CN114315991A - Competitive ELISA method based on duck flavivirus E protein and monoclonal antibody thereof - Google Patents
Competitive ELISA method based on duck flavivirus E protein and monoclonal antibody thereof Download PDFInfo
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Abstract
The invention relates to a competitive ELISA method based on duck flavivirus E protein and monoclonal antibody thereof, and also relates to duck flavivirus E protein expression and a monoclonal antibody preparation method thereof, belonging to the technical field of animal immunology detection. The invention utilizes the amplification of a primer pair C1 and C2 and a primer pair E1 and E2 to obtain a gene sequence of duck flavivirus E protein, constructs expression plasmid, introduces the expression plasmid into a prokaryotic expression host, performs induced expression and purification to obtain the duck flavivirus E protein, uses the duck flavivirus E protein as an antigen, obtains a duck flavivirus E protein monoclonal antibody by a hybridoma technology, and establishes a competitive ELISA method capable of being used for detecting the duck flavivirus antibody based on the duck flavivirus E protein and the monoclonal antibody thereof. The detection method has strong specificity and good stability, and can be used for detecting the duck flavivirus serum antibody. The duck flavivirus E protein 1 is used as a coating antigen, and the monoclonal antibody of the duck flavivirus E protein is used as a competitive antibody.
Description
Technical Field
The invention relates to the technical field of animal immunology detection, in particular to a competitive ELISA method based on duck flavivirus E protein and monoclonal antibody thereof.
Background
Duck flavivirus is a new infectious disease caused by duck flavivirus, mainly occurs to ducks, and can cause the laying rate of laying ducks to be reduced, and ducklings to grow slowly and die. The morbidity of the duck group can reach 80 percent, and the laying rate of laying ducks can be reduced from 90 percent to 10 percent. The duck which is endured after infection needs a long time to recover the production performance, and the production level before infection is difficult to reach. After the first case of duck flavivirus disease appears in China in 2010, the fact that the duck group is infected with DTMUV is reported in most duck breeding areas in China in succession, the disease brings great economic loss to duck breeding industry in China, seriously influences the healthy development of duck breeding industry in China, and brings a series of potential threats to public health in China.
At present, the pathogen detection of duck flavivirus is of various types, and mainly depends on virus separation and molecular biological detection. Virus isolation is a traditional detection method for duck flavivirus; the molecular biological detection method comprises RT-PCR, nested RT-PCR, fluorescent quantitative RT-PCR, loop-mediated isothermal amplification technology and the like. The antibody detection only adopts an indirect method, relatively speaking, the indirect ELISA specificity is not high, and in order to better carry out epidemic disease investigation and immune evaluation, the competitive ELISA development by using the monoclonal antibody is favorable for improving the detection accuracy.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a competitive ELISA method based on duck flavivirus E protein and monoclonal antibody thereof, relating to the preparation of duck flavivirus E protein monoclonal antibody, and establishing a monoclonal antibody competitive ELISA method to detect duck flavivirus antibody to form a duck flavivirus immune monitoring technology platform.
The technical scheme of the invention is as follows: a duck flavivirus E protein comprises a duck flavivirus E protein 1 and a foot-and-mouth disease A-type VP1 protein 2 which are obtained by two different expression modes, taking a duck flavivirus genome as a template, performing PCR amplification by using a pair of cloning primers and a pair of expression primers to obtain a DFV-E gene, connecting the DFV-E gene with pET-32a and pGEX-6p-1 plasmids respectively to construct expression plasmids PET-32a-E1 and pGEX-6p-1-E1, transferring the expression plasmids PET-32a-E1 and pGEX-6p-1-E1 into prokaryotic expression host escherichia coli BL21 respectively, and performing induction expression to obtain fusion protein 1 and fusion protein 2 respectively, and purifying the fusion protein 1 and the fusion protein 2 respectively to obtain the duck flavivirus E protein 1 and the duck flavivirus E protein 2.
Preferably, the pair of cloning primers comprises primers C1: 5'-gcaggattgtgcagaag-3', primer C2: 5'-ccatgcagtgtcaccca-3' are provided.
Preferably, the pair of expression primers comprises primers E1: 5'-ccggaattcaccgctgagatggagga-3', primer E2: 5'-ccgctcgagctggtacctgatctgtc-3' are provided.
Preferably, the duck flavivirus E protein 1 and the duck flavivirus E protein 2 are same sequence proteins obtained by different expression modes, and the protein sequences of the two are shown in SEQ ID NO: 1.
Preferably, as a further limitation of the present invention, the fusion protein is purified by His or GST column chromatography.
In order to fully disclose the invention, the steps of the method for constructing the recombinant gene expression vector, inducing and expressing the recombinant gene and obtaining the duck flavivirus E protein by chromatographic purification, which are related in the invention, can be realized according to the conventional existing method in a laboratory, wherein the specific steps of PCR amplification are as follows:
step A: taking duck flavivirus E gene as a template, utilizing a first pair of cloning primers C1 and C2 to amplify to obtain a sequence (900bp) containing the DFV-E gene, and then utilizing a second pair of expression primers E1 and E2 to amplify the sequence to obtain the DFV-E1 gene (660 bp).
And B: and D, purifying the DFV-E gene (900bp) obtained by amplification in the step A, and connecting with a pMD18-T vector to construct a pMD18-T-E cloning vector.
And C: and B, constructing pMD18-T-E in the step B as a template, performing PCR amplification by using expression primers E1 and E2, purifying and recovering a PCR product, and performing double enzyme digestion on the PCR purified product and expression vectors pET-32a and pGEX-6p-1 by using EcoRI and XholI endonucleases respectively.
The implementation process of the PCR amplification depends on the existing PCR technology, and realizes the prokaryotic expression of the protein through multiple times of PCR amplification and the gene engineering double enzyme digestion technology.
The invention also provides a method for preparing the duck flavivirus E protein and the monoclonal antibody thereof, and the preparation method comprises the following steps:
step a), animal immunization: immunizing a BALB/c female mouse with the age of 5-7 weeks by using duck flavivirus E protein 1 induced by an expression plasmid PET-32a-E1, and screening out a mouse with the titer of immunoassay being more than 1:12800 after 3 times of immunization;
step b), cell fusion: taking spleen cells of the immune mice obtained in the step a), carrying out cell fusion with myeloma cells SP2/0, using duck flavivirus E protein 2 obtained by induction of expression plasmids pGEX-6p-1-E1 as an envelope antigen, and carrying out indirect ELISA screening on the fused cells to obtain hybridoma cells.
Step c), mass cloning of monoclonal antibody: injecting the hybridoma cells obtained in the step b) into the abdominal cavity of a mouse, feeding the injected mouse for 1-3 weeks, collecting ascites of the mouse with the swollen abdomen, and purifying the ascites to obtain the duck flavivirus E protein monoclonal antibody.
As a further limitation of the invention, the duck flavivirus E protein 1 and the duck flavivirus E protein 2 are same sequence proteins obtained by different expression modes, and the protein sequences of the two are shown in SEQ ID NO. 1.
The invention also provides a competitive ELISA method based on duck flavivirus E protein and monoclonal antibody thereof, which comprises sample dilution, antigen coating, sealing, sample adding and monoclonal antibody adding, enzyme labeling antibody adding, color development and termination, measuring value and data processing, wherein the competitive ELISA detection method specifically comprises the following steps:
step 1) sample dilution: and (5) diluting the serum sample to be 2 times of the original volume to obtain the serum sample to be detected.
Step 2) antigen coating: the used coating antigen is duck flavivirus E protein 2 induced by expression plasmid pGEX-6p-1-E1, and the coating concentration of the coating antigen is 0.5 mu g/mL.
Step 3) sealing: and (3) sealing the antigen coated in the step 2) with sealing liquid for 40-60 min to obtain the sealed ELISA plate.
Step 4), sample adding and monoclonal antibody adding: adding the serum sample to be detected obtained in the step 1) into the closed ELISA plate obtained in the step 3), combining a serum antibody with the coating antigen, adding the duck flavivirus E protein monoclonal antibody with the dilution of 1:400, and combining the duck flavivirus E protein monoclonal antibody with the residual coating antigen which is not reacted with the serum antibody to obtain the ELISA plate after the sample and the monoclonal antibody are reacted.
Step 5) adding enzyme labeled antibody: adding an enzyme-labeled antibody into the enzyme-labeled plate after the reaction of the sample and the monoclonal antibody obtained in the step 4), wherein the dilution concentration of the enzyme-labeled antibody is 1:2000, and the enzyme-labeled antibody is combined with the duck flavivirus E protein monoclonal antibody but not with the serum antibody to obtain the enzyme-labeled plate after the enzyme-labeled monoclonal antibody is added.
Step 6) color development and termination: adding a freshly prepared substrate solution into the enzyme label plate added with the enzyme-labeled antibody obtained in the step 5), oscillating and mixing, incubating at room temperature for 10-20 min in a dark place for color development, adding a stop solution into each hole, and uniformly mixing and incubating for 5-10 min to stop reaction to obtain a solution capable of being measured.
Step 7) measuring: measurement of OD of the solution obtained in step 6) which can be measured450nmAbsorbance value A of450nm；
Step 8) data processing: using the absorbance A determined in step 7)450nmCalculating the inhibition rate of the sample to be detected, wherein the inhibition rate PI% of the sample to be detected is (1-A)450sample/A450Negative control) × 100.
Preferably, as a further limitation of the present invention, the confining liquid is a solution having a concentration of: 1% BSA solution, 1% gelatin solution, 5% fetal bovine serum solution, 5% skim milk powder solution.
Preferably, as a further limitation of the present invention, the antibody of the enzyme-labeled antibody is goat anti-mouse IgG-HRP; a is described450The negative control is absorbance value A determined by using serum without duck flavivirus infection450。
The invention has the beneficial effects that:
1. according to the invention, RT-PCR and protein prokaryotic expression technologies are utilized to successfully obtain duck flavivirus E protein 1 and duck flavivirus E protein 2 which have high purity, good specificity and immunogenicity;
2. by adopting a hybridoma technology, a duck flavivirus E protein 1 is used as an immunogen, a duck flavivirus E protein 2 is used as a coating antigen to establish an indirect ELISA screening method, hybridoma cell strains for resisting the duck flavivirus E protein are screened and prepared, monoclonal antibodies of the hybridoma cell strains can be blocked by duck flavivirus positive serum, and the identification result of the biological characteristics of the monoclonal antibodies shows that the monoclonal antibodies can be selected as detection antibodies of competitive ELISA, so that the difficulty of obtaining the monoclonal antibodies of specificity identification of the competitive ELISA detection method is solved;
3. according to the invention, the duck flavivirus E protein 1 is used as a coating antigen, and the monoclonal antibody of the duck flavivirus E protein is used as a competitive antibody, so that a monoclonal antibody competitive ELISA method for detecting the serum antibody of the duck flavivirus is established, and the fast and convenient duck flavivirus antibody detection method with strong specificity and high stability is obtained.
4. The invention determines the critical value of competitive ELISA yin-yang determination inhibition rate, wherein the inhibition rate (PI) is positive when the inhibition rate (PI) is more than or equal to 30%, and the inhibition rate (PI) is negative when the inhibition rate (PI) is less than 30%.
5. The competitive ELISA method for detecting the duck flavivirus antibody, which is established based on the duck flavivirus E protein and the antibody thereof, has the advantages of strong specificity, good stability and the like, can be used for detecting the duck flavivirus serum antibody, and provides a technical platform for evaluation of the immune effect of the duck flavivirus, monitoring of epidemic situations and epidemiological investigation.
Drawings
FIG. 1 shows the amplification result of the sequence (900bp) of the DFV-E gene of the present invention, in which the left arrow indicates the DFV-E gene (900 bp);
FIG. 2 shows the amplification result of the DFV-E1 gene (660bp) of the present invention, in which the left arrow indicates DFV-E1 gene (660 bp);
FIG. 3 is a SDS-PAG electrophoretogram of the E protein 1 of duck flavivirus of the present invention, in which the right arrow indicates the E protein 1 band of duck flavivirus;
FIG. 4 is a SDS-PAG electrophoretogram of the E protein 2 of duck flavivirus of the present invention, in which the right arrow indicates the band of the E protein 2 of duck flavivirus.
Detailed Description
The competitive ELISA method based on the monoclonal antibody against E protein of duck flavivirus and the application thereof are described in the following figures and examples, which are not intended to limit the content of the present invention, all reagents used in the following examples can be purchased commercially, and the procedures in the following examples can be performed by laboratory methods without specific reference.
Example 1
Obtaining of duck flavivirus E protein
(1) Primer design
According to the E gene sequence of duck flavivirus published by GenBank, a pair of cloning primers (C1 and C2) is designed to amplify a sequence containing DFV-E gene, and the size is 900 bp; a pair of specific expression primers (E1 and E2) aiming at a complete E gene (660bp) is designed according to a prokaryotic expression vector and a target gene sequence, and two enzyme cutting sites of EcoRI and XholI are inserted. The primer sequence was synthesized by Dalianbao bioengineering company.
TABLE 1 nucleotide sequence of primer for amplifying FMDV-A-VP1 gene
(2) Cloning of genes
The gene of the duck flavivirus virus is used as a template, a sequence (900bp) containing the DFV-E gene is obtained by utilizing a first pair of cloning primers C1 and C2 for amplification, and then the sequence is obtained by utilizing a second pair of expression primers E1 and E2 for amplification to obtain the DFV-E1 gene (660 bp). The cloning results of the DFV-E gene (900bp) are shown in FIG. 1, and the cloning results of the DFV-E1 gene (660bp) are shown in FIG. 2.
TABLE 2 Polymerase Chain Reaction (PCR) System
(3) Expression vector construction, induction expression and purification
FMDV-A-VP1 gene is connected with pET-32a and pGEX-6p-1 plasmids respectively to construct expression plasmids PET-32a-E1 and pGEX-6p-1-E1, the expression plasmids PET-32a-E1 and pGEX-6p-1-E1 are respectively transferred into prokaryotic expression host escherichia coli BL21 to be induced and expressed to obtain fusion protein 1 and fusion protein 2 respectively, and the fusion protein 1 and the fusion protein 2 are purified through His and GST column chromatography to obtain duck flavivirus E protein 1 and duck flavivirus E protein 2 respectively. The electrophoretograms of the fusion proteins are shown in FIGS. 3 and 4. The duck flavivirus E protein 1 and the duck flavivirus E protein 2 are same sequence proteins obtained by different expression modes, and the protein sequences of the two are shown in SEQ ID NO. 1.
Example 2
Obtaining monoclonal antibody of E protein of duck flavivirus
Step a), animal immunization: a7-week-old BALB/c female mouse is immunized by using duck flavivirus E protein 1 induced by an expression plasmid PET-32a-E1, and after 3 times of immunization, a mouse with the titer of each immunoassay larger than 1:12800 is screened out.
Step b), cell fusion: taking spleen cells of the immune mice obtained in the step a), carrying out cell fusion with myeloma cells SP2/0, using duck flavivirus E protein 2 obtained by induction of expression plasmids pGEX-6p-1-E1 as an envelope antigen, and carrying out indirect ELISA screening on the fused cells to obtain hybridoma cells.
Step c), identifying the stability of hybridoma cell strains: after hybridoma cells were continuously passaged and thawed after cryopreservation, culture supernatants were collected, respectively, and OD450 values were determined, as shown in Table 3. The results show that: the titer of the antibody secreted by the 3 hybridoma cell strains is kept to be changed among 1 gradient and is relatively stable.
Step d) monoclonal antibody subtype identification: and (3) detecting the subclasses of monoclonal antibody culture supernatants of hybridoma cell strains 2B6, 3D2 and 4E5 according to the operation of a mouse monoclonal antibody detection kit of SIGMA company, wherein the monoclonal antibodies are IgM subtype and kappa light chain.
TABLE 3 hybridoma cell antibody secretion stability assay
| 2B6 | | 4E5 | |
| Generation | |||
| 1 | 1：64 | 1：256 | 1：1024 |
| 5 th generation | 1：32 | 1：512 | 1：1024 |
| Generation 10 | 1：64 | 1：512 | 1：512 |
| After resuscitation | 1：64 | 1：512 | 1：1024 |
Example 3
Monoclonal antibody blocking ELISA assay and bulk cloning
Coating 0.5 mu g/mL recombinant protein pGEX-6p-1-E, adding diluted duck flavivirus negative and positive serum in a multiple ratio, and adding purified monoclonal antibody for monoclonal antibody blocking ELISA. The results are shown in Table 4, and as the dilution of the duck flavivirus E negative serum and the dilution of the duck flavivirus E positive serum are increased, the P/N values of the 2B6 monoclonal antibody supernatants, the 3D2 monoclonal antibody supernatants and the P/N values of the 4E5 monoclonal antibody supernatants are all increased, which indicates that the duck flavivirus E positive serum can block the 3 monoclonal antibody supernatants.
TABLE 4 monoclonal antibody blocking ELISA
Mass cloning of monoclonal antibody: according to the antibody titer, stability and monoclonal antibody blocking identification results, 4E5 with the best biological characteristics is finally selected as a competitive antibody of monoclonal antibody competitive ELISA. And injecting the obtained hybridoma cell 4E5 strain into the abdominal cavity of a mouse, feeding the injected mouse for 1 week, collecting ascites of the mouse with the enlarged abdomen, and purifying the ascites to obtain the duck flavivirus E protein monoclonal antibody.
Example 4
Determination of antigen coating concentration and dilution of serum to be tested
The pGEX-6p-1-E1 protein coating concentration and the dilution of the serum to be tested were determined by matrix titration (see Table 5). The results show that: the coating concentration of the protein antigen is 0.5 mug/mL, and the dilution multiple of the serum to be detected is 1:2, inhibition (PI%) was maximal and negative OD450 values were close to 1.0. Therefore, the coating concentration of pGEX-6p-1-E1 protein is determined to be 0.5 mug/mL, and the optimal dilution multiple of the serum to be detected is 1: 2.
TABLE 5 determination of antigen coating concentration and dilution of serum to be tested
Example 5
Monoclonal antibody dilution determination
The monoclonal antibody 4E5 was diluted to 1X 50, 1X 100, 1X 200, 1X 400, 1X 800, 1X 1600, 1X 3200, and 1X 6400 respectively for competition ELISA (see Table 6). The results show that: the inhibition rate (PI%) of mAb 4E5 was maximal at 1X 400, and the negative OD450 value was close to 1.0. Therefore, the dilution is 1X 400 times the optimal dilution of the monoclonal antibody 4E 5.
TABLE 6 monoclonal antibody optimum dilution
Example 6
Determination of critical value of yin and yang
45 parts of duck flavivirus negative serum is selected, competitive ELISA is carried out by using optimized conditions, and PI% average value and standard deviation value (SD) of all samples are calculated. The results are shown in Table 7, where the average inhibition was 7.73%, the standard deviation was 7.46%, and the cut-off value was 30%, which is the criterion X +3 SD. Therefore, the serum PI to be detected is more than or equal to 30 percent and is judged to be positive, and the PI is less than 30 percent and is judged to be negative.
TABLE 7 determination of the Yin-Yang Critical value of competitive ELISA
Example 7
Results of specificity test
Monoclonal antibody competition ELISA was performed with duck flavivirus negative and positive sera, newcastle disease negative and positive sera, Riemerella anatipestifer negative and positive sera, duck plague negative and positive, avian influenza H5 and H9 negative and positive sera as the sera to be tested (see Table 8 for results). The results show that: only the duck flavivirus positive serum can block the reaction of the monoclonal antibody and the antigen, and the rest of the sera are negative, which shows that the established monoclonal antibody competition ELISA has strong specificity.
TABLE 8 specificity test results
Example 8
Results compared to Duck flavivirus neutralization assay
The results are shown in table 9, and show that 100 parts of serum are detected by the monoclonal antibody competition ELISA and the monoclonal antibody competition ELISA, and the positive samples and the negative samples are respectively 51 parts and 46 parts, so that the positive detection coincidence rate of the established monoclonal antibody competition ELISA and the neutralization test is 96.23%, and the negative coincidence rate is 97.87%.
TABLE 9 coincidence rates detected by the two methods
Example 9
Competitive ELISA detection of duck flavivirus serum
Collecting 20 parts of duck serum of 4 duck farms respectively, and performing negative and positive judgment on the serum, wherein the method comprises sample dilution, antigen coating, sealing, sample adding and monoclonal antibody adding, enzyme labeling antibody adding, color development and termination, measuring value and data processing, and the competitive ELISA detection method comprises the following steps:
step 1) antigen coating: the used coating antigen is duck flavivirus E protein 2 induced by expression plasmid pGEX-6p-1-E1, and the coating concentration of the coating antigen is 0.5 ug/mL. And (3) sealing: and (3) sealing the antigen coated in the step 2) by using 1% BSA solution sealing solution for 40min to obtain a sealed ELISA plate.
Step 2) sample dilution: and (5) diluting the serum sample to be 2 times of the original volume to obtain the serum sample to be detected.
Step 3) sample adding: adding the serum sample to be detected obtained in the step 2) into the closed enzyme label plate obtained in the step 1), wherein the first 2 holes of the 1 st column are positive controls, the 3 and 4 holes are negative serum controls, and the sample to be detected is sequentially added from the 5 th hole, and each hole is 50 mu L. Placing into a constant temperature incubator at 37 deg.C for 45 min. Washing for 3 times, and patting on absorbent paper.
Step 4) adding monoclonal antibody: then adding 50 mu L of duck flavivirus E protein monoclonal antibody with the dilution of 1:400 into each well. Placing into a constant temperature incubator at 37 deg.C for 30 min. Washing for 3 times, and patting on absorbent paper.
Step 5) adding enzyme labeled antibody: adding an enzyme-labeled antibody into the enzyme-labeled plate obtained in the step 4), wherein the dilution concentration of the enzyme-labeled antibody is 1:2000, and each well is 50 mu L. Placing into a constant temperature incubator at 37 deg.C for 30 min. Washing for 3 times, and patting on absorbent paper. The antibody of the enzyme-labeled antibody used was goat anti-mouse IgG-HRP.
Step 6) color development and termination: adding a freshly prepared substrate solution into the enzyme label plate added with the enzyme-labeled antibody obtained in the step 5), wherein each well is 50 mu L, oscillating and mixing, incubating for 20min at room temperature in a dark place for color development, adding a stop solution, wherein each well is 50 mu L, and uniformly incubating for 5min to stop reaction to obtain a solution capable of being measured.
Step 7) measuring: measurement of OD of the solution obtained in step 6) which can be measured450nmAbsorbance value A of450nm；
Step 8) conditions for test establishment: a) the negative control OD value is more than 1.0; b) the inhibition rate of the positive control is more than 70%; c) the difference between the negative control OD value and the positive control is greater than 0.3.
Step 9) data processing: using the absorbance A determined in step 7)450nmCalculating the inhibition rate of the sample to be detected, wherein the inhibition rate PI% of the sample to be detected is (1-A)450sample/A450Negative control) × 100. The detection result is shown in 10. The test result shows that: the inhibition rate of positive serum is 72.39%, and the test is established; a positive sample with an OD value less than 0.8103 was obtained.
TABLE 10 test results of samples
| <> | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| A | 0.312 | 1.117 | 1.127 | 0.624 | 0.412 | 0.653 | 0.561 | 0.562 |
| B | 0.32 | 1.055 | 1.054 | 0.594 | 0.59 | 0.563 | 0.523 | 0.362 |
| C | 1.188 | 1.198 | 1.106 | 0.583 | 0.583 | 0.412 | 0.298 | 0.453 |
| D | 1.127 | 1.194 | 1.053 | 0.536 | 0.576 | 0.522 | 0.314 | 0.326 |
| E | 1.18 | 1.176 | 1.006 | 0.493 | 0.469 | 0.471 | 0.325 | 1.118 |
| F | 0.98 | 1.243 | 1.067 | 0.432 | 0.516 | 0.436 | 0.421 | 1.33 |
| G | 1.233 | 1.082 | 1.104 | 0.56 | 0.595 | 0.510 | 0.463 | 1.3 |
| H | 1.023 | 1.089 | 1.195 | 0.463 | 0.746 | 0.528 | 0.541 | 1.149 |
The foregoing embodiments and description have been presented only to illustrate the principles and preferred embodiments of the invention, and various changes and modifications may be made therein without departing from the spirit and scope of the invention as hereinafter claimed.
Claims (9)
1. A duck flavivirus E protein comprises a duck flavivirus E protein 1 and a duck flavivirus E protein 2 which are obtained by two different expression modes. Taking a duck flavivirus genome as a template, performing PCR amplification by using a pair of cloning primers and a pair of expression primers to obtain a DFV-E gene, connecting the DFV-E gene with pET-32a and pGEX-6p-1 plasmids respectively to construct an expression plasmid PET-32a-E, pGEX-6p-1-E, transferring the expression plasmids PET-32a-E and pGEX-6p-1-E into prokaryotic expression host escherichia coli BL21 respectively for induction expression to obtain fusion protein 1 and fusion protein 2 respectively, and purifying the fusion protein 1 and the fusion protein 2 respectively to obtain duck flavivirus E protein 1 and duck flavivirus E protein 2;
the method is characterized in that: the pair of cloning primers comprises a primer C1: 5'-gcaggattgtgcagaag-3', primer C2: 5'-ccatgcagtgtcaccca-3', respectively;
the pair of expression primers comprises a primer E1: 5'-ccggaattcaccgctgagatggagga-3', primer E2: 5'-ccgctcgagctggtacctgatctgtc-3', respectively;
the sequences of the duck flavivirus E protein 1 and the duck flavivirus E protein 2 are shown in SEQ ID NO 1.
2. The duck flavivirus E protein of claim 1, wherein: the duck flavivirus E protein 1 and the duck flavivirus E protein 2 are same sequence proteins obtained by different expression modes.
3. The duck flavivirus E protein of claim 1, wherein: the fusion protein 1 and the fusion protein 2 are purified by His and GST column chromatography.
4. The duck flavivirus E protein of claim 1, wherein: the PCR amplification comprises the following specific steps:
step A: taking duck flavivirus E gene as a template, amplifying by using a 1 st pair of cloning primers C1 and C2 to obtain a sequence containing the DFV-E gene, and amplifying the sequence by using a 2 nd pair of expression primers E1 and E2 to obtain the DFV-E1 gene;
and B: purifying the DFV-E gene obtained by amplification in the step A, and connecting with a pMD18-T vector to construct a pMD18-T-E cloning vector;
and C: and B, constructing pMD18-T-E in the step B as a template, performing PCR amplification by using expression primers E1 and E2, purifying and recovering a PCR product, and performing double enzyme digestion on the PCR purified product and expression vectors pET-32a and pGEX-6p-1 by using EcoRI and XholI endonucleases respectively.
5. A method for preparing the duck flavivirus E protein and the monoclonal antibody thereof of any one of claims 1-4, which comprises the following steps:
step a), animal immunization: immunizing a BALB/c female mouse with the age of 5-7 weeks by using duck flavivirus E protein 1 obtained by induction of expression plasmid PET-32a-E, and screening out a mouse with the immunoassay titer of more than 1:12800 after 3 times of immunization;
step b), cell fusion: taking spleen cells of the immunized mouse obtained in the step a), carrying out cell fusion with myeloma cells SP2/0, taking duck flavivirus E protein 2 obtained by induction of expression plasmids pGEX-6p-1-E as envelope antigen, and carrying out indirect ELISA screening on the fused cells to obtain hybridoma cells;
step c), mass cloning of monoclonal antibody: injecting the hybridoma cells obtained in the step b) into the abdominal cavity of a mouse, feeding the injected mouse for 1-3 weeks, collecting ascites of the mouse with the swollen abdomen, and purifying the ascites to obtain the duck flavivirus E protein monoclonal antibody.
6. The preparation method of the duck flavivirus E protein and the monoclonal antibody thereof according to claim 5, wherein the duck flavivirus E protein 1 and the duck flavivirus E protein 2 are the same sequence proteins obtained by different expression modes, and the protein sequences of the two are shown in SEQ ID NO. 1.
7. A competitive ELISA method based on the duck flavivirus E protein and the monoclonal antibody thereof of any one of claims 1-6, which comprises sample dilution, antigen coating, blocking, sample adding and monoclonal antibody adding, enzyme labeling of antibody, color development and termination, measurement, and data processing, and is characterized in that: in the competitive ELISA detection method:
step 1), sample dilution: diluting the serum sample to 2 times of the original volume to obtain a serum sample to be detected;
step 2), antigen coating: the used envelope antigen is duck flavivirus E protein 2 obtained by induction of expression plasmid pGEX-6p-1-E, and the envelope concentration of the envelope antigen is 0.5 mug/mL;
step 3), sealing: sealing the antigen coated in the step 2) with sealing liquid for 40-60 min to obtain a sealed ELISA plate;
step 4), sample adding and monoclonal antibody adding: adding the serum sample to be detected obtained in the step 1) into the closed enzyme label plate obtained in the step 3), so that a serum antibody is combined with the coating antigen, then adding the duck flavivirus E protein monoclonal antibody with the dilution of 1:400, so that the duck flavivirus E protein monoclonal antibody is combined with the residual coating antigen which is not reacted with the serum antibody, and obtaining the enzyme label plate after the reaction of the sample and the monoclonal antibody;
step 5), adding enzyme labeled antibody: adding an enzyme-labeled antibody into the enzyme-labeled plate after the reaction of the sample and the monoclonal antibody obtained in the step 4), wherein the dilution concentration of the enzyme-labeled antibody is 1:2000, and the enzyme-labeled antibody is combined with the duck flavivirus E protein monoclonal antibody but not with a serum antibody to obtain the enzyme-labeled plate after the enzyme-labeled antibody is added;
step 6), color development and termination: adding a freshly prepared substrate solution into the enzyme label plate added with the enzyme-labeled antibody obtained in the step 5), oscillating and mixing, incubating at room temperature for 10-20 min in a dark place for color development, adding a stop solution into each hole, and uniformly mixing to stop reaction to obtain a solution capable of being measured;
step 7), measuring: measurement of OD of the solution obtained in step 6) which can be measured450nmAbsorbance value A of450nm；
Step 8), data processing: using the absorbance A determined in step 7)450nmCalculating the inhibition rate of the sample to be detected, wherein the inhibition rate PI% of the sample to be detected is (1-A)450sample/A450Negative control) × 100.
8. The competitive ELISA method based on duck flavivirus E protein mab of claim 7, wherein: in the step 3), the concentration of the confining liquid is as follows: 1% BSA solution, 1% gelatin solution, 5% fetal bovine serum solution, 5% skim milk powder solution.
9. The competitive ELISA method based on duck flavivirus E protein mab of claim 7, wherein: in the step 5), the antibody of the enzyme-labeled antibody is goat anti-mouse IgG-HRP; a is described450The negative control is absorbance value A determined by using serum without duck flavivirus infection450。
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