CN110845582B - Preparation of feline parvovirus recombinant protein and monoclonal antibody thereof - Google Patents
Preparation of feline parvovirus recombinant protein and monoclonal antibody thereof Download PDFInfo
- Publication number
- CN110845582B CN110845582B CN201911334829.4A CN201911334829A CN110845582B CN 110845582 B CN110845582 B CN 110845582B CN 201911334829 A CN201911334829 A CN 201911334829A CN 110845582 B CN110845582 B CN 110845582B
- Authority
- CN
- China
- Prior art keywords
- recombinant protein
- parvovirus
- feline parvovirus
- monoclonal antibody
- feline
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Abstract
The invention belongs to the technical field of biological engineering. The invention relates to a cat parvovirus recombinant protein, which comprises two dominant epitopes of a cat parvovirus (FPV) antigen, wherein in order to improve the yield of the recombinant protein in a prokaryotic expression system, an amino acid sequence of the recombinant protein is converted into a corresponding nucleotide sequence by adopting an escherichia coli preferred codon, the nucleotide sequence is chemically synthesized, and a recombinant expression vector is constructed. The invention also relates to the preparation of the recombinant protein monoclonal antibody of the feline parvovirus, hybridoma cell strains are obtained after immunization, cell fusion and multi-round screening, the monoclonal antibody is purified and respectively marked with colloidal gold particles, and the optimal pairing combination of the monoclonal antibodies is determined through orthogonal experiments and can be used for early diagnosis of feline parvovirus infection.
Description
Technical Field
The invention belongs to the technical field of biological engineering. Specifically, the invention relates to a novel feline parvovirus recombinant protein, which is characterized in that a nucleotide sequence of the feline parvovirus recombinant protein is synthesized by coding through a genetic engineering technology, a plasmid vector containing the nucleotide sequence is constructed, an escherichia coli strain transformed with the plasmid vector is used for expressing the feline parvovirus recombinant protein; the recombinant protein is used for preparing a monoclonal antibody and is applied to the preliminary diagnosis of the feline parvovirus infection.
Background
Feline Parvovirus (FPV), also known as Feline panleukopenia virus, Feline infectious enteritis virus, Feline distemper virus, has clinical manifestations of hyperpyrexia, vomiting, diarrhea and enteritis after infection of cats with parvovirus, which can lead to severe reduction in the number of leukocytes. The virus mainly infects various animals of the family of cats and the family of weasels, particularly has higher morbidity and mortality rate of young animals below 6 months old, and is a virus with the widest infection range and the strongest pathogenicity in the parvovirus genus at present.
Under natural conditions, the excrement such as feces and urine, secretion such as nose, eyes and saliva and vomit of infected animals, particularly sick animals, are toxic and can pollute feed, drinking water, appliances and the surrounding environment. When FPV antibodies exist in the serum of infected animals, the excrement and urine can still be externally discharged for 6 weeks. As the feline parvovirus hazard increases, laboratory diagnostics have been developed by many researchers. The method mainly comprises the separation and identification of feline parvovirus, the method utilizes cultured cells to observe cytopathic effect, but some viruses are slow in proliferation and cannot grow in cell culture, the detoxification time can be very short compared with the clinical attack time, the infectivity can be lost during the sample storage and transportation process, and the method requires certain experience of operators and is high in cost. The electron microscope examination test has low sensitivity, different viruses with similar forms cannot be distinguished, and the sample treatment capacity is large, so that the application of the method is limited. The RT-PCR method for detecting the feline parvovirus nucleic acid has good specificity and sensitivity, can quickly distinguish viruses, is suitable for simultaneously detecting a large number of samples, but has expensive equipment, high requirement on operators, long detection time and high detection cost.
At present, the immunology method for detecting the feline parvovirus is a mainstream direction due to simple operation and easy judgment of results. The immunology method adopts the monoclonal antibody to identify and detect the feline parvovirus antigen in the blood sample, and the method has good specificity and sensitivity. If the colloidal gold platform is combined, an accurate result can be obtained within 10-15 minutes generally, the method is suitable for rapid detection of large-batch samples, special equipment and personnel requirements do not exist, and a master can detect the samples at home.
Disclosure of Invention
The design purpose is as follows: through designing a cat parvovirus recombinant protein and preparing a monoclonal antibody thereof, the specificity recognition detection of the cat parvovirus is realized, the detection sensitivity is enhanced, and the detection result is not distorted.
The design scheme is as follows: in order to achieve the above design objectives. The application: (1) the coat protein of the feline parvovirus is taken as a target antigen, two specific dominant epitopes of the antigen are analyzed and selected, and the sequence comparison result shows that the two selected antigen epitopes have no obvious homology with other protein sequences. (2) In order to promote stimulation of the selected dominant antigen epitope to the Balb/c mouse immune system and enhance the immune effect, the two selected dominant antigen epitope sequences are connected in series through flexible fragments (four continuous glycines) and then are repeated for four times to form a cat parvovirus recombinant protein amino acid sequence. (3) And E.coli preferred codons are adopted to convert the amino acid sequence of the feline parvovirus recombinant protein into a corresponding nucleotide sequence, so that the high-efficiency expression of the recombinant protein in the E.coli is facilitated. (4) And chemically synthesizing the nucleotide sequence obtained in the last step, carrying out enzyme digestion connection, inserting the synthesized nucleotide fragment into a prokaryotic expression vector pET-28a (+), and constructing a recombinant protein expression vector. (5) The cat parvovirus recombinant protein expression vector is used for transforming escherichia coli ER2566 competent cells, and a kanamycin resistance screening culture medium is used for screening to obtain a recombinant protein expression strain. (6) After the recombinant protein expression strain is cultured in a large scale, the strain is broken by ultrasonic waves and centrifuged at low temperature, the supernatant of the solution is taken to pass through a nickel agarose affinity chromatography column for affinity chromatography, and the purified recombinant protein of the feline parvovirus is obtained by elution. (7) After the purified feline parvovirus recombinant protein is used for immunizing a Balb/c mouse for multiple times, spleen cells of the feline parvovirus recombinant protein are taken to be fused with sp2/0 myeloma cells, and a hybridoma cell strain is finally obtained after multiple rounds of screening. (8) Preparing Balb/c mouse ascites from the hybridoma cell strain, purifying the monoclonal antibody in two steps by using an n-octanoic acid-ammonium sulfate precipitation method and Protein A affinity chromatography, and marking colloidal gold particles respectively. (9) Orthogonal experimental screening shows that the 3A6 monoclonal antibody coating and the 7B9 colloidal gold labeled monoclonal antibody are matched to be the best combination for detecting the feline parvovirus infection.
Detailed Description
Although the following embodiments describe the design concept of the present invention in more detail, these descriptions are only simple words for describing the design concept of the present invention, and are not intended to limit the design concept of the present invention, and any combination, addition or modification without departing from the scope of the design concept of the present invention will fall within the scope of the present invention.
Example 1: feline parvovirus dominant epitope selection
The coat protein of the feline parvovirus is taken as a target antigen, the hydrophilicity and the antigenicity of an epitope sequence of the feline parvovirus are analyzed by using biological software DNAssist2.0, and an A dominant epitope (SEQ ID No:2) and a B dominant epitope (SEQ ID No:3) are selected. The sequence comparison result shows that the selected A, B dominant antigen epitope sequences have high specificity and no obvious homology with other protein sequences.
Example 2: feline parvovirus dominant epitope tandem
In order to enhance the stimulation of the selected epitope to the mouse immune system to be beneficial to the subsequent experiment, A, B two dominant epitope sequences of the coat protein of the feline parvovirus are connected by flexible fragments (four continuous glycines) and then repeated for four times to obtain the amino acid sequence of the recombinant protein of the feline parvovirus, and the specific sequence of the amino acid sequence is shown as SEQ ID No:1 in the sequence table.
Example 3: nucleotide sequence for optimizing recombinant protein for coding feline parvovirus
In order to improve the expression quantity of the cat parvovirus recombinant protein in the escherichia coli, on the premise that the amino acid sequence of the recombinant protein is not changed, the amino acid sequence of the encoded cat parvovirus recombinant protein is converted into a corresponding nucleotide sequence according to an escherichia coli preferred codon, the specific sequence is shown as a sequence table SEQ ID No. 4, nucleotide sequences corresponding to enzyme cutting sites BamHI and EcoRI are respectively added at the upstream and the downstream of the nucleotide sequence, and the cat parvovirus recombinant protein is synthesized by Hangzhou xian to biological technology limited company. The synthesized target gene is cloned in pMD19-T vector (Takara Bio-engineering Co., Ltd.).
Example 4: construction of recombinant protein expression vector of Cat parvovirus
The pMD19-T vector containing the target gene and the pET-28a (+) vector (Novagen, Germany) were each double-digested with restriction enzymes BamHI and EcoRI (Bao bioengineering, Dalian, Co., Ltd.) at 37 ℃ for 12 hours, the digested products were subjected to 1% agarose gel electrophoresis, and the target gene and the pET-28a (+) vector were recovered by cutting the gel, respectively (the gel recovery kits used in the present invention were purchased from Ningbo Zhongding Biotechnology Co., Ltd.). Connecting the recovered target gene and pET-28a (+) vector at 25 ℃ for 3 hours by using T4 ligase (Bao bioengineering Dalian Co., Ltd.), transforming DH5 alpha competent cells by using the ligation product, coating the cells on an LB plate containing kanamycin resistance (50 mu g/mL), reversely culturing for 12 hours at 37 ℃, selecting a monoclonal strain on the plate to an LB liquid culture medium containing kanamycin resistance (50 mu g/mL), culturing for 12 hours at 37 ℃ by using a shaking table, extracting plasmids by using a plasmid purification kit (the plasmid purification kit used in the invention is purchased from Ningbo Zhongding Biotechnology Co., Ltd.), and performing double enzyme digestion identification by BamHI and EcoRI to obtain a correct recombinant expression vector.
Example 5: construction of recombinant protein expression Strain of Cat parvovirus
Coli ER2566 competent cells were transformed with the constructed recombinant expression vector, spread on a LB plate containing kanamycin resistance (50. mu.g/mL), and cultured overnight at 37 ℃. The next day, the monoclonal strains on the plates are picked to LB liquid culture medium containing kanamycin resistance (50 mug/mL), after shaking culture at the constant temperature of 37 ℃ for 8 hours, 1mL is taken for storage, and the rest is added with an inducer IPTG (isopropylthio-beta-D-galactoside) (the final concentration is 1.0mmol/L) for induction expression for 4 hours to prepare a protein electrophoresis sample. The result of 13% polyacrylamide gel electrophoresis shows that the recombinant protein is successfully expressed, and the recombinant protein expression strain of the feline parvovirus is obtained.
Example 6: purification of recombinant proteins of feline parvovirus
Inoculating a recombinant protein expression strain to an LB liquid culture medium, adding kanamycin to a final concentration of 50 mu g/mL, carrying out shake culture at a constant temperature of 37 ℃ for 8 hours, and then, adding the strain into the LB liquid culture medium containing 50 mu g/mL kanamycin to perform culture in a mode of mixing the strain with the LB liquid culture medium containing 50 mu g/mL kanamycin in a ratio of 1: diluting at a ratio of 100, subpackaging into bacteria culture bottles, shaking-culturing at 37 deg.C until OD600 is 0.8, adding inducer IPTG (isopropylthio-beta-D-galactoside) to final concentration of 1.0mmol/L, and further culturing and inducing for 4 hr. And after the thalli are collected by centrifugation, carrying out low-temperature ultrasonic bacteria breaking, carrying out low-temperature centrifugation, taking the supernatant, passing the supernatant through a nickel-agarose affinity chromatography column, washing and eluting to finally obtain the purified recombinant protein.
Example 7: obtaining of hybridoma cell lines
4-6-week-old female Balb/c mice were taken and basal immunization was performed by subcutaneous multiple injections of 100. mu.g of recombinant protein emulsified in Freund's complete adjuvant. A second booster immunization was performed 20 days later by emulsifying 80ug of recombinant protein in Freund's incomplete adjuvant and injecting subcutaneously at multiple sites. After 15 days, a third booster immunization was performed, and 80. mu.g of the recombinant protein was emulsified with Freund's incomplete adjuvant and injected subcutaneously in multiple spots. Two weeks later, 80ug of recombinant protein was intraperitoneally injected, blood was collected from the orbit 72 hours later, the mice were sacrificed, spleen cells were collected to prepare cell suspension, the cells were counted, sp2/0 mouse myeloma cells in good growth state were collected according to the number of 1/5 in the spleen cells, mixed and centrifuged, and polyethylene glycol (PEG-4000) was added to fuse the two. Equal volume of feeder cells was added, mixed well and plated in 96 well cell plates (200. mu.L/well) and cultured in 5% carbon dioxide incubator. After 5 days, the medium is changed for half, and after 10 days, the hybridoma cell culture supernatant in the 96-well cell culture plate is detected by adopting an indirect enzyme-linked immunosorbent assay. The specific method comprises the following steps:
diluting the recombinant protein by using a coating solution (the final concentration is 1 mu g/mL), adding an enzyme label plate (Stannless Seisaku bioengineering Co., Ltd.) into the diluted recombinant protein at a hole of 100 mu L/hole, coating the recombinant protein at 4 ℃ for 12 hours, and washing the recombinant protein for 1 time by using a washing solution through a DEM-3 type plate washing machine (Daan Gene of university of Zhongshan Co., Ltd.); adding sealing liquid, sealing at 180 μ L/hole at 37 deg.C for 1 hr, and washing plate with plate washing machine for 1 time; adding cell culture supernatant to be detected, positive control serum and negative control sample, incubating at 100 μ L/hole for 35 min at 37 deg.C, and washing with washing solution for 3 times; adding goat anti-mouse IgG labeled with HRP (horseradish peroxidase), incubating at 100. mu.L/well for 35 min at 37 ℃, and washing with washing solution 4 times; adding 50 mu L of color development liquid A and 50 mu L of color development liquid B into each hole, after shading and developing for 10 minutes at 37 ℃, adding stop solution to stop reaction, and reading OD value after zero calibration of blank holes with the wavelength of 450nm of an enzyme labeling instrument at 50 mu L/hole. The immune mouse eye serum is used as a positive control, and the formula of the related solution is as follows:
coating liquid: na (Na)2CO3 1.59g，NaHCO32.93g, and adding ultrapure water to a volume of 1000mL (pH 9.6).
Sealing liquid: na (Na)2HPO4.12H2O 2.68g,NaH2PO4.2H20.39g of O, 8.5g of NaCl, 20g of bovine serum albumin, and double distilled water to 1000mL (pH 7.4).
Washing liquid: na (Na)2HPO4.12H2O 2.68g，NaH2PO4.2H20.39g of O, 8.5g of NaCl, and 200.5 mL of Tween, and adding ultrapure water to the volume of 1000mL (pH7.4).
Color developing solution A: 200mg of TMB is dissolved in 100mL of absolute ethyl alcohol, and ultrapure water is added to the solution to reach the volume of 1000 mL.
Color developing solution B: citric acid 2.1g, Na2HPO4.12H2O71 g, and adding ultrapure water to the solution to make the volume reach 1000 mL.
When in use: 1mL of developing solution A +1mL of developing solution B + 0.4. mu.L of 30% H2O2
Stopping liquid: 2M H2SO421.7mL of concentrated H2SO4Adding ultrapure water to the solution until the volume is 1000 mL.
For positive hybridoma cells, subcloning was performed using limiting dilution. 5 hybridoma cell lines (2C1, 3A6, 5F3, 7B9 and 7D8) were obtained by co-screening through three times of subcloning.
Example 8: mass preparation and purification of monoclonal antibodies
Taking 6-8 week old healthy Balb/c mice, injecting liquid paraffin into abdominal cavity, each 500 μ L, injecting hybridoma cells (about 1 × 10) into abdominal cavity after 5 days6One/one), after 15 days, the mouse abdomen was swollen, ascites was collected, centrifuged at 12000rpm for 2 minutes, and the supernatant was collected as follows: adding 60mmol/L sodium acetate buffer solution with pH4.5 at a ratio of 2.5, adding n-octanoic acid 25 μ L per ml ascites supernatant, slowly adding n-octanoic acid under stirring with a magnetic stirrer, stirring at room temperature for 30min, centrifuging at 4 deg.C and 5000rpm for 30min, and removing precipitate. Precooling the supernatant at 4 ℃ to 1mAdding 0.227g of ammonium sulfate into the supernatant of L volume, slowly adding ammonium sulfate powder while stirring, continuously stirring for 30min at room temperature, centrifuging at 5000rpm for 15min, dissolving the precipitate in 1/10 volume of 10mmol/L PBS (pH7.4) buffer solution, and dialyzing the 10KDa dialysis bag in 10mmol/L PBS (pH7.4) buffer solution until no ammonium sulfate is contained. And purifying the dialyzed monoclonal antibody by Protein A affinity chromatography resin, washing and collecting an elution peak to obtain the purified monoclonal antibody.
Example 9: colloidal gold particle labeled monoclonal antibody
Adding 0.2mol/L potassium carbonate solution 20uL into 10ml of 0.01% colloidal gold solution, fully mixing uniformly, adding 100ug of antibody, reacting at room temperature for 2 hours, adding 10% Bovine Serum Albumin (BSA)1ml, sealing for 2 hours, centrifuging (7500rpm/min, 20 minutes), discarding the supernatant, fully dissolving the precipitate with 1ml of redissolution, uniformly spraying the redissolution onto glass fiber (width 6mm) according to 10uL/cm by using a gold spraying membrane drawing instrument (Shanghai gold mark Biotech Co., Ltd.), and placing in an electric heating constant temperature incubator (Shanghai one constant scientific apparatus Co., Ltd.) at 37 ℃ for standing for 2 hours.
The 2C1, 3A6, 5F3, 7B9 and 7D8 mabs were labeled with colloidal gold using the methods described above, respectively. The relevant solution formulation is as follows:
0.01% colloidal gold solution: 1ml of 1% chloroauric acid solution, 1.4ml of 1% trisodium citrate solution, adding ultrapure water, heating to dissolve, reacting and fixing the volume to 100 ml.
1% chloroauric acid solution: 1g AuCL3.HCl.4H2And dissolving the O powder by adding ultrapure water and fixing the volume to 100 ml.
1% trisodium citrate solution: 1g of trisodium citrate is dissolved by adding ultrapure water and the volume is adjusted to 100 ml.
Compounding the solution: 6.057g of Tris base is dissolved in 800ml of double distilled water, the pH value is adjusted to 8.0 by using a proper amount of HCL, and the double distilled water is added to reach the constant volume of 1000 ml.
Example 10: screening of paired monoclonal antibodies
The feline parvovirus monoclonal antibodies (2C1, 3A6, 5F3, 7B9, 7D8) were diluted with the coating solution (final concentration of 1mg/ml) respectively, and were uniformly coated on a nitrocellulose membrane (Sartorius) at 1ul/cm by a gold spraying and streaking instrument (Shanghai gold-labeled Biotech Co., Ltd.), which was taken as the T line. The goat anti-mouse solution (final concentration of 1mg/ml) was uniformly coated on the nitrocellulose membrane as line C by a gold spraying and streaking instrument (Shanghai gold-labeled Biotech Co., Ltd.) at 1 ul/cm. After the completion of the membrane scribing, the nitrocellulose membrane was left to stand at 37 ℃ for 12 hours in an electric-heating constant-temperature incubator (Shanghai-Hengyun scientific instruments Co., Ltd.).
And (3) sequentially assembling the sample pad, the glass fiber, the nitrocellulose membrane and the filter paper on a PVC (polyvinyl chloride) base plate according to a conventional process, cutting the assembly into strips with the width of 4mm, installing a reagent clamping strip shell and compacting the strips.
FPV positive serum samples and normal cat serum samples are loaded at 100 muL/hole, and after being placed for 15min at room temperature, the FPV positive serum samples and the normal cat serum samples are respectively read by a colloidal gold chromatography reading instrument (Shanghai Jihao scientific instruments Co., Ltd.) and the P/N value (the ratio of the positive sample detection value to the negative sample detection value) is calculated, which is detailed in Table 1.
The coated monoclonal antibodies and the colloidal gold-labeled monoclonal antibodies were subjected to orthogonal detection by the above-mentioned method, and the P/N value (the ratio of the mean value of the positive sample detection to the mean value of the negative sample detection) was obtained, as shown in Table 1.
TABLE 1 statistical P/N values of paired monoclonal antibodies
As can be seen from the above table, the optimal combination of the 3A6 monoclonal antibody coating and the 7B9 colloidal gold label pair for detecting feline parvovirus is shown.
SEQUENCE LISTING
<110> Hangzhou xian Zhi Biotechnology Co., Ltd
<120> preparation of recombinant protein of feline parvovirus and monoclonal antibody thereof
<130> 20191219
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 112
<212> PRT
<213> Artificial sequence (Artificial)
<400> 1
Asp Arg Thr Leu Ile Pro Ser His Thr Gly Gly Gly Gly Gly Asp Pro
1 5 10 15
Arg Tyr Ala Phe Gly Arg Gln His Gly Gly Gly Gly Gly Asp Arg Thr
20 25 30
Leu Ile Pro Ser His Thr Gly Gly Gly Gly Gly Asp Pro Arg Tyr Ala
35 40 45
Phe Gly Arg Gln His Gly Gly Gly Gly Gly Asp Arg Thr Leu Ile Pro
50 55 60
Ser His Thr Gly Gly Gly Gly Gly Asp Pro Arg Tyr Ala Phe Gly Arg
65 70 75 80
Gln His Gly Gly Gly Gly Gly Asp Arg Thr Leu Ile Pro Ser His Thr
85 90 95
Gly Gly Gly Gly Gly Asp Pro Arg Tyr Ala Phe Gly Arg Gln His Gly
100 105 110
<210> 2
<211> 9
<212> PRT
<213> Feline parvovirus (Feline Parvovirus)
<400> 2
Asp Arg Thr Leu Ile Pro Ser His Thr
1 5
<210> 3
<211> 12
<212> PRT
<213> Feline parvovirus (Feline Parvovirus)
<400> 3
Gly Asp Pro Arg Tyr Ala Phe Gly Arg Gln His Gly
1 5 10
<210> 4
<211> 336
<212> DNA
<213> Artificial sequence (Artificial)
<400> 4
gatcgcaccc tgattccgag ccataccggc ggcggcggcg gcgatccgcg ctatgccttt 60
ggccgccagc atggcggcgg cggcggcgat cgcaccctga ttccgagcca taccggcggc 120
ggcggcggcg atccgcgcta tgcctttggc cgccagcatg gcggcggcgg cggcgatcgc 180
accctgattc cgagccatac cggcggcggc ggcggcgatc cgcgctatgc ctttggccgc 240
cagcatggcg gcggcggcgg cgatcgcacc ctgattccga gccataccgg cggcggcggc 300
ggcgatccgc gctatgcctt tggccgccag catggc 336
Claims (4)
1. The feline parvovirus recombinant protein is characterized by being shown in a sequence table SEQ ID No: 1.
2. A polynucleotide, which has the nucleotide sequence shown in SEQ ID No. 4 of the sequence table and can encode the feline parvovirus recombinant protein of claim 1.
3. A plasmid vector comprising the polynucleotide of claim 2.
4. A strain transformed with the plasmid vector of claim 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911334829.4A CN110845582B (en) | 2019-12-23 | 2019-12-23 | Preparation of feline parvovirus recombinant protein and monoclonal antibody thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911334829.4A CN110845582B (en) | 2019-12-23 | 2019-12-23 | Preparation of feline parvovirus recombinant protein and monoclonal antibody thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110845582A CN110845582A (en) | 2020-02-28 |
| CN110845582B true CN110845582B (en) | 2021-07-23 |
Family
ID=69610244
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911334829.4A Active CN110845582B (en) | 2019-12-23 | 2019-12-23 | Preparation of feline parvovirus recombinant protein and monoclonal antibody thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110845582B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112553168B (en) * | 2020-12-23 | 2022-03-08 | 江苏省农业科学院 | Hybridoma cell 3A6 strain secreting monoclonal antibody against feline parvovirus VP2 protein and application thereof |
| CN114703204A (en) * | 2022-02-24 | 2022-07-05 | 深圳赫兹生命科学技术有限公司 | Feline parvovirus VP2 protein and resulting self-assembled virus-like particles |
| CN117700535B (en) * | 2024-02-05 | 2024-04-26 | 北京纳百生物科技有限公司 | Cat-panleukopenia virus VP2 protein monoclonal antibody and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001066568A2 (en) * | 2000-03-09 | 2001-09-13 | Heska Corporation | Use of recombinant antigens to determine the immune status of an animal |
| CN107254451A (en) * | 2010-07-16 | 2017-10-17 | 英特维特国际股份有限公司 | The parvovirus of attenuation living |
| CN109136198A (en) * | 2018-08-21 | 2019-01-04 | 华南农业大学 | A kind of expression Chicken Infectious Anemia Virus VP1, VP2 genetic recombination bird pox virus live vector vaccine |
-
2019
- 2019-12-23 CN CN201911334829.4A patent/CN110845582B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001066568A2 (en) * | 2000-03-09 | 2001-09-13 | Heska Corporation | Use of recombinant antigens to determine the immune status of an animal |
| CN107254451A (en) * | 2010-07-16 | 2017-10-17 | 英特维特国际股份有限公司 | The parvovirus of attenuation living |
| CN109136198A (en) * | 2018-08-21 | 2019-01-04 | 华南农业大学 | A kind of expression Chicken Infectious Anemia Virus VP1, VP2 genetic recombination bird pox virus live vector vaccine |
Non-Patent Citations (4)
| Title |
|---|
| "CAC88561.1";Jensen,W.A.;《Genbank》;20010928;全文 * |
| "CAA01829.1";佚名;《Genbank》;19950919;全文 * |
| "Molecular characterization of canine parvovirus in Vientiane,Laos";Soulasack Vannamahaxay等;《Arch Virol》;20170531;第162卷(第5期);全文 * |
| "猫细小病毒非结构蛋白基因克隆及抗原表位预测";黎明等;《黑龙江畜牧兽医》;20140210(第3期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110845582A (en) | 2020-02-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110845582B (en) | Preparation of feline parvovirus recombinant protein and monoclonal antibody thereof | |
| CN113150136B (en) | Preparation of novel coronavirus N protein monoclonal antibody | |
| CN111196857A (en) | Novel coronavirus multi-epitope recombinant antigen and preparation method thereof | |
| CN105542014B (en) | TP recombinant antigen and preparation method and application thereof | |
| CN114276445B (en) | Rotavirus recombinant protein specific antibody, plasmid vector and method | |
| CN112500479B (en) | Preparation of canine II type adenovirus recombinant protein monoclonal antibody | |
| CN109111507B (en) | Virus recombinant glycoprotein and eukaryotic cell high-efficiency expression method and application thereof | |
| CN109679970B (en) | Preparation method for rapidly detecting feline herpes type I virus | |
| CN110951703B (en) | Plasmodium vivax lactate dehydrogenase recombinant protein and preparation of monoclonal antibody thereof | |
| CN110903359B (en) | Preparation of campylobacter jejuni recombinant protein and monoclonal antibody thereof | |
| CN105906716B (en) | Preparation and application of echovirus 9 type VP1 protein specific epitope and fusion protein thereof | |
| CN110981969B (en) | ALV-K ELISA kit and detection method thereof | |
| CN112521461B (en) | Preparation of hepatitis A virus recombinant protein and rapid detection method thereof | |
| CN107586337B (en) | Preparation method of mouse anti-human KIAA0100 protein monoclonal antibody | |
| CN114409804B (en) | Escherichia coli enterotoxin multi-epitope fusion protein and preparation method and application thereof | |
| CN116333060B (en) | Porcine reproductive and respiratory syndrome virus GP5 protein, preparation method and application thereof, gene, kit and detection method | |
| CN108753734B (en) | Anti-tree shrew CD8 molecular monoclonal antibody, hybridoma cell strain secreting antibody and application | |
| CN111978382B (en) | Preparation method and application of recombinant protein of Sporothrix globosum Gp70 | |
| CN110054688B (en) | anti-TTC 36 monoclonal antibody and application thereof | |
| CN110054675B (en) | Immunogenic polypeptide, anti-TTC 36 antibody CP4-3 and application | |
| CN114085274B (en) | FIPV N recombinant protein and colloidal gold test strip for rapidly detecting FIPV infection | |
| CN111909949B (en) | Preparation method and application of recombinant protein HSP70_5 of Sporothrix globosum | |
| CN112920271A (en) | Polyclonal antibody of cabbage type rape drought-resistant gene BnatZF1A and preparation method thereof | |
| CN117903298A (en) | Porcine parvovirus 7-type Cap protein monoclonal antibody, preparation method and application | |
| CN113045674A (en) | Human adenovirus five serotype universal antigen epitope fusion protein and preparation and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |