CN112111006B - Antibody for resisting bovine sarcoidosis virus, detection test paper and kit - Google Patents
Antibody for resisting bovine sarcoidosis virus, detection test paper and kit Download PDFInfo
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Abstract
The invention relates to an antibody or an active fragment thereof specifically binding to bovine sarcoidosis virus (LSD), colloidal gold immunochromatographic test paper or a kit comprising the antibody, and a preparation method and application thereof. The test paper or the kit has the advantages of convenient production, stable effect, no non-specific reaction, low coating amount and contribution to mass production. The method for detecting the bovine sarcoidosis virus provided by the invention is simple and convenient to operate, has good reproducibility, realizes breakthrough in the field of on-site rapid detection of LSD, and enriches the existing LSD detection method.
Description
Technical Field
The invention relates to the field of immunity, in particular to an antibody for resisting bovine sarcoidosis virus, and a test paper and a kit for detecting the bovine sarcoidosis virus.
Background
Bovine nodular skin disease (LSD) is an acute and subacute contact infectious disease of cattle caused by bovine nodular skin disease virus (LSDV), the morbidity of the disease is between 5% and 45%, and the highest fatality rate can reach 10%. LSDV belongs to the genus capripoxvirus, a subfamily of chordopoxvirinae, and has up to 96% homology with the genomes of capripoxviruses and capripoxviruses. The disease is a notifiable disease (OIE 2014 version name book) of World Animal Health Organization (English: World Organization for Animal Health; French: Office international des pizooties, OIE), is a type of infectious disease determined by the name book of Animal quarantine diseases of the people's republic of China, can cause temporary reduction of milk yield, causes temporary or permanent sterility of the bull, damages skin, even causes death of animals due to secondary bacterial infection, and has great economic loss in the World. LSD is introduced into a plurality of provinces of China between 2019 and 2020, which causes huge economic loss to the local, brings certain pressure to the prevention and control work of China, and is the current work focus for quickly and effectively preventing, controlling, detecting and identifying the bovine sarcoidosis virus.
At present, methods for detecting bovine sarcoidosis (LSD) mainly include a separation culture identification method, a Polymerase Chain Reaction (PCR) method, a fluorescent quantitative PCR (FQ-PCR) method, immunofluorescence, agar immunodiffusion, enzyme-linked immunosorbent assay (ELISA) and the like. Since the isolation culture assay is complicated and time-consuming to operate, the PCR method and FQ-PCR and other methods require expensive instruments and reagents, and the assay cost is high, which is not suitable for the application in laboratories and field assays of basic units.
Disclosure of Invention
A first object of the present invention is to provide an antibody or an active fragment thereof that specifically binds bovine sarcoidosis virus.
Specifically, the antibody or active fragment provided by the invention can be specifically combined with the core protein P32 in the bovine sarcoidosis virus. According to the invention, through analyzing and comparing the LSDV strain and vaccine strain amino acid sequences, the amino acid sequence of the P32 protein is relatively conserved and has better immunogenicity, so that the P32 protein is selected as a target to detect LSD. The antibody or active fragment comprises at least one heavy chain variable region and at least one light chain variable region; wherein:
the heavy chain variable region has CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and/or CDR3 shown in SEQ ID NO. 3; alternatively, the heavy chain variable region has CDR1 of SEQ ID NO. 7, CDR2 of SEQ ID NO. 8 and/or CDR3 of SEQ ID NO. 9;
the light chain variable region has CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5 and/or CDR3 shown in SEQ ID NO. 6; alternatively, the light chain variable region has CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11 and/or CDR3 shown in SEQ ID NO. 12.
As a preferred embodiment of the present invention, in the antibody, the heavy chain variable region has CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and/or CDR3 shown in SEQ ID NO. 3; the light chain variable region has CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5 and/or CDR3 shown in SEQ ID NO. 6.
In a preferred embodiment of the present invention, in the antibody, the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 13, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids to the amino acid sequence shown in SEQ ID NO. 13; the light chain variable region has an amino acid sequence shown in SEQ ID NO. 14, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 14.
In a preferred embodiment of the present invention, the heavy chain variable region of the antibody has CDR1 shown in SEQ ID NO. 7, CDR2 shown in SEQ ID NO. 8 and/or CDR3 shown in SEQ ID NO. 9; the light chain variable region has CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11 and/or CDR3 shown in SEQ ID NO. 12.
In a preferred embodiment of the present invention, in the antibody, the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 15, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids to the amino acid sequence shown in SEQ ID NO. 15; the light chain variable region has an amino acid sequence shown in SEQ ID NO. 16, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 16.
In a preferred embodiment of the present invention, the antibody or active fragment is a monoclonal antibody and/or a genetically engineered antibody; the genetic engineering antibody is selected from one of a single-chain antibody, a single-chain antibody fragment, a chimeric monoclonal antibody fragment, a modified monoclonal antibody and a modified monoclonal antibody fragment.
The second purpose of the invention is to protect the nucleotide sequence which codes for the amino acid sequences shown in SEQ ID NO. 1-16 or the antibody or active fragment.
In the present invention, the nucleotide sequence shown by SEQ ID NO. 17 encodes the amino acid sequence shown by SEQ ID NO. 13; the nucleotide sequence shown as SEQ ID NO. 18 encodes the amino acid sequence shown as SEQ ID NO. 14; the nucleotide sequence shown as SEQ ID NO. 19 encodes the amino acid sequence shown as SEQ ID NO. 15; the nucleotide sequence shown as SEQ ID NO. 20 encodes the amino acid sequence shown as SEQ ID NO. 16.
The third purpose of the invention is to protect the immunity test paper for detecting the bovine sarcoidosis virus or a kit internally provided with the test paper, wherein the test paper contains the antibody or the active fragment provided by the invention. The test paper is preferably colloidal gold immunochromatographic assay test paper.
As a preferred embodiment of the present invention, the test strip comprises a binding pad, a detection line (T-line) and a control line (C-line); the combination pad is coated with a conjugate of the antibody or the active fragment and colloidal gold, the detection line is coated with the antibody or the active fragment, and the control line is coated with a goat anti-mouse IgG antibody.
Wherein, in the antibody or active fragment coated on the binding pad coupled with colloidal gold, the heavy chain variable region has CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and/or CDR3 shown in SEQ ID NO. 3; the light chain variable region has CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5 and/or CDR3 shown in SEQ ID NO. 6. Preferably, in the antibody or active fragment coated on the binding pad coupled with colloidal gold, the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 13, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 13; the light chain variable region has an amino acid sequence shown in SEQ ID NO. 14, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 14.
Wherein, in the antibody or active fragment coated on the detection line, the heavy chain variable region has CDR1 shown in SEQ ID NO. 7, CDR2 shown in SEQ ID NO. 8 and/or CDR3 shown in SEQ ID NO. 9; the light chain variable region has CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11 and/or CDR3 shown in SEQ ID NO. 12. Preferably, in the antibody or active fragment coated on the detection line, the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 15, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 15; the light chain variable region has an amino acid sequence shown in SEQ ID NO. 16, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 16.
As a preferred scheme of the invention, the colloidal gold immunochromatographic test strip is prepared by a method comprising the following steps:
the antibody or the active fragment provided by the invention is coupled with colloidal gold to obtain a solution, and the solution is sprayed on a bonding pad; after being dissolved by coating buffer solution, the antibody provided by the invention is coated on a T line of a nitrocellulose membrane; dissolving an anti-mouse IgG antibody by using a coating buffer solution, and coating the anti-mouse IgG antibody on a C line of a nitrocellulose membrane; and assembling the combination pad and the nitrocellulose membrane on a back plate to prepare a large plate, and cutting the large plate into a bare strip, namely the test paper.
The fourth purpose of the invention is to protect the application of the antibody or the active fragment provided by the invention, or the test paper or the kit in detecting bovine sarcoidosis virus in a sample, in particular to the application in detecting protein P32 in the bovine sarcoidosis virus. Preferably, the sample is bovine saliva, nodal tissue fluid or blood.
The fifth purpose of the invention is to protect the method for detecting the bovine sarcoidosis virus in a sample by using the antibody or the active fragment provided by the invention or the test paper or the kit, in particular to the method for detecting the protein P32 in the bovine sarcoidosis virus. Preferably, the sample is bovine saliva, nodal tissue fluid or blood.
As a preferred embodiment of the present invention, the method comprises the steps of: and dropwise adding the solution of the sample to be detected onto the colloidal gold immunochromatography detection test paper or into a sample adding hole of a kit internally provided with the test paper, standing, and observing the color development conditions of the T line and the C line on the test paper.
As a preferable embodiment of the present invention, if both the T line and the C line are red, the sample contains bovine sarcoidosis virus protein P32 at a concentration of 10ng/ml or more; if the C line is red and the T line is not developed, the sample contains the bovine sarcoidosis virus protein P32 with the concentration of less than 10ng/ml or does not contain the bovine sarcoidosis virus protein P32; if the C line does not develop color, the detection reagent is invalid.
Drawings
FIG. 1 is a schematic diagram showing the results of detection using the colloidal gold immunoassay chromatography test paper provided by the present invention.
Detailed Description
According to the technical scheme of the invention, certain amino acids in the amino acid sequence can be conservatively substituted without changing the activity or function of the protein, as shown in the following table 1:
TABLE 1
| Residue of | Conservative substitutions | Residue of | Conservative substitutions |
| Ala | Ser | Leu | Ile；Val |
| Arg | Lys | Lys | Arg；Gln |
| Asn | Gln；His | Met | Leu；Ile |
| Asp | Glu | Phe | Met；Leu；Tyr |
| Gln | Asn | Ser | Thr；Gly |
| Cys | Ser | Thr | Ser；Val |
| Glu | Asp | Trp | Tyr |
| Gly | Pro | Tyr | Trp；Phe |
| His | Asn；Gln | Val | Ile；Leu |
| Ile | Leu；Val |
Furthermore, because of the degeneracy of the bases, substitutions can be made to bases of a polynucleotide sequence without altering the activity or function of the polynucleotide sequence, see table 2 below:
TABLE 2
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1: prokaryotic expression and purification of P32 protein
In this example, prokaryotic expression and purification of P32 protein were performed by the following steps:
1. the genome sequence of LSDV is inquired from GeneBank, and the conserved gene LSDV-P32 is selected to express P32 protein through alignment analysis. The amino acid sequence of the P32 protein is analyzed by an online software TMHMM to find that a transmembrane region is arranged at the C terminal, and the transmembrane region at the C terminal is removed during expression and codon optimization is carried out because the transmembrane region protein is insoluble. The amino acid sequence of LSDV P32 protein is shown in Table 3.
Table 3: amino acid sequence of P32 protein
2. pGEX-6P-2-N280 plasmid with correct sequencing is selected and transformed into BL21(DE3) competent cells, and the cells are cultured in a constant temperature incubator at 37 ℃ overnight. Single colonies were picked and cultured overnight at 37 ℃ and 200rpm in 100mL of 2YT medium. Transferring into 2YT culture medium according to the ratio of 1:50, and placing into a constant temperature shaking table at 37 ℃ and 200 rpm. When OD600 is 0.6, 5-10 mL of bacterial liquid is taken, IPTG with the final concentration of 0.4mM/L is added into the rest bacteria, and the mixture is placed at 20 ℃ for induction overnight. And (3) taking 5-10 mL of the induced bacterial liquid, centrifuging the residual bacteria at 4 ℃ and 6000rpm for 20min, adding a precooled combined Buffer and 1mM/L PMSF, and carrying out ice-bath ultrasonic crushing. The mixture was ultracentrifuged at 45000g for 1h at 4 ℃ and the supernatant was collected and filtered. And (3) carrying out ultrasonic crushing on the taken out bacteria before and after the induction of 5-10 mL, running PAGE (polyacrylamide gel electrophoresis), carrying out counterstaining, and checking the protein expression condition.
3. GE Healthcare Glutathione Sepharose TM 4B was equilibrated 3 times with pre-cooled binding Buffer and bound to the disrupted protein. After washing the protein with precooled Wash Buffer until the protein fast stain did not change color (about 15 column volumes), DTT and PreScission protease with a final concentration of 1mM/L were added and the digestion was carried out overnight at 4 ℃. After leaving a sample of the enzyme-digested protein, centrifuging the sample at 4 ℃ and 4500rpm for 5min, collecting a supernatant, washing the medium with Wash Buffer for 2-3 times, collecting all the supernatants, dialyzing the supernatant in Buffer with 20mM Tris-HCl pH8.0 overnight, and passing through a GE Healthcare HiTraptM Q column. Samples of each peak were collected separately and examined on PAGE gels, 28kD protein was collected and concentrated, and PBS was iterated. Adding 10-15% of glycerol into the mixture, quickly freezing the mixture by liquid nitrogen, and storing the mixture at-80 ℃.
Example 2: preparation of P32 protein monoclonal antibody
8 female Balb/c mice with the age of 8-12 weeks are immunized by the P32 protein antigen purified in the example 1, after 3 times of immunization, orbital blood collection is carried out on the mice to obtain mouse serum, the P32 protein antibody titer in the mouse serum is detected by ELISA, and if the titer is less than 10000, the boosting immunization is carried out for 1-2 times until the antibody titer is greater than 10000.
Taking a mouse spleen cell with the antibody titer of more than 10000 to fuse with a myeloma SP2/0 cell, screening the fused cell by using an HAT selective culture medium, and carrying out ELISA positive screening and subcloning on the fused cell; and (3) selecting the screened positive monoclonal, taking ascites, purifying the antibody by using a Protein A/G antibody purification column, wherein the ELISA titer of the purified antibody is more than 1:128000, and the purity is more than 90%.
The cell lines corresponding to the two monoclonal antibodies are numbered 701 and 901 respectively.
Example 3: amplification and sequence determination of P32 protein monoclonal antibody CDR region sequence
Recovering hybridoma cell strains 701 and 901, culturing, and counting cells to 8 × 10 when the cells grow to logarithmic growth phase7At one/ml, cells were collected. Extracting total RNA of hybridoma cells according to TaKaRa MiniBEST Universal RNA Extraction Kit instruction, placing the reaction system shown in Table 4 at 65 ℃ for 5min, rapidly cooling on ice, and placing the reaction system shown in Table 5 at 42 ℃ for 60 min; 15min at 70 ℃; the 1st-Strand cDNA synthesis reaction was performed at 25 ℃ for 1 min.
Table 4: 1st-Strand cDNA Synthesis reaction mixture
| Reagent | Amount of the composition used |
| Oligo dT Primer(50μM) | 1μL |
| dNTP Mixture(10mM each) | 1μL |
| Template RNA | <5μg |
| RNase Free dH2O | Up to 10μL |
Table 5: reverse transcription reaction liquid
| Reagent | Amount of the composition used |
| The denatured reaction solution (from step 2) | 10μL |
| 5xPrimeScriptⅡbuffer | 4μL |
| RNase Inhibitor | 0.5μL(20U) |
| PrimeScriptⅡRTase(200U/μL) | 1μL(200U) |
| RNase Free dH2O | Up to 20μL |
Using cDNA as template, designing and synthesizing upstream and downstream primers of light chain and heavy chain variable region, the primer sequences are shown in Table 6, PCR amplifying light chain and heavy chain variable region nucleic acid sequences, obtaining DNA fragments by glue recovery, and detecting the concentration of recovered nucleic acid. Reference pMDTM19-T Vector Cloning Kit, Cloning of DNA fragment into pMDTMReacting in a 19-T vector at 16 ℃ for 30min, transforming the reaction product into TOP10 competent cells by heat shock, identifying PCR colonies, selecting positive colonies for amplification culture, extracting plasmids according to the specification of a Tiangen plasmid miniprep kit, measuring the concentration, identifying the PCR, and sequencing.
Table 6: variable region primer sequences
| Serial number | Name (R) | Primer sequence (5 '-3') |
| SEQ ID NO:22 | LSDV-P32-VLF | GTTAGATCTCCAGCTTGGTCCC |
| SEQ ID NO:23 | LSDV-P32-VLR | GACATTCAGCTGACCCAGTCTCCA |
| SEQ ID NO:24 | LSDV-P32-VHVR1 | CTCACCATGGRATGSAGCTGKGTMATS |
| SEQ ID NO:25 | LSDV-P32-VHCR2 | AYCTCCACACACAGGRRCCAGTGGATAGAC |
Example 4: monoclonal cell line 701 variable region sequences
And performing Blast comparison on the sequencing result in GenBank, wherein the result shows that the homology of the variable region sequences of the light chain and the heavy chain of the antibody and the submitted variable region sequence of the mouse IgG exceeds 96 percent, and the gene sequence obtained by sequencing is determined to be the sequence of the mouse antibody. The amino acid sequences of the variable regions of the light chain and the heavy chain of the antibody, and the division of the CDR region and the FR region are obtained by utilizing the IMGT/V-QUEST and ABYSIS software analysis. The results are as follows:
table 7: sequencing results of monoclonal cell line 701 antibody
Example 5: variable region sequences of monoclonal cell line 901
And performing Blast comparison on the sequencing result in GenBank, wherein the result shows that the homology of the variable region sequences of the light chain and the heavy chain of the antibody and the submitted variable region sequence of the mouse IgG exceeds 96 percent, and the gene sequence obtained by sequencing is determined to be the sequence of the mouse antibody. The amino acid sequences of the variable regions of the light chain and the heavy chain of the antibody, and the division of the CDR region and the FR region are obtained by utilizing the IMGT/V-QUEST and ABYSIS software analysis. The results are as follows:
table 8: sequencing result of monoclonal cell line 901 antibody
Example 5: colloidal gold immunoassay chromatography reagent detection
Coupling an antibody purified from ascites of a mouse with a cell strain number of 701 with colloidal gold with the particle size of 40nm, dissolving the antibody in a complex solution, and spraying the antibody on a pretreated bonding pad by using a gold-labeled three-dimensional film-drawing and point-spraying instrument; dissolving an antibody purified from ascites corresponding to a cell line number 901 by using a coating buffer solution, and coating the antibody on a TEST line (T line) of a pretreated Nitrocellulose (NC) membrane by using a gold-labeled three-dimensional membrane-scribing dot-spraying instrument; dissolving an outsourced goat anti-mouse IgG antibody with a coating buffer solution, and coating the dissolved antibody on a CONTROL line (C line) of a pretreated nitrocellulose membrane by using a gold-labeled three-dimensional membrane-scribing dot spraying instrument; assembling the bonding pad subjected to metal spraying treatment, the NC membrane subjected to coating treatment and the pretreated bonding pad on a back plate to manufacture a large plate. And cutting the large plate manufactured in the previous step into a bare strip with the width of 4mm by using a slitter, assembling the bare strip into a matched card shell, and preparing the colloidal gold detection reagent for detecting P32.
Preparing P32 sample solutions with different concentrations (the solvent is phosphate buffer solution with pH7.4 and 0.01M), respectively transferring 80 mu l of P32 sample solutions with different concentrations by a pipette, dripping the sample solutions in parallel into a sample adding hole of a colloidal gold detection reagent, observing the color development conditions of a T line and a C line when 10min is carried out, and if the T line and the C line are both red, the sample solution is positive; the C line is red, and the T line is negative when not developing color; the C line did not develop color and the detection reagent became ineffective. As shown in fig. 1.
The detection limit of the P32 colloidal gold detection reagent is 10 ng/ml.
The invention adopts prokaryotic expression to fuse with P32 protein of GST tag, greatly improves the expression quantity of P32 protein, utilizes glutathione agarose 4B medium to enrich GST-P32 protein, then uses PSP enzyme to carry out enzyme digestion to obtain P32 protein with purity of more than 90%, immunizes mice to prepare a plurality of anti-P32 monoclonal antibodies, carries out colloidal gold detection pairing on the prepared monoclonal antibodies, and respectively uses two successfully paired monoclonal antibodies (cell strain numbers are 701 and 901) for colloidal gold marking and coating, thereby establishing a colloidal gold test strip detection method for detecting P32. The method is simple and convenient to operate, good in reproducibility, and capable of achieving breakthrough in the field of LSD on-site rapid detection and enriching the existing LSD detection method.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
SEQUENCE LISTING
<110> Beijing Sanan Xinte Biotech Co., Ltd
<120> antibody for resisting bovine sarcoidosis virus, detection test paper and kit
<130> RYP2010790.9
<160> 25
<170> PatentIn version 3.5
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<213> Artificial Sequence
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<223> CDR1 of heavy chain variable region
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Thr Tyr Ala Val His
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<213> Artificial Sequence
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<400> 2
Val Ile Trp Ser Gly Gly Arg Thr Asp Tyr Asn Ala Ala Phe Met Cys
1 5 10 15
<210> 3
<211> 9
<212> PRT
<213> Artificial Sequence
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<223> CDR3 of heavy chain variable region
<400> 3
Asn Tyr Tyr Gly Gly Ser Leu Asp Tyr
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<210> 4
<211> 15
<212> PRT
<213> Artificial Sequence
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<223> CDR1 of light chain variable region of 701
<400> 4
Arg Ala Ser Asn Ser Val Ser Thr Ser Arg Tyr Ser Tyr Met His
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<211> 7
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<223> CDR2 of light chain variable region of 701
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Leu Val Ser Asn Leu Glu Ser
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Gln His Ile Arg Glu Leu Thr
1 5
<210> 7
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> 901 CDR1 of the heavy chain variable region
<400> 7
Thr Tyr Ala Val His
1 5
<210> 8
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> 901 CDR2 of the heavy chain variable region
<400> 8
Val Ile Trp Ile Gly Gly Arg Thr Asp Tyr Asn Ala Ala Phe Ile Ser
1 5 10 15
<210> 9
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> 901 CDR3 of the heavy chain variable region
<400> 9
Asn Tyr Tyr Gly Gly Ser Phe Asp Tyr
1 5
<210> 10
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR1 of the light chain variable region of 901
<400> 10
Arg Asp Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His
1 5 10 15
<210> 11
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR2 of the light chain variable region of 901
<400> 11
Leu Val Ser Asn Leu Glu Ser
1 5
<210> 12
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> CDR3 of the light chain variable region of 901
<400> 12
Gln His Ile Arg Glu Leu Thr
1 5
<210> 13
<211> 112
<212> PRT
<213> Artificial Sequence
<220>
<223> amino acid sequence of heavy chain variable region
<400> 13
Gln Ser Ala Pro Gly Leu Val Gln Ala Ser Gln Cys Leu Ser Ile Thr
1 5 10 15
Cys Thr Val Ser Gly Phe Ser Leu Thr Thr Tyr Ala Val His Trp Val
20 25 30
Arg Gln Pro Pro Gly Gln Gly Leu Glu Trp Gln Gly Val Ile Trp Ser
35 40 45
Gly Gly Arg Thr Asp Tyr Asn Ala Ala Phe Met Cys Arg Leu Ser Ile
50 55 60
Ser Lys Asp Asn Ser Asn Ser Gln Asp Phe Phe Lys Met Asn Ser Leu
65 70 75 80
Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Asn Tyr Tyr Gly
85 90 95
Gly Ser Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
100 105 110
<210> 14
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> amino acid sequence of light chain variable region
<400> 14
Asp Ile Val Leu Thr Gln Thr Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Ser Arg Ala Ser Asn Ser Val Ser Thr Ser
20 25 30
Arg 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 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 15
<211> 116
<212> PRT
<213> Artificial Sequence
<220>
<223> amino acid sequence of heavy chain variable region of 901
<400> 15
Gln Val Gln Leu Asn Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Leu Ser Gly Phe Ser Leu Thr Thr Tyr
20 25 30
Ala Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ile Gly Gly Arg Thr Asp Tyr Asn Ala Ala Phe Ile
50 55 60
Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Glu Met Asn Ser Leu Gln Pro Asn Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Arg Asn Tyr Tyr Gly Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr
100 105 110
Leu Thr Val Ser
115
<210> 16
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> 901 light chain variable region
<400> 16
Asp Ile 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 Asp 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 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 17
<211> 467
<212> DNA
<213> Artificial Sequence
<220>
<223> nucleotide sequence encoding 701 heavy chain variable region
<400> 17
tctcaccatg gctgtcttgg ggctgctctt ctgcctggtg acattcccaa gctgtgtcct 60
atcccaggcg caggtgaatc agtcagcacc aggcctagtg caggcctcac agtgcctgtc 120
catcacctgc acagtctctg gtttctcatt aactacctat gctgtacact gggttcgcca 180
gcctccagga cagggtctgg agtggcaggg agtgatatgg agtggtggaa ggacagacta 240
taatgctgct ttcatgtgca gactgagcat cagcaaggac aactccaaca gccaagattt 300
ctttaaaatg aacagtctac aagctgatga cacagccata tactactgtg ccagaaatta 360
ctacggtggt agccttgact actggggcca aggcaccact ctcacagtct cctcagccaa 420
aacgacaccc ccatctgtct atccactggc ccctgtgtgt ggagata 467
<210> 18
<211> 358
<212> DNA
<213> Artificial Sequence
<220>
<223> nucleotide sequence encoding 701 light chain variable region
<400> 18
tgacattgtg ctcacacaga ctcctgcttc cttagctgta tctctggggc agagggccac 60
gatctcatcc agggccagca atagtgtcag tacatctcgc tatagttata tgcactggaa 120
ccaacagaaa ccaggacagc cacccagact cctcatctat cttgtatcca acctagaatc 180
tggggtccct gccaggttca gtggcagtgg gtctgggaca gacttcaccc tcaacatcca 240
tcctgtggag gaggaggatg ctgcaaccta ttactgtcag cacattaggg agcttacacg 300
ttcggagggg ggaccaagct ggaaataaaa cgggctgatg ctgcaccaac tgtatcca 358
<210> 19
<211> 467
<212> DNA
<213> Artificial Sequence
<220>
<223> nucleotide sequence encoding the variable region of the 901 heavy chain
<400> 19
tctcaccatg gctgtcttgg ggctgctctt ctgcctggtg acattcccaa gctgtgtcct 60
ctcccaggtg cagctgaacc agtcaggtcc tggcctagtg cagccctcac agagcctgtc 120
catcacctgc acactctctg gtttctcctt aactacctat gctgtacact gggttcgcca 180
gtctccagga aagggtctgg agtggctggg agtgatttgg attggtggac gcacagacta 240
taatgcagct ttcatatcca gactgagcat cagcaaggac aattccaaga gccaagtttt 300
ctttgaaatg aacagtctgc aacctaatga cacagccata tattactgtg ccagaaatta 360
ctacggtggt agctttgact actggggcca aggcaccact ctcacagtct cctcagccaa 420
aacgacaccc ccatctgtct atccactggc ccctgtgtgt ggagata 467
<210> 20
<211> 358
<212> DNA
<213> Artificial Sequence
<220>
<223> nucleotide sequence encoding the variable region of the 901 light chain
<400> 20
tgacattgtg ctgacccagt ctcctgcttc cttagctgta tctctggggc agagggccac 60
catctcatac agggacagca aaagtgtcag tacatctggc tatagttata tgcactggaa 120
ccaacagaaa ccaggacagc cacccagact cctcatctat cttgtgtcca acctagaatc 180
tggggtccct gccaggttca gtggcagtgg gtctgggaca gacttcaccc tcaacatcca 240
tcctgtggag gaggaggatg ctgcaaccta ttactgtcag cacattaggg agcttacacg 300
ttcggagggg ggaccaagct ggaaataaaa cgggctgatg ctgcaccaac tgtatcca 358
<210> 21
<211> 280
<212> PRT
<213> Artificial Sequence
<220>
<223> P32 protein
<400> 21
Met Ala Asp Ile Pro Leu Tyr Val Ile Pro Ile Val Gly Arg Glu Ile
1 5 10 15
Ser Asp Val Val Pro Glu Leu Lys Ser Asp Asn Asp Ile Phe Tyr Lys
20 25 30
Lys Val Asp Thr Val Lys Asp Phe Lys Asn Ser Asp Val Asn Phe Phe
35 40 45
Phe Lys Asp Lys Lys Asp Ile Ser Leu Ser Tyr Lys Phe Leu Ile Trp
50 55 60
Glu Lys Val Glu Lys Ser Gly Gly Val Glu Asn Phe Thr Glu Tyr Phe
65 70 75 80
Ser Gly Leu Cys Asn Ala Leu Cys Thr Lys Glu Ala Lys Ser Ser Ile
85 90 95
Val Lys His Phe Ser Leu Trp Lys Ser Tyr Ala Asp Ala Asp Ile Lys
100 105 110
Asn Ser Glu Asn Lys Phe Ile Val Val Ile Glu Asp Asp Asn Thr Leu
115 120 125
Lys Asp Leu Ile Thr Ile His Asn Ile Ile Ile Glu Met Gln Glu Lys
130 135 140
Asn Ile Asp Ile Phe Gln Leu Arg Glu Thr Phe His Asn Ser Asn Ser
145 150 155 160
Arg Ile Leu Phe Asn Gln Glu Asn Asn Asn Phe Met Tyr Ser Tyr Thr
165 170 175
Gly Gly Tyr Asp Phe Thr Leu Ser Ala Tyr Val Ile Arg Leu Ser Ser
180 185 190
Ala Ile Lys Ile Ile Asn Glu Ile Ile Lys Asn Lys Gly Ile Ser Thr
195 200 205
Ser Leu Ser Phe Glu Met Tyr Lys Leu Glu Lys Glu Leu Lys Leu Asn
210 215 220
Arg Gln Val Leu Asn Asp Ser Ser Lys Tyr Ile Leu His Asn Thr Lys
225 230 235 240
Tyr Leu Ser Lys Lys Arg Ala Asn Glu Met Lys Asn Gly Ile Trp Asn
245 250 255
Arg Val Gly Lys Trp Met Ala His Arg Phe Pro Asp Phe Ser Tyr Tyr
260 265 270
Ile Ser His Pro Leu Val Ser Phe
275 280
<210> 22
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> LSDV-P32-VLF
<400> 22
gttagatctc cagcttggtc cc 22
<210> 23
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> LSDV-P32-VLR
<400> 23
gacattcagc tgacccagtc tcca 24
<210> 24
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> LSDV-P32-VHVR1
<400> 24
ctcaccatgg ratgsagctg kgtmats 27
<210> 25
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> LSDV-P32-VHCR2
<400> 25
ayctccacac acaggrrcca gtggatagac 30
Claims (17)
1. An antibody or an active fragment thereof that specifically binds bovine sarcoidosis virus, wherein the antibody or active fragment comprises a heavy chain variable region and a light chain variable region as follows;
the heavy chain variable region has CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and CDR3 shown in SEQ ID NO. 3, and the light chain variable region has CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5 and CDR3 shown in SEQ ID NO. 6; or
The heavy chain variable region has CDR1 shown in SEQ ID NO. 7, CDR2 shown in SEQ ID NO. 8 and CDR3 shown in SEQ ID NO. 9, and the light chain variable region has CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11 and CDR3 shown in SEQ ID NO. 12.
2. The antibody or active fragment thereof of claim 1, wherein the heavy chain variable region has the CDR1 of SEQ ID No. 1, CDR2 of SEQ ID No. 2 and CDR3 of SEQ ID No. 3;
the light chain variable region has CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5 and CDR3 shown in SEQ ID NO. 6.
3. The antibody or active fragment thereof according to claim 2,
the heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 13;
the variable region of the light chain has an amino acid sequence shown as SEQ ID NO. 14.
4. The antibody or active fragment thereof of claim 1, wherein the heavy chain variable region has the CDR1 of SEQ ID NO. 7, the CDR2 of SEQ ID NO. 8 and the CDR3 of SEQ ID NO. 9;
the light chain variable region has CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11 and CDR3 shown in SEQ ID NO. 12.
5. The antibody or active fragment thereof according to claim 4,
the heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 15;
the variable region of the light chain has an amino acid sequence shown as SEQ ID NO. 16.
6. The antibody or active fragment of any one of claims 1 to 5, wherein the antibody or active fragment is a monoclonal antibody or a genetically engineered antibody; the genetic engineering antibody is selected from one of a single-chain antibody, a single-chain antibody fragment, a chimeric monoclonal antibody fragment, a modified monoclonal antibody and a modified monoclonal antibody fragment.
7. A nucleic acid molecule encoding the antibody or active fragment of any one of claims 1 to 6.
8. An immunological test strip for bovine sarcoidosis virus or a kit comprising the test strip, wherein the test strip comprises the antibody or active fragment of any one of claims 1 to 6.
9. A test strip or kit according to claim 8, wherein the test strip is a colloidal gold immunochromatographic test strip.
10. A test strip or kit according to claim 8 wherein the test strip comprises a binding pad, a detection line and a control line; the conjugate of the antibody or the active fragment and colloidal gold is coated on the combination pad, the antibody or the active fragment is coated on the detection line, and a goat anti-mouse IgG antibody is coated on the control line;
wherein the antibody or active fragment coated on the conjugate pad, which is coupled to colloidal gold, has a heavy chain variable region having CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2 and CDR3 shown in SEQ ID NO. 3, and a light chain variable region having CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 5 and CDR3 shown in SEQ ID NO. 6.
11. The test paper or kit of claim 10, wherein the heavy chain variable region has an amino acid sequence shown by SEQ ID No. 13 and the light chain variable region has an amino acid sequence shown by SEQ ID No. 14.
12. The test paper or kit of claim 10, wherein the heavy chain variable region of the antibody or active fragment coated on the detection line has CDR1 shown in SEQ ID No. 7, CDR2 shown in SEQ ID No. 8 and CDR3 shown in SEQ ID No. 9, and the light chain variable region has CDR1 shown in SEQ ID No. 10, CDR2 shown in SEQ ID No. 11 and CDR3 shown in SEQ ID No. 12.
13. The test paper or kit of claim 12, wherein the heavy chain variable region has an amino acid sequence shown by SEQ ID No. 15, and the light chain variable region has an amino acid sequence shown by SEQ ID No. 16.
14. A test strip or kit according to claim 8, prepared by a method comprising the steps of:
spraying a solution obtained by coupling the antibody or active fragment according to claim 2 or 3 with colloidal gold onto a conjugate pad; coating the antibody or active fragment of claim 4 or 5 on a detection line of a nitrocellulose membrane after dissolving the antibody or active fragment with a coating buffer; dissolving an anti-mouse IgG antibody by using a coating buffer solution, and coating the anti-mouse IgG antibody on a control line of a nitrocellulose membrane; and assembling the combination pad and the nitrocellulose membrane on a back plate, and cutting the obtained large plate into bare strips, namely test paper.
15. Use of an antibody or active fragment according to any one of claims 1 to 6 in the preparation of a strip or kit for the detection of bovine sarcoidosis virus in a sample.
16. Use according to claim 15, wherein the sample is bovine saliva, nodal interstitial fluid or blood.
17. The use according to claim 15, characterized in that said method of detection comprises the steps of: dripping the solution of a sample to be detected on colloidal gold immunochromatography detection test paper or dripping the solution of the sample to be detected into a sample adding hole of a kit internally provided with the test paper, standing, and observing the color development conditions of a T line and a C line on the test paper;
if the detection line and the control line are both red, the bovine sarcoidosis virus protein P32 with the concentration of more than or equal to 10ng/ml is contained in the sample; if the control line is red and the detection line does not develop color, the sample contains the bovine sarcoidosis virus protein P32 with the concentration of less than 10ng/ml or does not contain the bovine sarcoidosis virus protein P32; if the control line does not develop color, the detection reagent is disabled.
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| CN114437209B (en) * | 2022-01-25 | 2022-09-20 | 北京弘进久安生物科技有限公司 | Monoclonal antibody specifically binding to abrin and application thereof |
| CN115260298A (en) * | 2022-04-22 | 2022-11-01 | 中国兽医药品监察所 | Monoclonal antibody of nodular skin disease virus and application thereof |
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