CN114853912A - Bovine viral diarrhea virus E2-E0 fusion protein, preparation method and application - Google Patents
Bovine viral diarrhea virus E2-E0 fusion protein, preparation method and application Download PDFInfo
- Publication number
- CN114853912A CN114853912A CN202210571495.8A CN202210571495A CN114853912A CN 114853912 A CN114853912 A CN 114853912A CN 202210571495 A CN202210571495 A CN 202210571495A CN 114853912 A CN114853912 A CN 114853912A
- Authority
- CN
- China
- Prior art keywords
- gly
- bovine viral
- viral diarrhea
- diarrhea virus
- ala
- 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.)
- Granted
Links
Images
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
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
- C12N2770/24322—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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
- C12N2770/24334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Virology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention belongs to the technical field of biology, and relates to bovine viral diarrhea virus E2-E0 fusion protein, a preparation method and application thereof. The invention firstly provides a method for fusing bovine viral diarrhea virus E0 truncated protein with bovine viral diarrhea virus E2 protein, which can promote the expression of E0 protein, does not influence the immunogenicity of E0 protein, and can be used for preparing a bovine viral diarrhea subunit vaccine; on the basis of the bovine viral diarrhea virus E0 truncated protein, the invention discovers that the expression level of the E2-E0 fusion protein can be further improved by more than 18 percent by mutating the amino acid at the position 160-162 of the bovine viral diarrhea virus E0 truncated protein from IAA to GGG, and the immunogenicity of the fusion protein is not influenced.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to bovine viral diarrhea virus E2-E0 fusion protein, a preparation method and application thereof.
Background
Bovine Viral Diarrhea (BVD) is an acute, febrile, highly contagious disease mainly characterized by diarrhea, reproductive disorders, immune dysfunction, etc. caused by Bovine Viral Diarrhea Virus (BVDV). Infection of cattle with BVDV results in decreased productivity, retarded growth, decreased milk production, and increased susceptibility to other diseases, resulting in early slaughter of animals. The disease has high morbidity, and the prevention and treatment difficulty lies in that the BVDV can cause immunosuppression and persistent infection after entering an organism, reduces the immunity of the organism, is easy to induce mixed infection or secondary infection of other pathogens, seriously influences the health state and the production performance of cattle flocks, and is one of the most important cattle infectious diseases in the world.
BVDV belongs to the flaviviridae (flaviviridae) Pestivirus genus (Pestivirus) and consists of a single-stranded positive sense RNA molecule of about 12.3kb in length on the genome. BVDV has 3 biotypes, usually based on 5' -UTR sequences, a genomic amino-terminal autoprotease (N) pro ) Or envelope glycoprotein (E2) region, into BVDV-1 and BVDV-2 types, BVDV-3 type being described as atypical BVDV. BVDV-1 type is further divided into 21 subtypes (1a to 1u), and BVDV-2 type is divided into 3 subtypes (2a to 2 c). At present, 8 subtypes are mainly prevalent in China: 1a, 1b, 1c, 1d, 1m, 1o, 1p and 1 q. The BVDV genome encodes 4 structural proteins, P14(C), gP48(E0), gP25(E1), gP53(E2), and the others are all non-structural proteins. Wherein E0, E1 and E2 form a virus envelope structure and are positioned on the surface of the virus.
Currently, there are two main approaches to controlling BVDV spread: eradicate persistent infection animals and vaccinate. Modified live or inactivated vaccines are mainly used in BVDV vaccination programs, but these vaccines have problems of biosafety risk or insufficient immune protective efficacy, respectively. The inactivated vaccine is safe for pregnant cows, but the immune period is short; although the attenuated vaccine has long immune period, the attenuated vaccine has safety risk to pregnant cows. Based on this, researchers have long been working on the development of BVD subunit vaccines. The E0 protein can induce organisms to generate virus neutralizing antibodies, is an important protective antigen of BVDV, and is also a candidate antigen for development of BVD subunit vaccines. The E0 protein has multiple glycosylation sites in the amino acid sequence and has important effect on maintaining the antigenicity. When E0 is prepared by prokaryotic Escherichia coli, the product exists in an insoluble inclusion body form, and lacks glycosylation modification, so that the immunogenicity of the antigen protein is seriously influenced; when E0 is prepared by eukaryotic CHO cells with complete glycosylation modification function, the target protein is not expressed or the expression level is extremely low, so that the research and development of BVD subunit vaccines are seriously hindered.
Disclosure of Invention
In order to solve the technical problem that the BVDV E0 protein is low in expression amount or not expressed in CHO cells, a great deal of research shows that the BVDV E0 protein can promote the expression of BVDV E0 in CHO cells after being truncated and fused with BVDV E2 protein, does not affect the expression of E2 protein and the immunogenicity of E0 protein and E2 protein, can be used for preparing BVD subunit vaccines, and specifically comprises the following contents:
in a first aspect, the invention provides a bovine viral diarrhea virus E2-E0 fusion protein, wherein the fusion protein comprises a bovine viral diarrhea virus E0 truncated protein and a bovine viral diarrhea virus E2 protein; the amino acid sequence of the truncated protein of the bovine viral diarrhea virus E0 is shown in SEQ ID NO. 1; the amino acid sequence of the bovine viral diarrhea virus E2 protein is shown in SEQ ID NO. 3.
Preferably, the nucleotide sequence for coding the truncated protein of the bovine viral diarrhea virus E0 is shown as SEQ ID NO. 2; the amino acid sequence of the protein E2 for encoding the bovine viral diarrhea virus is shown in SEQ ID NO. 4.
Preferably, the amino acid sequence of the bovine viral diarrhea virus E2-E0 fusion protein is shown as SEQ ID NO. 5.
Preferably, the gene sequence of the bovine viral diarrhea virus E2-E0 fusion protein is shown as SEQ ID NO. 6.
Preferably, the amino acid 160-162 of the bovine viral diarrhea virus E0 protein fragment is mutated from IAA to GGG; the amino acid sequence of the mutated bovine viral diarrhea virus E0 truncated protein is shown in SEQ ID NO. 7.
Preferably, the gene sequence of the mutant encoding the truncated protein of the bovine viral diarrhea virus E0 is shown as SEQ ID NO. 8.
Preferably, the amino acid sequence of the bovine viral diarrhea virus E2-E0 fusion protein is shown as SEQ ID NO. 9.
Preferably, the gene sequence of the bovine viral diarrhea virus E2-E0 fusion protein is shown as SEQ ID NO. 10.
In a second aspect, the invention provides an application of the bovine viral diarrhea virus E2-E0 fusion protein in preparing a bovine viral diarrhea virus vaccine.
Preferably, the vaccine is a subunit vaccine.
In a third aspect, the present invention provides a method for preparing the bovine viral diarrhea virus E2-E0 fusion protein of the first aspect, wherein the method comprises: genes for coding the bovine viral diarrhea virus E0 truncated protein and the bovine viral diarrhea virus E2 protein are connected in series and then cloned into a eukaryotic expression vector to obtain a recombinant plasmid for expressing the bovine viral diarrhea virus E0 truncated protein and the bovine viral diarrhea virus E2 protein; and transfecting the recombinant plasmid into a CHO cell, and culturing, screening and purifying to obtain the bovine viral diarrhea virus E2-E0 fusion protein.
Preferably, the eukaryotic expression vector is pcDNA3.1 vector.
Preferably, the CHO cells are CHO suspension cells.
Preferably, the method is:
(1) PCR amplifying a gene segment of a fusion protein of the bovine viral diarrhea virus E2-E0, wherein the gene segment is shown as SEQ ID NO.6 or SEQ ID NO. 10;
(2) carrying out double enzyme digestion on gene fragments of the vector pcDNA3.1 and the bovine viral diarrhea virus E2-E0 fusion protein by using restriction endonucleases Xho I and Hind III respectively, and connecting the enzyme digestion fragments by using DNA ligase to obtain a recombinant plasmid pcDNA3.1-BVDV-E2-E0 for expressing the bovine viral diarrhea virus E2-E0 fusion protein;
(3) transfecting the recombinant plasmid pcDNA3.1-BVDV-E2-E0 to CHO suspension cells, performing suspension culture, collecting cell culture supernatant, and purifying to obtain the bovine viral diarrhea virus E2-E0 fusion protein.
The invention has the beneficial effects that: when E0 is prepared by prokaryotic escherichia coli, the product exists in an insoluble inclusion body form, glycosylation modification is lacked, and the immunogenicity of antigen protein is seriously influenced; when E0 is prepared by eukaryotic CHO cells with complete glycosylation modification function, the target protein is not expressed or the expression level is extremely low, so that the research and development of BVD subunit vaccines are seriously hindered; the invention unexpectedly discovers that the protein E0 obtained by truncating the bovine viral diarrhea virus E0 protein is fused with the bovine viral diarrhea virus E2 protein, so that the expression of the E0 protein can be promoted, the immunogenicity of the original E0 protein and the original E2 protein is not influenced, and the BVD subunit vaccine can be prepared; based on the truncated protein of the bovine viral diarrhea virus E0, the invention discovers that the expression of the truncated protein of the bovine viral diarrhea virus E0 is further promoted by mutating the amino acid at the 160-162 th site of the protein of the bovine viral diarrhea virus E0 from IAA to GGG, the expression level of the protein is improved by more than 18 percent, and the immunogenicity of the protein is not influenced.
Drawings
FIG. 1 shows the expression and identification results of bovine viral diarrhea virus E2 protein;
FIG. 2 shows the purification result of bovine viral diarrhea virus E2 protein;
FIG. 3 shows the expression results of the E0 protein, the E0 truncated protein and the E0 truncated protein with mutated amino acids of the bovine viral diarrhea virus;
FIG. 4 shows the purification results of truncated protein of bovine viral diarrhea virus E0;
FIG. 5 the purification results of the E0 truncated protein of bovine viral diarrhea virus amino acid mutation;
FIG. 6 shows the expression identification results of truncated fusion protein of bovine viral diarrhea virus E2-E0;
FIG. 7 the purification results of truncated fusion protein of bovine viral diarrhea virus E2-E0;
FIG. 8 shows the expression identification results of truncated mutant fusion proteins of bovine viral diarrhea virus E2-E0; wherein, 1 is the expression identification of E0 truncated mutation; 2, expression identification is carried out after truncation mutation of E2-E0;
FIG. 9 the purification results of the truncated mutant fusion protein of bovine viral diarrhea virus E2-E0;
FIG. 10 shows the result of detection of antibodies in serum from immunized rabbit by indirect ELISA.
Detailed Description
The above-described scheme is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes and are not intended to limit the scope of the present invention. The conditions used in the examples may be further adjusted according to the conditions of the particular manufacturer, and the conditions not specified are generally the conditions in routine experiments.
The experiments described in the following examples obtain biosafety permits and foot and mouth disease laboratory activity permits:
according to the related requirements of biological safety 3-level laboratory (BSL-3) and related biological safety of bovine viral diarrhea disease, the Lanzhou veterinary research institute of the Chinese agricultural science institute reports the permission of the biological safety committee of the Lanzhou veterinary research institute, the ethical committee of experimental animals, the biological safety committee of the Chinese agricultural science institute, the ethical committee of experimental animals of the Lanzhou veterinary research institute and the biological safety committee of the Lanzhou veterinary research institute step by step, and the Lanzhou veterinary research institute records the permission and meets the requirements of the national biological safety level.
The standard strain of BVDV-NADL vaccine (purchased from China institute of veterinary medicine-China center for veterinary culture Collection of microorganisms), eukaryotic expression vector pcDNA3.1(+), and BVDV positive serum are preserved in the laboratory. CHO suspension cell expression system, Unstained protein MW marker (26610), RT-PCR amplification kit, 6 XHis Tag Monoclonal Antibody (HIS. H8), Unstained protein MW marker (26616), and BCA protein quantification kit, Alexa
488-labeled secondary goat anti-rabbit antibodies were purchased from Thermo Fisher Scientific, inc. Affinity chromatography Ni columns were purchased from GE. The RNA extraction kit and the DNA gel purification recovery kit are purchased from OMEGA GmbH. Xho I and Hind III restriction enzymes were purchased from New England Biolabs (NEB). Coli DH 5. alpha. competent cells were purchased from Hokko gold. A large number of plasmid extraction kits were purchased from MACHEREY-NAGEL. ECL color developing solutions were purchased from shanghai bi yunnan biotechnology limited. MD44 dialysis bags with a cut-off molecular weight of 8000-.
The term "vector" refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be transformed, transduced or transfected into a host cell to obtain expression of the genetic material element carried by the vector in the host cell. By way of example, the carrier includes: a plasmid; bacteriophage; cosmids, etc.
The term "vaccine" refers to a biological agent capable of providing a protective response in an animal, wherein the vaccine has been delivered and is not capable of causing serious disease. The vaccines of the present invention are genetically engineered subunit vaccines.
The vaccine of the present invention, further optionally comprises one or more adjuvants, excipients, carriers and diluents. The adjuvant can be any suitable adjuvant, chemical immune adjuvants such as aluminum hydroxide, Freund's adjuvant, mineral oil, span, etc.; microbial immune adjuvants such as mycobacteria, BCC, lipopolysaccharide, muramyl dipeptide, cytopeptide, fat-soluble waxy D, and short corynebacterium; the plant immunologic adjuvant is polysaccharides extracted from plant or large fungi, such as pachyman, carthamus tinctorius polysaccharide, Chinese herbal medicine, etc. And biochemical immune adjuvants such as thymosin, transfer factor, interleukin, etc. Preferred adjuvants may be nano-adjuvant biological adjuvants, interleukins, interferons, etc.
The vaccines of the present invention may also be used in combination vaccines, such as with other vaccines in pigs or cattle, but emphasis is placed on live attenuated vaccines, particularly in the integration of viral genes, such as bivalent, trivalent, etc.
The administration of the vaccines of the present invention may be by any convenient route, for example, intramuscular injection, intranasal, oral, subcutaneous, transdermal and vaginal routes. The vaccine may be administered after a prime-boost regimen. For example, after a first vaccination, the subject may receive a second booster administration after a period of time (e.g., about 7, 14, 21, or 28 days). Typically, the booster is administered at the same or a lower dose than the prime dose. In addition, a third booster immunization may be performed, for example 2-3 months, 6 months or a year after immunization.
Example 1 preparation of bovine viral diarrhea Virus E2-E0 truncated fusion protein and E2-E0 truncated mutant fusion protein
1. Gene fragment synthesis
Gene segments for coding bovine viral diarrhea virus E0 protein, E0 truncated protein, E0 truncated mutant protein, E2 protein, E2-E0 truncated fusion protein and E2-E0 truncated mutant fusion protein are synthesized by Beijing Liuhe Hua Dagenescience and technology Limited company, and are identified correctly by nucleic acid gel electrophoresis. Wherein, the gene segment of the protein of the encoding bovine viral diarrhea virus E0 is shown as SEQ ID NO.12, and the amino acid sequence is shown as SEQ ID NO. 11; the gene segment of the truncated protein of the encoding bovine viral diarrhea virus E0 is shown as SEQ ID NO.2, and the amino acid sequence is shown as SEQ ID NO. 1; the gene segment of the encoding bovine viral diarrhea virus E0 truncated mutation is shown as SEQ ID NO.8, the amino acid sequence is shown as SEQ ID NO.7, and the E0 truncated mutant protein means that the amino acids at the 160-162 th position of the E0 truncated protein are mutated from IAA to GGG; the gene segment of the protein of the encoding bovine viral diarrhea virus E2 is shown as SEQ ID NO.4, and the amino acid sequence is shown as SEQ ID NO. 3; the gene segment of the truncated fusion protein of the encoding bovine viral diarrhea virus E2-E0 is shown as SEQ ID NO.6, and the amino acid sequence is shown as SEQ ID NO. 5; the gene segment of the truncated mutant fusion protein of the encoding bovine viral diarrhea virus E2-E0 is shown as SEQ ID NO.10, and the amino acid sequence is shown as SEQ ID NO. 9.
2. Construction and characterization of expression plasmids
The recombinant plasmids are named pcDNA3.1-BVDV-E0 (full length of E0 protein), pcDNA3.1-BVDV-tE0(E0 truncated protein), pcDNA3.1-BVDV-mE0(E0 truncated protein mutation), pcDNA3.1-BVDV-E2(E2 protein), pcDNA3.1-BVDV-E2-tE0(E2-E0 truncated fusion protein), pcDNA3.1-BVDV-E2-mE0(E2-E0 truncated mutant fusion protein), and are synthesized by Beijing Heihua Dagenescience and technology Limited, and specifically comprise: the pcDNA3.1 vector is subjected to double enzyme digestion by restriction enzymes Xho I and Hind III, and the vector fragment is recovered and purified by cutting glue; simultaneously, carrying out double digestion on the recovered and purified E0 protein gene fragment, the E0 truncated protein gene fragment, the E0 truncated protein mutant gene fragment, the E2 protein fragment, the E2-E0 truncated fusion protein gene fragment and the E2-E0 truncated mutant fusion protein gene fragment by using restriction endonucleases Xho I and Hind III respectively, recovering and purifying the DNA fragment, then connecting the DNA fragment with the purified pcDNA3.1(+) vector fragment through T4 DNA ligase at the program of 16 ℃ for 12h, respectively transforming 58DH 5 alpha competent cells by the connecting products, picking up single colony enrichment culture, extracting a recombinant plasmid by using a plasmid extraction kit, wherein the recombinant plasmid is named as pcDNA3.1-BV-E0, pcDNA3.1-BVDV-tE0, pcDNA3.1-BVDV-mE0, pcDNA3.1-BVDV-E2, pcDNA3.1-BVDV-E-2-BVtE 6, pcDNA3.1-BVDV-3527-0; and carrying out double enzyme digestion identification on the recombinant plasmid by using Xho I and Hind III, carrying out sequencing identification on the recombinant plasmid with correct double enzyme digestion identification, determining the correct insertion of a target gene sequence, and ensuring that a reading frame is completely correct.
The size of the enzyme digestion product is consistent with the theoretical value after the constructed recombinant expression plasmids pcDNA3.1-BVDV-E0, pcDNA3.1-BVDV-tE0, pcDNA3.1-BVDV-mE0, pcDNA3.1-BVDV-E2, pcDNA3.1-BVDV-E2-tE0 and pcDNA3.1-BVDV-E2-mE0 are subjected to mutation fusion and are subjected to Xho I and Hind III double enzyme digestion, which shows that the amplified E0 fragment, E0 truncated fragment, E0 truncated mutant fragment, E2 fragment, E2-E0 truncated fragment and E2-E0 truncated mutant fragment are correctly connected with the vector, and the sequencing gene result also shows that the target fragment is successfully inserted into the expression vector.
3. Expression and purification of proteins
With reference to the ExpicHO expression system kit, the recombinant plasmids pcDNA3.1-BVDV-E0, pcDNA3.1-BVDV-tE0, pcDNA3.1-BVDV-mE0, pcDNA3.1-BVDV-E2, pcDNA3.1-BVDV-E2-tE0 and pcDNA3.1-BVDV-E2-mE0 were transfected into CHO suspension cells, respectively, and the transfected cells were transfected at 32 ℃ and 5% CO 2 And after suspension culture is continued for 12 days under the condition of humidity of 90%, centrifuging at 4000g for 10min, and collecting cell culture supernatant for SDS-PAGE identification. The cell culture supernatant collected by centrifugation was filtered through a 0.22 μm filter and purified according to the instructions of the GE affinity chromatography Ni column. The purified target protein was identified by SDS-PAGE, and the protein concentration was determined using BCA protein quantification kit.
And (3) centrifuging to collect cell culture supernatants of the transfected pcDNA3.1-BVDV-E0 plasmid, pcDNA3.1-BVDV-tE0 plasmid, pcDNA3.1-BVDV-mE0 plasmid, pcDNA3.1-BVDV-E2 plasmid, pcDNA3.1-BVDV-E2-tE0 plasmid and pcDNA3.1-BVDV-E2-mE0 plasmid, and performing SDS-PAGE identification. Purification was performed according to the instructions of GE affinity chromatography Ni column, the expressed protein supernatant was filtered and then loaded, the eluted sample was identified by SDS-PAGE, and the protein concentration was determined using BCA protein quantification kit.
As shown in FIG. 1, the soluble expression identification result of bovine viral diarrhea virus E2 protein shows that a specific protein band is observed between 45 kD and 66.2kD in the cell culture supernatant transfected with pcDNA3.1-BVDV-E2 plasmid, which indicates that the E2 protein is expressed; the SDS-PAGE identification of the purified samples is shown in FIG. 2: a specific protein band was observed at 45-66.2kD, consistent with the identification of cell supernatants, and the concentration of E2 protein after purification was 1.15mg/mL as determined by the BCA protein quantification kit.
The soluble expression identification results of the bovine viral diarrhea virus E0 protein, the tE0 protein and the mE0 protein are shown in FIG. 3, and a specific protein band is not observed in the cell culture supernatant of the pcDNA3.1-BVDV-E0 plasmid at about 30kD, which indicates that the E0 protein is not substantially expressed; and specific protein bands are observed at about 30kD in the cell culture supernatants transfected by the pcDNA3.1-BVDV-tE0 plasmid and the pcDNA3.1-BVDV-mE0 plasmid, which indicates that the tE0 protein and the mE0 protein can be expressed; the SDS-PAGE identification result of the purified sample of the bovine viral diarrhea virus tE0 protein is shown in FIG. 4: a specific protein band is observed at 25-35kD, and the concentration of the purified tE0 protein fragment is 0.35mg/mL according to the identification result of cell supernatant by a BCA protein quantitative kit; the SDS-PAGE identification result of the purified sample of the bovine viral diarrhea virus mE0 protein is shown in figure 5: a specific protein band was observed at 25-35kD, consistent with the identification of cell supernatants, and the BCA protein quantification kit determined the concentration of mE0 mutein after purification to be 0.47 mg/mL.
The soluble expression and identification results of the bovine viral diarrhea virus E2-tE0 fusion protein are shown in FIG. 6, and a specific protein band is observed in the cell culture supernatant of the pcDNA3.1-BVDV-E2-tE0 fusion protein plasmid at about 66.2-116kD, which indicates that the E2-tE0 fusion protein is expressed; the SDS-PAGE identification result of the purified sample of the bovine viral diarrhea virus E2-tE0 fusion protein is shown in FIG. 7: a specific protein band was observed at 66.2-116kD, consistent with the identification of cell supernatants, and the BCA protein quantification kit determined the concentration of E2-tE0 fusion protein to be 1.04mg/mL after purification.
The soluble expression and identification results of the bovine viral diarrhea virus E2-mE0 fusion protein are shown in FIG. 8, and a specific protein band is observed in the cell culture supernatant of the pcDNA3.1-BVDV-E2-mE0 transfected fusion plasmid at about 66.2-116kD, which indicates that the E2-mE0 fusion protein is expressed; the SDS-PAGE identification result of the purified sample of the bovine viral diarrhea virus E2-mE0 fusion protein is shown in FIG. 9: a specific protein band was observed at 66.2-116kD, consistent with the identification of cell supernatants, and the BCA protein quantification kit determined the concentration of E2-mE0 fragment mutant fusion protein to be 1.23mg/mL after purification.
Example 2 immunogenicity assays
And detecting the immune rabbit serum of the mE0 protein, the E2 protein, the E2-tE0 fusion protein and the E2-mE0 fusion protein by adopting indirect ELISA, and analyzing the immunogenicity of the immune rabbit serum. Purified BVDV-mE0 protein, BVDV-E2 protein, BVDV-E2-tE0 fusion protein, BVDV-E2-mE0 fusion protein (50 ng/well) were coated separately in ELISA plates with 50mM carbonate buffer and incubated overnight at 4 ℃. Blocking was performed with 1% Bovine Serum Albumin (BSA) at 37 ℃ for 2 h. Diluting the serum to be detected with serum diluent according to a ratio of 1:2000, adding the diluted serum to the reaction wells at a rate of 100 mu L/well, setting 3 multiple wells for each sample, and incubating for 1h at 37 ℃. HRP-labeled goat anti-rabbit IgG antibody is diluted with serum diluent at a ratio of 1:5000, added to reaction wells, 100. mu.L/well, and incubated at 37 ℃ for 1 h. TMB was developed at 50. mu.L/well for 10min, and then 50. mu.L of stop buffer was added to terminate the reaction, and the OD450nm value was read.
The indirect ELISA results are shown in FIG. 10, and the antibody in the serum can be detected 7 days after rabbit serum is immunized with mE0 protein, E2 protein, E2-tE0 fusion protein and E2-mE0 fusion protein, the antibody level is obviously increased 7 days after secondary immunization, and the antibody level is kept at a higher level 28 days after immunization. Compared with an inactivated BVDV antigen group, the antibody level of the BVDV-mE0 protein, the BVDV-E2 protein, the BVDV-E2-tE0 fusion protein and the BVDV-E2-mE0 fusion protein is obviously increased after immunization; compared with the BVDV-mE0 protein and the BVDV-E2 protein, the BVDV-E2-tE0 fusion protein and the BVDV-E2-mE0 fusion protein further increase the antibody level after immunization; the results prove that the fusion protein expressed by CHO cells and containing the E2-mE0 fragment and the mutant fusion protein have good immunogenicity.
Sequence listing
<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences
<120> bovine viral diarrhea virus E2-E0 fusion protein, preparation method and application
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 166
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Gly Gly Ala Ile Thr Gly Thr Ala Leu Gly Ala Ala Gly Thr Gly Gly
1 5 10 15
Ile Gly Ala Ala Met Pro Gly Ala Gly Val Ala Ala Ser Leu His Gly
20 25 30
Ile Thr Pro Gly Leu Ile Cys Thr Gly Val Pro Ser His Leu Ala Thr
35 40 45
Ala Met Gly Leu Leu Ala Ile His Gly Met Met Ala Ala Ser Gly Leu
50 55 60
Thr Ala Thr Thr Cys Cys Ala Leu Gly Ala His Gly Thr Ala Leu His
65 70 75 80
Gly Thr Cys Ala Thr Thr Ala Ile Gly Pro Thr Val Leu Ile Met Ala
85 90 95
Ala Thr Gly Ala Ala Leu Thr Gly Gly Gly Pro Pro Ala Gly Cys Ala
100 105 110
Val Thr Cys Ala Thr Ala Ala Ala Ala Ala Ile Ala Val Val Thr Gly
115 120 125
Ala Ala Ala Ala Pro Thr Leu Leu Thr Gly Cys Leu Leu Gly Leu Ala
130 135 140
Pro Ser Pro Ala Gly Ile Leu Met Gly Gly Pro Cys Ala Pro Gly Ile
145 150 155 160
Ala Ala Ser Ala Val Leu
165
<210> 2
<211> 498
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ggtgaaaaca taacacagtg gaacttgcaa gataatggga cagaagggat acaacgggcg 60
atgttccaaa ggggcgtgaa taggagccta catggaatct ggccagagaa aatctgtaca 120
ggtgtaccat cccatctagc cactgatatg gaattaaaag caattcacgg catgatggat 180
gcaagtgaaa agaccaacta cacatgttgc agacttcagc gccacgagtg gaacaaacat 240
ggttggtgca actggtacaa cattgaacct tgggtattga tcatgaatag gacccaagct 300
aatcttacag agggtcaacc accaagagag tgcgccgtca cgtgcaggta tgatagagat 360
aatgacataa atgttgtaac gcaagctaga gacagaccca cgctactgac aggctgtaag 420
aaagggaaga atttctcctt tgcaggaata ttgatgcagg gtccttgcaa ctttgagata 480
gcggcaagtg atgtgctg 498
<210> 3
<211> 338
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
His Leu Ala Cys Leu Pro Gly Pro Ser Thr Ala Ile Ala Leu Ala Gly
1 5 10 15
Ala Ile Gly Gly Leu Gly Ala Gly Gly Leu Thr Thr Thr Thr Leu Gly
20 25 30
Thr Ser Pro Gly Met Leu Leu Gly Ala Thr Met Val Ile Ala Thr Cys
35 40 45
Gly Ala Gly Leu Leu Met Thr Leu Gly Ala Cys Thr Ala Gly Thr Ala
50 55 60
Thr Leu Ala Ile Leu His Thr Ala Ala Leu Pro Thr Ser Val Val Pro
65 70 75 80
Leu Leu Leu Pro Ala Gly Ala Leu Gly Gly Ala Val Val Gly Met Ala
85 90 95
Ala Ala Pro Gly Pro Gly Leu Cys Pro Cys Ala Ala Leu Pro Ile Val
100 105 110
Ala Gly Leu Pro Ala Thr Thr Leu Leu Ala Gly Pro Ala Pro Gly Met
115 120 125
Val Cys Pro Ile Gly Thr Thr Gly Thr Val Ser Cys Thr Ser Pro Ala
130 135 140
Met Ala Thr Leu Ala Thr Thr Val Val Ala Thr Thr Ala Ala Ser Leu
145 150 155 160
Pro Pro Pro His Ala Gly Gly Cys Ile Thr Gly Leu Ala Leu Gly Gly
165 170 175
Ala Leu His Ala Cys Ile Leu Gly Gly Ala Thr Thr Cys Val Pro Gly
180 185 190
Ala Gly Leu Leu Thr Leu Gly Gly Ser Ile Gly Ser Cys Leu Thr Cys
195 200 205
Gly Thr Gly Pro Leu Gly Ser Gly Gly Leu Pro His Thr Pro Ile Gly
210 215 220
Leu Cys Leu Leu Gly Ala Gly Thr Gly Thr Ala Leu Val Ala Ser Thr
225 230 235 240
Ser Cys Ala Ala Gly Gly Val Ala Ile Val Pro Gly Gly Thr Leu Leu
245 250 255
Cys Leu Ile Gly Leu Thr Thr Val Gly Val Ile Ala Met Ala Thr Leu
260 265 270
Leu Gly Pro Met Pro Cys Ala Pro Thr Gly Ile Ile Ser Ser Gly Gly
275 280 285
Pro Val Gly Leu Thr Ala Cys Thr Pro Ala Thr Thr Leu Thr Leu Leu
290 295 300
Ala Leu Thr Pro Gly Pro Ala Ala Ser Thr Pro Gly Gly Thr Met Leu
305 310 315 320
Leu Gly Gly Thr Gly Thr Thr Pro Ala Leu Gly Val Thr Ala His His
325 330 335
Ala Ala
<210> 4
<211> 1014
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cacttggatt gcaaacctga attctcgtat gccatagcaa aggacgaaag aattggtcaa 60
ctgggggctg aaggccttac caccacttgg aaggaatact cacctggaat gaagctggaa 120
gacacaatgg tcattgcttg gtgcgaagat gggaagttaa tgtacctcca aagatgcacg 180
agagaaacca gatatctcgc aatcttgcat acaagagcct tgccgaccag tgtggtattc 240
aaaaaactct ttgatgggcg aaagcaagag gatgtagtcg aaatgaacga caactttgaa 300
tttggactct gcccatgtga tgccaaaccc atagtaagag ggaagttcaa tacaacgctg 360
ctgaacggac cggccttcca gatggtatgc cccataggat ggacagggac tgtaagctgt 420
acgtcattca atatggacac cttagccaca actgtggtac ggacatatag aaggtctaaa 480
ccattccctc ataggcaagg ctgtatcacc caaaagaatc tgggggagga tctccataac 540
tgcatccttg gaggaaattg gacttgtgtg cctggagacc aactactata caaagggggc 600
tctattgaat cttgcaagtg gtgtggctat caatttaaag agagtgaggg actaccacac 660
taccccattg gcaagtgtaa attggagaac gagactggtt acaggctagt agacagtacc 720
tcttgcaata gagaaggtgt ggccatagta ccacaaggga cattaaagtg caagatagga 780
aaaacaactg tacaggtcat agctatggat accaaactcg gacctatgcc ttgcagacca 840
tatgaaatca tatcaagtga ggggcctgta gaaaagacag cgtgtacttt caactacact 900
aagacattaa aaaataagta ttttgagccc agagacagct actttcagca atacatgcta 960
aaaggagagt atcaatactg gtttgacctg gaggtgactg accatcaccg ggat 1014
<210> 5
<211> 519
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
His Leu Asp Cys Lys Pro Glu Phe Ser Tyr Ala Ile Ala Lys Asp Glu
1 5 10 15
Arg Ile Gly Gln Leu Gly Ala Glu Gly Leu Thr Thr Thr Trp Lys Glu
20 25 30
Tyr Ser Pro Gly Met Lys Leu Glu Asp Thr Met Val Ile Ala Trp Cys
35 40 45
Glu Asp Gly Lys Leu Met Tyr Leu Gln Arg Cys Thr Arg Glu Thr Arg
50 55 60
Tyr Leu Ala Ile Leu His Thr Arg Ala Leu Pro Thr Ser Val Val Phe
65 70 75 80
Lys Lys Leu Phe Asp Gly Arg Lys Gln Glu Asp Val Val Glu Met Asn
85 90 95
Asp Asn Phe Glu Phe Gly Leu Cys Pro Cys Asp Ala Lys Pro Ile Val
100 105 110
Arg Gly Lys Phe Asn Thr Thr Leu Leu Asn Gly Pro Ala Phe Gln Met
115 120 125
Val Cys Pro Ile Gly Trp Thr Gly Thr Val Ser Cys Thr Ser Phe Asn
130 135 140
Met Asp Thr Leu Ala Thr Thr Val Val Arg Thr Tyr Arg Arg Ser Lys
145 150 155 160
Pro Phe Pro His Arg Gln Gly Cys Ile Thr Gln Lys Asn Leu Gly Glu
165 170 175
Asp Leu His Asn Cys Ile Leu Gly Gly Asn Trp Thr Cys Val Pro Gly
180 185 190
Asp Gln Leu Leu Tyr Lys Gly Gly Ser Ile Glu Ser Cys Lys Trp Cys
195 200 205
Gly Tyr Gln Phe Lys Glu Ser Glu Gly Leu Pro His Tyr Pro Ile Gly
210 215 220
Lys Cys Lys Leu Glu Asn Glu Thr Gly Tyr Arg Leu Val Asp Ser Thr
225 230 235 240
Ser Cys Asn Arg Glu Gly Val Ala Ile Val Pro Gln Gly Thr Leu Lys
245 250 255
Cys Lys Ile Gly Lys Thr Thr Val Gln Val Ile Ala Met Asp Thr Lys
260 265 270
Leu Gly Pro Met Pro Cys Arg Pro Tyr Glu Ile Ile Ser Ser Glu Gly
275 280 285
Pro Val Glu Lys Thr Ala Cys Thr Phe Asn Tyr Thr Lys Thr Leu Lys
290 295 300
Asn Lys Tyr Phe Glu Pro Arg Asp Ser Tyr Phe Gln Gln Tyr Met Leu
305 310 315 320
Lys Gly Glu Tyr Gln Tyr Trp Phe Asp Leu Glu Val Thr Asp His His
325 330 335
Arg Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
340 345 350
Ser Gly Glu Asn Ile Thr Gln Trp Asn Leu Gln Asp Asn Gly Thr Glu
355 360 365
Gly Ile Gln Arg Ala Met Phe Gln Arg Gly Val Asn Arg Ser Leu His
370 375 380
Gly Ile Trp Pro Glu Lys Ile Cys Thr Gly Val Pro Ser His Leu Ala
385 390 395 400
Thr Asp Met Glu Leu Lys Ala Ile His Gly Met Met Asp Ala Ser Glu
405 410 415
Lys Thr Asn Tyr Thr Cys Cys Arg Leu Gln Arg His Glu Trp Asn Lys
420 425 430
His Gly Trp Cys Asn Trp Tyr Asn Ile Glu Pro Trp Val Leu Ile Met
435 440 445
Asn Arg Thr Gln Ala Asn Leu Thr Glu Gly Gln Pro Pro Arg Glu Cys
450 455 460
Ala Val Thr Cys Arg Tyr Asp Arg Asp Asn Asp Ile Asn Val Val Thr
465 470 475 480
Gln Ala Arg Asp Arg Pro Thr Leu Leu Thr Gly Cys Lys Lys Gly Lys
485 490 495
Asn Phe Ser Phe Ala Gly Ile Leu Met Gln Gly Pro Cys Asn Phe Glu
500 505 510
Ile Ala Ala Ser Asp Val Leu
515
<210> 6
<211> 1557
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
cacttggatt gcaaacctga attctcgtat gccatagcaa aggacgaaag aattggtcaa 60
ctgggggctg aaggccttac caccacttgg aaggaatact cacctggaat gaagctggaa 120
gacacaatgg tcattgcttg gtgcgaagat gggaagttaa tgtacctcca aagatgcacg 180
agagaaacca gatatctcgc aatcttgcat acaagagcct tgccgaccag tgtggtattc 240
aaaaaactct ttgatgggcg aaagcaagag gatgtagtcg aaatgaacga caactttgaa 300
tttggactct gcccatgtga tgccaaaccc atagtaagag ggaagttcaa tacaacgctg 360
ctgaacggac cggccttcca gatggtatgc cccataggat ggacagggac tgtaagctgt 420
acgtcattca atatggacac cttagccaca actgtggtac ggacatatag aaggtctaaa 480
ccattccctc ataggcaagg ctgtatcacc caaaagaatc tgggggagga tctccataac 540
tgcatccttg gaggaaattg gacttgtgtg cctggagacc aactactata caaagggggc 600
tctattgaat cttgcaagtg gtgtggctat caatttaaag agagtgaggg actaccacac 660
taccccattg gcaagtgtaa attggagaac gagactggtt acaggctagt agacagtacc 720
tcttgcaata gagaaggtgt ggccatagta ccacaaggga cattaaagtg caagatagga 780
aaaacaactg tacaggtcat agctatggat accaaactcg gacctatgcc ttgcagacca 840
tatgaaatca tatcaagtga ggggcctgta gaaaagacag cgtgtacttt caactacact 900
aagacattaa aaaataagta ttttgagccc agagacagct actttcagca atacatgcta 960
aaaggagagt atcaatactg gtttgacctg gaggtgactg accatcaccg ggatggcggc 1020
ggcggttccg gaggtggcgg cagtggcggc ggtggttctg gtgaaaacat aacacagtgg 1080
aacttgcaag ataatgggac agaagggata caacgggcga tgttccaaag gggcgtgaat 1140
aggagcctac atggaatctg gccagagaaa atctgtacag gtgtaccatc ccatctagcc 1200
actgatatgg aattaaaagc aattcacggc atgatggatg caagtgaaaa gaccaactac 1260
acatgttgca gacttcagcg ccacgagtgg aacaaacatg gttggtgcaa ctggtacaac 1320
attgaacctt gggtattgat catgaatagg acccaagcta atcttacaga gggtcaacca 1380
ccaagagagt gcgccgtcac gtgcaggtat gatagagata atgacataaa tgttgtaacg 1440
caagctagag acagacccac gctactgaca ggctgtaaga aagggaagaa tttctccttt 1500
gcaggaatat tgatgcaggg tccttgcaac tttgagatag cggcaagtga tgtgctg 1557
<210> 7
<211> 166
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Gly Gly Ala Ile Thr Gly Thr Ala Leu Gly Ala Ala Gly Thr Gly Gly
1 5 10 15
Ile Gly Ala Ala Met Pro Gly Ala Gly Val Ala Ala Ser Leu His Gly
20 25 30
Ile Thr Pro Gly Leu Ile Cys Thr Gly Val Pro Ser His Leu Ala Thr
35 40 45
Ala Met Gly Leu Leu Ala Ile His Gly Met Met Ala Ala Ser Gly Leu
50 55 60
Thr Ala Thr Thr Cys Cys Ala Leu Gly Ala His Gly Thr Ala Leu His
65 70 75 80
Gly Thr Cys Ala Thr Thr Ala Ile Gly Pro Thr Val Leu Ile Met Ala
85 90 95
Ala Thr Gly Ala Ala Leu Thr Gly Gly Gly Pro Pro Ala Gly Cys Ala
100 105 110
Val Thr Cys Ala Thr Ala Ala Ala Ala Ala Ile Ala Val Val Thr Gly
115 120 125
Ala Ala Ala Ala Pro Thr Leu Leu Thr Gly Cys Leu Leu Gly Leu Ala
130 135 140
Pro Ser Pro Ala Gly Ile Leu Met Gly Gly Pro Cys Ala Pro Gly Gly
145 150 155 160
Gly Gly Ser Ala Val Leu
165
<210> 8
<211> 498
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ggtgaaaaca taacacagtg gaacttgcaa gataatggga cagaagggat acaacgggcg 60
atgttccaaa ggggcgtgaa taggagccta catggaatct ggccagagaa aatctgtaca 120
ggtgtaccat cccatctagc cactgatatg gaattaaaag caattcacgg catgatggat 180
gcaagtgaaa agaccaacta cacatgttgc agacttcagc gccacgagtg gaacaaacat 240
ggttggtgca actggtacaa cattgaacct tgggtattga tcatgaatag gacccaagct 300
aatcttacag agggtcaacc accaagagag tgcgccgtca cgtgcaggta tgatagagat 360
aatgacataa atgttgtaac gcaagctaga gacagaccca cgctactgac aggctgtaag 420
aaagggaaga atttctcctt tgcaggaata ttgatgcagg gtccttgcaa ctttgagggc 480
ggcggcagtg atgtgctg 498
<210> 9
<211> 519
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
His Leu Asp Cys Lys Pro Glu Phe Ser Tyr Ala Ile Ala Lys Asp Glu
1 5 10 15
Arg Ile Gly Gln Leu Gly Ala Glu Gly Leu Thr Thr Thr Trp Lys Glu
20 25 30
Tyr Ser Pro Gly Met Lys Leu Glu Asp Thr Met Val Ile Ala Trp Cys
35 40 45
Glu Asp Gly Lys Leu Met Tyr Leu Gln Arg Cys Thr Arg Glu Thr Arg
50 55 60
Tyr Leu Ala Ile Leu His Thr Arg Ala Leu Pro Thr Ser Val Val Phe
65 70 75 80
Lys Lys Leu Phe Asp Gly Arg Lys Gln Glu Asp Val Val Glu Met Asn
85 90 95
Asp Asn Phe Glu Phe Gly Leu Cys Pro Cys Asp Ala Lys Pro Ile Val
100 105 110
Arg Gly Lys Phe Asn Thr Thr Leu Leu Asn Gly Pro Ala Phe Gln Met
115 120 125
Val Cys Pro Ile Gly Trp Thr Gly Thr Val Ser Cys Thr Ser Phe Asn
130 135 140
Met Asp Thr Leu Ala Thr Thr Val Val Arg Thr Tyr Arg Arg Ser Lys
145 150 155 160
Pro Phe Pro His Arg Gln Gly Cys Ile Thr Gln Lys Asn Leu Gly Glu
165 170 175
Asp Leu His Asn Cys Ile Leu Gly Gly Asn Trp Thr Cys Val Pro Gly
180 185 190
Asp Gln Leu Leu Tyr Lys Gly Gly Ser Ile Glu Ser Cys Lys Trp Cys
195 200 205
Gly Tyr Gln Phe Lys Glu Ser Glu Gly Leu Pro His Tyr Pro Ile Gly
210 215 220
Lys Cys Lys Leu Glu Asn Glu Thr Gly Tyr Arg Leu Val Asp Ser Thr
225 230 235 240
Ser Cys Asn Arg Glu Gly Val Ala Ile Val Pro Gln Gly Thr Leu Lys
245 250 255
Cys Lys Ile Gly Lys Thr Thr Val Gln Val Ile Ala Met Asp Thr Lys
260 265 270
Leu Gly Pro Met Pro Cys Arg Pro Tyr Glu Ile Ile Ser Ser Glu Gly
275 280 285
Pro Val Glu Lys Thr Ala Cys Thr Phe Asn Tyr Thr Lys Thr Leu Lys
290 295 300
Asn Lys Tyr Phe Glu Pro Arg Asp Ser Tyr Phe Gln Gln Tyr Met Leu
305 310 315 320
Lys Gly Glu Tyr Gln Tyr Trp Phe Asp Leu Glu Val Thr Asp His His
325 330 335
Arg Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
340 345 350
Ser Gly Glu Asn Ile Thr Gln Trp Asn Leu Gln Asp Asn Gly Thr Glu
355 360 365
Gly Ile Gln Arg Ala Met Phe Gln Arg Gly Val Asn Arg Ser Leu His
370 375 380
Gly Ile Trp Pro Glu Lys Ile Cys Thr Gly Val Pro Ser His Leu Ala
385 390 395 400
Thr Asp Met Glu Leu Lys Ala Ile His Gly Met Met Asp Ala Ser Glu
405 410 415
Lys Thr Asn Tyr Thr Cys Cys Arg Leu Gln Arg His Glu Trp Asn Lys
420 425 430
His Gly Trp Cys Asn Trp Tyr Asn Ile Glu Pro Trp Val Leu Ile Met
435 440 445
Asn Arg Thr Gln Ala Asn Leu Thr Glu Gly Gln Pro Pro Arg Glu Cys
450 455 460
Ala Val Thr Cys Arg Tyr Asp Arg Asp Asn Asp Ile Asn Val Val Thr
465 470 475 480
Gln Ala Arg Asp Arg Pro Thr Leu Leu Thr Gly Cys Lys Lys Gly Lys
485 490 495
Asn Phe Ser Phe Ala Gly Ile Leu Met Gln Gly Pro Cys Asn Phe Glu
500 505 510
Gly Gly Gly Ser Asp Val Leu
515
<210> 11
<211> 1557
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
cacttggatt gcaaacctga attctcgtat gccatagcaa aggacgaaag aattggtcaa 60
ctgggggctg aaggccttac caccacttgg aaggaatact cacctggaat gaagctggaa 120
gacacaatgg tcattgcttg gtgcgaagat gggaagttaa tgtacctcca aagatgcacg 180
agagaaacca gatatctcgc aatcttgcat acaagagcct tgccgaccag tgtggtattc 240
aaaaaactct ttgatgggcg aaagcaagag gatgtagtcg aaatgaacga caactttgaa 300
tttggactct gcccatgtga tgccaaaccc atagtaagag ggaagttcaa tacaacgctg 360
ctgaacggac cggccttcca gatggtatgc cccataggat ggacagggac tgtaagctgt 420
acgtcattca atatggacac cttagccaca actgtggtac ggacatatag aaggtctaaa 480
ccattccctc ataggcaagg ctgtatcacc caaaagaatc tgggggagga tctccataac 540
tgcatccttg gaggaaattg gacttgtgtg cctggagacc aactactata caaagggggc 600
tctattgaat cttgcaagtg gtgtggctat caatttaaag agagtgaggg actaccacac 660
taccccattg gcaagtgtaa attggagaac gagactggtt acaggctagt agacagtacc 720
tcttgcaata gagaaggtgt ggccatagta ccacaaggga cattaaagtg caagatagga 780
aaaacaactg tacaggtcat agctatggat accaaactcg gacctatgcc ttgcagacca 840
tatgaaatca tatcaagtga ggggcctgta gaaaagacag cgtgtacttt caactacact 900
aagacattaa aaaataagta ttttgagccc agagacagct actttcagca atacatgcta 960
aaaggagagt atcaatactg gtttgacctg gaggtgactg accatcaccg ggatggcggc 1020
ggcggttccg gaggtggcgg cagtggcggc ggtggttctg gtgaaaacat aacacagtgg 1080
aacttgcaag ataatgggac agaagggata caacgggcga tgttccaaag gggcgtgaat 1140
aggagcctac atggaatctg gccagagaaa atctgtacag gtgtaccatc ccatctagcc 1200
actgatatgg aattaaaagc aattcacggc atgatggatg caagtgaaaa gaccaactac 1260
acatgttgca gacttcagcg ccacgagtgg aacaaacatg gttggtgcaa ctggtacaac 1320
attgaacctt gggtattgat catgaatagg acccaagcta atcttacaga gggtcaacca 1380
ccaagagagt gcgccgtcac gtgcaggtat gatagagata atgacataaa tgttgtaacg 1440
caagctagag acagacccac gctactgaca ggctgtaaga aagggaagaa tttctccttt 1500
gcaggaatat tgatgcaggg tccttgcaac tttgagggcg gcggcagtga tgtgctg 1557
<210> 11
<211> 228
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Gly Gly Ala Ile Thr Gly Thr Ala Leu Gly Ala Ala Gly Thr Gly Gly
1 5 10 15
Ile Gly Ala Ala Met Pro Gly Ala Gly Val Ala Ala Ser Leu His Gly
20 25 30
Ile Thr Pro Gly Leu Ile Cys Thr Gly Val Pro Ser His Leu Ala Thr
35 40 45
Ala Met Gly Leu Leu Ala Ile His Gly Met Met Ala Ala Ser Gly Leu
50 55 60
Thr Ala Thr Thr Cys Cys Ala Leu Gly Ala His Gly Thr Ala Leu His
65 70 75 80
Gly Thr Cys Ala Thr Thr Ala Ile Gly Pro Thr Val Leu Ile Met Ala
85 90 95
Ala Thr Gly Ala Ala Leu Thr Gly Gly Gly Pro Pro Ala Gly Cys Ala
100 105 110
Val Thr Cys Ala Thr Ala Ala Ala Ala Ala Ile Ala Val Val Thr Gly
115 120 125
Ala Ala Ala Ala Pro Thr Leu Leu Thr Gly Cys Leu Leu Gly Leu Ala
130 135 140
Pro Ser Pro Ala Gly Ile Leu Met Gly Gly Pro Cys Ala Pro Gly Ile
145 150 155 160
Ala Ala Ser Ala Val Leu Pro Leu Gly His Ala Cys Thr Ala Val Pro
165 170 175
Gly Ala Thr Ala His Thr Leu Val Ala Gly Met Thr Ala Thr Val Gly
180 185 190
Ser Ala Ala Gly Gly Thr Ala Leu Leu Thr Thr Thr Leu Gly Ala Gly
195 200 205
Leu Gly Ile Leu Gly Leu Leu Leu Gly Ala Leu Ser Leu Thr Thr Pro
210 215 220
Gly Ala Thr Ala
225
<210> 12
<211> 684
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ggtgaaaaca taacacagtg gaacttgcaa gataatggga cagaagggat acaacgggcg 60
atgttccaaa ggggcgtgaa taggagccta catggaatct ggccagagaa aatctgtaca 120
ggtgtaccat cccatctagc cactgatatg gaattaaaag caattcacgg catgatggat 180
gcaagtgaaa agaccaacta cacatgttgc agacttcagc gccacgagtg gaacaaacat 240
ggttggtgca actggtacaa cattgaacct tgggtattga tcatgaatag gacccaagct 300
aatcttacag agggtcaacc accaagagag tgcgccgtca cgtgcaggta tgatagagat 360
aatgacataa atgttgtaac gcaagctaga gacagaccca cgctactgac aggctgtaag 420
aaagggaaga atttctcctt tgcaggaata ttgatgcagg gtccttgcaa ctttgagata 480
gcggcaagtg atgtgctgtt taaagaacat gactgcacta atgtattcca ggatactgcc 540
cattaccttg tcgacgggat gaccaacacc gtagaaagtg ccaggcaagg gaccgcaaaa 600
ctaacaacct ggttaggcag acagcttggg atactgggaa aaaagctgga gaacaaaagt 660
aagacatggt tcggggcgta tgcg 684
Claims (10)
1. A bovine viral diarrhea virus E2-E0 fusion protein, wherein the E2-E0 fusion protein comprises a bovine viral diarrhea virus E0 truncated protein and a bovine viral diarrhea virus E2 protein; the amino acid sequence of the truncated protein of the bovine viral diarrhea virus E0 is shown in SEQ ID NO. 1; the amino acid sequence of the bovine viral diarrhea virus E2 protein is shown in SEQ ID NO. 3.
2. The bovine viral diarrhea virus E2-E0 fusion protein of claim 1 wherein the amino acid sequence of the bovine viral diarrhea virus E2-E0 fusion protein is set forth as SEQ ID No. 5.
3. A gene sequence encoding the bovine viral diarrhea virus E2-E0 fusion protein of claim 2, wherein the gene sequence is as shown in SEQ ID No. 6.
4. The bovine viral diarrhea virus E2-E0 fusion protein of claim 1, wherein the amino acid at position 160-162 of the truncated protein of bovine viral diarrhea virus E0 is mutated from IAA to GGG; the amino acid sequence of the mutant bovine viral diarrhea virus E0 truncated protein is shown in SEQ ID NO. 7.
5. The bovine viral diarrhea virus E2-E0 fusion protein of claim 4 wherein the amino acid sequence of the bovine viral diarrhea virus E2-E0 fusion protein is set forth as SEQ ID No. 9.
6. A gene sequence encoding the bovine viral diarrhea virus E2-E0 fusion protein of claim 5, wherein the gene sequence is as shown in SEQ ID No. 10.
7. Use of the bovine viral diarrhea virus E2-E0 fusion protein of any one of claims 1-2 or claims 4-5 in the preparation of a bovine viral diarrhea virus vaccine.
8. The method of producing a bovine viral diarrhea virus E2-E0 fusion protein of any one of claims 1-2 or claims 4-5, wherein the method comprises: cloning the gene of the fusion protein of the encoding bovine viral diarrhea virus E2-E0 into a eukaryotic expression vector to obtain a recombinant plasmid for expressing the fusion protein of the bovine viral diarrhea virus E2-E0; and transfecting the recombinant plasmid into a CHO cell, and culturing, screening and purifying to obtain the bovine viral diarrhea virus E2-E0 fusion protein.
9. The method of claim 8, wherein the eukaryotic expression vector is pcDNA3.1 vector; the CHO cells are CHO suspension cells.
10. The method of claim 9, wherein the method comprises:
(1) PCR amplifying a gene segment of a fusion protein of the bovine viral diarrhea virus E2-E0, wherein the gene segment is shown as SEQ ID NO.6 or SEQ ID NO. 10;
(2) carrying out double enzyme digestion on the gene segments of the vector pcDNA3.1 and the bovine viral diarrhea virus E2-E0 fusion protein by using restriction enzymes Xho I and Hind III, respectively, and connecting the enzyme digestion segments with the gene segments of the bovine viral diarrhea virus E2-E0 fusion protein by using DNA ligase to obtain a recombinant plasmid for expressing the bovine viral diarrhea virus E2-E0 fusion protein;
(3) and (3) transfecting the recombinant plasmid in the step (2) to CHO suspension cells, performing suspension culture, collecting cell culture supernatant, and purifying to obtain the bovine viral diarrhea virus E2-E0 fusion protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210571495.8A CN114853912B (en) | 2022-05-24 | 2022-05-24 | Bovine viral diarrhea virus E2-E0 fusion protein, preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210571495.8A CN114853912B (en) | 2022-05-24 | 2022-05-24 | Bovine viral diarrhea virus E2-E0 fusion protein, preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114853912A true CN114853912A (en) | 2022-08-05 |
| CN114853912B CN114853912B (en) | 2023-06-13 |
Family
ID=82639703
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210571495.8A Active CN114853912B (en) | 2022-05-24 | 2022-05-24 | Bovine viral diarrhea virus E2-E0 fusion protein, preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114853912B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1989240A (en) * | 2004-05-19 | 2007-06-27 | 贝林格尔·英格海姆维特梅迪卡有限公司 | Vaccine comprising an attenuated pestivirus |
| US20090068223A1 (en) * | 2005-11-15 | 2009-03-12 | Boehringer Ingelheim Vetmedica, Inc. | Combination vaccine comprising an attenuated bovine viral diarrhea virus |
| CN102517260A (en) * | 2011-12-29 | 2012-06-27 | 中国人民解放军军事医学科学院野战输血研究所 | Fusion protein carrying NS3 preponderant area of non-structural protein of bovine viral diarrhea virus as well as recombinant expression method and application thereof |
| CN112359023A (en) * | 2020-10-22 | 2021-02-12 | 中国农业大学 | Bovine viral diarrhea virus BVDV-BJ175170 and application thereof |
-
2022
- 2022-05-24 CN CN202210571495.8A patent/CN114853912B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1989240A (en) * | 2004-05-19 | 2007-06-27 | 贝林格尔·英格海姆维特梅迪卡有限公司 | Vaccine comprising an attenuated pestivirus |
| US20090068223A1 (en) * | 2005-11-15 | 2009-03-12 | Boehringer Ingelheim Vetmedica, Inc. | Combination vaccine comprising an attenuated bovine viral diarrhea virus |
| CN102517260A (en) * | 2011-12-29 | 2012-06-27 | 中国人民解放军军事医学科学院野战输血研究所 | Fusion protein carrying NS3 preponderant area of non-structural protein of bovine viral diarrhea virus as well as recombinant expression method and application thereof |
| CN112359023A (en) * | 2020-10-22 | 2021-02-12 | 中国农业大学 | Bovine viral diarrhea virus BVDV-BJ175170 and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 陈为宏等: "牛病毒性腹泻病毒E0和E2蛋白的融合表达及纯化" * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114853912B (en) | 2023-06-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2022121322A1 (en) | Recombinant subunit vaccine of novel coronavirus and application thereof | |
| CN108586618B (en) | Preparation and application of porcine epidemic diarrhea subunit vaccine | |
| WO2020238458A1 (en) | Cell strain for expressing e2 protein and application thereof, and e2 protein and application thereof | |
| CN113845576A (en) | Recombinant feline herpesvirus type 1 gB-gD protein and application thereof | |
| CN113248574A (en) | Method for efficiently expressing A-type seneca virus structural protein | |
| CN107353346A (en) | A kind of fusion protein being made up of pig albumin and Porcine interferon-gamma and preparation method thereof and a kind of Recombinant Swine long-acting interferon γ | |
| CN109705223B (en) | Recombinant subunit vaccine of orf virus and production method thereof | |
| CN111773383A (en) | O-type foot-and-mouth disease subunit vaccine and preparation method and application thereof | |
| CN111973738A (en) | Nucleic acid and recombinant protein co-immune vaccine based on hog cholera virus gene, preparation method and application | |
| CN107266587A (en) | A kind of recombinant bovine long-acting interferon α and prepare fusion protein of this long-acting interferon and preparation method thereof | |
| CN114853912B (en) | Bovine viral diarrhea virus E2-E0 fusion protein, preparation method and application | |
| CN114437235B (en) | Recombinant fusion protein of streptococcus equi subspecies 8 proteins, and preparation method and application thereof | |
| CN114773438B (en) | Bovine viral diarrhea virus E0 truncated protein, preparation method and application | |
| CN116240222A (en) | Codon-optimized bovine viral diarrhea virus type 1E2 protein gene and application thereof | |
| CN108671227A (en) | A kind of multi-joint subunit vaccine of wide spectrum preventing streptococcus suis infection | |
| CN112076314B (en) | A-type foot-and-mouth disease subunit vaccine and preparation method and application thereof | |
| CN111303301B (en) | Preparation method of mycobacterium tuberculosis immunogen protein ESAT6 | |
| CN114805609B (en) | Swine fever virus E2-E0 fusion protein, preparation method and application | |
| CN114835782B (en) | Swine fever virus E0 truncated protein, preparation method and application | |
| CN110747215A (en) | Recombinant baculovirus for efficiently expressing hog cholera E2 protein and construction method thereof | |
| CN111944028A (en) | Clostridium perfringens beta toxin mutant protein, preparation method, application and vaccine thereof | |
| CN116589538B (en) | Seven-component antigen African swine fever subunit vaccine | |
| CN111925449B (en) | Recombinant CHO cell strain expressing chicken VP2 and chicken GAL-1 fusion protein and construction method and application thereof | |
| CN116589539B (en) | Nine-component antigen African swine fever subunit vaccine | |
| CN111732667B (en) | Peste des petits ruminants virus genetic engineering subunit vaccine |
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 |