CN115304670A

CN115304670A - Murine monoclonal antibody of feline coronavirus nucleocapsid protein, and coding gene and application thereof

Info

Publication number: CN115304670A
Application number: CN202110514346.3A
Authority: CN
Inventors: 李守军; 姚淙文; 叶绍棠; 冯慧君; 徐亮
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-05-07
Filing date: 2021-05-07
Publication date: 2022-11-08
Anticipated expiration: 2041-05-07
Also published as: CN115304670B

Abstract

The invention discloses a murine monoclonal antibody of a feline coronavirus nucleocapsid protein, and a coding gene and application thereof. The monoclonal antibody prepared by the invention has strong specificity and high titer, can specifically detect domestic FCoV strains, can be applied to the detection of the domestic FCoV strains, or can be used for preparing products for detecting the domestic FCoV strains.

Description

Mouse-derived monoclonal antibody of feline coronavirus nucleocapsid protein, and coding gene and application thereof

Technical Field

The invention belongs to the technical field of cell engineering. More particularly, relates to a murine monoclonal antibody of a feline coronavirus nucleocapsid protein, and a coding gene and application thereof.

Background

Feline infectious peritonitis is a chronic lethal disease of cats caused by Feline coronavirus (FCoV), is mainly characterized by peritonitis and large amount of ascites accumulation, can be infected by cats of different ages, and has very high infection rate.

The existing detection methods for feline coronavirus comprise serum antibody screening, RT-PCR and the like, and the methods need to take blood from animals, but the blood taking of animals has the problems of difficulty in blood taking, small sample amount and the like. In addition, when the RT-PCR method is used to detect FCoV in the exudate, there is a false positive result due to the limited diagnostic specificity of the method. Chinese patent CN 112415202A discloses a test strip for detecting feline coronavirus, and a preparation method, a kit and a detection method thereof. The test strip is simple to operate, and can be used for simultaneously detecting the feline coronavirus antigens in excrement, secretion and ascites of the cats. However, FCoV is ubiquitous in cat flocks around the world, and FCoV strains infected by cats in different areas, breeding environments and breeds are greatly different, which brings difficulty to the detection of FCoV in different areas. Therefore, the FCoV detection method for detecting the domestic FCoV strain is of great significance.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects and shortcomings of the prior art and provide a murine monoclonal antibody of a feline coronavirus nucleocapsid protein, and a coding gene and application thereof.

The first purpose of the invention is to provide a murine monoclonal antibody of feline coronavirus nucleocapsid protein.

The second purpose of the invention is to provide the application of the monoclonal antibody in preparing a feline coronavirus detection product.

The third purpose of the invention is to provide the application of the feline coronavirus BS-8 strain nucleocapsid protein shown in SEQ ID NO.5 in the preparation of the murine monoclonal antibody of the feline coronavirus nucleocapsid protein.

The fourth purpose of the invention is to provide the application of the feline coronavirus BS-8 strain nucleocapsid protein shown in SEQ ID NO.5 in the preparation of feline coronavirus detection products.

It is a fifth object of the present invention to provide genes encoding murine monoclonal antibodies to the feline coronavirus nucleocapsid protein.

The sixth object of the present invention is to provide a recombinant expression vector comprising the above gene.

It is a seventh object of the present invention to provide a host cell comprising the above recombinant expression vector.

An eighth object of the invention is to provide a product for detecting feline coronavirus.

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

the invention firstly provides a murine monoclonal antibody of feline coronavirus nucleocapsid protein, which is prepared by taking nucleocapsid recombinant protein of feline coronavirus BS-8 strain as antigen, wherein the heavy chain amino acid sequence of the CDR region of the monoclonal antibody is shown as SEQ ID NO.1, and the light chain amino acid sequence of the monoclonal antibody is shown as SEQ ID NO. 3. Experiments show that the murine monoclonal antibody of the feline coronavirus nucleocapsid protein has strong specificity and high titer, and the antibody can be used for specifically detecting domestic FCoV strains. Therefore, the application of the invention protects the application of the monoclonal antibody in preparing a feline coronavirus detection product.

The invention also applies to protect the application of the feline coronavirus BS-8 strain nucleocapsid protein shown in SEQ ID NO.5 in the preparation of the murine monoclonal antibody of the feline coronavirus nucleocapsid protein.

The invention also applies to protect the application of the feline coronavirus BS-8 strain nucleocapsid protein shown in SEQ ID NO.5 in the preparation of feline coronavirus detection products.

The invention also provides a gene of the murine monoclonal antibody for coding the feline coronavirus nucleocapsid protein, wherein the gene sequence of the coding heavy chain amino acid is shown as SEQ ID NO.2, and the sequence of the coding light chain amino acid is shown as SEQ ID NO. 4.

The invention also provides a recombinant expression vector containing the gene.

The invention also provides a host cell containing the recombinant expression vector.

The invention also provides a product for detecting the feline coronavirus, and the monoclonal antibody thereof.

The invention also provides an ascites indirect immunofluorescence method for detecting the feline coronavirus, which takes the monoclonal antibody as a primary antibody and takes goat anti-mouse IgG as a secondary antibody.

Preferably, the Goat Anti-Mouse IgG is Goat Anti-Mouse IgG H from Abcam&L(Alexa

488)。

Preferably, the ascites indirect immunofluorescence method comprises the steps of:

s1, dripping fresh ascites to be detected on a glass slide, flattening the ascites by using a cover glass, drawing a circle along the edge by using an immunohistochemical pen, and drying in a 37 ℃ oven;

s2, placing the cover glass on a horizontal shaking table, washing 3 times with PBS at the lowest rotation speed, and washing for 3min each time. After the cleaning is finished, the PBS is discarded, and the PBS is fixed by 4 percent paraformaldehyde for 10min;

s3, after fixation is finished, washing for 3 times for 3min by PBS on a horizontal shaking table at the lowest rotating speed;

s4, after the cleaning is finished, removing the PBS, carrying out closed incubation for 15min by using immunofluorescence sealing fluid, and removing the sealing fluid;

s5.4 ℃ incubating the primary antibody for 12-14 h, recovering the primary antibody after the incubation is finished, and washing the primary antibody for 3 times by PBST, wherein each time lasts for 3min;

s6, incubating the secondary antibody for 1h at normal temperature in a dark place, discarding the secondary antibody after incubation, and washing the secondary antibody with PBST solution for three times, wherein each time lasts for 3min.

The invention also provides a frozen section indirect immunofluorescence method for detecting the feline coronavirus, which takes the monoclonal antibody as a primary antibody and takes goat anti-mouse IgG as a secondary antibody.

Preferably, the Goat Anti-Mouse IgG is Goat Anti-Mouse IgG H from Abcam&L(Alexa

488)。

Preferably, the frozen section indirect immunofluorescence method comprises the following steps:

s1, using a small intestine mesentery of a cat with a caesarean section to carry out frozen section, putting the cat on a horizontal shaking table, washing the cat with PBS for 3 times at the lowest rotating speed, and washing the cat for 3min each time;

s2, after the cleaning is finished, discarding PBS, and fixing for 10min by using 4% paraformaldehyde;

s3, after fixation is finished, washing for 3 times with PBS at the lowest rotating speed on a horizontal shaking table, wherein each time lasts for 3min;

s4, after washing is finished, removing PBS, carrying out closed incubation for 15min by using immunofluorescence confining liquid, and removing the confining liquid;

The invention has the following beneficial effects:

the monoclonal antibody prepared by the invention has strong specificity and high titer, and can be used for specifically detecting domestic FCoV strains, so that the monoclonal antibody can be applied to detection of domestic FCoV strains or used for preparing products for detecting domestic FCoV strains.

Drawings

FIG. 1 shows the amino acid homology analysis of 81 positive samples and domestic feline coronavirus strains.

FIG. 2 is an electrophoresis diagram of nucleocapsid recombinant protein of BS-8 strain, wherein pore channel 1 is unpurified recombinant protein, pore channel 2 is purified BS8-N-MBP recombinant protein, pore channel 3 is purified BS8-N-MBP recombinant protein which is cut by Xa Factor, and pore channel 4 is purified BS8-N-MBP recombinant protein which is cut by Xa Factor and then purified again.

FIG. 3 shows the titer test results after the first mouse three-immunization.

FIG. 4 shows the titer test results of the second mouse after the third immunization.

FIG. 5 is a graph showing the results of indirect immunization of 293T cell samples transfected with Myc-BS8N plasmid with supernatants of 1F9, 1G8, 1H6, 2A6, 2D3 and 2F5 cell lines.

FIG. 6 is a graph showing the results of indirect immunization of 293T cell samples transfected with Myc-BS8N plasmid with supernatants from 2H1, 2H11, 3A12, 3G6, 3H8, 4B11, 4B12, 4E1, 4E7, 5E2, 5F11 and 5G7C4 cell lines.

FIG. 7 is a graph showing the results of indirect immunization of 293T cell samples transfected with Myc-BS8N plasmid with supernatants of 5H3, 5H5, 6A6, 6A10, 6C4, 6C8, 6C11, 6D4, 6F12, 6G31E1, 7D3 cell lines and Thermo controls.

FIG. 8 is a graph showing the results of indirect immunization of CRFK1 cell samples transfected with Myc-BS8N plasmid and supernatants from 1G8, 2D3, 2F5, 2H1 and 3G6 cell lines.

FIG. 9 is a graph showing the results of indirect immunization of CRFK1 cell samples transfected with Myc-BS8N plasmid with supernatants from 4B11, 4B12, 4E1, 4E7 and 5E2 cell lines.

FIG. 10 is a graph showing the results of indirect immunization of CRFK1 cell samples transfected with Myc-BS8N plasmid with supernatants from 5F11, 6A6, 6A10, 6C8 and 6C11 cell lines.

FIG. 11 is a graph showing the results of indirect immunization of a sample of CRFK1 cells transfected with Myc-BS8N plasmid, supernatants from 6D4 and 6F12 cell lines, and Thermo controls.

FIG. 12 is a graph showing the results of indirect immunization of FCWF4 cell samples transfected with Myc-BS8N plasmid with supernatants from 1G8, 2D3, 2F5, 2H1 and 3G6 cell lines.

FIG. 13 is a graph showing the results of indirect immunization of FCWF4 cell samples transfected with Myc-BS8N plasmid with supernatants from 4B11, 4B12, 4E1, 4E7 and 5E2 cell lines.

FIG. 14 is a graph showing the results of indirect immunization of FCWF4 cell samples transfected with Myc-BS8N plasmid with supernatants from 5F11, 6A6, 6A10, 6C8 and 6C11 cell lines.

FIG. 15 is a graph showing the results of indirect immunization of FCWF4 cell samples transfected with Myc-BS8N plasmid with supernatants from 6D4 and 6F12 cell lines.

FIG. 16 is an electrophoresis image of the immunoblots of total proteins extracted after transfection of 293T, CRFK and FCWF-4 with Myc-BS8N plasmid.

FIG. 17 is an electrophoretogram of 4B11 cell line antibody after purification.

FIG. 18 is the amino acid sequence of the heavy chain of the 4B11 cell line antibody.

FIG. 19 is the light chain amino acid sequence of the 4B11 cell line antibody.

FIG. 20 shows the results of indirect immunofluorescence assay for feline coronavirus ascites.

FIG. 21 shows the results of indirect immunofluorescence assay on frozen sections of feline coronavirus.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

The BS-8 strain used in the invention is currently preserved in a virus seed bank of an ultra-low temperature refrigerator in a department of education and research in the department of orthopedics of the university of veterinary medicine of south China.

Example 1 preparation of BS-8 nucleocapsid recombinant protein

In order to realize the detection of domestic feline coronavirus (FCoV) strains, the partial N gene sequence obtained by sequencing 81 feline coronavirus positive samples is subjected to amino acid homology analysis by using MegAlign software together with the N gene sequences of 8 domestic and foreign feline coronavirus strains such as FCoV 79-1146, black, ucd-1 and the like and the N gene sequences of two CCoV1-71 and TN499 canine coronaviruses, and the analysis result shows that the homology of a BS-8 strain and the domestic strains is highest, even the homology of some strains is as high as 100%, and is shown in figure 1. Therefore, the invention selects the nucleocapsid recombinant protein of the BS-8 strain as the antigen to prepare the monoclonal antibody, and the gene sequence of the nucleocapsid protein of the coding BS-8 strain is shown as SEQ ID NO. 5.

1. Construction of recombinant expression vectors

The recombinant expression vector pmal-c5x-BS8N was constructed by homologous recombination method, with reference to the instruction manual of the homologous recombination Kit Cloneexpress II One Step Cloning Kit of Novowed Biotechnology Ltd.

Firstly, designing homologous recombination primers according to the sequences, amplifying the gene of the BS-8 nucleocapsid protein, wherein the sequences of the primers are shown as follows, and the primers are synthesized by Guangzhou Tianyihui gene technology Co., ltd.

pmal-c5x-osBS8-F：

CGCGATATCGTCGACGGATCCATGGCCACACAAGGACAACG

pmalc5x-osBS8-R：

TTAATTACCTGCAGGGAATTCTTAGTTCGAGACCTCATCAATCATCT

The expression vector pmal-c5x plasmid was cut in a double digestion system as follows:

after the system preparation was completed, the linearized vector was recovered by gel electrophoresis in a water bath at 37 ℃ for 15 minutes, and the recovered linearized vector was measured.

Entry cloning and single-fragment homologous recombination reaction:

the optimal usage amounts of the vector and the fragment are respectively calculated according to the following formula, and a homologous recombination system is prepared according to the calculated usage amounts.

Optimum cloning vector usage amount = [0.02 × cloning vector base number ] ng

Optimum amount of insert used = [ 0.04X number of bases of insert ] ng

Homologous recombination system

And after the system preparation is finished, reacting for 5min at 50 ℃, adding the obtained recombinant product into the unfrozen E.coli DH5 alpha competent cells after the reaction is finished, and gently and uniformly mixing. After mixing, the competent cells were kept on ice for 25min. After the completion of the standing, the competent cells were subjected to water bath at 42 ℃ for 45sec, and then immediately allowed to stand on ice for 2min after being taken out. After standing on ice, 700uL of LB broth without antibiotics is added into the competent cells, mixed evenly and then put into a constant temperature shaking table at 37 ℃ and recovered at 180rpm for 1h. The recovered competent cells were centrifuged at 3000rpm for 3min at room temperature, 600uL of the supernatant was aspirated with a pipette, and the cells were resuspended in the remaining liquid. The remaining liquid was spread evenly on ampicillin LB agar medium using a glass rod, and was cultured overnight in an incubator at 37 ℃ while being inverted. And (3) selecting a single colony, and selecting bacteria containing correct recombinant expression vectors for subsequent experiments after sequencing verification.

10mL of the suspension containing the recombinant plasmid pmal-c5X-BS8 (plasmid after recombination of FCoV-BS8-N and MBP tag) was inoculated into 1L of LB broth containing benzyl resistance. Glucose must be present in the growth medium to inhibit the maltose gene on the E.coli host chromosome because maltose is an amylase that degrades amylose on the affinity resin.

After the thalli grows to 2 multiplied by 10 ⁸ Adding IPTG to a final concentration of 3mM when the absorbance of each cell/mL or cell is greater than 0.6 at a wavelength of 630nm, and incubating the cells at 37 ℃ for 2 hours; centrifuging at 4000 Xg for 20 minutes after the incubation is finished, removing supernatant, and collecting thalli; resuspending the thallus with PBS buffer solution, rapidly collecting the thallus, placing the sample in ice water bath, and carrying out ultrasonic treatment for 15 seconds; protein release was monitored using Bradford assay, 5 μ l of sonication solution was added to 1mL of Bradford reagent and mixed, sonication continued for 2 minutes to maximize protein release; centrifuging at 20000 Xg for 20 min after ultrasonic treatment, and retaining supernatant, i.e. crude extract; diluted to 1mL crude extract with 6 XPBS and stored at-20 ℃.

2. Purification of recombinant proteins

The invention purifies protein by column chromatography.

(1) Filling a chromatographic column:

soaking the gasket with 20% ethanol for one night until no bubbles are generated on the surface; packing the pad into 2.5 × 10cm chromatographic column; pouring amylose resin into a chromatographic column of 2.5 multiplied by 10 cm; after the resin is precipitated, another gasket is inserted; washing the Column with 5 Column volumes of Column Buffer;

(2) And (3) purifying the recombinant protein:

loading the diluted crude extract at a flow rate of no more than 5mL/min, washing with 12 column volumes of PBS at a rate of no more than 10 mL/min; eluting the fusion protein with PBS containing 10mM maltose, collecting 10 to 20 fractions of 3ml each, the size of the fractions being equal to 1/5 of the column volume;

(3) Cleavage of the recombinant protein and purification of the target protein:

adding Xa Factor Protease into the eluent according to the proportion of adding 50 mu L of 1mg of recombinant protein, and reacting for 1h at normal temperature; the target protein was purified using an anion chromatography column, GE HiTrap Q FF.

The fusion protein lysis mixture was dialyzed against 20mM Tris-HCl,25mM NaCl, pH 8.0 (2 or 3 changes of 100 volumes each for at least 2 hours); washing the column with 15mL of the same buffer; loading the fusion protein lysis mixture onto a chromatographic column and collecting 2.5mL of column flow-through fraction; the column was washed with 3 to 5 volumes of the same buffer and 2.5mL of fractions were collected. In 20mM Tris-HCl, pH 8.0, a gradient elution from 25mM NaCl to 500mM NaCl (25 mL each) was started. Fractions of 1mL were collected and it was determined which fractions contained protein by measuring a280 or Bradford method. MBP eluted as a spike at 100-150 mM NaCl. Xa Factor eluted at about 400mM NaCl. The flow-through liquid was collected for western blot validation.

3. Western blot validation of the Effect of purified proteins

(1) Preparation of polyacrylamide gel:

preparing 10mL of 12% separation gel solution: 3.3mL of deionized water, 4mL of 30% acrylamide, 2.5mL of Tris pH 8.8, 100. Mu.L of 10% ammonium persulfate, and 100. Mu.5363. Mu.L of 10% SDS, L, TEMED. Mu.L were added in this order, and immediately and rapidly mixed. The separation gel solution was added rapidly into the gap between the two glass plates, leaving room for the perfusion of the concentrated gel, and 1ml of isopropanol (adherent addition) was carefully added to the separation gel solution. After the separation gel is completely polymerized, the liquid on the gel is drained as far as possible, and the residual liquid is sucked up by the edge of the filter paper.

Preparing 4mL of 5% concentrated glue solution: 2.7mL of deionized water, 670. Mu.L of 30% acrylamide, 500. Mu.L of Tris (pH6.8), 40. Mu.L of 10% ammonium persulfate and 40. Mu. L, TEMED6 of 10% SDS are added in sequence and immediately and rapidly mixed. The concentrated glue is quickly and directly poured into the polymerized separating glue, and a clean comb is immediately inserted into the concentrated glue solution to avoid mixing air bubbles. After the concentrated gel was completely polymerized, the comb was carefully removed and the protein electrophoresis buffer was added to the electrophoresis tank.

(2) Preparation of a sample:

taking 50 μ L of the crude extract, the sample after purification by a chromatographic column, the sample after cutting by Xa Factor and the sample after passing through an anion chromatographic column, adding 5 × Loading buffer, mixing uniformly, and carrying out boiling water bath for 10min.

(3) Electrophoresis

The sample mixture was slowly added to the sample tank using a pipette and pre-stained with 10. Mu.L of Marker.

And (3) turning on a power supply, carrying out 80V electrophoresis for 30min, and changing the voltage to 120V electrophoresis until the bromophenol blue loading buffer solution migrates to the bottom of the gel in the gel.

(4) Protein staining

After electrophoresis, the cut gel is put into deionized water for cleaning, shaken for 5min at normal temperature, the liquid is discarded, and the washing is repeated for three times. Adding bromophenol blue staining solution, covering the gel, and incubating for at least 1h at normal temperature by shaking. Followed by a rinse with deionized water for at least 30min until bands clearly appear.

The electrophoresis result is shown in FIG. 2, wherein the pore channel 1 is unpurified recombinant protein, the pore channel 2 is purified BS8-N-MBP recombinant protein, the pore channel 3 is purified recombinant protein and is cut by Xa Factor, and the pore channel 4 is purified BS8-N-MBP recombinant protein and is re-purified BS-8-N protein after being cut by Xa Factor. The purification effect of the protein is good, and the size of the obtained protein is consistent with the expectation.

EXAMPLE 2 preparation of monoclonal antibodies

1. Animal immunization

8 SPF-grade Balb/c mice were divided into two groups and immunized in batches for 4 times. The first immunization was performed by subcutaneous injection of mice with 50. Mu.g/antigen and PBS mixed with Freund's incomplete adjuvant. After 2 weeks of reaction, a second immunization was performed, and the above reagents were injected. After reacting for another 2 weeks, the third booster immunization was performed, the above reagent was injected, and after reacting for one week, the mice were bled, and the antibody ELISA titer was measured, and the fourth immunization was performed. After the injection of the above reagents, the antibody ELISA titer was measured 2 weeks after the reaction. Abdominal impact, mice were impacted intraperitoneally with 50 μ g/antigen and PBS.

2. ELISA for detecting immune titer

Antigen coating: diluting the antigen to 1ug/mL by using the coating solution, adding 100 muL/hole into a polystyrene 96-hole reaction plate, and standing overnight at 4 ℃;

washing: discarding the liquid in the holes the next day, and washing with washing solution for 1 time;

and (3) sealing: adding 200 mu L/hole sealing liquid, and standing at 37 ℃ for 2h;

washing: washed 3 times with the wash solution.

Adding the sample to be tested (primary antibody): the sample to be tested (the sample needs to be diluted, and is diluted proportionally) is added at 100uL per well and incubated for 1h.

Washing: discarding the sample to be tested, and washing for 3 times by using a washing solution;

adding an enzyme-labeled secondary antibody: adding HRP-labeled goat anti-mouse IgG (1 10000 diluted with enzyme), 100. Mu.L/well, and incubating at 37 ℃ for 40min;

washing: washing with washing solution for 5 times;

color development: add freshly prepared substrate solution 90. Mu.L/well and leave at 37 ℃ in the dark for 15min.

Terminating reaction and carrying out color comparison: add 50. Mu.L/well stop solution. The color turns yellow; the absorbance of each well at 450nm was measured using a microplate reader.

After the first and second batches of mice were inoculated with 3 times of immunogens, the serum titers thereof are shown in fig. 3 and 4, respectively, and it can be seen from the graphs that the serum titers of the experimental group are all greater than the dilution of the blank control group 1 at 8000, and the serum titers satisfy the following experimental conditions.

3. Cell fusion

Killing after removing eyeballs after 3 days of last impact, collecting positive control blood, taking out spleen, preparing single cell suspension, taking out SP2/0 cell in logarithmic phase, mixing with splenocyte at a certain ratio (1: 5-1: 10), reacting with 50% PEG1450 for 1min, diluting with basal medium DMEM, stopping, centrifuging at low speed, lightly suspending with HAT medium containing 20% fetal calf serum, mixing well, and performing 2 × 10 ⁷ The individual cells/plate are plated into a previously prepared feeder cell plate and placed at 5% CO ₂ The culture was carried out at 37 ℃.

The method comprises the following specific steps:

(1) Spleen cells: mice were dissected, immunized spleens were removed, and lymphocytes in the spleens were isolated

A. A1.5 mL tube was prepared in the clean bench. Adding 1mL of serum-free medium, two 3.5cm dish, 2mL of serum-free medium and two 15mL centrifuge tubes, wherein one centrifuge tube is added with 10mL of serum-free medium, surgical instruments (high pressure moist heat sterilization), silk net, a pipettor (1 mL) and a gun head;

B. an immunized Bal b/c mouse is taken, an eyeball is removed for blood collection, and serum is separated to serve as positive control serum during antibody detection. Simultaneously, killing the mice by neck breaking, soaking the mice in 75% alcohol for 5min, fixing the mice on a wax plate, cutting the skin on the spleen, taking out the spleen by using forceps, and placing the spleen in a 1.5mL tube;

C. the spleen was transferred to one of 3.5cm dish in a clean bench, fat and connective tissue on the spleen were removed, washed once, a silk net was laid on the dish lid, and the spleen was gently crushed and placed in the middle of the silk net. Folding the silk net twice, sucking the serum-free culture medium by a pipettor, gently blowing off the serum-free culture medium, grinding the serum-free culture medium by a grinding rod to enable the lymphocytes in the spleen to penetrate through the silk net to prepare single cell suspension, and collecting the single cell suspension in a 15mL centrifuge tube. Centrifuge at 1000rpm/min for 5min.

(2) SP2/0 preparation: separating tumor to prepare single cell suspension, centrifuging at 1000rpm for 5min, discarding supernatant, resuspending and mixing 10-20 mL of LDMEM (determined according to tumor size), separating by using lymphocyte separation liquid, wherein the volume ratio of the lymphocyte separation liquid to DMEM is 1:1, slowly adding the separation liquid into the resuspended cells dropwise, centrifuging at 2500rpm/min for 15min, carefully placing on a super-clean workbench, transferring the middle layer of milky halo to a new centrifuge tube (30 mL of DMEM is prepared in a tube in advance) by using a liquid transfer gun, centrifuging at 1000rpm/min for 5min, finally discarding supernatant, collecting cells in a10 cm culture dish, adjusting the state and expanding the culture by using 10% fetal calf serum, generally preparing 3-5 dishes on the same day for one mouse, centrifuging and expanding to 30 dishes on the next day, and then freezing and storing the 30-35 tubes. In preparation for fusion, the following steps may be followed:

A. resuscitating on the first day;

B. on the next day, the cells were passaged according to the number of fusions, 5 dishes/1 fusion;

C. after about two days, the cell status was observed, and the cells were collected for fusion, if logarithmic phase was reached.

(3) Preparing feeder layer cells: healthy Balb/c mice were aseptically removed from their spleens, and made into single spleen cell suspensions using HAT medium containing 20% fetal bovine serum, which were then plated in 96-well plates in advance according to the number of plates plated.

(4) Stopping liquid: the basal medium DMEM 20mL was placed in a 37 ℃ water bath in advance for incubation.

4. Cell establishment

(1) Detecting a fusion plate:

and (3) starting detection (the detection method is referred to as an attached table ELISA method) when the cells of the liquid change of the fusion plate grow to more than 1 ten thousand cells with medium size, and selecting a positive hole (generally OD450 is more than or equal to 0.5) as a subclone after the ELISA quality control is qualified (namely the negative control is less than 0.2 and the positive control is more than 1.0).

(2) Subcloning method and detection:

and (3) selecting holes with high detection positive values in the fusion plate for limited dilution, counting 60% of the number of the monoclonal holes in each plate as subclones, selecting the monoclonal holes with high positive values for limited dilution each time, performing ELISA detection for 5-7 days by each subclone, and performing amplification culture until finally screening monoclonal cell strains capable of stably secreting positive antibodies.

(3) Establishing a cell strain:

expanding and culturing cell strains which are screened in a subcloning stage and can stably secrete positive antibodies in a 24-hole plate, collecting supernatant after expansion for antigen detection, verifying the stability by adopting ELISA gradient dilution and WB (cell amplification) and collecting cells in a culture dish with the cell amplification being more than 10cm, collecting the supernatant again, detecting the titer of the antibodies, selecting 1-3 cell strains with higher titer, culturing the cell strains in a cell bottle, and freezing and storing the cell strains. The successfully established cell strains comprise 1F9, 1G8, 1H6, 2A6, 2D3, 2F5, 2H1, 2H11, 3A12, 3G6, 3H8, 4B11, 4B12, 4E1, 4E7, 5E2, 5F11, 5G7C4, 5H3, 5H5, 6A6, 6A10, 6C4, 6C8, 6C11, 6D4, 6F12, 6G31E1 and 7D3 according to the experimental sequence, and are used for subsequent antibody screening experiments.

(4) Cell line cryopreservation identification

After the cell strain is completely frozen, one cell in the same batch must be recovered for identification, and the identification standard is as follows:

(1) the number of revived living cells is more than or equal to 100 ten thousand cells/branch;

(2) the number of viable cells in the viable cells is more than or equal to 50 ten thousand per cell;

(3) the recovery cells can not be provided with other microorganisms (such as bacteria, fungi, mycoplasma and the like) except the cells of the cell strain;

(4) after the reviving cells grow to a certain number, selecting the grown cells as a monoclonal counting plate, and detecting whether the monoclonal antibody secretion capacity is full-positive or has antibody secretion;

(5) the cell culture supernatant is also used for ELISA to determine whether positive antibodies are secreted and Western blotting is carried out at the same time.

(5) Preparation of ascites

Ascites is prepared by intraperitoneal injection of mice with pristane or liquid paraffin, and the hybridoma cells are inoculated into the abdominal cavities of the mice one week later. After cell line is determined, 10% fetal calf serum culture medium is selected for expanding culture, and when the cell density reaches 1 × 10 ⁶ ～2×10 ⁶ at/mL, the mixture is centrifuged at 800rpm/min, the precipitate is collected and resuspended in PBS, the suspension is injected into mice (liquid paraffin) in the abdominal cavity, and after 7 to 10 days, the ascites is collected and prepared for purification.

5. Screening for antibodies

Construction of Myc-BS8N plasmid was performed according to the method of example 1, using the following primers:

Myc-bs8-F：

TGGCCATGGAGGCCCGAATTCAAATGGCCACACAAGGACAACG；

Myc-bs8-R：

the CCTCGAGAGATCTCGGTCGACAATTAGTTCGAGACCTCATCAATCATCT vector was replaced with pCMV-Myc, and the restriction sites were replaced with EcoRI and SalI.

And transfecting 293T, CRFK and FCWF-4 cells by using the constructed Myc-BS8N plasmid, and performing indirect immunofluorescence and protein extraction.

The cell sample transfected with the Myc-BS8N plasmid is subjected to indirect immunofluorescence verification by using the supernatant of the obtained hybridoma cell strain, and the result is shown in figures 5-15, wherein figures 5-7 are the experimental results of the hybridoma cell strain and 293T cells transfected with the Myc-BS8N plasmid, figures 8-11 are the experimental results of the hybridoma cell strain and CRFK cells transfected with the Myc-BS8N plasmid, figures 12-15 are the experimental results of the hybridoma cell strain and FCWF-4 cells transfected with the Myc-BS8N plasmid, and Thermo is a commercial antibody control. The result shows that the antibody obtained by screening can be specifically combined with Myc-BS8N recombinant protein, and green fluorescence is displayed.

WB verification was performed on the extracted protein sample according to the method of example 1, and the WB verification result is shown in fig. 16. The results show that Invitrogen commercial Antibody (Catalog # MA1-82189Coronavir pan Monoclonal Antibody (FIPV 3-70)) can only recognize phosphorylated nucleocapsid protein and can not recognize unmodified nucleocapsid protein, while cell strain supernatants screened by the invention can recognize both unmodified nucleocapsid protein and phosphorylated nucleocapsid protein.

6. Identification of antibody clonal subtypes

The identification of antibody clone subtype is carried out by ELISA method, which comprises the following steps:

(1) Coating with BS8 protein at a concentration of 1ug/ml, overnight at 4 ℃;

(2) Sealing with 5% skimmed milk powder at 37 deg.C;

(3) Washing 3 times with TBST at 200 uL/well;

(4) Adding cell supernatant at 100 uL/well, adding 6 wells in total, and incubating at 37 ℃ for 30min;

(5) Washing 3 times with TBST at 200 uL/well;

(6) 6-typing enzyme-labeled secondary antibodies (IgA, igM, igG1, igG2a, igG2b, igG 3) 1, diluted at 1000, incubated at 37 ℃ for 30min in 6 wells of step 5 at 100 ul/Kong Jiadao;

(7) Washing 5 times with TBST at 200 uL/well;

(8) Adding single-component TMB color development solution into the solution at 90 uL/hole, and incubating for 15min;

(9) The plate reader measures the OD450 readings, and the results are shown in the following table.

Microplate reader OD450 readings:

results of subtype detection

After carrying out immunofluorescence after transfection on 293T, CRFK and FCWF-4 cells and western immunoblotting after transfection for 4 rounds in total, all cell strains shown in the results are frozen, and a subclone hybridoma cell strain with the clone number of 4B11 is selected for antibody purification.

7. Antibody purification

The collected ascites is purified by Protein G-agarose affinity chromatography column after being pretreated, and the specific steps are as follows:

(1) Preparing a buffer solution: the initial buffer solution is phosphate buffer solution with pH7.0 and 20 mmol/L; the elution buffer is pH 2.7,0.1mmol/L glycine hydrochloric acid;

(2) Preparing a collecting pipe: taking 1.5mL centrifuge tubes, and adding 70 mu L of pH9,1mol/LTris-HCl into each centrifuge tube;

(3) Sample preparation: the sample obtained by 50% SAS precipitation was dialyzed overnight against the starting buffer and filtered through a 0.22 μm microfiltration membrane;

(4) And (3) purification process: the Protein G-Sepharose affinity column (HiTrap Protein G1 mL, pharmacia Biotech) was equilibrated with enough starting buffer (8-10 mL). Taking 15-25 mL of a sample to be purified (each milliliter of the sample contains 10.2-21.1 mg of protein) to be loaded on a column, wherein the flow rate is 0.5mL/min, then sequentially washing with 7-8 mL of initial buffer solution, 6-7 mL of elution buffer solution and 5mL of initial buffer solution at the same flow rate, and collecting 1mL of eluent in each tube;

(5) And (3) purity and activity identification: the purity of the purified MAb was confirmed by SDS-PAGE;

the purity identification result is shown in fig. 17, and the purity of the antibody is more than 90% by SDS verification, and the antibody can be used for the next experiment.

8. Antibody sequencing

mRNA from the 4B11 cell line was extracted, reverse-transcribed into cDNA, and CDR regions of heavy and light chains of the antibody were amplified and sequenced. The amino acid sequences of the heavy chains and the light chains of the 4B11 antibodies obtained by the deletion of the nonsense sequences in comparison are shown in FIG. 18 and FIG. 19 respectively.

The heavy chain amino acid sequence of the CDR region of the monoclonal antibody is shown as SEQ ID NO.1, the gene sequence of the coding heavy chain amino acid is shown as SEQ ID NO.2, the light chain amino acid sequence thereof is shown as SEQ ID NO.3, and the sequence of the coding light chain amino acid is shown as SEQ ID NO. 4.

Example 3 Virus detection

1. Indirect immunofluorescence of ascites

The method takes the monoclonal antibody as a primary antibody and uses Goat Anti-Mouse IgG H of Abcam company&L(Alexa

488 ) is a secondary antibody, and the specific steps are as follows:

(1) Dripping fresh ascites to be measured on a glass slide, flattening the ascites by using a cover glass, drawing a circle along the edge by using an immunohistochemical pen, and drying in a 37 ℃ oven;

(2) The coverslip was placed on a horizontal shaker and washed 3 times with PBS at the lowest rotation speed for 3min each. After the cleaning is finished, the PBS is discarded, and the PBS is fixed by 4 percent paraformaldehyde for 10min;

(3) After fixation, washing with PBS 3 times on a horizontal shaking table at the lowest rotation speed for 3min each time;

(4) After washing, discarding PBS, carrying out closed incubation for 15min by using immunofluorescence confining liquid, and discarding the confining liquid;

(5) Incubating the primary antibody at 4 ℃ for 12-14 h, recovering the primary antibody after the incubation is finished, and washing the primary antibody for 3 times by PBST, wherein each time lasts for 3min;

(6) Incubating the secondary antibody for 1h at normal temperature in a dark place, discarding the secondary antibody after incubation, and washing with PBST solution for three times, each time for 3min;

(7) After washing, the sample was observed with a fluorescence microscope and photographed.

The ascites indirect immunofluorescence assay results are shown in fig. 20, and cells infected with feline coronavirus in ascites were specifically labeled, showing bright green light.

2. Indirect immunofluorescence of frozen sections

488 ) is a secondary antibody, and the specific steps are as follows:

(1) Freezing and slicing intestinal mesentery of cat under caesarean section, washing with PBS for 3 times (3 min each time) at the lowest rotation speed on a horizontal shaker;

(2) After washing, discarding PBS, and fixing with 4% paraformaldehyde for 10min;

(4) After washing, removing PBS, carrying out closed incubation for 15min by using immunofluorescence confining liquid, and removing the confining liquid;

The indirect immunofluorescence results of the frozen sections are shown in FIG. 21, and cells infected with feline coronavirus in mesenteric and small intestinal tissues are specifically labeled, showing bright green light.

The strain to be detected is confirmed to be a positive sample of the feline coronavirus through PCR amplification verification, and the monoclonal antibody is indicated to be suitable for detecting the feline coronavirus.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.

Sequence listing

<120> murine monoclonal antibody of feline coronavirus nucleocapsid protein, and coding gene and application thereof

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 116

<212> PRT

<213> Feline coronavirus (Feline coronavirus)

<400> 1

Asp Val Lys Leu Gln Glu Ser Gly Ala Glu Leu Val Arg Ser Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Met Thr Ser Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Tyr Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Trp Asp Gly Leu Arg Ser Asp Tyr Trp Gly Gln Gly Thr Ser Val

100 105 110

Thr Val Ser Ser

115

<210> 2

<211> 348

<212> DNA

<213> Feline coronavirus (Feline coronavirus)

<400> 2

gatgtgaagc ttcaggagtc tggggcagag cttgtgaggt caggggcctc agtcaagttg 60

tcctgcacag cttctggctt caatattaaa gactactata tacactgggt gaggcagagg 120

cctgaacagg gcctggagtg gattggatgg attgatcctg agaatggtga tactgaatat 180

gccccgaagt tccagggcaa ggccactatg acttcagaca catcctccaa cacagcctac 240

ctgcagtaca gcagcctgac atctgaggac actgccgtct attactgtaa ttgggatggt 300

ttgaggtcgg actactgggg tcaaggaacc tcagtcaccg tctcctca 348

<210> 3

<211> 113

<212> PRT

<213> Feline coronavirus (Feline coronavirus)

<400> 3

Asp Val Leu Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Lys Ala Tyr Met Ser Trp Phe Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Val Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro His Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 4

<211> 339

<212> DNA

<213> Feline coronavirus (Feline coronavirus)

<400> 4

gatgttttga tgacccaaac tccactcact ttgtcggtaa ccattggaca accagcctcc 60

atctcttgca ggtcaagtca gagcctctta gatagtgatg gaaaggcata tatgagttgg 120

tttttacaga ggccaggcca gtctccaaag cgcctcatct atctggtgtc taaactggac 180

tctggagtcc ctgacagggt cactggcagt ggatcaggga cagacttcac actgaaaatc 240

agcagagtgg aggctgagga tttgggagtt tactattgct ggcaaggaac acattttccc 300

cacacgttcg gaggggggac caagctggaa ataaaacgg 339

<210> 5

<211> 1134

<212> DNA

<213> Feline coronavirus (Feline coronavirus)

<400> 5

atggccacac aaggacaacg cgtcaactgg ggagatgaac cttccaagag acgtggtcgt 60

tctaactctc gtggtcggaa gaataatgat atacctttgt cattctacaa ccccattacc 120

ctcgaacaag gatcaaagtt ttggaactta tgtccgagag actttgttcc caagggaata 180

ggtaataaag atcaacaaat tggttattgg aacagacaat ttcgctaccg tattgtcaaa 240

ggccagcgta aggaacttcc tgagagatgg ttcttctatt tcttaggtac aggacctcat 300

gctgatgcta aatttaaaga taaaattgat ggagtcttct gggttgcaag ggatggtgcc 360

atgaataagc caactacact tggcactcgt ggaaccaaca atgaatccaa accactgaaa 420

tttgatggta agataccacc gcaatttcag cttgaagtga accgatctag gaacaactca 480

agaagtggtt ctcagtctag atctgtttct agaaacaggt ctcaatctag aggaagacaa 540

caatccaatt accataacaa taatgttgag gatacaattg tagccgtgct tgataaatta 600

ggtgttactg acaagcaaag gtcacgttcc aaatcaaaag aacgtagtag ttccaactct 660

agggacacaa cacctaaaaa tgccaataaa cacacctgga agaaaactgc aggtaaggga 720

gatgtgacaa atttctatgg tgctagaagt gcttcagcta actttggtga tagtgatctc 780

gttgccaatg gtaacgctgc caaatgctac cctcagatag ctgaatgcgt tccatcagtg 840

tctagcgtgc tcttcggtag tcaatggtct gctgaagaag ctggagatca agtgaaagtc 900

acgctcactc atacctacta cctgccaaaa gatgatgcca aaaccagcca attcctagaa 960

cagattgacg cttacaagcg gccttcacaa gtggctaagg atcagaggca aagaaaaccc 1020

cgttctaagt ctgctgacaa aaagcctgag gaattgtctg taactctagt agaggcatac 1080

acagatgtgt ttgatgacac acaggttgag atgattgatg aggtctcgaa ctaa 1134

Claims

1. A murine monoclonal antibody of feline coronavirus nucleocapsid protein is characterized in that the heavy chain amino acid sequence of the CDR region of the monoclonal antibody is shown as SEQ ID NO.1, and the light chain amino acid sequence thereof is shown as SEQ ID NO. 3.

2. Use of the monoclonal antibody of claim 1 for the preparation of a feline coronavirus detection product.

Application of feline coronavirus BS-8 strain nucleocapsid protein shown in SEQ ID NO.5 in preparation of murine monoclonal antibody of feline coronavirus nucleocapsid protein.

Application of feline coronavirus BS-8 strain nucleocapsid protein shown in SEQ ID NO.5 in preparation of feline coronavirus detection products.

5. A gene encoding the murine monoclonal antibody to the feline coronavirus nucleocapsid protein of claim 1, wherein the gene encoding the heavy chain amino acid has the sequence shown in SEQ ID No.2 and the gene encoding the light chain amino acid has the sequence shown in SEQ ID No. 4.

6. A recombinant expression vector comprising the gene of claim 5.

7. A host cell comprising the recombinant expression vector of claim 6.

8. A product for detecting feline coronavirus comprising the monoclonal antibody of claim 1.

9. An ascites indirect immunofluorescence method for detecting feline coronavirus, comprising using the monoclonal antibody of claim 1 as a primary antibody and using goat anti-mouse IgG as a secondary antibody.

10. A frozen section indirect immunofluorescence method for detecting cat coronavirus, characterized in that the monoclonal antibody of claim 1 is used as a primary antibody, and goat anti-mouse IgG is used as a secondary antibody.