CN107167611A - A kind of Porcine epidemic diarrhea virus antibody sIgA ELISA detection method - Google Patents

A kind of Porcine epidemic diarrhea virus antibody sIgA ELISA detection method Download PDF

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CN107167611A
CN107167611A CN201710375327.0A CN201710375327A CN107167611A CN 107167611 A CN107167611 A CN 107167611A CN 201710375327 A CN201710375327 A CN 201710375327A CN 107167611 A CN107167611 A CN 107167611A
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siga
pedv
detection method
rabbit
antigen
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CN107167611B (en
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贾爱卿
邓胜朝
包银莉
冯晓声
王贵平
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Sichuan Hailinge Biological Pharmaceutical Co Ltd
GUANGDONG HAID ANIMAL HUSBANDRY VETERINARY RESEARCH INSTITUTE Co Ltd
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Sichuan Hailinge Biological Pharmaceutical Co Ltd
GUANGDONG HAID ANIMAL HUSBANDRY VETERINARY RESEARCH INSTITUTE Co Ltd
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Abstract

The invention discloses a kind of Porcine epidemic diarrhea virus antibody sIgA ELISA detection method, comprise the following steps：Add antigen：Antigen PEDV S1 albumen and coating buffer are added into ELISA Plate, are coated with；Closing：Add the PBS containing BSA to be closed as confining liquid, then discard confining liquid；Sample-adding：Add measuring samples；Add enzyme labelled antibody：Enzyme labelled antibody HRP rabbit-anti pig sIgA are added, uncombined enzyme labelled antibody is washed away after reaction with PBST；Colour developing and measure：Developer is added, terminate liquid is added after lucifuge colour developing, then determines OD450nm；The detection method is specific and reproducible, can provide accurate testing result to PEDV antibody sIgA, be also beneficial to the evaluation of PEDV immune effect of vaccine.

Description

A kind of Porcine epidemic diarrhea virus antibody sIgA ELISA detection method
Technical field
The present invention relates to a kind of Porcine epidemic diarrhea virus antibody sIgA ELISA detection method, belong to animal's antibody inspection Survey technology field.
Background technology
Pig epidemic diarrhea (Porcine epidemic diarrhea, PED) is by Porcine epidemic diarrhea virus A kind of acute high degree in contact enteric infectious disease of pig caused by (Porcine Epidemic Diarrhea Virus, PEDV), Using serious enteritis, vomiting and watery diarrhea as principal character, clinical change and symptom and transmissible gastroenteritis of swine (TGE) are extremely It is similar.PEDV route of transmission is mainly horizontal transmission, and main path is alimentary canal, and piglet is searching for food or contacted infected feeding Cause morbidity after material, excrement, drinking-water, low temperature, humidity, hygienic conditions can accelerate the transmission of disease when poor.PED flows in explosive OK, after most regional eruption and prevalences, this disease no longer occurs for the primary swinery of Second Year, the shelf for also having indivedual areas and pig farm Pig and wean pig, the annual autumn and winter continue morbidity.The pig at each age is susceptible, especially most susceptible with 2~7 age in days piglets, shows as height The incidence of disease and high fatal rate, huge economic loss is caused to pig industry.Cause under large quantities of death of piglet, slaughter rate of hogs Drop, causes market cut of pork not enough, and influence pork price is stable.
Because of the self immune system and imperfection of the piglet of 2-7 ages in days, if wanting to resist infecting for PEDV can only be by breast milk Come obtain in passive immunity, sow colostrum be mainly secretory immunoglobulin A (sIgA), sIgA can not by placental barrier, Newborn piglet can be obtained from breast milk, protect its own not infected by PEDV.
Current pig epidemic diarrhea vaccine carries out sow and is immunized, and piglet is obtained passive immunity, therefore evaluate in breast milk The titre of PEDVsIgA antibody is to evaluate the Main Means of PEDV vaccines, and the domestic detection method on pig colostrum sIgA is present Blank.Therefore the present invention establishes the ELISA method of PEDV sIgA antibody in detection sow milk, it would be desirable to sow milk Middle PEDV sIgA antibody provides objective appraisal, and the immune effect of PEDV vaccines is judged with this.
The content of the invention
In order to overcome the deficiencies in the prior art, first purpose of the invention is to provide a kind of Porcine epidemic diarrhea virus Antibody sIgA ELISA detection method, the detection method is specific and reproducible, and PEDV antibody sIgA can be provided accurately Testing result, be also beneficial to the evaluation of PEDV immune effect of vaccine.
Realize that the purpose of the present invention can reach by adopting the following technical scheme that：A kind of Porcine epidemic diarrhea virus antibody SIgA ELISA detection method, comprises the following steps：
Add antigen：Antigen PEDV S1 albumen and coating buffer are added into ELISA Plate, is coated with, is then washed away with PBST Uncombined antigen and impurity；
Closing：Add the PBS containing BSA to be closed as confining liquid, then discard confining liquid, wash away remnants' with PBST Confining liquid；
Sample-adding：Measuring samples are added, composition not with antigen binding is then washed away with PBST；
Add enzyme labelled antibody：Enzyme labelled antibody HRP- rabbit-anti pig sIgA are added, uncombined enzyme mark is washed away after reaction with PBST Antibody；
Colour developing and measure：Developer is added, terminate liquid is added after lucifuge colour developing, then determines OD450nm。
Further, preparing the method for the antigen PEDV S1 albumen includes：
PEDV truncates the amplification of S1 genes and the construction step of recombinant expression carrier：
The 435-789 amino acids sequences of S1 genes are truncated using PEDV as the PEDV where template amplification B cell antigen epi-position Row, reclaim and purifying purpose fragment, obtain PCR primer；PCR primer carries out double digestion respectively with prokaryotic expression carrier pGEX4T1, And digestion products reclaim and after purification, carries out the connection of digestion products under conditions of 16 DEG C by T4 ligases, obtains To recombinant expression carrier pGEX4T-S1；BL21 (DE3) competent cell is converted with recombinant expression carrier, blue hickie is then carried out Screening, picking colony carries out recombinant plasmid extraction, and the identification of BamHI and SalI double digestions is carried out to recombinant plasmid, to detect Whether restructuring is successful；
The induced expression and detecting step of recombinant plasmid：
BL21 (DE3) cell containing recombinant plasmid is inoculated in Amp+It is empty to convert pGEX4T1 in LB fluid nutrient mediums The Escherichia coli of carrier are negative control, collect thalline be resuspended, it is broken after, carry out SDS-PAGE electroresis appraisals and printed with protein The expression and reactionogenicity of mark method detection restructuring S1 albumen.
Further, PEDV is truncated in the amplification of S1 genes and the construction step of recombinant expression carrier, and the PCR expands B The step of PEDV where cell antigen epitope truncates the 435-789 amino acids sequences of S1 genes includes：
RT-PCR amplifications are first carried out, 10 μ L RT-PCR amplification systems are：
5 × RT-buffer 2 μ L, dNTPs 2 μ L, 1 μ L, RNase Inhibitor of primer S1-down 1 μ L, Rever Nucleic acid-templated 1 μ L, DEPC Water 2 μ L, the 42 DEG C of effect 50min of Tra Ace 1 μ L, PEDV, obtain cDNA；
Then performing PCR amplification is entered, 20 μ L pcr amplification reaction system is：
10 × KOD-Plus-Neo buffer, 2 μ L, 2mM dNTPs 2 μ L, 25mM MgSO41.2 1 μ L of μ L, S1-up, S1-down 1 μ L, KOD-Plus-Neo 0.5 μ L, cDNA 1 μ L, d2H2O 11.3μL；Amplification condition is：95 DEG C of pre-degenerations 5min；Into circulation：94 DEG C of denaturation 45sec, 50-62 DEG C of annealing 45sec, 72 DEG C of extension 1min, 30 circulations；72 DEG C re-extend After 10min, 4 DEG C of preservations obtain purpose fragment；
Wherein, specific primer used is：
Underscore part is restriction enzyme site.
Further, PEDV is truncated in the amplification of S1 genes and the construction step of recombinant expression carrier, 20 μ L double digestion bodies It is to be：10 × buffer-T 3,11 μ L of μ L, SalI of μ L, BamHI, PCR primer or pGEX4T1 10 μ L, d2H2O 5 μ L, 37 DEG C React 3h.
Further, PEDV is truncated in the amplification of S1 genes and the construction step of recombinant expression carrier, 10 μ L digestion products Linked system be：The μ L of 10 × T4 DNA ligases buffer, 1 μ L, S1 gene PCRs digestion products 5, prokaryotic expression carrier The μ L of 3 μ L, T4DNA ligase of pGEX4T1 digestion products 1；After each component is mixed, centrifuge, at least 8h is connected under the conditions of 16 DEG C.
Further, in the induced expression and detecting step of recombinant plasmid, by BL21 (DE3) cell containing recombinant plasmid It is inoculated in Amp+In LB fluid nutrient mediums, to convert the Escherichia coli of pGEX4T1 empty carriers as negative control, in 37 DEG C of condition It is lower to cultivate to OD600nmFor 0.6 when, add final concentration of 0.8mM IPTG under conditions of 37 DEG C, continue to cultivate 4h.
Further, preparing HRP- rabbit-anti pigs sIgA method includes：
SIgA purification procedures：
The colostrum of sow is collected, is centrifuged under conditions of 4~8 DEG C, discards upper-layer fat and lower sediment, intermediate layer breast is taken Clearly；Toward adding acetic acid in whey in the way of dropwise addition so that the pH of whey is 3.2-5.6, then albumen is sunken to a glass bottom；Then at 4 Centrifuged under conditions of DEG C, take supernatant, supernatant is adjusted to pH=7；Obtained supernatant is subjected to gel filtration chromatography, The eluent of each absworption peak is collected, SDS-PAGE electroresis appraisals are carried out to eluent；
The corresponding eluent in second peak is subjected to protein electrophorese, protein is then gone to by pvdf membrane by transferring film instrument On, the protein on film is carried out with the goat-anti pig IgA of 1000 times of horseradish peroxidase-labeled of dilution polyclonal antibody Be incubated, with immunoblotting detect separating resulting, from testing result draw sIgA in IgA heavy chains Western blotting, then show SIgA is separated successfully, obtains sIgA；
Rabbit-anti pig sIgA preparation and markers step：
New zealand rabbit is immunized in sIgA through isolating and purifying, head, which exempts from sIgA adding isometric Freund, not exclusively to be helped Multiple spot immune rabbit after agent emulsification, takes sIgA and multiple spot immune rabbit after the emulsification of isometric Freund's complete adjuvant, two exempt from after two weeks Carry out within three weeks three afterwards to exempt from, nape part multi-point injection；Three exempt from blood sampling separation serum after two weeks, obtain sIgA rabbit-anti pig positive serums； Serum is marked with HRP labelling kits, HRP- rabbit-anti pigs sIgA is obtained.
Further, add in antigen step, the concentration of antigen PEDV S1 albumen is 12.5 μ g/mL, and addition is 100 μ L/ is per hole；The 1h under conditions of 37 DEG C, is then coated with least 8h under conditions of 4 DEG C.
Further, in closing step, the PBS of the 1%BSA containing percent by volume is added as confining liquid, in 37 DEG C of bar 1.5h is closed under part, confining liquid is then discarded, and washes away with PBST remnants confining liquid.
Further, in load procedure, add after measuring samples and react 1h under conditions of 37 DEG C.
Further, enzyme labelled antibody HRP- rabbit-antis pig sIgA dilution factor is 1:5000-23000, addition is 100 μ L.
Further, add in enzyme labelled antibody step, diluted enzyme labelled antibody HRP- rabbit-antis pig sIgA is added, at 37 DEG C Under conditions of reaction 30~45min after, uncombined enzyme labelled antibody is washed away with PBST.
Further, in colour developing and determination step, 50 μ L terminations are added after adding the μ L of developer 100, lucifuge colour developing 10min Liquid, then determines OD450nm。
Compared with prior art, the beneficial effects of the present invention are：
The present invention is intended to provide the indirect ELISA detection method of a boar colostrum sIgA, detection method specificity and again Renaturation is good, can provide accurate testing result to PEDV antibody sIgA, be also beneficial to the evaluation of PE DV immune effect of vaccine.
Brief description of the drawings
Fig. 1 is the chromatographic purifying peak figure of embodiment 1；
Fig. 2 is embodiment 1SDS-PAGE electrophoretograms；
Fig. 3 is the Western blotting of embodiment 1 development figure；
Fig. 4 is embodiment 2PCR and double digestion electrophoretogram；
Fig. 5 embodiment 2SDS-PAGE electrophoretograms；
Fig. 6 is the Western blotting of embodiment 2 development figure.
Embodiment
Below, with reference to accompanying drawing and embodiment, the present invention is described further：
A kind of Porcine epidemic diarrhea virus antibody sIgA ELISA detection method, comprises the following steps：
First, antigen is added：By antigen PEDV S1 albumen and coating buffer addition ELISA Plate, the 1h under conditions of 37 DEG C, then At least 8h is coated with conditions of 4 DEG C, the concentration of antigen PEDV S1 albumen is 12.5 μ g/mL, and addition is 100 μ L/ per hole；So Uncombined antigen and impurity is washed away with PBST afterwards；
2nd, close：The PBS of the 1%BSA containing percent by volume is added as confining liquid, 1.5h is closed under conditions of 37 DEG C, Then confining liquid is discarded, and washes away with PBST remnants confining liquid；
3rd, it is loaded：With pig colostrum sample to be checked, after diluted, 1h, Ran Houyong are reacted under conditions of 37 DEG C PBST washes away composition not with antigen binding；
4th, enzyme labelled antibody is added：Enzyme labelled antibody HRP- rabbit-anti pig sIgA are added, enzyme labelled antibody HRP- rabbit-anti pigs sIgA's is dilute Degree of releasing is 1:5000-23000, addition is 100 μ L；After 30~45min of reaction under conditions of 37 DEG C, washed away and do not tied with PBST The enzyme labelled antibody of conjunction；
5th, develop the color and determine：50 μ L terminate liquids are added after adding the μ L of developer 100, lucifuge colour developing 10min, are then determined OD450nm。
In embodiment, prepareAntigen PEDV S1 albumenMethod include：
1st, PEDV truncates the amplification of S1 genes and the construction step of recombinant expression carrier：
The 435-789 amino acids sequences of S1 genes are truncated using PEDV as the PEDV where template amplification B cell antigen epi-position Row, be specially：
Using following primer as specific primer, upstream and downstream primer introduces restriction endonuclease sites BamHI respectively With SalI (underscore part),
RT-PCR amplifications are first carried out, 10 μ L RT-PCR amplification systems are：
5 × RT-buffer 2 μ L, dNTPs 2 μ L, 1 μ L, RNase Inhibitor of primer S1-down 1 μ L, Rever Nucleic acid-templated 1 μ L, DEPC Water 2 μ L, the 42 DEG C of effect 50min of Tra Ace 1 μ L, PEDV, obtain cDNA；
Then performing PCR amplification is entered, 20 μ L pcr amplification reaction system is：
10 × KOD-Plus-Neo buffer, 2 μ L, 2mM dNTPs 2 μ L, 25mM MgSO41.2 1 μ L of μ L, S1-up, S1-down 1 μ L, KOD-Plus-Neo 0.5 μ L, cDNA 1 μ L, d2H2O 11.3μL；Amplification condition is：95 DEG C of pre-degenerations 5min；Into circulation：94 DEG C of denaturation 45sec, 50-62 DEG C of annealing 45sec, 72 DEG C of extension 1min, 30 circulations；72 DEG C re-extend After 10min, 4 DEG C of preservations obtain purpose fragment, reclaim and purifying purpose fragment, obtain PCR primer.
PCR primer carries out double digestion respectively with prokaryotic expression carrier pGEX4T1, and 20 μ L double digestion systems are：10× The 11 μ L of μ L, SalI of μ L, BamHI of buffer-T 3, PCR primer or pGEX4T1 10 μ L, d2H2The μ L of O 5,37 DEG C are reacted 3h, right Digestion products are reclaimed and purified.
The connection of digestion products, the linked system of 10 μ L digestion products are carried out under conditions of 16 DEG C by T4 ligases For：The μ L of 10 × T4DNA ligases buffer, 1 μ L, S1 gene PCRs digestion products 5, prokaryotic expression carrier pGEX4T1 digestion products The μ L of 3 μ L, T4DNA ligase 1；After each component is mixed, centrifuge, connect at least 8h under the conditions of 16 DEG C of metal bath, recombinantly expressed Vector pGEX 4T-S1.
Metal bath refers to：A constant-temperature metal bath instrument for controlling to manufacture with semiconductor refrigerating technology using micro computer, instrument Device can configure multiple module, can be widely applied to the denaturation of the preservation, the preservation of various enzymes and reaction, nucleic acid and protein of sample Processing, PCR reactions, the pre-degeneration of electrophoresis and serum solidification etc..
BL21 (DE3) competent cell is converted with recombinant expression carrier, blue hickie screening is then carried out, picking colony is carried out Recombinant plasmid is extracted, and the identification of BamHI and SalI double digestions is carried out to recombinant plasmid, to detect whether to recombinate successfully.
2nd, the induced expression and detecting step of recombinant plasmid：
BL21 (DE3) cell containing recombinant plasmid is inoculated in Amp+It is empty to convert pGEX4T1 in LB fluid nutrient mediums The Escherichia coli of carrier are negative control, are cultivated under conditions of 37 DEG C to OD600nmFor 0.6 when, under conditions of 37 DEG C add Final concentration of 0.8mM IPTG, continues to cultivate 4h；Bacterium solution is taken, thalline is collected by centrifugation, is resuspended with PBS, after ultrasonication, is carried out SDS-PAGE electroresis appraisals；
After SDS-PAGE electroresis appraisals, the expression and reactionogenicity for recombinating S1 albumen are detected with immunoblotting.
In embodiment, prepareHRP- rabbit-anti pigs sIgAThe step of include：
1st, in pig colostrum sIgA purification procedures：
The colostrum of sow is collected, is centrifuged under conditions of 4~8 DEG C, discards upper-layer fat and lower sediment, intermediate layer breast is taken Clearly；Toward adding acetic acid in whey in the way of dropwise addition so that the pH of whey is 3.2-5.6, then albumen is sunken to a glass bottom；Then at 4 Centrifuged under conditions of DEG C, take supernatant, supernatant is adjusted to pH=7；Obtained supernatant is subjected to gel filtration chromatography, The eluent of each absworption peak is collected, SDS-PAGE electroresis appraisals are carried out to eluent；
The corresponding eluent in second peak is subjected to protein electrophorese, protein is then gone to by pvdf membrane by transferring film instrument On, the protein on film is carried out with the goat-anti pig IgA of 1000 times of horseradish peroxidase-labeled of dilution polyclonal antibody Be incubated, with immunoblotting detect separating resulting, from testing result draw sIgA in IgA heavy chains Western blotting, then show SIgA is separated successfully, obtains sIgA；
2nd, rabbit-anti pig sIgA preparation and markers step：
New zealand rabbit is immunized in sIgA through isolating and purifying, head exempts from the isometric Freund of 2mg sIgA additions is endless Multiple spot immune rabbit after full adjuvant emulsion, takes 4mg sIgA that rabbit is immunized with multiple spot after the emulsification of isometric Freund's complete adjuvant after two weeks Son, two, which exempt from progress three in latter three weeks, exempts from, 6mg/, nape part multi-point injection；Three exempt from blood sampling separation serum after two weeks, obtain sIgA rabbits Anti- pig positive serum；Serum is marked with HRP labelling kits, HRP- rabbit-anti pigs sIgA is obtained.
Embodiment 1：
Embodiment 1 is preparationHRP- rabbit-anti pigs sIgASpecific method：
1st, sIgA is isolated and purified：
The colostrum of sow is collected, under conditions of 4~8 DEG C, rotating speed 11000r/min centrifuges 25min, discards upper-layer fat And lower sediment, take intermediate layer whey；Toward addition 0.05-0.2M acetic acid and stirring in whey in the way of dropwise addition so that breast Clear final pH is 3.2-5.6, then albumen is sunken to a glass bottom；Under conditions of 4 DEG C, rotating speed 11000r/min, centrifugation 15min, takes supernatant, and supernatant is adjusted into pH=7；
Obtained supernatant is subjected to gel filtration chromatography：Flow velocity is first set as 0.5mL/min, post pressure alarm settings For 3MPa, first whole pipeline and affinity chromatography pillar are washed using deionized water, each 30mL is washed 2 times；PBS is used again Wash 2 times, each wash volumes are 30mL；
Take in 0.6mL sIgA crude product solutions, injection loading ring, setting flow velocity is 0.22mL/min, collects each absworption peak Eluent, peak figure are as shown in figure 1,1-3 peaks are respectively：IgM、IgA、IgG；
SDS-PAGE electroresis appraisals are carried out to eluent；The testing result of SDS-PAGE electrophoresis is as shown in Fig. 2 swimming lane 1-6 The eluent collected for different time, there it can be seen that having obvious 80KD bands in the 2nd swimming lane and the 3rd swimming lane, to this Albumen shows the secretory piece albumen that the albumen is sIgA through mass spectral analysis, it was demonstrated that the albumen is sIgA.
The corresponding eluent in 2nd peak is subjected to protein electrophorese, then gone to protein on pvdf membrane by transferring film instrument, The protein on film is incubated with the goat-anti pig IgA of 1000 times of horseradish peroxidase-labeled of dilution polyclonal antibody Educate, developed the color with DAB, separating resulting is detected with immunoblotting, as shown in Figure 3：Swimming lane 1 is positive control, can substantially be detected The Western blotting of the IgA heavy chains gone out in sIgA, the above results show that sIgA is separated successfully, and purity is higher.
Biotech firm is sent to carry out mass spectral analysis to the eluent that above immunoblotting result is positive, analysis result is simultaneously It is compared with the IgA of international publication sequence, comparison result has higher homology, it may be determined that succeeded from pig Colostrum in obtain the IgA of higher degree.
Due in the prior art, not there is the technology isolated and purified to the sIgA in pig colostrum, the present invention is in existing skill Improved on the basis of art, for pig colostrum substance characteristics, work out it is above-mentioned can efficient stable isolate and purify in pig colostrum SIgA method.
2nd, rabbit-anti pig sIgA preparation and mark：
New zealand rabbit is immunized in sIgA through isolating and purifying, head exempts from the isometric Freund of 2mg sIgA additions is endless Multiple spot immune rabbit after full adjuvant emulsion, takes 4mg sIgA that rabbit is immunized with multiple spot after the emulsification of isometric Freund's complete adjuvant after two weeks Son, two, which exempt from progress three in latter three weeks, exempts from, 6mg/, nape part multi-point injection；Three exempt from blood sampling separation serum after two weeks, obtain sIgA rabbits Anti- pig positive serum；Serum is marked with HRP labelling kits, HRP- rabbit-anti pigs sIgA is obtained.
Embodiment 2：
Embodiment 2 is preparationAntigen PEDV S1 albumenSpecific method：
1st, specific primer is designed：
(www.expasy.org) analysis online has been carried out to PEDV strains by using biological software, has finally been determined The 435-789 amino acids of PEDV S1 albumen are the potential B cell antigen epi-position region of the strain；Recycle The amplification of primer5.0 Software for Design truncates the specific primer of S1 gene 435-789 amino acids sequences, upstream and downstream primer difference Restriction endonuclease sites BamHI and SalI (underscore part) are introduced,
2nd, PEDV truncates the amplification of S1 genes and the structure of recombinant expression carrier：
The 435-789 amino acids sequences of S1 genes are truncated using PEDV as the PEDV where template amplification B cell antigen epi-position Row, be specially：
RT-PCR amplifications are first carried out, 10 μ L RT-PCR amplification systems are：
5 × RT-buffer 2 μ L, dNTPs 2 μ L, the μ L of 1 μ L, RNase Inhibitor of primer S1-down 1,
Nucleic acid-templated 1 μ L, DEPC Water 2 μ L, the 42 DEG C of effect 50min of Rever Tra Ace 1 μ L, PEDV, are obtained cDNA；
Then performing PCR amplification is entered, 20 μ L pcr amplification reaction system is：
10 × KOD-Plus-Neo buffer, 2 μ L, 2mM dNTPs 2 μ L, 25mM MgSO41.2 1 μ L of μ L, S1-up, S1-down 1 μ L, KOD-Plus-Neo 0.5 μ L, cDNA 1 μ L, d2H2O 11.3μL；Amplification condition is：95 DEG C of pre-degenerations 5min；Into circulation：94 DEG C of denaturation 45sec, 50-62 DEG C of annealing 45s ec, 72 DEG C of extension 1min, 30 circulations；72 DEG C are prolonged again Stretch after 10min, 4 DEG C of preservations obtain purpose fragment, detected through 1% agarose gel electrophoresis, purpose is occurred in that in 1560bp or so Band, is consistent with expected results；Purpose fragment is reclaimed and purified with DNA gel QIAquick Gel Extraction Kit, obtains PCR primer.
3rd, PCR primer and prokaryotic expression carrier pGEX4T1 carry out double digestion respectively：
20 μ L double digestion systems are：10 × buffer-T 3,11 μ L of μ L, SalI of μ L, BamHI, PCR primer or pGEX4T1 10 μ L, d2H2Digestion products are reclaimed and purified, digestion products are through 1% agarose gel electrophoresis by O 5 μ L, 37 DEG C of reaction 3h Detection.
4th, connect：
The connection of digestion products, the linked system of 10 μ L digestion products are carried out under conditions of 16 DEG C by T4 ligases For：The μ L of 10 × T4DNA ligases buffer, 1 μ L, S1 gene PCRs digestion products 5, prokaryotic expression carrier pGEX4T1 digestion products The μ L of 3 μ L, T4DNA ligase 1；After each component is mixed, centrifuge, connect at least 8h under the conditions of 16 DEG C of metal bath, recombinantly expressed Vector pGEX 4T-S1.
BL21 (DE3) competent cell is converted with recombinant expression carrier, blue hickie screening is then carried out, picking colony is carried out Recombinant plasmid is extracted, and the identification of BamHI and SalI double digestions, qualification result are carried out to recombinant plasmid as shown in figure 4, the 1st, There is 5300bp, 1560bp specific band occur in the 3rd swimming lane in 2 swimming lanes, it was demonstrated that recombinate successfully.
5th, the induced expression of recombinant plasmid and detection：
BL21 (DE3) cell containing recombinant plasmid is inoculated in Amp+It is empty to convert pGEX4T1 in LB fluid nutrient mediums The Escherichia coli of carrier are negative control, are cultivated under conditions of 37 DEG C to OD600nmFor 0.6 when, respectively at 30 DEG C, 37 DEG C of bar The concentration that 0.2-1.0mM scopes inside gradient sets IPTG is added under part, continues to cultivate 4h；1mL bacterium solutions are taken every 1h, are collected by centrifugation Thalline, is resuspended with PBS, after ultrasonication, is determined the expression quantity of albumen, is shown that the preferred experiment parameter of this step is：With 0.8mM IPTG, cultivate 4h under the conditions of 37 DEG C, highest expressing quantity can be obtained.
SDS-PAGE electroresis appraisals are carried out, as a result as shown in figure 5, the 1st, 2 swimming lanes is repeat, the 3rd swimming lane lures for empty carrier Lead control, the 1st, 2 swimming lanes occur in that one article of obvious purpose band in 74kD or so, molecular weight and expection are in the same size.
After SDS-PAGE electroresis appraisals, the protein on gel is transferred on nitrocellulose filter, confining liquid room temperature is used 1h is closed, adds and presses 1 with TBST:The anti-PEDV positive serums of pig of 500 dilutions, at 37 DEG C, are incubated after 1h with NC films, use TBST Washing 3 times；Add and use TBST1：The goat-anti pig IgG of the HRP marks of 10000 times of dilutions, at 37 DEG C, 1h is incubated with NC films, Washed with TBST 3 times；DAB nitrite ions are added in darkroom colour developing 5-10min, development is terminated；Immunoblotting result such as Fig. 6 institutes Show, the 2nd swimming lane is blank control, the 1st swimming lane occurs in that one article of specific band at 74KD, it was demonstrated that restructuring S1 albumen success tables Reach, and with good reactionogenicity.
Embodiment 3：
Embodiment 3 is the determination of the most suitable coating concentration of antigen and milk dilution factor
Take ELISA Plate (8*12), it is horizontally-arranged by antigen PEDV S1 albumen be diluted to 50 μ g/mL, 25 μ g/mL, 12.5 μ g/mL, 6.25 μ g/mL, 3.125 μ g/mL, 1.6 μ g/mL, 0.8 μ g/mL, 0.4 μ g/mL, 4 DEG C of coatings after 100 μ L, 37 DEG C of 1h are added per hole At least 8h.
The elisa plate closed is washed 3 times after taking out with PBST, each 1min；Add the 1%BSA's containing percent by volume PBS confining liquids, 150 μ L close 1.5h under conditions of 37 DEG C, then discard confining liquid per hole；Wash 3 times with PBST, every time 1min；Vertical setting of types is by yin and yang attribute milk from 1:10、1:20、1:40 times are serially diluted to 1:1280, per the μ L of hole 100, then 37 DEG C Under the conditions of react 1h；Washed with PBST 3 times, each 1min；100 μ L 1 are added per hole:It is prepared by the embodiment 1 of 10000 times of dilutions HR P- rabbit-anti pigs sIgA, react 30min under conditions of 37 DEG C；Washed with PBST 3 times, each 1min；100uL is added per hole After developer, 20 DEG C of lucifuge colour developing 10min, 50uL terminate liquids are added per hole, O D are determined450nm.Optimal antigen coat concentration and Antibody dilution selection standard is positive milk OD450nmValue is near 1, and positive OD450nmValue/feminine gender OD450nmIt is worth (P/N) ratio Value is maximum, OD450nmIt is worth result as shown in Table 1：
The OD value testing results of form 1
Drawn from form 1, dilution factor is 1:When 40, P/N ratio is maximum for 19.4 (1.243/0.064), determines antigen Most suitable coating concentration be that 12.5 μ g/mL, the optimum dilution degree of milk are 1:40.
Embodiment 4：
Selection and off-period of the embodiment 4 for confining liquid are determined
It is coated with using the concentration of 12.5 μ g/mL as antigen PEDV S1 albumen, indirect ELISA is carried out, respectively with containing 5% NBCS, 5% duck serum, 5% skimmed milk power, 1%BSA PBS react 1h as confining liquid under the conditions of 37 DEG C, develop the color 50uL terminate liquids are added after 10min per hole, OD is determined450nm, as a result as shown in Table 2, the OD of relatively more negative positive milk450nm Value and P/N values, selection P/N value maximums are as optimal confining liquid, it is determined that optimal confining liquid is 1%.
The OD value testing results of the different confining liquids of form 2
OD450nm>=0.4 is the positive, and ﹤ 0.4 is feminine gender.
It is coated with using the concentration of 12.5 μ g/mL as antigens, indirect ELISA is carried out, with the PBS containing 1%BSA respectively at 37 Closed under the conditions of DEG C after 30min, 60min, 90min, 120min, carry out ELISA reactions, determine OD450nm, relatively more negative positive breast The OD of juice450nmValue and P/N values, as a result as shown in Table 3, enzyme labelled antibody concentration when selection P/N values are maximum, it is determined that optimal closing Time is 1.5h.
The OD value testing results of the different off-periods of form 3
OD450nm>=0.4 is the positive, and ﹤ 0.4 is feminine gender.
Embodiment 5：
Embodiment 5 is the optimal diluted concentration of enzyme labelled antibody and the determination in reaction time
It is coated with using the concentration of 12.5 μ g/mL as antigens, carries out indirect ELISA, HRP- rabbit-anti pig IgA secondary antibodies is made respectively l:5000、l:10000、l:15000、1:20000 are diluted, and 50uL ends are added per hole after 1h, colour developing 10min are reacted under the conditions of 37 DEG C Only liquid, determines OD450nm.As a result as shown in Table 4, the OD of relatively more negative positive milk450nmValue and P/N values, selection P/N values are most Wonderful works enzyme labelled antibody optium concentration, it is 1 to determine optimum dilution degree:20000.
The OD value testing results of the different enzyme labelled antibody dilution factors of form 4
OD450nm>=0.4 is the positive, and ﹤ 0.4 is feminine gender.
30min, 45min, 60min, 90min under the conditions of the reaction condition of enzyme labelled antibody is set into 37 DEG C are relatively more cloudy Property positive milk OD450nmValue and P/N values, as a result as shown in Table 5, selection P/N values are maximum as optimum reacting time, it is determined that Optimum reacting time is 45min.
The OD value testing results of the enzyme labelled antibody differential responses time of form 5
OD450nm>=0.4 is the positive, and ﹤ 0.4 is feminine gender.
Embodiment 6：
Embodiment 6 is a kind of Porcine epidemic diarrhea virus antibody sIgA ELISA detection method, is comprised the following steps：
First, antigen is added：By antigen PEDV S1 albumen and coating buffer addition ELISA Plate, the 1h under conditions of 37 DEG C, then At least 8h is coated with conditions of 4 DEG C, the concentration of antigen PEDV S1 albumen is 12.5 μ g/mL, and addition is 100 μ L/ per hole；So Washed afterwards with PBST 3 times, each 1min；
2nd, close：The PBS of the 1%BSA containing percent by volume is added as confining liquid, 1.5h is closed under conditions of 37 DEG C, Then confining liquid is discarded, and is washed 3 times with PBST, each 1min；
3rd, it is loaded：Add after pig colostrum sample to be checked and react 1h under conditions of 37 DEG C, then wash 3 times with PBST, every time 1min；Pig colostrum sample concentration to be checked is 12.5 μ g/mL, and addition is 100 μ L/ per hole；
4th, enzyme labelled antibody is added：Enzyme labelled antibody HRP- rabbit-anti pig sIgA are added, enzyme labelled antibody HRP- rabbit-anti pigs sIgA's is dilute Degree of releasing is 1:20000, addition is 100 μ L；After reaction 45min under conditions of 37 DEG C, washed with PBST 3 times, each 1min；
5th, develop the color and determine：50 μ L terminate liquids are added after adding the μ L of developer 100, lucifuge colour developing 10min, are then determined OD450nm。
Sample is carried out in the same way to repeat detection, OD is determined450nmAs shown in Table 6：
Form 6 repeats the OD values of detection
Detect example 1：
Detection example 1 is replica test
Take and detect 3 portions of positive milk and 2 parts of negative milks respectively in the way of embodiment 6, as a result as shown in Table 7：3 parts The coefficient of variation of positive milk is 3.05%, 3.82%, 2.09%, respectively less than 10%, shows the reproducible of this method.
The replica test result of form 7
OD450nm>=0.4 is the positive, and ﹤ 0.4 is feminine gender.
Detect example 2：
Detection example 2 is specific test
Transmissible gastroenteritis of swine (TGEV), porcine rotavirus are detected with the ELISA detection method of embodiment 6 respectively (PoRV), pseudorabies (PRV), swine fever (HCV), the positive milk of porcine reproductive and respiratory syndrome (PRRSV) and PEDV are positive Milk, result of the test is as shown in Table 8：Transmissible gastroenteritis of swine, porcine rotavirus, pseudorabies, swine fever, pig breeding and breathing The positive milk ELISA testing results of syndrome are feminine gender, it was demonstrated that this method it is specific good.
The specific test result of form 8
Positive milk TGEV PoRV PRV HCV PRRSV PEDV
OD450nm 0.13 0.16 0.20 0.18 0.21 1.65
OD450nm>=0.4 is the positive, and ﹤ 0.4 is feminine gender.
For those skilled in the art, technical scheme that can be as described above and design, make other each It is kind corresponding to change and deform, and all these change and deformation should all belong to the protection model of the claims in the present invention Within enclosing.
SEQUENCE LISTING
<110>Guangdong Haida Husbandry and Veterinary Institute Co., Ltd.
<120>A kind of Porcine epidemic diarrhea virus antibody sIgA ELISA detection method
<130> 2017
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 28
<212> DNA
<213>It is artificial synthesized
<400> 1
aaggatccat gtgcactggt tacgctgc 28
<210> 2
<211> 28
<212> DNA
<213>It is artificial synthesized
<400> 2
aagtcgactt aatggtagaa gaaaccag 28

Claims (10)

1. a kind of Porcine epidemic diarrhea virus antibody sIgA ELISA detection method, it is characterised in that comprise the following steps：
Add antigen：Antigen PEDV S1 albumen and coating buffer are added into ELISA Plate, is coated with, is then washed away and do not tied with PBST The antigen and impurity of conjunction；
Closing：Add the PBS containing BSA to be closed as confining liquid, then discard confining liquid, remnants closing is washed away with PBST Liquid；
Sample-adding：Measuring samples are added, composition not with antigen binding is then washed away with PBST；
Add enzyme labelled antibody：Enzyme labelled antibody HRP- rabbit-anti pig sIgA are added, uncombined enzyme labelled antibody is washed away after reaction with PBST；
Colour developing and measure：Developer is added, terminate liquid is added after lucifuge colour developing, then determines OD450nm。
2. Porcine epidemic diarrhea virus antibody sIgA as claimed in claim 1 ELISA detection method, it is characterised in that system The method of the standby antigen PEDV S1 albumen includes：
PEDV truncates the amplification of S1 genes and the construction step of recombinant expression carrier：
The 435-789 amino acids sequences of S1 genes are truncated using PEDV as the PEDV where template amplification B cell antigen epi-position, are returned Receive and purifying purpose fragment, obtain PCR primer；PCR primer carries out double digestion respectively with prokaryotic expression carrier pGEX4T1, and right Digestion products reclaim and after purification, carry out the connection of digestion products under conditions of 16 DEG C by T4 ligases, obtain weight Group expression vector pGEX4T-S1；BL21 (DE3) competent cell is converted with recombinant expression carrier, blue hickie screening is then carried out, Picking colony carries out recombinant plasmid extraction, and the identification of BamHI and SalI double digestions is carried out to recombinant plasmid, to detect whether weight Constitute work(；
The induced expression and detecting step of recombinant plasmid：
BL21 (DE3) cell containing recombinant plasmid is inoculated in Amp+In LB fluid nutrient mediums, to convert pGEX4T1 empty carriers Escherichia coli be negative control, collect thalline be resuspended, it is broken after, carry out SDS-PAGE electroresis appraisals and with immunoblotting The expression and reactionogenicity of detection restructuring S1 albumen.
3. Porcine epidemic diarrhea virus antibody sIgA as claimed in claim 2 ELISA detection method, it is characterised in that PEDV is truncated in the amplification of S1 genes and the construction step of recombinant expression carrier, where the P CR amplifications B cell antigen epi-position PEDV truncate S1 genes 435-789 amino acids sequences the step of include：
RT-PCR amplifications are first carried out, 10 μ L RT-PCR amplification systems are：
5 × RT-buffer 2 μ L, dNTPs 2 μ L, 1 μ L, RNase Inhibitor of primer S1-down 1 μ L, Rever Tra Nucleic acid-templated 1 μ L, DEPC Water 2 μ L, the 42 DEG C of effect 50min of Ace 1 μ L, PEDV, obtain cDNA；
Then performing PCR amplification is entered, 20 μ L pcr amplification reaction system is：
10 × KOD-Plus-Neo buffer, 2 μ L, 2mM dNTPs 2 μ L, 25mM MgSO41.2 μ L, S1-up 1 μ L, S1- Down 1 μ L, KOD-Plus-Neo 0.5 μ L, cDNA 1 μ L, d2H2O 11.3μL；Amplification condition is：95 DEG C of pre-degeneration 5min；Enter Enter circulation：94 DEG C of denaturation 45sec, 50-62 DEG C of annealing 45sec, 72 DEG C of extension 1min, 30 circulations；72 DEG C re-extend 10min Afterwards, 4 DEG C of preservations, obtain purpose fragment；
Wherein, specific primer used is：
Underscore part is restriction enzyme site.
4. Porcine epidemic diarrhea virus antibody sIgA as claimed in claim 2 ELISA detection method, it is characterised in that PEDV is truncated in the amplification of S1 genes and the construction step of recombinant expression carrier, and the linked system of 10 μ L digestion products is：10× 1 μ L, S1 gene PCRs digestion products of T4DNA ligases buffer 5 μ L, the μ L of prokaryotic expression carrier pGEX4T1 digestion products 3, The μ L of T4DNA ligases 1；After each component is mixed, centrifuge, at least 8h is connected under the conditions of 16 DEG C.
5. Porcine epidemic diarrhea virus antibody sIgA as claimed in claim 2 ELISA detection method, it is characterised in that weight In the induced expression and detecting step of group plasmid, BL21 (DE3) cell containing recombinant plasmid is inoculated in Amp+LB liquid is trained Support in base, the Escherichia coli to convert pGEX4T1 empty carriers are cultivated to OD as negative control under conditions of 37 DEG C600nmFor 0.6 When, final concentration of 0.8mM IPTG is added under conditions of 37 DEG C, continues to cultivate 4h.
6. Porcine epidemic diarrhea virus antibody sIgA as claimed in claim 1 ELISA detection method, it is characterised in that system Standby HRP- rabbit-antis pig sIgA method includes：
SIgA purification procedures：
The colostrum of sow is collected, is centrifuged under conditions of 4~8 DEG C, is discarded upper-layer fat and lower sediment, take intermediate layer whey； Toward adding acetic acid in whey in the way of dropwise addition so that the pH of whey is 3.2-5.6, then albumen is sunken to a glass bottom；Then at 4 DEG C Under the conditions of centrifuge, take supernatant, supernatant be adjusted to pH=7；Obtained supernatant is subjected to gel filtration chromatography, collected The eluent of each absworption peak, SDS-PAGE electroresis appraisals are carried out to eluent；
The corresponding eluent in second peak is subjected to protein electrophorese, then protein is gone on pvdf membrane by transferring film instrument, used The goat-anti pig IgA of the horseradish peroxidase-labeled of 1000 times of dilution polyclonal antibody is incubated to the protein on film, Separating resulting is detected with immunoblotting, from testing result draw sIgA in IgA heavy chains Western blotting, then show sIgA point From success, sIgA is obtained；
Rabbit-anti pig sIgA preparation and markers step：
New zealand rabbit is immunized in sIgA through isolating and purifying, head exempts to add sIgA into isometric incomplete Freund's adjuvant breast Multiple spot immune rabbit after change, takes sIgA and multiple spot immune rabbit after the emulsification of isometric Freund's complete adjuvant, two exempt from rear three after two weeks Zhou Jinhang tri- exempts from, nape part multi-point injection；Three exempt from blood sampling separation serum after two weeks, obtain sIgA rabbit-anti pig positive serums；By blood It is marked with HRP labelling kits clearly, obtains HRP- rabbit-anti pigs sIgA.
7. Porcine epidemic diarrhea virus antibody sIgA as claimed in claim 1 ELISA detection method, it is characterised in that plus Enter in antigen step, the concentration of antigen PEDV S1 albumen is 12.5 μ g/mL, and addition is 100 μ L/ per hole；In 37 DEG C of condition Lower 1h, is then coated with least 8h under conditions of 4 DEG C.
8. Porcine epidemic diarrhea virus antibody sIgA as claimed in claim 1 ELISA detection method, it is characterised in that envelope Close in step, add the PBS of the 1%BSA containing percent by volume as confining liquid, 1.5h is closed under conditions of 37 DEG C, is then abandoned Deblocking liquid, and wash away with PBST remnants confining liquid.
9. Porcine epidemic diarrhea virus antibody sIgA as claimed in claim 1 ELISA detection method, it is characterised in that institute The dilution factor for stating enzyme labelled antibody HRP- rabbit-anti pigs sIgA is 1:5000-23000, addition is 100 μ L.
10. Porcine epidemic diarrhea virus antibody sIgA as claimed in claim 1 ELISA detection method, it is characterised in that institute State in addition enzyme labelled antibody step, add diluted enzyme labelled antibody HRP- rabbit-antis pig sIgA, 30 are reacted under conditions of 37 DEG C After~45min, uncombined enzyme labelled antibody is washed away with PBST.
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CN109856387A (en) * 2019-01-23 2019-06-07 南京农业大学 Pig epidemic diarrhea specificity SIgA antibody ELISA detection kit and its application
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