CN107011417B

CN107011417B - Recombinant protein, encoding gene and application thereof, and detection kit and detection method for porcine epidemic diarrhea virus antibody

Info

Publication number: CN107011417B
Application number: CN201710234819.8A
Authority: CN
Inventors: 宋勤叶; 姚作俊; 郭海勇; 逯纪成; 孙泰然
Original assignee: Heibei Agricultural University
Current assignee: Heibei Agricultural University
Priority date: 2017-04-12
Filing date: 2017-04-12
Publication date: 2020-04-03
Anticipated expiration: 2037-04-12
Also published as: CN107011417A

Abstract

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

Description

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

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant protein, a coding gene thereof, application thereof, a detection kit for porcine epidemic diarrhea virus antibodies and a detection method.

Background

Porcine Epidemic Diarrhea (PED) is one of the important infectious diseases that cause death of newborn piglets. The disease is caused by Porcine Epidemic Diarrhea Virus (PEDV) and is characterized by diarrhea, vomiting, dehydration and high mortality in suckling piglets. PED first occurred in 1971 in growing-finishing swinery in the uk, followed by subsequent occurrences in other countries in europe as well as asia. In 1976 PED occurred in Guangdong, Shanghai, Heilongjiang, etc. of China, and then the disease was endemic in domestic pig farms and PEDV was isolated from sick swine herds. In 10 months in 2010, the newborn piglet diarrhea caused by the PEDV variant strains is fulminantly epidemic in China and is rapidly spread all over the country. The piglets in 710 days old are seriously damaged, the morbidity of the piglets is up to 100 percent, the mortality rate of the piglets is up to 50 to 90 percent, and great economic loss is caused to the pig raising production in China.

PEDV belongs to a member of the genus coronavirus of the family Coronaviridae, has a genome length of about 28kb, and is a linear single-stranded positive-strand RNA virus. The viral genome encodes 4 structural proteins: spike protein (S), small membrane protein (E), membrane protein (M) and nucleocapsid protein (N), where the S protein is located on the surface of virions, is the most important structural protein of PEDV, and consists of 1383aa, including signal peptide, neutralizing epitope, transmembrane region and cytoplasmic region. The S protein can be divided into two regions of S1(1-789aa) and S2(790-1383aa), wherein the S1 region is positioned on the surface of the virus, can be combined with a receptor on a host cell and can induce the generation of a neutralizing antibody; the S2 region is located inside the virus and mediates fusion of the virus with the host cell. Therefore, the S protein is a main target protein for PEDV infection immunity research and vaccine design, the S protein antibody level and the change dynamics thereof are important bases for evaluating the immune effect and monitoring the immune state, and the S protein antibody level and the change dynamics thereof can also be used as important indexes for PEDV infection serum epidemiological investigation. Dingzhengjiang et al (2014) established an indirect ELISA method based on PEDV IgA antibody based on recombinant PEDV S1 protein, and HuaYao et al (2016) established an indirect ELISA method based on PEDV IgG based on PEDV S protein. Since the S1 protein region is the main region for inducing the protective immune response of the organism, the specific antibody level aiming at the region can indirectly reflect the immune protection level induced by the vaccine and the anti-infection immune level of the organism. It is known that serum PEDV antibodies have positive correlation with local mucosal immunity, and ELISA methods for detecting serum IgG antibodies have wider application range. Therefore, it is very necessary to establish an ELISA detection method for IgG antibody based on S1 protein.

Disclosure of Invention

In view of the above, the present invention aims to provide a recombinant protein, a coding gene thereof, applications thereof, and a detection kit and a detection method for porcine epidemic diarrhea virus antibodies, and the detection method provided by the present invention is simple, specific, sensitive, and stable.

The invention provides a recombinant protein, and the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1.

The invention also provides a gene for coding the recombinant protein, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

The recombinant protein provided by the invention can be used as a coating antigen, and an indirect ELISA method is used for detecting the porcine epidemic diarrhea virus antibody.

The invention also provides application of the recombinant protein in detecting the porcine epidemic diarrhea virus antibody, and the recombinant protein is mainly used as a coating antigen.

The invention also provides a kit for detecting the porcine epidemic diarrhea virus antibody, which comprises the following components: the recombinant protein, the coating solution, the washing solution, the confining solution, the diluent, the goat anti-pig IgG labeled by horseradish peroxidase and the substrate developing solution are described above.

Wherein, the sealing liquid is PBST solution containing 5% newborn calf serum.

The invention also provides a detection method of the porcine epidemic diarrhea virus antibody, which comprises the following steps:

and (2) after the recombinant protein is coated by the coating solution, washing by using a washing solution, adding a sealing solution for sealing, adding diluted serum to be detected for reaction, adding horse radish peroxidase-labeled goat anti-porcine IgG for secondary reaction after washing, and then washing, adding a substrate developing solution for developing to obtain a detection result.

The invention takes the recombinant protein shown in SEQ ID No.1 as the coating antigen, adopts indirect ELISA to detect the porcine epidemic diarrhea virus antibody, and has better specificity, sensitivity and repeatability, and visual and accurate result.

The invention designs and synthesizes specific primers aiming at the gene sequence of the Porcine Epidemic Diarrhea Virus (PEDV) S protein, amplifies corresponding nucleotide sequences, constructs prokaryotic expression plasmids expressing the region (S1), expresses recombinant S1 protein in vitro under IPTG induction, identifies the expressed protein by using Western blot, and proves that the expressed protein has good immunogenicity by immunizing a mouse with the protein and can stimulate the mouse to generate high-level specific antibodies. The recombinant protein provided by the invention is used as a detection antigen, an indirect ELISA antibody detection method based on PEDV S1 protein is established, and the method is used for detecting PEDV antibodies in colostrums of sows immunized by PEDV inactivated vaccines 4 weeks before delivery, serum at the day of delivery and serum of

piglets

1, 7, 14, 21, 28 and 35 days old (d). The result shows that the established indirect ELISA method can specifically detect the PEDV antibody, has no cross reaction with antiserum of hog cholera virus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine transmissible gastroenteritis virus and porcine rotavirus, can detect 8.3 mu g of total protein in every mL of PEDV antibody positive serum, has the coincidence rate with the immunoperoxidase single-layer test detection result of 97.14 percent, and has the variation coefficient of the batch-to-batch repeatability test of less than 10 percent. The positive rate of PEDV antibody in the serum of the immunized sow colostrum and the day of delivery is 100 percent, and the colostrum is lower than the average antibody level in the serum; the positive rate of the PEDV antibody in the piglet serum at 1d is 50%, the average antibody level is low and is close to the antibody level in colostrum, and all piglet sera are converted into negative PEDV antibody after 7 d. (conclusion) the established ELISA method was able to specifically detect PEDV antibodies; when the pregnant sow is immunized 1 time by the PEDV inactivated vaccine before delivery, the maternal specific antibody positive rate and the antibody level in the serum of the born piglet are low, and the maintenance time is short.

Drawings

FIG. 1 is an electrophoretogram of a target fragment amplified according to an embodiment of the present invention;

FIG. 2 shows the PCR and restriction enzyme identification of recombinant expression plasmid pET28 a-S1;

FIG. 3 is an SDS-PAGE and Western blot analysis of recombinant PEDV S1 protein;

FIG. 4 is a SDS-PAGE analysis of the expressed form of recombinant PEDV S1 protein;

FIG. 5 shows serum-specific antibodies of mice immunized with recombinant PEDV S1 protein;

FIG. 6 shows the kinetics of PEDV antibodies in the colostrum and serum of immunized sows and the specific antibodies in the serum of piglets born.

Detailed Description

Example 1

1 materials and methods

1.1 bacterial species and expression vectors

Coli DH5 α and BL21 competent cells, prokaryotic expression vector pET-28a (+), all stored in the animal infectious disease laboratory of Hebei agricultural university.

1.2 Primary reagents and antibodies

TRIzol total RNA extraction reagent, dNTPs, Taq DNA polymerase and a centrifugal column type DNA gel purification recovery kit which are purchased from Tiangen Biochemical technology (Beijing) Co., Ltd; reverse transcriptase, a product of Promega corporation; restriction endonucleases BamH I and Xho I, available from Bao bioengineering (Dalian) Inc.; a Ni-Agarose His tag protein purification kit, purchased from Beijing kang as a reagent biotechnology limited; a protein quantitative test kit purchased from Nanjing to build a bioengineering institute. HRP-goat anti-porcine IgG and HRP-goat anti-mouse IgG were purchased from Beijing Solaibao Biotechnology Ltd. Porcine anti-PEDV, porcine circovirus type 2 (PCV2), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Classical Swine Fever Virus (CSFV), porcine pseudorabies virus (PRV), porcine transmissible gastroenteritis virus (TGEV), porcine type A Rotavirus (RVA) and other virus antibodies, and mouse anti-PEDV positive and negative serum are prepared and stored in animal infectious disease laboratories of North and Hebei agricultural universities.

1.3 construction and identification of prokaryotic expression plasmid of PEDV S1 protein gene

1.3.1 primer design and Synthesis

Specific primers for amplifying partial fragment S1 were designed by using the biology software Oligo6.0 against the PEDV S protein gene:

U1：5′-ACTGAATTCATGGTACTGGGCGGTTATCTA-3′，

l1: 5'-ATTCTCGAGTTAGGCTAAGTGAGGATCTGA-3' are provided. The primers were synthesized by Biotechnology engineering (Shanghai) Inc.

1.3.2 PCR amplification and recovery of target genes

PEDVRNA was extracted, reverse transcribed into cDNA, and then PCR was performed using primers U1-L1 to amplify the desired nucleic acid fragment. The PCR amplification conditions were: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, extension at 72 ℃ for 1min, and 35 cycles; extension at 72 ℃ for 10 min. The amplified target fragment is 945bp in size. After the PCR product is detected by 1% agarose gel electrophoresis that the size of the amplified fragment is consistent with the expectation, a centrifugal plant type DNA gel purification recovery kit is used for recovering the target gene.

1.3.3 construction and identification of recombinant expression plasmid pET28a-S1

Digesting and recovering PCR products and prokaryotic expression vector pET28a (+) by using restriction endonucleases EcoRI and Xho I, respectively recovering and purifying and recovering restriction enzyme digestion products by using a centrifugal strain type DNA gel purification recovery kit after electrophoresis of the restriction enzyme digestion products, connecting the PCR products and pET28a (+) by using T4 DNA ligase, transforming the connection products pET28a-S1 into DH5 α competent cells, and coating the DH5 α competent cells with the ligation products⁺Culturing on an LB flat plate at 37 ℃ for 16-18 h; selecting a single colony for carrying out PCR identification on the bacterial liquid, extracting bacterial liquid plasmids, carrying out double enzyme digestion identification on the bacterial liquid plasmids by using restriction endonucleases EcoRI and Xho I, and sending the bacterial liquid with correct identification to a biological engineering (Shanghai) company Limited for sequencing.

1.4 inducible expression and identification of recombinant protein S1

1.4.1 expression of recombinant S1 protein

The constructed recombinant expression plasmid pET28a-S1 of the PEDV S1 protein is transformed into E.coli BL21 competent cells and coated on Kan⁺Culturing on LB solid medium at 37 deg.C for 24h, picking positive colony (pET28a-S1/BL21) and inoculating into Kan⁺In LB liquid medium, shaking culture is carried out overnight at 37 ℃, and then Kan is transferred according to the proportion of 1:100⁺In a LB liquid culture medium, shaking culture is carried out at 37 ℃ and 230r/min until the logarithmic growth phase (OD) of flora_600nm0.6 to 0.8), adding the final concentration0.5mmol/L IPTG, inducing expression at 35 ℃ for 5h, collecting 1mL of thalli before and after induction and 12000r/min, centrifuging for 1min, collecting thalli precipitation, adding 50 mu L of PBS and 50 mu L of 2 xSDS loading buffer solution, mixing uniformly, centrifuging for 1min at 12000r/min, carrying out SDS-PAGE electrophoresis, and detecting the expression condition and molecular weight of protein. Meanwhile, BL21(pET-28a/BL21) cells transformed with the empty plasmid pET-28a were used as controls.

1.4.2 expression forms of the recombinant protein S1

And (3) taking the bacteria liquid after induction expression, centrifuging for 10min at 7500r/min, discarding the supernatant, adding 2mL of bacteria lysate into each gram of bacteria precipitate, mixing uniformly, carrying out ice bath for 10min, carrying out intermittent ultrasonic lysis on ice until the bacteria liquid is clear, centrifuging for 10min at 12000r/min at 4 ℃, collecting the supernatant and the precipitate, carrying out SDS-PAGE electrophoresis, and determining the expression form of the protein.

1.4.3 Western blot identification of recombinant protein S1

Taking the induced expression bacterial liquid, after SDS-PAGE electrophoresis, transferring the protein onto a PVDF membrane, putting the membrane into 5% skimmed milk powder sealing liquid, and standing overnight at 4 ℃; adding mouse anti-PEDV serum diluted at a ratio of 1:320, and shaking on a shaking table for 1h at room temperature; after washing the membrane, adding HRP-labeled goat anti-mouse IgG diluted by 1:2000, and oscillating for 1-2 h at room temperature; and after washing, putting the film into a DAB substrate developing solution for developing for 3-10 min, stopping reaction by using deionized water after strips are clear, and photographing to record results.

1.5 immunogenicity of recombinant protein S1

And (4) taking the bacteria liquid expression protein of the induction expression, and purifying the target protein according to the instruction of the Ni-Agarose His tag protein purification kit. The protein was purified by SDS-PAGE for the presence of bands, and the protein content was determined by UV spectrophotometry. The recombinant protein is taken to immunize 6 mice with age of 6 weeks for 3 times, the interval of each time is 2 weeks, and the inoculation dose and the route are shown in table 1.2 non-immunized mice were also set up as negative controls. Blood was collected from the tail vein 2 weeks after each inoculation, and serum was isolated and specific antibodies were detected by ELISA. The main steps of ELISA antibody detection are as follows: coating a 96-well enzyme label plate with 1 mu g/mL recombinant S1 protein, repeating 2 wells with each well being 100 mu L/well; after sealing, adding 1:160 times diluted serum to be detected, incubating for 1h at 37 ℃, and washing; adding into each hole100 mu L of goat anti-mouse IgG-HRP diluted 1:2000 times, incubating at 37 ℃ for 45min, and washing; adding 100 mu L of a freshly prepared TMB substrate solution into each hole, and reacting for 15min at room temperature in a dark place; add 50. mu.L of 2M H per well₂SO₄The reaction was terminated. Determination of OD Using an enzyme-Linked Detector_450nmThe production of mouse specific antibodies induced by the recombinant protein was analyzed.

TABLE 1 immunization procedure for recombinant proteins

1.6 establishment and preliminary application of indirect ELISA antibody detection method based on recombinant S1 protein

1.6.1 determination of antigen coating concentration and confining liquid

Forming a square matrix by using the purified recombinant PEDV S1 protein and different confining liquids, screening the optimal antigen coating concentration and the optimal confining liquid, namely diluting the purified recombinant PEDV S1 protein into 0.25, 0.5 and 1 mu g/mL by using the coating liquid (carbonate buffer solution with pH 9.6), respectively adding the diluted protein into each hole of a 96-hole enzyme label plate, and repeating the steps for 2 holes at 100 mu L/hole; incubating the ELISA plate at 37 ℃ for 1h, and then standing at 4 ℃ for overnight; the coating solution was discarded and each well was washed 3 times, 3 min/time, patted dry with 280. mu.L of LPBST (0.01 mol/LpH value 7.4PBS of 0.05% Tween-20); adding different sealing solutions (PBST containing 5% newborn calf serum, 5% horse serum, 5% skimmed milk powder, 2% BSA and 1% gelatin) into antigen wells with corresponding concentrations, incubating at 37 deg.C for 1h with 100 μ L/well, discarding the sealing solution, and patting to dry; adding 100 mu L of pig anti-PEDV positive serum and negative serum diluted by 1:40 times into corresponding antigen coated holes; incubating the ELISA plate at 37 ℃ for 1h, and washing; adding 100 μ L of 1:1000 times diluted goat anti-pig IgG-HRP into each well, incubating the ELISA plate at 37 deg.C for 45min, and washing; adding 100 mu L of a freshly prepared TMB substrate solution into each hole, and reacting for 15min at room temperature in a dark place; add 50. mu.L of 2MH per well₂SO₄The reaction was terminated. Determination of OD Using an enzyme-Linked Detector_450nmThe OD values of the positive and negative sera were compared, and the ratio of the OD values of the positive serum (P) to the negative serum (N) (P/N value). The antigen coating concentration and the blocking liquid when the P/N value is maximum are the optimal antigen coating concentration and blockingClosing the liquid.

1.6.2 determination of coating Condition and blocking Condition

Adding a proper amount of recombinant PEDV S1 protein into corresponding holes of an ELISA plate, and performing antigen coating under the following 5 conditions of 1h action at 37 ℃, overnight at 4 ℃, 1h action at 37 ℃, 1h action at 25 ℃, overnight at 4 ℃, 1h action at 25 ℃ and overnight at 4 ℃. The antigen wells were then blocked under 4 conditions, i.e., incubation at 37 ℃ for 30min, 37 ℃ for 1h, 25 ℃ for 30min, and 25 ℃ for 1h, respectively. Then, according to the reaction conditions in 1.4.1, the serum to be detected and the enzyme-labeled antibody are sequentially added to carry out ELISA, and the optimal antigen coating condition and the optimal sealing condition are determined.

1.6.3 determination of the serum concentration to be examined and the concentration of the enzyme-labeled antibody

Respectively diluting anti-PEDV positive serum and anti-PEDV negative serum by 1:40, 1:80, 1:160, 1:320, 1:640 and 1:1280 times, diluting goat anti-pig IgG-HRP by 1:500 and 1:1000 times to form a square matrix, performing ELISA according to the reaction conditions in 1.4.1, and determining the optimal working concentration of the serum to be detected and the enzyme-labeled antibody.

1.6.4 determination of reaction time between blood serum to be detected and enzyme-labeled antibody

Taking the recombinant PEDV S1 protein coated ELISA plate, respectively adding 100 mu L of anti-PEDV positive serum and anti-PEDV negative serum (diluted by 1:40 times) into corresponding holes, and respectively acting for 45min and 1h at 37 ℃; after washing, 100. mu.L of goat anti-porcine IgG-HRP (1:1000 fold dilution) was added to each well and allowed to act at 37 ℃ for 30min and 45min, respectively; developing color, terminating reaction, measuring the OD value of the negative serum and the positive serum, comparing the P/N value and determining the optimal reaction time of the serum to be detected and the enzyme-labeled antibody.

1.6.5 determination of seronegative-positive cut-off value

50 parts of PEDV antibody negative serum are detected by the established method, and the OD of the sample is calculated_450nmMean (X) and Standard Deviation (SD) of the values. OD of the sample according to statistical principles_450nmWhen the value is more than or equal to X +3SD, the product is judged to be positive, OD_450nmWhen the value is less than or equal to X +2SD, the result is judged to be negative, and the result between the two is judged to be suspicious.

1.6.6 specificity and sensitivity assays

The established indirect ELISA method is used for detecting virus antibodies such as PCV2, PRRSV, CSFV, PRV, TGEV, RVA and the like, and simultaneously, PEDV negative and positive antibody controls are set for evaluating the specificity of the method.

After 5 parts of PEDV antibody positive serum are diluted by 1:80, 1:160, 1:320, 1:640, 1:1280 and 1:2560 times, the specific antibody is detected by the established ELISA method. 3 replicates were made for each dilution, while 3 dilution controls and 3 blank well controls were set up. The sensitivity of the method was evaluated as the mean protein content in serum at the highest dilution factor at which the PEDV antibody could be detected.

1.6.7 comparative test 70 pig sera were tested by the established ELISA method and Immunoperoxidase monolayered assay (IPMA), and the results of the two methods were compared to evaluate the compliance of the ELISA method with IPMA.

1.6.8 repeatability test the same batch of recombinant PEDV S1 protein coated ELISA plate, and 10 pig sera were tested simultaneously, each serum was repeated 3 wells for 3 times. And (3) coating 4 batches of recombinant PEDV S1 protein on an enzyme label plate, and detecting 10 parts of pig serum respectively, wherein each part of serum is subjected to 3-hole repeated assay. The within-and between-batch reproducibility of the established ELISA method was analyzed according to the Coefficient of Variation (CV).

1.6.9 selecting randomly 5 pregnant sows, immunizing PED inactivated vaccine in the acupoint of sea 4 weeks before birth, and feeding each newborn piglet with colostrum by natural lactation method after the sow is born. The established ELISA method is used for detecting specific antibodies in serum of colostrum, the day of farrowing of the sow and the serum of piglets born by the colostrum (10-13 heads per litter) at 1d, 7d, 14d, 21d, 28d and 35d, and the dynamic change of the specific maternal antibody in the serum of newborn piglets is preliminarily analyzed.

2 results

2.1 construction and identification of recombinant expression plasmid pET28a-S1

Extracting PEDV RNA, performing reverse transcription, and performing PCR amplification to obtain a target fragment with an expected size (945bp), wherein the result is shown in figure 1, figure 1 is an electrophoresis diagram of the target fragment obtained by amplification in the embodiment of the invention, and 1 is a relative molecular mass standard of DNA; 2 is PCR product. Recovering a target gene by using a DNA gel purification recovery kit, digesting by restriction endonucleases EcoR I and Xho I, connecting with a prokaryotic expression vector pET28a (+) in vitro to construct a recombinant expression plasmid pET28a-S1, and after PCR, enzyme digestion and sequencing, proving that a target gene fragment (S1) is successfully cloned to the corresponding position of the recombinant expression plasmid pET28a (+), see figure 2, and figure 2 is the PCR and enzyme digestion identification of the recombinant expression plasmid pET28a-S1, wherein 1 is the relative molecular mass standard of DNA; 2 is a recombinant plasmid bacterial liquid PCR product; 3 is pET28a plasmid PCR control; 4 is pET28a-S1 which is not cut by enzyme; 5 is the EcoRI and XhoI double cleavage product of pET28 a-S1. The nucleic acid sequence of the amplified PEDV S1 is shown as SEQ ID No.2, and the amino acid sequence thereof is shown as SEQ ID No. 2.

2.2 expression of recombinant protein S1 and Westernblot identification

After 5h of IPTG induction at 35 ℃, the results are shown in figure 3, and figure 3 shows SDS-PAGE and Westernblot analysis of the recombinant PEDV S1 protein, wherein a is the expression and purification of the recombinant S1 protein. M, protein low molecular mass standard; before induction, pET-28a/BL 21; after induction of pET28a/BL 21; before induction, pET28a-S1/BL 21; pET28a-S1/BL21 induced for 5 h; 5. and (3) purifying the recombinant protein. B is Westernblot analysis of recombinant S1 protein. M, protein low molecular mass standard; 1. reacting the recombinant protein with mouse anti-PEDV negative serum; 2. the recombinant protein reacts with mouse anti-PEDV positive serum. The results show that the molecular weight of the recombinant protein expressed by pET28a-S1/BL21 is about 38kDa, which is consistent with the expected size (FIG. 3a), and the protein can be specifically combined with the mouse anti-PEDV serum by the detection of Westernblot (FIG. 3 b).

2.3 expression of recombinant protein S1 in the form of Inclusion bodies

BL21(pET28a-S1/BL21) carrying the recombinant expression plasmid is induced by IPTG for 5h, bacteria are collected, after ultrasonic lysis, the precipitate and SDS-PAGE of the supernatant show that protein with expected size appears in the precipitate, but almost no protein with expected size appears in the supernatant, and the result is shown in figure 4, figure 4 is SDS-PAGE analysis of the expression form of the recombinant S1 protein, wherein M is a protein low molecular mass standard; 1 is cracking precipitation; 6 is the lysis supernatant. The recombinant S1 protein was shown to be expressed in E.coli as inclusion bodies.

2.4 the recombinant protein S1 has good immunogenicity

Specific antibodies were detected in three sera of 6 mice 14 days after the first immunization of the mice with the expressed recombinant protein; high-level specific antibodies were detected in the serum of all immunized mice 14 days after the second immunization, and the antibody level 14 days after the third immunization was almost maintained at the same level as that of the second immunization, as shown in fig. 5, wherein fig. 5 shows the specific antibodies in the serum of the mice immunized with the recombinant S1 protein, and V1-V6 show the mice immunized with the recombinant protein; neg.1 and neg.2, non-immunized control mice; 0, 1-3, which refer to serum samples collected before immunization and 14 days after the first, second and third immunizations, respectively. The results show that the expressed recombinant S1 protein can stimulate mice to produce specific antibodies and has good immunogenicity.

2.5 Indirect ELISA antibody detection method for PEDV based on recombinant S1 protein

2.5.1 working conditions and criteria for Indirect ELISA

The PEDV indirect ELISA antibody detection method is established by taking the recombinant S1 protein as a coating antigen. The ELISA method comprises the following main operation steps and judgment standards: incubating the enzyme-linked immunosorbent assay plate with 1 mu g/mL recombinant PEDV S1 protein at 37 ℃ for 1h, coating the enzyme-linked immunosorbent assay plate at 4 ℃ overnight, washing, and blocking the enzyme-linked immunosorbent assay plate with PBST (bovine serum albumin) of 5% newborn calf serum at 37 ℃ for 1 h; washing, adding 1:40 times diluted pig serum to be detected, and reacting for 1h at 37 ℃; washing, adding HRP-labeled goat anti-pig IgG diluted by 1:1000 times, and reacting at 37 ℃ for 45 min; washing, adding a freshly prepared TMB substrate solution, and reacting for 15min at room temperature in a dark place; 2M H was added₂SO₄The reaction was terminated, and the results are shown in tables 2 to 5. Determination of OD Using an enzyme-Linked Detector_450nmAnd (4) judging the result. When the sample OD_450nmWhen the value is more than or equal to 0.373, the specific antibody is judged to be positive; when OD is reached_450nmWhen the value is less than or equal to 0.305, judging the test result as negative; when OD is reached_450nmAnd when the value is between the two values, the result is judged to be suspicious.

TABLE 2 determination of antigen coating concentration and confining liquid

Note: +, positive serum; -, negativeSerum; P/N, positive serum OD_450nmValue/negative serum OD_450nmThe value is obtained.

TABLE 3 determination of optimal antigen coating and blocking conditions

Note: +, positive serum; -, negative serum; P/N, positive serum OD_450nmValue/negative serum OD_450nmThe value is obtained.

TABLE 4 determination of optimal dilution concentration of serum to be tested and enzyme-labeled antibody

TABLE 5 determination of optimal action time between serum to be detected and enzyme-labeled secondary antibody

2.5.2 specificity and sensitivity of Indirect ELISA

When the established method is used for detecting the known PCV2, PRRSV, CSFV, PRV, TGEV and RVA antibodies, the OD value is less than 0.305, and the OD value is shown in the table 6, which shows that the established ELISA method has no cross reaction with other virus serum and has good specificity. The PEDV antibody positive serum is diluted in a multiple ratio, and when each mL of the porcine anti-PEDV serum contains 8.3 mu g of protein, the built ELISA can detect the specific antibody of the PEDV, which indicates that the ELISA method can detect at least < 8.3 mu g of specific antibody.

TABLE 6 specific detection of ELISA

Note: +, positive serum; negative serum.

2.5.3 compliance of Indirect ELISA with immunoperoxidase monolayer assay (IPMA)

34 parts and 35 parts of PEDV antibody positive serum and 36 parts and 35 parts of PEDV antibody negative serum are respectively detected from 70 parts of serum by using IPMA and ELISA, the positive coincidence rate of the detection result of the IPMA and the PEDV antibody negative serum is 97.06%, the negative coincidence rate of the PEDV antibody negative serum is 97.22%, and the total coincidence rate of the PEDV antibody negative serum is 97.14%.

2.5.4 repeatability

When the same batch of the ELISA plate is coated with the heavy S1 protein and the same serum sample is detected for 3 times repeatedly, or when the same serum sample is detected by coating the ELISA plate with 4 batches of the recombinant S1 protein, the variation coefficients are respectively 3.75% -9.30% and 1.49% -7.84%, and are both lower than 10% (tables 7 and 8). The results show that recombinant PEDV S1 protein has good reproducibility as a detection antigen, both in-batch and in-batch repeat assays.

TABLE 7 repeat test in batches

TABLE 8 repeat test between lots

2.5.5 preliminary application

PEDV specific antibodies were detected in both colostrum and serum of 5 prenatal vaccinated pregnant sows, wherein colostrum was at a lower level than the mean antibody level in serum and OD was lower_450nmThe values were 0.508. + -. 0.058 and 0.824. + -. 0.093, respectively (FIG. 6 a). The serum antibody positive rate of 5-litter piglet 1-day old is 50%, and the average antibody level (OD) of the antibody positive piglet is low_450nmValues of 0.481 ± 0.057), all sera of all piglets turned antibody-negative after 7 days of age (fig. 6 b). FIG. 6 shows the PEDV antibodies in the colostrum and serum of immunized sows and the specific antibodies in the serum of piglets bornIsoantibody dynamic, wherein a is PEDV antibody in colostrum and serum of immunized sows, and 1, 2, 3, 4 and 5 are sow numbers; b is the PEDV antibody profile in serum of piglets per litter.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

<110> university of agriculture in Hebei

<120> recombinant protein, encoding gene thereof, application thereof, detection kit for porcine epidemic diarrhea virus antibody and detection method

<160>2

<170>PatentIn version 3.3

<210>1

<211>311

<212>PRT

<213> Artificial sequence

<400>1

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

1 5 10 15

Thr Trp Tyr Cys Ala Gly Gln His Pro Thr Ala Ser Gly Val His Gly

20 25 30

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

35 4045

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Ser Ser Asp Pro His Leu Ala

305 310

<210>2

<211>936

<212>DNA

<213> Artificial sequence

<400>2

gtactgggcg gttatctacc tattggtgaa aaccagggtg tcaattcaac ttggtactgt 60

gctggccaac atccaactgc tagtggcgtt catggtatct ttgttagcca tattagaggt 120

ggtcatggct ttgagattgg catttcgcaa gagccttttg accctagtgg ttaccagctt 180

tatttacata aggctactaa cggtaacact aatgctactg cgcgactgcg catttgccag 240

tttcctagca ttaaaacatt gggccccact gctaataatg atgttacaac aggtcgtaat 300

tgcctattta acaaagccat cccagctcat atgagtgaac atagtgttgt cggcataaca 360

tgggataatg atcgtgtcac tgtcttttct gacaagatct attattttta ttttaaaaat 420

gattggtccc gtgttgcgac aaagtgttac aacagtggag gttgtgctat gcaatatgtt 480

tacgaaccca cctactacat gcttaatgtt actagtgctg gtgaggatgg tatttcttat 540

caaccctgta cagctaattg cattggttat tctgccaatg tatttgctac tgagcccaat 600

ggccacatac cagaaggttt tagttttaat aattggtttc ttttgtccaa tgattccact 660

ttggtgcatg gtaaggtggt ttccaaccaa ccattgttgg tcaattgtct tttggccatt 720

cctaagattt atggactagg ccaatttttt tcctttaatc aaacgatcga tggtgtttgt 780

aatggagctg ctgtgcagcg tgcaccagag gctctgaggt ttaatattaa tgacacctct 840

gtcattcttg ctgaaggctc aattgtactt catactgctt taggaacaaa tttttctttt 900

gtttgcagta attcctcaga tcctcactta gcctaa 936

Claims

1. A recombinant protein is characterized in that the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1.

2. A gene encoding the recombinant protein of claim 1.

3. The gene of claim 2, wherein the nucleotide sequence is represented by SEQ ID number 2.

4. Use of the recombinant protein of claim 1 for the preparation of a kit for the detection of antibodies to porcine epidemic diarrhea virus.

5. A kit for detecting porcine epidemic diarrhea virus antibodies, comprising: the recombinant protein, the coating solution, the washing solution, the blocking solution, the diluent, the horseradish peroxidase-labeled goat anti-pig IgG and the substrate developing solution of claim 1.

6. The kit according to claim 5, wherein the blocking solution is PBST solution containing 5% newborn calf serum.