CN107011417A - Recombinant protein, encoding gene and application thereof, and detection kit and detection method for porcine epidemic diarrhea virus antibody - Google Patents

Recombinant protein, encoding gene and application thereof, and detection kit and detection method for porcine epidemic diarrhea virus antibody Download PDF

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CN107011417A
CN107011417A CN201710234819.8A CN201710234819A CN107011417A CN 107011417 A CN107011417 A CN 107011417A CN 201710234819 A CN201710234819 A CN 201710234819A CN 107011417 A CN107011417 A CN 107011417A
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recombinant protein
serum
antibody
pedv
diarrhea virus
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宋勤叶
姚作俊
郭海勇
逯纪成
孙泰然
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Heibei Agricultural University
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Abstract

The invention provides a recombinant protein, and the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1. The invention also provides a gene shown as SEQ ID No. 2. The invention also provides application of the protein in detecting the porcine epidemic diarrhea virus antibody, and the protein is mainly used as a coating antigen. The invention also provides a kit for detecting the porcine epidemic diarrhea virus antibody, which comprises the following components: the recombinant protein, the coating solution, the washing solution, the confining solution, the diluent, the goat anti-pig IgG labeled by horseradish peroxidase and the substrate developing solution are described above. The invention takes the recombinant protein shown in SEQ ID No.1 as the coating antigen, adopts indirect ELISA to detect the porcine epidemic diarrhea virus antibody, and has better specificity, sensitivity and repeatability, and visual and accurate result.

Description

Recombinant protein, its encoding gene, its application and the inspection of Porcine epidemic diarrhea virus antibody Test agent box and detection method
Technical field
The invention belongs to gene engineering technology field, more particularly to a kind of recombinant protein, its encoding gene, its application and pig The detection kit and detection method of epidemic diarrhea virus antibody.
Background technology
Pig epidemic diarrhea (Porcine epidemic diarrhea, PED) is the important biography for causing newborn piglet dead One of catch an illness.The disease is caused by Porcine epidemic diarrhea virus (PEDV), to suffer from diarrhoea, vomit, be dehydrated and suckling pig high mortality It is characterized.PED betided the growing and fattening swinery of Britain in 1971 first, then successive in European other countries and Asia Occur.PED occurs for the ground such as China Guangdong, Shanghai, Heilungkiang in 1976, the subsequent disease at home pig farm in endemicity form Prevalence, and it is isolated to PEDV from ill swinery.In October, 2010, newborn piglet diarrhoea is in China as caused by PEDV variants Outbreak of epidemic, is propagated rapidly throughout the country.This time prevalence is with the aggrieved most serious of suckling pig in 7 10d ages, its incidence of disease Up to 100%, the death rate causes huge economic losses up to 50%~90% to China's Swine Production.
PEDV belongs to coronaviridae coronavirus genus member, and full-length genome about 28kb is linear single-stranded positive RNA diseases Poison.4 kinds of structural proteins of viral genome codes：Spike protein (S), small membrane gene (E), memebrane protein (M) and nucleocapsid protein (N), wherein S protein is located at virion surface, is the most important structural proteins of PEDV, is made up of 1383aa, including signal peptide, Neutralizing epitope, transmembrane region and cytoplasmic domain.S protein can be divided into two regions of S1 (1-789aa) and S2 (790-1383aa), S1 areas Positioned at virus surface, it can be combined with the acceptor on host cell, the generation of neutralizing antibody can be induced；S2 areas are located at viral internal, Mediate retroviral is merged with host cell.Therefore, S protein is the major target proteins of the research of PEDV infection immunities and vaccine design, S Protein antibodies level and its change dynamic are to evaluate the important evidence of immune effect and monitoring immune state, while can also conduct PEDV infects the important indicator of seroepidemiological survey.Ding Zhenjiang etc. (2014) is based on restructuring PEDV S1 albumen and established PEDV IgA antibody indirect ELISA methods, China credit etc. (2016) establish the PEDVIgG indirect ELISAs based on PEDV S proteins Method.Because S1 protein domains are the main regions of induction body protective immune response, so for the special anti-of the region Body level can more reflect vaccine-induced immunoprotection level and body anti-infectious immunity level indirectly.Known blood-serum P EDV resists Body has positive correlation with local mucosa-immune level, and detects that the application of serum IgG antibody ELISA method is wider It is general.Therefore, it is highly desirable to set up the IgG antibody ELISA detection method based on S1 albumen.
The content of the invention
In view of this, it is an object of the invention to provide a kind of recombinant protein, its encoding gene, its application and pig are popular The detection kit and detection method of diarrhea virus antibody, the detection method that provides of the present invention is easy, specificity, sensitivity and steady It is qualitative strong.
The invention provides a kind of recombinant protein, its amino acid sequence is as shown in SEQ ID NO.1.
Present invention also offers a kind of gene for encoding recombinant protein as described above, its nucleotide sequence such as SEQ ID Shown in NO.2.
The recombinant protein that the present invention is provided can be as envelope antigen, using indirect elisa method to Porcine epidemic diarrhea virus Antibody is detected.
Present invention also offers a kind of recombinant protein as described above answering in detection Porcine epidemic diarrhea virus antibody With being used primarily as envelope antigen.
Present invention also offers a kind of kit for detecting Porcine epidemic diarrhea virus antibody, including：Weight as described above Histone, coating buffer, cleaning solution, confining liquid, dilution, the goat-anti pig IgG and substrate nitrite ion of horseradish peroxidase-labeled.
Wherein, the confining liquid is the PBST solution containing 5% NBCS.
Present invention also offers a kind of detection method of Porcine epidemic diarrhea virus antibody, comprise the following steps：
After recombinant protein as described above is coated with coating buffer, washed with cleaning solution, add confining liquid closing, add dilute The test serum released is reacted, and the goat-anti pig IgG that horseradish peroxidase-labeled is added after washing is washed after secondary response again, plus Enter substrate nitrite ion to be developed the color, obtain testing result.
The present invention detects that pig is popular using the recombinant protein shown in SEQ ID No.1 as envelope antigen using indirect ELISA Property diarrhea virus antibody, with preferable specificity, sensitiveness and repeatability, visual result is accurate.
The present invention has designed and synthesized specific primer for Porcine epidemic diarrhea virus (PEDV) S protein gene order, Corresponding nucleotide sequence has been expanded, the prokaryotic expression plasmid for expressing the region (S1) is constructed, the vivoexpression under IPTG inductions Restructuring S1 albumen, is identified expressing protein using Western blot, and by by protein immunization mouse, it was demonstrated that table The albumen reached has good immunogenicity, and mouse can be stimulated to produce high-level specific antibody.The weight provided using the present invention Histone establishes the indirect ELISA antibody detection method based on PEDV S1 albumen, and examined with this method as detection antigen The colostrum, childbirth same day serum and its piglet 1,7,14,21,28 and 35 that sow is immunized for antenatal 4 weeks with PEDV inactivated vaccines are surveyed PEDV antibody during age in days (d) in serum.As a result show, the indirect ELISA method of foundation can specifically detect PEDV antibody, With CSFV, porcine circovirus 2 type, porcine reproductive and respiratory syndrome virus, pseudorabies virus, transmissible gastroenteritis of swine disease The antiserum no cross reaction of poison and porcine rotavirus is minimum in every mL PEDV Positive Seras to detect 8.3 μ g Total protein, be 97.14% with the coincidence rate of immunoperoxidase monolayer assay testing result, in batch and batch between repeatability examination The coefficient of variation tested is less than 10%.PEDV antibody positive rates in immune sow colostrum, childbirth same day serum are 100%, colostrum It is lower than mean antibody levels in serum；1d piglet blood-serum P EDV antibody positive rates are 50%, and mean antibody levels are low, with colostrum In antibody level approach, all piglet serum all switch to PEDV negative antibodies after 7d.The ELISA method that (conclusion) is set up PEDV antibody can specifically be detected；When antenatal use PEDV inactivated vaccines are immunized 1 time to farrowing sow, it is farrowed in Swine serum Source of parents specific antibody positive rate and antibody level it is low, and it is of short duration to hold time.
Brief description of the drawings
Fig. 1 is the electrophoretogram that the embodiment of the present invention expands obtained purpose fragment；
Fig. 2 is recombinant expression plasmid pET28a-S1 PCR and digestion identification；
Fig. 3 analyzes for SDS-PAGE and the Western blot of restructuring PEDV S1 albumen；
Fig. 4 analyzes for the SDS-PAGE of restructuring PEDV S1 protein expression forms；
Fig. 5 is the blood serum special antibody after restructuring PEDV S1 protein immunization mouse；
Fig. 6 is dynamic by the PEDV antibody in sow colostrum, serum and its specific antibody in institute's farrowing Swine serum is immunized.
Embodiment
Embodiment 1
1 materials and methods
1.1 strains and expression vector
E.coli DH5 α and BL21 competent cell, prokaryotic expression carrier pET-28a (+), it is dynamic by Agricultural University Of Hebei Thing Infectious Diseases Lab is preserved.
1.2 main agents and antibody
TRIzol total RNA extraction reagents, dNTPs, Taq archaeal dna polymerase, centrifugation column type DNA gel purifying reclaim reagent Box, purchased from TIANGEN Biotech (Beijing) Co., Ltd.；Reverse transcriptase, is Promega Products；Restriction endonuclease BamH I and Xho I, purchased from precious bioengineering (Dalian) Co., Ltd；Ni-Agarose His label protein purification kits, It is reagent bio tech ltd purchased from Beijing health；Protein quantification testing cassete, Bioengineering Research Institute is built up purchased from Nanjing. HRP- goat-antis pig IgG and HRP- sheep anti-mouse iggs, purchased from Beijing Suo Laibao Bioisystech Co., Ltd.The anti-PEDV of pig, pig circular ring virus 2 Malicious 2 types (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), CSFV (CSFV), PRV (PRV), pig The antiviral antibody such as infectious gastroenteritis virus (TGEV) and pig A types rotavirus (RVA) and the anti-PEDV of mouse are positive and negative Serum, is prepared by Agricultural University Of Hebei's zoonosis laboratory and is preserved.
The structure of 1.3 PEDV S1 GFP prokaryotic expression plasmids and identification
1.3.1 design of primers and synthesis
For PEDV S protein genes, special the drawing of applied biology software Oligo6.0 design amplification S1 Partial Fragments Thing：
U1：5 '-ACTGAATTCATGGTACTGGGCGGTTATCTA-3 ',
L1：5′-ATTCTCGAGTTAGGCTAAGTGAGGATCTGA-3′.Primer is by giving birth to work bioengineering (Shanghai) share Co., Ltd synthesizes.
1.3.2 the PCR amplifications of target gene are with reclaiming
PEDVRNA is extracted, reverse transcription expands purpose nucleic acid fragment for after cDNA, performing PCR is entered with primer U1-L1.PCR expands Increasing condition is：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 1min, 55 DEG C of annealing 1min, 72 DEG C of extension 1min, 35 circulations；72℃ Extend 10min.The purpose fragment size of amplification is 945bp.PCR primer detects that amplified fragments are big through 1% agarose gel electrophoresis It is small with after expected be consistent, QIAquick Gel Extraction Kit recovery target gene purify with centrifugation plant type DNA gel.
1.3.3 recombinant expression plasmid pET28a-S1 structure and identification
The PCR primer and prokaryotic expression carrier pET28a reclaimed with restriction endonuclease EcoR I and Xho I digestion (+), digestion products after electrophoresis, respectively with centrifugation plant type DNA gel purify QIAquick Gel Extraction Kit recovery purifying recovery digestion products, PCR primer and pET28a (+) are connected with T4DNA ligases；Connection product pET28a-S1 is transformed into DH5 α competent cells, It is coated on Kan+On/LB flat boards, 37 DEG C of 16~18h of culture；Picking single bacterium colony does bacterium solution PCR identifications, extracts bacterium solution matter Grain carries out double digestion identification with restriction endonuclease EcoR I and Xho I, identifies that correct bacterium solution is sent to the biological work of raw work Journey (Shanghai) Co., Ltd. is sequenced.
1.4 recombinant protein S1 induced expression and identification
1.4.1 the expression of S1 albumen is recombinated
The PEDV S1 Protein reconstitution expression plasmids pET28a-S1 of structure is transformed into E.coli BL21 competent cells In, it is coated on Kan+On/LB solid mediums, 24h is cultivated in 37 DEG C, picking positive bacterium colony (pET28a-S1/BL21) is inoculated into Kan+In/LB fluid nutrient mediums, 37 DEG C overnight shaking culture, then in 1: 100 ratio transfer Kan+In/LB fluid nutrient mediums, 37 DEG C of 230r/min shakings are cultivated to flora exponential phase (OD600nm=0.6~0.8) when, add final concentration of 0.5mmol/L IPTG, 35 DEG C of induced expression 5h, collect induction before and induction after thalline 1mL 12000r/min, centrifugation 1min collect thalline sink Form sediment, plus 50 μ L PBS and 50 μ L 2 × SDS sample-loading buffers, mix, 12000r/min centrifugation 1min, carry out SDS-PAGE electricity Swimming, detects the expression and its molecular size range of albumen.Conversion empty plasmid pET-28a BL21 (pET-28a/ are set up simultaneously BL21) thalline is used as control.
1.4.2 recombinant protein S1 expression-form
The bacterium solution after induced expression is taken, 7500r/min centrifugation 10min, supernatant discarding, every gram of bacterial sediment adds 2mL bacterium Body lysate is mixed, and ice bath 10min is interrupted ultrasonic degradation on ice, and until bacterium solution is limpid, 4 DEG C of 12000r/min are centrifuged 10min, collects supernatant precipitation and carries out SDS-PAGE electrophoresis, determine the expression-form of albumen.
1.4.3 recombinant protein S1 Western blot identifications
Take after the bacterium solution of induced expression, SDS-PAGE electrophoresis, albumen be transferred on pvdf membrane, by film be put into 5% it is de- In fat milk powder confining liquid, 4 DEG C overnight；Add 1:1h is shaken on the anti-PEDV serum of mouse of 320 dilutions, room temperature, shaking table；Wash film Afterwards, the goat anti-mouse IgG of the HRP marks of 1: 2000 dilution, 1~2h of shaken at room temperature are added；After washing, film is put into DAB bottoms Develop the color 3~10min in thing nitrite ion, and band clearly uses deionized water terminating reaction, photographs to record result afterwards.
1.5 recombinant protein S1 immunogenicity
The bacterium solution expressing protein of induced expression is taken, is illustrated according to Ni-Agarose His label proteins purification kit, it is pure Change destination protein.Miscellaneous band is whether there is through SDS-PAGE analysis purifying proteins, protein content is determined with ultraviolet specrophotometer.Take restructuring 66 week old mouse of protein immunization, are immunized 3 times altogether, every minor tick 2 weeks, and dosage of inoculation and approach are shown in Table 1.Simultaneously set up 2 it is non- Immune mouse, is used as negative control.2 weeks tail vein bloods after inoculation, separate serum every time, and specific antibody is detected with ELISA. ELISA detects mainly comprising the following steps for antibody：96 hole elisa Plates are coated with 1 μ g/mL restructuring S1 albumen, per the μ L/ holes of hole 100, weight Multiple 2 holes；After closing, 1 is added:The serum to be checked of 160 times of dilutions, 1h, washing are incubated in 37 DEG C；100 μ L 1 are added per hole:2000 Dilute sheep anti-mouse igg-HRP, 45min, washing are incubated in 37 DEG C again；The tmb substrate solution of 100 μ L Fresh is added per hole, Room temperature lucifuge reacts 15min；50 μ L 2M H are added per hole2SO4Terminating reaction.OD is determined with enzyme detector450nmValue, analysis The production of recombinant protein inducing mouse specific antibody.
The immunization program of the recombinant protein of table 1
The foundation of the 1.6 indirect ELISA antibody detection methods based on restructuring S1 albumen and Preliminary Applications
1.6.1 the determination of antigen coat concentration and confining liquid
The restructuring PEDV S1 albumen of purifying is constituted into square formation from different confining liquids, optimal antigen coat concentration and envelope is screened Liquid is closed, i.e., the restructuring PEDV S1 albumen of purifying is diluted to 0.25,0.5 and 1 μ with coating buffer (pH9.6 carbonate buffer solution) G/mL, is separately added into each hole of 96 hole elisa Plates, 100 μ L/ holes, repeats 2 holes；ELISA Plate is put into 37 DEG C of incubation 1h, 4 DEG C are then put Effect is stayed overnight；Coating buffer is discarded, 3 are washed with 280 μ LPBST (0.05%Tween-20 0.01mol/LpH value 7.4PBS) per hole It is secondary, 3min/ times, pat dry；Added in the antigen hole of respective concentration different confining liquids (containing 5% NBCS, 5% horse serum, The PBST of 5% skimmed milk power, 2%BSA and 1% gelatin), 100 μ L/ holes put 37 DEG C of incubation 1h, discard confining liquid, pat dry；In phase Answer 100 μ L 1 of addition in antigen coat hole:The anti-PEDV positive serums of pig and negative serum of 40 times of dilutions；ELISA Plate is put 37 DEG C It is incubated 1h, washing；100 μ L 1 are added per hole:1000 times of dilute goat-anti pig IgG-HRP, 37 DEG C of incubation 45min are put by ELISA Plate, Washing；The tmb substrate solution of 100 μ L Fresh, room temperature lucifuge reaction 15min are added per hole；50 μ L 2M is added per hole H2SO4Terminating reaction.OD is determined with enzyme detector450nmValue, compares the OD values of positive and negative serum, and positive serum (P) and The ratio (P/N values) of negative serum (N) OD values.Antigen coat concentration and confining liquid when P/N values are maximum are optimal antigen bag By concentration and confining liquid.
1.6.2 the determination of coating condition and sealing condition
Appropriate restructuring PEDV S1 albumen is added in ELISA Plate respective aperture, antigen is carried out under the conditions of following 5 kinds respectively Coating, i.e., 37 DEG C effect 1h, 4 DEG C overnight, 37 DEG C effect 1h, 25 DEG C effect 1h, 4 DEG C overnight, 25 DEG C effect 1h, 4 DEG C overnight.So Afterwards respectively under the conditions of following 4 kinds blocking antigen hole, i.e., 37 DEG C be incubated 30min, 37 DEG C be incubated 1h, 25 DEG C be incubated 30min, 25 DEG C It is incubated 1h.Then serum to be checked is sequentially added according to the reaction condition in 1.4.1 and enzyme labelled antibody carries out ELISA, it is determined that most preferably Antigen coat condition and sealing condition.
1.6.3 serum-concentration to be checked and the determination of enzyme labelled antibody concentration
The anti-PEDV positives and negative serum are made 1 respectively:40、1:80、1:160、1:320、1:640 and 1:1280 times dilute Release, goat-anti pig IgG-HRP is made 1:500 and 1:1000 times of dilutions, constitute square formation, according to the reaction condition in 1.4.1, carry out ELISA, determines the best effort concentration of serum to be checked and enzyme labelled antibody.
1.6.4 the determination in serum and enzyme labelled antibody reaction time to be checked
The ELISA Plate for taking restructuring PEDV S1 albumen to be coated with, is separately added into the anti-PEDV of 100 μ L positive and cloudy in respective aperture Property serum (1:40 times of dilutions), in acting on 45min and 1h respectively at 37 DEG C；After washing, 100 μ L goat-antis pig IgGs of every hole addition- HRP(1:1000 times of dilutions), in acting on 30min and 45min respectively at 37 DEG C；Colour developing, terminating reaction determines positive and negative serum OD values, compare P/N values, determine the optimum reacting time of serum to be checked and enzyme labelled antibody.
1.6.5 the determination of serum yin and yang attribute critical value
50 parts of PEDV negative antibody serum are detected with the method for foundation, sample OD is calculated450nmThe average value (X) and standard of value Variance (SD).According to Principle of Statistics, the OD of sample450nmDuring value >=X+3SD, the positive, OD are judged to450nmDuring value≤X+2SD, sentence For feminine gender, it is judged to therebetween suspicious.
1.6.6 specificity and sensitivity tests
The antiviral antibodies such as PCV2, PRRSV, CSFV, PRV, TGEV and RVA are detected with the indirect ELISA method of foundation, simultaneously Set up PEDV positive and negative antibody controls, the specificity of evaluation method.
5 parts of PEDV Positive Seras are made 1:80、1:160、1:320、1:640、1:1280、1:2560 doubling dilutions Afterwards, specific antibody is detected with the ELISA method of foundation.Each dilution factor does 3 repetitions, while setting up 3 dilution controls and 3 Individual blank well control.To be able to detect that the protein content average value in the highest extension rate serum of PEDV antibody come the side of evaluation The sensitiveness of method.
1.6.7 contrast test is respectively with the ELISA method set up and immunoperoxidase monolayer assay (Immunoperoxidase monolayer assay, IPMA) detects 70 parts of Swine serums, compares the testing result of two methods, Evaluate ELISA method and IPMA coincidence rate.
1.6.8 replica test recombinates PEDV S1 albumen coated elisa plates with same batch, while detecting 10 parts of pig bloods Clearly, every part of serum repeats 3 holes, and experiment is repeated 3 times altogether.The restructuring PEDV S1 albumen coated elisa plates of 4 batches are separately taken, respectively 10 parts of Swine serums are detected, every part of serum repeats 3 holes.According to the coefficient of variation (CV), analysis set up ELISA method batch in criticize between Repeatability.
1.6.9 Preliminary Applications randomly select 5 farrowing sows, and epidemic disease was inactivated through Houhai acupoint immunity inoculation PED in antenatal 4 weeks Seedling, after sow production, makes every newborn piglet have colostrum by natural lactation method.Detected using the ELISA method of foundation Serum and its farrowed pig (per 10~13, nest) serum in 1d, 7d, 14d, 21d, 28d and 35d on the day of colostrum, Farrowing In specific antibody, the dynamic change of special maternal antibody in initial analysis newborn piglet serum.
2 results
2.1 recombinant expression plasmid pET28a-S1 structure and identification
Extract PEDV RNA, through PCR after reverse transcription, expected size (945bp) purpose fragment has been arrived in amplification, as a result referring to Fig. 1, Fig. 1 are the electrophoretogram that the embodiment of the present invention expands obtained purpose fragment, wherein, 1 is DNA relative molecular mass standards；2 For PCR primer.With DNA gel purify QIAquick Gel Extraction Kit reclaim target gene, through restriction endonuclease EcoR I and Xho I It is connected in vitro with prokaryotic expression carrier pET28a (+) after digestion, constructs recombinant expression plasmid pET28a-S1, through PCR, enzyme After cutting and being sequenced, it was demonstrated that target gene fragment (S1) successful clone to recombinant expression plasmid pET28a (+) relevant position, referring to Fig. 2, Fig. 2 are recombinant expression plasmid pET28a-S1 PCR and digestion identifications, wherein, 1 is DNA relative molecular mass standards；2 are Recombinant plasmid bacterium solution PCR primer；3 be that pET28a plasmid PCRs are compareed；4 be the pET28a-S1 of non-digestion；5 be pET28a-S1's EcoR I and the double digestion products of Xho I.The PEDV S1 nucleotide sequences of amplification are as shown in SEQ ID No.2, and its amino acid sequence is such as Shown in SEQ ID No.2.
2.2 recombinant protein S1 expression is identified with Western blot
At 35 DEG C through IPTG induce 5h after, as a result referring to Fig. 3, Fig. 3 for restructuring PEDV S1 albumen SDS-PAGE with Western blot are analyzed, wherein, a is the expression and purification of restructuring S1 albumen.M. protein low molecule quality standard；1.pET- Before 28a/BL21 inductions；After 2.pET28a/BL21 inductions；Before 3.pET28a-S1/BL21 inductions；4.pET28a-S1/BL21 is lured Lead 5h；5. recombinant protein after purification.B analyzes for the Western blot of restructuring S1 albumen.M. protein low molecule quality mark It is accurate；1. recombinant protein reacts with the anti-PEDV negative serums of mouse；2. recombinant protein reacts with the anti-PEDV positive serums of mouse.As a result Show, the recombinant protein molecular weight about 38kDa of pET28a-S1/BL21 expression, with being expected (Fig. 3 a) in the same size, through Western Blot detects that the albumen can be combined (Fig. 3 b) with the anti-PEDV blood serum specials of mouse.
2.3 recombinant protein S1 are expressed with inclusion bodies
The BL21 (pET28a-S1/BL21) of recombinant expression plasmid is carried after IPTG inductions 5h, bacterium is collected, through ultrasound After ripple cracking, it is precipitated and the SDS-PAGE of supernatant is shown, the albumen for occurring expected size in precipitation, and in supernatant almost It is not anticipated that big little albumen, as a result referring to Fig. 4, Fig. 4 analyzes for the SDS-PAGE of restructuring S1 protein expression forms, wherein, M is egg White matter low molecule quality standard；1 is cracking precipitation；6 be cracking supernatant.Show restructuring S1 albumen in Escherichia coli with inclusion body Form is expressed.
2.4 recombinant protein S1 have good immunogenicity
With 14 days after the recombinant protein first immunisation mouse of expression, detected in three serum of 6 mouse special anti- Body；High-caliber specific antibody is detected in whole immune serums within second immune latter 14 days, after third time is immune The antibody level of 14 days almost maintains phase same level with secondary, referring to Fig. 5, after Fig. 5 is restructuring S1 protein immunization mouse Blood serum special antibody, wherein, V1~V6 be recombinant protein be immunized mouse；Neg.1 and Neg.2, nonimmune control mice；0,1 ~3, refer to immune preceding and first and second and three immune serum samples gathered for latter 14 days respectively.The above results show, the weight of expression Group S1 albumen can stimulate mouse to produce specific antibody, with good immunogenicity.
The 2.5 PEDV indirect ELISA antibody detection methods based on restructuring S1 albumen
2.5.1 the condition of work of indirect ELISA and criterion
To recombinate S1 albumen as envelope antigen, PEDV indirect ELISA antibody detection methods are established.ELISA method Main operational steps are with criterion：It is coated with overnight at 37 DEG C of incubation 1h, 4 DEG C with 1 μ g/mL restructuring PEDV S1 albumen ELISA Plate, washing, 1h is closed with the PBST of 5% NBCS at 37 DEG C；Washing, adds 1:The pig blood to be checked of 40 times of dilutions Clearly, 1h is reacted at 37 DEG C；Washing, adds 1:The goat-anti pig IgG of the HRP marks of 1000 times of dilutions, in reaction at 37 DEG C 45min；Washing, adds the tmb substrate solution of Fresh, room temperature lucifuge reaction 15min；Add 2M H2SO4Terminating reaction, knot Fruit is referring to 2~table of table 5.OD is determined with enzyme detector450nmValue, result of determination.As sample OD450nmDuring value >=0.373, judge It is positive for specific antibody；Work as OD450nmDuring value≤0.305, it is determined as feminine gender；Work as OD450nmWhen value falls between, it is determined as It is suspicious.
The determination of the antigen coat concentration of table 2 and confining liquid
Note：+, positive serum；-, negative serum；P/N, positive serum OD450nmValue/negative serum OD450nmValue.
The determination of the optimal antigen coat condition of table 3 and sealing condition
Note：+, positive serum；-, negative serum；P/N, positive serum OD450nmValue/negative serum OD450nmValue.
The serum to be checked of table 4 and the determination of the optimal diluted concentration of enzyme labelled antibody
Note：+, positive serum；-, negative serum；P/N, positive serum OD450nmValue/negative serum OD450nmValue.
The serum to be checked of table 5 and the determination of ELIAS secondary antibody the best use of time
Note：+, positive serum；-, negative serum；P/N, positive serum OD450nmValue/negative serum OD450nmValue.
2.5.2 the specificity and sensitiveness of indirect ELISA
During PCV2, PRRSV, CSFV, PRV, TGEV and RVA antibody known with the method detection of foundation, the equal ＜ of OD values 0.305, referring to table 6, show that the ELISA method set up and other serum virus do not have cross reaction, with good specificity. By PEDV Positive Sera doubling dilutions, when containing 8.3 μ g albumen in the anti-PEDV serum of every mL pigs, the ELISA of foundation can Detect PEDV special
Xenoantibody, shows that the ELISA method is at least able to detect that the μ g of ＜ 8.3 specific antibody.
The ELISA of table 6 specific detection
Note：+, positive serum；-, negative serum.
2.5.3 the coincidence rate of indirect ELISA and immunoperoxidase monolayer assay (IPMA)
Detect 34 parts and 35 parts of PEDV Positive Seras respectively from 70 parts of serum using IPMA and ELISA, detect 36 parts and 35 parts of PEDV negative antibodies serum, the positive coincidence rate of the two testing result is 97.06%, and negative match-rate is 97.22%, total coincidence rate is 97.14%.
2.5.4 it is repeated
When with detection identical blood serum sample with a batch of heavy S1 albumen coated elisa plate, is repeated 3 times, or with 4 The restructuring S1 albumen coated elisa plates of batch, when detecting identical blood serum sample, its coefficient of variation is respectively 3.75%~9.30% With 1.49%~7.84%, 10% (table 7 and table 8) is below.The result shows, either criticize in or batch between repeat to test, Restructuring PEDV S1 albumen is respectively provided with good repeatability as detection antigen.
Repeat to test in 7 batches, table
Repeat to test between 8 batches, table
2.5.5 Preliminary Applications
In the farrowing sow colostrum and serum of 5 passive immunity inoculation PED inactivated vaccines, PEDV can be detected special Xenoantibody, wherein colostrum are lower than the mean antibody levels in serum, OD450nmBe worth for respectively 0.508 ± 0.058 and 0.824 ± 0.093 (Fig. 6 a).The positive rate of antibody of the age in days piglet of 5 nest 1 is 50%, and the mean antibody levels of antibody positive piglet are low (OD450nmBe worth for 0.481 ± 0.057), the serum of all piglets all switchs to negative antibody (Fig. 6 b) after 7 ages in days.Fig. 6 is immune Specific antibody dynamic in PEDV antibody and its farrowed Swine serum in sow colostrum, serum, wherein, at the beginning of a is immune sow PEDV antibody in breast and serum, 1,2,3,4 and 5 be that sow is numbered；B is per the PEDV Antibody dynamics in nest piglet serum.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should It is considered as protection scope of the present invention.
<110>Agricultural University Of Hebei
<120>Recombinant protein, its encoding gene, its application and detection kit and the detection of Porcine epidemic diarrhea virus antibody Method
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 311
<212> PRT
<213>Artificial sequence
<400> 1
Val Leu Gly Gly Tyr Leu Pro Ile Gly Glu Asn Gln Gly Val Asn Ser
1 5 10 15
Thr Trp Tyr Cys Ala Gly Gln His Pro Thr Ala Ser Gly Val His Gly
20 25 30
Ile Phe Val Ser His Ile Arg Gly Gly His Gly Phe Glu Ile Gly Ile
35 40 45
Ser Gln Glu Pro Phe Asp Pro Ser Gly Tyr Gln Leu Tyr Leu His Lys
50 55 60
Ala Thr Asn Gly Asn Thr Asn Ala Thr Ala Arg Leu Arg Ile Cys Gln
65 70 75 80
Phe Pro Ser Ile Lys Thr Leu Gly Pro Thr Ala Asn Asn Asp Val Thr
85 90 95
Thr Gly Arg Asn Cys Leu Phe Asn Lys Ala Ile Pro Ala His Met Ser
100 105 110
Glu His Ser Val Val Gly Ile Thr Trp Asp Asn Asp Arg Val Thr Val
115 120 125
Phe Ser Asp Lys Ile Tyr Tyr Phe Tyr Phe Lys Asn Asp Trp Ser Arg
130 135 140
Val Ala Thr Lys Cys Tyr Asn Ser Gly Gly Cys Ala Met Gln Tyr Val
145 150 155 160
Tyr Glu Pro Thr Tyr Tyr Met Leu Asn Val Thr Ser Ala Gly Glu Asp
165 170 175
Gly Ile Ser Tyr Gln Pro Cys Thr Ala Asn Cys Ile Gly Tyr Ser Ala
180 185 190
Asn Val Phe Ala Thr Glu Pro Asn Gly His Ile Pro Glu Gly Phe Ser
195 200 205
Phe Asn Asn Trp Phe Leu Leu Ser Asn Asp Ser Thr Leu Val His Gly
210 215 220
Lys Val Val Ser Asn Gln Pro Leu Leu Val Asn Cys Leu Leu Ala Ile
225 230 235 240
Pro Lys Ile Tyr Gly Leu Gly Gln Phe Phe Ser Phe Asn Gln Thr Ile
245 250 255
Asp Gly Val Cys Asn Gly Ala Ala Val Gln Arg Ala Pro Glu Ala Leu
260 265 270
Arg Phe Asn Ile Asn Asp Thr Ser Val Ile Leu Ala Glu Gly Ser Ile
275 280 285
Val Leu His Thr Ala Leu Gly Thr Asn Phe Ser Phe Val Cys Ser Asn
290 295 300
Ser Ser Asp Pro His Leu Ala
305 310
<210> 2
<211> 936
<212> DNA
<213>Artificial sequence
<400> 2
gtactgggcg gttatctacc tattggtgaa aaccagggtg tcaattcaac ttggtactgt 60
gctggccaac atccaactgc tagtggcgtt catggtatct ttgttagcca tattagaggt 120
ggtcatggct ttgagattgg catttcgcaa gagccttttg accctagtgg ttaccagctt 180
tatttacata aggctactaa cggtaacact aatgctactg cgcgactgcg catttgccag 240
tttcctagca ttaaaacatt gggccccact gctaataatg atgttacaac aggtcgtaat 300
tgcctattta acaaagccat cccagctcat atgagtgaac atagtgttgt cggcataaca 360
tgggataatg atcgtgtcac tgtcttttct gacaagatct attattttta ttttaaaaat 420
gattggtccc gtgttgcgac aaagtgttac aacagtggag gttgtgctat gcaatatgtt 480
tacgaaccca cctactacat gcttaatgtt actagtgctg gtgaggatgg tatttcttat 540
caaccctgta cagctaattg cattggttat tctgccaatg tatttgctac tgagcccaat 600
ggccacatac cagaaggttt tagttttaat aattggtttc ttttgtccaa tgattccact 660
ttggtgcatg gtaaggtggt ttccaaccaa ccattgttgg tcaattgtct tttggccatt 720
cctaagattt atggactagg ccaatttttt tcctttaatc aaacgatcga tggtgtttgt 780
aatggagctg ctgtgcagcg tgcaccagag gctctgaggt ttaatattaa tgacacctct 840
gtcattcttg ctgaaggctc aattgtactt catactgctt taggaacaaa tttttctttt 900
gtttgcagta attcctcaga tcctcactta gcctaa 936

Claims (7)

1. a kind of recombinant protein, it is characterised in that its amino acid sequence is as shown in SEQ ID NO.1.
2. a kind of gene for encoding recombinant protein as claimed in claim 1.
3. gene according to claim 2, it is characterised in that its nucleotide sequence is as shown in SEQ ID NO.2.
4. application of the recombinant protein in detection Porcine epidemic diarrhea virus antibody described in claim 1.
5. a kind of kit for detecting Porcine epidemic diarrhea virus antibody, including：Recombinant protein, coating described in claim 2 Liquid, cleaning solution, confining liquid, dilution, the goat-anti pig IgG and substrate nitrite ion of horseradish peroxidase-labeled.
6. kit according to claim 5, it is characterised in that the confining liquid is the PBST containing 5% NBCS Solution.
7. a kind of detection method of Porcine epidemic diarrhea virus antibody, comprises the following steps：
After recombinant protein described in claim 2 is coated with coating buffer, washed with cleaning solution, add confining liquid closing, add The test serum of dilution is reacted, and the goat-anti pig IgG that horseradish peroxidase-labeled is added after washing is washed after secondary response again, Add substrate nitrite ion to be developed the color, obtain testing result.
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