CN114751979A

CN114751979A - Monoclonal antibody of anti-transmissible gastroenteritis virus NSP14 protein, preparation method and application thereof

Info

Publication number: CN114751979A
Application number: CN202210384741.9A
Authority: CN
Inventors: 朱记平; 谢立兰; 李毅; 王小梅; 李么明
Original assignee: Wuhan Chenpeng Yongjia Biotechnology Co ltd
Current assignee: Wuhan Chenpeng Yongjia Biotechnology Co ltd
Priority date: 2022-04-13
Filing date: 2022-04-13
Publication date: 2022-07-15

Abstract

The invention provides a monoclonal antibody of anti-transmissible gastroenteritis virus NSP14 protein, a preparation method and application thereof, belongs to the field of genetic engineering, and can solve the technical problem that the monoclonal antibody of TGEVNSP14 protein is lacked in the prior art. The swine transmissible gastroenteritis virus NSP14 protein monoclonal antibody is a swine transmissible gastroenteritis virus NSP14 protein monoclonal antibody, the amino acid sequence of the monoclonal antibody is shown as SEQ ID NO.1, and the nucleotide sequence of the monoclonal antibody for encoding the swine transmissible gastroenteritis virus NSP14 protein is shown as SEQ ID NO. 2; the monoclonal antibody for resisting the porcine transmissible gastroenteritis virus NSP14 protein is prepared by the following method: target gene interception, primer design and synthesis, construction of a truncation mutant, transformation of a truncation mutant host bacterium, induction expression and purification of target protein and the like. The invention can be applied to the aspects of resisting the transmissible gastroenteritis of swine and preparing preparations or medicaments for resisting the transmissible gastroenteritis of swine.

Description

Monoclonal antibody of anti-transmissible gastroenteritis virus NSP14 protein, preparation method and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a swine transmissible gastroenteritis virus NSP14 protein monoclonal antibody, a preparation method and application thereof.

Background

Porcine Transmissible Gastroenteritis (TGE) is an acute intestinal infectious disease caused by porcine transmissible gastroenteritis virus (TGEV). The clinical manifestations of the disease are severe vomiting, diarrhea and the like, and the disease has the characteristics of high morbidity and mortality, and the mortality rate of piglets within 10 days of age is up to 100%. Is popular in the world, and brings great economic loss to the pig raising industry.

The Non-structural protein 14 (NSP 14) in coronavirus has high conservation. Studies have shown that the NSP14 protein of SARS coronavirus (SARS-CoV) and mouse hepatitis coronavirus (MHV) has 3'-5' Exonuclease (Exonuclease, ExoN) activity and N7-methyltransferase (N7-methyltransferase, N7-MTase) function, and plays an important role in the replication, transcription and translation processes of the virus, and is expected to become a new target of the medicine for treating the virus. At present, the research on monoclonal antibodies against TGEV S protein, M protein and N protein, and the report on monoclonal antibodies against TGEV NSP5 protein and NSP8 protein are both available at home and abroad, but the monoclonal antibodies against TGEV NSP14 protein and TGE early diagnosis methods based on the monoclonal antibodies are not reported yet.

Therefore, the invention provides a monoclonal antibody for preparing anti-TGEV NSP14 protein and a preparation method thereof to fill the blank of the prior art.

Disclosure of Invention

The invention provides a monoclonal antibody of anti-transmissible gastroenteritis virus NSP14 protein, a preparation method and application thereof, aiming at the technical problem that the monoclonal antibody of TGEV NSP14 protein is lack in the prior art, has the characteristics of simple preparation process, strong operability, remarkable treatment effect and the like, and fills the blank of the prior art in the monoclonal antibody of TGEV NSP14 protein and subsequent application thereof.

In order to achieve the purpose, the invention adopts the technical scheme that:

the monoclonal antibody of the protein NSP14 for resisting the transmissible gastroenteritis virus is a monoclonal antibody of the protein NSP14 for resisting the transmissible gastroenteritis virus, and the amino acid sequence of the monoclonal antibody is shown as SEQ ID No. 1.

In one embodiment, the nucleotide sequence of the monoclonal antibody encoding the NSP14 protein of the porcine transmissible gastroenteritis virus is shown as SEQ ID NO. 2.

The invention provides a preparation method of a monoclonal antibody of anti-transmissible gastroenteritis virus NSP14 protein, which comprises the following steps:

designing and synthesizing a primer: designing and synthesizing primers according to a nsp14 gene sequence of the transmissible gastroenteritis virus disclosed by an NCBI database to obtain a primer 1 and a primer 2;

And (3) intercepting and selecting target genes: performing hydrophilicity, immunogenicity and surface exposure analysis on NSP14 protein of the porcine transmissible gastroenteritis virus, and selecting a target gene from an NSP14 gene sequence according to an analysis result;

construction of truncation mutants: amplifying the target gene by taking the primer 1 and the primer 2 as upstream and downstream primers and taking the nsp14 gene sequence of the transmissible gastroenteritis virus as a template, and constructing a truncated mutant pET-nsp14a containing the target gene;

and (3) conversion of the truncation mutants: converting the truncated mutant pET-nsp14a into host bacteria, and determining whether the target gene is inserted successfully or not after plate coating and culturing, double enzyme digestion identification and sequencing;

and (3) inducing expression of the target protein: adding the successfully inserted host bacteria into a liquid culture medium containing IPTG (isopropyl-beta-D-thiogalactoside) to perform protein induction expression;

and (3) target protein purification: and after the induction expression of the protein is finished, collecting host bacterium bacteria for protein purification to obtain purified NSP14a protein.

In one embodiment, the nucleotide sequences of primer 1 and primer 2 are shown in SEQ ID NO. 3-4.

In one embodiment, in the target gene-intercepting step, the hydrophilicity, immunogenicity and surface exposure of the amino acid sequence at position 201-460 of the NSP14 protein are determined to be expected by performing hydrophilicity, immunogenicity and surface exposure analysis on the NSP14 protein of the porcine transmissible gastroenteritis virus, and therefore, the nucleotide sequence encoding the amino acid sequence is used as the target gene.

In one embodiment, the vector used to construct the truncated mutant pET-nsp14a is pET-28a (+).

In one embodiment, in the step of inducing and expressing the target protein, the inducing and expressing conditions are 16-37 ℃, 6-8h, the working concentration of IPTG is 0.2-1.0mmol/L, and the target protein purification is carried out by combining the denaturation of inclusion body protein, dialysis concentration and gel cutting recovery.

In one embodiment, the preparation method further comprises performing serum antibody titer detection, specificity detection, immunofluorescence detection, subclass identification and epitope identification tests on the purified NSP14a protein.

The invention provides an application of another monoclonal antibody for resisting swine transmissible gastroenteritis virus NSP14 protein in resisting swine transmissible gastroenteritis.

The invention provides an application of another monoclonal antibody of the NSP14 protein for resisting the transmissible gastroenteritis virus in preparing preparations or medicaments for resisting the transmissible gastroenteritis virus.

Compared with the prior art, the invention has the advantages and positive effects that:

1. the monoclonal antibody of the NSP14 protein for resisting the transmissible gastroenteritis virus of swine provided by the invention is prepared by intercepting and selecting a target gene meeting the standard in an NSP14 gene sequence in a manner of analyzing hydrophilicity, immunogenicity and surface exposure, and then carrying out steps of constructing a truncated mutant, transforming host bacteria, expressing protein, purifying and the like, and the monoclonal antibody prepared by the method has the characteristics of remarkable treatment effect and the like;

2. The monoclonal antibody for resisting the transmissible gastroenteritis virus NSP14 protein has wide application prospects in resisting the transmissible gastroenteritis of swine and preparing preparations or medicaments for resisting the transmissible gastroenteritis of swine, and fills the blank of the prior art in the monoclonal antibody of the TGEV NSP14 protein and subsequent application thereof to a certain extent.

Drawings

FIG. 1 shows the PCR amplification result of nsp14a gene (FIG. 1A) and the double digestion identification result of recombinant plasmid pET-nsp14a (FIG. 1B);

FIG. 2 shows the purified NSP14a protein band provided by the present invention;

FIG. 3 shows the result of the serum antibody titer of BALB/c mice immunized with NSP14a protein after purification as provided in the present invention;

FIG. 4 is a schematic diagram showing the Western Blot detection result of the purified NSP14a protein according to the embodiment of the present invention;

FIG. 5 shows the indirect immunofluorescence assay of the purified NSP14a protein according to the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

The embodiment of the invention provides a monoclonal antibody of a protein NSP14 for resisting a transmissible gastroenteritis virus, wherein the monoclonal antibody of the protein NSP14 for resisting the transmissible gastroenteritis virus is a monoclonal antibody of a protein NSP14 for resisting the transmissible gastroenteritis virus, and the amino acid sequence of the monoclonal antibody is shown as SEQ ID No. 1.

In a specific embodiment, the nucleotide sequence of the monoclonal antibody encoding the transmissible gastroenteritis virus NSP14 protein is shown as SEQ ID NO. 2.

The invention provides a preparation method of the monoclonal antibody for resisting the NSP14 protein of the porcine transmissible gastroenteritis virus, which comprises the following steps:

s1, target gene interception: performing hydrophilic, immunogenic and surface exposure analysis on NSP14 protein of the porcine transmissible gastroenteritis virus, and selecting a target gene in the NSP14 gene sequence according to an analysis result;

in the step S1, DNAstar software is used for analyzing hydrophilicity, immunogenicity and surface exposure of the NSP14 protein when the target gene is truncated, and the NSP14 protein is found to have better hydrophilicity, immunogenicity and surface exposure at the 201-460 amino acid positions through analysis, so the fragment is selected for construction of the truncation mutant.

S2, primer design and synthesis: combining the analysis result of the target gene interception step, and carrying out primer design and synthesis according to the nsp14 gene sequence of the transmissible gastroenteritis virus of swine, which is published by NCBI database, to obtain a primer 1 and a primer 2;

s3, construction of truncation mutants: amplifying the target gene by taking the primer 1 and the primer 2 as upstream and downstream primers and taking the nsp14 gene sequence of the transmissible gastroenteritis virus as a template, and constructing a truncated mutant pET-nsp14a containing the target gene;

s4, conversion of truncation mutants: converting the truncated mutant pET-nsp14a into host bacteria, and determining whether the target gene is inserted successfully or not after plate coating and culturing, double enzyme digestion identification and sequencing;

s5, inducing expression of target protein: adding the successfully inserted host bacteria into a liquid culture medium containing IPTG (isopropyl-beta-D-thiogalactoside) to perform protein induction expression;

s6, purification of target protein: and after the induction expression of the protein is finished, collecting host bacterium bacteria for protein purification to obtain purified NSP14a protein.

In a specific embodiment, the nucleotide sequences of the primer 1 and the primer 2 are shown as SEQ ID NO. 3-4.

In a specific embodiment, the vector used to construct the truncation mutant pET-nsp14a includes, but is not limited to, pET-28a (+).

In a specific embodiment, in the step of inducing and expressing the target protein, the inducing and expressing conditions are 16-37 ℃, 6-8h, the working concentration of IPTG is 0.2-1.0mmol/L, and the target protein is purified by combining denaturation of inclusion body protein, dialysis concentration and recovery of gel cutting.

In a specific embodiment, the preparation method further comprises performing serum antibody titer detection, specificity detection, immunofluorescence detection, subclass identification and epitope identification tests on the purified NSP14a protein.

The invention provides an application of a monoclonal antibody of anti-transmissible gastroenteritis virus NSP14 protein in resisting transmissible gastroenteritis of swine.

The invention provides application of a monoclonal antibody of anti-transmissible gastroenteritis virus NSP14 protein in preparation of a preparation or a medicament for resisting transmissible gastroenteritis of swine.

In order to more clearly and specifically describe the monoclonal antibody against the porcine transmissible gastroenteritis virus NSP14 protein, the preparation method and the application thereof, the following description will be made in conjunction with specific examples.

Example 1

The embodiment provides a preparation method of a monoclonal antibody against porcine transmissible gastroenteritis virus NSP14 protein, which specifically comprises the following steps:

(1) and (3) intercepting and selecting target genes: performing hydrophilicity, immunogenicity and surface exposure analysis on NSP14 protein of the porcine transmissible gastroenteritis virus by using DNAstar software, and finding that the 201 th to 460 th amino acids of the NSP14 protein have better hydrophilicity, immunogenicity and surface exposure, so that a nucleotide sequence for coding the amino acid fragment is selected as a target gene constructed by a truncated mutant;

(2) designing and synthesizing primers: according to the analysis result of the step (1), by referring to the nsp14 gene sequence (HQ462571.1) published by NCBI, designing a Primer by using Primer-Premier 5.0 software, and sending the Primer to the company of Biotechnology engineering (Shanghai) GmbH to synthesize a Primer (the sequence information is shown in Table 1, and the sequence information is shown in SEQ ID NO.3-4 in the sequence table);

TABLE 1 primer sequences

Name of primer	Sequence information	Cleavage site
			28a-nsp14aF	5'-gcgGGATCCaaaattggaagaccacaa-3'	BamHI
28a-nsp14aR	5'-gcgGTCGACgtcatgacttaagtggtacata-3'	SalI

(3) Construction of truncation mutants: 28a-nsp14aF and 28a-nsp14aR in the table 1 are respectively used as upstream and downstream primers, nsp14 is used as a template to amplify the target gene, the amplification system and the amplification conditions are shown in the tables 2-3, agarose gel electrophoresis is carried out on a PCR product, ultraviolet detection and analysis results (shown in a figure 1A) are carried out, pET-28a (+) is used as a vector to construct a truncated mutant pET-nsp14a, double digestion identification is carried out, the identification result is shown in a figure 1B, the result shown in the figure can indicate that the size of an nsp14a gene is 777bp, the size of a pET-28a (+) vector is 5369bp, and the result is consistent with the expectation, a plasmid sample which is identified correctly by digestion is sent to a Hanwu sequencing department of the Biotechnology engineering (Shanghai) Limited company for sequencing, and the result indicates that no base mismatch exists and the plasmid construction is correct;

TABLE 2PCR amplification System

TABLE 3PCR amplification conditions

TABLE 4 double digestion System

Component	20μLReaction
		Plasmid
	400～600ng
		Restrictionenzyme1	1μL
Restrictionenzyme2	1μL
		10×Buffer	2μL
ddH₂O	To20μL

(4) And (3) inducing expression of the target protein: after the recombinant plasmid pET-nsp14a is transformed into BL21(DE3) cells, 3.6L of bacterial liquid is induced under the optimal induction condition (16-37 ℃ and IPTG working concentration of 0.2-1.0mmol/L for 6h) for protein purification. Protein purification is carried out by adopting an inclusion body protein denaturation method and a dialysis concentration method, a large amount of protein is separated out, and most of protein still remains in a precipitate. Therefore, in this experiment, the inclusion body protein of NSP14a was purified by a gel cutting recovery method, and the purification effect is shown in fig. 2, which indicates that the NSP14a protein with high purity was obtained.

Example 2

This example provides a purified NSP14a protein mouse immunoassay, specifically:

(1) firstly, exempting from: adding equivalent volume of Freund's Complete Adjuvant (FCA) into purified NSP14a protein, completely emulsifying by pipette, injecting into healthy BALB/c mouse (8 weeks old) with backside subcutaneous multiple spot according to 50 μ g/mouse antigen amount, immunizing for 6 mice totally, and performing second immunization after two weeks;

(2) and (2) avoiding: adding equivalent volume of Freund incomplete adjuvant (FICA) into purified NSP14a protein, completely emulsifying by pipette, injecting 200 μ g/mouse back subcutaneous multiple points with 200 μ g antigen amount, and performing three-immunization two weeks later;

(3) And (3) three-step (I): taking purified NSP14a protein, adding no adjuvant, and injecting BALB/c mouse intraperitoneally according to 100 ug/mouse antigen amount;

(4) the immunization mode is the same as three immunization modes;

(5) after 1 week of each immunization, the serum antibody titer is detected by an indirect ELISA method, the measurement result is shown in figure 3, the result is the serum antibody titer test result after one week of quadruplicate immunization, and the figure shows that the serum antibody titer of the mouse M2 meets the fusion requirement; in addition, when the antibody titer is less than or equal to 10^-4Then, the booster immunization before the fusion is carried out;

(6) pre-fusion booster: 3 days before fusion, injecting BALB/c mouse into abdominal cavity according to 200 mug/mouse antigen amount, and continuously enhancing immunity for 3 days;

wherein, the indirect ELISA method for detecting the serum antibody titer in the step (5) specifically comprises the following steps:

after one week of secondary immunization, blood is collected from the canthus of the mouse, the blood is taken as a primary antibody to carry out immunoreaction with the antigen coated on the ELISA plate, a secondary antibody is added after reaction, the secondary antibody is combined with the primary antibody, and finally the color is developed with TMB color developing solution, and an ELISA reader is used for measuring OD450_nmWhen the ratio of the OD value of the positive serum at the wavelength of 450nm to the OD value of the negative serum at the wavelength of 450nm is more than 2.5, the maximum dilution factor of the sample is the antibody titer of the serum, and the specific steps are as follows:

(5-1) coating antigen: diluting the purified protein with coating solution according to 1 μ g/mL, adding to ELISA plate at 100L/well, standing overnight at 4 deg.C, washing with PBST for 3 times, 5 min/time the next day;

(5-2) sealing: adding 5% skimmed milk powder diluted by PBST into the ELISA plate, sealing at 200 μ L/hole, washing with PBST at 37 deg.C for 1h for 3 times, 5 min/time;

(5-3) primary antibody incubation: taking blank mouse serum as a negative control, taking no serum as a blank control, diluting immune mouse serum according to the proportion of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400 and 1:12800, longitudinally adding the diluted immune mouse serum into an enzyme label plate at a rate of 100 mu L/hole, repeating the dilution for 3 times, washing the immune mouse serum for 3 times at 37 ℃ for 1h, and washing the immune mouse serum for 5 min/time;

(5-4) Secondary antibody incubation: goat anti-mouse IgG-HRP (100. mu.L/well) diluted at 1:5000 was added and incubated at 37 ℃ for 1 h. PBST is washed for 3 times and 5 min/time;

(5-5) color development: adding TMB color development solution into the mixture, performing 100 mu L/hole incubation for 5-10 min in a 37 ℃ wet box in a dark place;

(5-6) terminating: adding stop solution (2mol/L HCl), 50 μ L/well, and measuring OD with microplate reader₄₅₀And (5) nm value.

Example 3

This example provides a specific detection test of purified NSP14a protein, while in examples 1-2, prokaryotic TGEV NSP14 protein was used in the screening process of preparing monoclonal antibody (i.e. purified NSP14a protein) in order to detect McAb reactivity, this example uses a reaction with eukaryotic TGEV NSP14 protein to verify whether antibody can specifically and sensitively resist TGEV NSP14 protein, and the specific steps are as follows:

(1) Recovering PK-15 cells, inoculating to 6-well plate containing fresh culture medium, standing at 37 deg.C with 5% CO₂Subculturing in a cell incubator;

(2) and (3) inoculation of toxin: when the cells grow to 80-90% of the cell pores, removing a culture medium (the dead cells can be omitted when few are used), inoculating 0.01MOI TGEV WH-1 strain virus maintenance liquid, covering the cells, changing the liquid (removing unadsorbed dead virus particles) after 1h of induction, supplementing a certain amount of maintenance culture medium, performing the next operation after 70-80% of cells are diseased, and simultaneously establishing a PK-15 cell control without virus inoculation;

(3) sample treatment: absorbing and discarding the culture medium in the 6-well plate, digesting cells by using pancreatin, transferring the cell suspension into a centrifugal tube of 1.5mL, centrifuging for 3min at 5000r/min, discarding the supernatant, washing for 2 times by using PBS, discarding the PBS, and completely absorbing residual liquid by using a white pipette head at-80 ℃ for storage;

(4) boiling samples: and (4) taking the cell sediment in the step (3), adding 100 mu L of cell lysate, simultaneously adding a protease inhibitor, after the cells are fully cracked, centrifuging for 3-5 min at 12000g, adding 5 xSDS Loading Buffer into supernatant, carrying out boiling water bath for 10min, immediately carrying out ice bath for 3min, centrifuging for 2min at 4 ℃ at 12000r/min, and taking supernatant for Western Blot detection.

And (3) analysis of test results: the Western Blot assay results are shown in fig. 4, and the assay shows that the cell culture supernatant can specifically react with TGEV NSP14 protein (57KD), indicating that the obtained monoclonal antibody can specifically recognize TGEV NSP14 protein.

Example 4

This example provides an indirect immunofluorescence assay for purified NSP14a protein, specifically including:

(2) and (3) inoculation of toxin: the inoculation is carried out as described in example 3;

(3) washing: the culture medium is sucked and discarded, and washed with PBS for 2 times and 5 min/time, and the PBS is discarded;

(4) fixing the cells: adding 4% paraformaldehyde fixing solution into cells, standing at 50 μ L/well for 15min at room temperature, washing with PBS for 3 times and 5 min/time, and discarding PBS (note: when the time is not allowed, washing with PBS, soaking with PBS, and storing at 4 deg.C);

(5) adding 0.2% (or 0.5%) TritonX-100 at a concentration of 50. mu.L/well, and standing at room temperature for 10-15 min. Washing with PBS for 3 times (5 min/time), and discarding PBS;

(6) and (3) sealing: sealing with special sealing liquid, 50 mu L/hole, incubating at room temperature or 37 ℃ for 30-60 min, or overnight at 4 ℃. Removing the blocking solution, washing with PBS for 3 times and 5 min/time;

(7) Incubation of primary antibody: using the positive monoclonal strain cell culture supernatant as a primary antibody, incubating at 37 ℃ for 1h or overnight at 4 ℃ for 50 mu L/hole, and washing with PBS for 3 times and 5 min/time;

(8) incubation of secondary antibody: cy 3-labeled goat anti-mouse IgG (red light) was diluted with PBS at a ratio of 1:500, 50. mu.L/well, and incubated at 37 ℃ for 1h (this step was protected from light). Washing with PBS for 3 times, 5 min/time;

(9) and (3) cell nucleus staining: adding 50 mu L/hole of DAPI (containing a blocking tablet for preventing quenching) for staining a nucleus, and storing at 4 ℃ (keeping the step away from light);

(10) and (4) observation: the results were observed with a fluorescence microscope in the dark and photographed.

And (3) analysis of test results: an indirect immunofluorescence detection test is shown in fig. 5, and the test shows that the monoclonal antibody (abbreviated as 'McAb') obtained by the invention has obvious fluorescence reaction with TGEV but no reaction with normal PK-15 cells, and further proves that the obtained McAb can specifically recognize TGEV NSP14 protein.

Example 5

In this example, a large amount of anti-TGEV NSP14 protein McAb was prepared and tested for potency assay, specifically:

in the test, a method for inducing ascites in mice is adopted to prepare a large amount of anti-TGEV NSP14 protein McAb, indirect ELISA is used to detect the antibody titer (the specific operation steps are the same as in example 2), and the test results are shown in the following table:

TABLE 5 Titers of McAb against TGEV NSP14 protein

Sample	Antibody titer	OD450nm
			12F1
	100×2⁹	0.549
			Negative control	/	0.214
Positive control	/	1.230
			Blank control	/	0.089

As can be seen from the data shown in the above table, the monoclonal antibody 12F1 prepared by the method of inducing ascites in mice in the examples of the present invention has an antibody titer of 100X 2⁹。

Example 6

In this example, a subclass identification test of anti-TGEV NSP14 protein McAb was performed, specifically:

the 12F1 monoclonal antibody is used as a primary antibody, goat anti-mouse IgG1-HRP and IgG2a-HRP are used as secondary antibodies, indirect ELISA detection is carried out, and the measurement results are shown in the table 6:

TABLE 6 subclass identification of McAb against TGEV NSP14 protein

Second antibody	IgG1-HRP	IgG2a-HRP	Negative of	Positive for
					Clone strain	12F1	12F1	/	/
OD₄₅₀nm	2.076	0.137	0.214	1.28

As can be seen from the data shown in the above table, the subclass of 12F1 mAb is IgG 1.

In conclusion, the monoclonal antibody prepared by the preparation method of the monoclonal antibody for resisting the transmissible gastroenteritis virus NSP14 protein provided by the invention has the serum antibody titer meeting the fusion requirement, can specifically recognize the TGEV NSP14 protein, can be prepared in a large scale in a mode of inducing ascites in mice, and has the antibody titer as high as 100 multiplied by 2⁹Further lays a foundation for the batch production and subsequent application of the monoclonal antibody.

Sequence listing

<110> Wuhan Chengpo Yongjia Biotech Co., Ltd

<120> monoclonal antibody of NSP14 protein for resisting transmissible gastroenteritis virus of swine, preparation method and application thereof

<130> 20220413

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 780

<212> DNA

<213> Artificial sequence ()

<400> 1

aaaattggaa gaccacaaaa atgtgaatgc ggcaaaagtg caacttgtta tagtagctct 60

caatctgttt atgcttgctt caagcatgca ttaggatgtg attatttata taacccttac 120

tgcattgaca tacagcaatg gggttacaca ggatctttga gcatgaatca tcatgaagtt 180

tgcaacattc atagaaatga gcatgtagct agtggtgatg ctatcatgac tagatgtctc 240

gctatacatg actgttttgt caaacgtgtt gattggtcaa ttgtgtaccc ttttattgac 300

aatgaagaaa agatcaataa agctggtcgc atagtgcagt cacatgtcat gaaagctgct 360

ctgaagattt ttaatcctgc tgcaattcac gatgtgggta atccaaaagg catccgttgt 420

gctacaacac caataccatg gttttgttat gatcgtgatc ctattaataa caatgttaga 480

tgtctggatt atgactatat ggtacatggt caaatgaatg gtcttatgtt attttggaac 540

tgtaatgtag acatgtaccc agagttttca attgtttgta gatttgatac tcgcactcgc 600

tctaaattgt ctttagaagg ttgtaatggt ggtgcattgt atgttaataa ccatgctttc 660

cacacaccag cttatgatag aagagctttt gctaagctta aacctatgcc attcttttac 720

tatgatgata gtaattgtga acttgttgat gggcaaccta attatgtacc acttaagtca 780

<210> 2

<211> 260

<212> PRT

<213> Artificial sequence ()

<400> 2

Lys Ile Gly Arg Pro Gln Lys Cys Glu Cys Gly Lys Ser Ala Thr Cys

1 5 10 15

Tyr Ser Ser Ser Gln Ser Val Tyr Ala Cys Phe Lys His Ala Leu Gly

20 25 30

Cys Asp Tyr Leu Tyr Asn Pro Tyr Cys Ile Asp Ile Gln Gln Trp Gly

35 40 45

Tyr Thr Gly Ser Leu Ser Met Asn His His Glu Val Cys Asn Ile His

50 55 60

Arg Asn Glu His Val Ala Ser Gly Asp Ala Ile Met Thr Arg Cys Leu

65 70 75 80

Ala Ile His Asp Cys Phe Val Lys Arg Val Asp Trp Ser Ile Val Tyr

85 90 95

Pro Phe Ile Asp Asn Glu Glu Lys Ile Asn Lys Ala Gly Arg Ile Val

100 105 110

Gln Ser His Val Met Lys Ala Ala Leu Lys Ile Phe Asn Pro Ala Ala

115 120 125

Ile His Asp Val Gly Asn Pro Lys Gly Ile Arg Cys Ala Thr Thr Pro

130 135 140

Ile Pro Trp Phe Cys Tyr Asp Arg Asp Pro Ile Asn Asn Asn Val Arg

145 150 155 160

Cys Leu Asp Tyr Asp Tyr Met Val His Gly Gln Met Asn Gly Leu Met

165 170 175

Leu Phe Trp Asn Cys Asn Val Asp Met Tyr Pro Glu Phe Ser Ile Val

180 185 190

Cys Arg Phe Asp Thr Arg Thr Arg Ser Lys Leu Ser Leu Glu Gly Cys

195 200 205

Asn Gly Gly Ala Leu Tyr Val Asn Asn His Ala Phe His Thr Pro Ala

210 215 220

Tyr Asp Arg Arg Ala Phe Ala Lys Leu Lys Pro Met Pro Phe Phe Tyr

225 230 235 240

Tyr Asp Asp Ser Asn Cys Glu Leu Val Asp Gly Gln Pro Asn Tyr Val

245 250 255

Pro Leu Lys Ser

260

<210> 3

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 3

gcgggatcca aaattggaag accacaa 27

<210> 4

<211> 31

<212> DNA

<213> Artificial sequence ()

<400> 4

gcggtcgacg tcatgactta agtggtacat a 31

<210> 5

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 5

gcgggatcca tacatgactg ttttgtc 27

<210> 6

<211> 31

<212> DNA

<213> Artificial sequence ()

<400> 6

gcggtcgacg tcatgactta agtggtacat a 31

<210> 7

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 7

gcgggatcca aaattggaag accacaa 27

<210> 8

<211> 31

<212> DNA

<213> Artificial sequence ()

<400> 8

gcggtcgacg tcagtcaata aaagggtaca c 31

<210> 9

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 9

gcgggatcca aaattggaag accacaa 27

<210> 10

<211> 31

<212> DNA

<213> Artificial sequence ()

<400> 10

gcggtcgacg tcaaccatta caaccttcta a 31

Claims

1. The monoclonal antibody for resisting the transmissible gastroenteritis virus NSP14 protein is characterized in that the monoclonal antibody for resisting the transmissible gastroenteritis virus NSP14 protein is a monoclonal antibody for resisting the transmissible gastroenteritis virus NSP14 protein, and the amino acid sequence of the monoclonal antibody is shown as SEQ ID No. 1.

2. The monoclonal antibody against swine transmissible gastroenteritis virus NSP14 protein of claim 1, wherein the nucleotide sequence encoding the monoclonal antibody against swine transmissible gastroenteritis virus NSP14 protein is shown as SEQ ID No. 2.

3. The method for preparing the monoclonal antibody against the NSP14 protein of porcine transmissible gastroenteritis virus of claim 1 or 2, comprising the steps of:

intercepting and selecting target genes: after hydrophilic, immunogenic and surface exposure analysis is carried out on NSP14 protein of the porcine transmissible gastroenteritis virus, a target gene is intercepted in the NSP14 gene sequence according to the analysis result;

Designing and synthesizing primers: combining the analysis result of the target gene interception step, and carrying out primer design and synthesis according to the nsp14 gene sequence of the transmissible gastroenteritis virus of swine, which is published by NCBI database, to obtain a primer 1 and a primer 2;

construction of truncation mutants: the primer 1 and the primer 2 are used as upstream and downstream primers, the target gene is amplified by taking the gene sequence of the transmissible gastroenteritis virus nsp14 of the pig as a template, and a truncated mutant pET-nsp14a containing the target gene is constructed;

and (3) target protein purification: and after the protein induction expression is finished, collecting host bacterium bacteria for protein purification to obtain purified NSP14a protein.

4. The method for preparing the monoclonal antibody against the NSP14 protein of porcine transmissible gastroenteritis virus of swine virus of claim 3, wherein the nucleotide sequences of the primer 1 and the primer 2 are shown in SEQ ID NO. 3-4.

5. The method for preparing the monoclonal antibody against porcine transmissible gastroenteritis virus NSP14 protein as claimed in claim 3, wherein in the step of target gene selection, after analyzing the hydrophilicity, immunogenicity and surface exposure of the NSP14 protein of porcine transmissible gastroenteritis virus, the amino acid sequence at position 201-460 of NSP14 protein is determined to have the hydrophilicity, immunogenicity and surface exposure as expected, so that the nucleotide sequence encoding the amino acid sequence is used as the target gene.

6. The method for preparing the monoclonal antibody against the NSP14 protein of the porcine transmissible gastroenteritis virus of the swine, wherein the vector for constructing the truncated mutant pET-NSP14a is pET-28a (+).

7. The method for preparing the monoclonal antibody against porcine transmissible gastroenteritis virus NSP14 protein according to claim 3, wherein in the step of inducing and expressing the target protein, the inducing and expressing conditions are 16-37 ℃ for 6-8h, the working concentration of IPTG is 0.2-1.0mmol/L, and the purification of the target protein is performed by combining the denaturation of inclusion body protein, dialysis concentration and recovery of gel cutting.

8. The method for preparing the NSP14 protein monoclonal antibody against transmissible gastroenteritis virus of swine according to claim 3, wherein the preparation method further comprises the steps of performing serum antibody titer detection, specificity detection, immunofluorescence detection, subclass identification and epitope identification tests on the purified NSP14a protein.

9. The monoclonal antibody against transmissible gastroenteritis virus NSP14 protein of claim 1 or claim 2, wherein the monoclonal antibody is used for resisting transmissible gastroenteritis of swine.

10. The monoclonal antibody against transmissible gastroenteritis virus NSP14 protein of claim 1 or claim 2, wherein the monoclonal antibody is used for preparing preparations or medicaments against transmissible gastroenteritis of swine.