CN113337525A - Encoding gene of porcine epidemic diarrhea virus S1D fragment protein and application thereof - Google Patents

Encoding gene of porcine epidemic diarrhea virus S1D fragment protein and application thereof Download PDF

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CN113337525A
CN113337525A CN202110363023.9A CN202110363023A CN113337525A CN 113337525 A CN113337525 A CN 113337525A CN 202110363023 A CN202110363023 A CN 202110363023A CN 113337525 A CN113337525 A CN 113337525A
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protein
diarrhea virus
epidemic diarrhea
porcine epidemic
inclusion body
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郭霄峰
李洁森
莫炜钰
邝燕齐
莫梅君
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South China Agricultural University
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    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • G01—MEASURING; TESTING
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    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
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    • C12N2800/00—Nucleic acids vectors
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    • C12N2800/101—Plasmid DNA for bacteria
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus

Abstract

The invention discloses a coding gene of S1D fragment protein of porcine epidemic diarrhea virus, and the nucleotide sequence is shown as SEQID NO. 1. The invention establishes a preparation method for expressing a large amount of porcine epidemic diarrhea virus S1D fragment protein by utilizing the coding gene of the S1D fragment protein after codon optimization, the S1D fragment protein can be efficiently expressed by utilizing the preparation method, the purity is high, the biological activity is good, and the monoclonal antibody prepared by utilizing the preparation method has high titer and strong specificity; the whey IgA ELISA detection method established as the antigen has good repeatability, sensitivity and specificity.

Description

Encoding gene of porcine epidemic diarrhea virus S1D fragment protein and application thereof
Technical Field
The invention relates to the technical field of porcine epidemic diarrhea, in particular to a coding gene of porcine epidemic diarrhea virus S1D fragment protein and application thereof.
Background
Epidemic diarrhea (Porcine epidemic diarrhea) is a highly contagious disease in which infection with Porcine Epidemic Diarrhea Virus (PEDV) causes vomiting, diarrhea, dehydration and death in piglets, all of which are infected with pigs of different ages, but the mortality rate is highest in piglets of 10 days of age. In China, the disease is first reported in autumn 2010 and then spreads across the country. At present, a large number of suckling piglets die due to the disease, serious economic loss is caused to farmers, and the disease becomes one of important epidemic diseases affecting the healthy development of the pig industry in China.
PEDV has a typical coronavirus structure, containing 4 major structural proteins, nucleocapsid protein (N), membrane protein (M), spike protein (S), and vesicle membrane protein (E). Wherein the S protein carries a major B lymphocyte epitope which can induce the body to produce neutralizing antibodies. Thus, the S protein plays a key role in viral infection, pathogenicity, and host cell tropism. Before PEDV enters cells, S protein is split into S1 (1-789 aa) and S2 (790-1383 aa) under the action of cellular protease, and S1 is folded into a spherical domain. The COE (collagenase equivalent domain) region (499-638 aa) of the S1 protein is the main antigen for inducing the organism to produce neutralizing antibodies, while the S1D (636-789 aa) region of the S1 protein comprises 1 linear epitope (697-742 aa) and 2B cell epitopes (744-759 aa and 756-771 aa), both of which are considered as excellent vaccine candidate antigens.
To further develop PEDV vaccines, a large amount of S1D fragment protein is required to be available as an antigen. The most mature technology which is adopted by people at first and mastered at present is a prokaryotic expression system. It mainly uses the carrier with cloned target gene segment to transform bacteria (usually colibacillus) to obtain target protein by induction expression. Because of its clear genetic background, low cost and short cycle, the most commonly used expression system is E.coli. However, when exogenous proteins are expressed in E.coli, the expression level is often low, mainly because the codons of the exogenous genes are non-preferred codons of E.coli, bacteria lack tRNA recognizing the codons, the translation speed of the proteins is often dependent on tRNA, and the reduction of the translation speed leads to the reduction of the synthesis rate of the proteins, so that the expression level is not high, and even the translation can be stopped early.
Meanwhile, after a large amount of protein is expressed, the selection of a purification method is particularly important, the commonly used method is nickel column affinity purification or gel cutting purification, and the two methods have respective advantages and disadvantages. The former method can obtain a target protein which is relatively pure and has good activity, but the target protein is more lost in the column passing process, so that the concentration of the purified protein is not high, and the subsequent immunization of mice is not facilitated; the latter method also allows to obtain a purer target protein without too much loss, but the purified protein is not biologically active.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a coding gene of the S1D fragment protein of the porcine epidemic diarrhea virus and application thereof. The technical problem of low expression quantity of the recombinant protein in a prokaryotic expression system (escherichia coli) is solved, so that the purpose of large-quantity expression of the recombinant protein in cells is achieved, and the preparation of the monoclonal antibody prepared by the recombinant protein and the establishment of a whey IgA ELISA detection method are realized.
The first purpose of the invention is to provide a coding gene of S1D fragment protein of porcine epidemic diarrhea virus.
The second objective of the invention is to provide a recombinant vector.
The third purpose of the invention is to provide an engineering bacterium.
The fourth purpose of the invention is to provide a preparation method for expressing the S1D fragment protein of the porcine epidemic diarrhea virus in a large amount.
The fifth purpose of the invention is to provide the S1D fragment protein of the porcine epidemic diarrhea virus prepared by any one of the preparation methods.
The sixth purpose of the invention is to provide the application of the porcine epidemic diarrhea virus S1D fragment protein in the preparation of a porcine epidemic diarrhea virus detection kit and/or an anti-porcine epidemic diarrhea virus antibody.
In order to achieve the purpose, the invention is realized by the following scheme:
the invention successfully constructs a recombinant plasmid pET-delta S1D containing a sequence delta S1D gene after codon optimization of S1D fragment protein, successfully expresses recombinant protein by utilizing an escherichia coli exogenous gene expression system, and the recombinant protein is porcine epidemic diarrhea virus S1D fragment protein through SDS-PAGE and Western blotting identification. After codon optimization, compared with the non-optimized S1D fragment protein, the codon-optimized delta S1D fragment protein has obviously improved expression level. The adopted method is an optimized inclusion body purification method, a washing buffer solution, a dissolving buffer solution and a renaturation buffer solution of the inclusion body are prepared, and the pH value is strictly regulated; washing the crude inclusion body with washing buffer solution at room temperature for 3 times, and washing away much impure protein in the process to prepare a refined inclusion body; dissolving refined inclusion bodies for 2-12 h at 4 ℃ by using a dissolving buffer solution, determining the specific time according to the dissolving degree of the inclusion bodies, centrifuging and taking supernatant for dialysis renaturation when most of the inclusion bodies are dissolved in the dissolving buffer solution, recovering the activity of target protein at 4 ℃ by using the renaturation buffer solution according to a method of reducing renaturation in a gradient manner, finally successfully preparing delta S1D fragment protein with activity and good purity, determining by a BCA method, wherein the concentration of the purified delta S1D fragment protein can reach about 1mg/mL, and the purified delta S1D fragment protein can be used as a good antigen for immunizing a mouse to prepare a monoclonal antibody or coating an ELISA plate for ELISA detection.
Further, a monoclonal antibody of the delta S1D fragment protein is prepared, a BALB/c mouse is immunized by the purified delta S1D fragment protein, a monoclonal cell strain is successfully screened out by adopting cell fusion, screening and subcloning methods, and the mouse ascites is prepared by utilizing an in vivo induction method. The ELISA results showed that the antibody titer of ascites reached 1:1000000, the ascites reacts positively with PEDV virion and purified delta S1D fragment protein, and reacts negatively with PEDV N protein, pET-32a idle protein and 5 virions of PRRSV, TGEV, CSFV, PDCoV and SADs. Western-blotting shows that specific bands exist in the reaction of ascites with the delta S1D fragment protein and PEDV S protein, and no specific band appears in the reaction of ascites with pET-32a no-load protein and normal Vero cell protein. IFA results show that ascites and positive control groups can enable cells to generate specific green fluorescent signals, and green fluorescent signals are not seen in blank and negative groups.
And establishing a whey IgA ELISA detection method, and determining the optimal reaction condition of the method by optimizing different conditions based on the purified delta S1D fragment protein as a coating antigen. The optimal antigen coating concentration and conditions are 4 mu g/mL and overnight coating at 4 ℃, the optimal whey dilution multiple is 1: 160, the optimal blocking liquid and blocking time are respectively 5% (w/v) BSA and 90min, the optimal action time of whey is 120min, the optimal dilution concentration and action time of HRP-goat anti-pig IgA are respectively 1: 10000. 60min, and the optimal color development time of the substrate color development liquid (TMB) is 25 min. The critical value of the positive whey is determined to be 0.144 by using a combination method of an average value and a standard deviation, the repeatability experiments show that the variation coefficient of the repeatability experiments in batches and between batches does not exceed 10%, and the sensitivity experiments show that 4 positive whey samples are diluted to 1: 160, the result is still positive, and a comparison experiment shows that the sensitivity of the method established in the research is 85.4%, the specificity is 80.0%, and the coincidence rate of the two methods is 85.0%, which indicates that the detection method established in the research has good sensitivity and specificity, the positive detection rate of the method is slightly lower than that of a domestic kit, and the method provides technical support for clinically monitoring the anti-PEDV IgA level of sow colostrum whey.
Therefore, the invention claims a coding gene of the S1D fragment protein of porcine epidemic diarrhea virus, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1.
Codon-optimized gene sequence Δ S1D (438bp), SEQ ID NO: 1:
ATG ACC CTG GAT GTG TGC ACC AAA TAT ACC ATT TAT GGC TTT AAA GGC GAA GGC ATT ATT ACC CTG ACC AAT AGC AGC TTT CTG GCG GGC GTG TAT TAT ACC AGC GAT AGC GGC CAG CTG CTG GCG TTT AAA AAT GTG ACC AGC GGC GCG GTG TAT AGC GTG ACC CCG TGC AGC TTT AGC GAA CAG GCG GCG TAT GTG GAT GAT GAT ATT GTG GGC GTG ATT AGC AGC CTG AGC AGC AGC ACC TTT AAT AGC ACC CGC GAA CTG CCG GGC TTT TTT TAT CAT AGC AAT GAT GGC AGC AAT TGC ACC GAA CCG GTG CTG GTG TAT AGC AAT ATT GGC GTG TGC AAA AGC GGC AGC ATT GGC TAT GTG CCG AGC CAG AGC GGC CAG GTG AAA ATT GCG CCG ATG GTG ACC GGC AAT ATT AGC ATT CCG ACC AAT TTT AGC
the invention also claims a recombinant vector which is connected with the coding gene.
Preferably, the vector is an expression vector.
More preferably, the vector is pET-32 a.
The invention also claims an engineering bacterium containing the strain of the recombinant vector.
Preferably, the strain is escherichia coli.
The invention also claims a method for expressing the porcine epidemic diarrhea virus S1D fragment protein in large quantity, which utilizes a bacterial strain containing the plasmid carrying the coding gene for expression.
Preferably, the vector is an expression vector.
More preferably, the vector is pET-32 a.
Preferably, the strain is escherichia coli.
Preferably, when the bacterial liquid OD600When the value is 0.6-1.0, IPTG with the final concentration of 0.1-1.5 mmol/L is added, and the induction expression is carried out for 2-6 hours at the temperature of 16-42 ℃.
More preferably, when the bacterial liquid OD600When the value reached 0.6, IPTG was added to a final concentration of 0.1mmol/L, and expression was induced at 37 ℃ for 6 hours.
Preferably, the bacteria solution after induction expression is centrifuged at 6000-8000 r/min for 10-20 min, the supernatant is discarded to obtain thalli, the thalli is resuspended by PBS containing 1-2% (v/v) PMSF (phenylmethylsulfonyl fluoride) at 0.01mol/L and pH of 7.4, the bacteria are ultrasonically crushed in an ice bath, and precipitates are respectively harvested after centrifugation at 11000-12000 r/min for 15-20 min, so as to obtain an inclusion body crude product.
More preferably, the bacterial liquid after induction expression is centrifuged at 6000r/min for 15min, the supernatant is discarded to obtain thalli, the thalli is resuspended by PBS containing 1% (v/v) PMSF (phenylmethylsulfonyl fluoride) at the pH of 7.4 at 0.01mol/L, the bacteria are ultrasonically crushed in ice bath, and the precipitates are respectively harvested after centrifugation at 12000r/min for 20min, so as to obtain the inclusion body crude product.
Preferably, an inclusion body washing solution and Tris-HCl are used for washing the inclusion body crude product in sequence, wherein the inclusion body washing solution contains Tris-HCl, NaCl, urea and Triton X-100; dissolving with inclusion body denaturation dissolving solution, wherein the inclusion body denaturation dissolving solution contains Tris-HCl, NaCl, urea, Triton X-100 and DTT. .
More preferably, the inclusion body washing solution contains 15-30 mmol/L Tris-HCl with pH 7.5-8.0, 0.4-0.6 mol/L NaCl, 1-2 mol/L urea and 1-2% (v/v) Triton X-100.
Further preferably, the inclusion body wash solution contains 20mmol/L Tris-HCl pH 8.0, 0.5mol/L NaCl, 2mol/L urea, and 2% Triton X-100.
More preferably, the inclusion body crude product is washed twice by using an inclusion body washing solution in sequence, washed for 2-4 h, and then washed by using Tris-HCl.
More preferably, the inclusion body denaturation solution contains 15-30 mmol/L of Tris-HCl with pH 7.5-8.0, 0.4-0.6 mol/L of NaCl, 6-8 mol/L of urea, 1-2% of Triton X-100, and 0.1-0.3 mmol/L of DTT.
Further preferably, the inclusion body-denatured solution contains 20mmol/L of Tris-HCl pH 8.0, 0.5mol/L of NaCl, 6mol/L of urea, 2% Triton X-100, and 0.2mmol/L of DTT.
Preferably, the dialysis is performed sequentially with TGE buffer containing decreasing concentrations of urea.
More preferably, 6-8M, 4-6M, 2-4M and 0-2M urea TGE buffer solution is used for dialysis in sequence.
Further preferably, dialysis is performed sequentially using a urea TGE buffer containing 6M, 4M, 2M, and 0M.
The application of the coding gene, the recombinant vector and/or the engineering bacterium in preparing the porcine epidemic diarrhea virus S1D fragment protein also belongs to the protection scope of the invention.
The invention also claims the protein of the porcine epidemic diarrhea virus S1D fragment prepared by any one of the preparation methods.
The application of the porcine epidemic diarrhea virus S1D fragment protein in the preparation of a porcine epidemic diarrhea virus detection kit and/or an anti-porcine epidemic diarrhea virus antibody also belongs to the protection scope of the invention.
Preferably, the antibody is a monoclonal antibody.
The invention also provides a detection kit for the porcine epidemic diarrhea virus, which is established by the monoclonal antibody prepared from the S1D fragment protein of the porcine epidemic diarrhea virus and contains the monoclonal antibody as a primary antibody.
Also contained were PBS and a FITC-labeled goat anti-mouse IgG secondary antibody.
The invention also provides a whey IgA ELISA detection kit prepared from the porcine epidemic diarrhea virus S1D fragment protein.
It also contained diluent (phosphate buffer), blocking solution (PBST containing 5% (w/v) BSA), PBST wash (0.05% (v/v) Tween-20 in 0.01mol/L PBS, pH 7.4), TMB substrate solution, 2mol/L sulfuric acid.
The using method comprises the following steps: coating the enzyme-labeled plate with purified delta S1D protein at a coating concentration of 4 mu g/mL for overnight at 4 ℃, and sealing the enzyme-labeled plate with 5% (w/v) PBST of BSA as a sealing liquid for 90 min; sealing, adding whey at a ratio of 1: 160, performing action at 37 deg.C for 120min, respectively performing dilution at a ratio of 1:10000 on HRP-goat anti-pig IgA for 60min at 37 deg.C, and performing light-shielding action on TMB at room temperature for 25 min.
More preferably, the purified Δ S1D protein is coated on a microplate at a coating concentration of 4 μ g/mL, each well is coated with 100 μ L, incubated at 37 ℃ for 1h, and coated overnight at 4 ℃; PBST cleaning solution is washed for 4 times, each time is 1min, the enzyme label plate is sealed by 5% (w/v) PBST of BSA as sealing solution, 200 mu L/hole, and the sealing time is 90 min; after sealing, PBST washing liquid is washed for 4 times, each time is 1min, the PBST washing liquid is dried after washing, whey is added according to the dilution ratio of 1: 160, and the PBST washing liquid acts for 120min at 37 ℃; washing with PBST washing solution for 4 times, each time for 1min, drying, diluting HRP-goat anti-pig IgA with a ratio of 1:10000, treating with 100 μ L/well, and treating at 37 deg.C for 60 min; PBST washing solution is washed for 4 times, each time is 1min, the PBST washing solution is dried by patting, each hole is 100 mu L, and TMB is reacted at room temperature of 37 ℃ in a dark place and is protected from light for 25 min; the reaction was stopped by adding 50. mu.L/well of 2mol/L sulfuric acid, and the reading was carried out at a wavelength of 630nm using a microplate reader.
When in use
Figure BDA0003006332790000041
That is, OD450nm was determined to be positive at 0.144 or more. When in use
Figure BDA0003006332790000042
Namely OD450nm is less than or equal to 0.126, the judgment is negative,
compared with the prior art, the invention has the following beneficial effects:
the invention establishes a preparation method for expressing a large amount of porcine epidemic diarrhea virus S1D fragment protein by utilizing the coding gene of the S1D fragment protein after codon optimization, the S1D fragment protein can be efficiently expressed by utilizing the preparation method, the purity is high, the biological activity is good, and the monoclonal antibody prepared by utilizing the preparation method has high titer and strong specificity; the whey IgA ELISA method established as the antigen has good repeatability, sensitivity and specificity.
Drawings
FIG. 1 shows PCR amplification of the S1D and Δ S1D genes; m: DNA molecular mass standard; 1: the S1D gene; 2: Δ S1D gene; 3: and (5) negative control.
FIG. 2 is a double restriction enzyme identification of recombinant plasmid; m: DNA molecular mass standard; 1: pET-S1D; 2: pET-. DELTA.S 1D; 3: and (5) negative control.
FIG. 3 is an SDS-PAGE analysis of S1D and Δ S1D recombinant proteins; m: protein molecular mass standard; 1 BL21(DE3) host bacteria; 2: pET-32a is unloaded; 3, inducing no pET-S1D; 4: no induction of pET-. DELTA.S 1D; 5: induction of pET-S1D; 6 induced pET-. DELTA.S 1D.
FIG. 4 shows Western blotting analysis of S1D and. DELTA.S 1D recombinant proteins; m: protein molecular mass standard; 1 BL21(DE3) host bacteria; 2: pET-32a is unloaded; 3: pET-S1D was not induced; 4: no induction of pET-. DELTA.S 1D; 5: induction of pET-S1D; 6: pET-. DELTA.S 1D was induced.
FIG. 5 shows Western blotting analysis (PEDV S protein monoclonal antibody) of S1D and. DELTA.S 1D recombinant proteins; m: protein molecular mass standard; 1: BL21(DE3) host bacteria; 2: pET-32a is unloaded; 3: pET-S1D was not induced; 4: no induction of pET-DeltaS 1D; 5: induction of pET-S1D; 6: induction of pET-DeltaS 1
FIG. 6 is an optimization of induction temperature; m is protein molecular mass standard; 1-5: the induction temperatures of the bacterial liquid containing PET-S1D are respectively 16 ℃, 22 ℃, 28 ℃, 37 ℃ and 42 ℃; 6-10: the induction temperatures of the PET-Delta S1D-containing bacterial liquid are respectively 16 ℃, 22 ℃, 28 ℃, 37 ℃ and 42 ℃.
FIG. 7 is an optimization of induction time; m: protein molecular mass standard; 1-5: the induction time of the bacterial liquid containing PET-S1D is 2h, 3h, 4h, 5h and 6h respectively; the induction time of 6-10 bacterial liquid containing PET-delta S1D is 2h, 3h, 4h, 5h and 6h respectively.
FIG. 8 is an optimization of IPTG induction concentration; m: protein molecular mass standard; 1-6: the concentration of induced IPTG in the bacterial liquid containing PET-S1D is 0.1mmol/L, 0.5mmol/L, 0.8mmol/L, 1.0mmol/L, 1.2mmol/L and 1.5mmol/L respectively; the induced IPTG concentrations of the 7-12 PET-containing Delta S1D bacteria liquid are respectively 0.1mmol/L, 0.5mmol/L, 0.8mmol/L, 1.0mmol/L, 1.2mmol/L and 1.5 mmol/L.
FIG. 9 is a solubility analysis of S1D and Δ S1D fragment proteins; m: protein molecular mass standard; 1. 7: pET-32a supernatant; 2. 8: pET-32a precipitation; 3. 9: S1D supernatant; 4. 10: S1D precipitation; 5.Δ S1D supernatant: 6. 12: Δ S1D precipitated.
FIG. 10 shows the difference between the expression levels of S1D and Δ S1D fragments; **: the protein expression quantity of the S1D and the delta S1D fragments is obviously different (p is less than 0.01 by a t test method).
FIG. 11 is a washing of inclusion bodies; m: protein molecular mass standard; 1: washing the supernatant for the first time; 2: washing the precipitate for the first time; 3: washing the supernatant for the second time; 4: washing the precipitate for the second time; 5: washing the supernatant for the third time; 6: the precipitate was washed for a third time.
FIG. 12 shows inclusion body solubilization; m: protein molecular mass standard; 1: the Δ S1D fragment proteolytically solubilized supernatant.
FIG. 13 shows dialytic renaturation of inclusion bodies; m is protein molecular mass standard; 1: the Δ S1D fragment proteolytically solubilized supernatant.
FIG. 14 is a mass spectrometry analysis of Δ S1D.
Figure 15 is mouse antibody levels after triage.
FIG. 16 shows the results of different antigen coating amounts.
FIG. 17 is a negative mouse serum assay.
FIG. 18 shows the growth status of hybridoma cells at different stages after fusion (x 100, 500 um); a: 4 d; b is the 7 th; c: and (10 d).
FIG. 19 is the antibody titer of ascites.
FIG. 20 is ascites-specific assay.
FIG. 21 shows the specificity of the reaction of ascites with the Δ S1D fragment protein detected by Western-blotting; m: protein molecular mass standard; 1: pET-32 a; 2: purified Δ S1D fragment protein.
FIG. 22 shows the specificity of the reaction of ascites with PEDV detected by Western-blotting; m: protein molecular mass standard; 1: vero cells; 2: PEDV.
FIG. 23 shows the specificity of FA for detecting ascites; a ascites: 1:500 dilution; b, ascites: 1:1000 dilution; c: ascites 1:2000 dilution; d: a positive control; e: negative control; f: blank control.
FIG. 24 shows optimal antigen coating conditions.
FIG. 25 shows the selection of the optimal blocking solution.
Fig. 26 shows the optimal closing time.
Figure 27 is the optimal whey effect time.
FIG. 28 shows the optimal enzyme-labeled secondary antibody action time.
FIG. 29 shows the dilution of the optimal enzyme-labeled secondary antibody.
FIG. 30 shows the optimal color development time of the substrate.
Fig. 31 is a determination of the critical value.
FIG. 32 is a sensitivity experiment.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 optimization of the sequence of the S1D Gene
First, experiment method
1. Optimization of S1D Gene sequence
According to the codon preference of escherichia coli, the S1D gene of the PEDV epidemic wild strain ZH02 is subjected to complete-sequence nucleotide synonymous substitution, and the optimized DNA is synthesized by Guangzhou branch of the biological engineering (Shanghai) GmbH and named as PUC-delta S1D.
2. Synthesis of primers
Specific primers are respectively designed for amplification of S1D and delta S1D genes by utilizing Oligo software according to sequences of PEDV S1D gene and optimized delta S1D gene. The 5' ends of the two pairs of primers are introduced with enzyme cutting sites BamHI and XhoI. Primer sequences are shown in Table 1 (underlined cleavage sites).
Table 1 primer sequences:
Figure BDA0003006332790000071
3. PCR amplification of S1D and Δ S1D
The S1D gene was amplified using cDNA of PEDV ZH02 strain as a template, and a PCR reaction system (25. mu.L) was: 13 mu L of 2 XTaq Plus Master Mix enzyme, 0.5 mu L of upstream primer and downstream primer, 1 mu L of DNA template and 10 mu L of deionized water. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15 s; annealing at 63.5 ℃ for 15 s; extension for 1min at 72 ℃ for 35 cycles; finally, the extension is carried out for 5min at 72 ℃.
The plasmid PUC-delta S1D is used as a template to amplify the delta S1D gene, and a PCR reaction system (25 mu L) comprises: 13 mu L of 2 XTaq Plus Master Mix enzyme, 0.5 mu L of upstream primer and downstream primer, 1 mu L of DNA template and 10 mu L of deionized water. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15 s; annealing at 67.5 ℃ for 15 s; extension for 1min at 72 ℃ for 35 cycles; finally, the extension is carried out for 5min at 72 ℃.
The PCR products are identified by 1.5% agarose gel electrophoresis, and the target gene is recovered according to the steps of the gel recovery kit.
Second, experimental results
The S1D and. DELTA.S 1D genes were amplified by PCR using the cDNA of PEDV ZH02 strain and the plasmid PUC-. DELTA.S 1D as templates and the primers shown in Table 1, and bands appeared at about 456bp (FIG. 1), corresponding to the expected sizes.
Example 2 construction of prokaryotic expression vectors pET-S1D and pET-DeltaS 1D
First, experiment method
After the S1D and delta S1D DNA fragments are subjected to double enzyme digestion of BamH I and Xho I and gel cutting recovery, the DNA fragments are respectively connected with pET-32a vectors subjected to the same enzyme digestion to construct recombinant plasmids pET-S1D and pET-delta S1D, and the recombinant plasmids are transformed into BL21(DE3) competent cells. The positive plasmids identified by PCR and double enzyme digestion are sent to the company Limited of engineering bioengineering (Shanghai) for sequencing.
Second, experimental results
The prokaryotic expression vectors pET-S1D and pET-delta S1D are subjected to double enzyme digestion, and then bands appear at 5.7kb and 456bp (figure 2), and the sequencing result shows that the bands are correct, so that recombinant strains pET-S1D and pET-delta S1D are obtained.
Example 3 prokaryotic expression and characterization of S1D and Δ S1D fragment proteins
First, experiment method
Positive recombinant strains pET-S1D and pET-delta S1D, which were correctly sequenced in example 2, were inoculated into a liquid LB medium containing ampicillin when the OD of the bacterial solution was equal600When the concentration is 0.6, IPTG with the final concentration of 1.0mmol/L is added, the induction expression is carried out at 37 ℃ and 200r/min, and meanwhile, an expression bacterium of an empty vector pET-32a is set as a negative control. After inducing for 6h, collecting mycoprotein, and detecting whether the S1D and delta S1D fragment proteins are correctly expressed by SDS-PAGE and Western-blotting.
The specific method of SDS-PAGE is as follows:
preparing SDS-polyacrylamide gel according to a method for preparing a kit by adopting Yazyme PAGE gel, and then carrying out electrophoresis, wherein the upper layer gel is 80V for 20-30 min, and the lower layer gel is 120V for 60 min. And after the electrophoresis is finished, taking down the gel, dyeing the gel for 30-40 min by using Coomassie brilliant blue dyeing liquid, repeatedly decoloring after the electrophoresis is finished, and observing the result when the color background of the gel is lighter.
The Western-blotting method comprises the following specific steps:
SDS-polyacrylamide gel is prepared according to the method of an Yazyme PAGE gel kit, and then electrophoresis is carried out, wherein the upper layer gel is 80V and 20-30 min, and the lower layer gel is 120V and 60 min. After electrophoresis is finished, the membrane is subjected to electric conversion by a conventional method, a constant current is set to be 200mA, the time is 40min, after the electrophoresis is finished, the PVDF membrane is taken down, PBST is washed for four times and then sealed for 1-2 h by 5% (w/v) skimmed milk powder, and after PBST is washed for four times, 1:1000 dilutions of His-tag-tagged murine mab with 1:1000 dilutions of the PEDV S protein murine mAb were incubated overnight at 4 deg.C, washed four times with PBST and added 1: and (3) incubating a 5000-diluted HRP-goat anti-mouse IgG secondary antibody at 37 ℃ for 1h, washing PBST for four times, adding an exposure liquid in a dark place, exposing in an FCL chemical exposure instrument for 2-5 min, and observing the result.
Second, experimental results
SDS-PAGE detection of IPTG-induced bacteria liquid shows that 1 obvious protein band at 34kDa corresponds to the expected size, and no protein band exists in the uninduced group, the host original bacteria BL21(DE3) and pET-32a, which indicates that S1D fragment protein is successfully expressed.
Western-blotting detection is carried out again on the mouse anti-His-tag monoclonal antibody and the PEDV S protein monoclonal antibody, and the result shows that the induced recombinant proteins have obvious blotting bands at 34kDa, the uninduced groups have micro-expression, and BL21(DE3) and pET-32a have no blotting bands in no-load (figure 4 and figure 5), which indicates that the target protein consistent with the expectation is obtained.
Example 4 expression level of Δ S1D fragment protein
First, effect of Induction temperature on protein expression of Δ S1D fragment
1. Experimental methods
The positive recombinant strains pET-S1D and pET-Delta S1D which were correctly sequenced in example 2 were inoculated into a liquid LB medium containing ampicillin, respectively, after the concentration of the bacterial solution reached OD600 of 0.5, IPTG was added to the final concentration of 1.0mmol/L, and the expression was induced at five induction temperatures of 42 ℃, 37 ℃, 28 ℃, 22 ℃ and 16 ℃ for 4 hours, and the expressed products were treated in the same manner as in example 3 and subjected to SDS-PAGE gel electrophoresis.
2. Results of the experiment
As shown in FIG. 6, the expressed protein amounts of the bacterial solutions containing pET-S1D and PET-DeltaS 1D at 28 ℃ are significantly higher than those at 16 ℃, 22 ℃, 37 ℃ and 42 ℃, which indicates that the optimal induced expression temperatures of the bacterial strains containing pET-S1D and PET-DeltaS 1D are 28 ℃.
Secondly, influence of induction time concentration on expression of Delta S1D fragment protein
1. Experimental methods
The positive recombinant strains pET-S1D and pET-Delta S1D which were correctly sequenced in example 2 were inoculated into a liquid LB medium containing ampicillin, respectively, after the concentration of the bacterial liquid reached OD 600-0.5, IPTG was added to the final concentration of 1.0mmol/L, and the resultant was induced at 37 ℃ for 2 hours, 3 hours, 4 hours, 5 hours, and 6 hours, respectively, and the expression products were treated in the same manner as in example 3 and subjected to SDS-PAGE gel electrophoresis.
2. Results of the experiment
As shown in FIG. 7, the expression protein amounts of the bacterial solutions containing pET-S1D and PET-DeltaS 1D were significantly higher than those of the bacterial solutions containing pET-S1D and PET-DeltaS 1D at 6h of induction, indicating that the optimal induction expression time of the bacterial strains containing pET-S1D and PET-DeltaS 1D was 6 h.
Third, the effect of IPTG induction concentration on the expression of the protein of the Delta S1D fragment
1. Experimental methods
The positive recombinant strains pET-S1D and pET-Delta S1D which were correctly sequenced in example 2 were inoculated into a liquid LB medium containing ampicillin, and after the concentration of the bacterial solution reached OD600 ═ 0.5, IPTG was added to the final concentrations of 0.1mmol/L, 0.5mmol/L, 0.8mmol/L, 1.0mmol/L, 1.2mmol/L and 1.5mmol/L, respectively, and expression was induced at 37 ℃ for 4 hours, and the expressed products were treated as in example 3 and examined by SDS-PAGE gel electrophoresis.
2. Results of the experiment
As shown in FIG. 8, the amounts of protein expressed by the bacterial solutions containing pET-S1D and PET- Δ S1D at the IPTG induction concentration of 0.1mmol/L were significantly higher than those expressed at the induction concentrations of 0.5mmol/L, 0.8mmol/L, 1.0mmol/L, 1.2mmol/L and 1.5mmol/L, indicating that the strains containing pET-S1D and PET- Δ S1D all had the optimum IPTG induction concentration of 0.1 mmol/L.
Example 5 solubility analysis of Δ S1D fragment protein and comparison of expression levels before and after sequence optimization
First, experiment method
Recombinants containing pET-32a, pET-S1D and pET-delta S1D were cultured in a large amount in a liquid LB medium containing ampicillin, respectively, after induction expression, the resulting mixture was centrifuged at 6000r/min for 15min, the supernatant was discarded, and the bacterial sludge was resuspended in PBS containing 1% (v/v) PMSF (phenylmethylsulfonyl fluoride) at 0.01mol/L and pH 7.4. Ultrasonically crushing bacteria in ice bath, centrifuging at 12000r/min for 20min, and respectively harvesting supernatant and precipitate (the precipitate is a crude inclusion body). The S1D and Δ S1D fragment proteins were analyzed by SDS-PAGE to determine whether the expressed protein was present in soluble form or in the form of inclusion bodies. Meanwhile, the difference of the expression levels of the S1D fragment protein before codon optimization and the Delta S1D fragment protein after codon optimization is analyzed after gray scanning is carried out on the expression levels by using Image J software.
Second, experimental results
SDS-PAGE (figure 9) shows that the protein expressed by pET-32a in no-load is mainly in the supernatant, the position is about 20kDa, and a small amount of protein is expressed in the sediment; the S1D and the Delta S1D fragment proteins are expressed in a small amount in cell supernatant, and exist mainly in the form of inclusion bodies. The differences of the expression levels of S1D and the protein of the Delta S1D fragment are analyzed after gray-scale scanning by using Image J software (figure 10), and the expression level of the Delta S1D of the optimized recombinant is obviously higher than that of the protein of the S1D fragment compared with that of the non-optimized recombinant, and the difference between the expression levels is obvious (p is less than 0.01).
Example 6 purification of Δ S1D fragment protein
First, experiment method
1. Washing and solubilization of Inclusion bodies
The crude inclusion bodies were added to 10ml of cold wash solution (20mmol/L Tris-HCl, pH 8.0, 0.5mol/L NaCl, 2mol/L urea, 2% Triton X-100) and stirred for 20 min. Centrifuging at 12000rpm for 25min at 4 deg.C, discarding supernatant, and collecting precipitate. Repeating the steps (washing for 2-4 h to remove membrane fragments and membrane proteins). Washing the obtained precipitate with 50mmol/L Tris-HCl for 1 time (to remove residual EDTA), centrifuging under the same centrifugation condition, and discarding supernatant to obtain the washed inclusion body. A small portion of the supernatant and pellet from each wash was analyzed by SDS-PAGE gel electrophoresis.
10ml of inclusion body-denatured solution (20mmol/L Tris-HCl pH 8.0, 0.5mol/L NaCl, 6mol/L urea, 0.2mmol/L DTT, 2% Triton X-100) was added to the above-mentioned precipitate to dissolve it, and the mixture was stirred at room temperature for 30 to 60min to dissolve it sufficiently, centrifuged at 12000rpm at 4 ℃ for 15min, and the supernatant was collected by discarding the precipitate. The supernatant was filtered through a 0.45um filter and stored at-20 ℃ while a small sample was taken for SDS-PAGE gel electrophoresis analysis.
2. Dialytic renaturation of inclusion bodies
The renaturation of the target protein is restored by renaturation buffer solution at 4 ℃ according to a gradient renaturation reduction method, and the specific method is as follows:
(1) cutting dialysis membrane with proper length (determined by dialysis protein volume)
(2) Placing the dialysis membrane in a beaker, adding appropriate amount of double distilled water, and boiling for 10min
(3) Washing the dialysis membrane with room-temperature double-distilled water for 2-3 times
(4) Clamping one end of the dialysis membrane by a clamp, adding protein, removing bubbles, clamping the other end, putting into a TGE buffer solution beaker containing a proper volume of 6M urea, and dialyzing at 4 ℃ for 4-6 h or overnight
(5) Changing TGE buffer solution of 6M urea into TGE buffer solution of 4M urea, dialyzing at 4 ℃ for 4-6 h or staying overnight
(6) Changing TGE buffer solution of 4M urea into TGE buffer solution of 2M urea, dialyzing at 4 ℃ for 4-6 h or staying overnight
(7) Changing TGE buffer solution of 2M urea into urea TGE buffer solution of 0M, dialyzing at 4 ℃ for 4-6 h or staying overnight
(8) Collecting renatured protein after dialysis, subpackaging and storing in a refrigerator at-80 ℃, taking a small part for SDS-PAGE gel electrophoresis analysis, and observing the purification effect of the protein.
3. Purified protein concentration assay (BCA)
The concentration of the purified protein obtained was determined according to the method of the BCA protein quantification kit
4. Mass spectrometric analysis of the purified product after expression
The purified expression product delta S1D fragment protein is sent to the company of Biotechnology engineering (Shanghai) for Maldi-TOF-TOF mass spectrometry identification.
Second, experimental results
The obtained crude inclusion body delta S1D fragment protein is washed for three times, the washing supernatant and the precipitate are collected each time, and the purification effect is analyzed by SDS-PAGE Coomassie staining, the result is shown in figure 11, the supernatant washed each time has some impure proteins, and the impure proteins washed for the first time are more, and after 3 times of washing, the obtained refined inclusion body has good purity and less impure proteins, which indicates that the washing method of the inclusion body can effectively remove the impure proteins.
The refined inclusion body prepared by the method is dissolved by using a dissolving buffer solution at the temperature of 4 ℃, the supernatant is collected, and after SDS-PAGE cooma staining, the purification effect of the inclusion body is analyzed, the result is shown in figure 12, the obtained Delta S1D fragment protein has high purity, and basically has no other impurity protein, which indicates that the prepared Delta S1D fragment protein can be subjected to dialysis renaturation in the next step.
Dialyzing and renaturing the obtained dissolved protein of the Delta S1D fragment of the supernatant, collecting the supernatant, analyzing the purification effect after protein renaturation after SDS-PAGE cooma staining, and obtaining a result shown in figure 13, wherein the purity of the obtained protein of the Delta S1D fragment after dialysis is good, no obvious hybrid protein appears, and the protein concentration reaches 1.0mg/mL after being determined by a BCA method, which proves that the purity and the concentration of the renatured protein are both suitable for the immunization of a mouse in the next step.
The result of Maldi-TOF-TOF mass spectrometry identification of the Delta S1D fragment protein by Biotechnology (Shanghai) corporation is shown in FIG. 14, the coverage rate of the peptide fragment is 31%, the red font is the identified peptide fragment, the total of 6 fragments, and further proves that the Delta S1D recombinant protein is PEDV S1 protein, which is consistent with the expectation.
Example 7 preparation of antibody against Δ S1D fragment protein
Immunization of BALB/c mice
1. Experimental methods
The immunization program is shown in table 2. 25 female SPF BALB/c mice of 6 weeks old, 10 positive control groups and 15 negative control groups were selected. Emulsifying the protein of the Delta S1D fragment purified in example 6 with Freund' S complete adjuvant (CFA) in equal volume, injecting the protein into the neck and back of the mouse at multiple points, and leaving the negative group of mice untreated; carrying out secondary immunization and tertiary immunization by emulsifying with equivalent volume of antigen by Freund incomplete adjuvant emulsion (IFA) at 14d and 28d respectively; detecting the titer of the mouse antibody one week after the three-immunization, and when the titer of the positive antibody reaches 1:105Then splenocytes can be taken for cell fusion, and the non-emulsified antigen is used for strengthening immunity once 3d before the fusion; blood is collected from the marginal veins of the eyes after the mice are subjected to the three-immunity, the mice are kept still for 30min in an incubator at 37 ℃, and then the mice are centrifuged for 15min at 4000r/min, and supernatant is sucked for later use.
TABLE 2 BABL/c mouse immunization procedure
Number of immunizations Time of immunization Immunization dose Immunological pathways
One need not 0d th 300 ug/piece (emulsified with equal CFA) Subcutaneous part of the back
Two exempt from 14d th 300 ug/piece (mixed emulsion with equal amount of IFA) Subcutaneous part of the back
Sanwu 28d th 300 ug/piece (mixed emulsion with equal amount of IFA) Subcutaneous part of the back
Exempt from 3d before fusion 150 ug/only (without adjuvant) Abdominal cavity
2. Results of the experiment
The results are shown in FIG. 15, and the antibody titers of 10 positive mice all reached 1: 150000 it was demonstrated that the antigen Δ S1D immunized mice had a good effect and were ready for the next cell fusion, i.e. 1 positive mouse boosted 3d before fusion.
Two, indirect ELISA method condition groping
1. Experimental methods
And (3) taking the serum of the mice 7d after the three-immunization for indirect ELISA, and optimizing the concentration of the coating antigen.
The OD of 15 negative control mice sera was measured by indirect ELISA using the Δ S1D protein as the envelope antigen450And the statistical method is used as the positive and negative judgment standard.
Coating: diluting antigen with coating solution at concentrations of 1 μ g/mL, 2 μ g/mL, 4 μ g/mL, 8 μ g/mL, 12 μ g/mL, 16 μ g/mL, 24 μ g/mL, 32 μ g/mL, adding to ELISA plate at 100 μ L/well, adsorbing at 37 deg.C for 1h, and standing at 4 deg.C overnight; washing: spin-drying the coating liquid, adding 200 μ L of washing liquid into each hole, and washing with vibrationWashing for 3 times and 30 s/time; and (3) sealing: adding sealing liquid, sealing with 100 μ L/hole in a 37 deg.C wet box for 3 hr, drying, and washing as above; sample adding: diluting serum with antibody diluent, adding diluted serum 100 μ L/hole into the above enzyme labeling plate, reacting at 37 deg.C for 60min, draining off liquid, and washing; adding a secondary antibody: diluting enzyme-labeled goat anti-mouse IgG with PBS at a concentration of 1:10000 times, adding 100 μ L/well of the enzyme-labeled plate, allowing the plate to act at 37 ℃ for 30min in a wet box, draining off the liquid in the plate, and washing the plate as above; adding a substrate: adding 100 mu L of substrate solution into each hole, and reacting for 10min at room temperature; adding a stop solution: add 50. mu. L H to each well2SO4Terminating the reaction; measuring: the absorbance value of each well was measured using two wavelengths (where OD450nm is the actual measurement wavelength and OD450nm is the reference wavelength). An indirect ELISA standard determination experiment was performed simultaneously: sera of 15 blank SPF mice were collected and negative values were determined using Δ S1D protein as the envelope antigen. And (3) measuring by using a microplate reader, wherein the detection wavelength is 450nm, the correction wavelength is 630nm, zeroing is carried out by using a blank control, and the OD value of each well is read.
Reading and result judgment: the result of the sample reading is denoted S, if
Figure BDA0003006332790000111
Namely, the test result is judged to be positive; if it is
Figure BDA0003006332790000112
Is determined to be negative if
Figure BDA0003006332790000113
Then the test is judged to be suspicious and needs to be carried out again. Wherein the content of the first and second substances,
Figure BDA0003006332790000114
mean values of the sera of the negative mice, SD standard deviation.
2. Results of the experiment
The results are shown in FIG. 16. When the antigen coating concentration is fixed, the OD450 value is gradually reduced along with the increase of the dilution multiple of positive serum; under other conditions, when the antigen concentration is lower than 12ug/mL, the OD450 value is increased along with the increasing concentration, but when the antigen concentration is higher than 12ug/mL, the OD450 value is slightly reduced and then kept constant, and meanwhile, in consideration of saving the antigen, the antigen with the concentration of 8ug/mL is used for coating in the subsequent ELISA detection.
As shown in fig. 17, the dilution in serum was calculated according to statistical methods to be 1: average value of OD450 of 15 serum at 100
Figure BDA0003006332790000121
0.022, SD 0.007,
Figure BDA0003006332790000122
when the OD450 is more than or equal to 0.043, the sample can be judged to be positive, when the OD450 is less than or equal to 0.036, the sample can be judged to be negative, and when the OD450 is between the OD450 and the sample, the sample can be judged to be suspicious; at serum dilution 1: mean value of 15 serum OD450 at 5000
Figure BDA0003006332790000123
0.014, SD 0.004,
Figure BDA0003006332790000124
when OD450 is more than or equal to 0.027, the product can be judged to be positive, when OD450 is less than or equal to 0.023, the product can be judged to be negative, and when OD450 is between the two products, the product can be judged to be suspicious.
EXAMPLE 8 preparation of monoclonal antibody hybridoma secreting Δ S1D fragment protein
First, cell fusion
1. Experimental methods
(1) Preparation of SP2/0 myeloma cells
Recovering SP2/0 myeloma cells by using a 1640 complete culture medium 4d before fusion, culturing for 1-2 generations under the conditions of 37 ℃ and 5% CO2 to recover the growth performance of the cells, and selecting cell seeds with good growth state for amplification culture; the SP2/0 cells were guaranteed to grow at least over 3 plates with a diameter of 10cm on the day of fusion.
(2) Preparation of spleen cells
The day before cell fusion, the neck of the mouse of example 7, which is over 8 weeks old and homologous with BALB/c, is dislocated and killed, the mouse is soaked in 75% alcohol for 5min, and the mouse is fixed in a dissecting plate soaked with benzalkonium bromide in an ultra-clean bench with the abdomen upward; are respectively provided withCutting two thighs, cutting forwards and backwards to a certain length, pressing the tail part, clamping fur with a pair of tweezers, tearing forwards, exposing the right spleen, clamping peritoneum at the spleen with the tweezers, cutting a small opening (only the spleen can be seen), taking a half of the spleen, clamping the spleen with the tweezers, pulling out the spleen from the small opening, and simultaneously removing fat connective tissues and the like attached to the spleen with the scissors; placing spleen in a petri dish containing 1640 (with lid, pouring culture medium before dissecting mouse), gently washing, and carefully stripping off connective tissue; transferring to another plate containing 1640, and gently squeezing spleen with penicillin bottle bottom or syringe core to fully release splenocytes; adding 5mL HAT culture medium, gently purging all splenocytes in the plate, transferring to a 50mL centrifuge tube, adding HAT culture medium to 30mL, subpackaging in 6 96-well plates with 50 μ L of each well, 37 deg.C, and 5% CO2Culturing under the condition for later use.
(3) Fusion
Taking a BABL/c mouse after 3d of boosting immunity, removing an eyeball and taking blood; preparing a spleen cell suspension; myeloma cells and splenocytes were mixed in a 1:10, transferring the mixture into a 50mL centrifugal tube, centrifuging for 10min at 1000r/m, removing supernatant, and completely sucking residual liquid by a dropper; gently stirring the precipitate with a dropper to make the precipitate loose and uniform to be pasty; fusion in 37 ℃ water bath: uniformly rotating the centrifuge tube by one hand, sucking 1mL of 50% (w/v) PEG1450 solution by the other hand by using a pipette, adding the solution along the rotating tube wall (approaching the cell as much as possible), controlling the time from the addition to the completion of the addition to be about 1min while stirring, adding 1mL of incomplete DMEM culture solution preheated to 37 ℃ by using the pipette within 1min, repeating the step for 3 times, and adding 3mL of incomplete DMEM culture solution in 1min till the solution is full of 25 mL (note that the operation is gentle at this time, the addition is performed while rotating, and the cell is not stirred as much as possible); standing at 37 deg.C for 10min, centrifuging at 800r/m for 10min, and removing supernatant; adding 10mL HAT culture solution, and slightly blowing, sucking and uniformly mixing; according to the number of the used 96-hole culture plates, 15mL of liquid for one 96-hole culture plate is used for calculating and supplementing HAT culture solution to the required amount; the fused cell suspension was added to a 96-well plate containing feeder cells at 150. mu.L/well, 37 ℃ and 5% CO2Culturing in an incubator.
2. Results of the experiment
The positive mice were boosted once 3d before fusion, and splenocytes from the positive mice were taken out on the day of fusion and mixed with myeloma cells SP2/0 according to a ratio of 10: 1, and adding 50% (w/v) PEG1450 to complete the fusion of the two. The under-the-lens results of cell fusion are shown in fig. 18, and the hybridoma cells successfully fused at the 4d (a) begin to divide and proliferate to form a cell mass consisting of 20-30 cells; the hybridoma cells have good activity and rapid proliferation in the 7d (b) and form a cell cluster consisting of 100-200 cells; and 10d, (c) the hybridoma cells grow to 1/5-1/2 of the bottom of the hole, and the cell culture supernatant can be sucked to detect the antibody by using the established indirect ELISA method.
Screening of Positive hybridoma
1. Experimental methods
(1) Initial detection
And detecting the antibody in the fusion cell supernatant by using an established indirect ELISA method, eliminating negative holes during primary detection, and performing secondary detection on positive holes and suspicious positive holes.
(2) Screening for false positives
And (3) screening false positive of the detected positive by taking the purified empty pET-32a vector expression product as an antigen, and removing the monoclonal antibody aiming at the antigen site of the non-target protein on the vector.
(3) Cloning of Positive hybridoma cells
Cloning is carried out on the positive hybridoma cell wells obtained by screening by a limiting dilution method to obtain cell strains capable of stably secreting the antibody.
Cloning: 130 cells were taken and placed in 6.5mL of HT medium containing feeder cells, i.e.20 cells/mL, 100. mu.L/well plus A, B, C rows, 2 cells per well. The remaining 2.9mL of cell suspension was supplemented with 2.9mL of HT medium containing feeder cells, with 10 cells/mL, and 100. mu.L/well in D, E, F triplicate, for 1 cell per well. The remaining 2mL of cell suspension was supplemented with 2.2mL of HT medium containing feeder cells, 5 cells/mL, 100. mu.L/well, and G, H two rows of 0.5 cells per well. After culturing for 4-5 days, small cell clones can be seen on an inverted microscope, and 200. mu.L/well of complete culture solution is supplemented. And after subcloning for 9d, observing the growth condition of cells in the culture plate, marking the number of cell colonies, detecting by using an indirect ELISA (enzyme-linked immunosorbent assay) method when the supernatant of a cell hole becomes yellowish or yellow, and subcloning the cells with higher reading and the cell clone number in the hole of 1-2 again until obtaining the single-cell clone stably secreting the monoclonal antibody.
(4) Expanding culture
While subcloning, the excess hybridoma cells were transferred to a 24-well cell culture plate for expansion, i.e., primary wells.
(5) Positive hybridoma cell cryopreservation
And (4) performing amplification culture on all the remaining cells except the subclones of the cells of the positive hole to be detected before subcloning, performing cryopreservation, and storing the original hole. Finally, the positive hybridoma cell strain which is 100% positive is obtained and is also subjected to expanded culture and frozen storage. Blowing down hybridoma cells during freezing, centrifuging to remove supernatant, resuspending the precipitate with DMEM culture solution containing 10% (v/v) DMSO and 50% (v/v) calf serum, and subpackaging in freezing tubes (1 mL/tube) with cell density not less than 2 × 106Marking, placing at 4 ℃ for 30min, at-20 ℃ for 1h, at-70 ℃ for 1h, and transferring into a liquid nitrogen tank for preservation.
(6) Cell resuscitation
Thawing after freezing for one week to detect cell survival rate and loss of antibody secretion characteristic, taking out the frozen tube from the liquid nitrogen tank during thawing, quickly placing in a 37 deg.C water bath, gently shaking for thawing within 1min, taking out from the water bath after thawing, placing on an ice box, centrifuging at 1000r/min for 5min, transferring to a super clean bench, aseptically opening the frozen tube, transferring the cell suspension to a cell bottle, suspending in preheated HT complete culture solution, placing at 37 deg.C and 5% CO2Culturing in an incubator.
2. Results of the experiment
8ug/mL of Delta S1D fragment protein is used as an antigen coated ELISA plate, an established indirect ELISA method is adopted, and according to the negative, blank and positive control results obtained by the judgment standard and the growth state of fused hybridoma cells, the OD450 of a hole to be detected is judged to be preliminary positive, the OD450 of the hole to be detected is not less than 0.1, the OD450 of the hole to be detected is not less than 0.07, the hole to be detected is preliminary weak positive, and the OD450 of the hole to be detected is less than 0.07, and the hole to be detected is negative. And finally obtaining the monoclonal cell strain which stably secretes the delta S1D antibody through subsequent false positive screening and 3-5 times of cloning experiments of positive hybridoma cells.
Preparation of ascites of mice and detection of potency
1. Experimental methods
After the screened cell strain is cultured stably, the ascites is prepared by adopting an in vivo induction method for the patient with good growth state and strong antibody secretion capacity. Freund's incomplete adjuvant was used at 500. mu.L/peritoneal BALB/c mice, 7 days later. The cells were aspirated from the flask and counted, adjusted to 1X 106And (4) washing the mice per mL by DMEM or normal saline for 2-3 times, and injecting the treated mice into the abdominal cavity. And after about 10-15 days, extracting ascites when the abdomen of the mouse is obviously enlarged. The harvested mouse ascites fluid was diluted with antibody according to 1: 1000. the antibody titer of ascites was measured by an indirect ELISA method using a negative control in which the antibody titer of ascites was diluted at 1:10000, 1:30000, 1:50000, 1:200000, 1:300000, 1:500000, 1:700000, and 1: 1000000.
2. Results of the experiment
8ug/mL of delta S1D fragment protein was used as an antigen coated ELISA plate, ascites of different dilutions was used as a primary antibody, HRP-goat anti-mouse IgG was used as a secondary antibody, and a negative and a control were set simultaneously, and detection was performed by an indirect ELISA method, and the results are shown in FIG. 19. The monoclonal antibody is prepared by the method that 1: after 1000000 dilution, the antibody is still weak positive, which proves that the prepared ascites antibody has higher titer.
Fourth, specific detection
1. Experimental methods
PEDV, PRRSV, TGEV, CSFV, PDCoV, SADS, PEDV delta S1D fragment protein, PEDV N protein and pET-32a no-load protein are taken as antigen coated enzyme label plates, positive and negative serum controls are set at the same time, and the specificity of the obtained ascites is detected by an indirect ELISA method.
2. Results of the experiment
The results are shown in FIG. 20. The ascites can react positively with PEDV virus and purified delta S1D fragment protein, and react negatively with PEDV N protein, 32a no-load protein, PRRSV, TGEV, CSFV, PDCoV and 5 viruses of SADs, and the ascites prepared by the method is consistent with the expectation.
Fifth, Western blot analysis
1. Experimental methods
Respectively taking purified delta S1D, pET-32a, PEDV and Vero cell protein as antigens to prepare the ascites 1: a Western-blotting test was performed using 1000 dilutions as primary antibodies and HRP-labeled goat anti-mouse IgG as secondary antibodies. PEDV was infected to Vero cells at an MOI of 1.0, and 48h after infection, the cells were harvested and protein samples were prepared. Meanwhile, the purified delta S1D and pET-32a empty carrier protein are used as antigens. The prepared ascites is used as a primary antibody, and goat anti-mouse IgG marked by HRP is used as a secondary antibody to carry out Western-blotting test, and the reactivity of the ascites with delta S1D and PEDV S protein is detected.
2. Results of the experiment
The results showed that ascites fluid was not reactive with empty carrier protein pET-32a, and strongly cleaved with the Δ S1D fragment protein, resulting in a specific band at 34kDa (FIG. 21). Ascites did not respond to Vero cells and strongly produced a 180kDa specific band with PEDV infected Vero cells (FIG. 22). The results were in agreement with expectations, indicating that the monoclonal antibody specificity in ascites was good.
Six, indirect Immunofluorescence (IFA)
1. Experimental methods
After Vero cells of a 96-well plate grow into a monolayer, PEDV is inoculated. After 20h, the medium was discarded and pre-cooled 80% (v/v) acetone was added 150. mu.L/well and fixed at-20 ℃ for 30 min. After 3 washes with sterile PBS, add 1:500, 1: 1000. 1:50 μ L/well of Δ S1D mab diluted in 2000PBS, incubated at 37 ℃ for 2h, set to 1:1000 PBS diluted PEDV N mab as positive control, 1: the serum of a negative mouse diluted by 1000 is used as a negative control; no antibody was added as a blank. Sterilized PBS was washed 3 times, and a 1:500 PBS dilution of 50. mu.L/well FITC-labeled secondary goat anti-mouse IgG antibody was added and incubated at 37 ℃ for 1 h. The sterilized PBS was washed 3 times, and 100. mu.L of PBS remained in the wells, and the results were observed by a fluorescence inverted microscope.
2. Results of the experiment
As shown in FIG. 23, 3 dilutions of ascites and the positive control group showed specific green fluorescence signals, while the blank and negative groups showed no green fluorescence signals, further indicating that the prepared monoclonal antibody specifically recognizes the S1D fragment protein of PEDV.
Example 9 establishment of whey IgA ELISA detection method
Selection of optimal antigen coating concentration and whey dilution
1. Experimental methods
The Δ S1D protein purified in example 6 was diluted to a final concentration of 16. mu.g/mL, 8. mu.g/mL, 4. mu.g/mL, 2. mu.g/mL, 1. mu.g/mL using phosphate buffered saline as a diluent by a matrix titration method, two columns were coated at each concentration, 100. mu.L was coated in each well, and 1 hour at 37 ℃ was followed by overnight coating at 4 ℃. PBST washing solution for 4 times, each time for 1min, after washing and dried, PBST washing solution containing 0.05% (v/v) Tween-20 0.01mol/L PBS, pH 7.4. PBST containing 0.5% (v/v) bovine whey albumin (BSA) was added at 200. mu.L/well, blocked at 37 ℃ for 2h, and washed as above. And (5) drying after washing. Respectively mixing the negative whey and the positive whey according to the ratio of 1:20, 1: 40, 1: 80 and 1: 160; 1: after 320 five dilutions were added to ELISA plates at 100. mu.L/well and allowed to react at 37 ℃ for 70min, and then removed and washed as above. After dried, adding HRP-labeled goat anti-pig IgA enzyme-labeled secondary antibody diluted by 1:10000 times, reacting at 37 ℃ for 40min at a concentration of 100 mu L/hole, and washing as above. After washing, TMB substrate solution is added, 100 mu L/hole, and reaction is carried out for 10min at 37 ℃ in a dark place. The reaction was stopped by adding 50. mu.L/well of 2mol/L sulfuric acid, and the reading was carried out at a wavelength of 630nm using a microplate reader. The antigen concentration and whey dilution of the positive whey with OD value close to 1 and the largest P/N value are used as the optimal antigen coating concentration and whey dilution.
2. Results of the experiment
As shown in Table 3, the OD450nm value of the positive latex was close to 1.0 and the P/N value was the greatest at a dilution of whey at a dilution factor of 1: 160 at an antigen coating concentration of 4. mu.g/mL. Therefore, the optimal antigen coating concentration was selected to be 4. mu.g/mL, and the optimal whey dilution factor was 1: 160.
TABLE 3 determination of optimal antigen coating concentration and whey dilution
Figure BDA0003006332790000151
Figure BDA0003006332790000161
Second, selection of optimal coating time of antigen
1. Experimental methods
The purified delta S1D protein of example 6 was coated on the ELISA plate at the optimal antigen coating concentration (4. mu.g/mL) under 4 different conditions; overnight at 4 ℃; 4 ℃ overnight, and 37 ℃ for 1 h; incubating at 37 ℃ for 1 h; incubating at 37 ℃ for 1h, and standing overnight at 4 ℃; after the coating is completed, other steps are the same as above. The P/N values of the positive and negative whey of each group were compared to select the optimal coating time.
2. Results of the experiment
The optimal coating concentration was used for antigen coating, and the antigen was coated at different times, as shown in FIG. 24, in which P/N was the largest at 4 ℃ overnight, and the optimal coating conditions for antigen were determined at 4 ℃ overnight.
Selection of optimal confining liquid
1. Experimental methods
The purified Δ S1D protein from example 6 was coated on an ELISA plate at optimal antigen coating concentration (4 μ g/mL) and optimal coating time (overnight at 4 ℃), blocking solution set 5, 5% (w/v) PBST for BSA, 10% (w/v) PBST for BSA, 5% (w/v) PBST for Skim Milk Powder (SMP), 10% (w/v) PBST for Skim Milk Powder (SMP), and 1 × protein-free blocking solution, respectively. Add 100. mu.L of blocking solution to each well and block for 2h at 37 ℃. After blocking was complete, whey was added at the optimal dilution of 1: 160 and the other steps were as above. The P/N values of the positive and negative whey of each group were compared to select the best blocking solution.
2. Results of the experiment
As shown in FIG. 25, the P/N value was the largest when PBST of 5% (w/v) BSA was used as a blocking solution. The PBST of 5% (w/v) BSA was determined as the optimal blocking solution.
Selection of optimum closure time
1. Experimental methods
The purified Δ S1D protein of example 6 was coated on the microplate at the optimum antigen coating concentration (4. mu.g/mL) and the optimum coating time (overnight at 4 ℃), the microplate was sealed with 5% (w/v) PBST containing BSA as the blocking solution, and the plate was sealed at 37 ℃ for 30min, 60min, 90min, and 120min, after sealing, whey was added at the determined optimum dilution of 1: 160, and the other steps were as above. The P/N values of the positive and negative whey of each group were compared to select the appropriate blocking time.
2. Results of the experiment
As a result, as shown in FIG. 26, the P/N value was the largest when the sample was sealed at 37 ℃ for 90 min. The optimal sealing time was determined to be 90 min.
Selection of optimal primary antibody action time
1. Experimental methods
The Δ S1D protein purified in example 6 was coated on the microplate at the optimum antigen coating concentration (4. mu.g/mL) and the optimum coating time (overnight at 4 ℃), and the microplate was blocked with 5% BSA in PBST as blocking solution for 90 min. After sealing, whey was added at the determined optimal dilution of 1: 160 and allowed to act at 37 ℃ for 30min, 60min, 90min, and 120min, respectively, with the other steps being as above. Comparing P/N values of the negative whey and the positive whey of each group to select proper whey acting time.
2. Results of the experiment
As shown in FIG. 27, the P/N value was the highest when whey acted for 120 min. The optimal reaction time for whey was determined to be 120 min.
Sixthly, selection of optimal action time of enzyme-labeled antibody
1. Experimental methods
The Δ S1D protein purified in example 6 was coated on the microplate at the optimum antigen coating concentration (4. mu.g/mL) and the optimum coating time (overnight at 4 ℃), and the microplate was blocked with 5% (w/v) PBST containing BSA as the blocking solution for 90 min. After blocking, whey was added at the determined optimal dilution 1: 160, allowed to act at 37 ℃ for 120min, and the enzyme-labeled antibody was added at 1:10000 after the enzyme label plate is added, the reaction is carried out for 30min, 45min, 60min and 90min at 37 ℃, other steps are the same as the above, and the P/N value of each group of negative and positive whey is compared to select the proper action time of the enzyme-labeled antibody.
2. Results of the experiment
As shown in FIG. 28, the P/N value was the highest at 60min after the enzyme-labeled antibody had been allowed to act thereon. The optimal reaction time of the enzyme-labeled antibody is determined to be 60 min.
Seventhly, determining the optimal acting concentration of enzyme-labeled secondary antibody
1. Experimental methods
The Δ S1D protein purified in example 6 was coated on the microplate at the optimum antigen coating concentration (4. mu.g/mL) and the optimum coating time (overnight at 4 ℃), and the microplate was blocked with 5% (w/v) PBST containing BSA as the blocking solution for 90 min. After sealing, adding whey according to the determined optimal dilution of 1: 160, acting at 37 ℃ for 120min, and respectively carrying out 1:5000, 1:10000 and 1: 15000. and 1, 20000 gradient dilution, action at 37 ℃ for 40min, and other steps are the same as above, and the P/N values of the negative whey and the positive whey of each group are compared to select proper action time of the enzyme-labeled antibody.
2. Results of the experiment
As shown in FIG. 29, the enzyme-labeled secondary antibody showed the highest P/N value when diluted 1: 10000. Determining the optimal action dilution of the enzyme-labeled secondary antibody as 1: 10000.
eighthly, determining the color development time of the substrate
1. Experimental methods
The Δ S1D protein purified in example 6 was coated on the microplate at the optimum antigen coating concentration (4. mu.g/mL) and the optimum coating time (overnight at 4 ℃), and the microplate was blocked with 5% (w/v) PBST containing BSA as the blocking solution for 90 min. And after sealing, adding whey according to the determined optimal dilution of 1: 160, acting at 37 ℃ for 120min, respectively diluting HRP-goat anti-pig IgA at 1:10000 for 60min at 37 ℃, acting TMB at room temperature in a dark place for 10min, 15min, 20min and 25min, and comparing the P/N values of the negative whey and the positive whey to select the substrate color development time in the same other steps.
2. Results of the experiment
As a result, as shown in FIG. 30, the P/N value was the highest when the substrate (TMB) color development time was 25 min. The optimal color development time of the substrate is determined to be 25 min.
Ninthly, determining a critical value
1. Experimental methods
Taking 10 parts of PE produced by Shanghai enzyme-linked biotechnology limited companyDetecting whey OD450nm value of negative whey identified by DV IgA ELISA kit, and calculating average value of sample OD450nm values
Figure BDA0003006332790000182
And standard deviation SD, according to statistical principles, of the sample
Figure BDA0003006332790000185
If the result is positive,
Figure BDA0003006332790000184
if so, the result is negative, and if the result is between the two, the result is suspicious.
The established detection conditions were:
the Δ S1D protein purified in example 6 was coated on the microplate at the optimum antigen coating concentration (4. mu.g/mL) and the optimum coating time (overnight at 4 ℃), and the microplate was blocked with 5% (w/v) BSA in PBST as blocking solution for 90 min. Sealing, adding whey according to determined optimal dilution of 1: 160, acting at 37 deg.C for 120min, diluting HRP-goat anti-pig IgA at 1:10000 respectively, acting at 37 deg.C for 60min, and acting TMB at room temperature in dark place for 25min, and performing other steps as above.
2. Results of the experiment
The results are shown in FIG. 31. The average of 10 samples was calculated to be 0.090 with a standard deviation of 0.018. According to the statistical principle, when
Figure BDA0003006332790000186
That is, OD450nm was determined to be positive at 0.144 or more. When in use
Figure BDA0003006332790000187
That is, OD450nm ≦ 0.126 was judged negative, and between the two, it was suspicious.
Example 10 reproducibility of whey IgA ELISA detection method
One, batch repeat test
1. Experimental methods
Coating an ELISA plate with the protein of the DeltaS 1D fragment purified in the same batch in example 6, taking 4 positive whey samples and 1 negative whey sample, repeating 5 holes for each sample to perform ELISA detection, performing detection according to the detection method established in example 9, and calculating the average value, standard deviation and coefficient of variation of 5 repetitions of each sample according to the result.
2. Results of the experiment
The results are shown in table 4, and the coefficient of variation of the repeated tests in the batch is less than 10%, which indicates that the established indirect ELISA method has good repeatability.
4 in-batch repeat experiments
Figure BDA0003006332790000181
Repeat test between two batches
1. Experimental methods
And (3) coating 5 ELISA plates of different batches with the purified protein antigen of the same batch, taking 4 positive whey samples and 1 negative whey sample for ELISA detection, and calculating the average value, standard deviation and variation coefficient of each sample on the ELISA plates of 5 different batches according to the result.
2. Results of the experiment
The results are shown in table 5, and the coefficient of variation of the batch-to-batch repeated tests is less than 10%, which indicates that the established indirect ELISA method has good stability.
TABLE 5 repeated experiments between batches
Figure BDA0003006332790000191
Example 11 sensitivity of whey IgAELISA detection method
First, experiment method
Taking 4 parts of positive whey samples and 1 part of negative whey samples to respectively carry out 6 gradient dilutions in the ratio of 1: 40, 1: 80, 1: 160, 1: 320, 1: 640 and 1: 1280, detecting according to the indirect ELISA method established in the example 9, and carrying out sensitivity analysis on the results.
Second, experimental results
The results are shown in FIG. 32. When the positive whey is diluted to 160 times, 4 positive whey samples can still detect positive, which proves that the ELISA detection method established in the experiment has good sensitivity.
Example 12 comparison of whey IgAELISA assay with commercially available products
First, experiment method
The whey samples of 133 examined sows with clinical diarrhea were examined by the IgA antibody detection method established in example 9 and the PEDV IgAELISA kit produced by shanghai enzyme-linked biotechnology limited, respectively, and the coincidence rate of the two detection methods, i.e., the ratio of the sum of the number of positive or negative samples detected by the two methods at the same time to the total number of samples, was calculated.
Second, experimental results
The results of the detection are shown in FIG. 6. The sensitivity of the method constructed in the research is 85.4%, the specificity is 80.0%, and the coincidence rate of the two methods is 85.0%.
TABLE 6
Figure BDA0003006332790000192
Figure BDA0003006332790000201
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Sequence listing
<110> southern China university of agriculture
<120> encoding gene of porcine epidemic diarrhea virus S1D fragment protein and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 438
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgaccctgg atgtgtgcac caaatatacc atttatggct ttaaaggcga aggcattatt 60
accctgacca atagcagctt tctggcgggc gtgtattata ccagcgatag cggccagctg 120
ctggcgttta aaaatgtgac cagcggcgcg gtgtatagcg tgaccccgtg cagctttagc 180
gaacaggcgg cgtatgtgga tgatgatatt gtgggcgtga ttagcagcct gagcagcagc 240
acctttaata gcacccgcga actgccgggc tttttttatc atagcaatga tggcagcaat 300
tgcaccgaac cggtgctggt gtatagcaat attggcgtgt gcaaaagcgg cagcattggc 360
tatgtgccga gccagagcgg ccaggtgaaa attgcgccga tggtgaccgg caatattagc 420
attccgacca attttagc 438

Claims (10)

1. The encoding gene of the S1D fragment protein of the porcine epidemic diarrhea virus is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.
2. A recombinant vector comprising the coding gene of claim 1 linked thereto.
3. An engineered bacterium comprising the recombinant vector of claim 2.
4. A preparation method for expressing a large amount of S1D fragment protein of porcine epidemic diarrhea virus is characterized in that engineering bacteria containing recombinant plasmids carrying the coding genes of claim 1 are used for induction expression; taking and crushing thalli, carrying out solid-liquid separation and taking precipitate to obtain an inclusion body crude product; washing the inclusion body crude product, dissolving, dialyzing and renaturing to obtain the inclusion body.
5. The method according to claim 4, wherein the bacterial liquid OD is600When the value is 0.6-1.0, IPTG with the final concentration of 0.1-1.5 mmol/L is added, and the induction expression is carried out for 2-6 hours at the temperature of 16-42 ℃.
6. The method of claim 4, wherein the crude inclusion body is washed with an inclusion body washing solution and Tris-HCl in sequence, wherein the inclusion body washing solution comprises Tris-HCl, NaCl, urea, and Triton X-100; dissolving with inclusion body denaturation dissolving solution, wherein the inclusion body denaturation dissolving solution contains Tris-HCl, NaCl, urea, Triton X-100 and DTT.
7. The process of claim 4, wherein the dialysis is performed sequentially with TGE buffer containing urea in decreasing concentration.
8. Use of the coding gene of claim 1, the recombinant vector of claim 2 and/or the engineered bacterium of claim 3 for preparing the S1D fragment protein of porcine epidemic diarrhea virus.
9. The porcine epidemic diarrhea virus S1D fragment protein prepared by the preparation method of any one of claims 4 to 7.
10. The use of the porcine epidemic diarrhea virus S1D fragment protein of claim 9 in the preparation of a porcine epidemic diarrhea virus detection kit and/or an anti-porcine epidemic diarrhea virus antibody.
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