CN114853912B - Bovine viral diarrhea virus E2-E0 fusion protein, preparation method and application - Google Patents
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Abstract
The invention belongs to the technical field of biology, and relates to a bovine viral diarrhea virus E2-E0 fusion protein, a preparation method and application thereof. The invention firstly provides a method for fusing the truncated protein of the bovine viral diarrhea virus E0 and the bovine viral diarrhea virus E2 protein, which not only can promote the expression of the E0 protein, but also does not influence the immunogenicity of the E0 protein, and can be used for preparing subunit vaccine of the bovine viral diarrhea; based on the bovine viral diarrhea virus E0 truncated protein, the invention discovers that the 160-162 amino acids of the bovine viral diarrhea virus E0 truncated protein are mutated from IAA to GGG, so that the expression quantity of the E2-E0 fusion protein can be further improved by more than 18 percent, 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 a bovine viral diarrhea virus E2-E0 fusion protein, a preparation method and application thereof.
Background
Bovine viral diarrhea (bovine viral diarrhea, BVD) is an acute, febrile, highly contagious disease caused by bovine viral diarrhea virus (bovine viral diarrhea virus, BVDV) and characterized by diarrhea, reproductive disorders, immune dysfunction, etc. 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 incidence rate, the prevention and treatment difficulty is that BVDV can cause immunosuppression and persistent infection after entering the body, reduce the immunity of the body, easily induce mixed infection or secondary infection of other pathogens, seriously influence the health state and the production performance of cattle, and is one of the most important cattle infectious diseases worldwide.
BVDV belongs to the Flaviviridae (Flaviviridae) Pestivirus genus (Pestiviruses), and the genome consists of a single-stranded sense RNA molecule of about 12.3kb in length. BVDV has 3 biotypes, usually based on the 5' -UTR sequence, the genomic amino-terminal autoprotease (N pro ) Or the envelope glycoprotein (E2) region is divided into BVDV-1 and BVDV-2 types, BVDV-3 type is described as atypical BVDV. BVDV-1 is further classified into 21 subtypes (1 a to 1 u), and BVDV-2 is classified into 3 subtypes (2 a to 2 c). Currently, 8 subtypes are mainly popular in China: 1a, 1b, 1c, 1d, 1m, 1o, 1p and 1q. BVDV genomes encode 4 structural proteins, namely P14 (C), gP48 (E0), gP25 (E1) and gP53 (E2), and the other structural proteins 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 methods for controlling BVDV transmission: eliminate continuously infected animals and vaccinate. The modified live vaccine or the inactivated vaccine is mainly used for BVDV vaccination programs, but the vaccines respectively have the problems of biological safety risk or insufficient immune protection efficacy and the like. The inactivated vaccine is safe to pregnant cows, but has a shorter immunization period; attenuated vaccines, although long in immunization period, present a safety risk to pregnant cattle. Based on this, researchers have long been working on the development of BVD subunit vaccines. The E0 protein can induce the organism to generate virus neutralizing antibodies, is an important protective antigen of BVDV and is also a candidate antigen for developing BVD subunit vaccine. The presence of multiple glycosylation sites in the amino acid sequence of the E0 protein plays an important role in maintaining its 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 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 quantity is extremely low, and the situation seriously hinders the research and development of BVD subunit vaccine.
Disclosure of Invention
In order to solve the technical problem that BVDV E0 protein is expressed in a CHO cell in low or non-expressed amount, the invention discovers that BVDV E0 protein is truncated and fused with BVDV E2 protein, and then BVDV E0 expression can be promoted in the CHO cell, thus not affecting E2 protein expression and immunogenicity of E0 protein and E2 protein, and the invention can be used for preparing BVDsubunit vaccine, and specifically comprises the following steps:
in a first aspect, the present invention provides a bovine viral diarrhea virus E2-E0 fusion protein, said fusion protein comprising a bovine viral diarrhea virus E0 truncated protein and a bovine viral diarrhea virus E2 protein; the amino acid sequence of the bovine viral diarrhea virus E0 truncated protein is shown as SEQ ID NO. 1; the amino acid sequence of the E2 protein of the bovine viral diarrhea virus is shown as SEQ ID NO. 3.
Preferably, the nucleotide sequence of the truncated protein of the E0 of the bovine viral diarrhea virus is shown as SEQ ID NO. 2; the amino acid sequence of the E2 protein of the bovine viral diarrhea virus is shown as 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 E2-E0 fusion protein of the bovine viral diarrhea virus is shown as SEQ ID NO. 6.
Preferably, amino acids 160-162 of the E0 protein fragment of the bovine viral diarrhea virus are mutated from IAA to GGG; the amino acid sequence of the mutated bovine viral diarrhea virus E0 truncated protein is shown as SEQ ID NO. 7.
Preferably, the mutated bovine viral diarrhea virus E0 truncated protein has a gene sequence shown in 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 the application of the bovine viral diarrhea virus E2-E0 fusion protein in the first aspect in preparing 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 according to the first aspect, the method comprising: the method comprises the steps of connecting genes encoding the bovine viral diarrhea virus E0 truncated protein and the bovine viral diarrhea virus E2 protein in series, and cloning the genes into a eukaryotic expression vector to obtain recombinant plasmids for expressing the bovine viral diarrhea virus E0 truncated protein and the bovine viral diarrhea virus E2 protein; and then transfecting the recombinant plasmid into CHO cells, culturing, screening and purifying to obtain the bovine viral diarrhea virus E2-E0 fusion protein.
Preferably, the eukaryotic expression vector is a pcDNA3.1 vector.
Preferably, the CHO cells are CHO suspension cells.
Preferably, the method is as follows:
(1) Amplifying a gene fragment encoding bovine viral diarrhea virus E2-E0 fusion protein by PCR, wherein the gene fragment is shown as SEQ ID NO.6 or SEQ ID NO. 10;
(2) Double enzyme cutting is carried out on the 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 the enzyme cut fragments are connected by using DNA ligase to obtain 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 into CHO suspension cells, performing suspension culture, collecting cell culture supernatant, and purifying to obtain the bovine viral diarrhea virus E2-E0 fusion protein.
The beneficial effects of the invention are as follows: (1) 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 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 quantity is extremely low, thus seriously impeding the research and development of BVD subunit vaccine under the conditions; the invention surprisingly discovers that the E0 protein is obtained by truncating the E0 protein of the bovine viral diarrhea virus and is fused with the E2 protein of the bovine viral diarrhea virus, so that the expression of the E0 protein can be promoted, the immunogenicity of the original E0 protein and the immunogenicity of the E2 protein are not influenced, and the E0 protein can be used for preparing BVD subunit vaccines; (2) based on the truncated protein of the bovine viral diarrhea virus E0, the invention discovers that the 160 th-162 th amino acid of the bovine viral diarrhea virus E0 protein is mutated from IAA to GGG, so that the expression of the truncated protein of the bovine viral diarrhea virus E0 is further promoted, the protein expression quantity is improved by more than 18 percent, and the immunogenicity of the protein is not influenced.
Drawings
FIG. 1 shows the results of the expression and identification of E2 protein of bovine viral diarrhea virus;
FIG. 2 purification results of bovine viral diarrhea virus E2 protein;
FIG. 3 results of E0 truncated protein expression of bovine viral diarrhea virus E0 protein, E0 truncated protein and amino acid mutation;
FIG. 4 purification results of E0 truncated protein of bovine viral diarrhea virus;
FIG. 5 purification results of E0 truncated protein of bovine viral diarrhea virus amino acid mutation;
FIG. 6 shows the results of expression identification of truncated fusion proteins of bovine viral diarrhea virus E2-E0;
FIG. 7 purification results of truncated fusion proteins of bovine viral diarrhea virus E2-E0;
FIG. 8 shows the results of expression identification of a truncated mutant fusion protein of bovine viral diarrhea virus E2-E0; wherein 1 is E0 truncated mutation expression identification; 2 is expression identification after E2-E0 truncated mutation;
FIG. 9 purification results of a truncated mutant fusion protein of bovine viral diarrhea virus E2-E0;
FIG. 10 results of indirect ELISA immune rabbit serum antibody detection.
Detailed Description
The above-described aspects are further described below in conjunction with specific embodiments. It should be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The implementation conditions used in the examples may be further adjusted according to the conditions of the specific manufacturer, and the implementation conditions not specified are generally those in routine experiments.
The experiments described in the examples below obtained biosafety permissions and foot and mouth disease laboratory activity permissions:
the national institute of agricultural sciences was issued with the national institutes of agricultural sciences biosafety, such as the national institutes of agricultural sciences biosafety committee, the laboratory animal ethics committee, the national institutes of veterinary sciences biosafety, and the national institutes of veterinary sciences biosafety committee, according to the related requirements of biosafety class 3 laboratory (BSL-3) and bovine viral diarrhea, and has been documented at the agricultural rural department, conforming to the requirements of the national biosafety class.
BVDV-NADL vaccine standard strain (purchased from China veterinary microbiological culture collection center, china veterinary drug administration), eukaryotic expression vector pcDNA3.1 (+), BVDV positive serum were preserved by the present laboratory. CHO suspension cell expression system, unstained protein MW marker (26610), RT-PCR amplification testKit, 6X His Tag Monoclonal Antibody (HIS.H8), unstained protein MW marker (26616) and BCA protein quantitative kit, alexa
488-labeled goat anti-rabbit secondary antibodies were purchased from Thermo Fisher Scientific company. Affinity chromatography Ni columns were purchased from GE company. Both the RNA extraction kit and the DNA gel purification recovery kit were purchased from OMEGA Inc. Xho I and Hind III restriction endonucleases were purchased from New England Biolabs (NEB). Coli DH 5. Alpha. Competent cells were purchased from full gold company. A large number of plasmid extraction kits were purchased from MACHEREY-NAGEL company. ECL color solutions were purchased from shanghai bi yun biotechnology limited. MD44 dialysis bags with cutoff molecular weight of 8000-10000 Dalton were purchased from Beijing Soy Bao technology Co.
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 bring about expression of the genetic material elements carried thereby in the host cell. For example, the carrier comprises: a plasmid; a bacteriophage; cosmids, and the like.
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 vaccine of the invention is a genetically engineered subunit vaccine.
The vaccine of the present invention further optionally comprises one or more adjuvants, excipients, carriers and diluents. The adjuvant can be any suitable adjuvant, such as chemical immune adjuvants like aluminum hydroxide, freund's adjuvant, mineral oil, span, etc.; microbial immunoadjuvants such as mycobacteria, BCC, lipopolysaccharide, muramyl dipeptide, cytopeptide, liposoluble waxy D, and corynebacterium pumilum; the plant immunoadjuvant is polysaccharides extracted from plants or large fungi, such as pachyman, safflower polysaccharide, chinese herbal medicines, etc. And biochemical immune adjuvants such as thymus peptide, transfer factor, interleukin, etc. Preferred adjuvants may be nanoadjuvant biological adjuvants, interleukins, interferons, etc.
The vaccine of the invention can also be used in combination vaccines, such as with other vaccines for pigs or cattle, but emphasis is placed on attenuated live vaccines, in particular on integration of viral genes, such as bivalent, trivalent etc.
The vaccine of the present invention may be administered by any convenient route, such as intramuscular injection, intranasal, oral, subcutaneous, transdermal and vaginal. 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 dose for booster administration is the same or lower than the dose for priming administration. In addition, a third boost may be performed, for example, 2-3 months, 6 months or one 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 fragments encoding the bovine viral diarrhea virus E0 protein, E0 truncated mutant protein, E2-E0 truncated fusion protein and E2-E0 truncated mutant fusion protein are synthesized by Beijing Liuhua big gene technology Co. Wherein, the gene fragment for encoding the E0 protein of the bovine viral diarrhea virus is shown as SEQ ID NO.12, and the amino acid sequence is shown as SEQ ID NO. 11; the gene fragment for encoding the bovine viral diarrhea virus E0 truncated protein is shown as SEQ ID NO.2, and the amino acid sequence is shown as SEQ ID NO. 1; the gene fragment for encoding the E0 truncated mutation of the bovine viral diarrhea virus is shown as SEQ ID NO.8, the amino acid sequence is shown as SEQ ID NO.7, and the E0 truncated mutant protein refers to the mutation of 160 th-162 th amino acid of the E0 truncated protein from IAA to GGG; the gene fragment for encoding the E2 protein of the bovine viral diarrhea virus is shown as SEQ ID NO.4, and the amino acid sequence is shown as SEQ ID NO. 3; the gene fragment for encoding the truncated fusion protein of the 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 fragment for encoding the truncated mutant fusion protein of the 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 identification of expression plasmids
The recombinant plasmids were designated 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 mutation fusion protein), and were synthesized by Beijing Liuhua macrogene technologies Co., ltd: the pcDNA3.1 vector is subjected to double enzyme digestion by using restriction enzymes Xho I and Hind III, and the purified vector fragment is recovered after gel cutting; simultaneously, the recovered and purified E0 protein gene fragment, E0 truncated protein mutant gene fragment, E2 protein fragment, E2-E0 truncated fusion protein gene fragment and E2-E0 truncated fusion protein gene fragment are respectively cut by restriction enzymes Xho I and Hind III, the purified DNA fragments are recovered and then are connected with purified pcDNA3.1 (+) carrier fragments through T4 DNA ligase, the procedure is 16 ℃ for 12 hours, DH5 alpha competent cells are respectively transformed by the connection products, single colony enrichment culture is selected, recombinant plasmids are extracted by a plasmid extraction kit, and the recombinant plasmids are named pcDNA3.1-BVDV-E0, pcDNA3.1-BVDV-tE0, pcDNA3.1-BVDV-mE0, pcDNA3.1-DV-E2, pcDNA3.1-BVDV-E2-mE0; the recombinant plasmid is subjected to double restriction enzyme identification by Xho I and Hind III, and the recombinant plasmid with correct double restriction enzyme identification is subjected to sequencing identification, so that the correct insertion of the target gene sequence is determined, and the complete correct reading frame is ensured.
The recombinant expression plasmids pcDNA3.1-BVDV-E0, pcDNA3.1-BVDV-tE0, pcDNA3.1-BVDV-mE0, pcDNA3.1-BVDV-E2-tE0 and pcDNA3.1-BVDV-E2-mE0 are subjected to double digestion by Xho I and Hind III, the enzyme digestion products are consistent with the theoretical values in size, the amplified E0 fragment, E0 truncated mutant fragment, E2-E0 truncated fragment and E2-E0 truncated mutant fragment are correctly connected with the vector, and the gene sequencing result also shows that the target fragment is successfully inserted into the expression vector.
3. Expression and purification of proteins
Recombinant plasmids pcDNA3.1-BVDV-E0, pcDNA3.1-BVDV-tE0, pcDNA3.1-BVDV-mE0, pcDNA3.1-BVDV-E2-tE0, pcDNA3.1-BVDV-E2-mE0 were transfected into CHO suspension cells at 32℃with 5% CO, respectively, according to the instructions of the ExpiCHO expression system kit 2 After continuing the suspension culture at 90% humidity for 12 days, the cell culture supernatant was collected by centrifugation at 4000g for 10min and subjected to SDS-PAGE identification. The cell culture supernatant collected by centrifugation was filtered through a 0.22 μm filter, and then purified according to the GE company affinity chromatography Ni column. SDS-PAGE identifies the purified target protein and protein concentration is determined using BCA protein quantification kit.
Cell culture supernatants transfected with pcDNA3.1-BVDV-E0 plasmid, pcDNA3.1-BVDV-tE0 plasmid, pcDNA3.1-BVDV-mE0 plasmid, pcDNA3.1-BVDV-E2-tE0 plasmid, pcDNA3.1-BVDV-E2-mE0 plasmid were collected by centrifugation, and SDS-PAGE identification was performed. Purification was performed according to the GE company affinity chromatography Ni column protocol, the expressed protein supernatant was filtered and loaded, and eluted samples were identified by SDS-PAGE, and protein concentration was determined using BCA protein quantification kit.
The result of the identification of the soluble expression of the E2 protein of the bovine viral diarrhea virus is shown in figure 1, and a specific protein band is observed between 45 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 BCA protein quantification kit.
The identification results of the soluble expression of the E0 protein, the tE0 protein and the mE0 protein of the bovine viral diarrhea virus are shown in figure 3, and no specific protein band is observed in the cell culture supernatant transfected with pcDNA3.1-BVDV-E0 plasmid at about 30kD, which indicates that the E0 protein is basically not expressed; whereas cell culture supernatants transfected with pcDNA3.1-BVDV-tE0 plasmid and pcDNA3.1-BVDV-mE0 plasmid observed specific protein bands around 30kD, indicating tE0 protein and mE0 protein were able to be expressed; the SDS-PAGE identification result of the bovine viral diarrhea virus tE0 protein purified sample is shown in FIG. 4: specific protein bands are observed at 25-35kD, and are consistent with the identification result of cell supernatants, and the concentration of the purified tE0 protein fragment is 0.35mg/mL by the BCA protein quantitative kit; the SDS-PAGE identification result of the bovine viral diarrhea virus mE0 protein purified sample is shown in FIG. 5: a specific protein band was observed at 25-35kD, consistent with the identification of cell supernatants, and the concentration of purified mE0 mutein was 0.47mg/mL as determined by BCA protein quantification kit.
The identification result of the soluble expression of the bovine viral diarrhea virus E2-tE0 fusion protein is shown in FIG. 6, and a specific protein band is observed at about 66.2-116kD in the cell culture supernatant transfected with the pcDNA3.1-BVDV-E2-tE0 fusion protein plasmid, which indicates that the E2-tE0 fusion protein is expressed; SDS-PAGE identification result of the bovine viral diarrhea virus E2-tE0 fusion protein purified sample is shown in FIG. 7: a specific protein band was observed at 66.2-116kD, consistent with the identification of cell supernatants, and the concentration of the purified E2-tE0 fusion protein was 1.04mg/mL as determined by BCA protein quantification kit.
The identification result of the soluble expression of the bovine viral diarrhea virus E2-mE0 fusion protein is shown in FIG. 8, and a specific protein band is observed at about 66.2-116kD in the cell culture supernatant transfected with the pcDNA3.1-BVDV-E2-mE0 fusion plasmid, which indicates that the E2-mE0 fusion protein is expressed; SDS-PAGE identification result of the bovine viral diarrhea virus E2-mE0 fusion protein purified sample is shown in FIG. 9: a specific protein band was observed at 66.2-116kD, consistent with the identification of cell supernatants, and the concentration of the purified E2-mE0 fragment mutant fusion protein was 1.23mg/mL as determined by BCA protein quantification kit.
Example 2 immunogenicity detection
The immunogenicity of the serum of rabbits immunized with mE0 protein, E2-tE0 fusion protein and E2-mE0 fusion protein is analyzed by indirect ELISA. Purified BVDV-mE0 protein, BVDV-E2-tE0 fusion protein, BVDV-E2-mE0 fusion protein (50 ng/well) were coated in ELISA plates with 50mM carbonate buffer, respectively, and incubated overnight at 4 ℃. Blocking was performed with 1% Bovine Serum Albumin (BSA) at 37℃for 2h. Serum to be tested was diluted 1:2000 with serum dilution and added to the reaction wells, 100. Mu.L/well, 3 multiplex wells per sample and incubated at 37℃for 1h. HRP-labeled goat anti-rabbit IgG antibodies were diluted 1:5000 in serum diluent and added to the wells and incubated at 37℃for 1h at 100. Mu.L/well. TMB was developed, 50. Mu.L per well, after 10min development, the reaction was stopped by adding 50. Mu.L of stop solution, and the OD450nm was read.
The indirect ELISA results are shown in FIG. 10, and serum antibodies can be detected 7 days after rabbit serum is immunized by mE0 protein, E2-tE0 fusion protein and E2-mE0 fusion protein, and the antibody level is obviously increased after 7 days of secondary immunization and is kept high at the 28 th day after immunization. Compared with the inactivated BVDV antigen group, BVDV-mE0 protein, BVDV-E2-tE0 fusion protein and BVDV-E2-mE0 fusion protein have obviously raised antibody level after immunization; compared with BVDV-mE0 protein and BVDV-E2 protein, the BVDV-E2-tE0 fusion protein and BVDV-E2-mE0 fusion protein adopted by the invention have the advantage that the antibody level is further increased after immunization; the results prove that the E2-mE0 fragment fusion protein expressed by CHO cells has good immunogenicity.
Sequence listing
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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. The bovine viral diarrhea virus E2-E0 fusion protein is characterized in that 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 bovine viral diarrhea virus E0 truncated protein is shown as SEQ ID NO. 1; the amino acid sequence of the E2 protein of the bovine viral diarrhea virus is shown as SEQ ID NO. 3.
2. The bovine viral diarrhea virus E2-E0 fusion protein of claim 1 wherein the bovine viral diarrhea virus E2-E0 fusion protein has an amino acid sequence depicted in SEQ ID No. 5.
3. A gene encoding the bovine viral diarrhea virus E2-E0 fusion protein of claim 2, wherein the gene sequence is depicted in SEQ ID No. 6.
4. The bovine viral diarrhea virus E2-E0 fusion protein of claim 1 wherein the bovine viral diarrhea virus E0 truncate protein comprises an IAA mutation to GGG at amino acids 160-162; the mutated amino acid sequence of the mutated E0 truncated protein of the bovine viral diarrhea virus is shown as SEQ ID NO. 7.
5. The bovine viral diarrhea virus E2-E0 fusion protein of claim 4 wherein the bovine viral diarrhea virus E2-E0 fusion protein has an amino acid sequence depicted in SEQ ID NO. 9.
6. A gene encoding the bovine viral diarrhea virus E2-E0 fusion protein of claim 5, wherein the gene sequence is shown in SEQ ID NO. 10.
7. Use of a bovine viral diarrhea virus E2-E0 fusion protein according to any one of claims 1-2 or claims 4-5 in the preparation of a bovine viral diarrhea virus vaccine.
8. A method of preparing a bovine viral diarrhea virus E2-E0 fusion protein according to any one of claims 1-2 or claims 4-5, comprising: cloning a gene for encoding the bovine viral diarrhea virus E2-E0 fusion protein into a eukaryotic expression vector to obtain a recombinant plasmid for expressing the bovine viral diarrhea virus E2-E0 fusion protein; and then transfecting the recombinant plasmid into CHO cells, 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 a pcdna3.1 vector; the CHO cells are CHO suspension cells.
10. The method of preparation according to claim 9, wherein the method is:
(1) Amplifying a gene fragment encoding bovine viral diarrhea virus E2-E0 fusion protein by PCR, wherein the gene fragment is shown as SEQ ID NO.6 or SEQ ID NO. 10;
(2) Carrying out double enzyme digestion on the 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 fragments with the gene fragments of the bovine viral diarrhea virus E2-E0 fusion protein by using DNA ligase to obtain recombinant plasmid for expressing the bovine viral diarrhea virus E2-E0 fusion protein;
(3) And (3) transfecting the recombinant plasmid in the step (2) into CHO suspension cells, carrying out suspension culture, collecting cell culture supernatant, and purifying to obtain the bovine viral diarrhea virus E2-E0 fusion protein.
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Citations (1)
| 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 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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| 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 |
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