CN114409772B

CN114409772B - Detection kit for M protein of porcine reproductive and respiratory syndrome virus and application thereof

Info

Publication number: CN114409772B
Application number: CN202210045288.9A
Authority: CN
Inventors: 梁荣乾; 闵运
Original assignee: Zhejiang Orient Gene Biotech Co Ltd
Current assignee: Zhejiang Orient Gene Biotech Co Ltd
Priority date: 2022-01-15
Filing date: 2022-01-15
Publication date: 2023-07-21
Anticipated expiration: 2042-01-15
Also published as: CN114409772A

Abstract

The invention discloses a monoclonal antibody of porcine reproductive and respiratory syndrome virus, wherein the heavy chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO.2, and the light chain variable region sequence is shown as SEQ ID NO. 3. The invention also discloses a kit for detecting porcine reproductive and respiratory syndrome virus, which comprises an effective amount of monoclonal antibody, PRRSV M protein and a matched detection reagent. The monoclonal antibody prepared by the invention has good specificity and sensibility, and is suitable for preparing different porcine reproductive and respiratory syndrome virus diagnostic reagents, such as colloidal gold, ELISA, chemiluminescence and other detection kits. The kit has good sensitivity and specificity, and is suitable for screening PRRSV and evaluating vaccine immunity.

Description

Detection kit for M protein of porcine reproductive and respiratory syndrome virus and application thereof

Technical Field

The invention belongs to the technical field of diagnosis of viral epidemic diseases, and particularly relates to a detection kit for porcine reproductive and respiratory syndrome virus M protein and application thereof.

Background

Porcine reproductive and respiratory syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS for short), commonly known as Blue Ear Disease, is an infectious Disease that severely harms the pig industry caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). PRRS can cause reproductive dysfunction in sows and severe respiratory diseases in piglets, and is a swine infectious disease which seriously affects economic benefits.

PRRSV is a member of the genus arterivirus, a single-stranded positive strand RNA virus with a envelope, of about 15kb in size, and the virion is spherical with a diameter of 55-60 nm. There are 2 serotypes of virus, namely American and European, and strains isolated in China are American. Viruses are sensitive to acids and alkalis, and are particularly not alkali-resistant, and general disinfectants have effects on the viruses, but can keep the infectivity in the air for about 3 weeks. The RNA of PRRSV contains 9 Open Reading Frames (ORFs), 1a, 1b, 2a, 2b, 3, 4, 5, 6, 7, respectively. Major structural proteins of PRRSV include GP5 protein (envelope glycoprotein), M protein (matrix protein), N protein (nucleocapsid protein). Thus, GP5 protein and M protein are target proteins for PRRSV diagnosis. The invention mainly establishes a set of diagnostic kit aiming at PRRSV M protein, which is used for detecting PRRSV antibody.

Disclosure of Invention

In order to make up for the defects of the prior art, one of the purposes of the invention is to provide a monoclonal antibody of PRRSV M protein and a preparation method thereof; the invention also provides a kit for detecting the PRRSV M protein and monoclonal antibody prepared by the method.

Therefore, on the one hand, the invention discloses a monoclonal antibody of porcine reproductive and respiratory syndrome virus, which can specifically bind PRRSV M protein, the heavy chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO.2, and the light chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO. 3.

Preferably, the heavy chain constant region of the monoclonal antibody is lgG1 type, and the light chain constant region is Kappa type.

Preferably, the epitope recognized by the monoclonal antibody is located in aa1-aa14 of PRRSV M protein, and the amino acid sequence of aa1-aa14 of PRRSV M protein is MGSSIDDFCNDSTA.

In another aspect, the present invention also provides a method for preparing the monoclonal antibody, the method comprising the steps of: 1) Preparing PRRSV M protein; 2) Preparing hybridoma cells for specifically expressing the anti-PRRSV M protein monoclonal antibody; 3) Preparing ascites fluid of the mouse by using the hybridoma prepared in the step 2), and purifying the anti-PRRSV M protein monoclonal antibody from the ascites fluid.

Preferably, when PRRSV M protein is prepared in the step 1) of the invention, the nucleotide sequence of the expressed M protein is shown as SEQ ID NO. 1.

In yet another aspect, the present invention provides a kit for detecting antibodies to porcine reproductive and respiratory syndrome virus, said kit comprising an effective amount of said monoclonal antibody and PRRSV M protein; and a matched detection reagent.

Preferably, the kit is a blocking ELISA antibody detection kit, and the coating concentration of the PRRSV M protein is 1 mug/ml.

Preferably, the kit of the present invention further comprises a sample diluent, a 25 x concentrated wash solution, a substrate solution, a stop solution, a positive control, and a negative control.

In still another aspect, the invention also provides an application of the monoclonal antibody against PRRSV M protein in a diagnostic reagent for porcine reproductive and respiratory syndrome virus antibody.

The PRRSV M protein prepared by the invention is suitable for preparing different diagnostic reagents for porcine reproductive and respiratory syndrome virus, such as colloidal gold, chemiluminescence detection kit and the like. The monoclonal antibody of the PRRSV M protein has good specificity and sensibility, and is suitable for preparing different diagnostic reagents of porcine reproductive and respiratory syndrome virus, such as detection kits of colloidal gold, ELISA, chemiluminescence and the like.

The kit for detecting the porcine reproductive and respiratory syndrome virus antibody is suitable for detecting the porcine reproductive and respiratory syndrome virus antibody in porcine serum, has the advantages of strong specificity, high sensitivity, good stability and high detection speed, and can be used for screening porcine reproductive and respiratory syndrome virus, evaluating vaccine immunity and the like.

Drawings

FIG. 1 SDS-PAGE of PRRSV M protein after purification, 1 is the PRRSV M protein after purification.

FIG. 2 shows SDS-PAGE patterns after purification of monoclonal antibodies, and FIG. 1 shows the purified monoclonal antibodies.

FIG. 3BCA kit calibration curve.

FIG. 4 cross-reactivity assay, wherein M is Marker,1-5 are PCV2, CSFV, PRRSV, PRV, PEDV/TGEV vaccine samples, respectively, and 6 is PRRSV M protein.

FIG. 5PRRSV M protein transmembrane region prediction results. Wherein an Inside indicates an intracellular region, a larger Inside number indicates a greater likelihood that the amino acid is located in the intracellular region; outlide indicates an extracellular region, and the larger the outlide number, the greater the likelihood that the amino acid will be located in the extracellular region; transmembrane represents a Transmembrane region, and a larger value of Transmembrane represents a greater probability of the amino acid being in the Transmembrane region.

FIG. 6 is a graph showing the results of ELISA detection of monoclonal antibody-recognizing antigen epitope.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

Example 1: preparation of porcine reproductive and respiratory syndrome virus M protein

The complete coding sequence (CDR) of PRRSV M gene (GenBank: MN 913537.1) is subjected to codon optimization, the nucleotide sequence (shown as SEQ ID NO. 1) of PRRSV M protein after the codon optimization is subcloned into pET-30a plasmid to construct a prokaryotic expression vector pET-30a-M, recombinant clone pET-30a-M is transformed into E.coli BL21 (DE 3), single colony is selected and cultured in LB culture medium containing kanamycin (final concentration is 50 mu g/ml), when the logarithmic phase (OD 600 is 0.6-1.0), IPTG is added to transfer to 16 ℃, and induction is carried out for 16h, so as to express target protein. Purifying by nickel column purification method, eluting target protein with 350mM imidazole, concentrating the eluate with 10kD ultrafiltration tube, dialyzing the eluate, changing into PBS, and detecting 20 μl PRRSV M protein with SDS-PAGE protein gel, wherein there is a clear band with purity of about 19.9kDa or more (figure 1). Protein content was measured using BCA kit at a protein concentration of 1.45mg/ml.

Example 2: preparation of anti-PRRSV M protein monoclonal antibody

BALB/c mice were immunized with PRRSV M protein prepared in example 1 as antigen. After the PRRSV M protein and Freund's complete adjuvant are subjected to isovolumetric emulsification, muscle is subjected to multipoint injection to obtain BALB/c female mice of 6-8 weeks old, and 100 mug of each female mouse is obtained. After two weeks, the mixture was emulsified with incomplete Freund's adjuvant, and the mixture was subjected to intramuscular multipoint immunization at 100. Mu.g/dose, and the mixture was repeated once at two-week intervals, and after the third immunization, the antibody titer was determined. Titers above 1:10 ⁵ Spleen cells from a mouse were collected, ground and isolated, fused with myeloma Sp2/0 cell lines by the PEG method, and the fused cells were resuspended in complete RPMI 1640 medium containing 20% fresh bovine serum of HAT, and then plated in 96-well plates at 100. Mu.l/well, 37℃and 5% CO ₂ Culturing. After 15 days, the cells were cultured in complete RPMI 1640 medium containing 20% calf serum with HT. The culture supernatants were taken and tested for positive clones by ELISA coated with PRRSV M protein (100 ng/well coating). And (3) carrying out cloning culture on the screened cell strain with high antibody titer and good morphology by adopting a limiting dilution method until monoclonal is obtained, and carrying out expanded culture and preservation on the hybridoma cells.

Example 3: purification of anti-PRRSV M protein monoclonal antibodies

Taking 6-8 week-old BALB/c female mice, injecting pristane, 0.5 ml/mouse, and injecting hybridoma cells 1×10 per mouse 7 days later ⁶ And after 7-10 days, extracting ascites of the mice, centrifuging at 8000rpm at 4 ℃ for 30min, and collecting supernatant. Monoclonal antibodies were purified using caprylic acid-ammonium sulfate precipitation, briefly described as follows: taking 10ml of ascites supernatant, adding 40ml of acetate buffer (0.06 mol/L, pH value is 4.5), adding 330 μl of octanoic acid at room temperature after uniformly mixing, mixing at room temperature for 30min, standing at 4deg.C for 2 hours, centrifuging at 4deg.C for 30min at 10000rpm, collecting supernatant, adding the same volume of precooled saturated ammonium sulfate solution in ice bath, standing at 4deg.C for 14+ -2 hours, centrifuging at 4deg.C for 5000rpmAnd collecting the precipitate after 30 min. The pellet was dissolved in 5ml PBS and dialyzed against PBS overnight. The dialyzed monoclonal antibody was sterilized by filtration through a 0.22 μm filter, and was aliquoted at 0.1 ml/tube. Mu.l of the monoclonal antibody was assayed using SDS-PAGE protein gel with two clear bands having a purity of 90% or more (FIG. 2), and the concentration of the monoclonal antibody was assayed using BCA kit (FIG. 3) at 1.36mg/ml.

Example 4: detection of monoclonal antibodies

Reactivity determination: ELISA titers of monoclonal antibodies were determined by indirect ELISA (coating amount of PRRSV M protein 100 ng/well), and monoclonal antibodies (initial concentration 10. Mu.g/ml) were diluted in a 1:10 ratio of fold to 1:10 ⁷ As a result, the ratio was 1:10 ⁶ (positive when the P/N value is greater than 2.1, wherein P is the OD450nm value of the sample and N is the OD450nm value of the blank). The titers of monoclonal antibodies are shown to be good.

Cross-reactivity identification: PCV2 subunit vaccine, CSFV live vaccine, PRRSV live vaccine, PRV live vaccine and PEDV/TGEV live vaccine are purchased from the market, and after dilution according to the specification, total protein is extracted for the detection of the wersterin blot (1:1000 times dilution of monoclonal antibody, 1:10000 times dilution of HRP-labeled goat anti-mouse IgG secondary antibody). The results (FIG. 4) show that the monoclonal antibodies have no specific reaction, indicating that the monoclonal antibodies have good specificity.

Subclass determination: the subtype of the monoclonal antibody was identified using a mouse monoclonal antibody subtype identification kit (Sigma, mouse Monoclonal Antibody Isotyping Kit), and the identification result shows that the heavy chain constant region of the monoclonal antibody is lgG1 type and the light chain constant region of the monoclonal antibody is Kappa type.

Example 5: monoclonal antibody sequencing

Total RNA of hybridoma cells was extracted according to the instructions using Trizol reagent (Thermo Co.). Mu.g of total RNA was taken, 1.0. Mu.l of oligo (dT) (10. Mu.M) was added, 1. Mu.l of dNTPs (10 mM) was added, the total volume was brought to 13. Mu.l with DECP water, and the mixture was homogenized, incubated at 65℃for 5min and then placed on ice for 1min, then 4.0. Mu.l of RT buffer (5X), 1.0. Mu.l of DTT (100 mM), 1.0. Mu. l Ribonuclease Inhibitor and 1.0. Mu.l of reverse transcriptase (takara Co.) were added, reacted at 50℃for 10min, and incubated at 80℃for 10min to terminate the reaction, and the obtained cDNA was stored at-20 ℃. Specific nested PCR primers are designed, the primer sequence used in the amplification reaction is complementary with the first framework region and the constant region of the antibody variable region, and a target gene is amplified by adopting a conventional PCR method. Among them, the primer sequence was designed according to the literature (CN 111393525B). The sequences of the heavy chain variable region and the light chain variable region of the antibody are shown as SEQ ID NO.2 and SEQ ID NO.3 after sequencing. The amino acid sequence information of the variable region of the monoclonal antibody is shown in Table 1.

TABLE 1 amino acid sequence information for monoclonal antibodies

Example 6: determination of monoclonal antibody recognition epitopes

To investigate the recognition region of the monoclonal antibodies, the transmembrane region of the expressed PRRSV M protein was predicted and the results are shown (fig. 5). According to the predicted result, 7-segment polypeptide is designed and synthesized to cover the whole PRRSV M protein. The amino acid sequences of the polypeptide fragments are shown in Table 2.

TABLE 2 predicted transmembrane region results for PRRSV M protein

Diluting the polypeptide with coating buffer (0.05M sodium carbonate solution with pH of 9.6), coating the ELISA plate, and standing at 4deg.C overnight; PBST washes the plate 3 times; blocking with 5% skimmed milk, and incubating at 37deg.C for 2 hr; the monoclonal antibody obtained by the invention is used as a primary antibody to carry out indirect ELISA, and the monoclonal antibody is incubated for 1h at 37 ℃; after washing the plate, adding HRP-labeled goat anti-mouse IgG, and incubating for 30min at 37 ℃; after washing the plate, TMB substrate chromogenic solution is added, after incubation for 10min at 37 ℃, chromogenic is stopped, and reading is performed by an enzyme-labeled instrument. The results (FIG. 6) show that the screened monoclonal antibodies can react specifically with the polypeptide P1 (MGSSIDDFCNDSTA).

Example 7: detection of PRRSV antibodies (blocking ELISA method)

The PRRSV M protein prepared in example 1 and the monoclonal antibodies prepared in examples 2, 3 were used to establish a porcine reproductive and respiratory syndrome virus blocking ELISA antibody detection method for serum antibody detection of porcine reproductive and respiratory syndrome virus.

1 preparation of the kit

1.1 preparation of an ELISA plate: PRRSV M protein was diluted to 1. Mu.g/ml with coating buffer (0.05M pH9.6 sodium carbonate solution), and the ELISA plate was added at 100. Mu.l/well overnight at 4 ℃; taking out, discarding the liquid in the hole, washing 3 times by using a washing liquid, and beating to dry; blocking solution (1% BSA in PBST) was added, 200. Mu.l/well and incubated at 37℃for 2 hours; taking out, discarding the liquid in the hole, beating to dry, and vacuum packaging;

1.2 positive control: the commercially available vaccine (purchased from Boringer) was used to immunize healthy and susceptible 28-day-old piglets (ASFV, CSFV, FMDV, PRV, PCV, PRRSV, TGEV antibody antigen negative, and their antigens and antibodies were detected using a commercial kit), and the two and three-way-free were performed on days 14 and 28, respectively, 2 ml/head/time, 7 days after three-way-free were collected, detected using an indirect ELISA method, and pigs with titers higher than 1:10000 were selected for serum preparation. Carotid artery exsanguination is carried out on experimental pigs meeting the conditions, centrifugation is carried out for 10 minutes at 3000r/min, supernatant is taken, proClin300 with the final concentration of 0.1% is added, and filtration sterilization is carried out by a 0.22 mu m filter membrane after uniform mixing. And (5) quantitatively subpackaging, wherein 1 ml/tube is the positive control.

1.3 negative control: healthy and susceptible 28-day-old piglets (ASFV, CSFV, FMDV, PRV, PCV, PRRSV and TGEV antibody antigen are negative, and the antigens and antibodies are detected by using a commercial kit), carotid artery bloodletting is carried out, centrifugation is carried out for 10 minutes at 3000r/min, the supernatant is taken, proClin300 with the final concentration of 0.1% is added, and after uniform mixing, a 0.22 mu m filter membrane is used for filtration sterilization. And (5) quantitatively packaging, wherein 1 ml/tube is taken as a negative control.

1.4 preparation of sample dilutions: 1 XPBST buffer containing 1% BSA and 0.1% ProClin300, and filtering with 0.22 μm filter membrane for sterilization, and packaging quantitatively.

1.5 Preparation of 25 x concentrated washes: proClin300 with the final concentration of 0.1% is added into 25 XPBST solution, and the mixture is uniformly mixed, filtered and sterilized by a 0.22 mu m filter membrane, and the mixture is quantitatively packaged.

1.6 preparation of enzyme-labeled antibody: to dilute 1 ten thousand-fold with sample dilutions (using the ELISA plate prepared in this way, HRP-labeled monoclonal antibody was detected by direct ELISA, maximum dilution at OD450nm greater than 1.1). Wherein the HRP label is prepared with reference to the instructions of the HRP conjugate kit (ab 102890).

1.7 preparation of substrate solution: is a single-component TMB color development liquid of Beijing Soxhaust biological technology Co., ltd.

1.8 preparation of stop solution: is 2M H ₂ SO ₄ 。

1.9 assembly of the kit: the kit was assembled as follows.

Name of the name	Quantity of
		Antigen coated plate	2 pieces of board (192 parts/box)
Positive control	1 mL/tube
		Negative control	1 mL/tube
Sample diluent	12 mL/bottle
		25 Xconcentrated washing liquid	30 mL/bottle
Enzyme-labeled antibody	25 mL/bottle
		Substrate solution	25 mL/bottle
Stop solution	12 mL/bottle
		Description	1 part of

2 detection of the kit

2.1 sample addition: add 50. Mu.l of sample dilution to each well, and then add 50. Mu.l of positive control, negative control and serum to be tested to the corresponding well, mix well by shaking. Incubate at 37℃for 60 min.

2.2 washing: after the incubation is finished, the liquid in the hole is thrown away, the washing liquid is added into the hole, 300 mu l/hole is washed for 3 to 5 times, and the hole is dried by beating.

2.3 incubation of enzyme-labeled antibody: enzyme-labeled antibody was added at 100. Mu.l/well and incubated at 37℃for 30 minutes.

2.4 washing: the method is the same as 2.2.

2.5 color development: substrate solution, 100. Mu.l/well, was added and incubated at 37℃for 10 minutes in the absence of light.

2.6 termination: stop solution, 50. Mu.l/well, was added and mixed with gentle shaking.

2.7 reading: immediately after the termination solution is added, the coating plate is placed in an enzyme labeling instrument to read OD _450nm Values.

2.8S/N value calculation the S/N value is calculated according to the following calculation formula.

2.9 determination

2.9.1 test establishment conditions: the OD450nm reading per well of the negative control wells should be greater than 0.7 and the maximum difference between wells should be <0.3, and the OD450nm reading per well of the positive control wells should be <0.3.

2.9.2, when the S/N value is more than 0.6, judging as negative; and judging positive when the S/N value is less than or equal to 0.6.

3, verifying the performance of the kit: 3 kits were continuously produced according to the above method, with lot numbers 211101, 211102, 211103, respectively, and performance verification was performed using these three kits.

3.1 sensitivity verification: the three batches of kits carry out gradient detection on the prepared positive control, and the minimum detection gradient can reach 1600 times, so that the sensitivity of the kit is good. The specific data are shown in Table 3.

TABLE 3 sensitivity test results (S/N value)

3.2 specificity verification: the three batches of the kit detect 7 parts of swine-origin specific quality control serum, and the results are negative, which indicates that the specificity of the kit is good. The specific data are shown in Table 4.

TABLE 4 specificity test results

3.3 repeatability verification: the variation coefficient of the three batches of kits for the positive serum and the negative serum is less than 10% in batches, which indicates that the repeatability of the kit is good. See in particular tables 5 and 6.

TABLE 5 repeatability test results (in batch)

TABLE 6 repeatability test results (batch to batch)

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Sequence listing

<110> Liang Rongqian

<120> detection kit for M protein of porcine reproductive and respiratory syndrome virus and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 522

<212> DNA

<213> nucleotide sequence of PRRSV M protein after codon optimization (Artificial Sequence)

<400> 1

atgggttctt ccatcgatga tttctgcaac gactccaccg ctgcccagaa agtactgctg 60

gctttcagca tcacctatac cccgatcatg atctacgccc tgaaagtttc tcgtggtcgt 120

ctgctgggtc tgctgcacct gctgatcttc ctgaactgca ccttcacctt tggttacatg 180

acctttgtac acttccagtc taccaacaaa gtggcgctga ccatgggcgc tgtcgttgca 240

ctgctgtggg gtgtttacag cgccatggaa acctggcgtt tcatcacttc tcgttgccgt 300

ctgtgtctgc tgggtcgcaa atacatcctg gcaccggcgc accacgttga atctgctgcg 360

ggtttccacc ctatcaccgc gagcgacaac catgccttcg tcgtacgtcg cccgggttcc 420

actaccgtta acggtactct ggttccgggt ctgaaatccc tggtactggg cggtcgtcgt 480

gcagtgaaac gtggtgtagt taacctggtt aaatacgcga aa 522

<210> 2

<211> 369

<212> DNA

<213> anti-PRRSV M protein monoclonal antibody heavy chain variable region sequence (Artificial Sequence)

<400> 2

gaaaccctgg tggaaagcgg cgatctgcag atgaacagcc tgaaaagcga agatagctgc 60

gcggcgagcg gctttacctt tagccattat ggcagctggg tgcgccagac cccggataaa 120

cgcctggaat gggtggcgac cattggcagc cgcggcacct atacccatta tccggatagc 180

gtgaaaggcc gctttaccat tagccgcgat aacgataaaa acgcgctgta tctggtgaaa 240

ccgggcggca gcctgaaact gaccgcgatg tattattgcg cgcgccgcag cgaattttat 300

ctggtgaaac cgagcaacaa cggcggcagc ctgaaactgg gccagggcac caccgtgacc 360

gtgagcagc 369

<210> 3

<211> 327

<212> DNA

<213> anti-PRRSV M protein monoclonal antibody light chain variable region sequence (Artificial Sequence)

<400> 3

gatattctga cccagagccc ggcgagcctg gcggtgagcc tgaccagcgt gggcgatcgc 60

gtgagcatta cctgcaaagc gagccagaac gtgggcaccg cggtgcagaa accgggccag 120

ccgccgaaac tgatttatgc gattagcaac cgcggcagcg gcgtgccggc gcgctttagc 180

ggcagcggca gcggcaccga ttttagcctg aacattcatc cggtggaaga agatgatccg 240

gcgatgtatt tttgccagca gaccagcggc accgatacca aagaagtgcc gtggaccttt 300

ggcgcgggca ccaaactgga aattaaa 327

Claims

1. The monoclonal antibody of the porcine reproductive and respiratory syndrome virus can specifically bind PRRSV M protein, and is characterized in that the heavy chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO.2, and the light chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO. 3.

2. The monoclonal antibody of claim 1, wherein the heavy chain constant region of the monoclonal antibody is lgG1 and the light chain constant region is Kappa.

3. The monoclonal antibody of claim 1, wherein the epitope recognized by the monoclonal antibody is located between aa1 and aa14 of PRRSV M protein, and the amino acid sequence of aa1 to aa14 of PRRSV M protein is MGSSIDDFCNDSTA.

4. A kit for detecting antibodies to porcine reproductive and respiratory syndrome virus, said kit comprising an effective amount of the monoclonal antibody of claim 1 and PRRSV M protein; and a matched detection reagent.

5. The kit according to claim 4, wherein the kit is a blocking ELISA antibody detection kit, and the coating concentration of the PRRSV M protein is 1 [ mu ] g/ml.

6. The kit of claim 4, further comprising a sample diluent, a 25 x concentrated wash, a substrate solution, a stop solution, a positive control, and a negative control.

7. Use of the monoclonal antibody against PRRSV M protein according to claim 1 in a diagnostic reagent for porcine reproductive and respiratory syndrome virus antibodies.