CN113817070B - ELISA detection kit for type 1 duck hepatitis A virus specific fusion protein S1 antigen and type 1 duck hepatitis A virus antibody - Google Patents

ELISA detection kit for type 1 duck hepatitis A virus specific fusion protein S1 antigen and type 1 duck hepatitis A virus antibody Download PDF

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CN113817070B
CN113817070B CN202110926349.8A CN202110926349A CN113817070B CN 113817070 B CN113817070 B CN 113817070B CN 202110926349 A CN202110926349 A CN 202110926349A CN 113817070 B CN113817070 B CN 113817070B
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duck hepatitis
virus
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fusion protein
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胡玉立
刘国兴
祝春花
徐松
顾素云
肖爱芳
周平
谢红玲
石宝兰
漆世华
冯钊
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National Pharmaceutical Group Animal Health Co ltd
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Abstract

The invention provides an ELISA detection kit for a type 1 duck hepatitis A virus specific fusion protein S1 antigen and a type 1 duck hepatitis A virus antibody, wherein the amino acid sequence of the type 1 duck hepatitis A virus specific fusion protein S1 antigen is shown as SEQ ID NO. 1. The ELISA detection kit comprises: an ELISA plate coated with a type 1 duck hepatitis A virus specific fusion protein S1 antigen. The invention adopts 6 specific polypeptide epitopes and obtains the amino acid sequence shown in SEQ ID NO.1 by optimizing the sequence among different specific epitopes and the copy number of the specific epitopes, and the type 1 duck hepatitis A virus specific fusion protein S1 has good antigen specificity, does not generate cross reaction with other common duck virus antibodies, and does not generate cross reaction with type 3 duck hepatitis A virus serum. Can be used for detecting type 1 duck hepatitis A virus antibody in duck serum and duck yolk.

Description

ELISA detection kit for type 1 duck hepatitis A virus specific fusion protein S1 antigen and type 1 duck hepatitis A virus antibody
Technical Field
The invention belongs to the technical field of biology, and relates to an ELISA detection kit for a type 1 duck hepatitis A virus specific fusion protein S1 antigen and a type 1 duck hepatitis A virus antibody.
Background
Duck Hepatitis A Virus (DHAV) belongs to Picornaviridae and avian hepatovirus (Avihepatovirus) and can cause acute, contact and high-lethality diseases of ducklings. According to antigenicity and genome difference, duck hepatitis A virus can be divided into DHAV-1, DHAV-2 and DHAV-3. Clinically, symptoms caused by DHAV-1, DHAV-2 and DHAV-3 cannot be distinguished, and are expressed as acute onset, high lethality and characteristic liver lesions, and the differential diagnosis can be carried out by molecular biological methods such as PCR and the like.
Immunological diagnostic methods such as ELISA have also been reported, especially the immunological cross-reaction of DHAV-3 with DHAV-1 antibody. Through genome analysis, the amino acid homology of DHAV structural proteins VP0, VP1 and VP3 is about 90%, and the amino acid homology is probably the reason for the immunological cross reaction among DHAV 3 genotypes (or serotypes). These factors make it easy for the immunological detection method originally applied to detecting DHAV-1 to show false positive, i.e., DHAV-3 stimulates the antibody produced by the host, and the immunological detection method for detecting DHAV-1 shows false positive.
Therefore, how to develop an ELISA detection kit for the duck hepatitis A virus type 1 antibody with good specificity has important significance.
Disclosure of Invention
In order to solve the technical problem, the invention provides an ELISA detection kit for a type 1 duck hepatitis A virus specific fusion protein S1 antigen and a type 1 duck hepatitis A virus antibody, which can specifically identify the type 1 duck hepatitis A virus, has good specificity, does not generate cross reaction with other common duck virus antibodies, and does not generate cross reaction with type 3 duck hepatitis A virus serum.
In the first aspect of the invention, the invention provides a type 1 duck hepatitis A virus specific fusion protein S1 antigen, and the amino acid sequence of the type 1 duck hepatitis A virus specific fusion protein S1 antigen is shown in SEQ ID NO. 1.
In a second aspect of the invention, the invention provides a nucleic acid molecule for encoding the duck hepatitis A virus type 1 specific fusion protein S1 antigen, and the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 8.
In a third aspect of the invention, an expression vector of the type 1 duck hepatitis A virus specific fusion protein S1 antigen is provided, and the nucleotide sequence of an expression region of the expression vector is shown as SEQ ID No. 8.
The expression vector is capable of expressing the nucleic acid in a prokaryotic or eukaryotic host cell. The carrier may be a conventional carrier; can be plasmid vector, phage vector, virus vector;
in a fourth aspect of the invention, there is provided an engineered bacterium or eukaryotic host cell comprising said expression vector.
In a fifth aspect of the present invention, there is provided a method for preparing a type 1 duck hepatitis a virus specific fusion protein S1 antigen, the method comprising:
obtaining an expression vector of the type 1 duck hepatitis A virus specific fusion protein S1 antigen;
transforming the expression vector of the type 1 duck hepatitis A virus specific fusion protein S1 antigen into an escherichia coli competent cell to obtain an expression engineering bacterium;
and performing induction expression and purification on the expression engineering bacteria to obtain the type 1 duck hepatitis A virus specific fusion protein S1 antigen.
In a sixth aspect of the present invention, there is provided an ELISA detection kit for type 1 duck hepatitis a virus antibodies, comprising: an ELISA microplate coated with the type 1 duck hepatitis A virus specific fusion protein S1 antigen of claim 1.
Furthermore, the coating amount of the type 1 duck hepatitis A virus specific fusion protein S1 antigen is 0.05-2 mug/hole.
Further, the ELISA detection kit further comprises:
horseradish peroxidase-labeled enzyme-labeled secondary antibody;
a standard negative serum;
standard positive serum;
detection reagent: coating solution, coating lotion, diluent, washing solution, confining solution, antibody diluent, developing solution and stop solution.
In the technical scheme, the negative control serum is derived from an SPF duck, and the positive serum is collected after the SPF duck is immunized with a type 1 duck hepatitis A virus live vaccine; the enzyme-labeled secondary antibody is rabbit anti-duck IgY labeled by horseradish peroxidase.
In a seventh aspect of the present invention, there is provided a method for using the ELISA detection kit using the type 1 duck hepatitis a virus antibody, the method comprising:
diluting the type 1 duck hepatitis A virus specific fusion protein S1 antigen, adding the diluted type 1 duck hepatitis A virus specific fusion protein S1 antigen into an ELISA plate for adsorption, and sealing after air drying to obtain an ELISA plate coated with the type 1 duck hepatitis A virus specific fusion protein S1 antigen;
adding the serum to be detected, the negative serum and the positive serum which are respectively used as primary antibodies into plate holes of the ELISA plate coated with the type 1 duck hepatitis A virus specific fusion protein S1 antigen for incubation; washing and spin-drying the waste liquid, adding an enzyme-labeled secondary antibody labeled by horseradish peroxidase, incubating, washing and spin-drying;
adding a developing solution for incubation at room temperature in a dark place, then adding a stop solution for stopping the reaction, and measuring the OD value on an enzyme-linked immunosorbent assay (ELISA) instrument at the wavelength of 450 nm.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
1. the invention provides a 1-type duck hepatitis A virus specific fusion protein S1, wherein the amino acid sequence of the S1 protein is shown in SEQ ID NO.1, 6 specific epitopes distributed in 1-type duck hepatitis A virus structural proteins VP0, VP1 or VP3 are connected in series to form the fusion protein, different specific epitopes are connected through a polypeptide GPG, the amino acid sequences of the 6 specific polypeptide epitopes are shown in SEQ ID NO. 2-7, so that the different specific epitopes are not interfered with each other, the amino acid sequence shown in SEQ ID NO.1 is obtained by optimizing the sequence among the different specific epitopes and the copy number of the specific epitopes, and the S1 fusion protein has extremely added specificity aiming at 1-type duck hepatitis A virus antibodies;
further, the sequence of the S1 protein is optimized by the codon preference of escherichia coli, and the S1 protein coding sequence is shown as SEQ ID No. 8; the fusion protein S1 can realize soluble expression through an escherichia coli expression system, and the fusion protein can be purified through metal affinity chromatography to obtain recombinant protein with higher purity and activity;
the 1-type duck hepatitis A virus specific fusion protein S1 has good antigen specificity, does not generate cross reaction with other common duck virus antibodies, and does not generate cross reaction with 3-type duck hepatitis A virus serum. Can be used for detecting the duck hepatitis A virus antibody type 1 in duck serum and duck egg yolk.
2. The ELISA detection kit for the type 1 duck hepatitis A virus antibody provided by the invention can be used for detecting the type 1 duck hepatitis A virus antibody in duck serum and duck egg yolk antibody, has strong specificity and high sensitivity, and can be used for differential diagnosis with the type 3 duck hepatitis A virus antibody.
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In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is an SDS-PAGE electrophoresis chart of the type 1 duck hepatitis A virus specific fusion protein S1 antigen expression and purification provided by the embodiment of the present invention; m, protein marker; lane 1, supernatant after e.coli induced expression; lane 2, precipitation after e.coli induced expression; lane 3, recombinant S1 protein after metal affinity chromatography;
FIG. 2 is a schematic diagram of tandem connection of 6 specific epitopes of a type 1 duck hepatitis A virus specific fusion protein S1 antigen provided by an embodiment of the present invention;
FIG. 3 is an SDS-PAGE electrophoresis chart showing the expression and purification of type 1 duck hepatitis A virus specific fusion protein S1 antigen provided in comparative example 1; lane 1 is post-induction supernatant, lane 2 is pellet, lane 3 is purified protein, and M is protein marker.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are illustrative of the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
The technical scheme of the invention has the following general idea:
according to a typical implementation manner of the embodiment of the invention, the type 1 duck hepatitis A virus specific fusion protein S1 antigen is provided, and the amino acid sequence of the type 1 duck hepatitis A virus specific fusion protein S1 antigen is shown as SEQ ID No. 1.
The duck hepatitis A virus structural protein antigen is characterized by being formed by connecting 6 specific epitopes distributed in type 1 duck hepatitis A virus structural proteins VP0, VP1 or VP3 in series (and the 6 specific polypeptide epitopes have 2 copies), wherein different specific epitopes are connected through a polypeptide GPGPG (as shown in figure 2) (6 × His represents a histidine tag for metal affinity chromatography, L1-L6 represent specific polypeptides 1-6, 6 specific polypeptide epitope amino acid sequences are shown as SEQ ID NO. 2-7), the selection and arrangement sequence of the specific polypeptides and a connecting peptide GPG among the polypeptides are optimized. The amino acid sequence of the finally obtained type 1 duck hepatitis A virus specific fusion protein S1 antigen is shown in SEQ ID NO. 1. The use of GPGPG as a linker peptide between polypeptides is beneficial to maintain the structure of adjacent specific polypeptides, while the use of flexible GSGS and the like is not beneficial to the expression of recombinant proteins (FIG. 3, lane 1 is post-induction supernatant, lane 2 is precipitate, lane 3 is purified protein, M is protein marker).
In order to make the type 1 duck hepatitis A virus specific fusion protein S1 antigen soluble and efficiently expressed, the codon is optimized to obtain the nucleotide sequence shown in SEQ ID NO.8, which is suitable for expression in Escherichia coli.
Constructing an expression plasmid: the DNA sequence T1 for coding S1 is synthesized by Beijing Optimalaceae biotechnology, inc., the synthesized gene sequences T1, pET28 (a) + vector are purified after double enzyme digestion by NcoI and XhoI, then T4 DNA ligase is used for connecting the double enzyme digested T3 and pET28 (a) + vector, and the obtained product is transformed into BL21 (DE 3) competent cells, and the recombinant expression bacterium BL21 (DE 3) -S1 for expressing S1 is obtained.
The expression strain BL21 (DE 3) -S1 is induced and expressed and then purified by metal affinity chromatography. In the process of protein expression, the IPTG induction concentration is 0.5mmol/L, the culture temperature of the engineering bacteria of the escherichia coli is 37 ℃, the culture temperature of the escherichia coli in the stage of protein induction expression is 16-25 ℃, and under the condition, the S1 expression quantity is high and exists mainly in a soluble form.
And then preparing an ELISA detection kit from the purified 1-type duck hepatitis A virus specific fusion protein S1 antigen: and coating the type 1 duck hepatitis A virus specific fusion protein S1 antigen to obtain the ELISA plate.
Preferably, the coating amount of the type 1 duck hepatitis A virus specific fusion protein S1 antigen is 0.05-2 mug/hole.
The ELISA detection kit further comprises:
horseradish peroxidase-labeled enzyme-labeled secondary antibody: rabbit anti-duck IgY marked by horseradish peroxidase;
a standard negative serum;
standard positive sera;
detection reagent: coating solution, coating lotion, diluent, washing solution, confining solution, antibody diluent, developing solution and stop solution.
The ELISA detection kit for the type 1 duck hepatitis A antibody comprises the following steps in the using process:
(1) Diluting the sample to be detected by using a sample diluent according to the instruction, adding the sample to the antigen-coated hole, and then adding a negative control and a positive control.
(2) And (3) placing the antigen-coated plate containing the sample to be detected and the negative and positive controls in a thermostat for incubation for 30 minutes, so that the antibody in the sample to be detected is fully combined with the antigen in the antigen-coated plate to form an antigen-antibody compound.
(3) The liquid in the antigen-coated plate was discarded, and the antigen-coated wells were then washed 4 to 6 times with a washing solution.
(4) Adding rabbit anti-duck IgY marked by HRP, and placing the mixture in a thermostat for incubation for 30 minutes.
(5) The liquid in the antigen-coated plate was discarded, and then the antigen-coated wells were washed 4 to 6 times with a washing solution.
(6) Adding TMB substrate, and placing in an incubator to be protected from light and develop for 10 minutes.
(7) Stop solution was added and absorbance was read at a wavelength of 450nm using a microplate reader.
The following will describe in detail an ELISA detection kit of a duck hepatitis a virus type 1 specific fusion protein S1 antigen and duck hepatitis a virus type 1 antibody according to the present application with reference to examples and experimental data.
Example 1 expression and purification of recombinant S1 protein
(1) Construction of expression vectors
Amplifying a template which is synthesized by Beijing Optimalaceae biotechnology and is optimized by the codon preference of escherichia coli by using primers S1F and S1R to obtain a DNA fragment with a nucleotide sequence shown as SEQ ID NO. 8;
the DNA fragment was used as a template for amplification using a 50. Mu.L system containing 10. Mu.L of 5 XPCR buffer, 4. Mu.L of dNTP mix (2.5 mM), 1. Mu.L of each of primers S1F and S1R (10. Mu.M), high fidelity DNA polymerase PrimeStar HS 0.5. Mu.L, ddH 2 O, 0.5. Mu.L of template diluted 10-fold. Wherein the sequence of the primer S1F is
<xnotran> 5'-cgcgccatgggtagtcatcatcatcatca-3' ( SEQ ID NO.9 ); </xnotran> The sequence of the primer S1R is
5 'cgcggctcgagtatttcaggctaccaaaaggtggc-3' (shown as SEQ ID NO. 10).
The amplification reaction was performed on an EppendorffPCR instrument with the program: 95 ℃ for minutes. 35 cycles of 95 ℃ for 10 seconds, 55 ℃ for 30 seconds and 68 ℃ for 30 seconds. 10 minutes at 68 ℃. The DNA fragment was purified using the Omega cycle pure kit.
After the purified DNA fragment and pET28 (a) + empty carrier are double-digested by NcoI and XhoI, the DNA fragment and the carrier are respectively purified, then the DNA fragment and the carrier are connected by T4 DNA ligase, and are transformed into BL21 (DE 3) competence to obtain a positive transformant, and plasmid sequencing is extracted to confirm that the constructed sequence is consistent with the expected sequence.
(2) Inducible expression of recombinant proteins
Positive transformants after sequencing verification were picked, inoculated into fresh Luria-Bertani medium, shake-cultured at 37 ℃ at 180rpm to OD600 of about 0.6, and added with 0.5mM IPTG to induce expression at 16 ℃ for 16h. The resulting suspension was then centrifuged to collect a bacterial solution, disrupted by ultrasonication, centrifuged to remove the precipitate, and purified by metal affinity chromatography (Ni-IDA resin, genetic script) according to the instructions of the reagents.
(3) An SDS-PAGE electrophoresis chart of the type 1 duck hepatitis A virus specific fusion protein S1 antigen expression and purification provided by the embodiment of the invention is shown in figure 1, and the Image J software is used for analyzing the result in the figure 1, so that the purity of the obtained type 1 duck hepatitis A virus specific fusion protein S1 antigen is more than 90%.
Comparative example 1
The fusion protein S1 in this comparative example uses the flexible peptide GSGS instead of "GPGPG" of example 1 as a linker peptide between polypeptides; the method comprises the following specific steps:
amplifying a DNA fragment with a nucleotide sequence shown in SEQ ID NO.11 synthesized by Beijing Strataceae Biotechnology Limited by using primers S2F (sequence 5-cgcgcctgggttctctccaccaccaccaccaccaccaccac-3'; shown in SEQ ID NO. 12) and S2R (sequence 5-cgcgctcgagttatttcagagagaaccgaaggtagct-3'; shown in SEQ ID NO. 13); the other steps were the same as in example 1.
The SDS-PAGE electrophoresis Image of the fusion protein S1 expression purification in the comparative example is shown in FIG. 3, and as can be seen from FIG. 3, the band is thinner than that of the Marker, the expression level is lower, and the result of FIG. 1 is analyzed by Image J software, so that the purity of the obtained duck hepatitis A virus type 1 specific fusion protein S1 antigen is about 80%, which indicates that the adoption of other flexible peptides is not beneficial to the expression and purification of recombinant proteins.
Example 2 serum type 1 duck hepatitis A virus antibody ELISA detection kit and preparation method thereof
1. Serum 1 type duck hepatitis A virus antibody ELISA detection kit
1. Coated enzyme label plate
(1) Coating liquid: 0.01M PBS (pH 7.2);
(2) Coating lotion: 0.01M PBS (pH 7.2);
(3) The coating method comprises the following steps: the fusion protein S1 purified in example 1 was diluted with the coating solution until the protein content was 5. Mu.g/ml, and 100. Mu.L of the protein was added to each well of a 96-well microplate, and the mixture was adsorbed at 2-8 ℃ for 24 hours. The coating solution was emptied and the plate was washed 3 times with wash solution using coating.
When determining the coating concentration of the antigen protein, firstly using the coating solution to dilute the purified fusion protein S1 until the protein content is 0.05 mu g/ml,0.1 mu g/ml,0.2 mu g/ml,0.3 mu g/ml,0.4 mu g/ml,0.5 mu g/ml,1 mu g/ml,2 mu g/ml,3 mu g/ml,4 mu g/ml,5 mu g/ml,6 mu g/ml,7 mu g/ml,8 mu g/ml,9 mu g/ml,10 mu g/ml and 20 mu g/ml, respectively adding the fusion protein S1 into micropores of a 96-well enzyme label plate, adding 100 mu L of the fusion protein S into each pore, and adsorbing the fusion protein S1 at 2-8 ℃ for 24 hours. From this, an optimized coating concentration, i.e., 5. Mu.g/ml, was selected.
2. And (3) sealing:
(1) Sealing liquid for coating: BSA or skim milk, concentration 2% -10%;
(2) And (3) sealing operation: the coating solution was air-dried, 150. Mu.L/well of the coating blocking solution was added, and the mixture was left at 2 to 8 ℃ overnight. And removing the sealing liquid and then naturally drying for 24 hours.
3. Incubation of Positive and negative serum (Primary antibody incubation)
(1) Sample diluent: 1% BSA, 0.05% NaN3 in 0.01M PBS (pH 7.2).
(2) Preparation of standard negative serum: diluting SPF duck serum.
(3) Preparation of standard positive serum: the Specific Pathogen Free (SPF) duck hepatitis A virus vaccine is prepared by collecting SPF duck immune inactivated type 1 duck hepatitis A virus.
(4) Washing liquid: 10 XWash in 0.1M PBS (pH 7.2) containing 0.5% Tween 20, with ddH before use 2 Diluted 10 times with O.
(5) The operation is as follows: the serum to be detected is diluted by a sample diluent and added into a coated plate hole with the concentration of 100 mu L/hole. The standard positive serum or the standard negative serum is directly sucked and added into a coated plate hole, and the volume is 100 mu L/hole. The plate was placed on an ELISA plate at 37 ℃ for 30min.
The plate was washed 5 times with washing solution after emptying the serum dilution.
4. Incubation of enzyme-labeled Secondary antibodies
The horseradish peroxidase-labeled secondary enzyme-labeled antibody (HRP-labeled rabbit anti-duck IgY is a commercial reagent) is diluted to a working concentration (12000 times diluted by 0.01M PBS) by using a serum diluent, 100 mu L/hole is added into the hole of the enzyme-labeled plate, and the mixture is placed at 37 ℃ for 30min.
5. Color development:
adding 100 mu L of TMB substrate solution, mixing with gentle shaking, and reacting at 37 ℃ for 10min.
TMB substrates are commercial reagents.
6. And (4) terminating:
stopping liquid: 2M sulfuric acid.
After the color development is finished, 100. Mu.L/well of stop solution is added.
7. Reading a plate:
reading the absorbance of the microplate at a wavelength of 450 nm.
The type 1 duck hepatitis A virus specific fusion protein S1 antigen provided by the invention is applied to preparation of an ELISA kit, and can also be used for preparation of products in various forms such as immune colloidal gold detection test strips, immunofluorescence, immunoturbidimetry, chemiluminescence and the like.
2. Operation steps of ELISA detection kit for serum type 1 duck hepatitis A virus antibody
(1) Adding 148.5 mu L of serum diluent into 1.5 mu L of sample to be detected, shaking and mixing uniformly, transferring 100 mu L of sample to an antigen coated plate, adding 100 mu L of negative control and positive control to other antigen coated holes respectively, setting 3 times of repetition for each sample to be detected and control, and then standing and incubating the antigen coated plate for 1h at 37 ℃. The antigen coated plate is washed 4 times by a plate washing machine, 100 mu L of rabbit anti-duck IgY marked by HRP is added into each hole, and the mixture is kept stand and incubated for 30min at 37 ℃. Washing the antigen coated plate for 4 times by using a plate washing machine, adding 100 mu L of TMB substrate into each hole, developing for 10min in a dark place at 37 ℃, adding 100 mu L of stop solution, and reading a light absorption value at a wavelength of 450nm by using an enzyme labeling instrument.
(2) And (5) judging a result:
if OD450 is larger than or equal to negative control OD450 multiplied by 2.5, the judgment is positive;
if OD450 is less than or equal to negative control OD 450X 2, judging as negative;
if the negative control OD 450X 2.5 >.
Example 3 detection of samples by serum type 1 Duck hepatitis A Virus antibody ELISA detection kit
1. Preparation of serum 1 type and serum 3 type duck hepatitis A virus serum and yolk antibody:
diluting duck embryo attenuated virus seed 1 and serum 3 type duck hepatitis A virus with sterile normal saline to 10% 2.5 ELD 50 and/mL, inoculating 0.1mL of diluted virus solution to well-developed SPF duck embryos of 10 days old, and collecting allantoic fluid, allantoic membranes and embryos of the dead duck embryos for 72-96 h. Homogenizing the collected sample, filtering with 0.5mm copper gauze to collect supernatant, adding 0.2% formaldehyde, inactivating for 24 hr, and recovering supernatantAdding white oil, and emulsifying to obtain inactivated vaccine.
The SPF duckling is immunized with type 1 and serum type 3 duck hepatitis A virus inactivated vaccines respectively for 3 times, and is immunized once at 7 days old, 14 days old and 21 days old respectively. Sera were collected 7 days after the last immunization.
A duck hepatitis A virus antigen and an antibody negative duck of 140 days old are immunized with duck hepatitis A virus inactivated vaccines of type 1 and serum type 3 respectively for three times, the two immunizations are separated by one week, laid eggs are collected for one week continuously 7 days after the last immunization, and the eggs are used for preparing a yolk antibody. The yolk antibody is extracted by a chloroform method, namely, the yolk antibody is obtained by stirring physiological saline with 5 times of yolk liquid volume, uniformly mixing by vortex oscillation, adding chloroform with the same volume as the mixed liquid, uniformly mixing by oscillation, centrifuging and collecting supernatant fluid phase.
2. The serum type 1 duck hepatitis a virus serum (DHAV-1S 1 to DHAV-1S 3) and the serum type 3 duck hepatitis a virus serum (DHAV-3S 1 to DHAV-3S 3), the serum type 1 duck hepatitis a virus egg yolk antibody (DHAV-1E 1 to DHAV-1E 3), the serum type 3 duck hepatitis a virus egg yolk antibody (DHAV-3E 1 to DHAV-3E 3), and the pathogenic escherichia coli serum (e.coli, agar titer 1, 128) and the listeria anatipestifolia positive serum (r.anatipestififer, agar titer 1. The results are shown in Table 1.
TABLE 1 ELISA test results for serum and yolk antibody samples
Figure GDA0003343393320000091
From the data in table 1, it can be seen that:
negative control OD450 × 2.5=0.1475; negative control OD450 × 2=0.118;
in serum of the type 1 duck hepatitis A virus, DHAV-1S1 to DHAV-1S3 are positive;
DHAV-3S 1-DHAV-3S 3 in serum of the type 3 duck hepatitis A virus are all negative;
in the serum type 1 duck hepatitis A virus egg yolk antibody, DHAV-1E 1-DHAV-1E 3 are positive;
serum 3 type duck hepatitis A virus yolk antibodies (DHAV-3E 1-DHAV-3E 3 are all negative;
pathogenic Escherichia coli serum and Riemerella anatipestifer positive serum are negative; the ELISA detection kit for detecting the duck hepatitis A virus type 1 antibody provided by the embodiment of the invention is good in specificity, and does not generate cross reaction with other common duck virus antibodies or generate cross reaction with duck hepatitis A virus type 3 serum.
Meanwhile, the ELISA detection kit for detecting the duck hepatitis A virus type 1 antibody provided by the embodiment of the invention can be used for detecting the duck hepatitis A virus type 1 antibody level and diagnosing duck hepatitis A virus type 1 infection, and can also be used for differential diagnosis of duck hepatitis A virus type 1 and duck hepatitis A virus type 3 infection.
Finally, it should be further noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Sequence listing
<110> national drug group animal health Provisions Ltd
ELISA detection kit for <120> type 1 duck hepatitis A virus specific fusion protein S1 antigen and type 1 duck hepatitis A virus antibody
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Val Asp Gly Pro Gly Pro Gly Gln Trp Val Pro Asn Asp Asp Tyr Tyr
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Ala Cys Leu Arg Trp Gly Pro Gly Pro Gly Ile Thr Thr Ser Asp Thr
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Thr Gly Gly Gly Pro Gly Pro Gly Ile Leu Ala Ser Phe Gln Trp Gln
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Ser Thr Leu Thr Pro Ser Ala Arg Ile Asn Arg Tyr Gln Leu Ile Phe
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Arg Asn Ile Pro Gly Pro Gly Pro Gly Arg Pro Ile Pro Gly Thr Ser
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Glu Ala Thr Phe Gly Gly Pro Gly Pro Gly Lys Arg Arg Trp Lys Pro
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Arg Gly Tyr Phe Arg Gly Pro Gly Pro Gly Ala Pro Thr Ser Thr Thr
145 150 155 160
Leu Ser Arg Gly Ser Thr Gly Val Ile Pro Thr Leu Asp Gln Ser Gly
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Asp Glu Val Asp Gly Pro Gly Pro Gly Gln Trp Val Pro Asn Asp Asp
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Tyr Tyr Ala Cys Leu Arg Trp Gly Pro Gly Pro Gly Ile Thr Thr Ser
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Asp Thr Thr Gly Gly Gly Pro Gly Pro Gly Ile Leu Ala Ser Phe Gln
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Trp Gln Ser Thr Leu Thr Pro Ser Ala Arg Ile Asn Arg Tyr Gln Leu
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Ile Phe Arg Asn Ile Pro Gly Pro Gly Pro Gly Arg Pro Ile Pro Gly
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Thr Ser Glu Ala Thr Phe Gly Ser Leu Lys
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ggtagtcatc atcatcatca tcataaacgt cgttggaaac cgcgtggtta ttttcgtggt 60
ccgggtccgg gtgcgccgac aagtacaaca ctgagccgtg gtagcacagg tgttattccg 120
accctggatc agtcaggcga tgaagttgat ggtccgggtc ctggtcagtg ggttccgaat 180
gatgattatt atgcatgcct gcgctggggt ccgggtccag gtattacgac cagcgatacc 240
accggtggcg gtccgggtcc gggcattctg gcaagctttc agtggcagag taccctgaca 300
ccgtccgcac gtattaatcg ttatcagctg atttttcgta acattccggg tccgggtccg 360
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cgttggaaac ctcgtggtta ttttcgcggt ccgggtccgg gtgcaccgac cagcacaaca 480
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ggcccgggtc ctggccagtg ggttcctaat gatgattatt atgcttgcct gcgttggggt 600
ccgggcccgg gtattacaac gagcgataca accggtggtg gtccgggtcc gggtattctg 660
gcaagttttc agtggcagtc aaccctgacc ccgtccgcac gcattaatcg ttatcagctg 720
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aacgacgact actacgcttg cctgcgttgg ggtccgggtc cgggtatcac cacctctgac 240
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cgtcgttgga aaccgcgtgg ttacttccgt ggtccgggtc cgggtgctcc gacctctacc 480
accctgtctc gtggttctac cggtgttatc ccgaccctgg accagtctgg tgacgaagtt 540
gacggtccgg gtccgggtca gtgggttccg aacgacgact actacgcttg cctgcgttgg 600
ggtccgggtc cgggtatcac cacctctgac accaccggtg gtggtccggg tccgggtatc 660
ctggcttctt tccagtggca gtctaccctg accccgtctg ctcgtatcaa ccgttaccag 720
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Claims (9)

1. The specific fusion protein S1 antigen of the duck hepatitis A virus type 1 is characterized in that the amino acid sequence of the specific fusion protein S1 antigen of the duck hepatitis A virus type 1 is shown as SEQ ID No. 1.
2. A nucleic acid molecule encoding the duck hepatitis A virus type 1 specific fusion protein S1 antigen according to claim 1, wherein the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID No. 8.
3. An expression vector of a type 1 duck hepatitis A virus specific fusion protein S1 antigen is characterized in that the nucleotide sequence of an expression region of the expression vector is shown as SEQ ID No. 8.
4. A preparation method of an expression vector of a type 1 duck hepatitis A virus specific fusion protein S1 antigen is characterized by comprising the following steps:
obtaining the nucleic acid molecule of claim 2;
and carrying out purification and enzyme linkage on the nucleic acid molecule and an expression vector pET28 (a) + after double enzyme digestion by NcoI and XhoI respectively to obtain an expression vector of the type 1 duck hepatitis A virus specific fusion protein S1 antigen.
5. An engineering bacterium for expressing a type 1 duck hepatitis A virus specific fusion protein S1 antigen, which is characterized by comprising an expression vector of the type 1 duck hepatitis A virus specific fusion protein S1 antigen according to claim 3.
6. A preparation method of a type 1 duck hepatitis A virus specific fusion protein S1 antigen is characterized by comprising the following steps:
obtaining an expression vector of the type 1 duck hepatitis A virus specific fusion protein S1 antigen of claim 3;
transforming the expression vector of the type 1 duck hepatitis A virus specific fusion protein S1 antigen into an escherichia coli competent cell to obtain expression engineering bacteria;
and performing induction expression and purification on the expression engineering bacteria to obtain the type 1 duck hepatitis A virus specific fusion protein S1 antigen.
7. An ELISA detection kit for type 1 duck hepatitis A virus antibody is characterized in that the ELISA detection kit comprises: an ELISA plate coated with the type 1 duck hepatitis A virus specific fusion protein S1 antigen described in claim 1.
8. The ELISA detection kit for a type 1 duck hepatitis A virus antibody according to claim 7, characterised in that the amount of the coating of the type 1 duck hepatitis A virus specific fusion protein S1 antigen is 0.05-2 μ g/well.
9. The ELISA detection kit for a type 1 duck hepatitis A virus antibody according to claim 7, further comprising:
horseradish peroxidase-labeled enzyme-labeled secondary antibody;
a standard negative serum;
standard positive serum;
detection reagent: coating solution, washing solution for coating, diluent, washing solution, confining solution, antibody diluent, developing solution and stop solution.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104448005A (en) * 2014-12-13 2015-03-25 青岛宏昊生物科技有限公司 Fusion protein antigen of duck hepatitis A virus-3 VP1 protein and LTB and application of fusion protein antigen
CN105273065A (en) * 2015-11-02 2016-01-27 四川农业大学 1-type duck hepatitis A virus VP2 recombinant protein, ELISA kit and preparation method thereof
CN106370854A (en) * 2016-08-17 2017-02-01 中国科学院微生物研究所 Duck hepatitis A virus 1 antibody detection method and special-purpose kit thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104448005A (en) * 2014-12-13 2015-03-25 青岛宏昊生物科技有限公司 Fusion protein antigen of duck hepatitis A virus-3 VP1 protein and LTB and application of fusion protein antigen
CN105273065A (en) * 2015-11-02 2016-01-27 四川农业大学 1-type duck hepatitis A virus VP2 recombinant protein, ELISA kit and preparation method thereof
CN106370854A (en) * 2016-08-17 2017-02-01 中国科学院微生物研究所 Duck hepatitis A virus 1 antibody detection method and special-purpose kit thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
High yield expression of duck hepatitis A virus VP1 protein in Escherichia coli, and production and characterization of polyclonal antibody;Chuanfeng Li等;《Journal of Virological Methods》;20130410;第69-75页 *

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