CN113956353A

CN113956353A - Monoclonal antibody of anti-porcine acute diarrhea syndrome coronavirus N protein, recognition region of monoclonal antibody and application of monoclonal antibody

Info

Publication number: CN113956353A
Application number: CN202111182911.7A
Authority: CN
Inventors: 王�琦; 曹丽艳; 郑海学; �田宏; 孔祥雨; 袁聪; 段月月; 李想通; 索学鹏
Original assignee: Lanzhou Veterinary Research Institute of CAAS; Institute of Urban Agriculture of Chinese Academy of Agricultural Sciences
Current assignee: Lanzhou Veterinary Research Institute of CAAS; Institute of Urban Agriculture of Chinese Academy of Agricultural Sciences
Priority date: 2021-10-11
Filing date: 2021-10-11
Publication date: 2022-01-21
Anticipated expiration: 2041-10-11
Also published as: CN113956353B

Abstract

The invention belongs to the technical field of biology, and particularly relates to an N protein monoclonal antibody for resisting porcine acute diarrhea syndrome coronavirus (SADS-CoV). The invention clones the full-length N protein gene into a pET32a vector, constructs a prokaryotic expression recombinant plasmid pET32a-N, and obtains purified N protein through induction expression and protein purification. The purified N protein is used for immunizing Balb/c mice, spleen cells of the immunized mice are taken to be fused with mouse myeloma SP2/0 cells, and two anti-N protein monoclonal antibodies are obtained by screening and are named as 4B10 and 6E8 respectively. Through truncated expression, the antibody is found to be capable of specifically recognizing and binding to the N-terminal region of the N protein, and the recognition regions of 4B10 and 6E8 are 64-84 aa. The monoclonal antibody provided by the invention can be obtained by using a genetic engineering or protein engineering method. The antibody can specifically recognize SADS-CoV, has neutralizing activity on viruses, and provides a new raw material for researching the pathogenic mechanism of SADS-CoV, developing an early detection kit and a therapeutic antibody.

Description

Monoclonal antibody of anti-porcine acute diarrhea syndrome coronavirus N protein, recognition region of monoclonal antibody and application of monoclonal antibody

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an anti-porcine acute diarrhea syndrome coronavirus (SADS-CoV) N protein monoclonal antibody, a recognition region of the monoclonal antibody and application of the monoclonal antibody.

Background

Pig acute diarrhea syndrome (SADS) is an acute infectious disease caused by pig acute diarrhea syndrome coronavirus (SADS-CoV). The clinical manifestations mainly include acute diarrhea and vomiting of infected piglets, the newborn piglets within 5 days of age die quickly due to sudden weight loss and severe dehydration, and the death rate is up to 90%. The homology of the porcine enteric coronavirus and the HKU2 nucleotide sequence from bat reaches more than 90%. As no effective SADS vaccine and antiviral drug is available so far, the main measures for preventing and controlling the disease are to control the infection source and cut off the potential transmission path.

Coronaviruses (CoVs) are enveloped, single-stranded, positive-strand RNA viruses belonging to the order Nidovirales, the family Coronaviridae, the genus Coronavirus (Coronavirus). The SADS-CoV genome is approximately 27kb in size, has a cap structure at the 5 'end and a Poly (A) tail at the 3' end. The genome consists of 5 'non-coding regions (UTRs), 9 Open Reading Frames (ORFs) and 3' UTR regions. The 5' end 2/3 genome is ORF1a and ORF1b, encoding 16 nonstructural proteins (nsp 1-16). The 3' end 1/3 genome sequence includes 7 ORFs encoding 4 structural proteins: spike protein (S), envelope protein (E), membrane glycoprotein (M), and nucleocapsid protein (N); there is an ORF3 between the S and E proteins; n is followed by two overlapping ORFs encoding the nonstructural proteins NS7a and NS7 b. The length of the coding region of the N gene is 1128 bases, the coding region totally encodes 375 amino acids, and the molecular weight of the protein is about 41.7 ku. The N protein is a nucleoplasm shuttle protein, is a main binding site of serum antibodies and has good immunogenicity. The N protein has good conservation and can be expressed in large quantity in the whole process of virus infection, so the N protein can be used as a candidate protein for virus diagnosis.

Disclosure of Invention

The invention provides an N protein monoclonal antibody for resisting porcine acute diarrhea syndrome coronavirus, and a region of the monoclonal antibody for recognizing the N protein and application of the monoclonal antibody.

In a first aspect of the present invention, a monoclonal antibody against porcine acute diarrhea syndrome coronavirus N protein is provided, wherein the monoclonal antibody comprises a heavy chain constant region, a heavy chain variable region, a light chain constant region and a light chain variable region;

the heavy chain variable region comprises CDR1 with an amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 4, CDR2 with an amino acid sequence shown as SEQ ID NO. 2 or SEQ ID NO. 5, and CDR3 with an amino acid sequence shown as SEQ ID NO. 3 or SEQ ID NO. 6; the heavy chain variable region of the monoclonal antibody 4B10 prepared by the invention comprises a CDR1 with an amino acid sequence shown as SEQ ID NO. 1, a CDR2 with an amino acid sequence shown as SEQ ID NO. 2 and a CDR3 with an amino acid sequence shown as SEQ ID NO. 3, and the heavy chain variable region of the monoclonal antibody 6EB prepared by the invention comprises a CDR1 with an amino acid sequence shown as SEQ ID NO. 4, a CDR2 with an amino acid sequence shown as SEQ ID NO. 5 and a CDR3 with an amino acid sequence shown as SEQ ID NO. 6.

The light chain variable region comprises CDR1 with an amino acid sequence shown as SEQ ID NO. 7 or SEQ ID NO. 10, CDR2 with an amino acid sequence shown as SEQ ID NO. 8 or SEQ ID NO. 11, and CDR3 with an amino acid sequence shown as SEQ ID NO. 9 or SEQ ID NO. 12. The light chain variable region of the monoclonal antibody 4B10 prepared by the invention comprises a CDR1 with an amino acid sequence shown as SEQ ID NO. 7, a CDR2 with an amino acid sequence shown as SEQ ID NO. 8 and a CDR3 with an amino acid sequence shown as SEQ ID NO. 9, and the light chain variable region of the monoclonal antibody 6EB prepared by the invention comprises a CDR1 with an amino acid sequence shown as SEQ ID NO. 10, a CDR2 with an amino acid sequence shown as SEQ ID NO. 11 and a CDR3 with an amino acid sequence shown as SEQ ID NO. 12.

The amino acid sequence of the heavy chain variable region of the monoclonal antibody for resisting the porcine acute diarrhea syndrome coronavirus N protein is shown as SEQ ID NO. 13 or SEQ ID NO. 14; the DNA sequence of the light chain variable region of the monoclonal antibody for resisting the porcine acute diarrhea syndrome coronavirus N protein is shown as SEQ ID NO. 17 or SEQ ID NO. 18.

In a second aspect, the invention provides a DNA sequence for encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody against the porcine acute diarrhea syndrome coronavirus N protein.

The gene nucleotide sequence of the variable region of the heavy chain of the monoclonal antibody for encoding the N protein of the porcine acute diarrhea syndrome is shown as SEQ ID NO. 15 or SEQ ID NO. 16; the gene nucleotide sequence of the variable region of the light chain of the monoclonal antibody for encoding the anti-porcine acute diarrhea syndrome coronavirus N protein is shown as SEQ ID NO. 19 or SEQ ID NO. 20.

The heavy chain constant region of the monoclonal antibody against the porcine acute diarrhea syndrome coronavirus N protein is IgG1 or IgG2 a.

The light chain constant region of the monoclonal antibody for resisting the porcine acute diarrhea syndrome coronavirus N protein is Kappa type.

In a third aspect of the invention, a monoclonal antibody against the porcine acute diarrhea syndrome coronavirus N protein can specifically identify a region of the porcine acute diarrhea syndrome coronavirus N protein; the monoclonal antibody for resisting the porcine acute diarrhea syndrome coronavirus N protein can specifically identify the N-terminal region of the porcine acute diarrhea syndrome coronavirus N protein and has a sequence shown as SEQ ID NO. 21.

The fourth aspect of the invention provides a preparation method of an anti-porcine acute diarrhea syndrome coronavirus N protein monoclonal antibody, which comprises the steps of cloning the porcine acute diarrhea syndrome coronavirus N gene into a pET-32a vector to construct a prokaryotic expression vector, purifying the protein by using imidazole elution modes with different concentrations, taking the purified N protein as an antigen to immunize a Balb/c mouse, taking mouse spleen cells and myeloma cells SP2/0 to fuse, preparing hybridoma cells, carrying out indirect ELISA and indirect immunofluorescence verification on cell supernatants, screening to obtain positive clones, injecting the hybridoma cells into the mouse to prepare ascites after three times of subcloning, and finally purifying the obtained ascites to obtain the anti-porcine acute diarrhea syndrome coronavirus N protein monoclonal antibody.

In the fifth aspect of the invention, the application of the monoclonal antibody of the porcine acute diarrhea syndrome coronavirus N protein in preparing a medicament for treating the porcine acute diarrhea syndrome coronavirus infection is provided.

The invention has the beneficial effects that:

the invention utilizes a prokaryotic expression system to express and purify recombinant SADS-CoV N protein, takes the recombinant SADS-CoV N protein as an immune source to immunize a mouse, and successfully obtains two monoclonal antibodies aiming at the N protein through cell fusion and subcellular screening. The N gene was expressed truncated and found to specifically recognize and bind to the N-terminal region of the N protein. Specifically, the protein can specifically identify and combine 64-84 aa of the N-terminal region of the N protein, and a reliable research tool is provided for researching the function of the N protein.

The monoclonal antibody provided by the invention can be obtained by using a conventional genetic engineering or protein engineering method, avoids the loss of the antibody of the hybridoma cells in long-term cryopreservation, is also favorable for optimizing the antibody on the gene and protein levels, and further improves the specificity and the affinity of the antibody.

The monoclonal antibody prepared by the invention can specifically recognize and neutralize porcine acute diarrhea syndrome coronavirus, so that the antibody provides a new raw material for researching the pathogenic mechanism of SADS-CoV, developing an early detection kit and a therapeutic antibody.

Drawings

FIG. 1 shows the results of PCR amplification of SADS-CoV N gene and restriction enzyme digestion of pET32a-N recombinant plasmid;

A.M, DL2000 relative molecular mass standard; 1: N gene, about 1000bp in size; 2: control water.

B.M, DL5000 relative molecular mass standard; 1: the pET32a-N recombinant plasmid was digested with BamHI and XhoI.

FIG. 2 shows the results of recombinant protein expression and purification of pET32 a-N;

A.M: a protein Marker; 1: before induction, pET32 a-N; 2: pET32a-N cleavage of the supernatant; 3: precipitation after pET32a-N cleavage;

B.M: a protein Marker; 1-2: the purified pET32a-N protein.

FIG. 3 shows the antibody titer of mice detected by ELISA after immunization with N protein;

1-3: 1.2 and 3 immunized mice, NC: and (5) negative control.

FIG. 4 is an IFA identification of monoclonal antibody reactivity;

A.4B10 monoclonal antibody;

B.6E8 monoclonal antibody;

SP2/0 cell culture fluid.

FIG. 5 is a Western blot to identify the reactivity of monoclonal antibodies;

A.4B10 monoclonal antibody;

B.6E8 monoclonal antibody.

M: protein Marker, 1: purifying the protein by pET32 a-N; 2: huh7 cell controls; 3: SADS-CoV infected Huh7 cells.

FIG. 6 shows the result of subclass identification.

FIG. 7 shows the results of neutralization of SADS-CoV virus at 0.01MOI after 10, 20 and 40 fold dilutions of 4B10 and 6E8 monoclonal antibodies;

FIG. 8 shows the result of identifying the N protein region recognized by an antibody;

A.N protein truncation expression scheme;

the regions of R1 and R2 induced expression of SDS-PAGE identification. M: protein marker; 1: before R1 induction; 2: r1 lysis supernatant; 3: r1 cleavage of the precipitate; 4: before R2 induction; 5: after induction of R2.

C.4B10 western blot identification results. M: protein marker; 1: r1 expression bacteria; 2: r2 expression strain.

D.6E8 western blot identification results. M: protein marker; 1: r1 expression bacteria; 2: r2 expression strain.

The E.R1.1 and R1.2 regions induce expression of SDS-PAGE identification results. M: protein marker; 1: r1.1 before induction; 2: after R1.1 induction; 3: r1.2 before induction; 4: after R1.2 induction.

F.4B10 western blot identification results. M: protein marker; 1: r1.1 expression bacteria; 2: r1.2 expression bacteria.

G.6E8 western blot identification results. M: protein marker; 1: r1.1 expression bacteria; 2: r1.2 expression bacteria.

The regions of H.R1.2.1 and R1.2.2 induced expression of SDS-PAGE identification. M: protein marker; 1: r1.2.1 before induction; 2: r1.2.1 after induction; 3: r1.2.2 before induction; 4: r1.2.2 after induction.

I.4B10 western blot identification results. M: protein marker; 1: r1.2.2 expression bacteria; 2: r1.2.1 expressing the bacteria.

And (3) identifying the result of the J.6E8 western blot. M: protein marker; 1: r1.2.1 expression bacteria; 2: r1.2.2 expressing the bacteria.

FIG. 9 shows the result of variable region PCR amplification;

A. and (3) carrying out PCR amplification on the heavy chain variable region. M: DL2000 marker; 1: water control; 2: 4B10 heavy chain variable region PCR amplification; 3: 6E8 heavy chain variable region was PCR amplified.

B. And (3) performing PCR amplification on the light chain K variable region. M: DL2000 marker; 1: water control; 2: 4B10 light chain variable region PCR amplification; 3: 6E8 light chain variable region was PCR amplified.

Detailed Description

The present invention will be described in more detail with reference to the following embodiments for understanding the technical solutions of the present invention, but the present invention is not limited to the scope of the present invention.

The strain SADS-CoV GDS04 was given by professor Cao Yongchang, university of Zhongshan.

Construction, expression and purification of SADS-CoVN protein recombinant plasmid

1.1 construction of the pET32a-N recombinant plasmid

Specific primers were designed with reference to SADS-CoV GDS04 strain N protein (Genbank accession No.: MF167434.1), and the upstream P1: 5' -CGCGGATCCATGGCCACTGTTAATTGG-3', downstream P2: 5' -CCGCTCGAGCTAATTAATAATCTCATC-3 ', and BamHI and XhoI cleavage sites (underlined) were introduced at the 5' ends of the upstream and downstream primers. After primer synthesis, PCR amplification was performed using SADS-CoV GDS04 strain cDNA as a template. The PCR reaction system is as follows: PrimeSTAR Max Premix (2X) 25. mu.L, each of P1 and P2 1. mu.L, cDNA 1. mu.L, ddH₂And O is supplemented to 50 mu L. The reaction procedure is as follows: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min, and 30 cycles; extension at 72 ℃ for 10 min. The PCR product was detected by electrophoresis on a 1% agarose gel. The gel recovery was carried out according to the instruction of the EZNAGEL Extraction Kit of Omega.

The recovered product of the N gene PCR gel and pET32a vector are double digested with BamH I and XhoI, and then connected to transform DH5 alpha competent cell. And carrying out double enzyme digestion identification on the constructed recombinant plasmid, carrying out sequencing identification on the plasmid identified as positive by enzyme digestion, and naming the positive recombinant plasmid as pET32 a-N.

1.2 inducible expression and purification of N recombinant proteins

The recombinant plasmid pET32a-N was transformed into E.coli BL21(DE3), and a single colony was picked up in 5mL of ampicillin-resistant LB as OD₆₀₀When the concentration is about 0.8, IPTG is added to the medium to a final concentration of 1mmol/L for induction expression. Centrifuging at 12000rpm for 2min 5h after induction, collecting precipitate, re-suspending the precipitate with appropriate amount of PBS, performing ultrasonic treatment for 5min (3 s over, stopping for 3s), centrifuging at 12000rpm for 10min at 4 deg.C after ultrasonic treatment, and collecting supernatant and precipitate respectively. Taking the supernatantAdding 5 × loading buffer to the solution and precipitate, boiling in boiling water for 10min, and performing SDS-PAGE identification.

The induced expression level of pET32a-N protein was expanded as described above, and the supernatant was lysed after sonication and purified by nickel column. The specific operation steps are as follows:

1) resin filling: adding 2mL of nickel column NTA resin into an empty column, washing the column once by using distilled water with the volume 5 times of that of the column when a preservation solution descends to the surface of the resin, and then balancing the column by using a balancing solution (20mM Tris-HCl, 500mM NaCl, 5mM imidazole, pH7.4) with the volume 5 times of that of the column;

2) loading: when the balance liquid is reduced to the surface of the resin, adding 3mL of cracking supernatant containing recombinant protein, repeatedly loading for 2-3 times, acting for 2min each time, and collecting sample flow-through liquid; adding 5 times of column volume of balancing solution to wash the column for 1 time;

3) protein elution: protein samples were eluted with different concentrations of imidazole (25mM, 50mM, 75mM, 100mM, 150mM, 200mM and 300mM) in equilibration solutions, 1mL each time, twice for each concentration, in order to determine the optimal wash-out and protein elution concentrations of imidazole;

4) and (3) column cleaning and preservation: the column was washed once with 5 volumes of 0.5M NaOH, then once with distilled water, followed by addition of an appropriate amount of 70% absolute ethanol and storage in a refrigerator at 4 ℃. Each collected sample was identified by SDS-PAGE.

2 animal immunization

3 female BALB/c mice of 6W age were selected, and the mice were immunized with the purified N recombinant protein in an amount of 30. mu.g/mouse. For the first immunization, the N protein and Freund's complete adjuvant are mixed in equal volume and emulsified, and a mouse is injected subcutaneously by multiple points (one point on the back and two points on the abdomen). And performing boosting immunization every 2W for four times, wherein the boosting immunization is to uniformly mix the N recombinant protein and Freund's incomplete adjuvant in equal volume for emulsification, and the immunization method is the same as the first immunization. And 7d after the four-immunization, tail blood collection is carried out to determine the antibody titer.

3 ELISA method for detecting polyclonal antibody titer

SADS-CoV virus solution and coating solution are diluted 1:1 to coat ELISA plates, 50 mu L/hole, coated overnight at 4 ℃, and washed four times with PBSTAfter that, 2% trehalose was added and blocked at 4 ℃ for 10 hours. Diluting positive serum and negative serum with PBST at multiple ratio of 1:100 to 1:12800, diluting 8 gradients, acting at 37 deg.C for 1h, washing PBST for four times, adding goat anti-mouse IgG labeled with HRP (1:20000 dilution), washing PBST for four times, developing TMB color, and measuring OD on microplate reader₄₅₀。

4 preparation of monoclonal antibodies

The specific operation steps are as follows:

(1) preparing feeder layer cells: HAT culture solution is injected into abdominal cavity of mouse, and is slowly and repeatedly sucked, and the liquid is spread in 96-well plate after being sucked out.

(2) Cell fusion: the spleen cells and a proper amount of SP20 cells are subjected to cell fusion under the action of a fusion agent PEG. The fused cells were plated in 96-well plates containing feeder cells.

(3) Screening of positive clones:

a) and (3) an indirect ELISA detection method, wherein the purified N protein is coated on an ELISA plate, and the condition that the fused cells secrete the antibody is detected.

b) An indirect Immunofluorescence (IFA) detection method comprises the steps of adding SADS-CoV into supernatant in ELISA antibody positive cell wells to infect Huh7 cells, then adding FITC-labeled goat anti-mouse IgG, and observing results under a fluorescence microscope after reaction.

(4) Subcloning of positive hybridoma cells: ELISA and IFA positive wells were selected and the screened positive hybridoma cells were subcloned by limiting dilution. Wells in which only a single clone grew were marked by observation under an inverted microscope, and the supernatant was taken and subjected to antibody detection using the ELISA and IFA methods described above. Positive cells were entered into the next round of subcloning for a total of three times.

(5) Preparing ascites: taking 10-12W Balb/c mice, injecting 0.5mL of Freund's incomplete adjuvant into the abdominal cavity of each mouse, and injecting 5 multiplied by 10 into the abdominal cavity of each mouse after 1W⁵And (3) carrying out hybridization on the hybridoma cells (0.2mL) for 7-10 days, then obviously bulging the abdominal cavity of the mouse body, collecting ascites, detecting the titer of the mouse body by ELISA, subpackaging and storing at-80 ℃.

(6) Purification of ascites: according to PIERCE company NAb^TMProtein G Spin Purification KitPerforming affinity chromatography purification, and then identifying the purity by SDS-PAGE electrophoresis.

5 identification of monoclonal antibodies

(1) And (3) specific identification: including ELISA, IFA and Western blot verification.

a) And (3) ELISA verification: purified N protein and His-tagged irrelevant protein (2. mu.g/mL) were diluted with coating solution and coated on ELISA plates at 50. mu.L/well overnight at 4 ℃ and washed four times with PBST, and then blocked with 2% trehalose at 4 ℃ for 10 h. Adding monoclonal antibody into protein N and His-labeled irrelevant protein coated plate, acting at 37 deg.C for 1h, washing with PBST for four times, adding HRP-labeled goat anti-mouse IgG (1:20000 dilution), washing with PBST for four times, developing TMB color, and measuring OD on enzyme labeling instrument₄₅₀。

b) IFA verification: SADS-CoV was used to infect Huh7 cells, cells were fixed with paraformaldehyde 36h after virus infection, ascites was diluted with PBS (500-fold) and added to virus-infected cells, followed by incubation with FITC-anti-mouse secondary antibody (100-fold dilution), and after the reaction was completed, observation was performed under a fluorescence microscope.

c) Western blot verification: purified N protein or collection of virus-infected and uninfected Huh7 cells were run on SDS-PAGE, followed by transfer of the protein gel to NC membranes for wersternblot validation. The primary antibody is a monoclonal antibody, and the secondary antibody is HRP-anti-mouse IgG.

(2) Subclass identification: according to SBA Clonotyping of Southern Biotech^TMSystem/HRP antibody subclass identification kit operating instructions for the antibody subclass identification of the obtained monoclonal antibody.

(3) And (3) identification of neutralizing activity: ascites fluid was diluted at different ratios (10, 20 and 40 fold) and mixed with equal volume of 0.01MOI SADS-CoV, incubated for 1h at 37 ℃ and then 100. mu.L per well of Huh7 cells confluent in a single 96 well plate was added. After 48h cells were fixed with 4% paraformaldehyde for IFA validation.

(4) Epitope identification: and (3) after the N gene is truncated, cloning the N gene to a prokaryotic expression vector, performing induced expression, performing SDS-PAGE, transferring the SDS-PAGE to an NC membrane, and verifying the reactivity of the monoclonal antibody through Western blot so as to determine the epitope recognized by the monoclonal antibody.

6 monoclonal antibody variable region gene PCR amplification and sequence determination

First, RNA of a monoclonal antibody hybridoma was extracted and reverse-transcribed to synthesize cDNA using Oligo-dt or random primers (PrimeScript II 1st Strand cDNA Synthesis Kit, TAKARA, 6210A), respectively.

The variable region gene of the antibody is amplified by nested PCR. Firstly, the cDNA is taken as a template, a first round of mouse antibody IgG1 and kappa light chain primers are used for amplifying antibody variable region genes, and then a second round of mouse antibody IgG1 and kappa light chain primers are used for amplifying antibody variable region genes by taking a first round of products as a template. The PCR reaction system is as follows: PrimeSTAR Max Premix (2X) 25. mu.L, each of P1 and P2 1. mu.L, cDNA 1. mu.L, ddH₂And O is supplemented to 50 mu L. The reaction procedure is as follows: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extension at 72 ℃ for 10 min. Primer reference for antibody variable region gene amplification (von Boehmer, L., Liu, C., Ackerman, S., Gitlin, A.D., Wang, Q., Gazumyan, A., Nussenzweig, M.C.,2016.Sequencing and fastening of anti-specific antibodies from mouse B cells. Nature protocols 11,1908-1923.)

After the amplification is finished, 1% agarose gel electrophoresis is carried out, the size of the heavy chain and kappa light chain variable region genes is about 300bp, and the target fragments are recovered by cutting gel. The recovered target fragment was inserted into the pMD-19T vector and subjected to sequencing.

7 results

7.1 amplification of N Gene and construction of recombinant plasmid

The N gene was amplified using SADS-CoV cDNA as a template, and the PCR product was identified by 1% agarose gel electrophoresis, as shown in FIG. 1A, with the N fragment approximately 1000bp in size.

The recovered product of the N gene PCR gel and pET32a vector are double digested with BamH I and XhoI, and then connected to transform DH5 alpha competent cell. The constructed recombinant plasmid pET32a-N is subjected to double enzyme digestion identification, the enzyme digestion result is shown in figure 1B, and the double enzyme digestion product has bands at positions of more than 5000bp (vector band) and about 1000bp (target fragment), and the double enzyme digestion product is consistent with the expectation. And (4) sequencing and identifying the plasmid which is identified as positive by enzyme digestion, wherein the sequencing result is consistent with the target sequence.

7.2 inducible expression and purification of recombinant protein

The recombinant plasmid pET32A-N is transformed into escherichia coli BL21(DE3), IPTG with the final concentration of 1mmol/L is added for induction expression, and SDS-PAGE identification is carried out after the induction product is subjected to ultrasonic treatment, and the result shows that pET32A-N is expressed in both supernatant and sediment (figure 2A), which indicates that the N protein is partially soluble, so that the protein is purified by a nickel column purification method. As can be seen from FIG. 2B, the N protein was obtained with higher purity.

7.3 ELISA for the detection of the potency of the polyclonal antibodies

Balb/c mice were immunized four times with purified N recombinant protein and subsequently tested for antibody titer. The ELISA plate is coated with virus liquid after SADS-CoV inactivation, antibody titer is detected, and nonimmunized mouse serum is used as a Negative Control (NC), and the result shows that the OD of No. 1, No. 2 and No. 3 immunized mice is OD when the serum is diluted 1:12800₄₅₀/NC>2.0, indicating that the antibody titer can reach more than 1:12800 (FIG. 3).

7.4 monoclonal antibody specificity identification

Monoclonal antibodies specifically recognizing the SADS-CoVN protein were obtained by screening using cell fusion technology and named 4B10 and 6E 8. The results of ELISA identification showed that both monoclonal antibodies reacted with N protein but not His-tagged unrelated protein, indicating that the screened monoclonal antibody specifically recognized N protein (Table 1). IFA results showed that 4B10 and 6E8 acted on SADS-CoV infected Huh7 cells were able to detect specific green fluorescent signals (FIGS. 4A and B), while SP2/0 cell supernatant acted on SADS-CoV infected cells did not see fluorescent signals (FIG. 4C). Westernblot results showed that both 4B10 and 6E8 recognized purified N protein as well as N protein in virus-infected cells (FIGS. 5A and B).

TABLE 1 monoclonal antibody ELISA test results

7.5 monoclonal antibody subclass identification

Subtype identification was performed on 4B10 and 6E8 monoclonal antibodies according to the mouse monoclonal antibody subclass identification kit, and the identification results showed that the heavy chain constant region of 4B10 monoclonal antibody was IgG1 type, 6E8 was IgG2a type, and their light chain constant regions were Kappa type (FIG. 6).

7.6 characterization of neutralizing Activity of monoclonal antibodies

To verify whether 4B10 and 6E8 have the ability to neutralize the virus, the collected ascites fluid was first diluted at different ratios (10, 20 and 40 fold) and then mixed with equal volumes of 0.01MOI of SADS-CoV. By IFA verification, it can be seen that 4B10 and 6E8 both neutralize the virus and are dose-dependent with ascites fluid, with lower dilution times being more potent in neutralization (FIG. 7).

7.7 identification of N protein region recognized by monoclonal antibody

To identify the region of the monoclonal antibody that recognizes the N protein, the N protein was subjected to truncated recombinant plasmids as shown in fig. 8A, designated R1, R2, R3, R1.1 and R1.2 regions, respectively. The insertion of the R3 region into the prokaryotic expression vector pET32a did not induce the expression of the protein, while both the R1 and R2 regions were expressed (FIG. 8B). Western blot results showed that 4B10 and 6E8 mAbs recognized the R1 region and failed to recognize the R2 region (FIGS. 8C and D). The R1 region is truncated and expressed (FIG. 8E), and Western blot results show that 4B10 and 6E8 can recognize the R1.1 and R1.2 regions (FIGS. 8F and G), which indicates that the regions of 4B10 and 6E8 recognizing the N protein are the overlapped parts of the two regions of R1.1 and R1.2, namely 43-95 aa. The R1.2 region was further truncated and expressed (FIG. H), as shown in FIGS. 8I and J, 4B10 and 6E8 reacted only with R1.2.1 but not with R1.2.2, so that the recognition regions of 4B10 and 6E8 mAbs were non-overlapping portions of R1.2.2, i.e., 64-84 aa, as shown in SEQ ID NO: 21.

7.8 monoclonal antibody variable region PCR amplification

PCR products of about 300bp in size were amplified from 4B10 and 6E8 hybridoma cDNA (FIG. 9), consistent with the expected amplification product size; after recovery from the gel cuts, the clones were cloned into the pMD19T vector for sequencing. And comparing the sequencing result with an antibody gene library (IMGT) for analysis, and confirming that the amplified sequences are the DNA sequence of the heavy chain variable region and the DNA sequence of the light chain variable region of the monoclonal antibody. Specifically, the DNA sequence of the heavy chain variable region of the mouse anti-SADS-CoV N protein monoclonal antibody 4B10 is shown as SEQ ID NO. 15; the DNA sequence of the light chain variable region of the mouse anti-SADS-CoV N protein monoclonal antibody 4B10 is shown in SEQ ID NO. 19. The DNA sequence of the heavy chain variable region of the mouse anti-SADS-CoV N protein monoclonal antibody 4B10 is shown as SEQ ID NO. 16; the DNA sequence of the light chain variable region of the mouse anti-SADS-CoV N protein monoclonal antibody 6E8 is shown in SEQ ID NO: 20.

The above-described embodiments are merely preferred embodiments of the present invention, and not intended to limit the scope of the invention, so that equivalent changes or modifications in the structure, features and principles described in the present invention should be included in the claims of the present invention.

SEQUENCE LISTING

<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences

Lanzhou veterinary research institute of Chinese academy of agricultural sciences, and City agricultural research institute of Chinese academy of agricultural sciences

<120> monoclonal antibody against porcine acute diarrhea syndrome coronavirus N protein, recognition region of the monoclonal antibody

And uses thereof

<130> do not

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<210> 8

<211> 3

<212> PRT

<213> Artificial Sequence

<400> 8

Leu Thr Ser

1

<210> 9

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 9

Gln Gln Trp Asn Ser Asn Pro Leu Thr

1 5

<210> 10

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 10

Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr

1 5 10

<210> 11

<211> 3

<212> PRT

<213> Artificial Sequence

<400> 11

Leu Val Ser

1

<210> 12

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 12

Gln His Ile Arg Glu Leu Thr Arg

1 5

<210> 13

<211> 121

<212> PRT

<213> Artificial Sequence

<400> 13

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr

20 25 30

Asn Met Phe Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Tyr Phe Asp Pro Tyr Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe

65 70 75 80

Met His Leu Asn Asn Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asn Asp Met Thr Thr Thr Val Tyr Tyr Ala Met Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Thr Val Thr Val

115 120

<210> 14

<211> 120

<212> PRT

<213> Artificial Sequence

<400> 14

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Val His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile

35 40 45

Gly Val Phe Ser Thr Tyr Tyr Gly Asn Ala Asn Tyr Asn Gln Asn Phe

50 55 60

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

65 70 75 80

Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Asp Tyr Tyr Gly Ser Ser Asn Val Asp Tyr Ala Met

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Ser

115 120

<210> 15

<211> 365

<212> DNA

<213> Artificial Sequence

<400> 15

gaggtccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta 60

tcctgcaagg cttctggtta ttcattcact gactacaaca tgttctgggt gaagcagagc 120

catggaaaga gccttgagtg gattggatat tttgatcctt acaatggtgg tactaactac 180

aaccagaagt tcaagggcaa ggccacattg actgttgaca agtcctccag cacagccttc 240

atgcatctca acaacctgac atctgaggac tctgcagtct attactgtgc aagagagaac 300

gatatgacta cgacggttta ctatgctatg gactactggg gtcaaggaac cacggtcact 360

gtctc 365

<210> 16

<211> 360

<212> DNA

<213> Artificial Sequence

<400> 16

caggtgcaac tgaagcagtc tggggctgag ctggtgaggc ctggggtctc agtgaagatt 60

tcctgcaagg gttctggcta cacattcact gattatgctg tgcactgggt gaagcagagt 120

catgcaaaga gtctagagtg gattggagtt tttagtactt actatggtaa tgctaactac 180

aaccagaact tcaagggcaa ggccacaatg accgtagaca aatcctccaa cacagcctat 240

atggaacttg ccagactgac atctgaggat tctgccatct attactgtgc aagaggaggg 300

gattactacg gtagtagcaa cgtagactat gctatggact actggggtca aggaacctca 360

<210> 17

<211> 105

<212> PRT

<213> Artificial Sequence

<400> 17

Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly Glu

1 5 10 15

Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Ile Tyr

20 25 30

Trp Tyr Gln Gln Arg Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr Leu

35 40 45

Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly

50 55 60

Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp

65 70 75 80

Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Ser Asn Pro Leu Thr Phe

85 90 95

Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 18

<211> 108

<212> PRT

<213> Artificial Sequence

<400> 18

Glu Asn Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg

85 90 95

Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Ser

100 105

<210> 19

<211> 315

<212> DNA

<213> Artificial Sequence

<400> 19

attgttctca cccagtctcc agcactcatg tctgcatctc caggggagaa ggtcaccatg 60

acctgcagtg ccagctcaag tgtgaattac atttactggt accagcagag gccaagatcc 120

tcccccaaac cctggattta tctcacatcc aacctggctt ctggagtccc tgctcgcttc 180

agtggcagtg ggtctgggac ctcttactct ctcacaatta gcagcatgga ggctgaagat 240

gctgccactt attactgcca gcagtggaat agtaacccgc tcacgttcgg tgctgggacc 300

aagctggagc tgaaa 315

<210> 20

<211> 327

<212> DNA

<213> Artificial Sequence

<400> 20

gaaaatgtgc tcacccagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60

atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 120

caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180

ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240

cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 300

tcggaggggg gaccaagctg gagctga 327

<210> 21

<211> 21

<212> PRT

<213> swine acute diarrhea syndrome coronavirus

<400> 21

Lys Arg Trp Arg Met Gln Lys Gly Gln Arg Lys Asp Gln Pro Ser Asn

1 5 10 15

Trp His Phe Tyr Tyr

20

Claims

1. The monoclonal antibody of the N protein of the anti-porcine acute diarrhea syndrome coronavirus comprises a heavy chain constant region, a heavy chain variable region, a light chain constant region and a light chain variable region; it is characterized in that the preparation method is characterized in that,

the heavy chain variable region comprises CDR1 with an amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 4, CDR2 with an amino acid sequence shown as SEQ ID NO. 2 or SEQ ID NO. 5, and CDR3 with an amino acid sequence shown as SEQ ID NO. 3 or SEQ ID NO. 6;

the light chain variable region comprises CDR1 with an amino acid sequence shown as SEQ ID NO. 7 or SEQ ID NO. 10, CDR2 with an amino acid sequence shown as SEQ ID NO. 8 or SEQ ID NO. 11, and CDR3 with an amino acid sequence shown as SEQ ID NO. 9 or SEQ ID NO. 12.

2. The monoclonal antibody against N protein of porcine acute diarrhea syndrome coronavirus according to claim 1,

the amino acid sequence of the heavy chain variable region of the monoclonal antibody for resisting the porcine acute diarrhea syndrome coronavirus N protein is shown as SEQ ID NO. 13 or SEQ ID NO. 14; the amino acid sequence of the light chain variable region of the monoclonal antibody for resisting the porcine acute diarrhea syndrome coronavirus N protein is shown as SEQ ID NO. 17 or SEQ ID NO. 18.

3. The DNA sequence of the heavy chain variable region and the light chain variable region of the monoclonal antibody for resisting the porcine acute diarrhea syndrome coronavirus N protein of claim 2, wherein the DNA sequence of the heavy chain variable region of the monoclonal antibody for resisting the porcine acute diarrhea syndrome coronavirus N protein is shown as SEQ ID NO. 15 or SEQ ID NO. 16; the DNA sequence of the light chain variable region of the monoclonal antibody for encoding the anti-porcine acute diarrhea syndrome coronavirus N protein is shown as SEQ ID NO. 19 or SEQ ID NO. 20.

4. The application of the monoclonal antibody for resisting the porcine acute diarrhea syndrome coronavirus N protein in the claim 1 in specifically identifying the porcine acute diarrhea syndrome coronavirus N protein, which is characterized in that the monoclonal antibody for resisting the porcine acute diarrhea syndrome coronavirus N protein can specifically identify the N-terminal region of the porcine acute diarrhea syndrome coronavirus N protein as shown in SEQ ID NO. 21.

5. The use of the monoclonal antibody against porcine acute diarrhea syndrome coronavirus N protein of claim 1 in the preparation of a medicament for treating porcine acute diarrhea syndrome coronavirus infection.

6. A recombinant expression vector comprising the DNA segment of claim 3.

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