CN111537729B - Rapid diagnosis test paper for Rhabdoviral disease of paralichthys olivaceus and preparation method thereof - Google Patents

Rapid diagnosis test paper for Rhabdoviral disease of paralichthys olivaceus and preparation method thereof Download PDF

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CN111537729B
CN111537729B CN202010323726.4A CN202010323726A CN111537729B CN 111537729 B CN111537729 B CN 111537729B CN 202010323726 A CN202010323726 A CN 202010323726A CN 111537729 B CN111537729 B CN 111537729B
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paralichthys olivaceus
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rhabdovirus
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CN111537729A (en
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唐小千
战文斌
官曼玉
龚娇娇
绳秀珍
邢婧
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Ocean University of China
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Abstract

The invention discloses a rapid diagnosis test paper for a Rhabdoviral disease of a paralichthys olivaceus and a preparation method thereof. The diagnosis test paper comprises a carrier plate, a sample pad, a gold mark 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 carries 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; two detection lines T1 and T2 and a quality control line C are arranged on the nitrocellulose membrane, 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 olivaceus 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 and convenient, sensitive and accurate; the cost is low, and the stability is good; is suitable for rapid and accurate diagnosis of the Rhabdoviral disease of the paralichthys olivaceus in the culture process of the paralichthys olivaceus.

Description

Rapid diagnosis test paper for Rhabdoviral disease of paralichthys olivaceus and preparation method thereof
Technical Field
The invention relates to a diagnosis test strip and a preparation method thereof, in particular to a rapid diagnosis test strip for a Paralichthys rhabdovirus disease and a preparation method thereof, belonging to the technical fields of immunology, microbiology and diagnosis reagent crossing.
Background
The Paralichthys rhabdovirus (Hirame novirhabdovirus, HIRRV) is a single-stranded negative strand RNA virus with a non-segmented envelope and is also a main virus causing the Paralichthys olivaceus diseases. The Paralichthys olivaceus rhabdovirus disease caused by Paralichthys olivaceus rhabdovirus has the characteristics of strong transmissibility, short disease course and high mortality, is one of the most common diseases in the Paralichthys olivaceus breeding process, causes serious harm to the Paralichthys olivaceus breeding industry, and simultaneously brings potential threat to the global fish breeding industry. Therefore, the rapid diagnosis of the Rhabdoviral disease of the paralichthys olivaceus can help farmers to take corresponding prevention and control measures in time, effectively control outbreaks and epidemic diseases and reduce the breeding risk.
The existing detection methods for HIRRV mainly comprise a cell culture virus isolation 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 (LAMP) technology, an enzyme-linked immunosorbent assay (ELISA) and the like. The above detection methods have respective advantages, but they are limited in that specialized equipment and complicated operation techniques are required, rapid detection is difficult to achieve on the cultivation site, and the methods cannot be applied to detection of parent fish diseases because they all require killing fish to sample for detection. Therefore, the rapid, accurate and sensitive method for detecting the Paralichthys rhabdovirus, which can be applied to the breeding production field, is not only beneficial to the farmers to detect the diseases, but also plays a vital role in effectively preventing and controlling the diseases. Based on the existence or non-existence of the pathogenic specific antibody in the serum of the fish body, the infection state and the immune state of the fish body can be evaluated. Along with the development of bioinformatics and proteomics technologies in recent years, serological detection methods established by using specific antigens of pathogens have been reported. In addition, the colloidal gold immunochromatography technology has the characteristics of rapidness, simplicity, visual results and the like, and has been 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 on a culture site and cannot be applied to parent fish disease detection, the invention aims to provide rapid diagnosis test paper for the Paralichthys olivaceus rhabdovirus diseases, which is rapid, simple and convenient, has strong specificity and high sensitivity, is suitable for rapid diagnosis of the Paralichthys olivaceus rhabdovirus on the culture site, and has visible diagnosis results without professional instruments and equipment and complex operation technology.
The invention also aims to provide a preparation method of the rapid diagnosis test paper for the Rhabdoviral disease of paralichthys olivaceus.
The invention is based on immunoblotting experimental analysis, discovers that the anti-HIRRV serum of the paralichthys olivaceus can generate stronger immune reaction with the paralichthys olivaceus rhabdovirus nucleoprotein and matrix protein, which indicates that the paralichthys olivaceus rhabdovirus nucleoprotein and matrix protein can effectively induce the paralichthys olivaceus to generate specific antibodies and generate immune reaction with the paralichthys rhabdovirus nucleoprotein and matrix protein, thus selecting the structural protein and the matrix protein of the paralichthys olivaceus rhabdovirus as specific antigens, preparing recombinant paralichthys olivaceus rhabdovirus nucleoprotein and matrix protein through a prokaryotic expression technology and a His tag protein purification technology, and combining colloid Jin Jianjie immune chromatography technology to realize rapid diagnosis of the paralichthys olivaceus rhabdovirus.
The invention aims at realizing the following specific technical scheme:
the rapid diagnosis test paper for the Rhabdoviral disease comprises a carrier plate, a sample pad, a gold-labeled 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 mark pad carrying a gold mark mouse 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 mark pad is overlapped below the sample pad, and the other end edge is overlapped above the nitrocellulose membrane; the nitrocellulose membrane is provided with two detection lines T1 and 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 turbot rhabdovirus Nucleoprotein (N protein for short), the detection line T2 is coated with recombinant turbot rhabdovirus Matrix protein (Matrix protein for short M protein) and the quality control line C is coated with goat anti-mouse IgG.
The mouse anti-paralichthys olivaceus immunoglobulin (IgM) monoclonal antibody can specifically bind to the heavy chain of the paralichthys olivaceus IgM, the antibody type is IgG, the antibody is secreted by hybridoma cell strain JF-IgM-H, the preservation number of the hybridoma cell strain is CCTCC-C200631, the preservation unit is China center for type culture collection, and the preservation date is 7 months and 6 days in 2006. The hybridoma cell line JF-IgM-H has been disclosed in the patent application CN 107607718A.
The recombinant turbot rhabdovirus nucleoprotein and matrix protein are prepared by taking the turbot rhabdovirus nucleoprotein and matrix protein as specific antigens through a prokaryotic expression technology and a His tag protein purification technology.
The recombinant nucleoprotein and the matrix protein are structural proteins of the Paralichthys olivaceus rhabdovirus, have molecular weights of 60kDa and 42kDa respectively, and can be specifically identified by the serum of the Paralichthys olivaceus rhabdovirus.
The paralichthys olivaceus rhabdovirus nucleoprotein and the matrix protein are structural proteins of the paralichthys olivaceus rhabdovirus, and the molecular weights of the structural proteins are 42kDa and 24kDa respectively.
A preparation method of a rapid diagnosis test paper for Rhabdoviral disease of paralichthys olivaceus comprises the following steps:
(1) Production and purification of ascites of mouse anti-paralichthys olivaceus immunoglobulin monoclonal antibody:
resuscitating and culturing hybridoma cell strain JF-IgM-H secreting anti-paralichthys olivaceus IgM monoclonal antibodies, injecting BALB/c mice into the abdominal cavity, collecting ascites, and purifying by an octanoic acid-ammonium sulfate method to obtain the monoclonal antibodies;
(2) Preparation and purification of the Paralichthys olivaceus rhabdovirus N protein and M protein:
selecting the structural proteins N and M of the turbot rhabdovirus as specific antigens through a western immunoblotting experiment, and preparing the recombinant proteins N and M of the turbot rhabdovirus through a prokaryotic expression technology and a His tag protein purification technology;
(3) Preparation of a gold mark pad:
preparing colloidal gold with particle size of 20-30 nm by adopting a trisodium citrate reduction method, and regulating the pH value of the colloidal gold to 8.2 by adopting a potassium carbonate solution; adding anti-paralichthys olivaceus immunoglobulin monoclonal antibody into colloidal gold, adding bovine serum albumin, centrifuging and purifying, and suspending with gold-labeled preservation solution to obtain 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 carrying 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 carrying the gold-labeled monoclonal antibody;
(4) Preparation of nitrocellulose membrane:
spraying purified recombinant turbot rhabdovirus nucleoprotein and matrix protein on a nitrocellulose membrane according to a certain concentration, interval and sequence to serve as detection lines T1 and T2, spraying goat anti-mouse IgG on the nitrocellulose membrane to serve as a quality control line, airing, soaking in sealing liquid, rinsing with PBST, and drying;
(5) Preparation of a rapid serological diagnosis test paper for Rhabdoviral disease:
the nitrocellulose membrane, the water absorption pad, the gold mark pad and the sample pad are sequentially stuck on a carrier plate, the nitrocellulose membrane, the water absorption pad, the gold mark pad and the sample pad are put into a groove of a slitter to be cut into test paper with the width of 3.7 and mm, the cut test paper is put into an aluminum foil bag filled with a drying agent to be sealed, and the aluminum foil bag is filled with a packaging card shell to be sealed and preserved, thus obtaining the test paper.
The preparation method of the recombinant Paralichthys olivaceus rhabdovirus nucleoprotein and matrix protein comprises the following steps:
(1) Designing a specific primer according to the gene coding sequences of the turbot rhabdovirus nucleoprotein and the matrix protein in the NCBI database, wherein the sequence of the primer for amplifying the nucleoprotein coding sequence is as follows:
HIRRV-N F：5′-CGAATTCGCGAACCTTAAGGAAGAATTTG -3′；
HIRRV-N R：5′-TGTCGACGTAGTACTCTTCGTCCTCTC -3′；
the matrix protein coding sequence amplification primers are as follows:
HIRRV-M-F：5’-CGGGATCCATGTCTCTCTTCAAGCGAAC-3’；
HIRRV-M-R：5’-GCGTCGACTTTCCCCTTTTTGGTTG-3’；
after the amplified products are purified, respectively constructing prokaryotic recombinant expression plasmids of the amplified products and the pET-32a plasmid;
(2) Respectively transforming the constructed plasmids into escherichia coli, screening positive clone strains, and performing sequencing verification, and then inducing recombinant protein expression by IPTG;
(3) Purifying recombinant nucleoprotein and matrix protein respectively by using a nickel agarose affinity chromatographic column, dialyzing the purified protein suspension step by step, sequentially reducing the concentration of urea in the dialysate from 8 mol/L, 6mol/L, 4 mol/L and 2 mol/L to 0 mol/L, and finally dialyzing by using PBS (phosphate buffer solution), thereby finally obtaining the recombinant turbot rhabdovirus nucleoprotein and matrix protein.
The method for detecting the Rhabdoviral disease by using the test paper comprises the following steps:
the test paper detection card is horizontally placed, 100 mu L of the pretreated sample liquid to be detected is dripped into the sample hole, the sample liquid is waited for 10-15 min, the detection result is observed by naked eyes, and the fish disease condition 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:
taking 1mL blood from the tail vein of the paralichthys olivaceus, standing the blood sample at room temperature, and sucking the supernatant after the serum is naturally separated out to obtain the serum of the fish to be detected. Diluting the serum 10 times to obtain the sample liquid to be detected.
The invention adopts the principle of indirect immunochromatography. When a sample is diffused to the gold-labeled pad, the gold-labeled monoclonal antibody is combined with the flounder IgM in the sample to form a large number of gold-labeled monoclonal antibody-IgM complexes, and due to siphoning, the complexes enter a nitrocellulose membrane along with the sample liquid, if the sample contains antibodies against the flounder rhabdovirus, when the sample liquid reaches a detection line T2, a part of the gold-labeled monoclonal antibody-IgM complexes are specifically combined with M protein coated at the detection line T2 to form a gold-labeled monoclonal antibody-IgM composite structure, and colloidal gold particles marked on the monoclonal antibody are fixed and accumulated at the detection line T2 to enable the detection line T2 to show macroscopic red. The rest of the gold-labeled monoclonal antibody-IgM complex continues to advance along the membrane along with the sample liquid, and when the rest of the gold-labeled monoclonal antibody-IgM complex reaches a detection line T1, the other part of the gold-labeled monoclonal antibody-IgM complex is specifically combined with the N protein coated at the detection line T1, and the detection line T1 also has macroscopic red color. Finally, when the sample liquid moves forward to a quality control line, free gold-labeled monoclonal antibody which is not combined with IgM in serum and gold-labeled monoclonal antibody-IgM complex which is not reacted with antigen on a detection line are combined on the quality control line which is pre-coated with goat anti-mouse IgG to form gold-labeled monoclonal antibody-goat anti-mouse IgG complex or gold-labeled monoclonal antibody-IgM-goat anti-mouse IgG complex, and the gold-labeled monoclonal antibody-IgM complex is fixed by colloidal gold particles and accumulates at the quality control line to show macroscopic red. If the detection sample does not contain the paralichthys olivaceus rhabdovirus antibody or the antibody concentration, the two detection lines are not red at the same time, and the quality control line shows red; if the quality control line and the detection line are not red, the test paper is invalid, and the detection result is invalid.
The invention has the following advantages: the detection is quick, and the naked eye direct vision result appears about 10 min; the operation is simple and convenient, and no professional is needed; the detection cost is low, no special instrument or equipment is needed, and the method is suitable for on-site detection of cultivation; the sensitivity is high, and the kit can be used for diagnosing 160-fold diluted paralichthys olivaceus antiserum; the specificity is strong, and the kit does not have cross reaction with the antiserum of the paralichthys necrosis virus, viral hemorrhagic septicemia virus, lymphocystis virus, edwardsiella tarda, vibrio anguillarum, vibrio fish intestinal tract, vibrio parahaemolyticus, vibrio alginolyticus and Streptococcus iniae; the repeatability is realized; the storage is convenient, the stability is good, the effective period is 6 months at room temperature, and the effective period is 12 months at 4 ℃; is suitable for rapid, simple and accurate diagnosis of the Rhabdoviral disease of the paralichthys olivaceus in the culture process of the paralichthys olivaceus.
Drawings
FIG. 1 is a graph showing the results of ELISA for determining the serum titer of the anti-Paralichthys olivaceus rhabdovirus.
FIG. 2 is a diagram showing the composition of purified HIRRV structural proteins by SDS-PAGE analysis.
Wherein M: protein molecular weight standard; 1: purifying HIRRV.
FIG. 3 is a Western blotting identification of immunogenic proteins of Paralichthys olivaceus rhabdovirus.
Wherein M: protein molecular weight standard; v: the reaction result of HIRRV structural protein and anti-Paralichthys olivaceus rhabdovirus serum; c: results of the reaction of HIRRV structural protein with healthy paralichthys olivaceus serum.
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 and M proteins.
Wherein M: protein molecular weight standard; 1: the reaction result of the anti-HIRRV serum of the paralichthys olivaceus and the recombinant N protein; 2: the reaction result of healthy paralichthys olivaceus serum and recombinant N protein; 3: the reaction result of the anti-HIRRV serum of the paralichthys olivaceus and the recombinant M protein; 4: and (3) reaction results of healthy paralichthys olivaceus serum and recombinant M protein.
Fig. 5 is a schematic structural diagram of the rapid diagnostic test strip of the present invention.
1, a sample pad; 2. a gold mark pad; 3. a nitrocellulose membrane; 4. a water absorbing pad; 5. a carrier plate; 6. a detection line T2; 7. a detection line T1; 8. and a quality control line.
FIG. 6 is a schematic diagram of the rapid diagnostic test paper according to the invention for detecting the serum sample of Paralichthys olivaceus.
Wherein, I: serum of Paralichthys olivaceus infected with HIRRV; II, III: the paralichthys olivaceus is not infected by the paralichthys olivaceus rhabdovirus, but other pathogens which have immunological cross reaction with antigen N protein or antigen M protein exist; IV: the paralichthys olivaceus is not infected with the paralichthys olivaceus rhabdovirus; v and VI: the test paper fails or the operation is problematic.
FIG. 7 is a graph showing the actual detection results of the rapid diagnostic test paper of the present invention on different dilutions of the paralichthys olivaceus sample.
Wherein, I-V: the dilutions are 1:10, 1:20, 1:40, 1:80, 1:160 respectively of paralichthys olivaceus serum; VI: paralichthys olivaceus serum with dilution of 1:320; VII: healthy paralichthys olivaceus serum.
FIG. 8 is a graph showing the actual detection result of the rapid diagnosis test paper of the present invention on the paralichthys olivaceus sample.
I: the test strip of the anti-HIRRV serum of the paralichthys olivaceus detects positive; II, III: the detection results are weak positive respectively of the anti-Infectious Haematopoietic Necrosis Virus (IHNV) serum and the anti-Viral Hemorrhagic Septicemia Virus (VHSV) serum of the paralichthys olivaceus; IV-XII: the detection results are negative respectively, namely, the anti-lymphocystis disease virus serum of the paralichthys olivaceus, the healthy paralichthys olivaceus serum, PBS, the anti-Edwardsiella tarda of the paralichthys olivaceus, the vibrio anguillarum, the vibrio fish intestinal tract, the vibrio parahaemolyticus, the vibrio alginolyticus and the streptococcus iniae serum.
Detailed Description
The invention will be further illustrated by the following specific examples in conjunction with the accompanying drawings.
The materials and instruments used in the following examples:
the experimental paralichthys olivaceus is purchased from a Shandong sun-shine paralichthys olivaceus farm, has a weight of 500g-800g, is temporarily cultured for 1 week in an environment with a water temperature of 20-22 ℃, is changed once daily at regular time, and is fed with pellet feed, so that continuous oxygenation during the culture period is ensured.
Paralichthys rhabdovirus (CNPo 2015), infectious Haematopoietic Necrosis Virus (IHNV), viral Hemorrhagic Septicemia Virus (VHSV), paralichthys lymphocystis disease virus (LCDV), edwardsiella tarda, vibrio anguillarum, vibrio fish intestinal tract, vibrio parahaemolyticus, vibrio alginolyticus, streptococcus iniae virus strains and strains were identified and preserved by the present laboratory.
Inverted microscope (available from Olympus corporation); ultracentrifuge (available from Hitachi corporation); high speed refrigerated centrifuge (Sigma); nanoDrop8000 (available from Thermo company); PCR instrument, agarose gel electrophoresis system, SDS-PAGE gel electrophoresis system, and electrotransfer system (available from Bio-rad company); microplate reader (purchased from TECAN); protein purification systems (available from GE); film spraying instrument XYZ3060, semi-automatic film sticking instrument LM5000, 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 Co.); IPTG, urea, dialysis bags (from Solarbio); hisTrapTMHAP affinity column (5 mL), trisodium citrate, chloroauric acid, goat anti-mouse IgG, bovine serum albumin, tween-20 (from Sigma Co.); nitrocellulose membranes (available from Whatman company).
Example 1: screening of Rhabdoviral immunogenic proteins
1. Preparation of antisera
Inoculating the preserved Paralichthys Rhabdoviral suspension into a cell culture bottle filled with monolayer EPC cells, 75cm 2 Cell monolayers were inoculated with 1mL of HIRRV virus suspension. The cell culture broth (containing 10% fetal bovine serum) was replaced with cell maintenance broth (containing 2% fetal bovine serum), and the virus-infected cell culture flask was transferred to a 20 ℃ incubator. And (3) periodically observing the morphological change of the cells, waiting for 3-4 days, completely curing CPE, collecting cell suspension in a culture bottle, and storing in a refrigerator at-80 ℃. Taking HIRRV-infected cell suspension, thawing the cell suspension on ice before use, and inactivating 72 h by using formalin solution with the final concentration of 0.5% in centrifugation of the cell suspension at 4 ℃; centrifuging the virus solution at 4deg.C with an ultracentrifuge for 3 hr at 120000g, collecting precipitate, concentrating by 10 timesThe rows are resuspended. The copy number of the virus is 10 6.3 TCID 50 100uL. According to the following steps of 1:1 mixing HIRRV virus with Freund's complete adjuvant, injecting into the abdominal cavity to infect Paralichthys, injecting into the control group 1mL each, and injecting equal amount of PBS. Tail vein blood sampling is carried out every other week after immunization, and the paralichthys olivaceus blood is collected. The blood sample was first placed obliquely at room temperature for 1h, then placed in a refrigerator at 4 ℃ overnight, and the next day, the centrifuge temperature was set to 4 ℃, centrifuged at 5000g for 15min, and the supernatant was carefully aspirated. And (5) placing the serum in a refrigerator at the temperature of minus 20 ℃ for freezing and storing, and waiting for subsequent experiments.
2. Indirect ELISA determination of antiserum titers
A96-well ELISA plate was used, and 100. Mu.L of the HIRRV virus suspension concentrated by ultracentrifugation was added to each well to coat the wells overnight at 4 ℃. The next day, the coating solution was discarded, and 200. Mu.L of PBST was added to each well and washed 3 times for 5 minutes, and after the washing was completed, 200. Mu.L of 5% BSA was added to each well, and the wells were placed in an incubator 1h at 37℃to be blocked. The blocking solution was discarded per well and washed 3 times with 5min each with PBST. The anti-HIRRV serum of paralichthys olivaceus was subjected to gradient dilution 10-fold, 20-fold, 40-fold, 80-fold, 160-fold, 320-fold and 640-fold, 100. Mu.L of the serum was added to each well, and incubated in an incubator at 37℃for 1.5 h, and an equal amount of PBS was used as a control. The cells were washed 3 times with PBST for 5min each, and 100. Mu.L of murine anti-Paralichthys IgM mab was added to each well and incubated at 37℃for 1h, and the cells were washed 3 times with PBST for 5min each. 100. Mu.L of AP secondary antibody (AP-labeled goat anti-mouse IgG) was added to each well, and the mixture was left at 37℃for 45min and washed with PBST three times for 5min each. The prepared pNppNa color-developing solution is added with 100 mu L of the solution in each hole, the solution is placed in an enzyme labeling instrument, OD value is read at 405 and nm, and the ratio (P/N) of positive value to negative control light absorption value is calculated, wherein the result is positive when the P/N is more than or equal to 2.1. The maximum dilution of P/N not less than 2.1 is the serum titer, and ELISA results (figure 1) show that the serum titer is 1:160, and the ELISA results can be used for the next experiment for screening HIRRV immunogenic proteins.
3. Purification of HIRRV virions
100mL of HIRRV-infected cell suspension is taken out from a refrigerator at-80 ℃ and subjected to repeated freeze thawing for 3 times, ultrasonic crushing is carried out for 30min, centrifugation is carried out for 20min at 6000g of centrifugal force under the condition of 4 ℃, and the supernatant is collected to remove cell debris. Using a centrifuge, centrifugation was performed at 12000g for 30min at 4℃to remove cell debris, and the supernatant was collected. At 4℃and by ultracentrifugation for 3h at 120000g, the precipitate was collected, and concentrated 10-fold with an appropriate amount of TNE (10 mmol/L Tris-HCl,1mmol/L NaCl,1mmol/L EDTA, pH 7.4) buffer was added for resuspension. Sucrose solutions of 20%, 30%, 40% and 50% were prepared and sucrose solutions of 20%, 30%, 40% and 50% were carefully sequentially spread into centrifuge tubes. An interface (a bright band) formed by a distinct density gradient difference 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 fitted with an ultracentrifuge, and the ultracentrifuge was started and centrifuged at 120000g for 3 hours at 4 ℃. After centrifugation, the ultracentrifuge was run to remove the vacuum, the centrifuge was opened, and the centrifuge tube was gently removed by action to rapidly collect the turbid solution (HIRRV solution density approximately equal to 37% sucrose density) that appeared between 30% and 40% sucrose interface layers. To the collected liquid, an appropriate amount of PBS buffer was added, and the mixture was centrifuged at 120000g for 3 hours at 4℃with an ultracentrifuge to remove sucrose, and the precipitate was collected. Dissolving the precipitate in a proper amount of PBS buffer solution, detecting, and freezing in a refrigerator at-20deg.C for subsequent experiment.
4. Polyacrylamide gel electrophoresis experiment
Taking 15 μl of purified virus solution, adding appropriate amount of 5×SDSbuffer, heating in boiling water for 15min, and cooling. SDS-PAGE gels consisting of 3% (V/V) concentrate and 12% (V/V) separator were prepared; sample 15 ul is added into each hole, a power supply is switched on, a constant current of 30 mA is set, and when a blue indicator of SDS buffer overflows to the bottom edge, the power supply is immediately turned off, and electrophoresis is stopped. Gently pry the glass apart, carefully remove the gel, stain in coomassie brilliant blue R250 solution for 40min, and then leave in the wash solution on a shaker overnight. In FIG. 2, 4 distinct protein bands can be seen, G protein (60 kDa), N protein (42 kDa), P protein (29 kDa) and M protein (24 kDa), respectively. The L protein has a molecular weight of 224kDa, but is not shown in the picture due to the minimal L protein content and the relatively high molecular weight. NV protein (13 kDa) was present only in the infected cells and not in the mature virions.
5. Immunoblotting experiments
SDS-PAGE was performed on the purified HIRRV supernatant, procedure reference 4, and the gel was immersed in a pre-formulated transfer buffer. The gel size is measured, a PVDF film with the same size as the gel is cut, the PVDF film is pretreated, soaked in methanol for 15 seconds, soaked in ultrapure water for 2 minutes, and transferred into buffer for 5 minutes. The method is characterized in that the method is carried out in sequence from bottom to top according to the sequence of filter paper, PVDF film, gel and filter paper, and air bubbles between the PVDF film and the gel are avoided during operation. The clamps are tightened and placed in the electrophoresis tank with the PVDF membrane facing the anode. The transfer membrane buffer is filled up in the electrophoresis tank, and the electrophoresis apparatus is set to have constant pressure of 30 to V for 90 minutes. And after the transfer of the film is completed, taking out the PVDF film, putting the PVDF film into methanol for slightly wetting, and then putting the PVDF film into ponceau dyeing liquid for dyeing for 3-5 min. Rinsing in distilled water, a red-colored protein band was observed with the dye liquor. Cutting off the protein Marker, sucking the water with filter paper, and preserving for later use. Adding 3-5% BSA, sealing 1h in 37 deg.C incubator or overnight at 4 deg.C, PBST washing three times each for 5min, adding healthy Paralichthys olivaceus serum as primary antibody, adding healthy Paralichthys olivaceus serum as negative control, and incubating at 37 deg.C for 90 min. PBST is washed three times, each time for 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 secondary antibody is taken out for washing in the same step. AP-labeled goat anti-mouse IgG was added as a tertiary antibody, incubated at 37℃for 45min, and then removed for washing in the same manner as above. Adding NBT/BCIP color development liquid, developing for 3-5 min in dark, observing whether bands appear, and immediately stopping developing in pure water after the bands appear. The results are shown in FIG. 3, and show that protein bands appear in the vicinity of the 60kDa, 42kDa and 24kDa molecular weights of the proteins according to the protein Maker. The specific reaction of the G protein, the N protein and the M protein of the HIRRV after cell culture, separation and purification can be proved to occur with the anti-HIRRV serum of the paralichthys olivaceus, wherein the reaction strips of the N protein and the M protein are thicker, which shows that the N protein and the M protein have higher abundance in viruses or have stronger immunogenicity to induce the paralichthys olivaceus to generate higher-level specific antibody level, therefore, the N protein and the M protein are determined to be suitable to be used as capture antigens of serological diagnostic test paper to be coated on a detection line for detecting the virus specific antibodies in the serum of the fish body.
6. Preparation of recombinant antigens 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, specific primers are designed by using Primer 5.0 software, and the selective restriction enzymes are SalI, eoR I, bamH I and SalI, wherein the sequences of the primers 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 cleavage sites are Sal I, eoR I and BamH I, sal I, respectively.
PCR reaction System (25. Mu.L System): DEPC water 17 u L,10 x Ex Taq Buffer 2.5 u L, dNTP mix (2.5 mmol/L) 2 u L, F-Primer 1 u L, R-Primer 1 u L, template ((reverse transcription HIRRV DNA) 1 u L, ex Taq 0.5 u L amplification procedure 95 ℃ 5min,1 cycle, 95 ℃ 30s, 55 ℃ 30s,72 ℃ 1 min,35 cycles, 72 ℃ 10 min using 1.0% agarose gel electrophoresis to identify PCR products, agarose gel recovery kit to recover purified DNA fragment, and performing double enzyme digestion experiment, specific reaction system is that recovery PCR products 1 u g, K Buffer 2 u L, salI 1 u L, bamHI 1 u L, eoRI 1 u L, adding ddH2O to final volume 20 u L,37 ℃ enzyme digestion 5 h, directly using PCR product purification kit to purify agarose gel, determining purity of the agarose gel by freeze-drying the product 8000, determining the purity of the product.
(2) Double enzyme digestion, connection, transformation and positive bacteria detection: the product and pET-32a plasmid are respectively subjected to double digestion by restriction enzymes Sal I, eoR I, bamH I and Sal I, and the concentration ratio of the product and the pET-32a plasmid is adjusted according to the target gene concentration and the pET-32a plasmid concentration. The final reaction system was as follows: 6 mu L of target gene, T4 DNA Ligase: 2. mu L, pET-32a plasmid: 2. mu L, T4 DNA Ligase buffer: 2. mu L, ddH2O: 8. mu.L, the ligation system was ligated overnight at 16 ℃.1 tube DH 5. Alpha. Competent cells (200. Mu.l) were removed from-80℃and immediately thawed by placing them on ice for 7 min; adding a connection product (< 50 ng) into the ultra-clean workbench, and placing on ice for 30min after the connection product is uniformly flicked; immediately carrying out ice bath for 2min after heat shock at 42 ℃ for 50 s; then 890 mu L of LB liquid medium without ampicillin is added into the tube, and the mixture is stirred and cultured for 1h in a constant temperature incubator at 37 ℃; after the cultivation is finished, the mixture is centrifuged for 2min at 4000r, 200 mu L of supernatant is reserved in an ultra-clean bench, uniformly mixed and precipitated, then the mixture is uniformly coated on an LB solid medium containing 50 mu g/mL ampicillin, and after 3min of standing on the front side, the mixture is placed in a constant temperature incubator at 37 ℃ for inverted cultivation for overnight. The following day single colonies on the plates were randomly picked and placed in new plates and marked. Then, as a template, the upstream primer of the target gene and the T7 downstream primer are placed in a PCR reaction system to be uniformly mixed, and then the PCR reaction is carried out. The PCR products are subjected to agarose electrophoresis, single-strip and uniform-size positive bacteria, the positive bacteria are selected to be cultured in a 1.5mL centrifuge tube containing 1mL LB liquid medium at 37 ℃ until the logarithmic phase, and then the positive bacteria are sent to the Optimago company for sequencing. BLAST on-line comparison is carried out on the sequencing result in NCBI, and the colony with the correct matching of the sequencing result and the target gene is selected as a positive colony. The corresponding positive colonies on the plates were transferred to ampicillin-containing liquid medium and incubated overnight with shaking at 37 ℃. The plasmid of the target gene contained in the positive bacterial liquid cultivated overnight is extracted by using a plasmid small-amount extraction kit and is transformed into BL21 (DE 3) competent cells, and the transformation and the positive bacterial detection steps are the same. Inoculating positive bacteria into LB liquid culture medium containing ampicillin antibiotics, shake culturing at 37deg.C overnight, mixing 500 μl of bacterial liquid with 30% glycerol, and storing in a refrigerator at-80deg.C.
(3) Recombinant bacteria induced expression and purification:
inoculating recombinant expression bacteria into 500 mL LB liquid medium containing 0.1 ampicillin antibiotics, shake culturing at 37deg.C for 6 h, when OD600 reaches about 0.6,1mL of the cells were collected, and after the induction of expression 12 h by adding IPTG at a final concentration of 1.0 mmol/L, 1mL cells were collected. After centrifugation of the remaining culture at 8000 g for 10 min, 50: 50 mL Binding buffer (8 mol/L urea, 0.5 mol/L NaCl,20 mmol/L Na) was added 3 PO 4 30 mmol/L imidazole, pH was adjusted to 7.4). After ultrasonication, 12000 and g were centrifuged for 5 min. The sample is filtered by a filter membrane with the size of 0.45 mu m, then a nickel agarose affinity chromatography column is added, and after being balanced by Binding buffer, the sample is filtered by an absorption buffer (8 mol/L urea, 0.5 mol/L NaCl,20 mmol/L Na 3 PO 4 500 mmol/L imidazole, adjusting the pH value to 7.4) as eluent, collecting the eluted proteins step by step, then dialyzing step by step, sequentially reducing the concentration of urea in the dialysate from 8 mol/L, 6mol/L, 4 mol/L, 2 mol/L to 0 mol/L, replacing the dialysate with a middle interval of 12 h, and finally dialyzing with PBS. SDS-PAGE detects samples before and after IPTG induction and after purification. The dialyzed sample is first tested for purity by electrophoresis and the high purity protein is then tested and stored in a-80℃refrigerator for further use.
7. Immunoblotting experiment to analyze antigen characteristics of recombinant N protein and M protein
And (3) carrying out electrophoresis and membrane transfer on the N protein and the M protein of the recombinant antigen with molecular weights of 60kDa and 42kDa, wherein the specific operation steps are the same as 2. The antibody is incubated with prepared anti-HIRRV serum or healthy serum, a secondary anti-mouse anti-flounder IgM monoclonal antibody 2D8 is incubated for 60min at 37 ℃, AP-labeled goat anti-mouse IgG antibody is added after the washing is performed in the same step, the prepared NBT/BCIP color development liquid is added after the washing is completed, the color development is protected from light for 3-5 min, and the strip is observed to immediately stop the color development. As shown in FIG. 4, the recombinant N protein and the recombinant M protein only react with the anti-HIRRV serum of the paralichthys olivaceus and do not react with the healthy paralichthys olivaceus serum, which indicates that the recombinant antigen can well retain the characteristics of the virus antigen, can be recognized by the anti-HIRRV serum of the paralichthys olivaceus, and is suitable for being used as a capture antigen of diagnostic test paper to be coated on a detection line.
Example 2: preparation of rapid diagnosis test paper for Rhabdoviral disease of paralichthys olivaceus
1. Preparation and purification of ascites of mouse anti-paralichthys olivaceus IgM monoclonal antibody
(1) Ascites fluidPreparation: taking out hybridoma cell strain JF-IgM-H from a refrigerator with ultralow temperature of-80 ℃, immediately putting into a water bath kettle with the temperature of 37 ℃ and rapidly shaking to defrost the cells within 1 min. The thawed cells were centrifuged at 200℃ 200 g for 4 min and the supernatant was discarded under sterile conditions. Add 500. Mu.L GIT medium to the cryopreservation tube to resuspend cells, transfer the cells into the cell culture wells at 5% CO 2 Culturing in a concentration incubator. The growth state of the hybridoma cells is observed under an inverted microscope, and the hybridoma cells in the logarithmic growth phase are round in appearance, transparent, uniform in size, orderly arranged and distributed in a semi-compact mode. Typically, the passaging is performed every 3-4 days, and cells in the growth-promoting log phase with good morphology are selected for subsequent use. Hybridoma cells were collected, washed 5 times with sterile RPMI1640, and resuspended with an appropriate amount of RPMI 1640. BALB/c mice of 8-12 weeks old were intraperitoneally injected with 0.5. 0.5 mL sterile liquid paraffin. One week later, a suspension of 0.5X 10 hybridoma cells was intraperitoneally injected at about 5X 0.5 mL 5 - 1×10 6 Individual cells. The abdominal condition of the mice is observed every day, if the abdomen of the mice is observed to be obviously enlarged, the skin is tense when the mice are touched by hands, and the ascites can be collected.
(2) Purification of ascites: standing the obtained ascites in a refrigerator at 4deg.C overnight, centrifuging at 4deg.C and 3 rpm for 10 min, removing upper lipid substances, removing lower cell precipitate, and collecting intermediate layer ascites. Measuring ascites
Adding 0.06mol/L acetic acid buffer solution with pH of 4.8 according to the volume ratio of 1:2, and regulating with 0.1mol/L HCl
The pH was brought to 4.5. The solution was added with 33ul of n-octanoic acid per ml of ascites, and an appropriate amount of n-octanoic acid was sucked up and slowly added drop by drop. After stirring for 30min, a white precipitate was seen to appear. The mixture was placed in a refrigerator at 4℃overnight, and the supernatant was collected by centrifugation at 12,000 r/min for 30min on the next day. The supernatant was filtered with 0.45um filter, 1/10 volumes of 10 XPBS containing 0.2 mmol/LEDTA (200. 200 mL of 10 XPBS was added with 0.015g EDTA. PH was adjusted to 7.4 with 1mol/L NaOH. 0.258 g of ammonium sulphate powder was slowly added to each mL of liquid with stirring under ice bath conditions at 4℃for 30min, precipitation was continuously generated in the liquid, and then left to stand 2 h. The separated precipitate was dissolved in the corresponding PBS with a centrifuge at 12000 r/min at 4℃for 30min with 200ul PBS added per mL of ascites, the solution was poured into a dialysis bag, placed in a refrigerator at 4℃for dialysis, the dialysate was replaced every 4h, the lyophilized solution was lyophilized in a lyophilizer to powder, and after that, the protein concentration of anti-flounder IgM monoclonal antibody was 6 mg/mL with BCA protein concentration measuring kit.
2. Preparation of colloidal gold marked mouse anti-paralichthys olivaceus IgM monoclonal antibody
(1) Preparation of colloidal gold: weighing 20mL of ultrapure water, and pouring into a beaker which is clean in advance; adding chloroauric acid solution with the concentration of 1% to make the final concentration of the chloroauric acid solution be 0.01%, adding 200uL of chloroauric acid solution into each 20mL of ultrapure water, and covering a preservative film to reduce evaporation of water; the beaker was placed on a magnetic stirrer, stirred and heated, and after the solution began to boil, 300uL of 1% sodium citrate solution was rapidly added; continuously stirring and heating for 2-3min, wherein the color of the solution is observed to change obviously, the solution turns from colorless to grey black, then turns into mauve, finally turns into red or orange red, and the heating is stopped at the moment; continuously stirring for 3min, standing in dark at room temperature, and cooling; according to the mark made at the beaker before the reaction, adding ultrapure water to make up the ultrapure water evaporated during the heating process. After cooling, 0.1mol/L K was added dropwise with stirring 2 CO 3 Adjusting the pH value of the solution to 8.2; the colloidal gold with good quality is clear and transparent red liquid, light can pass through, the surface has no floaters and the solution has no turbidity, and the colloidal gold is preserved at 4 ℃.
(2) Preparing the colloidal gold labeled mouse anti-paralichthys olivaceus IgM monoclonal antibody: adding 18 mug of monoclonal antibody into 1mL of colloidal gold as the optimal labeling proportion, taking the colloidal gold, adding the monoclonal antibody according to the proportion, stirring for 20min, and standing for 10 min. 5mL of 5% BSA was added to 20mL of colloidal gold to give a final BSA concentration of 1%, stirring was continued for 15min, standing for 10 min, centrifuging at 4000r/min at 4℃for 20min, discarding the precipitate, collecting the supernatant, centrifuging at 13500r/min at 4℃for 30min, collecting the precipitate, and discarding the supernatant. The pellet was resuspended in gold-labeled antibody wash solution and centrifuged at 13500r/min at 4℃for 30min, and the supernatant was discarded. The colloidal gold was concentrated 10 times, the precipitate was resuspended in 2mL of gold-labeled antibody preservation 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 obtained 2mL of gold-labeled antibody.
3. Preparation of gold mark pad 2
And (3) uniformly spraying the gold-labeled monoclonal antibody liquid on glass fibers, freeze-drying, and sealing and storing at a low temperature in a dark place.
4. Preparation of nitrocellulose membrane 4
The concentration of recombinant antigen N protein and M protein is respectively adjusted to 1.5mg/mL and 1.0 mg/mL by using sterile PBS, N with the concentration of 1.5mg/mL is sprayed on a nitrocellulose membrane by using a film spraying instrument to be used as T1, M with the concentration of 1.0 mg/mL is sprayed on the nitrocellulose membrane to be used as a detection line T2, and the distance between the detection lines T1 and T2 is 3 mm; the concentration of goat anti-mouse IgG is adjusted to 250 mug/mL, the goat anti-mouse IgG is sprayed on a nitrocellulose membrane to serve as a quality control line 8, the distance between the quality control line 8 and a detection line T1 is 10mm, the quality control line 8 is close to the water absorption pad 4, and the detection line is close to the sample pad 1. After drying the coated nitrocellulose membrane, it was immersed in a blocking solution (0.01 mol/L PBS containing 2% BSA, pH 7.4) at 37℃for 60min, rinsed with PBST, and dried.
5. The invention relates to a preparation method of a serological rapid diagnosis test paper for Rhabdoviral disease of paralichthys olivaceus
A nitrocellulose membrane 3, a gold label pad 2, a sample pad 1 and a water absorbing pad 4 are sequentially adhered to one side of a carrier plate 5 by wearing gloves, and a test paper plate is assembled. Wherein the upper edge of the nitrocellulose membrane 3 is overlapped under the lower edge of the water absorbing pad 4, the lower edge of the nitrocellulose membrane 3 is overlapped under the upper edge of the gold mark pad 2, and the upper edge of the sample pad 1 is overlapped over the lower edge of the gold mark pad 2 (see fig. 5). The assembled test paper board is put into a groove of a slitter to be cut into test paper with the width of 3.7 and mm, and is put into a packaging card shell and sealed in an aluminum foil bag filled with a drying agent, thus obtaining the test paper.
Example 3: detection method of serological rapid diagnosis test paper for Rhabdoviral disease of paralichthys olivaceus
1. Preparation of test samples
Taking diseased paralichthys olivaceus, taking 1mL of blood from tail veins, standing the blood sample at room temperature for 1h, and then sucking the supernatant to obtain the serum of the fish to be detected. Diluting the serum 10 times to obtain the detection sample liquid.
2. Application of serological rapid diagnosis test paper for Rhabdoviral disease of paralichthys olivaceus
The test paper detection card is horizontally placed, 100 mu L of the processed detection sample liquid is dripped into the sample hole, the detection result is observed by naked eyes after about 10 minutes, and the disease condition is judged according to the color development condition of the detection lines T1 and T2.
3. Result judgment
I: both the detection line T, T and the quality control line appear red, and are positive results, which indicate that the paralichthys olivaceus rhabdovirus antibodies exist after infection.
II, III: the detection lines T1 or T2 and the quality control line appear red, a negative result, indicating that there is no flounder rhabdovirus antibody, but there is an antibody of other pathogens that have cross-reactivity with the antigen N protein or M protein.
IV: only the quality control line appeared red, which is a negative result, indicating that there was no flounder rhabdovirus antibody.
V: neither the test line nor the quality control line appears red, and is an invalid result, indicating that the test paper is invalid or has a problem in operation (see fig. 6).
Example 4: sensitivity, specificity, repeatability and stability test paper for rapid diagnosis of Paralichthys rhabdovirus disease
1. Sensitivity of
The Paralichthys olivaceus serum infected with the Paralichthys olivaceus rhabdovirus is subjected to gradient dilution (1:10, 1:20, 1:40, 1:80, 1:160, 1:320) by using PBS, and the Paralichthys olivaceus serum is detected by using a test strip. The detection result shows that: the test strips were tested positive by red lines (I-V) at the detection line and the quality control line of the test strips with dilutions of 1:10, 1:20, 1:40, 1:80 and 1:160 of paralichthys olivaceus serum. Only the quality control lines of the paralichthys olivaceus serum and healthy paralichthys olivaceus serum with the dilution of 1:320 showed color development, and the results were negative (VI, VII) (see FIG. 7).
2. Specificity (specificity)
Selecting 12 groups of samples, wherein the samples are respectively Paralichthys olivaceus anti-rhabdovirus (HIRRV) serum, paralichthys olivaceus anti-Infectious Haematopoietic Necrosis Virus (IHNV) serum, antiviral hemorrhagic septicemia virus (VHSV) serum, paralichthys olivaceus anti-lymphocystis virus (LCDV) serum, healthy Paralichthys olivaceus serum, PBS, edwardsiella tarda antiserum, vibrio anguillarum antiserum, vibrio fish intestinal antiserum, vibrio parahaemolyticus antiserum, vibrio alginolyticus antiserum and Streptococcus iniae antiserum
The detection result shows that: the samples of group 1 showed red color (I) on both T1, T2 and quality control lines, the serum of anti-Infectious Haematopoietic Necrosis Virus (IHNV) from paralichthys olivaceus of group 2 and 3 showed pale red color (II, III) on the serum test result of anti-Viral Hemorrhagic Septicemia Virus (VHSV), and the samples of groups 4-12 showed red color (IV-XII) on only the quality control lines (see FIG. 7).
3. Repeatability of
The 12 groups of samples are detected by test paper of different batches, the test paper of each batch is repeatedly detected for 5 times, and the results have 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 every 15 days with the test paper. The results show that: the effective period is 6 months when the product is stored at room temperature, and 12 months when the product is stored at 4 ℃.
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 (5)

1. The rapid diagnosis test paper for the Rhabdoviral disease comprises a carrier plate, a sample pad, a gold-labeled 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; the method is characterized in that the nitrocellulose membrane is provided with two detection lines T1 and 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 olivaceus rhabdovirus nucleoprotein; the detection line T2 is coated with recombinant flounder rhabdovirus matrix protein; the quality control line C is coated with goat anti-mouse IgG; the recombinant Paralichthys rhabdovirus nucleoprotein and matrix protein are prepared by the following method:
(1) Designing a specific primer according to the gene coding sequences of the turbot rhabdovirus nucleoprotein and the matrix protein in the NCBI database, wherein the sequence of the primer for amplifying the nucleoprotein coding sequence is as follows:
HIRRV-N F：5′-CGAATTCGCGAACCTTAAGGAAGAATTTG -3′；
HIRRV-N R：5′-TGTCGACGTAGTACTCTTCGTCCTCTC -3′；
the matrix protein coding sequence amplification primers are as follows:
HIRRV-M-F：5′-CGGGATCCATGTCTCTCTTCAAGCGAAC-3′；
HIRRV-M-R：5′-GCGTCGACTTTCCCCTTTTTGGTTG-3′；
after the amplified products are purified, respectively constructing prokaryotic recombinant expression plasmids of the amplified products and the pET-32a plasmid;
(2) Respectively transforming the constructed plasmids into escherichia coli, screening positive clone strains, and performing sequencing verification, and then inducing recombinant protein expression by IPTG;
(3) Purifying recombinant nucleoprotein and matrix protein respectively by using a nickel agarose affinity chromatographic column, dialyzing the purified protein suspension step by step, sequentially reducing the concentration of urea in the dialysate from 8 mol/L, 6mol/L, 4 mol/L and 2 mol/L to 0 mol/L, and finally dialyzing by using PBS (phosphate buffer solution), thereby finally obtaining the recombinant turbot rhabdovirus nucleoprotein and matrix protein.
2. The rapid diagnostic test paper according to claim 1, wherein the mouse anti-paralichthys olivaceus immunoglobulin monoclonal antibody can specifically bind to the heavy chain of the paralichthys olivaceus immunoglobulin, and is secreted by hybridoma cell strain JF-IgM-H, wherein the hybridoma cell strain has a preservation number of CCTCC-C200631, the preservation unit is China center for type culture collection, and the preservation date is 2006, 7, and 6.
3. The rapid diagnostic test paper according to claim 1, wherein the recombinant turbot rhabdovirus nucleoprotein and the matrix protein have molecular weights of 60kDa and 42kDa, respectively.
4. The rapid diagnostic test paper according to claim 1, wherein the recombinant paralichthys olivaceus rhabdovirus nucleoprotein and matrix protein are capable of specifically binding to the paralichthys olivaceus anti-rhabdovirus serum.
5. A method for preparing the rapid diagnosis test paper for the rhabdovirus diseases of paralichthys olivaceus as claimed in claim 1, which is characterized by comprising the following steps:
(1) Production and purification of ascites of mouse anti-paralichthys olivaceus immunoglobulin monoclonal antibody:
resuscitating and culturing hybridoma cell strain JF-IgM-H secreting anti-paralichthys olivaceus immunoglobulin monoclonal antibody, injecting BALB/c mice into the abdominal cavity, collecting ascites, and purifying by octanoic acid-ammonium sulfate method to obtain monoclonal antibody;
(2) Preparation and purification of Rhabdoviral nucleoprotein and matrix protein
Selecting structural proteins of the turbot rhabdovirus, namely nucleoprotein and matrix protein, as specific antigens through a western immunoblotting experiment, and preparing recombinant turbot rhabdovirus nucleoprotein and matrix protein through a prokaryotic expression technology and a His tag protein purification technology;
(3) Preparation of gold mark pad
Preparing colloidal gold with particle size of 20-30 nm by adopting a trisodium citrate reduction method, and regulating the pH value of the colloidal gold to 8.2 by adopting a potassium carbonate solution; adding anti-paralichthys olivaceus immunoglobulin monoclonal antibody into colloidal gold, adding bovine serum albumin, centrifuging and purifying, and suspending with gold-labeled preservation solution to obtain 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 carrying the gold-labeled monoclonal antibody;
(4) Preparation of nitrocellulose membranes
Spraying purified structural proteins of the turbot rhabdovirus nuclear proteins and matrix proteins with certain concentrations on a nitrocellulose membrane according to a certain interval and concentration sequence to serve as detection lines T1 and T2, spraying goat anti-mouse IgG on the nitrocellulose membrane to serve as a quality control line, airing, soaking in sealing liquid, rinsing with PBST, and drying;
(5) Preparation of serological rapid diagnosis test paper for Paralichthys olivaceus rhabdovirus
And (3) sequentially attaching a nitrocellulose membrane, a water absorption pad, a gold label pad and a sample pad on a carrier plate, putting into a slitter groove for cutting into test paper with the width of 3.7 and mm, putting the cut test paper into an aluminum foil bag filled with a drying agent for sealing, loading into a packaging card shell, and sealing in the aluminum foil bag for preservation.
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