CN111537729A

CN111537729A - Paralichthys rhabdovirus disease rapid diagnosis test paper and preparation method thereof

Info

Publication number: CN111537729A
Application number: CN202010323726.4A
Authority: CN
Inventors: 唐小千; 战文斌; 官曼玉; 龚娇娇; 绳秀珍; 邢婧
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2020-04-22
Filing date: 2020-04-22
Publication date: 2020-08-14
Anticipated expiration: 2040-04-22
Also published as: CN111537729B

Abstract

The invention discloses a test paper for rapidly diagnosing paralichthys rhabdovirus disease and a preparation method thereof. The diagnostic test paper comprises a carrier plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water absorption pad, wherein the water absorption pad, the nitrocellulose membrane and the sample pad are respectively positioned at the upper end, the middle part and the lower end of the carrier plate; the gold-labeled pad is loaded with a gold-labeled mouse anti-paralichthys olivaceus immunoglobulin monoclonal antibody, one end of the edge of the gold-labeled pad is overlapped below the sample pad, and the other end of the edge of the gold-labeled pad is overlapped above the nitrocellulose membrane; the nitrocellulose membrane is provided with two detection lines T1 and T2 and a quality control line C, and the quality control line C is close to the water absorption pad; the detection lines T1 and T2 are respectively coated with recombinant paralichthys rhabdovirus nucleoprotein and matrix protein, and the quality control line C is coated with goat anti-mouse IgG. The invention has the following advantages: the detection is quick, simple, convenient, sensitive and accurate; the cost is low, and the stability is good; is suitable for the rapid and accurate diagnosis of the paralichthys rhabdovirus disease in the paralichthys olivaceus culture process.

Description

Paralichthys rhabdovirus disease rapid diagnosis test paper and preparation method thereof

Technical Field

The invention relates to a diagnostic test strip and a preparation method thereof, in particular to a paralichthys olivaceus rhabdovirus disease rapid diagnostic test strip and a preparation method thereof, belonging to the cross technical field of immunology, microbiology and diagnostic reagents.

Background

The Hirame rhabdovirus (HIRRV) is a single-stranded negative-strand RNA virus with an envelope that is not segmented, and is also a major virus causing flounder disease. The flounder rhabdovirus disease caused by the flounder rhabdovirus has the characteristics of strong transmissibility, short disease course and high lethality rate, is one of the most common diseases in the flounder culture process, causes serious harm to the flounder culture industry and brings potential threat to the global fish culture industry. Therefore, the rapid diagnosis of the paralichthys rhabdovirus disease can help farmers to take corresponding prevention and control measures in time, effectively control the outbreak and prevalence of the disease and reduce the breeding risk.

At present, the detection method aiming at the HIRRV mainly comprises a cell culture isolated virus method, a histopathological section method, an electron microscope observation method, a Polymerase Chain Reaction (PCR), a real-time quantitative PCR, a loop-mediated isothermal amplification technology (LAMP), an enzyme-linked immunosorbent assay (ELISA) and the like. The above detection methods have their advantages, but they are limited by the need of professional equipment and complicated operation techniques, are difficult to realize rapid detection in the culture field, and cannot be applied to the detection of diseases of parent fish, because the above methods all require killing the fish and sampling for detection. Therefore, a fast, accurate and sensitive method for detecting the paralichthys rhabdovirus, which can be applied to a culture production field, is established, and not only is the disease detection of farmers facilitated, but also the important effect on the effective prevention and control of the disease is played. Based on the existence or nonexistence of the pathogen specific antibody in the fish serum, the infection state and the immune state of the fish can be evaluated. With the development of bioinformatics and proteomics technology in recent years, serological detection methods established by using specific antigens of pathogens have been reported. In addition, the colloidal gold immunochromatographic assay has the characteristics of rapidness, simplicity, convenience, visual results and the like, and is widely applied to various aspects such as food additive detection, veterinary drug residue detection, pathogenic microorganism detection and the like in recent years.

Disclosure of Invention

Aiming at the problems that the existing detection method is limited by the need of professional equipment and complex operation technology, is difficult to realize rapid detection in a culture field and cannot be applied to the detection of parent fish diseases, the invention aims to provide the test paper for rapidly diagnosing the paralichthys olivaceus rhabdovirus diseases, which is rapid, simple, convenient, strong in specificity and high in sensitivity, is suitable for rapidly diagnosing the paralichthys olivaceus rhabdovirus in the culture field, and has a diagnosis result visible to naked eyes without the need of professional instruments and complex operation technology.

The invention also aims to provide a preparation method of the test paper for rapidly diagnosing the paralichthys rhabdovirus disease.

The invention discovers, based on immunoblot analysis, that anti-HIRRV serum of the paralichthys olivaceus can generate stronger immunoreaction with rhabdovirus nucleoprotein and matrix protein of the paralichthys olivaceus, which indicates that the rhabdovirus nucleoprotein and the matrix protein of the paralichthys olivaceus can effectively induce the paralichthys olivaceus to generate specific antibodies and generate immunoreaction with the rhabdovirus nucleoprotein of the paralichthys olivaceus, so that the structural protein and the matrix protein of the rhabdovirus of the paralichthys olivaceus are selected as specific antigens, the recombinant rhabdovirus nucleoprotein and the matrix protein of the paralichthys olivaceus are prepared by a prokaryotic expression technology and a His tag protein purification technology, and the colloidal gold indirect immunochromatography technology.

The purpose of the invention is realized by the following specific technical scheme:

a test paper for rapidly diagnosing paralichthys olivaceus rhabdovirus diseases comprises a carrier plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad and the water absorption pad are positioned at two ends of the carrier plate, and the nitrocellulose membrane is positioned in the middle of the carrier plate; a gold-labeled pad loaded with a gold-labeled anti-paralichthys olivaceus immunoglobulin (IgM) monoclonal antibody is arranged at the junction of the sample pad and the nitrocellulose membrane, one end edge of the gold-labeled pad is overlapped below the sample pad, and the other end edge of the gold-labeled pad is overlapped above the nitrocellulose membrane; the nitrocellulose membrane is provided with two detection lines T1, T2 and a quality control line C which are sequentially arranged, and the quality control line C is close to the water absorption pad; the detection line T1 is coated with recombinant paralichthys rhabdovirus Nucleoprotein (N protein for short), the detection line T2 is coated with recombinant paralichthys rhabdovirus Matrix protein (Matrix protein for short), and the quality control line C is coated with goat anti-mouse IgG.

The mouse anti-flounder immunoglobulin (IgM) monoclonal antibody can be specifically combined with a heavy chain of flounder IgM, the antibody type is IgG, the monoclonal antibody is obtained by secreting a hybridoma cell strain JF-IgM-H, the preservation number of the hybridoma cell strain is CCTCC-C200631, the preservation unit is the China center for type culture preservation, and the preservation date is 2006, 7 and 6 days. The hybridoma cell strain JF-IgM-H is disclosed in the patent application CN 107607718A.

The recombinant paralichthys rhabdovirus nucleoprotein and the matrix protein are prepared by taking the paralichthys rhabdovirus nucleoprotein and the matrix protein as specific antigens and adopting a prokaryotic expression technology and a His tag protein purification technology.

The recombinant nucleoprotein and the matrix protein are structural proteins of the paralichthys rhabdovirus, the molecular weights of the recombinant nucleoprotein and the matrix protein are 60kDa and 42kDa respectively, and the recombinant nucleoprotein and the matrix protein can be specifically identified by anti-paralichthys rhabdovirus serum of the paralichthys olivaceus.

The paralichthys rhabdovirus nucleoprotein and the matrix protein are structural proteins of the paralichthys rhabdovirus, and the molecular weights of the structural proteins are 42kDa and 24kDa respectively.

A preparation method of test paper for rapidly diagnosing paralichthys rhabdovirus diseases comprises the following steps:

(1) ascites production and purification of the mouse anti-flounder immunoglobulin monoclonal antibody:

recovering and culturing hybridoma cell strain JF-IgM-H secreting bastard halibut IgM monoclonal antibody, injecting BALB/c mouse into abdominal cavity, collecting ascites, and purifying by caprylic acid-ammonium sulfate method to obtain monoclonal antibody;

(2) preparing and purifying the flounder rhabdovirus N protein and M protein:

selecting a structural protein N protein and an M protein of the paralichthys rhabdovirus as specific antigens through a protein immunoblotting experiment, and preparing the recombinant paralichthys rhabdovirus N protein and the recombinant paralichthys rhabdovirus M protein through a prokaryotic expression technology and a His tag protein purification technology;

(3) preparing a gold label pad:

preparing colloidal gold with the particle size of 20-30 nm by adopting a trisodium citrate reduction method, and adjusting the pH value of the colloidal gold to 8.2 by using a potassium carbonate solution; adding the anti-flounder immunoglobulin monoclonal antibody into colloidal gold, adding bovine serum albumin, centrifugally purifying, and suspending by using a gold-labeled preservative solution to obtain a gold-labeled monoclonal antibody liquid; spraying the prepared gold-labeled monoclonal antibody liquid on glass fiber, and freeze-drying to obtain a gold-labeled pad loaded with the gold-labeled monoclonal antibody; spraying the prepared gold-labeled monoclonal antibody liquid on glass fiber, and freeze-drying to obtain a gold-labeled pad loaded with the gold-labeled monoclonal antibody;

(4) preparation of nitrocellulose membrane:

respectively spraying the purified recombinant paralichthys rhabdovirus nucleoprotein and matrix protein on a nitrocellulose membrane according to a certain concentration, a certain distance and a certain sequence to be used as detection lines T1 and T2, spraying goat anti-mouse IgG on the nitrocellulose membrane to be used as a quality control line, airing, soaking in a sealing solution, rinsing PBST, and drying;

(5) the production of serological quick diagnosis test paper for paralichthys rhabdovirus disease:

sequentially sticking a nitrocellulose membrane, a water absorption pad, a gold label pad and a sample pad on a carrier plate, putting the carrier plate into a groove of a slitting machine, cutting the carrier plate into test paper with the width of 3.7 mm, putting the cut test paper into an aluminum foil bag filled with a drying agent, sealing the aluminum foil bag, putting the aluminum foil bag into a packaging card shell, and sealing the aluminum foil bag for preservation to obtain the test paper.

The preparation method of the recombinant paralichthys rhabdovirus nucleoprotein and the matrix protein comprises the following steps:

(1) designing a specific primer according to gene coding sequences of the rhabdovirus nucleoprotein and the matrix protein of the paralichthys olivaceus in an NCBI database, wherein the sequence of a nucleoprotein coding sequence amplification primer is as follows:

HIRRV-N F：5′-CGAATTCGCGAACCTTAAGGAAGAATTTG -3′；

HIRRV-N R：5′-TGTCGACGTAGTACTCTTCGTCCTCTC -3′；

the matrix protein coding sequence amplification primer is as follows:

HIRRV-M-F：5’-CGGGATCCATGTCTCTCTTCAAGCGAAC-3’；

HIRRV-M-R：5’-GCGTCGACTTTCCCCTTTTTGGTTG-3’；

after the amplification product is purified, pET-32a plasmids are used for respectively constructing prokaryotic recombinant expression plasmids of the amplification product and the prokaryotic recombinant expression plasmids;

(2) respectively transforming the constructed plasmids into escherichia coli, and inducing recombinant protein expression by IPTG after screening and sequencing verification of positive clone strains;

(3) purifying the recombinant nucleoprotein and the matrix protein respectively by using a nickel agarose affinity chromatography column, dialyzing the purified protein suspension step by step, sequentially reducing the urea concentration in dialysate from 8 mol/L, 6mol/L, 4 mol/L, 2 mol/L to 0 mol/L, and finally dialyzing by using PBS to finally obtain the recombinant paralichthys rhabdovirus nucleoprotein and the matrix protein.

The method for detecting the paralichthys rhabdovirus disease by using the test paper of the invention comprises the following steps:

the test paper detection card is horizontally placed, 100 mu L of the sample liquid to be detected after pretreatment is dripped into a sample hole, the waiting time is 10-15 min, the detection result is observed by naked eyes, and the disease condition of the fish body is judged according to the color development conditions of the detection lines T1 and T2.

The pretreatment method of the sample to be detected comprises the following steps:

collecting 1mL of blood from the tail vein of the paralichthys olivaceus, standing the blood sample at room temperature, and sucking supernatant after the serum is naturally separated out, namely the fish serum to be detected. Diluting the serum by 10 times to obtain the sample solution to be detected.

The invention adopts the principle of indirect immunochromatography. When the sample is diffused to the gold-labeled pad, the gold-labeled monoclonal antibodies are combined with the bastard halibut IgM in the sample to form a large amount of gold-labeled monoclonal antibody-IgM complexes, the complexes enter the nitrocellulose membrane along with the sample liquid due to siphoning, if the sample contains antibodies against bastard halibut rhabdoviruses, when the sample liquid reaches the detection line T2, a part of the gold-labeled monoclonal antibody-IgM complexes are specifically combined with the M protein coated thereon to form a gold-labeled monoclonal antibody-IgM-M composite structure, and the colloidal gold particles labeled on the monoclonal antibodies are fixed and accumulated at the detection line T2 to show a macroscopic red color. The rest of the gold-labeled monoclonal antibody-IgM complex continues to move along the membrane along with the sample liquid, and when reaching the detection line T1, another part of the gold-labeled monoclonal antibody-IgM complex is specifically bound to the N protein coated thereon, and the detection line T1 also shows a visible red color. And finally, when the sample solution moves forward to a quality control line, the free gold-labeled monoclonal antibody which is not combined with the IgM in the serum and the gold-labeled monoclonal antibody-IgM compound which is not reacted with the antigen on the detection line are combined on the quality control line which is coated with goat anti-mouse IgG in advance to form a gold-labeled monoclonal antibody-goat anti-mouse IgG compound or a gold-labeled monoclonal antibody-IgM-goat anti-mouse IgG compound, and the red color which is fixed by colloidal gold particles and can be seen by naked eyes is formed at the quality control line in an accumulated mode. If the detection sample does not contain the paralichthys rhabdovirus antibody or the antibody concentration, the two detection lines are not red at the same time, and the quality control line is red; if the quality control line and the detection line do not display red, the test paper is invalid, and the detection result is invalid.

The invention has the following advantages: the detection is rapid, and a direct-vision result appears after about 10 min; the operation is simple and convenient, and no professional is needed; the detection cost is low, professional instruments and equipment are not needed, and the method is suitable for detection in a culture site; the sensitivity is high, and the method can be used for diagnosing 160-time diluted paralichthys olivaceus antiserum; the specificity is strong, and the antibody does not have cross reaction with the paralichthys antiserum of infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus, lymphocystis virus, edwardsiella tarda, vibrio anguillarum, vibrio piscicola, vibrio parahaemolyticus, vibrio alginolyticus and streptococcus iniae; the repeatability is good; the storage is convenient, the stability is good, the validity period is 6 months at room temperature, and the validity period is 12 months at 4 ℃; is suitable for the rapid, simple and accurate diagnosis of the paralichthys rhabdovirus disease in the paralichthys olivaceus culture process.

Drawings

FIG. 1 is a graph showing the results of measuring the anti-paralichthys rhabdovirus serum titer of paralichthys olivaceus by ELISA.

FIG. 2 is a structural protein composition of purified HIRRV analyzed by SDS-PAGE.

Wherein, M: protein molecular weight standards; 1: purifying the HIRRV.

FIG. 3 is a Western blotting image for identifying the immunogenicity protein of paralichthys rhabdovirus.

Wherein, M: protein molecular weight standards; v: reaction results of the HIRRV structural protein and anti-paralichthys rhabdovirus serum of paralichthys olivaceus; c: the reaction result of the HIRRV structural protein and the serum of healthy lefteye flounder.

FIG. 4 is a graph showing the results of Western blotting analysis of the reaction characteristics of anti-HIRRV serum from Paralichthys olivaceus with recombinant N protein and M protein.

Wherein, M: protein molecular weight standards; 1: the reaction result of anti-HIRRV serum of the paralichthys olivaceus and the recombinant N protein; 2: the reaction result of the healthy lefteye flounder serum and the recombinant N protein; 3: the reaction result of anti-HIRRV serum of the paralichthys olivaceus and the recombinant M protein; 4: the reaction result of the healthy lefteye flounder serum and the recombinant M protein.

FIG. 5 is a schematic structural diagram of the rapid diagnostic test strip of the present invention.

Wherein, 1, a sample pad; 2. a gold label pad; 3. a nitrocellulose membrane; 4. a water absorbent pad; 5. a carrier plate; 6. a detection line T2; 7. a detection line T1; 8. and (4) quality control line.

FIG. 6 is a schematic diagram of the result of the test paper for rapid diagnosis of Paralichthys olivaceus serum sample.

Wherein, I: HIRRV infected bastard halibut serum; II, III: the paralichthys olivaceus is not infected with the rhabdovirus, but other pathogens which have immunological cross reaction with antigen N protein or M protein exist; IV: the paralichthys olivaceus is not infected with the rhabdovirus; v and VI: the test paper fails or the operation is problematic.

FIG. 7 is a graph showing the actual test results of the rapid diagnostic test paper of the present invention on Paralichthys olivaceus samples of different dilutions.

Wherein, I-V: the dilution is 1:10, 1:20, 1:40, 1:80 and 1:160 of the paralichthys olivaceus serum respectively; VI: the flounder serum with the dilution of 1: 320; VII: healthy paralichthys olivaceus serum.

FIG. 8 is a graph showing the actual test result of the rapid diagnostic test paper of the present invention on a Paralichthys olivaceus sample.

I: detecting the anti-HIRRV serum of the paralichthys olivaceus by a test strip to be positive; II, III: the serum is anti-Infectious Hematopoietic Necrosis Virus (IHNV) serum and antiviral hemorrhagic septicemia virus (VHSV) serum of the paralichthys olivaceus respectively, and the detection result is weak positive; IV-XII: the serum is the serum of flounder anti-lymphocystis disease virus, the serum of healthy flounder, PBS, the serum of flounder anti-Edwardsiella tarda, Vibrio anguillarum, Vibrio pisciformis, Vibrio parahaemolyticus, Vibrio alginolyticus and Streptococcus iniae, and the detection results are all negative.

Detailed Description

The invention is further illustrated by the following specific embodiments in conjunction with the accompanying drawings.

Materials and equipment used in the following examples:

the experimental paralichthys olivaceus is purchased from a certain paralichthys olivaceus farm in the east, the weight of the experimental paralichthys olivaceus is 500g-800g, the experimental paralichthys olivaceus is cultured for 1 week in the environment with the water temperature of 20-22 ℃, water is changed once every day at regular time, granulated feed is fed, and continuous oxygenation is ensured during the culture period.

Paralichthys rhabdovirus (CNPo 2015), Infectious Hematopoietic Necrosis Virus (IHNV), Viral Hemorrhagic Septicemia Virus (VHSV), Paralichthys lymphocystis virus (LCDV), Edwardsiella tarda, Vibrio anguillarum, Vibrio piscine, Vibrio parahaemolyticus, Vibrio alginolyticus, Streptococcus iniae strains and strains were identified and deposited by the laboratory.

Inverted microscope (available from Olympus); ultracentrifuge (available from Hitachi corporation); high speed refrigerated centrifuge (Sigma corporation); NanoDrop8000 (available from Thermo corporation); a PCR instrument, agarose gel electrophoresis system, SDS-PAGE gel electrophoresis system, and electric transfer system (purchased from Bio-rad); enzyme reader (from TECAN); protein purification system (available from GE); film spraying instrument XYZ3060, semi-automatic film sticking instrument LM5000, and slitting instrument (purchased from Bidot corporation); BCA protein concentration assay kit (purchased from Beyotime corporation); ex Taq, dNTP mix, competent cell BL21, agarose gel recovery kit, PCR product recovery kit (purchased from Transgen); IPTG, urea, dialysis bag (available from Solarbio corporation); HisTratpHAP affinity chromatography column (5 mL), trisodium citrate, chloroauric acid, goat anti-mouse IgG, bovine serum albumin, Tween-20 (purchased from Sigma); nitrocellulose membrane (available from Whatman).

Example 1: screening of paralichthys rhabdovirus immunogenic protein

1. Preparation of antiserum

Inoculating the preserved hirame rhabdovirus suspension into a cell culture bottle full of a monolayer of EPC cells at 75cm²Cell monolayers were inoculated with 1mL of HIRRV virus suspension. The cell culture solution (containing 10% fetal bovine serum) was replaced with a cell maintenance solution (containing 2% fetal bovine serum), and the virus-infected cell culture flask was transferred to a 20 ℃ incubator. Periodically observing cell morphology change, and collecting cell suspension in culture flask after 3-4 days to complete CPE lesion, and storing in-80 deg.C refrigerator. Taking cell suspension infected with HIRRV, thawing on ice before use, centrifuging the cell suspension, and inactivating the cell suspension for 72 h at 4 ℃ by using formalin solution with the final concentration of 0.5%; the virus solution was centrifuged at 120000g for 3 hours at 4 ℃ in an ultracentrifuge, and the precipitate was collected, concentrated 10-fold and resuspended. The number of copies of the virus was 10^6.3TCID₅₀Per 100 uL. According to the following steps: 1, mixing the HIRRV virus and a Freund's complete adjuvant, injecting 1mL per tail into the abdominal cavity to infect the paralichthys olivaceus, and injecting PBS with the same amount into a control group to inject the PBS. And (4) collecting blood from the tail vein every other week after immunization, and collecting the blood of the paralichthys olivaceus. The blood sample was first placed in an inclined manner at room temperature for 1h, then placed in a refrigerator at 4 ℃ overnight, the next day, the centrifuge temperature was set to 4 ℃, centrifuged at 5000g for 15min, and the supernatant carefully aspirated. And (3) placing the serum in a refrigerator at the temperature of-20 ℃ for freezing storage, and using the serum for subsequent experiments.

2. Indirect ELISA for determining antiserum titer

A96-well plate was used, and 100. mu.L of the ultracentrifuge-concentrated HIRRV virus suspension was added to each well and coated overnight at 4 ℃. The next day, the coating solution was discarded, 200. mu.L of PBST was added to each well and washed 3 times for 5min each, and after washing was completed, 200. mu.L of 5% BSA was added to each well and placed in an incubator at 37 ℃ for 1h for sealing. Blocking solution was discarded from each well and washed 3 times with PBST for 5min each time. The anti-HIRRV serum of the paralichthys olivaceus is diluted by 10 times, 20 times, 40 times, 80 times, 160 times, 320 times and 640 times in a gradient way, 100 mu L of the anti-HIRRV serum is added into each hole, the mixture is incubated for 1.5 h in an incubator at 37 ℃, and the same amount of PBS is used as a control. Washing with PBST for 3 times (5 min each time), adding 100 μ L of mouse anti-Paralichthys IgM monoclonal antibody into each well, incubating at 37 deg.C for 1h, and washing with PBST for 3 times (5 min each time). mu.L of AP secondary antibody (AP-labeled goat anti-mouse IgG) was added to each well, left at 37 ℃ for 45min, and washed three times with PBST for 5min each. Adding 100 mu L of prepared pNppNa coloring solution into each hole, placing the pNppNa coloring solution into an enzyme labeling instrument, reading an OD value at 405 nm, and calculating the ratio (P/N) of a positive value to a negative control light absorption value, wherein when the P/N is more than or equal to 2.1, the result is positive. The maximum dilution with P/N more than or equal to 2.1 is the serum titer, and the ELISA result (figure 1) shows that the serum titer is 1:160, so that the method can be used for the next experiment for screening the HIRRV immunogenic protein.

3. Purification of HIRRV virions

Taking 100mL of cell suspension infected with HIRRV out of a refrigerator at-80 deg.C, repeatedly freezing and thawing for 3 times, ultrasonically crushing for 30min, centrifuging at 4 deg.C under 6000g for 20min, collecting supernatant, and removing cell debris. Centrifugation was performed at 12000g for 30min at 4 ℃ using a centrifuge to remove cell debris, and the supernatant was collected. At 4 ℃, 120000g of the mixture was ultracentrifuged for 3h, the precipitate was collected and concentrated 10-fold by adding an appropriate amount of TNE (10 mmol/L Tris-HCl, 1mmol/L NaCl, 1mmol/L EDTA, pH 7.4) buffer for resuspension. 20%, 30%, 40% and 50% sucrose solutions were prepared and carefully placed into centrifuge tubes in order of concentration 20%, 30%, 40% and 50%. A distinct density gradient difference forming interface (a bright band) was observed, and the virus concentrate was slowly and gently added to the 20% sucrose interface. The centrifuge tube was assembled into a horizontal rotor of an ultracentrifuge, the ultracentrifuge was started, and centrifugation was performed at a centrifugal force of 120000g for 3 hours at 4 ℃. After centrifugation was complete, the ultracentrifuge was de-vacuumed, the centrifuge was opened and the action gently disassembled the centrifuge tube to quickly collect the cloudy solution that appeared between the 30% and 40% sucrose boundary layers (the HIRRV solution density was approximately equal to 37% sucrose density). An appropriate amount of PBS buffer was added to the collected liquid, and centrifuged at 120000g at 4 ℃ for 3 hours using an ultracentrifuge to remove sucrose, and the precipitate was collected. Dissolving the precipitate in a proper amount of PBS buffer solution, detecting, freezing and storing in a refrigerator at-20 ℃ for subsequent experiments.

4. Polyacrylamide gel electrophoresis experiment

Taking 15 μ L of purified virus liquid, adding appropriate amount of 5 XSDSBuffer, heating in boiling water for 15min, and cooling for use. Preparing an SDS-PAGE gel consisting of 3% (V/V) of the concentrated gel and 12% (V/V) of the separation gel; and (3) adding samples, adding 15 ul of samples into each hole, switching on a power supply, setting a constant current of 30 mA, and immediately turning off the power supply to stop electrophoresis when the blue indicator of the SDS buffer reaches the bottom edge and overflows. The glass was gently pried open, the gel carefully removed, stained in Coomassie Brilliant blue R250 for 40min, and then placed in a wash solution to destain on a shaker overnight. In FIG. 2, 4 clear protein bands can be seen, which are the G protein (60 kDa), the N protein (42 kDa), the P protein (29 kDa) and the M protein (24 kDa), respectively. The molecular weight of the L protein is 224kDa, but it is not shown in the figure because of the minimal content of L protein and the large molecular weight. The NV protein (13 kDa) is present only in the infected cells and not in the mature virions.

5. Immunoblotting experiments

The purified HIRRV supernatant was subjected to SDS-PAGE, protocol 4, and the gel was soaked in a pre-prepared membrane transfer buffer. Measuring the size of the gel, shearing a PVDF membrane with the size equal to that of the gel, pretreating the PVDF membrane, soaking in methanol for 15s, soaking in ultrapure water for 2min, and transferring into buffer solution for 5 min. The filter paper-PVDF membrane-gel-filter paper are placed in sequence from bottom to top, and bubbles between the PVDF membrane and the gel are avoided during operation. The clamp was tightened and placed in an electrophoresis tank with the PVDF membrane facing the anode. The electrophoresis tank is filled with a rotating film buffer, and the electrophoresis apparatus is set to be at a constant voltage of 30V for 90 min. After the membrane transfer is completed, the PVDF membrane is taken out, placed in methanol for slightly wetting, and then placed in ponceau dyeing solution for dyeing for 3-5 min. Rinsing in distilled water, a protein band stained red by the dye liquor was observed. Shearing the protein Marker, sucking water by using filter paper, and storing for later use. Adding 3-5% BSA, sealing at 37 deg.C for 1 hr or at 4 deg.C overnight, washing with PBST for three times (5 min each time) for using anti-HIRRV serum of Paralichthys olivaceus as primary antibody, adding serum of healthy Paralichthys olivaceus as negative control, and incubating at 37 deg.C for 90 min. PBST is washed for three times, each time is 5min, a mouse anti-paralichthys olivaceus IgM monoclonal antibody 2D8 is added as a secondary antibody, incubation is carried out for 60min at 37 ℃, and the washing is carried out in the same way after the secondary antibody is taken out. Adding AP-labeled goat anti-mouse IgG as a tertiary antibody, incubating at 37 ℃ for 45min, taking out, and washing in the same way. Adding NBT/BCIP coloring solution, coloring for 3-5 min in dark, observing whether a band appears, and stopping coloring in pure water immediately after the band appears. As a result, as shown in FIG. 3, protein bands appeared in the vicinity of the protein molecular weights of 60kDa, 42kDa and 24kDa according to the protein marker. The G protein, the N protein and the M protein of the HIRRV after cell culture, separation and purification are proved to be capable of specifically reacting with anti-HIRRV serum of the paralichthys olivaceus, wherein the reaction bands of the N protein and the M protein are thicker, which indicates that the N protein and the M protein have higher abundance in virus or stronger immunogen performance to induce the paralichthys olivaceus to generate higher-level specific antibody level, so that the N protein and the M protein are determined to be suitable for being used as capture antigens of serological diagnosis test paper to be coated on a detection line for detecting the virus specific antibody in fish serum.

6. Preparation of recombinant antigen and specificity analysis

(1) Cloning of the fragment of interest: according to genome sequences of the N protein and the M protein of the paralichthys rhabdovirus published by NCBI database, a Primer 5.0 software is used for designing specific primers, restriction endonucleases are selected as SalI, EoRI, BamH I and SalI, and the Primer sequences are as follows:

HIRRV-N F：5′-CGAATTCGCGAACCTTAAGGAAGAATTTG -3′；

HIRRV-N R：5′-TGTCGACGTAGTACTCTTCGTCCTCTC -3′；

HIRRV-M-F：5’-CGGGATCCATGTCTCTCTTCAAGCGAAC-3’ ；

HIRRV-M-R：5’-GCGTCGACTTTCCCCTTTTTGGTTG-3’；

the restriction sites are Sal I, EoR I, BamH I and Sal I.

PCR reaction system (25. mu.L system): DEPC water 17. mu.L, 10 XEx Taq Buffer 2.5. mu.L, dNTPMixture (2.5 mmol/L) 2. mu.L, F-Primer 1. mu.L, R-Primer 1. mu.L, template ((DNA of reverse transcription HIRRV) 1. mu.L, Ex Taq 0.5. mu.L. the amplification procedure was 95 ℃ 5min, 1 cycle, 95 ℃ 30s, 55 ℃ 30s, 72 ℃ 1min, 35 cycles, 72 ℃ 10 min. the PCR product was identified by electrophoresis using 1.0% agarose gel, the purified DNA fragment was recovered using agarose gel recovery kit and a double digestion experiment was performed, in particular, the reaction system was that 1. mu.g of PCR product was recovered, 2. mu.L of Kbuffer, 1. mu.L of enzyme digestion I, 1. mu.L of BamHI, 1. mu.L of BamH, 1. mu.L of EoRI, 2O was added to the final volume 20. mu.L, 5h at 37 ℃ and was taken out and purified directly by using PCR product purification kit, purity was determined by agarose electrophoresis and concentration was determined using NanoDrop 8000. The purified product is frozen at-20 ℃ for standby.

(2) Double enzyme digestion, connection, transformation and positive bacteria detection: the product and the pET-32a plasmid are subjected to double enzyme digestion by using restriction enzymes Sal I, EoR I, BamH I and SalI respectively, and the concentration ratio of the product and the pET-32a plasmid is adjusted according to the concentration of the target gene and the concentration of the pET-32a plasmid. The final reaction system was as follows: 6 μ L of the target gene, T4 DNA Ligase: 2 μ L, pET-32a plasmid: 2 μ L, T4 DNA Ligase buffer: 2 μ L, ddH 2O: 8 μ L, ligation was performed overnight at 16 ℃. 1-tube DH 5. alpha. competent cells (200. mu.l) were removed from-80 ℃ and immediately thawed on ice for 7 min; adding a connecting product (less than 50ng) into an ultra-clean workbench, uniformly bouncing, and then placing on ice for 30 min; performing heat shock at 42 ℃ for 50s, and immediately performing ice bath for 2 min; then 890 mul L of LB liquid culture medium without ampicillin is added into the tube and evenly mixed, and then the mixture is cultured for 1 hour in a constant temperature incubator at 37 ℃ by shaking; after the culture is finished, centrifuging for 2min at 4000r, leaving 200 mu L of supernatant in an ultra-clean bench, uniformly mixing and precipitating, uniformly coating the supernatant on an LB solid culture medium containing 50 mu g/mL ampicillin, placing the mixture on the front surface for 3min, marking, and placing the mixture in a constant-temperature incubator at 37 ℃ for inverted culture overnight. The next day, single colonies on the plates were randomly picked and placed on new plates and marked. Then as a template, an upstream primer of the target gene and a downstream primer of T7 are put into a PCR reaction system and mixed evenly for PCR reaction. And (3) performing agarose electrophoresis on the PCR product to obtain positive bacteria with single bands and consistent sizes, selecting the positive bacteria, culturing the positive bacteria in a 1.5mL centrifuge tube containing 1mL LB liquid culture medium at 37 ℃ until logarithmic phase, and sending the positive bacteria to the department of Ongjingke company for sequencing. And carrying out BLAST on-line comparison on the sequencing result by using NCBI (national center for Biotechnology information) and selecting a colony with the correct sequencing result and target gene as a positive colony. The corresponding positive colonies on the plate were transferred to a liquid medium containing ampicillin and cultured overnight with shaking at 37 ℃. Extracting the plasmid of the target gene contained in the cultured overnight positive bacterial liquid by using a plasmid miniprep kit, transforming the plasmid into BL21 (DE 3) competent cells, and carrying out the transformation and positive bacteria detection steps as above. Inoculating the positive bacteria to LB liquid culture medium containing ampicillin antibiotic, performing shaking culture at 37 deg.C overnight, mixing 500 μ L of the bacterial liquid with equal volume of 30% glycerol, and storing in a refrigerator at-80 deg.C.

(3) Inducing expression and purification of recombinant bacteria:

the recombinant expression strain is inoculated into 500 mL LB liquid culture medium containing 0.1 ampicillin antibiotic, shaking culture is carried out for 6 h at 37 ℃, 1mL of strain is collected when OD600 reaches about 0.6, then IPTG with the final concentration of 1.0 mmol/L is added for induction expression for 12 h, and then 1mL of strain is collected. The remaining culture was centrifuged at 8000 g for 10min, and 50 mL binding buffer (8 mol/L urea, 0.5 mol/L NaCl, 20 mmol/L Na) was added₃PO₄30 mmol/L imidazole, pH adjusted to 7.4). After ultrasonication, the mixture was centrifuged at 12000g for 5 min. The sample needs to be filtered by a filter membrane of 0.45 mu m, then a nickel agarose affinity chromatography column is added, and after being balanced by a Binding buffer, the sample is subjected to Elution buffer (8 mol/L urea, 0.5 mol/L NaCl, 20 mmol/L Na)₃PO₄500 mmol/L imidazole, pH value is adjusted to 7.4) is taken as eluent, eluted protein is collected step by step, then dialysis step by step is carried out, the concentration of urea in dialysate is reduced from 8 mol/L, 6mol/L, 4 mol/L and 2 mol/L to 0 mol/L in sequence, the dialysate is replaced at the interval of 12 h, and finally, PBS is used for dialysis. SDS-PAGE detects IPTG before and after induction and after purification. The dialyzed sample is firstly checked for purity by electrophoresis, and the high-purity protein can be subjected to subsequent tests and stored in a refrigerator at-80 ℃ for later use.

7. Immunoblotting assay for antigenic characterization of recombinant N and M proteins

The molecular weight of the N protein and the molecular weight of the M protein of the recombinant antigen are 60kDa and 42kDa, and the specific operation steps are the same as 2. And incubating the primary antibody with the prepared anti-HIRRV serum of the paralichthys olivaceus or the healthy serum, incubating the anti-mouse anti-paralichthys olivaceus IgM monoclonal antibody 2D8 for 60min at 37 ℃, washing in the same step, adding the AP-labeled goat anti-mouse IgG antibody, washing, adding the prepared NBT/BCIP color development liquid, performing color development in a dark place for 3-5 min, and observing a strip to immediately stop color development. The result is shown in figure 4, the recombinant N protein and M protein only react with anti-HIRRV serum of the paralichthys olivaceus, but do not react with healthy paralichthys olivaceus serum, which indicates that the recombinant antigen can well retain the characteristics of virus antigen, can be identified by the anti-HIRRV serum of the paralichthys olivaceus, and is suitable for being used as a capture antigen of diagnosis test paper to be coated on a detection line.

Example 2: preparation of test paper for rapidly diagnosing paralichthys olivaceus rhabdovirus disease

1. Preparation and purification of ascites of mouse anti-flounder IgM monoclonal antibody

(1) Preparing ascites: and (3) taking out the hybridoma cell strain JF-IgM-H from an ultralow-temperature refrigerator at minus 80 ℃, immediately putting the hybridoma cell strain JF-IgM-H into a water bath kettle at 37 ℃, and quickly shaking to unfreeze the cells within 1 min. Thawed cells were centrifuged at 200 g for 4 min and the supernatant discarded under sterile conditions. Add 500. mu.L of GIT medium to the cryopreserved tubes to resuspend the cells, transfer the cells to cell culture wells in 5% CO₂Culturing in an incubator with concentration, observing the growth state of hybridoma cells under an inverted microscope, selecting the cells in logarithmic growth phase with vigorous growth and good shape for subsequent use after passage for once every 3-4 days, collecting the hybridoma cells, cleaning the cells for 5 times with sterile RPMI1640, resuspending the cells with proper amount of RPMI1640, taking BALB/c mice of 8-12 weeks old, injecting 0.5 mL of sterile liquid paraffin into the abdominal cavity, injecting 0.5 mL of hybridoma cell suspension into the abdominal cavity after one week, wherein the suspension contains about 5 × 10⁵- 1×10⁶And (4) cells. The abdominal condition of the mouse is observed every day, if the abdominal part of the mouse is obviously enlarged and the skin feels tense when the mouse is touched by hands, the ascites can be collected.

(2) Purification of ascites: standing the obtained ascites in a refrigerator at 4 deg.C overnight, centrifuging at 3000 rpm for 10min at 4 deg.C the next day, removing upper lipid substance, removing lower cell precipitate, and collecting middle ascites. Measuring ascites

Volume, 0.06mol/L acetate buffer pH 4.8 was added in a volume ratio of 1:2, adjusted with 0.1mol/L HCl

The pH value is 4.5. 33ul of octanoic acid was added per ml of ascites, and an appropriate amount of octanoic acid was sucked up and slowly added dropwise. After stirring for 30min, a white precipitate was visible. The mixture was placed in a refrigerator at 4 ℃ overnight, centrifuged at 12000 r/min for 30min the next day, and the supernatant was collected. Filtering the supernatant with 0.45um filter membrane, adding 1/10 volume of 10 XPBS containing 0.2 mmol/LEDTA (200 mL of 10 XPBS is added with 0.015g EDTA, adjusting pH value to 7.4 with 1mol/L NaOH, slowly adding 0.258 g ammonium sulfate powder into each milliliter of liquid while stirring under the condition of ice bath at 4 ℃, continuously stirring for 30min to generate precipitates in the liquid, standing for 2 h, centrifuging for 30min at 12000 r/min at 4 ℃ by using a centrifuge, adding 200ul PBS into each ascites to dissolve the separated precipitates into the corresponding PBS, pouring the solution into a dialysis bag, placing the dialysis bag in a refrigerator at 4 ℃, dialyzing in ultrapure water, replacing dialyzate once every 4h, replacing 3-4 times, freeze-drying the dialyzate solution in a freeze-drying machine to obtain powder, dissolving the powder in ultrapure water, and measuring the protein concentration of the anti-flounder IgG monoclonal antibody by using BCA protein concentration measuring kit mg/mL.

2. Preparation of colloidal gold labeled mouse anti-flounder IgM monoclonal antibody

(1) Preparing colloidal gold: measuring 20mL of ultrapure water, and pouring into a beaker which is cleaned in advance; adding a chloroauric acid solution with the concentration of 1% to ensure that the final concentration is 0.01%, adding 200uL of the chloroauric acid solution into each 20mL of ultrapure water, and covering a preservative film to reduce the evaporation of water; the beaker was placed on a magnetic stirrer and heated, and when the solution started to boil, 300uL of 1% sodium citrate solution was added quickly; stirring and heating for 2-3min, wherein the solution color is changed from colorless to gray black, then to purple red, and finally to red or orange red, and the heating is stopped; continuing stirring for 3min, standing in dark at room temperature, and cooling; adding ultrapure water according to the mark made at the position of the beaker before the reaction, and complementing the steam in the heating processThe emitted ultrapure water. After cooling, 0.1mol/L of K is added dropwise with stirring₂CO₃Adjusting the pH value of the solution to 8.2; the colloidal gold with good quality is clear and transparent red liquid observed by naked eyes, light can penetrate through the colloidal gold, no floating substances exist on the surface of the colloidal gold, the solution is free from turbidity, and the colloidal gold is stored at 4 ℃.

(2) Preparing the colloidal gold labeled mouse anti-flounder IgM monoclonal antibody: adding 18 mu g of monoclonal antibody into 1mL of colloidal gold as the optimal labeling proportion, adding the monoclonal antibody into the colloidal gold according to the optimal labeling proportion, stirring for 20min, and standing for 10 min. Adding 5mL of 5% BSA into 20mL of colloidal gold to make the final concentration of BSA be 1%, stirring for 15min, standing for 10min, centrifuging at 4 ℃ for 20min at 4000r/min, discarding the precipitate, taking the supernatant, centrifuging at 4 ℃ for 13500r/min for 30min, keeping the precipitate, and discarding the supernatant. The precipitate was resuspended in a gold-labeled antibody wash, centrifuged at 13500r/min at 4 ℃ for 30min, and the supernatant was discarded. The colloidal gold was concentrated 10-fold, and the precipitate was resuspended in 2mL of gold-labeled antibody stock solution (0.01 mol/L PBS containing 1% sucrose, 1% BSA, 0.1% Tween-20 and 0.02% sodium azide, pH 7.4), stored at 4 ℃, and 20mL of colloidal gold finally yielded 2mL of gold-labeled antibody.

3. Preparation of gold-labeled pad 2

And (3) uniformly spraying the gold-labeled monoclonal antibody liquid on glass fiber, freeze-drying, and storing at low temperature in a dark and sealed manner.

4. Preparation of nitrocellulose Membrane 4

Adjusting the concentrations of recombinant antigen N protein and M protein to 1.5mg/mL and 1.0 mg/mL respectively by using sterile PBS, spraying N with the concentration of 1.5mg/mL on a nitrocellulose membrane by using a membrane spraying instrument to serve as T1, spraying M with the concentration of 1.0 mg/mL on the nitrocellulose membrane to serve as a detection line T2, wherein the distance between the detection lines T1 and T2 is 3 mm; adjusting the concentration of goat anti-mouse IgG to 250 mug/mL, spraying the goat anti-mouse IgG on a nitrocellulose membrane to serve as a quality control line 8, wherein the distance between the quality control line 8 and a detection line T1 is 10mm, the quality control line 8 is close to a water absorption pad 4, and the detection line is close to a sample pad 1. The coated nitrocellulose membrane was dried, soaked in 37 ℃ blocking solution (0.01 mol/L PBS containing 2% BSA, pH 7.4) for 60min, rinsed with PBST, and dried.

5. The invention relates to a method for preparing serological quick diagnosis test paper for paralichthys rhabdovirus disease

Wearing gloves, sticking a nitrocellulose membrane 3, a gold label pad 2, a sample pad 1 and a water absorption pad 4 on one side of a carrier plate 5 in sequence, and assembling into a test paper board. In which the upper edge of the nitrocellulose membrane 3 overlaps below the lower edge of the bib pad 4, the lower edge of the nitrocellulose membrane 3 overlaps below the upper edge of the gold-labeled pad 2, and the upper edge of the sample pad 1 overlaps above the lower edge of the gold-labeled pad 2 (see fig. 5). And putting the assembled test paper board into a groove of a slitting machine, cutting the assembled test paper board into test paper with the width of 3.7 mm, putting the test paper board into a packaging card shell, and sealing the packaging card shell in an aluminum foil bag filled with a drying agent to obtain the test paper.

Example 3: detection method of serological quick diagnosis test paper for paralichthys rhabdovirus disease

1. Preparation of test samples

Taking 1mL of blood from the tail vein of the diseased paralichthys olivaceus, standing the blood sample at room temperature for 1h, and then sucking the supernatant, namely the fish serum to be detected. Diluting the serum by 10 times to obtain the detection sample solution.

2. Application of serological quick diagnosis test paper for paralichthys rhabdovirus disease

The test paper detection card is horizontally placed, 100 mu L of the processed detection sample liquid is dripped into the sample hole, the waiting time is about 10min, the detection result is observed by naked eyes, and the disease condition is judged according to the color development conditions of the detection lines T1 and T2.

3. Result judgment

I: the detection line T, T2 and the quality control line are both red, and a positive result shows that the paralichthys rhabdovirus is infected and the paralichthys rhabdovirus antibody exists.

II, III: the detection lines T1 or T2 and the quality control line appear red, which is a negative result, indicating that there is no antibody to the paralichthys rhabdovirus but there are antibodies to other pathogens that cross-react with the antigen N protein or M protein.

IV: only the control line shows red color, which is a negative result, indicating that the paralichthys rhabdovirus antibody does not exist.

V: neither the detection line nor the quality control line appeared red, which is an invalid result indicating that the test paper failed or that there was a problem with the operation (see fig. 6).

Example 4: sensitivity, specificity, repeatability and stability test of test paper for rapidly diagnosing paralichthys olivaceus rhabdovirus disease

1. Sensitivity of the probe

Paralichthys olivaceus serum infected with Paralichthys rhabdovirus was diluted in PBS in a gradient (1: 10, 1:20, 1:40, 1:80, 1:160, 1: 320) and tested with a test strip. And (3) displaying a detection result: the flounder serum with the dilution degree of 1:10, 1:20, 1:40, 1:80 and 1:160 ensures that red lines (I-V) appear at the detection line and the quality control line of the test strip, and the result is positive. The flounder serum with the dilution of 1:320 and the healthy flounder serum only have quality control line color development, and the result is negative (VI, VII) (as shown in figure 7).

2. Specificity of

Selecting 12 groups of samples, which are Paralichthys anti-rhabdovirus (HIRRV) serum, Paralichthys anti-Infectious Hematopoietic Necrosis Virus (IHNV) serum, antiviral hemorrhagic septicemia virus (VHSV) serum, Paralichthys anti-lymphocystis virus (LCDV) serum, healthy Paralichthys serum, PBS, Edwardsiella tarda antiserum, Vibrio anguillarum antiserum, Vibrio parahaemolyticus antiserum, Vibrio alginolyticus antiserum, Streptococcus iniae antiserum

And (3) displaying a detection result: the samples in group 1 showed red (I) in T1, T2 and control line, the detection results of anti-Infectious Hematopoietic Necrosis Virus (IHNV) serum and anti-Viral Hemorrhagic Septicemia Virus (VHSV) serum in

group

2 and 3 showed light red (II, III), and only the control line in group 4-12 showed red (IV-XII) (see FIG. 7).

3. Repeatability of

The 12 groups of samples are detected by using test paper of different batches, and the test paper of each batch is repeatedly detected for 5 times, so that the result has no obvious difference.

4. Stability of

The test paper was placed at room temperature and 4 ℃ and 12 groups of samples in (1) were tested with the test paper every 15 days. The results show that: the shelf life at room temperature is 6 months, and the shelf life at 4 deg.C is 12 months.

Those skilled in the art will appreciate that modifications, additions and substitutions are possible, without departing from the scope of the invention as disclosed in the accompanying claims.

Claims

1. A test paper for rapidly diagnosing paralichthys olivaceus rhabdovirus diseases comprises a carrier plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad and the water absorption pad are positioned at two ends of the carrier plate, and the nitrocellulose membrane is positioned in the middle of the carrier plate; a gold-labeled pad loaded with a gold-labeled mouse anti-paralichthys immunoglobulin monoclonal antibody is arranged at the junction of the sample pad and the nitrocellulose membrane, the edge of one end of the gold-labeled pad is overlapped below the sample pad, and the edge of the other end of the gold-labeled pad is overlapped above the nitrocellulose membrane, and the gold-labeled pad is characterized in that the nitrocellulose membrane is provided with two detection lines T1, T2 and a quality control line C which are sequentially arranged, and the quality control line C is close to the water absorption pad; the detection line T1 is used for coating recombinant paralichthys rhabdovirus nucleoprotein; the detection line T2 is used for coating recombinant paralichthys rhabdovirus matrix protein; the quality control line C is coated with goat anti-mouse IgG.

2. The rapid diagnostic test paper according to claim 1, wherein the monoclonal antibody against the paralichthys olivaceus immunoglobulin is capable of specifically binding to the heavy chain of the paralichthys olivaceus immunoglobulin and is obtained by secreting a hybridoma cell strain JF-IgM-H, wherein the hybridoma cell strain has a preservation number of CCTCC-C200631, the preservation unit is the China center for type culture Collection, and the preservation date is 2006, 7, 6.

3. The rapid diagnostic test paper according to claim 1, wherein the recombinant bast-fish rhabdovirus nucleoprotein and matrix protein are prepared by taking the bast-fish rhabdovirus nucleoprotein and matrix protein as specific antigens and adopting a prokaryotic expression technology and a His tag protein purification technology.

4. The rapid diagnostic test strip according to claim 1, wherein the recombinant paralichthys rhabdovirus nucleoprotein and the matrix protein have molecular weights of 60kDa and 42kDa, respectively.

5. The rapid diagnostic test strip according to claim 1, wherein said recombinant Paralichthys rhabdovirus nucleoprotein and matrix protein are capable of specifically binding to Paralichthys anti-Paralichthys rhabdovirus serum.

6. A preparation method of test paper for rapidly diagnosing paralichthys rhabdovirus diseases is characterized by comprising the following steps:

recovering and culturing hybridoma cell strain JF-IgM-H secreting bastard halibut immunoglobulin monoclonal antibody, injecting BALB/c mouse into abdominal cavity, collecting ascites, and purifying by caprylic acid-ammonium sulfate method to obtain monoclonal antibody;

(2) preparation and purification of bastard halibut rhabdovirus nucleoprotein and matrix protein

Selecting structural protein nucleoprotein and matrix protein of the paralichthys rhabdovirus as specific antigens through a protein immunoblotting experiment, and preparing recombinant nucleoprotein and matrix protein of the paralichthys rhabdovirus through a prokaryotic expression technology and a His tag protein purification technology;

(3) preparation of gold label pad

Preparing colloidal gold with the particle size of 20-30 nm by adopting a trisodium citrate reduction method, and adjusting the pH value of the colloidal gold to 8.2 by using a potassium carbonate solution; adding the anti-flounder immunoglobulin monoclonal antibody into colloidal gold, adding bovine serum albumin, centrifugally purifying, and suspending by using a gold-labeled preservative solution to obtain a gold-labeled monoclonal antibody liquid; spraying the prepared gold-labeled monoclonal antibody liquid on glass fiber, and freeze-drying to obtain a gold-labeled pad loaded with the gold-labeled monoclonal antibody;

(4) preparation of nitrocellulose membranes

Respectively spraying purified structural protein nucleoprotein and matrix protein of the paralichthys rhabdovirus with certain concentration on a nitrocellulose membrane according to a certain distance and a certain concentration sequence to be used as detection lines T1 and T2, spraying goat anti-mouse IgG on the nitrocellulose membrane to be used as a quality control line, airing, soaking in a closed solution, rinsing with PBST, and drying;

(5) production of serological quick diagnosis test paper for paralichthys rhabdovirus