CN104777312A - ELISA kit used for detecting swine transmissible gastroenteritis virus antibody - Google Patents
ELISA kit used for detecting swine transmissible gastroenteritis virus antibody Download PDFInfo
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Abstract
The invention provides an ELISA kit used for detecting a swine transmissible gastroenteritis virus antibody. The kit is an ELISA strip using purified recombinant swine transmissible gastroenteritis virus N protein as an envelope antigen, and further comprises a cleaning solution, a serum diluent, a substrate color-developing solution, a second enzyme-labeled sheep antibody against pig, a stop buffer, TGEV positive serum and TGEV negative serum. The kit is used for detecting an antibody against the swine transmissible gastroenteritis virus; and according to detection results, the kit has the advantages of good specificity, sensitivity and repeatability, accurate detection, simple operation and applicability to popularization in clinical application, and provides reliable means for rapid detection of swine transmissible gastroenteritis.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of ELISA kit detecting transmissible gastro-enteritis virus antibody.
Background technology
Transmissible gastroenteritis of swine (transmissible gastroenteritis, TGE) be by the transmissible gastro-enteritis virus of coronaviridae, coronavirus genus (transmissible gastroenteritis virus, TGEV) one caused is acute, high degree in contact sexually transmitted disease, to vomit, serious watery diarrhea, dehydration and to piglet within 2 weeks age highly fatal rate be feature.TGE is a kind of disease of world-wide prevalence, and International Office of Epizootics (OIE) is classified as the pig infectious disease must examined in category-B disease, and therefore, the research of TGE diagnostic techniques has great importance.
Diagnosis at present for TGEV mainly relies on RT-PCR method, the method to experimental facilities and conditional request higher, be not suitable for extensive quick diagnosis clinically.Apply various immune serum learn a skill epidemic disease made accurately, diagnosis is rapidly one of important measures of this disease of prevention and corntrol.ELISA method has the advantages such as easy, quick, high specificity, and ELISA detects the immune effect that antibody can evaluate vaccine, but needs antigen purity higher and immunogenicity is good.TGEV N protein molecular mass is the nucleocapsid protein of 35 ~ 48kDa, phosphorylation, N protein is a kind of protein of virion inside, the nucleoprotein of main composition virus, it holds geneome RNA, helically formula structure, diameter 9 ~ 16nm, makes it easily mix in virus, during replication in vitro, antiserum prepared by N protein can suppress the synthesis of the gene RNA of 90%.N protein contains proteolysis sites, by proteolysis and dephosphorylation, plays an important role to the assembling of virion.N gene can induce body to produce cellular immunity, and well-conserved due to nucleoprotein, the research of nucleoprotein as diagnostic antigen is also attracted widespread attention.
Summary of the invention
The object of the present invention is to provide a kind of ELISA kit and the application thereof that detect TGEV antibody.This kit is with low cost, easy and simple to handle, and the detection for TGEV provides a kind of fast easy-to-use serological diagnostic method, good market prospects.
The present invention realizes especially by following technical scheme:
Detect an ELISA kit for transmissible gastro-enteritis virus antibody, comprise following component:
1) elisa plate bar: using transmissible gastro-enteritis virus recombinant N protein as envelope antigen, elisa plate bar is closed through 5% (M/V) skimmed milk, packs in 4 DEG C of preservations with packaging bag;
2) cleansing solution: containing the pH7.4 phosphate buffer (PBS-T) of 0.05% Tween-20;
3) serum dilution: 5% (M/V) skimmed milk;
4) substrate nitrite ion: add H
2o
2tMB solution;
5) goat-anti pig ELIAS secondary antibody: the goat-anti pig ELIAS secondary antibody being diluted to working concentration 1:10000 with serum dilution;
6) stop buffer: 2M H
2sO
4solution;
7) TGEV positive serum;
8) TGEV negative serum.
Transmissible gastro-enteritis virus recombinant N protein of the present invention is prepared by following steps:
1) using the RNA of transmissible gastro-enteritis virus TGEV as material, its N gene is obtained by RT-PCR method amplification, gene accession number JX827607.1, amplified fragments size is 816bp, and using pET-28a plasmid as vector construction recombinant plasmid pET-28a-N, after N gene and pET-28a carrier use BamH I and Sal I double digestion respectively, connect construction recombination plasmid pET-28a-N, double digestion qualification recombinant plasmid;
2) positive recombinant expression plasmid pET-28a-N is transformed BL21 (DE3) competent cell, obtain positive plasmid bacterium monoclonal pET-28a-N/BL21, this monoclonal is in 37 DEG C of cultivations, when A600 value reaches 0.4 ~ 0.6, adding IPTG to final concentration is that 0.8mmol/L carries out abduction delivering, the thalline of 6h after collection abduction delivering, carry out SDS-PAGE qualification, get the thalline of 6h after abduction delivering, ultrasonication, centrifugally get supernatant respectively and precipitation carries out SDS-PAGE electroresis appraisal afterwards, according to electrophoresis result, get supernatant expression liquid and carry out Western blot qualification, a large amount of abduction delivering N protein, ultrasonication, get supernatant and express liquid, Ni-TED post is used to carry out purifying, collect 1xElution Buffer wash-out gained purifying protein.
ELISA kit concrete operation step of the present invention is as follows:
1) blood serum sample is added:
After serum to be checked being done 1:80 dilution with serum dilution 5% (M/V) skimmed milk, every hole adds 100 μ L, and each blood serum sample adds holes, sets up TGEV positive and negative serum in contrast simultaneously, respectively adds holes; Act on 30 minutes under room temperature, discard the liquid in reacting hole: each hole 200 μ L cleansing solutions fully clean 5 times, each 5 minutes, by the liquid removing in reacting hole after each washing, after last removing cleansing solution, the liquid that fully removing is residual;
2) goat-anti pig ELIAS secondary antibody is added:
Every hole adds 100 μ L goat-anti pig ELIAS secondary antibody, room temperature effect 30 minutes, by method washing in step (1);
3) substrate nitrite ion is added:
Every hole adds 100 μ L and adds H
2o
2tMB solution, lucifuge effect 10 minutes under room temperature;
4) cessation reaction:
Every hole adds 100 μ L 2M H
2sO
4stop buffer, color development stopping is reacted;
5) OD value is measured by microplate reader:
Use microplate reader under 450nm wavelength, measure each hole absorption photometric (OD) value;
6) result judges:
Indirect ELISA adds positive serum, negative serum when detecting, and measures OD value by microplate reader under 450nm wavelength.Test set up condition be: Positive control wells OD450 value all should≤1.0 and <3.0, negative control hole OD450 value all should≤0.2.Calculating the ratio K Q of each part serum OD450 and Positive control wells OD450, is positive when sample KQ value >=26.768, is negative during < 26.768.
Beneficial effect of the present invention is: ELISA kit of the present invention, use transmissible gastroenteritis of swine virus (TGEV) recombinant N protein as envelope antigen, this albumen is the prokaryotic expression product of TGEV N genetic recombination pET-28a-N expression vector, be easy to preparation and purification, immunoreactivity is good, as envelope antigen after this protein purification, its concentration is easy to deterministic finite automata, is conducive to a large amount of production and the cost control of this kit; Kit of the present invention is for detecting the antibody of transmissible gastro-enteritis virus, and through inspection, this kit specificity, susceptibility, repeatability are all good.This kit detects accurate, easy and simple to handle, is suitable for promoting in clinical practice, for the quick detection of transmissible gastroenteritis of swine disease provides reliable means.
Accompanying drawing explanation
Fig. 1 is that pET-28a-N/BL21 expresses the qualification of bacterium positive monoclonal; M:DS 15000Marker; 1: positive pET-28a-N/BL21 bacterium monoclonal; 2: negative pET-28a-N/BL21 bacterium monoclonal;
Fig. 2 is the abduction delivering of TGEV N protein; M:Prestained Peotein Ruler; 1: pET-28a-N/BL21 bacterium liquid sample after induction; 2: the pET-28a-N/BL21 bacterium liquid sample of not inducing;
Fig. 3 is the qualification of N protein solubility expression; M:BlueRay Prestained Peotein ladder; 1: supernatant samples after ultrasonication; 2: inclusion body sample after ultrasonication;
Fig. 4 is that Western Blot identifies; M:BlueRay Prestained Peotein ladder; 1: pET-28a-N/BL21 bacterium liquid sample after induction;
Fig. 5 is the purifying of N protein; M:BlueRay Prestained Peotein ladder; 1: the 1mL eluent; 2: the 2mL eluents; 3: the 3mL eluents.
Embodiment
Below in conjunction with embodiment, the present invention is described further, the following stated, only to preferred embodiment of the present invention, not do other forms of restriction to the present invention, any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed to the Equivalent embodiments of equal change.Everyly do not depart from the present invention program's content, any simple modification done following examples according to technical spirit of the present invention or equivalent variations, all drop in protection scope of the present invention.
The preparation of embodiment 1 kit
The structure of 1.TGEV N protein expression vector
Extract transmissible gastro-enteritis virus and infect disease pig (Xin Xiang pig farm, Hongze, Jiangsu Province) enteron aisle pathological material of disease RNA, amplification obtains its N gene (accession number JX827607.1).N gene connects pMD19-T carrier (TaKaRa), and 16 DEG C of connections are spent the night.Connect product conversion Trans5 α competent cell (TaKaRa), and the LB being applied to amicillin resistance (100mg/ml) is dull and stereotyped, 37 DEG C of overnight incubation.Picking monoclonal, in the LB nutrient culture media of amicillin resistance after shaken cultivation 10h, extract plasmid, enzyme cuts qualification.Check order being initially identified as positive bacterium liquid.Choose the pMD18-T-N plasmid that order-checking is correct, with pET-28a empty plasmid (Novagen), use BamH I and Sal I double digestion respectively.Digestion products carries out agarose gel electrophoresis, cuts N gene object band and pET-28a carrier ribbon respectively, cuts glue and reclaims.Reclaim product 16 DEG C of connections to spend the night.Connect product conversion Trans5a competent cell, routinize and choose positive colony, extract the qualification of thalline plasmid enzyme restriction, as shown in Figure 1, obtain pET-28a-N recombinant plasmid.
2. the abduction delivering of recombinant expression plasmid
Restructuring pET-28a-N Plastid transformation BL21 (DE3) competent cell (TaKaRa), routinizes and chooses positive colony, extracts the qualification of thalline plasmid enzyme restriction, called after pET-28a-N/BL21.PET-28a-N/BL21 expresses bacterium activation culture, to A600=0.6, gets 1ml bacterium liquid and preserves, add 0.1%IPTG and induce.1ml bacterium liquid is got every 1h, 6h to induction after induction.By bacterium liquid 4 DEG C, the centrifugal 5min collecting precipitation of 12000r/min collected.Often pipe precipitation adds 10 microlitre SDS-PAGE sample-loading buffers after having hanged with the PBS of 40 microlitres, and boiling water bath 10min, uses SDS-PAGE electrophoretic analysis.Electrophoresis result as shown in Figure 2, with reference to molecular mass standard, visible abduction delivering obtains albumen size and is about 45kDa, and to add his-tag (gehealthcare) in the same size with the N protein of prediction, proves that this recombinant plasmid obtains expression in Escherichia coli.
Get the bacterium liquid that 1ml induces 5h, the centrifugal 2min of 8000rpm, with the resuspended thalline of 300 μ L PBS.Freeze thawing 3 times, ultrasonication, centrifuging supernatant and inclusion body.By the resuspended inclusion body precipitation of 300 μ l PBS.Respectively get 40 μ L supernatants and inclusion body re-suspension liquid, add 10 microlitre SDS-PAGE sample-loading buffers, boiling water bath 10min, carries out SDS-PAGE electrophoresis.Electrophoresis result as shown in Figure 3, express in supernatant by visible N protein.
3. the TGEV N protein Western Blot that recombinates tests
After getting induction, 5h bacterium liquid carries out SDS-PAGE electrophoresis, as shown in Figure 4.By SDS-PAGE gel good for electrophoresis, not dyed, directly with transfer device, albumen is transferred on nitrocellulose filter (NC film) with 20V electricity, transfer printing 1.5h.Take out NC film, in TBST, rinsing once, adds confining liquid (TBST containing 2% skimmed milk power), lie against on shaking table and slowly shake 1h.NC film is put into the primary antibodie (TGEV Positive Sera) with confining liquid dilution, lies against room temperature effect 1h on shaking table.Incubation well takes out NC film afterwards, washes 5 times, each 5min with TBST.NC film is put into two anti-(1:5000 dilutes the anti-pig IgG two of enzyme mark and resists) with confining liquid dilution, lies against room temperature effect 0.5h on shaking table.Incubation well takes out NC film afterwards, washes 5 times, each 5min with TBST.NC film is put into 10mL substrate solution, lucifuge colour developing 1min ~ 15min, once there is band, take out immediately and immerse ddH
2in O, rinsing to be taken pictures with cessation reaction.
4. the purifying of recombinant protein
PET-28a-N/BL21 expresses bacterium and adds 5ml LB nutrient culture media, and activation culture is spent the night.Get 2ml bacterium liquid to add in 200ml LB nutrient culture media, be cultured to A600=0.6, after adding 0.1%IPTG, Fiber differentiation 5h.Collect the centrifugal 10min of bacterium liquid 8000rpm, abandon supernatant, thalline is weighed.It is resuspended that every gram of precipitation adds 10ml LEW solution, and the centrifugal 10min of 8000rpm, abandons supernatant again.
It is resuspended that every gram of precipitation adds 5ml LEW solution, ultrasonication.The centrifugal 10min of 10000rpm, collects supernatant, after using 0,45 μm of frit, joins Protino Ni-TED post and carry out purifying, collects 1ml filtered solution.Use 8ml LEW to wash Ni-TED post, collect 1ml cleansing solution.With 9ml Elution buffer wash-out, each 1ml, washes 9 times, collects each eluent.SDS-PAGE checks, and visible purification effect is good as shown in Figure 5, and this N protein is easy to preparation and purification.
The preparation of sample diluting liquid, cleansing solution, stop buffer: cleansing solution is the 0.01M pH7.4 phosphate buffer containing 0.05% Tween-20: KH
2pO
40.2g, Na
2hPO
412H
2o 2.9g, NaCl 8g, is settled to 1000mL, adds 0.5mL Tween-20; Sample diluting liquid is the cleansing solution containing 1% (M/V) BSA: 1g BSA adds in 100mL cleansing solution; Stop buffer is 2M sulfuric acid solution: the 111.2mL concentrated sulphuric acid, and dilution is settled to 1000mL.
5. the suitableeest bag of antigen is by the determination of concentration and serum optimal dilution
Adopt the suitableeest bag of upright titrimetry determination antigen by concentration and serum optimal dilution.
With coating buffer, the TGEV recombinant N protein of purifying is gradient dilution 1:80 (390ug/ml), 1:160,1:320,1:640,1:1280,1:2560,1:5120,1:10240, be added in 96 hole polystyrene micro-reaction plates with the minim in 100 μ L/ holes, composition square formation, 4 DEG C of bags are spent the night; Discard antigen liquid, fully wash three times with cleansing solution, 200 μ l/ holes, 5min/ time, use the skimmed milk of 5% to close, 200 μ L/ holes, 37 DEG C of closed 1h; Discard confining liquid, with the washing of above-mentioned method of operating, TGEV positive and negative serum is taken turns doing 1:5,1:10 and is diluted to 1:640, join in polystyrene micro-reaction plate respectively with the amount of every hole 100 μ L, under the condition of 37 DEG C, act on 60min; Discard serum, with the washing of above-mentioned method of operating, add the ELIAS secondary antibody of 1:10000 dilution, 100 μ l/ holes, act on 30min under the condition of 37 DEG C; Discard ELIAS secondary antibody, wash by above-mentioned method of operating, discard liquid in hole as far as possible, add substrate nitrite ion 100 μ L/ hole, 37 DEG C of lucifuge colour developing 10min, finally add stop buffer cessation reaction; Choose positive serum OD450nm value about 1, negative serum OD450nm is lower than 0.2, and antigen coated concentration when positive serum OD450nm/ negative serum OD450nm is maximum and serum dilution are best effort concentration.
Upright titration results is as shown in table 1, and when recombinant N protein dilution 1:2560 (0.152 μ g/mL) coated elisa plate, during serum-dilution 1:80, positive serum OD value is close to 2, and negative serum OD value is less than 0.2, and P/N value is 10.06 larger.Therefore determine that the bag of recombinant N protein is 0.152 μ g/mL by optium concentration, serum optimum dilution degree is 1:80.
The upright titration results of table 1ELISA
6. bag is by the selection of condition
Best antigen concentration coated elisa plate, spends the night 4 DEG C of reactions respectively, 37 DEG C of reaction 1h adds that 4 DEG C of reactions are spent the night, 37 DEG C of reaction 1h, 37 DEG C of reaction 2h, carry out bag quilt.Carry out ELISA detection with known positive and negative serum, measure OD450 and analyze P/N situation of change, evaluate its bag by effect.Finally determine that best bag is 37 DEG C of reaction 2h by condition.
7. the selection of confining liquid
Best antigen concentration coated elisa plate, bag is moved to end rear washing, uses 5% skimmed milk power, 1%BSA, 1% gelatin as confining liquid respectively, closes 1h.When other conditions are identical, carry out ELISA detection with known positive and negative serum, measure OD450 and analyze P/N situation of change, evaluating its sealing effect.Finally determine that best confining liquid is 5% skimmed milk.
8. the determination of sealing condition
When other conditions are fixing, with selected confining liquid respectively 37 DEG C close 30,60,120,360min, carry out ELISA detection with known positive and negative serum, measure OD450 and also analyze P/N situation of change, evaluate its sealing effect.Finally determine that best sealing condition is 37 DEG C of reaction 30min.
9. the determination of the suitableeest antigen-antibody reaction time
Best confining liquid is closed, after adding the serum of optimum dilution degree, 37 DEG C hatch 15 respectively, 30,45,60min.Carry out ELISA detection when other conditions are constant, measure OD450 value and analyze P/N situation of change, evaluating its effect.Finally determine that the suitableeest antigen-antibody reaction time is 30min.
10. the determination of the suitableeest two anti-working concentrations
After ELIAS secondary antibody is diluted to 1:5000,1:10000,1:15000,1:20000, carry out ELISA detection when other conditions are constant, according to OD450 value and P/N value situation of change, evaluate its effect.Finally determine that the suitableeest two anti-working concentrations are 1:10000.
The determination of 11. the suitableeest developing times
Act on 5 respectively in room temperature after adding TMB, 10,15,20min, be above-mentioned optimized top condition in other conditions, according to OD450 value and P/N value situation of change, determine best developing time.Result is as shown in table 2, and best developing time is 10min.
The determination of the suitableeest developing time of table 2
The determination of 12. yin and yang attribute critical values
Detect without TGEV immunity history with the indirect ELISA method set up and infect history 80 parts of piglet serum, read 0D450 value, calculate mean value (X) and the standard variance (SD) of the 0D450 value of 80 negative part serum, calculate yin and yang attribute critical value according to formula (yin and yang attribute critical value=X+3SD).X=0.149, SD=0.029 as calculated, therefore ELISA yin and yang attribute critical value=0.149+3*0.029=0.236.
During for eliminating the detection of each indirect ELISA, the impact of experimental situation and operation, adds standard positive and negative serum during each detection.The condition that test is set up is: the OD450 of Positive control wells should≤1.0, and <3.0, and the OD450 of negative control hole all should the KQ value of < 0.2. calculation sample.
If KQ >=26.768, be judged to be transmissible gastro-enteritis virus antibody positive; If KQ < 26.768, be judged to be transmissible gastro-enteritis virus negative antibody.
The determination of 13.ELISA running program
By above every determined optimum operation condition, determine that ELISA running program is: take out and wrapped by and the elisa plate bar closed, with serum dilution, serum to be checked is done 1:80 dilution, add each hole of ELISA Plate, every hole 100 μ L, every part of serum to be checked and TGE feminine gender, positive serum add 2 holes, and 37 DEG C are reacted 30 minutes; Discard liquid in hole, every hole 200 μ L cleansing solutions wash 5 times, each 5 minutes, after having washed for the last time, thieving paper has been patted, abandons liquid in most hole; Every hole adds 100 μ L ELIAS secondary antibody, and 37 DEG C are reacted 30 minutes; Discard liquid in hole, every hole 200 μ L cleansing solutions wash 5 times, each 5 minutes, after having washed for the last time, thieving paper has been patted, abandons liquid in most hole; After every hole adds 100 μ LTMB substrate reactions liquid, after room temperature lucifuge develops the color 10 minutes, add 100 μ L stop buffers; Read each hole OD value by microplate reader at 450nm wavelength, calculating mean value, KQ value be result of determination also.
Embodiment 2 specificity experiments
By the indirect ELISA method operation of having set up, detect known PEDV, CSFV, PRRSV, PRV, TGEV, FMDV, PCV2 positive serum (IDEXX kit provides) respectively, every part of serum does 3 parallel repetitions.Under 450nm wavelength, measure each hole OD value by microplate reader, determine the specificity of this indirect ELISA method.
Result shows, the KQ of PEDV, CSFV, PRRSV, FMDV, PCV2 is all less than 20, is judged to be feminine gender; To be 0.386> critical value 0.236, KQ be the OD value of PRV 33.84 is greater than 20, is judged to be the positive, but the ratio 2.955>2 of itself and positive control, affect less.
Embodiment 3 sensitivity tests
4 parts of TGEV positive serums doubling dilution from 1:80 respectively, carries out ELISA detection by optimal detection condition, and result as shown in table 3 shows that 4 parts of positive serums become feminine gender when 1:2560,1:2560,1:2560,1:1280 respectively.Show that kit has good sensitivity.
Table 3 sensitivity Detection result
Embodiment 4 replica test
1) criticize in repeat experiment:
Use with batch TGEV N protein of induction purification, by best antigen coated concentration coated elisa plate, by the ELISA method of operating set up, detect the different serum of the TGE antibody horizontal of 8 parts of Stochastic choice and standard serum respectively at different time, under 450nm wavelength, measure OD value with microplate reader instrument.Calculate each part serum KQ value, and the mean value X of KQ value, standard deviation SD, coefficient of variation CV, concrete condition is in table 4.
Repeated experiment result in table 4 batch
The coefficient of variation of 8 parts of serum KQ values is between 0.45%-5.71% as shown in Table 4, good in different time operation detection result repeatability with batch proteantigen.
2) criticize between repeat experiment:
Use the TGEV N protein of different batches induction purification, by best antigen coated concentration coated elisa plate, by the ELISA method of operating set up, detect the serum that the TGE antibody horizontal of 8 parts of Stochastic choice is different simultaneously, under 450nm wavelength, measure OD value with microplate reader instrument.Calculate each part serum KQ value, and the mean value X of KQ value, standard deviation SD, coefficient of variation CV, concrete condition is in table 5.
Repeated experiment result between table 5 batch
The coefficient of variation of 8 parts of serum OD450 is between 1.25-5.55% as shown in Table 5, and different batches antigen testing result repeatability is good.
The clinical Swine serum of embodiment 5 detects
With the TGEV ELISA set up, 200 parts of pig anteserum samples that pig farm, Xiuqian City of Jiangsu Province gathers are detected.Blood sample 100 parts wherein before immune swine epidemic diarrhea and transmissible gastroenteritis of swine bivalent inactivated vaccine, immune vaccine month post-sampling 100 parts.Testing result shows, and before vaccine immunity, TGEV seroprevalence is that 25%, after vaccine immunity, TGEV seroprevalence reaches 100%, and swinery is in a good state of health.Show that TGEV serum antibody ELISA kit that this patent is set up can be used for assessing the immune effect of vaccine, for the prevention and control of pig farm TGEV epidemic situation provide technical support.
Claims (4)
1. detect an ELISA kit for transmissible gastro-enteritis virus antibody, it is characterized in that comprising following component:
1) elisa plate bar: using transmissible gastro-enteritis virus recombinant N protein as envelope antigen, elisa plate bar is closed through 5%M/V skimmed milk, packs in 4 DEG C of preservations with packaging bag;
2) cleansing solution: containing the pH7.4 phosphate buffer of 0.05% Tween-20;
3) serum dilution: 5%M/V skimmed milk;
4) substrate nitrite ion: add H
2o
2tMB solution;
5) goat-anti pig ELIAS secondary antibody: the goat-anti pig ELIAS secondary antibody being diluted to working concentration 1:10000 with serum dilution;
6) stop buffer: 2M H
2sO
4solution;
7) TGEV positive serum;
8) TGEV negative serum.
2. ELISA kit according to claim 1, is characterized in that: described transmissible gastro-enteritis virus recombinant N protein is prepared by following steps:
1) using the RNA of transmissible gastro-enteritis virus TGEV as material, its N gene is obtained by RT-PCR method amplification, gene accession number JX827607.1, amplified fragments size is 816bp, and using pET-28a plasmid as vector construction recombinant plasmid pET-28a-N, after N gene and pET-28a carrier use BamH I and Sal I double digestion respectively, connect construction recombination plasmid pET-28a-N, double digestion qualification recombinant plasmid;
2) positive recombinant expression plasmid pET-28a-N is transformed BL21 (DE3) competent cell, obtain positive plasmid bacterium monoclonal pET-28a-N/BL21, this monoclonal is in 37 DEG C of cultivations, when A600 value reaches 0.4 ~ 0.6, adding IPTG to final concentration is that 0.8mmol/L carries out abduction delivering, the thalline of 6h after collection abduction delivering, carry out SDS-PAGE qualification, get the thalline of 6h after abduction delivering, ultrasonication, centrifugally get supernatant respectively and precipitation carries out SDS-PAGE electroresis appraisal afterwards, according to electrophoresis result, get supernatant expression liquid and carry out Western blot qualification, a large amount of abduction delivering N protein, ultrasonication, get supernatant and express liquid, Ni-TED post is used to carry out purifying, collect 1xElution Buffer wash-out gained purifying protein.
3. ELISA kit according to claim 1, is characterized in that: described ELISA kit concrete operation step is as follows:
1) blood serum sample is added:
After serum to be checked being done 1:80 dilution with serum dilution 5%M/V skimmed milk, every hole adds 100 μ L, and each blood serum sample adds holes, sets up TGEV positive and negative serum in contrast simultaneously, respectively adds holes; Act on 30 minutes under room temperature, discard the liquid in reacting hole: each hole 200 μ L cleansing solutions fully clean 5 times, each 5 minutes, by the liquid removing in reacting hole after each washing, after last removing cleansing solution, the liquid that fully removing is residual;
2) goat-anti pig ELIAS secondary antibody is added:
Every hole adds 100 μ L goat-anti pig ELIAS secondary antibody, room temperature effect 30 minutes, by method washing in step (1);
3) substrate nitrite ion is added:
Every hole adds 100 μ L and adds H
2o
2tMB solution, lucifuge effect 10 minutes under room temperature;
4) cessation reaction:
Every hole adds 100 μ L 2M H
2sO
4stop buffer, color development stopping is reacted;
5) OD value is measured by microplate reader:
Use microplate reader under 450nm wavelength, measure each hole absorption photometric OD value;
6) result judges.
4. ELISA kit according to claim 1, is characterized in that: described result judges to be specially: add positive serum, negative serum when indirect ELISA detects, measure OD value by microplate reader under 450nm wavelength; Test set up condition be: Positive control wells OD450 value all should≤1.0 and <3.0, negative control hole OD450 value all should≤0.2; Calculating the ratio K Q of each part serum OD450 and Positive control wells OD450, is positive when sample KQ value >=26.768, is negative during < 26.768.
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Cited By (3)
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| CN110607402A (en) * | 2019-10-11 | 2019-12-24 | 扬州大学 | Kit for detecting porcine transmissible gastroenteritis virus PLA |
| CN112834744A (en) * | 2020-12-31 | 2021-05-25 | 天津瑞普生物技术股份有限公司 | ELISA kit for positive rate of adenovirus type 3 neutralizing antibody in pig population and detection method thereof |
| CN113740528A (en) * | 2020-12-08 | 2021-12-03 | 山东畜牧兽医职业学院 | Swine transmissible gastroenteritis virus antibody detection kit |
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|---|---|---|---|---|
| CN110607402A (en) * | 2019-10-11 | 2019-12-24 | 扬州大学 | Kit for detecting porcine transmissible gastroenteritis virus PLA |
| CN110607402B (en) * | 2019-10-11 | 2023-01-03 | 扬州大学 | Kit for detecting porcine transmissible gastroenteritis virus PLA |
| CN113740528A (en) * | 2020-12-08 | 2021-12-03 | 山东畜牧兽医职业学院 | Swine transmissible gastroenteritis virus antibody detection kit |
| CN113740528B (en) * | 2020-12-08 | 2024-08-16 | 山东畜牧兽医职业学院 | Swine transmissible gastroenteritis virus antibody detection kit |
| CN112834744A (en) * | 2020-12-31 | 2021-05-25 | 天津瑞普生物技术股份有限公司 | ELISA kit for positive rate of adenovirus type 3 neutralizing antibody in pig population and detection method thereof |
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