CN107664697B

CN107664697B - Expression vector and preparation method thereof, PEDV-S1 protein and indirect ELISA detection kit containing protein

Info

Publication number: CN107664697B
Application number: CN201710704142.XA
Authority: CN
Inventors: 黄耀伟; 雷喜梅; 赵鹏伟
Original assignee: Hangzhou Jie Meng Animal Epidemic Prevention And Control Technology Co Ltd; Zhejiang University ZJU
Current assignee: Hangzhou Jie Meng Animal Epidemic Prevention And Control Technology Co Ltd; Zhejiang University ZJU
Priority date: 2017-08-16
Filing date: 2017-08-16
Publication date: 2020-02-07
Anticipated expiration: 2037-08-16
Also published as: CN107664697A

Abstract

The invention provides an expression vector and a preparation method thereof, PEDV-S1 protein and an indirect ELISA detection kit containing the protein, and relates to the technical field of molecular biology. The expression vector for expressing the porcine epidemic diarrhea virus S1 protein is constructed by a eukaryotic expression vector or an insect expression vector, and the eukaryotic expression vector for expressing the porcine epidemic diarrhea virus S1 protein is used for constructing the porcine epidemic diarrhea virus S1 protein, so that the expression level is high, the purification is easy, and the antigenicity is good. In addition, the ELISA detection kit established by taking the S1 protein provided by the invention as the coating antigen can accurately detect the anti-porcine epidemic diarrhea virus antibody in a clinical sample. Meanwhile, the kit provided by the invention has the advantages of strong specificity, high sensitivity, simplicity, rapidness, simple preparation method and low cost, provides a new choice for diagnosis, general survey and immune monitoring of PEDV, and has a good clinical application prospect.

Description

Expression vector and preparation method thereof, PEDV-S1 protein and indirect ELISA detection kit containing protein

Technical Field

The invention relates to the technical field of molecular biology, in particular to an expression vector and a preparation method thereof, PEDV-S1 protein and an indirect ELISA detection kit containing the protein.

Background

Porcine Epidemic Diarrheia (PED) is caused by Porcine Epidemic Diarrheic Virus (PEDV), a highly-contact enteric infectious disease characterized mainly by diarrhea, vomiting, dehydration and high lethality to suckling piglets. By 2017, PED has spread to most pig raising countries around the world, causing huge economic loss to the pig industry all over the world, and becoming a problem which is commonly concerned and needs to be solved by the pig raising industry all over the world.

Currently, the clinical diagnosis method for PEDV is mainly ELISA, i.e. enzyme linked immunosorbent assay, which detects antigen or specific antibody to evaluate the PEDV vaccine condition in pig farms, serological diagnosis of infected pigs, etc.

PEDV is a single-stranded positive-stranded RNA virus, capped at the 5 'end and a poly-A tail at the 3' end. The genome comprises 5 '-UTR, 3' -UTR and 7 ORFs. Of these 7 ORFs, 4 encode structural proteins: spike protein (S), small envelope protein (E), envelope protein (M) and nucleocapsid protein (N), the remaining 3 encode non-structural proteins: replicase 1a, replicase 1b and ORF3 protein. Wherein the S protein primarily mediates binding of the virus to the receptor, inducing production of neutralizing antibodies; at present, ELISA diagnostic kits based on PEDV M and N, S, ORF3 proteins and PEDV holovirus are reported, but the preparation of antigens, particularly S protein antigens, is mainly based on prokaryotic expression system for expression and purification, and the expression products can not effectively simulate the natural conformation of virus proteins, so that the kit is relatively time-consuming and labor-consuming, low in expression amount, and low in purity and specificity.

Therefore, it is important to develop a preparation method with high expression, high purity and strong specificity for the S protein antigen, and an indirect ELISA kit using the protein as a coating antigen.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the present invention is to provide an expression vector for expressing the porcine epidemic diarrhea virus S1 protein, the second purpose of the present invention is to provide a preparation method of the expression vector, and the third purpose of the present invention is to provide the porcine epidemic diarrhea virus S1 protein prepared by the method, so as to alleviate the technical problems of time and labor waste, low expression quantity, purity and specificity of S1 protein expressed and purified based on a prokaryotic expression system in the prior art.

The fourth purpose of the invention is to provide an indirect ELISA detection kit for porcine epidemic diarrhea virus S1 protein, so as to alleviate the technical problems of low specificity and poor sensitivity of the ELISA diagnostic kit based on PEDV-S protein in the prior art.

The expression vector for expressing the porcine epidemic diarrhea virus S1 protein is based on a eukaryotic expression vector, and is integrated with a PEDV-S1 gene optimized by codons to obtain the eukaryotic expression vector for expressing the porcine epidemic diarrhea virus S1 protein;

wherein the sequence of the PEDV-S1 gene optimized by the codon is shown in SEQ ID NO. 1.

The invention also provides a preparation method of the expression vector, which comprises the following steps:

artificially synthesizing a PEDV-S1 gene containing EcoRI and XhoI enzyme cutting sites by taking the PEDV-S1 gene optimized by the codon as a template, and integrating the PEDV-S1 gene containing the EcoRI and XhoI enzyme cutting sites into EcoRI/XhoI sites of a eukaryotic expression vector pFase-hLgG 1-Fc1 to obtain a pFase-PEDV-S1-Fc eukaryotic expression vector for expressing the porcine epidemic diarrhea virus S1 protein.

Further, the artificial synthesis is carried out on the PEDV-S1 gene by PCR amplification by taking PEDV-S1-F1 and PEDV-S1-R1 as a primer combination;

wherein, the PEDV-S1-F1 has a sequence shown in SEQ ID NO.2, and the PEDV-S1-R1 has a sequence shown in SEQ ID NO. 3.

The invention also provides a porcine epidemic diarrhea virus S1 protein prepared by the expression vector.

The invention also provides an expression vector for expressing the porcine epidemic diarrhea virus S1 protein, which is based on an insect expression vector and is integrated with a PEDV-S1 gene optimized by codons to obtain the insect expression vector for expressing the porcine epidemic diarrhea virus S1 protein;

wherein the sequence of the PEDV-S1 gene optimized by the codon is shown in SEQ ID NO. 7.

artificially synthesizing a PEDV-S1 gene containing BamHI and XhoI enzyme cutting sites by using the PEDV-S1 gene optimized by the codon as a template, and integrating the PEDV-S1 gene containing the BamHI and XhoI enzyme cutting sites into the BamHI/XhoI sites of an insect expression vector pFastbac1 to obtain a pFastbac1-PEDV-S1 insect expression vector for expressing the S1 protein of the porcine epidemic diarrhea virus.

Further, the artificial synthesis uses PEDV-S1-BamHIF and PEDV-S1-XhoIR-6His as primer combination to carry out PCR amplification on the PEDV-S1 gene;

wherein, the PEDV-S1-BamHIF has a sequence shown in SEQ ID NO.4, and the PEDV-S1-XhoIR-6His has a sequence shown in SEQ ID NO. 5.

In addition, the invention also provides an indirect ELISA detection kit for the porcine epidemic diarrhea virus S1 protein, wherein the coating antigen in the indirect ELISA detection kit is the porcine epidemic diarrhea virus S1 protein.

Further, the kit also comprises a washing solution, a coating solution, a blocking solution, a sample diluent, a substrate, a stop solution, a negative control, a positive control and a secondary antibody.

The expression vector for expressing the porcine epidemic diarrhea virus S1 protein is constructed on the basis of a eukaryotic expression vector and is integrated with a PEDV-S1 gene optimized by codons. The eukaryotic expression vector for expressing the porcine epidemic diarrhea virus S1 protein is used for constructing the porcine epidemic diarrhea virus S1 protein, and has the advantages of high expression level, easy purification and good antigenicity. The invention also provides an expression vector for expressing the porcine epidemic diarrhea virus S1 protein, which is constructed on the basis of an insect expression vector and is integrated with the PEDV-S1 gene optimized by codons. The insect expression vector for expressing the porcine epidemic diarrhea virus S1 protein provided by the invention is used for constructing the porcine epidemic diarrhea virus S1 protein, and has the advantages of high expression level, easiness in purification and good antigenicity. In addition, the ELISA detection kit established by taking the porcine epidemic diarrhea virus S1 protein prepared by the expression vector provided by the invention as the coating antigen can accurately detect the anti-porcine epidemic diarrhea virus antibody in a clinical sample. Meanwhile, the kit provided by the invention has the advantages of strong specificity, high sensitivity, simplicity, rapidness, simple preparation method and low cost, provides a new choice for diagnosis, general survey and immune monitoring of PEDV, and has a good clinical application prospect.

Drawings

FIG. 1 is a diagram showing the result of identification of the recombinant pFase-PEDV-S1-Fc plasmid provided in example 1 of the present invention;

FIG. 2 is a Western blot detection result chart of the PEDV-S1-Fc1 recombinant protein provided in example 1 of the present invention;

FIG. 3 is a diagram showing the results of PCR identification of white spots on KTG plates according to example 2 of the present invention;

FIG. 4A is a diagram showing the result of cell morphology 0h after the recombinant baculovirus plasmid provided in example 2 of the present invention transfects sf9 insect cells;

FIG. 4B is a diagram showing the results of cell morphology of sf9 insect cells transfected with the recombinant baculovirus plasmid provided in example 2 of the present invention for 72 h;

FIG. 5A is a graph showing the results of IFA detection of uninfected recombinant baculovirus sf9 cells provided in example 2 of the present invention;

FIG. 5B is a graph showing the results of IFA detection of infected recombinant baculovirus sf9 cells provided in example 2 of the present invention;

FIG. 6 is a Western blot detection result chart of the recombinant baculovirus provided in example 2 of the present invention;

FIG. 7 is a graph showing the results of detection of anti-PEDV IgG antibody in clinical pig serum by indirect ELISA according to Experimental example 9 of the present invention;

FIG. 8 is a graph showing the results of detection of anti-PEDV IgA antibodies in clinical pig serum by indirect ELISA according to Experimental example 9 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides an expression vector for expressing porcine epidemic diarrhea virus S1 protein, which is constructed on the basis of a eukaryotic expression vector and is integrated with a PEDV-S1 gene optimized by codons to obtain the eukaryotic expression vector for expressing the porcine epidemic diarrhea virus S1 protein;

wherein, the sequence of the PEDV-S1 gene optimized by the codon is shown in SEQ ID NO. 1; the amino acid sequence of the PEDV-S1 protein is shown in SEQ ID NO. 6.

The S1 gene sequence of the eukaryotic expression vector for expressing the porcine epidemic diarrhea virus S1 protein is combined with the eukaryotic expression vector to carry out codon optimization, so that compared with the prior art, the eukaryotic expression vector for expressing the porcine epidemic diarrhea virus S1 protein provided by the invention has higher expression efficiency.

the PEDV-S1 gene is subjected to PCR amplification by taking a codon-optimized PEDV-S1 gene (SEQ ID NO.1) as a template and PEDV-S1-F1 (SEQ ID NO.2) and PEDV-S1-R1(SEQ ID NO.3) as primer combinations to obtain the PEDV-S1 gene with two ends respectively containing EcoRI and XhoI enzyme cutting sites, and the PEDV-S1 gene containing the EcoRI and XhoI enzyme cutting sites is cloned into the EcoRI/XhoI site of a eukaryotic expression vector pFase-hLgG 1-Fc1 to obtain a pFase-PEDV-S1-Fc eukaryotic expression vector for expressing the porcine epidemic diarrhea virus S1 protein.

In the invention, the method for preparing the PEDV-S1 protein by using the expression vector comprises the following steps: transfecting the eukaryotic expression vector into 293T cells, harvesting cell supernatants, and performing affinity purification to obtain the PEDV-S1 protein.

The invention also provides a porcine epidemic diarrhea virus S1 protein prepared by applying the eukaryotic expression vector.

The eukaryotic expression system provided by the invention is used for expression and purification, and the expression product of the porcine epidemic diarrhea virus S1 protein can effectively simulate the natural conformation of the virus protein, so that the eukaryotic expression system is time-saving and labor-saving, and has high expression quantity, high purity and strong specificity.

The invention also provides an expression vector for expressing the porcine epidemic diarrhea virus S1 protein, which is constructed on the basis of an insect expression vector and is integrated with a PEDV-S1 gene optimized by codons to obtain the insect expression vector for expressing the porcine epidemic diarrhea virus S1 protein.

Wherein, the sequence of the PEDV-S1 gene optimized by the codon is shown in SEQ ID NO. 7.

At present, an insect cell expression system is rarely used in China, and the insect cell expression system provided by the invention can efficiently express the S1 protein by combining with an optimized S1 sequence, namely, the expression level is high, the purity is higher, and the purification is convenient.

the PEDV-S1 gene optimized by codon is used as a template, the PEDV-S1-BamHIF (SEQ ID NO.4) and the PEDV-S1-XhoIR-6His (SEQ ID NO.5) are used as primer combinations to carry out PCR amplification on the PEDV-S1 gene to obtain the PEDV-S1 gene of which both ends respectively comprise BamHI and XhoI enzyme cutting sites, the PEDV-S1 gene comprising the BamHI and XhoI enzyme cutting sites is cloned into the BamHI/XhoI site of an insect expression vector pFastgab 1 to obtain a pFastgab 1-PEDV-S1 insect expression vector for expressing the porcine epidemic diarrhea virus S1 protein.

The method for preparing the PEDV-S1 protein by using the expression vector comprises the steps of transfecting the eukaryotic expression vector into a DH5 α competent cell for amplification and plasmid extraction, adding target plasmid DNA into a DH10Bac competent cell, selecting correct clone to transfect an sf9 cell to obtain baculovirus, infecting the sf9 cell after the baculovirus is subjected to amplification culture, harvesting cell supernatant, and obtaining the PEDV-S1 protein after affinity purification.

The invention also provides a porcine epidemic diarrhea virus S1 protein prepared by the insect expression vector.

The expression purification is carried out based on the insect expression system provided by the invention, and the expression product of the porcine epidemic diarrhea virus S1 protein can effectively simulate the natural conformation of the virus protein, so that the insect expression system is time-saving and labor-saving, and has high expression quantity, high purity and strong specificity.

In addition, the invention also provides an indirect ELISA detection kit for the porcine epidemic diarrhea virus S1 protein, wherein the coating antigen is the porcine epidemic diarrhea virus S1 protein obtained by expression and purification based on the eukaryotic expression system provided by the invention and/or the porcine epidemic diarrhea virus S1 protein obtained by expression and purification based on the insect expression system provided by the invention.

In the invention, the kit further comprises a washing solution, a coating solution, a confining solution, a sample diluent, a substrate, a stop solution, a negative control, a positive control and a secondary antibody.

Wherein, the washing solution is PBST (PBS added with 0.05 percent Tween-20 pH-7.4), the content is (20mL multiplied by 20 times) multiplied by 1 bottle; coating liquid is 0.05M Na₂CO₃-NaHCO₃The pH value of the solution is 9.6, and the content of the solution is (10mL multiplied by 10 times) multiplied by 1 bottle; the confining liquid is 5% skimmed milk powder, and is diluted by PBST (0.05% Tween-20 pH-7.4), the content is (10mL multiplied by 10) multiplied by 1 bottle; the sample diluent is a confining liquid and is used for diluting a serum sample (pig serum), and the content of the sample diluent is (10mL multiplied by 10 times) multiplied by 1 bottle; the substrate is a single-component TMB color development liquid (Beijing Soilebao), and the content is 10mL multiplied by 1 bottle; the stop solution is 2M concentrated H₂SO₄6mL multiplied by 1 bottle; the negative control is PEDV negative pig serum with the content of 0.5mL multiplied by 1 bottle; the positive control is PEDV positive pig serum with the content of 0.5mL multiplied by 1 bottle; the second antibody is goat anti-pig IgA second antibody, conjugated by horseradish peroxidase, and numbered as follows: PA184625, Saimer Feishale, 6mL × 1 bottle; goat anti-pig IgG secondary antibody, horseradish peroxidase conjugate, number: PA184628, Saimer Feishale, 6mL × 1 bottles.

The operation program of the indirect ELISA kit provided by the invention comprises the following steps:

step (a): adding the coating antigen into an enzyme label plate in an amount of 0.44 ng/hole/100 mu L for coating the enzyme label plate;

step (b): adding a sealing liquid into the coated enzyme label plate for sealing;

step (c): adding the diluted sample, the negative control and the positive control into an enzyme label plate, adding a secondary antibody after incubation, and continuing incubation;

step (d): adding substrate into the enzyme label plate with sample, reacting in dark place, adding stop solution to stop reaction, measuring with enzyme label instrument, and recording OD of each well₄₅₀A value;

a step (e): judging the result, namely calculating an S/P value according to the OD value of each hole, and judging the result to be positive when the S/P value is more than or equal to the S/P average value X +3 SD; when the S/P value of the sample is less than X +2SD, judging the sample to be negative; when the S/P value of the sample is more than or equal to X +2SD, the sample is suspicious; and (4) retesting is required, and the retesting result S/P value is more than or equal to X +2SD and is positive, otherwise, the retesting result S/P value is negative.

Note: S/P ═ Sample-Negative)/(Positive-Negative)

The porcine epidemic diarrhea virus S1 protein provided by the invention has high purity and good antigenicity; the indirect ELISA detection kit established by taking the S1 protein as the coating antigen can accurately detect the anti-porcine epidemic diarrhea virus IgG and IgA antibodies; meanwhile, the antigen amount of the S1 protein used by the kit can be as low as 0.44 ng/well/100 mu L, namely 44ng of antigen is needed for one 96-well plate; that is, 1mg of antigen may be sufficient to coat 22700 pieces of 96-well plates. Meanwhile, only 1 million yuan RMB is needed for producing 1mg of antigen by applying the method provided by the invention. The cost is far lower than that of most of the current diagnostic kits for detecting the PEDV antibody in China. In conclusion, the kit has the advantages of strong specificity, high sensitivity, simplicity, rapidness, simple preparation method, long storage life, low cost and good clinical application prospect.

Example 1 construction of eukaryotic expression System and preparation of PEDV-S1 protein

Construction of S1 Gene expression vector

1.1 primer design

A pair of specific primers is designed and synthesized by referring to PEDV (GenBank accession No. KU558701) strain in GenBank, wherein PEDV-S1-F1 is shown as SEQ ID NO.2, PEDV-S1-R1 is shown as SEQ ID NO.3, and the primers are synthesized by Huada biology Limited.

1.2 construction of PEDV-S1-Fc recombinant plasmid

And (3) taking the codon-optimized recombinant plasmid for expressing the PEDV-S as a template (constructed and stored in the laboratory), carrying out double enzyme digestion on the amplified PEDV-S1 and the amplified pFase-hLgG 1-Fc1 by EcoRI and XhoI respectively, and connecting to construct a pFase-PEDV-S1-Fc expression plasmid. The positive identified recombinant plasmid (as shown in FIG. 1) was sent to Huada biology for sequence determination, and the determined nucleotide sequence and the encoded amino acid sequence were analyzed and aligned by software to check the correctness of the reading frame.

1.3 eukaryotic expression and purification of PEDV-S1 protein

1.3.1 the correct pFase-PEDV-S1-Fc expression plasmid was identified and transiently transfected into 293T cells, after 72 hours the cell culture supernatant was harvested and Western blot was used to detect S1 protein expression (FIG. 2, lane A).

1.3.2 affinity purification of secretory PEDV-S1-Fc fusion Protein with HiTrap Protein A Sepharose High Performance column (GE healthcare; catalog No. 17-0402-01); the BCA kit (Byunnan) is used for determining the concentration of the purified PEDV-S1-Fc protein; then, carrying out enzyme digestion on the purified PEDV-S1-Fc1 protein by TEV protease to obtain PEDV-S1 protein without an Fc tag; after Western blot detection (shown in lane B of FIG. 2), the concentration of the purified PEDV-S1 protein was determined using BCA kit (Biyun day); subpackaging and freezing at-80 ℃. As can be seen from FIG. 2, the protein has high concentration and good purity, and can be used as a subsequent ELISA antigen.

The PEDV recombinant S1 eukaryotic protein obtained in the embodiment has good expression, high purity and good reactivity.

Example 2 construction of insect expression System and preparation of PEDV-S1 protein

Construction of pFastbac1-PEDV-S1 recombinant plasmid

1.1 primer design

Through sequence alignment analysis, primers are designed by using biological software DNAstar and Primer Premier, and respective enzyme cutting sites are added to the primers, wherein PEDV-S1-BamHIF is shown as SEQ ID NO.4, and PEDV-S1-XhoIR-6His is shown as SEQ ID NO. 5.

1.2 cloning of fragments of interest

Amplifying a target sequence PEDV-S1 containing a preset enzyme cutting site by using conventional PCR

(1) The following reagents were added to a 200 μ L PCR reaction tube:

reactants	Dosage (mu L)
		Q5High-Fidelity 2×Master Mix	25
10μM Forward Primer	2.5
		10μM Reverse Primer	2.5
Template DNA	1
		Nuclease-Free Water	19

(2) After mixing well, the mixture was centrifuged at low speed for a short time.

(3) Setting reaction parameters:

(4) electrophoresis was performed on a 1% agarose gel at 120V for 30 min.

1.3 construction of recombinant plasmid by double digestion

After recovering the target gene and the vector amplified by PCR using a gel recovery kit, they were digested with BamHI and XhoI and recovered, and ligated with T4 ligase at 16 ℃ overnight.

1.4 transformation and characterization

(1) Adding all the ligation products into DH5 α competent cells, and carrying out ice bath for 30 min;

(2) taking out, thermally shocking at 42 deg.C for 90s, and immediately ice-cooling for 2 min;

(3) adding 600 μ L LB culture medium, placing at 37 deg.C, 220rpm incubator, shaking for 1 h;

(4) taking 150 mu L of LB plate coated with ampicillin, placing the plate in a constant-temperature incubator at 37 ℃, and carrying out inverted culture for 16-18 h;

(5) PCR identification is carried out on the extracted plasmid by using a small quantity of kit extracted plasmid (AxyPrep plasmid DNA small quantity kit), and positive Boshang sequencing is carried out.

1.5 swivel base

(1) Transposition reaction

1) Adding target plasmid DNA into DH10Bac competent cells, and carrying out ice bath for 30 min;

2) heat shock at 42 deg.C for 1min, immediately taking out and ice bath for 2 min;

3) adding 800 μ L LB culture medium, shaking at 37 deg.C and 220rpm for 4 min;

4) preparing a bacterial culture plate (50 mu g/mL kana, 7 mu g/mL genta, 10 mu g/mL tet, 100 mu g/mLBluo-gal, 40 mu g/mL IPTG), coating the diluted product on the plate, culturing the plate in an inverted mode at the temperature of 37 ℃ for 48 hours, and picking white spots for analysis;

(2) identification

1) Selecting a plurality of clones to be cultured in LB liquid culture medium containing 50 mu g/mL kana, 7 mu g/mL genta and 10 mu g/mL tet;

2) extraction of PEDV-S1-pFastBac DNA (AxyPrep plasmid DNA minikit);

3) and (3) performing PCR identification on PEDV-S1-pFastBac DNA, amplifying a target gene by using a specific primer, and verifying that the plasmid is positive.

1.6 transfection of Sf9 cells with recombinant plasmids

(1) Culturing mature cells with sf 900-III serum-free medium at 27 ℃ for at least 1h before transfection;

(2) preparation of transfection reagent mixture:

1) add 3. mu.g of plasmid to 100. mu.L of serum-free sf 900-III medium;

2) turning Cellffectin Reagent (Invitrogen company) upside down for 5-10 times, mixing uniformly, taking 6 mu L, and adding 100 mu L of serum-free sf 900-III culture medium;

3) mixing the two solutions (the total volume should be less than 220 μ L), lightly mixing, and standing at room temperature for 35 min;

(3) removing the cell culture medium, and adding a new serum-free sf 900-III culture medium;

(4) adding 1.8mL of sf 900-III culture medium into the mixture, reversing, uniformly mixing, and adding into a cell culture solution;

(5) no CO at 27 ℃₂Culturing for 5h in the incubator, sucking the culture medium away, and changing into sf 900-III culture medium;

(6) continued at 27 ℃ without CO₂The culture box is used for culturing, and the cytopathic condition is observed during the culture;

(7) after 72-96 h, if the cells have obvious lesions (become large and round), collecting the supernatant as the P0 virus.

(8) A small number of P0 samples were processed and WB was used to verify the expression of the target protein.

1.7 amplification culture of baculovirus

(1) Preparing sf9 cells in advance, paving the cells in a 6-well plate, and culturing for 1h at room temperature;

(2) observing whether the cells adhere to the wall;

(3) adding proper amount of P1 virus, infecting with MOI 0.1;

(4)27 ℃ and no CO₂Culturing for 72-96 h in an incubator, collecting supernatant, and centrifuging to obtain P2 virus;

(5) repeating the steps to obtain P3, P4 virus and the like;

(6) titre detection by IFA.

1.8 baculovirus expression of purified proteins

(1)1×10⁷The above titers of virus infected sf9 cells (6T 75 cell culture flasks) without CO₂Culturing at 27 ℃ for 72-96 h;

(2) collecting supernatant, respectively loading into a 50mL sterile centrifuge tube, centrifuging at 6000rpm at 4 ℃ for 10min, and collecting supernatant for later use;

(3) purification of proteins by nickel column

a) Taking 1mL of nickel sepharose gel affinity column, and washing the purification column on the column by using distilled water with 3-5 times volume;

b) equilibrating the purification column with 5 volumes of binding buffer (10mM PBS, 10mM imidazole) at a flow rate of 1 mL/min;

c) injecting the sample into a purification column, and repeating for 1-2 times;

d) gradient eluting with 20mM,40mM,60mM,80mM,100mM,250mM,500mM imidazole solution, collecting eluate in tubes;

(4) performing SDS-PAGE protein electrophoresis and WB to determine the target protein;

(5) carrying out protein concentration on the eluent by using a protein displacement tube and displacing imidazole in the eluent;

(6) protein concentration was measured by BCA method.

2. Results

2.1 identification of recombinant plasmids

Positive recombinant plasmid pFastbac1-PEDV-S1 with correct sequencing is transformed into DH10Bac competent cells, and the white spots on the KTG plates are identified by PCR with specific primers (PEDV-S1-BamHIF and PEDV-S1-XhoIR-6 His). The results are shown in FIG. 3, which indicates that the recombinant plasmid was successfully constructed initially.

2.2 recombinant baculovirus detection

Recombinant baculovirus plasmids were transfected into sf9 insect cells. Morphological changes of insect cells before and after transfection were observed, and basically no obvious change was observed 48h before infection, and after 72h, cells became large and round, vacuolated, and a small amount of cells were broken and floated, as shown in FIGS. 4A and 4B. The virus infection liquid is collected, the titer of the virus is determined by IFA, and the virus is identified by using the specific His monoclonal antibody, the result is shown in figure 5A and figure 5B (200 ×), and as can be seen from figure 5A and figure 5B, the insect expression system can efficiently express the PEDV specific S1 protein.

2.3 WB identification of recombinant baculoviruses

A small amount of first-generation infected cells were collected, treated with MLB to obtain the supernatant and precipitate of the infected virus, and subjected to denaturation treatment such as centrifugal boiling, followed by WB analysis. As shown in FIG. 6, the results are shown in FIG. 6, wherein 1 is infected cell lysis supernatant, 2 is infected cell lysis precipitate, and 3 is negative control.

Example 3 procedure for Indirect ELISA method

(1) Enzyme label plate coating

PEDV-S1 protein provided in example 1 and/or example 2 of the present invention was used as an antigen coating buffer (Na having a pH of about 9.6)₂CO₃-NaHCO₃Solution) to 0.44 ng/hole/100 muL, adding an enzyme label plate, 100 muL/hole, incubating in a 37 ℃ wet box (namely keeping the incubation humidity at 65-70%) for 1h, then taking out overnight at 4 ℃ (12-14 h), discarding liquid in the hole, washing by PBST for 3 times, 3min each time, and patting dry;

(2) sealing of

Adding a sealing solution (300 mu L/hole) into the enzyme label plate, and incubating for 90min in a 37 ℃ wet box (namely, keeping the incubation humidity at 65-70%). Taking out, discarding the blocking solution, washing with PBST for 3 times, 3min each time, and drying;

(3) sample application

Adding the sample diluted by the confining liquid 1:100 into an enzyme label plate, simultaneously adding a negative control sample and a positive control sample, 100 mu L/hole, and incubating for 1h at 37 ℃ in a wet box. Taking out and discarding liquid in the hole, washing for 3 times by PBST (PBST), 3min each time, and patting to dry;

(4) adding a second antibody

Adding a secondary antibody diluted by a sealing solution 1:10,000 into an enzyme label plate, incubating for lh at a temperature of 37 ℃ in a wet box at a concentration of 100 mu L/hole, taking out the secondary antibody and discarding liquid in the hole, washing for 5 times by PBST (Poly-beta-phenylenebenzobisthiazole), 3min each time, and patting dry;

(5) adding substrate

Adding single-component TMB color development liquid into an enzyme label plate, carrying out a light-shielding reaction for 10-15min in a 37 ℃ wet box at 100 mu L/hole;

(6) termination of the reaction

Adding 2M concentrated H into the ELISA plate₂SO₄Stopping solution, 50 μ L/well, measuring with enzyme-labeling instrument within 15min after adding, and recording OD450 value of each well;

(7) interpretation of results

Calculating an S/P value according to the OD value of each hole, and judging the hole to be positive when the S/P value is more than or equal to the S/P average value X +3 SD; when the S/P value of the sample is less than X +2SD, judging the sample to be negative; when the S/P value of the sample is more than or equal to X +2SD, the sample is suspicious; and (4) retesting is required, and the retesting result S/P value is more than or equal to X +2SD and is positive, otherwise, the retesting result S/P value is negative.

Note: S/P ═ Sample-Negative)/(Positive-Negative)

Experimental example 1 optimization of antigen coating concentration and serum dilution ratio

And determining the optimal working concentration of the recombinant protein and the optimal dilution multiple of the serum to be detected by a square matrix titration method. The purified S1 protein was diluted with the coating solution to a concentration of 1.375. mu.g/mL, 0.68. mu.g/mL, 0.34. mu.g/mL, 0.172. mu.g/mL, 0.086. mu.g/mL, 0.043. mu.g/mL, 0.021. mu.g/mL, 0.001. mu.g/mL, and the microplate was coated at 100. mu.L/well. PEDV negative and positive sera were diluted at 1:100, 1:200, 1:400 and 1:800 fold ratio, respectively, each serum dilution corresponding to 8 antigen coating concentrations. Horse radish peroxidase-labeled goat anti-pig IgG was diluted 10,000-fold. According to the procedure of the indirect ELISA method provided in example 3 of the present invention, the OD450 reading of each well was measured by a microplate reader, and the P/N (positive/negative) value was calculated.

The result of the square matrix titration method shows that: when the concentration of the coating antigen is 0.043 mug/mL and the optimal dilution multiple of the serum to be detected is 1:100, the P/N value obtained by detection is as high as 13.495, and the optimal detection effect is achieved. Therefore, the optimal working concentration of the antigen was 0.043. mu.g/mL, and the optimal dilution factor of the serum to be tested was 1:100 (Table 3).

TABLE 3 optimal coating concentration of PEDV-S1 protein antigen and optimal dilution of positive serum

Note: (+) denotes positive serum, and (-) denotes negative serum

Experimental example 2 optimization of working concentration of enzyme-labeled Secondary antibody

The ELISA plate is coated with the optimal working concentration of the recombinant protein, the plate is closed, negative serum and positive serum with the optimal dilution times are added, the enzyme-labeled secondary antibody is diluted according to the proportion multiple ratio of 1:1000, 1:2000, 1:4000, 1:8000 and 1:10,000, the known PEDV negative and positive serum is detected according to the operation procedure of the indirect ELISA method provided by the embodiment 3 of the invention, an enzyme-labeling instrument is used for measuring the OD450 value of each hole, and the P/N value is calculated so as to determine the optimal dilution of the enzyme-labeled secondary antibody.

And coating the ELISA plate with the antigen with the optimal coating concentration, adding the negative and positive serum with the optimal dilution times for reaction, and performing gradient dilution on the secondary antibody. The results show that the optimal secondary antibody dilution is 1:10,000:

TABLE 4 determination of optimal dilution factor of enzyme-labeled Secondary antibody

Experimental example 3 selection of blocking conditions

ELISA detection was performed by incubation with a 37 ℃ wet cassette for 1h, overnight at 4 ℃ after incubation, and overnight at 4 ℃ directly, respectively, and evaluated according to P/N values.

The antigen was coated according to the conditions determined above, and the results showed that the optimal blocking conditions were 37 ℃ for 1h and then overnight at 4 ℃ (Table 5)

TABLE 5 determination of optimal blocking conditions

According to the indirect ELISA principle, the optimal antigen coating concentration, the serum dilution factor, the enzyme-labeled secondary antibody dilution factor and the optimal blocking solution are determined, and an indirect ELISA program of the S1 protein is established.

Experimental example 4 determination of negative and positive judgment criteria by Indirect ELISA

30 portions of pig negative serum are taken, detection is carried out by an established indirect ELISA method, each sample is repeated for 2 times, the OD450 value is determined, the S/P value is calculated according to the OD value of each hole, and the average value is taken. According to the statistical principle, the average value (X) of S/P values of 30 negative sera and 3 times of Standard Deviation (SD) are the critical values of negative and positive sera; (Sample-Negative)/(Positive-Negative); when the S/P value of the sample is more than or equal to the S/P average value X +3SD, judging the sample to be positive; when the S/P value of the sample is less than X +2SD, judging the sample to be negative; when the S/P value of the sample is more than or equal to X +2SD, the sample is suspicious; and (4) retesting is required, and the retesting result S/P value is more than or equal to X +2SD and is positive, otherwise, the retesting result S/P value is negative.

Detecting 30 parts of pig negative serum by using an indirect ELISA method established in the experiment, and calculating the average value X and standard deviation SD of the negative serum according to the OD450 value;

for anti-PEDV IgG antibody detection: x +2SD is 0.150, and X +3SD is 0.184. Therefore, the indirect ELISA for detecting anti-PEDV IgG antibody in serum by PEDV-S1 protein has the following criteria: when the OD450 value is more than 0.184, the sample can be judged to be positive, when the OD450 value is less than 0.150, the sample can be judged to be negative, and when the OD450 value is between the two values, the sample is a suspicious sample and needs to be retested; if the new value is still not more than 0.184, the result is negative, otherwise, the result is positive.

For anti-PEDV IgA antibody detection: x +2SD is 0.144, and X +3SD is 0.192. Therefore, the indirect ELISA for detecting anti-PEDV IgA antibodies in serum by PEDV-S1 protein has the following criteria: when the OD450 value is more than 0.192, the sample can be judged to be positive, when the OD450 value is less than 0.144, the sample can be judged to be negative, and when the OD450 value is between the two values, the sample is a suspicious sample and needs to be retested; if the new value is still not more than 0.192, the result is negative, otherwise, the result is positive.

Experimental example 5 specific detection

According to the indirect ELISA method established by the experiment, standard positive serum and pig negative serum of TGEV, PDCOV and PRRSV are respectively detected under the same condition, and positive serum and negative serum of PEDV and blank control are set at the same time. OD450 was measured, and the results were evaluated.

The indirect ELISA detection method constructed by the experiment is used for respectively detecting standard positive serum and pig negative serum of TGEV, PDCOV, PEDV and PRRSV under the same condition, and the result shows that the OD450 values of the other sera are less than 0.1 except the PEDV positive serum, which indicates that the constructed indirect ELISA detection method does not have a serological cross reaction (see table 6) and has good specificity.

TABLE 6 specificity test

Envelope antigens	TGEV	PDCOV	PRRSV	PEDV
					anti-PEDV positive serum	0.327	0.313	0.432	1.236
anti-PEDV negative serum	0.069	0.058	0.059	0.065
					P/N	8.99	5.07	5.53	15.17

Experimental example 6 sensitivity test

PEDV positive serum is diluted by 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400 and 1:12800 times, and the titer of the PEDV positive serum is detected by an established indirect ELISA method.

PEDV positive serum is taken to be diluted in a multiple proportion, and the titer of the PEDV positive serum is detected by an established indirect ELISA method, wherein the minimum detectable titer is 1:6400, and the sensitivity is high.

Experimental example 7 repeatability test

(one) in-batch repeatability test

The same batch of plates was coated with the protein PEDV-S1 antigen, and three positive sera were diluted appropriately and tested in 4 replicates at different times according to the formula: the coefficient of variation (CV%) was [ standard deviation (S)/average (X) ] × 100%, and the intra-batch coefficient of variation was calculated to determine the intra-batch reproducibility.

As shown by table 7: the variation coefficients of 3 positive serums are 2.1-3.8% and are less than 5%, which shows that the variation degree is smaller and the repeatability is good when the same batch of ELISA plates are used for detecting the same sample.

TABLE 7 results of in-batch repeatability tests

(II) test of repeatability between batches

Coating PEDV-S1 protein antigen with different batches of plates, diluting three positive serums properly, repeating the test for 4 batches at the same time, according to the formula: the coefficient of variation (CV%) was [ standard deviation (S)/average (X) ] × 100%, and the batch-to-batch coefficient of variation was calculated to determine the batch-to-batch reproducibility.

When ELISA plates of different batches are used for detecting 3 positive serums, the variation coefficient is 2.2% -3.8%, and is less than 10%, which shows that the variation degree is small when ELISA plates of different batches are used for detecting the same sample, and the repeatability is good.

TABLE 8 results of the repeatability tests between batches

Experimental example 8 shelf life test

And (3) coating the ELISA plate according to the optimal antigen coating concentration, storing at 4 ℃ after sealing, detecting known positive and negative samples in one week, two weeks and four weeks respectively, and determining the storage period of the kit according to the change of OD 450.

The results (Table 9) show that the P/N values decreased with longer storage time, but were still higher, i.e.the samples were still effectively tested after one month storage at 4 ℃ after blocking.

TABLE 9 shelf life test results

Experimental example 9 clinical application of Indirect ELISA

The indirect ELISA method established in the research is used for detecting the PEDV IgG antibody in 951 serum samples of pigs with diarrhea symptoms in 24 provinces of pig farms, namely, 24 pig farms, collected from Shandong, Henan, Jiangxi, Hunan, Jiangsu, Heilongjiang and Zhejiang in 2015-2016 in the laboratory, and the result is shown in FIG. 7, which indicates that the kit can effectively detect the PEDV IgG antibody in clinical samples.

The indirect ELISA method established in the research is used for detecting the existence of the anti-PEDV IgA antibody in part of clinical serum from the source, and the result is shown in figure 8, which shows that the kit can effectively detect the anti-PEDV IgA antibody in a clinical sample.

In conclusion, the experimental results show that the ELISA detection kit consisting of the PEDV-S1 protein can accurately, conveniently, quickly and specifically detect the anti-porcine epidemic diarrhea virus antibody in clinical samples.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Zhejiang university

<120> expression vector and preparation method thereof, PEDV-S1 protein and indirect ELISA detection kit containing protein

<160>7

<170>PatentIn version 3.5

<210>1

<211>2475

<212>DNA

<213> Artificial sequence

<400>1

atgaagtctc tcacatactt ctggctgttc ctgccagtgc tgtccacact gtccctgcct 60

caggacgtga caagatgcag cgctaacaca aacttccgca gattcttctc taagttcaac 120

gtgcaggccc ccgccgtggt ggtgctgggg ggatacctgc ctattgggga gaaccagggg 180

gtgaacagca catggtactg cgccggaagg cacccaaccg cttccggggt gcacggaatt 240

ttcgtgtctc acattcgcgg cgggcacgga ttcgagattg gaattagcca agagccattc 300

gacccatctg gataccagct gtacctccac aaggccacca acggaaacac aaacgctacc 360

gctagactga gaatttgcca gttcccaagc attaagacac tcgggccaac cgctaacaac 420

gacgtgacca ccggaagaaa ctgcctgttc aacaaggcta tccccgccca catgtccgag 480

cactctgtgg tgggaattac atgggacaac gacagagtga cagtgttcag cgacaagata 540

tactacttct acttcaagaa cgactggagt agggtggcta ccaagtgcta caacagcggc 600

ggatgcgcta tgcaatatgt gtacgagcct acatactaca tgctgaacgt gacatccgcc 660

ggggaggacg gaatctctta ccagccttgc accgctaact gcattgggta cgccgctaac 720

gtgttcgcca ccgagcctaa cggccacatc cccgagggat tctccttcaa caactggttc 780

ctcctgagca acgactctac actggtgcac ggcaaggtgg tgtctaacca gccactcctg 840

gtgaactgcc tcctcgctat tcctaagata tatggcctcg ggcagttctt ctctttcaac 900

cagaccattg acggggtgtg caacggagcc gctgtgcaga gggcccccga ggctctgagg 960

ttcaacatta acgacacatc tgtgatcctc gccgagggat ctattgtgct ccacaccgcc 1020

ctgggaacaa acttctcctt cgtgtgctct aactctaccg agccccacct ggctaccttc 1080

gctatcccac tcggggctat ccaggtgcca tactactgct tcctgaaggt ggacacatac 1140

aactctacag tgtacaagtt cctcgccgtg ctccccccta cagtgagaga gattgtgatt 1200

acaaagtacg gcgacgttta tgtgaacggg ttcggatacc tccacctcgg gctcctggac 1260

gccgtgacaa ttaacttcac cgggcacgga accgacgacg acgtgtctgg gttctggaca 1320

atcgcaagca ccaacttcgt ggacgccctg attgaggtgc agggcacagc tattcagcgg 1380

atactgtact gcgacgaccc agtgtctcag ctcaagtgct ctcaggtggc tttcgacctc 1440

gacgacgggt tctacccaat tagcagtcgt aacctcctgt cccacgagca gcctatttcc 1500

ttcgtggccc tcccatcttt caacgaccac tctttcgtga acatcacagt gagcgcatcg 1560

ttcggcgggc actccggcgc taacctgatc gcctcggaca ccacaattaa cggattctct 1620

tctttctgcg tggacacccg gcagttcacc attagcctgt tctacaacgt gacaaactct 1680

tacggatatg tatctaactc ccaggactct aactgccctt tcacactcca gtccgtgaac 1740

gactacctgt ctttcagcaa gttctgcgtg tccacctctc tgctggcctc cgcttgcaca 1800

attgacctgt tcggataccc cgagttcgga agcggagtga agttcacatc cctgtacttc 1860

cagttcacaa agggagaatt gattaccgga accccaaagc cacttgaagg tgtgaccgac 1920

gtgtctttca tgaccctcga cgtgtgcacc aagtacacca tatacggatt caagggggag 1980

ggaattatta cactgaccaa ctctagcttc ctggccgggg tgtactacac atctgacagc 2040

gggcagctcc tggctttcaa gaacgtgaca tctggggctg tgtactctgt gaccccttgc 2100

tctttcagcg agcaggccgc ctatgtcgat gacgacattg tgggagtgat tagcagcctg 2160

tctaacagca ccttcaacag cacacgcgaa cttccgggct tcttctacca ctctaacgac 2220

ggatctaact gcacagagcc agtgctggtg tactctaaca ttggggtgtg caagtccggg 2280

tctatcggat acgttccatc ccagtccggg caggtgaaga ttgcccctac agtgaccgga 2340

aacattagca ttcctaccaa cttcagcatg tccatcagaa ccgagtactt acagctgtac 2400

aacacccccg tgtctgtgga ctgcgcaacc tatgtctgca acggaaattc aagggagaac 2460

ctgtacttcc agagc 2475

<210>2

<211>37

<212>DNA

<213> Artificial sequence

<400>2

ccggaattcg ccaccatgaa gtctctcaca tacttct 37

<210>3

<211>53

<212>DNA

<213> Artificial sequence

<400>3

ccgctcgaga tatcgctctg gaagtacagg ttctcccttg aatttccgtt gca 53

<210>4

<211>25

<212>DNA

<213> Artificial sequence

<400>4

gtcggatcca tgaagtcttt aacct 25

<210>5

<211>61

<212>DNA

<213> Artificial sequence

<400>5

ccgctcgagt cagtgatgat gatgatgatg agaaataaca cccacttttc aacttcgcca 60

t 61

<210>6

<211>825

<212>PRT

<213> Porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus)

<400>6

Met Lys Ser Leu Thr Tyr Phe Trp Leu Phe Leu Pro Val Leu Ser Thr

1 5 10 15

Leu Ser Leu Pro Gln Asp Val Thr Arg Cys Ser Ala Asn Thr Asn Phe

20 25 30

Arg Arg Phe Phe Ser Lys Phe Asn Val Gln Ala Pro Ala Val Val Val

35 40 45

Leu Gly Gly Tyr Leu Pro Ile Gly Glu Asn Gln Gly Val Asn Ser Thr

50 55 60

Trp Tyr Cys Ala Gly Arg His Pro Thr Ala Ser Gly Val His Gly Ile

65 70 75 80

Phe Val Ser His Ile Arg Gly Gly His Gly Phe Glu Ile Gly Ile Ser

85 90 95

Gln Glu Pro Phe Asp Pro Ser Gly Tyr Gln Leu Tyr Leu His Lys Ala

100 105 110

Thr Asn Gly Asn Thr Asn Ala Thr Ala Arg Leu Arg Ile Cys Gln Phe

115 120 125

Pro Ser Ile Lys Thr Leu Gly Pro Thr Ala Asn Asn Asp Val Thr Thr

130 135 140

Gly Arg Asn Cys Leu Phe Asn Lys Ala Ile Pro Ala His Met Ser Glu

145 150 155 160

His Ser Val Val Gly Ile Thr Trp Asp Asn Asp Arg Val Thr Val Phe

165 170 175

Ser Asp Lys Ile Tyr Tyr Phe Tyr Phe Lys Asn Asp Trp Ser Arg Val

180 185 190

Ala Thr Lys Cys Tyr Asn Ser Gly Gly Cys Ala Met Gln Tyr Val Tyr

195 200 205

Glu Pro Thr Tyr Tyr Met Leu Asn Val Thr Ser Ala Gly Glu Asp Gly

210 215 220

Ile Ser Tyr Gln Pro Cys Thr Ala Asn Cys Ile Gly Tyr Ala Ala Asn

225 230 235 240

Val Phe Ala Thr Glu Pro Asn Gly His Ile Pro Glu Gly Phe Ser Phe

245 250 255

Asn Asn Trp Phe Leu Leu Ser Asn Asp Ser Thr Leu Val His Gly Lys

260 265 270

Val Val Ser Asn Gln Pro Leu Leu Val Asn Cys Leu Leu Ala Ile Pro

275 280 285

Lys Ile Tyr Gly Leu Gly Gln Phe Phe Ser Phe Asn Gln Thr Ile Asp

290 295 300

Gly Val Cys Asn Gly Ala Ala Val Gln Arg Ala Pro Glu Ala Leu Arg

305 310 315 320

Phe Asn Ile Asn Asp Thr Ser Val Ile Leu Ala Glu Gly Ser Ile Val

325 330 335

Leu His Thr Ala Leu Gly Thr Asn Phe Ser Phe Val Cys Ser Asn Ser

340 345 350

Thr Glu Pro His Leu Ala Thr Phe Ala Ile Pro Leu Gly Ala Ile Gln

355 360 365

Val Pro Tyr Tyr Cys Phe Leu Lys Val Asp Thr Tyr Asn Ser Thr Val

370 375 380

Tyr Lys Phe Leu Ala Val Leu Pro Pro Thr Val Arg Glu Ile Val Ile

385 390 395 400

Thr Lys Tyr Gly Asp Val Tyr Val Asn Gly Phe Gly Tyr Leu His Leu

405 410 415

Gly Leu Leu Asp Ala Val Thr Ile Asn Phe Thr Gly His Gly Thr Asp

420 425 430

Asp Asp Val Ser Gly Phe Trp Thr Ile Ala Ser Thr Asn Phe Val Asp

435 440 445

Ala Leu Ile Glu Val Gln Gly Thr Ala Ile Gln Arg Ile Leu Tyr Cys

450 455 460

Asp Asp Pro Val Ser Gln Leu Lys Cys Ser Gln Val Ala Phe Asp Leu

465 470 475 480

Asp Asp Gly Phe Tyr Pro Ile Ser Ser Arg Asn Leu Leu Ser His Glu

485 490 495

Gln Pro Ile Ser Phe Val Ala Leu Pro Ser Phe Asn Asp His Ser Phe

500 505 510

Val Asn Ile Thr Val Ser Ala Ser Phe Gly Gly His Ser Gly Ala Asn

515 520 525

Leu Ile Ala Ser Asp Thr Thr Ile Asn Gly Phe Ser Ser Phe Cys Val

530 535 540

Asp Thr Arg Gln Phe Thr Ile Ser Leu Phe Tyr Asn Val Thr Asn Ser

545 550 555 560

Tyr Gly Tyr Val Ser Asn Ser Gln Asp Ser Asn Cys Pro Phe Thr Leu

565 570 575

Gln Ser Val Asn Asp Tyr Leu Ser Phe Ser Lys Phe Cys Val Ser Thr

580 585 590

Ser Leu Leu Ala Ser Ala Cys Thr Ile Asp Leu Phe Gly Tyr Pro Glu

595 600 605

Phe Gly Ser Gly Val Lys Phe Thr Ser Leu Tyr Phe Gln Phe Thr Lys

610 615 620

Gly Glu Leu Ile Thr Gly Thr Pro Lys Pro Leu Glu Gly Val Thr Asp

625 630 635 640

Val Ser Phe Met Thr Leu Asp Val Cys Thr Lys Tyr Thr Ile Tyr Gly

645 650 655

Phe Lys Gly Glu Gly Ile Ile Thr Leu Thr Asn Ser Ser Phe Leu Ala

660 665 670

Gly Val Tyr Tyr Thr Ser Asp Ser Gly Gln Leu Leu Ala Phe Lys Asn

675 680 685

Val Thr Ser Gly Ala Val Tyr Ser Val Thr Pro Cys Ser Phe Ser Glu

690 695 700

Gln Ala Ala Tyr Val Asp Asp Asp Ile Val Gly Val Ile Ser Ser Leu

705 710 715 720

Ser Asn Ser Thr Phe Asn Ser Thr Arg Glu Leu Pro Gly Phe Phe Tyr

725 730 735

His Ser Asn Asp Gly Ser Asn Cys Thr Glu Pro Val Leu Val Tyr Ser

740 745 750

Asn Ile Gly Val Cys Lys Ser Gly Ser Ile Gly Tyr Val Pro Ser Gln

755 760 765

Ser Gly Gln Val Lys Ile Ala Pro Thr Val Thr Gly Asn Ile Ser Ile

770 775 780

Pro Thr Asn Phe Ser Met Ser Ile Arg Thr Glu Tyr Leu Gln Leu Tyr

785 790 795 800

Asn Thr Pro Val Ser Val Asp Cys Ala Thr Tyr Val Cys Asn Gly Asn

805 810 815

Ser Arg Glu Asn Leu Tyr Phe Gln Ser

820 825

<210>7

<211>2343

<212>DNA

<213> Artificial sequence

<400>7

atgaagtctt taacctactt ctggttgttc ttaccagtac tttcaacact tagcctacca 60

caagatgtca ccaggtgctc agctaacact aattttaggc gtttcttttc aaaatttaat 120

gttcaggcgc ctgcagttgt tgtactgggc ggttatctac ctattggtga aaaccagggt 180

gtcaattcaa cttggtactg tgctggccga catccaactg ctagtggcgt tcatggtatc 240

tttgttagcc atattagagg tggtcatggc tttgagattg gcatttcgca agagcctttt 300

gaccctagtg gttaccagct ttatttacat aaggctacta acggtaacac taatgctact 360

gcgcgactgc gcatttgcca gtttcctagc attaaaacat tgggccccac tgctaataat 420

gatgttacaa caggtcgtaa ttgcctattt aacaaagcca tcccagctca tatgagtgaa 480

catagtgttg tcggcataac atgggataat gatcgtgtca ctgtcttttc tgacaagatc 540

tattattttt attttaaaaa tgattggtcc cgtgttgcga caaagtgtta caacagtgga 600

ggttgtgcta tgcaatatgt ttacgaaccc acctattaca tgcttaatgt tactagtgct 660

ggtgaggatg gtatttctta tcaaccctgt acagctaatt gcattggtta tgctgccaat 720

gtatttgcta ctgagcccaa tggccacata ccagaaggtt ttagttttaa taattggttt 780

cttttgtcca atgattccac tttggtgcat ggtaaggtgg tttccaacca accattgttg 840

gtcaattgtc ttttggccat tcctaagatt tatggactag gccaattttt ctcctttaat 900

caaacgatcg atggtgtttg taatggagct gctgtgcagc gtgcaccaga ggctctgagg 960

tttaatatta atgacacctc tgtcattctt gctgaaggct caattgtact tcatactgct 1020

ttaggaacaa atttttcttt tgtttgcagt aattccacag agcctcattt agccaccttc 1080

gccatacctc tgggtgctat ccaagtaccc tattactgtt ttcttaaagt ggatacttac 1140

aactccactg tttataaatt cttggctgtt ttacctccaa ccgtcaggga aattgtcatc 1200

accaagtatg gtgatgttta tgtcaatggg tttggctatt tgcatctcgg tttgttggac 1260

gctgtcacaa ttaatttcac tggtcatggc actgacgatg acgtttcagg tttctggacc 1320

atagcatcga ctaattttgt tgatgcactt atcgaagttc aaggaactgc cattcagcgt 1380

attctttatt gtgatgatcc tgttagccaa ctcaagtgtt ctcaggttgc ttttgacctt 1440

gacgatggtt tctaccctat ttcttctaga aaccttctga gtcatgaaca gccaatttct 1500

tttgttgctt tgccatcatt taatgatcat tcttttgtta acattactgt ctctgcgtcc 1560

tttggtggtc atagtggtgc caaccttatt gcatctgaca ctactatcaa tgggtttagt 1620

tctttctgtg ttgacactag acaatttacc atttcactgt tttataacgt tacaaacagt 1680

tatggttatg tgtctaattc acaggacagt aattgtcctt tcaccttgca atctgttaat 1740

gattacctgt cctttagtaa attttgtgtt tccaccagcc ttttggctag tgcctgtacc 1800

atagatcttt ttggttaccc tgagttcggt agtggtgtta agtttacgtc cctttacttt 1860

caattcacaa agggtgagtt gattactggc acgcctaaac cacttgaagg tgtcacggac 1920

gtttctttta tgactctgga tgtgtgtacc aagtatacta tctatggctt taaaggtgag 1980

ggtatcatta cccttacaaa ttctagcttt ttggcaggtg tttattacac atctgattct 2040

ggacagttgt tagcctttaa gaatgtcact agtggtgctg tttattctgt tacgccatgt 2100

tctttttcag agcaggctgc atatgttgat gatgatatag tgggtgttat ttctagtttg 2160

tctaactcca cttttaacag cactagggag ttgcctggtt tcttctacca ttctaatgat 2220

ggctctaatt gtacagagcc tgtgttggtg tatagtaaca taggtgtttg taaatctggc 2280

agtattggct acgtcccatc tcagtctggc caagtcaaga ttgcacccac ggttactggg 2340

aat 2343

Claims

1. An indirect ELISA detection kit for porcine epidemic diarrhea virus S1 protein is characterized in that the envelope antigen in the indirect ELISA detection kit is porcine epidemic diarrhea virus S1 protein obtained by expression and purification of a eukaryotic expression system and/or porcine epidemic diarrhea virus S1 protein obtained by expression and purification of an insect expression system;

the porcine epidemic diarrhea virus S1 protein obtained by expression and purification of the eukaryotic expression system is based on an eukaryotic expression vector and is integrated with a PEDV-S1 gene optimized by a codon to obtain the eukaryotic expression vector for expressing the porcine epidemic diarrhea virus S1 protein; wherein, the sequence of the PEDV-S1 gene optimized by the codon of the eukaryotic expression system is shown as SEQ ID NO. 1; the eukaryotic expression system is used for expressing S1 protein by using a eukaryotic expression vector pFase-hLgG 1-Fc 1;

the porcine epidemic diarrhea virus S1 protein obtained by expression and purification of the insect expression system is based on an insect expression vector and is integrated with a PEDV-S1 gene optimized by a codon to obtain an insect expression vector for expressing the porcine epidemic diarrhea virus S1 protein; the insect expression system is used for expressing S1 protein by using an insect expression vector pFastbac 1;

wherein, the sequence of the PEDV-S1 gene optimized by the codon of the insect expression system is shown as SEQ ID NO. 7;

the use method of the indirect ELISA detection kit comprises the following steps:

step (a): adding the coating antigen into the ELISA plate in an amount of 0.44 ng/hole/100 mu L for coating the ELISA plate;

step (b): adding a sealing liquid into the coated ELISA plate for sealing;

step (c): adding the diluted sample, the negative control and the positive control into an enzyme label plate, adding a second antibody after incubation, and continuing incubation;

step (d): adding substrate into the enzyme label plate with sample, and reacting in darkThen, stop solution was added to terminate the reaction, and OD per well was measured and recorded by microplate reader₄₅₀A value;

2. The indirect ELISA detection kit for the porcine epidemic diarrhea virus S1 protein according to claim 1, wherein the preparation method of the eukaryotic expression system comprises:

artificially synthesizing a PEDV-S1 gene containing EcoRI and XhoI enzyme cutting sites by taking the codon optimized PEDV-S1 gene of the eukaryotic expression system as a template, and integrating the PEDV-S1 gene containing the EcoRI and XhoI enzyme cutting sites into the EcoRI/XhoI sites of a eukaryotic expression vector pFase-hLgG 1-Fc1 to obtain the pFase-PEDV-S1-Fc eukaryotic expression vector for expressing the porcine epidemic diarrhea virus S1 protein.

3. The indirect ELISA detection kit for the porcine epidemic diarrhea virus S1 protein according to claim 2, wherein the artificial synthesis uses PEDV-S1-F1 and PEDV-S1-R1 as primer combination to perform PCR amplification on the PEDV-S1 gene optimized by codons of the eukaryotic expression system;

wherein, the PEDV-S1-F1 has a sequence shown as SEQ ID NO.2, and the PEDV-S1-R1 has a sequence shown as SEQ ID NO. 3.

4. The indirect ELISA detection kit for the porcine epidemic diarrhea virus S1 protein according to claim 1, wherein the preparation method of the insect expression system comprises:

artificially synthesizing PEDV-S1 gene containing BamHI and XhoI enzyme cutting sites by using a codon optimized PEDV-S1 gene of an insect expression system as a template, and integrating the PEDV-S1 gene containing the BamHI and XhoI enzyme cutting sites into the BamHI/XhoI sites of an insect expression vector pFastbac1 to obtain the pFastbac1-PEDV-S1 insect expression vector for expressing the porcine epidemic diarrhea virus S1 protein.

5. The indirect ELISA detection kit for S1 protein of porcine epidemic diarrhea virus of claim 4, wherein the artificial synthesis uses PEDV-S1-BamHIF and PEDV-S1-XhoIR-6His as primer combination to perform PCR amplification on PEDV-S1 gene optimized by codons of insect expression system;

6. The indirect ELISA detection kit for the protein of porcine epidemic diarrhea virus S1 of claim 1, wherein the kit further comprises a washing solution, a coating solution, a blocking solution, a sample diluent, a substrate, a stop solution, a negative control, a positive control and a secondary antibody.