL containing in the LB fluid nutrient medium of Kan respectively with the ratio of 1:100,220r/min 37 DEG C of shaken cultivation are about 0.4 ~ 0.6 to bacterium liquid OD600 value, get 1mL 4 DEG C preservation, using this as pre-induction sample;

(2) in 6 pipe bacterium liquid, adding IPTG makes it be respectively to final concentration: 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L, 1.0mmol/L, 1.2mmol/L, IPTG is added to final concentration 1.0mmol/L in pET28a contrast, the centrifugal 2min of 1mL bacterium liquid 12000r/min is got after Fiber differentiation 4h, to abandon after supernatant by Sample storage in 4 DEG C, as postinduction sample;

(3) 10 μ L 5 × SDS LoadingBuffer are added after getting the resuspended sample of 40 μ L aqua sterilisa more wherein, denatured by boiling 10min after mixing, the centrifugal 2min of 12000r/min after cooling;

(4) get 10 μ L supernatants and carry out SDS-PAGE electrophoresis, the best induced concentration of electrophoresis post analysis protein expression.

(iii) the optimization of different inducing temperature

PET-28a-N and pET28 α bacterium liquid is inoculated in 5mL containing in the LB fluid nutrient medium of Kan respectively in the ratio of 1:100, shaken cultivation is about 0.4 ~ 0.6 to bacterium liquid OD600, add IPTG to suitable final concentration, the centrifugal 2min of 1mL bacterium liquid 12000r/min is got after Fiber differentiation 4h, collecting precipitation, 10 μ L 5 × SDS Loading Buffer are added again wherein, denatured by boiling 10min after mixing, the centrifugal 2min of 12000r/min after cooling after getting the resuspended sample of 40 μ L aqua sterilisa; Get 10 μ L supernatants and carry out SDS-PAGE electrophoresis, the best inducing temperature of electrophoresis post analysis protein expression.

(iv) the soluble analysis of recombinant protein

(1) contained in the LB fluid nutrient medium of Kan in 5mL by mono-for pET-28a-N good for grow on plates colony inoculation, 37 DEG C of 220r/min shaken cultivation are spent the night;

(2) the 5mL bacterium liquid of previous step incubated overnight is inoculated in 500mL containing in the LB fluid nutrient medium of Kan in the ratio of 1:100, shaken cultivation bacterium liquid is about 0.4 ~ 0.6 to OD600, add IPTG to optium concentration 0.8mM, 37 DEG C of 220r/min are cultured to optimum optimization time 4h;

(3) by the centrifugal 10min of bacterium liquid 12000r/min after abduction delivering, collecting precipitation;

(4) by 100mL cellular lysate liquid (containing 1mg/mL lysozyme, now with the current) resuspended precipitation, 4 DEG C are spent the night;

(5) by the thalline re-suspension liquid of spending the night at-20 DEG C and room temperature multigelation 3 times, be placed on ice with the condition that the intensity of 40% and the time interval of 1sec are ultrasonic disruption, ultrasonication 10min;

(6) thalline after ultrasonic disruption, cleer and peaceful precipitation in 12000r/min centrifugal 10min collection, carries out SDS-PAGE electrophoresis by upper cleer and peaceful precipitation, to determine that pET-28a-N exists with the form of inclusion body or exists with the form of soluble protein.

(v) the purifying of recombinant protein

1. glue purification is cut in the KCl dyeing of recombinant protein

(1) get two pieces of glass plates to assemble, be fixed on glue platform, add the separation gel of 12% to its 2/3 volume place, drive bubble out of with the compacting of water seal mouth; After gelling to be separated is solid, water is sucked, add 5% concentrated glue, do not insert comb, wait to coagulate.Protein sample after gel-like fluid process, direct loading.Voltage 60 ~ 80V during concentrated glue, after about 30min, voltage 120 ~ 150V during separation gel, until bromophenol blue moves to the bottommost of separation gel completely, stops electrophoresis.

(2) take out gel and be placed in a clean large plate, dye with appropriate 0.25M KCl dyeing liquor, about 5min;

(3) cut the object band gel be painted silvery white with scalpel blade, put it into (advanced processing is crossed and PBS is housed) in bag filter; Be placed in the Horizontal electrophoresis tank electrophoresis containing tris-glycocoll, about 30min; Its destination protein is entered in PBS can stop.

(4) super filter tube PBS liquid containing destination protein being placed in 30KDa carries out centrifugal, and the centrifugal 10min of 7000r/min, can obtain the destination protein that purifying is good, and-20 DEG C save backup.

2. the nickel ion affinity chromatograph purifying of recombinant protein

(1) filter (0.45 μm of filter membrane) after sample being regulated pH to 8.0, be sub-packed in 10mL centrifuge tube and save backup in-20 DEG C;

(2) add in glass chromatography column after getting the mixing of 5mL filler, after compacting of spending the night, successively wash post to balance with ultrapure water, loading Buffer, flow velocity is 2mL/min;

(3) loading 10mL, flow velocity is 0.5mL/min;

(4) loading Buffer wash-out foreign protein balances again to Ni-NTA post, and flow velocity is 1mL/min;

(5) wash-out is carried out with the wash-out Buffer containing imidazole concentration being 20mmol/L, 40mmol/L, 60mmol/L, 80mmol/L, 100mmol/L, 250mmol/L, 500mmol/L successively, flow velocity is 1mL/min, collect the eluent occurring eluting peak, for SDS-PAGE electrophoresis;

(6) collect the good protein eluate of purification effect to save backup in-20 DEG C.

(vi) the immunoblotting assay of recombinant protein

(1) get two pieces of supporting glass plates to assemble, be fixed on glue platform, add separation gel to the 2/3 volume place of 12%, drive bubble out of with hydraulic pressure glue simultaneously; After gelling to be separated is solid, water is sucked, add 5% concentrated glue, insert comb, wait to coagulate.

(2) the protein sample loading that 10 μ L handle well is got.Voltage 80V is set, 30min; 120V, 90min, move to from separation gel bottommost about 1cm to bromophenol blue, stops electrophoresis.

(3) take out running gel, cut containing Marker and have the gel lane of protein sample to carry out dyeing-decolorzing, in contrast; Residue has the gel lane of protein sample to be placed in transfering buffering liquid to balance 30min.

(4) they are all placed in transfering buffering liquid and balance 30min by cellulose nitrate (NC) film that the whatman 3mm filter paper that shearing 6 is slightly less than glue is identical with glue size with 1.

(5) transfer printing box is opened, spreading 3 the whatman 3mm filter paper, cellulose nitrate (NC) film, separation gel and other 3 the whatman 3mm filter paper that have balanced near anode-side is overlapping successively from down to up, get rid of bubble with glass rod in the process of putting, and NC film front is connected with glue.

(6) cover electrode cap and transfer box, 18V shifts 45min.

(7), after electrophoretic blotting terminates, gripping NC film, in deionized water, soaks 5min, gets rid of the bubble on film.

(8) be transferred to by film in the plate containing confining liquid, guarantee that film contacts completely with confining liquid, 37 DEG C of 60r/min on air table, reaction 1h, outwells confining liquid, washes film 4 times, each 5min with TBS cleansing solution.

(9) abandon cleansing solution to the greatest extent, add the anti-PEDV positive serum of pig of 200 times of PBS dilutions, soak NC film completely, 37 DEG C of 60r/min on air table, reaction 1h, repeats washing step above.

(10) add the anti-pig IgG of horseradish peroxidase-labeled rabbit of 2000 times of PBS dilutions, 37 DEG C of 60r/min react 1h, repeat washing step above.

(11) outwell cleansing solution, add substrate (DAB), reaction 5 ~ 15min, observes colour developing situation.

(12) band on NC film taken a picture and analyze.

Two, experimental result

The clone of 1.1 N genes and analysis of biological information

1.1.1 N gene magnification result

Utilize Auele Specific Primer to carry out RT-PCR to PEDV N gene, amplified the cDNA fragment that size is about 1326bp, its length and expection size consistent (see Fig. 1).

1.1.2 the double digestion qualification of recombinant plasmid PGEM-T-N

From recombinant plasmid, the specific fragment of 1326bp is amplified by PCR method, again by plasmid DNA through BamH I and XhoI double digestion, product electrophoresis obtains the DNA band (see Fig. 2) of two sizes about 3000bp and 1326bp respectively, consistent with expected result.

1.1.3 the sequencing of recombinant plasmid PGEM-T-N

Send Hua Da biotech firm to check order by being accredited as positive recombinant plasmid, sequencing result display PEDV N gene has successfully been cloned on PGEM-T carrier, and by recombinant plasmid called after PGEM-T-N.

(1) N gene order of the present invention (SEQ ID NO:1) is as follows:

ATGGCTTCTG TCAGCTTTCA GGATCGTGGC CGCAAACGGG TGCCATTATCTCTCTATGCCCCTCTTAGGG TTACTAATGA CAAGCCCCTT TCTAAGGTACTTGCAAACAA CGCTGTACCCACTAACAAGG GGAATAAGGA CCAGCAAATTGGGTACTGGA ATGAGCAAAT TCGCTGGCGCATGCGCCGTG GTGAGCGAATTGAACAACCT TCCAATTGGC ATTTCTACTA CCTCGGAACAGGACCTCACGGCGACCTCCG TTATAGGACT CGTACTGAGG GTGTTTTCTGGGTTGCTAAAGAAGGCGCAA AGACTGAACC CACTAATTTG GGTGTCAGAAAGGCGTCTGA AAAGCCAATCATTCCAAAAT TCTCTCAACA GCTCCCCAGTGTAGTTGAGA TTGTTGAACC TAACACACCTCCTGCTTCAC GTGCAAATTCGCGTAGCAGG AGTCGTGGCA ATGGCAACAA TAGGTCTAGATCTCCAAGTAACAACAGAGG CAATAACCAG TCCCGTGGTA ATTCACAGAA TCGTGGAAATAACCAGGGTC GTGGAGCTTC TCAGAACAGA GGAGGCAATA ATAATAACAATAACAAGTCTCGTAACCAGT CCAATAACAG GAACCAGTCA AATGACCGTGGTGGTGTAAC ATCACGCGATGATCTGGTGG CTGCTGTCAA GGATGCACTTAAATCTTTGG GTATTGGAGA AAATCCTGACAGGCATAAGC AACAGCAGAAGCCTAAGCAG GAAAAGTCTG ACAACAGCGG CAAAAATACACCTAAGAAGAACAAATCCAG GGCCACTTCG AAGGAACGTG ACCTCAAAGACATCCCAGAGTGGAGGAGAA TTCCCAAGGG CGAAAATAGC GTAGCAGCTTGCTTCGGACC CAGAGGGGGCTTCAAAAACT TTGGAGATGC GGAATTTGTCGAAAAAGGTG TTGATGCGTC AGGCTATGCTCAGATCGCCA GTTTAGCACCAAATGTTGCA GCATTGCTCT TTGGTGGTAA TGTGGCTGTTCGTGAGCTAGCGGACTCTTA CGAGATTACA TACAACTATA AAATGACTGTGCCAAAGTCAGATCCAAATG TTGAGCTTCT TGTTTCACAG GTGGATGCATTTAAAACTGG GAATGCAAAACTCCAGAGAA AGAAGGAAAA GAAGAACAAGCGTGAAACCA CGCTGCAGCA GCATGAAGAGGCCATCTACG ATGATGTGGGTGCGCCATCT GATGTGACCC ATGCCAATCT GGAATGGGACACAGCTGTTGATGGTGGTGA TACGGCCGTT GAAATTATCA ACGAGATCTTCGATACAGGAAATTAA

(2) amino acid (SEQ ID NO:2) of encoding is as follows:

MASVSFQDRGRKRVPLSLYAPLRVTNDKPLSKVLANNAVPTNKGNKDQQIGYWNEQIRWRMRRGERIEQPSNWHFYYLGTGPHGDLRYRTRTEGVFWVAKEGAKTEPTNLGVRKASEKPIIPKFSQQLPSVVEIVEPNTPPASRANSRSRSRGNGNNRSRSPSNNRGNNQSRGNSQNRGNNQGRGASQNRGGNNNNNNKSRNQSNNRNQSNDRGGVTSRDDLVAAVKDALKSLGIGENPDRHKQQQKPKQEKSDNSGKNTPKKNKSRATSKERDLKDIPEWRRIPKGENSVAACFGPRGGFKNFGDAEFVEKGVDASGYAQIASLAPNVAALLFGGNVAVRELADSYEITYNYKMTVPKSDPNVELLVSQVDAFKTGNAKLQRKKEKKNKRETTLQQHEEAIYDDVGAPSDVTHANLEWDTAVDGGDTAVEIINEIFDTGN

1.1.4 the component of N gene coding amino acid and analysis

Sequencing result shows: street strain's N gene order is made up of 1326bp, 441 amino acid of encoding.Amino acid whose isoelectric point is 9.918, and the molecular weight of albumen is 48.9KDa.

1.1.5 the analysis of N gene nucleotide series chadogram

AHYS-2012 is downloaded from Genebank database, Chinju99, CH-S, CQ12-2, CV777, GDYE11, JS-2004-2, LJB-03, the N gene order of SC and YT12-4 strain, order-checking strain N gene order is carried out nucleotide with it and amino acid sequence compares, result shows: the increase nucleotide homology of the PEDV N gene order that obtains and above-mentioned strain of the present invention is respectively 97.5%, 94.9%, 96.2%, 99.5%, 95.9%, 99.3%, 98.6%, 97.6%, 95.6%, 99.3%, amino acid identity is respectively: 94.8%, 95.7%, 97.1%, 99.3%, 96.6%, 98.9%, 98.6%, 98.0%, 95.7%, 98.9%.(see Fig. 3,4)

The N gene molecule chadogram of AHYS-2012, Chinju99, CH-S, CQ12-2, CV777, GDYE11, JS-2004-2, LJB-03, SC, YT12-4 and SC-D strain is constructed by MEGA5.1 method, find that the sibship of SC-D and CQ12-2 is nearest, comparatively near with the sibship of YT12-4, GDYE11, and with the sibship (see Fig. 5) comparatively far away of classical strains CV777 and SC.

The prokaryotic expression of 1.2 N genes

1.2.1 the qualification of recombinant expression plasmid pET-28a-N

The correct clone bacterium of qualification being transformed with being connected with expression vector pET28a after BamH I and XhoI double digestion, cultivating, extracting plasmid, carry out plasmid PCR and BamH I and XhoI double digestion to recombinant expression plasmid DNA to identify, electrophoresis and enzyme are cut result and are shown as the DNA band that two sizes are about 5400bp and 1326bp, conform to expected results (see Fig. 6,7).

1.2.2 the sequencing of recombinant expression plasmid pET-28a-N

Send Hua Da biotech firm to carry out sequencing recombinant expression plasmid pET-28a-N, it is the open reading frame of 1326bp that result display N gene order contains a length, derivation coding 441 amino acid.

1.2.3 the optimization of recombinant protein abduction delivering

(i) the determination of recombinant protein different abduction delivering time

The recombinant protein pET-28a-N of abduction delivering and empty carrier pET-28a is carried out SDS-PAGE electrophoresis, result display (see Fig. 8): different time sections all energy abduction delivering goes out the protein band of a treaty 52kDa size, but expression is different, front 4h can increase in time and increasing, after 4 hours, expression starts to reduce slowly, empty carrier pET-28a does not have onesize band to occur, so be defined as inducing rear 4h by the best induction time of expression of recombinant proteins.

(ii) the different abduction delivering temperature analysis of recombinant protein

SDS-PAGE electrophoresis result display (see Fig. 9), when 25,30 DEG C and 37 DEG C of inductions, the expression of recombinant protein is almost suitable, therefore the temperature of protein expression is defined as 37 DEG C.

(iii) recombinant protein different abduction delivering IPTG concentration analysis

The recombinant protein pET-28a-N of abduction delivering and empty carrier pET-28a is carried out SDS-PAGE electrophoresis, result display (see Figure 10): along with the difference adding IPTG concentration, the expression of recombinant protein is not identical yet, in rising trend during 0.2 ~ 0.8mM, during 0.8 ~ 1.2mM, the expression of recombinant protein is almost suitable, so we are decided to be 0.8mM IPTG concentration.

1.2.4 the soluble analysis of recombinant protein and purifying

By the bacterium liquid after induction 4h after ultrasonication, upper cleer and peaceful precipitation carries out SDS-PAGE electrophoresis, and result display (see Figure 11) recombinant protein mainly exists with the form of supernatant.Supernatant KCl dyeing is cut glue purification and nickel ion affinity chromatograph purifying and compared it simultaneously, the effect that glue purification is cut in result display KCl dyeing is better than nickel ion affinity chromatograph purifying (see Figure 13), so select KCl dyeing to cut glue method carry out purifying (see Figure 12), adopt Coomassie Brilliant Blue, with normal concentration albumen for reference, software automatic analysis draws, after purifying, the concentration of recombinant protein is about 2.0mg/mL.

1.2.5 recombinant protein immunoblotting assay

Transfer on nitrocellulose filter by after the recombinant N protein sample electrophoresis after expression and purification with half dry type electricity escape (18V 45min), carry out Western blot analysis.After the colour developing of DAB nitrite ion, nitrocellulose filter occurs the specific band of 1 treaty 52.0KDa size, has illustrated that the recombinant N protein of prokaryotic expression has good immunogenicity (see Figure 14).

Embodiment 2 ELISA detection kit of the present invention

One, experimental technique

The foundation of 1.1 indirect ELISA methods

1.1.1 the concrete operations of indirect ELISA method

Cleansing solution, dilution: PBST solution.

(1) bag quilt: wrapped after antigen coating buffer (carbonate solution of pH9.6) dilution by recombinant N protein and pulled by 96 hole enzyme marks, 100 μ l/ holes, are transferred to 4 DEG C after 37 DEG C of placement 1h and spend the night.

(2) wash: dry the liquid in hole, every hole adds 200 μ l cleansing solutions as far as possible, whirlpool concussion washing 3 times, each 3min.

(3) close: every hole adds the confining liquid of 100 μ l, 37 DEG C of reaction 1h, repeated washing step.

(4) increase serum: the serum to be checked adding optimum dilution degree, 100 μ l/ holes, 37 DEG C of incubation 1h, repeated washing step.

(5) add two to resist: every hole adds the anti-pig IgG of horseradish peroxidase-labeled rabbit (1:2000) 100 μ l, 37 DEG C of reaction 30min, repeated washing step.

(6) substrate is added: every hole adds the TMB 100 μ l of substrate nitrite ion 0.208mmol/L, room temperature lucifuge reaction 10 ~ 15min.

(7) cessation reaction: every hole adds stop buffer (2M H ₂sO ₄solution) 50 μ l, cessation reaction, measures its OD450 value.

1.1.2 the determination of antigen and serum best effort concentration

When other conditions are all constant, recombinant expressed N protein coating buffer (carbonate solution of pH9.6) after purifying is diluted according to 10ug/ml, 5ug/ml, 2.5ug/ml, 1.25ug/ml, 0.625ug/ml, 0.313ug/ml six dilutabilitys respectively, 100 μ l/ hole coated elisa plates; Pig TGEV and PEDV Combined vaccine positive serum and negative serum are pressed 1:10,1:20,1:40,1:80,1:160,1:320 doubling dilution respectively, carries out the test of square formation drop reaction.Microplate reader measures OD450 reading, calculates P/N value, determines that antigen the best bag is by concentration and serum optimum dilution degree.

1.1.3 the determination of confining liquid

When other conditions are identical, close ELISA Plate with skim milk, the BSA of 1%, the gelatin of 3% of 5% respectively, do blank, microplate reader measures the reading of OD450 simultaneously, to determine best confining liquid.

1.1.4 the determination of the anti-pig IgG optimum dilution degree of HRP-rabbit

With the suitableeest antigen concentration coated elisa plate, 100 μ l/ holes, after washing plate, close with the suitableeest confining liquid and off-period, yin and yang attribute serum reacts after adding by the dilutability of the best, and the anti-pig IgG of HRP-rabbit adds ELISA Plate successively with 1:1000,1:2000,1:4000,1:6000,1:8000 five dilutabilitys and reacts, and adds substrate reactions liquid 100 μ l/ hole, colour developing 10 ~ 15min, every hole adds 2mol/L H2SO4 50 μ l cessation reaction.Microplate reader reads OD450 value to determine the anti-pig IgG optimum dilution degree of enzyme mark rabbit.

1.1.5 the determination of best off-period

At identical conditions, with suitable confining liquid, it is closed, close 45min, 60min, 90min, 120min each reaction repetition 2 hole respectively, microplate reader reads OD450 value for 37 DEG C, to determine best off-period.

1.1.6 the determination in best serum and rabbit anti-pig IgG reaction time

With the suitableeest antigen concentration and antigen coated condition coated elisa plate, 100 μ l/ holes, after washing plate, close with the suitableeest confining liquid and off-period, add best dilute serum, 37 DEG C are terminated reaction respectively at after 30min, 60min, 90min, 120min, after adding the anti-pig IgG of rabbit, 37 DEG C are reacted respectively at 30min, 60min, 90min, and each reaction work 2 hole is repeated.Microplate reader reads OD450 value to determine the optimum reacting time of serum and the anti-pig IgG of rabbit.

1.1.7 the determination of criterion

The indirect ELISA method getting 65 parts of pig negative serums foundation that laboratory is preserved detects, and each sample repeats 2 times, measures OD450 value, gets its mean value.Add that 3 times of standard deviations (S) are the critical value of yin and yang attribute according to the average OD450 value (X) of principle of statistics 65 parts of negative serums, when the OD450 of sample is greater than yin and yang attribute critical value, the positive can be judged in the level of 99.9%, otherwise be then negative.

1.1.8 specific detection

By the indirect ELISA method that this experiment is set up, respectively the standard positive serum of TGEV, RV, PEDV, PRSSV, APP, HPS is detected under identical conditions, establish the positive of PEDV, negative serum and blank simultaneously.Microplate reader is measured OD450 value, carries out result judgement.

1.1.9 replica test

(i) interior revision test is criticized

Randomly draw PEDV positive serum 4 parts, negative serum 1 part, utilize indirect ELISA within 4 time periods, carry out repeatability and detect, record OD450 value, calculates variation within batch coefficient (CV), if CV<10%, then reproducible.

(ii) a revision test is criticized

Utilize the ELISA Plate of 4 kinds of different batches to carry out repeatability to 4 parts of PEDV positive serums and 1 part of negative serum to detect, the result of record OD450, calculates interassay coefficient of variation (CV), if CV<10%, then reproducible.

1.1.10 storage life test

According to best antigen concentration coated elisa plate, close rear 4 DEG C of preservations, after one month, the known positive, negative sample are detected, determine the storage life of kit according to the change of OD450.

1.1.11 the detection of coincidence rate

With the indirect ELISA method that this research is set up, the 105 parts of Swine serum preserved in this laboratory Serum Bank are detected, detect with U.S.'s R & B company PEDV antibody assay kit simultaneously, calculate positive rate, negative rate and both coincidence rates.

1.1.12 the clinical practice of indirect ELISA method

Detect from 326 parts of blood serum samples in Sichuan gathering from 2013-2014 years with the indirect ELISA method set up, and establish PEDV positive and negative serum control, microplate reader reads OD450 value.

Two, experimental result

1.1 the foundation of ELISA antibody detection method

1.1.1 the determination of antigen and serum best effort concentration

With dilutability be 10 μ g/mL, the recombinant N protein of 5 μ g/mL, 2.5 μ g/mL, 1.25 μ g/mL, 0.625 μ g/mL, 0.313 μ g/mLde is that the serum to be checked of 1:10,1:20,1:40,1:80,1:160,1:320 carries out square formation burette test respectively with extension rate, result shows: when envelope antigen concentration is 2.5 μ g/mL, when serum diluting multiple to be checked is 1:20, the P/N value that detection obtains, up to 4.178, is optimum detection effect.Therefore, the best effort concentration of antigen is decided to be 2.5 μ g/mL, and the optimum diluting multiple of serum to be checked is decided to be 1:20 (table 1).

The determination of table 1 antigen and serum best effort concentration

1.1.2 the determination of best confining liquid

According to the above-mentioned condition envelope antigen determined, close respectively with the gelatin of the skim milk of 5%, the BSA of 1% and 3%, the confining liquid that result display is best is the BSA (see table 2) of 1%.

The determination of the best confining liquid of table 2

1.1.3 the determination of the anti-pig IgG optimum dilution degree of HRP-rabbit

With the best bag by the antigen coated ELISA Plate of concentration, the yin and yang attribute serum adding optimum diluting multiple reacts, and the dilutability selecting the anti-pig IgG of HRP-rabbit is respectively that 1:1000,1:2000,1:4000,1:6000,1:8000 react.The anti-pig IgG dilutability of HRP-rabbit that result display is best is 1:2000 (see table 3).

The determination of table 3 ELIAS secondary antibody optimum dilution degree

1.1.4 the determination of best off-period

When other reaction conditionss are all constant, utilize concentration be 1% BSA close after 45min, 1h, 1.5h, 2h respectively, carry out ELISA detection and compare its result, the off-period that result display is best is 1 hour (see table 4)

The determination of table 4 best off-period

1.1.5 the determination of best sera incubation time

When other reaction conditionss are all constant, after serum is hatched 45min, 1h, 1.5h, 2h respectively, carry out ELISA detection and compare its result, the best incubation time of result display serum is 1h (see table 5).

The determination of table 5 optimum antibody incubation time

1.1.6 the determination of the best incubation time of the anti-pig IgG of HRP-rabbit

When other reaction conditionss are all constant, after anti-for HRP-rabbit pig IgG is hatched 45min, 1h, 1.5h, 2h respectively, carry out ELISA detection and compare its result, the anti-pig IgG incubation time of HRP-rabbit that result display is best is 30min (see table 6).

The determination of the best incubation time of the anti-pig IgG of table 6 enzyme mark rabbit

1.1.7 the determination of criterion

65 parts of pig negative serums are detected with the indirect ELISA antibody detection method that this experiment is set up, the mean value X=0.1329 of negative serum as can be seen from Table 7, standard variance S=0.07226, then X+3S=0.1329+3x0.07226=0.35 is so be positive when OD450 >=0.35 of sample, otherwise is then negative.

The determination of table 7 criterion

1.1.8 specific detection

With the indirect ELISA detection method of this experimental construction, respectively the standard positive serum of TGEV, RV, PEDV, PRSSV, APP, HPS is detected under the same conditions, result display is except PEDV positive serum, all the other OD450 values detecting serum are all less than 0.35, show constructed indirect ELISA detection method serum-free cross reaction (see table 8).

Table 8 specific test

1.1.9 replica test

(i) interior replica test is criticized

Shown by table 9: the coefficient of variation of 4 parts of positive serums and 1 part of negative serum is all less than 10%, this shows that same sample is less with the degree of variation in a collection of test, and repeatability is good

Replica test result in table 9 batch

(ii) a repeated experiment is criticized

The coefficient of variation of the ELISA method set up with the elisa plate of different batches to 4 parts of positive serums and 1 part of negative serum is all less than 10%, and this shows that the degree of variation of sample in same batch is less, and repeatability is good.

Replica test result between table 10 batch

1.1.10 stability

The indirect ELISA method set up with this test is placed in well 4 DEG C of ELISA Plate of preserving to bag, detect with a serum after one month, record OD450 change not obvious, this illustrates that the ELISA Plate of recombinant N protein bag quilt deposits one month on testing result impact not quite at 4 DEG C, i.e. at least one moon of storage life.

1.1.11 coincidence rate

The indirect ELISA method set up with this experiment and the Porcine-PEDV kit of R & B company compare, and result negative rate is 87.7%, positive rate 95.0%, and both coincidence rates are 90.48% (see table 11).

Table 11 indirect ELISA and the comparative analysis of ELISA detection kit testing result

1.1.12 the clinical practice of indirect ELISA

The indirect ELISA method set up with this research detects (baseline results is in table 13) 326 parts of blood serum samples that this laboratory gathered 11, Sichuan regional from 2013-2014 years, the total positives rate of result display serum is 57.4%, randomly draw 100 parts of serum and carry out commercial kit detection, result display detects 46 parts of positive serums simultaneously, 52 parts of negative serums, both degrees of conformity are 98% [(46+52)/100] (see table 12).

Table 12 PEDV clinical sample testing result

The clinical sample baseline results that table 13. indirect ELISA detects

Experimental result explanation, basically identical by the result of the testing result of kit of the present invention and the Porcine-PEDV kit of R & B company, illustrate that kit that recombinant N protein of the present invention forms accurately can detect the Porcine epidemic diarrhea virus in clinical sample.

To sum up, invent the ELISA detection kit comprising recombinant N protein and accurately can detect Porcine epidemic diarrhea virus, basically identical with the testing result of the Porcine-PEDV kit of commercially available R & B company, may be used for alternative commercially available kit to use, the simultaneously high specificity, highly sensitive, simple, quick of kit of the present invention, preparation method is simple, with low cost.

Claims (11)

1. one kind is detected the ELISA detection kit of porcine epidemic diarrhea resisting antiviral antibody, it is characterized in that: it is with the Recombinant Swine epidemic diarrhea virus N protein of amino acid sequence as shown in SEQ ID NO:2 for antigen, detects the porcine epidemic diarrhea resisting antiviral antibody in measuring samples.

2. detection kit according to claim 1, is characterized in that: described ELISA detection kit is indirect ELISA testing kit, and it comprises following component:

(1) the Recombinant Swine epidemic diarrhea virus N protein of amino acid sequence as shown in SEQ ID NO:2;

(2) ELISA Plate;

(3) antigen coated liquid;

(4) cleansing solution;

(5) confining liquid;

(6) sample diluting liquid;

(7) ELIAS secondary antibody;

(8) substrate solution;

(9) stop buffer.

3. detection kit according to claim 2, is characterized in that: described antigen coated liquid is carbonate solution.

4. detection kit according to claim 2, is characterized in that: described cleansing solution or sample diluting liquid are PBST solution.

5. detection kit according to claim 2, is characterized in that: described confining liquid is the gelatin of the skim milk of 5%, the BSA of 1% or 3%.

6. detection kit according to claim 2, is characterized in that: described ELIAS secondary antibody is the anti-pig IgG of horseradish peroxidase-labeled rabbit.

7. detection kit according to claim 2, is characterized in that: the TMB solution of described substrate solution to be concentration be 0.208mmol/L.

8. detection kit according to claim 2, is characterized in that: the H of described stop buffer to be concentration be 2mol/L ₂sO ₄solution.

9. the using method of claim 1 ~ 8 any one detection kit, is characterized in that: it comprises the steps:

(1) bag quilt: get the Recombinant Swine epidemic diarrhea virus N protein of amino acid sequence as shown in SEQ ID NO:2, being diluted to protein concentration with coating buffer is 0.313 ~ 10 μ g/mL, coated elisa plate, washing;

(2) close: add confining liquid and close, washing;

(3) add measuring samples: get measuring samples, by diluted 10 ~ 320 times, add in microwell plate, 37 DEG C of incubation 1h, washing;

(4) add ELIAS secondary antibody: get ELIAS secondary antibody, by diluted 2000 times, add in microwell plate, 37 DEG C of reaction 30min, washing;

(5) substrate is added: add substrate solution, room temperature lucifuge reaction 10 ~ 15min;

(6) cessation reaction: add stop buffer, cessation reaction, measures its OD450 value.

10. using method according to claim 9, is characterized in that:

In step (1), be 2.5 μ g/mL by coating buffer diluted protein to concentration;

In step (3), measuring samples diluted 20 times.

The purposes of 11. claim 1 ~ 8 any one detection kit in the detection kit of preparation Porcine epidemic diarrhea virus.