CN114835782B - Swine fever virus E0 truncated protein, preparation method and application - Google Patents
Swine fever virus E0 truncated protein, preparation method and application Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a swine fever virus E0 truncated protein, a preparation method and application thereof. The invention firstly provides a swine fever virus E0 truncated protein, which remarkably promotes the expression of E0 protein, has the yield of 0.38mg/ml, does not influence the immunogenicity of the E0 protein, and can be used for preparing swine fever subunit vaccine; based on the hog cholera virus E0 truncated protein, the invention discovers that the 160 th, 162 th, 165 th and 166 th amino acids of the E0 truncated protein are all mutated into glycine G, so that the expression quantity of the E0 truncated protein can be further improved by more than 30%, and the immunogenicity of the E0 truncated protein is not influenced.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a swine fever virus E0 truncated protein, a preparation method and application thereof.
Background
Hog cholera (classical swine fever, CSF), also known as Hog Cholera (HC), is an acute febrile fatal disease caused by hog cholera virus (Hog Cholera virus, HCV or Classical swine fever virus, CSFV), which is highly contagious, prevalent, high in morbidity and mortality, and extremely harmful. The International animal epidemic agency (OIE) previously identified it as a type A infectious disease, and now listed as a notification epidemic, and China listed it as a type of animal epidemic.
CSFV is a enveloped virus with a virion size of about 40-60nm. The viral genome is a single-stranded positive-strand RNA, is about 12.3kb long, contains a large Open Reading Frame (ORF), encodes a polyprotein of 3898 amino acid residues, and has a molecular weight of about 438kD. The polyprotein is processed into 12 mature viral proteins by proteases of viruses and host cells simultaneously and after translation, and comprises structural proteins and non-structural proteins, wherein the order from N to C ends of the polyprotein is as follows: n (N) pro 、C、E0(E rns ) E1, E2, P7, NS2-3, NS4A, NS4B, NS5A, NS5B. NS2-3 can be processed into NS2, NS3 (P80), except C, E, E1 and E2 which are structural proteins, the rest are non-structural proteins. Processing of the structural proteins is mediated by the host cell's signal peptidase which cleaves the multimeric protein first between the nucleocapsid protein C and the E012 precursor, then cleaves at the C-terminus of E2, and finally E012 is rapidly cleaved into E01 and E2. After release of E2 from the E012 precursor, E01 is processed into E0 and E1, and finally the 3 envelope glycoproteins form complexes via intramolecular or intermolecular disulfide bonds, assembling into a virion structure.
At present, a comprehensive prevention and control strategy mainly comprising prevention is adopted to prevent and control the swine fever, wherein an inactivated vaccine is prepared by a large amount of processes of amplification, inactivation, emulsification and the like of a swine fever field strain through a virus culture system, but the traditional inactivated vaccine has the problems of short immunization duration, risk of virus dispersion due to incomplete virus inactivation, high production cost and the like in the production and preparation process. Based on this, researchers have long been working on the development of CSF subunit vaccines. The E0 protein can induce the organism to generate virus neutralizing antibodies, is an important protective antigen of CSFV, and is also a candidate antigen for the development of CSF 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 CSF subunit vaccine.
Disclosure of Invention
In order to solve the technical problem that CSFV E0 is expressed in CHO cells in low or non-expressed amount, the invention finds that CSFV E0 protein fragments obtained by truncating CSFV E0 protein can be expressed in CHO cells in large amount without affecting immunogenicity, and can be used for preparing CSF subunit vaccine, which comprises the following steps:
in a first aspect, the invention provides a swine fever virus E0 truncated protein, wherein the amino acid sequence of the swine fever virus E0 truncated protein is shown as SEQ ID NO. 1.
In a second aspect, the invention provides a gene sequence for encoding the swine fever virus E0 truncated protein of the first aspect, wherein the gene sequence is shown in SEQ ID NO. 2.
In a third aspect, the present invention provides a swine fever virus E0 truncated protein, wherein amino acids 160, 162, 165 and 166 of the swine fever virus E0 truncated protein of the first aspect are mutated to glycine G; the amino acid sequence of the mutated swine fever virus E0 truncated protein is shown as SEQ ID NO. 3.
In a fourth aspect, the invention provides a gene sequence for encoding the swine fever virus E0 truncated protein of the third aspect, which is characterized in that the gene sequence is shown in SEQ ID NO. 4.
In a fifth aspect, the present invention provides an application of the swine fever virus E0 truncated protein of the first aspect and the third aspect in preparation of swine fever vaccines.
Preferably, the vaccine is a subunit vaccine.
In a sixth aspect, the present invention provides a method for preparing a truncated protein of classical swine fever virus E0 according to the first and third aspects, the method comprising: cloning a gene for encoding the swine fever virus E0 truncated protein into a eukaryotic expression vector to obtain a recombinant plasmid for expressing the swine fever virus E0 truncated protein; and then transfecting the recombinant plasmid into CHO cells, culturing, screening and purifying to obtain the swine fever virus E0 truncated 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 a swine fever virus E0 truncated protein by PCR, wherein the gene fragment is shown as SEQ ID NO.2 or SEQ ID NO. 4;
(2) Double enzyme cutting is carried out on the gene fragments of the vector pcDNA3.1 and the truncated protein of the classical swine fever virus E0 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-CSFV-tE0 for expressing the truncated protein of the classical swine fever virus E0;
(3) The recombinant plasmid pcDNA3.1-CSFV-tE0 is subjected to truncated transfection on CHO suspension cells, suspension culture is carried out, cell culture supernatant is collected, and the swine fever virus E0 truncated protein is obtained after purification.
The beneficial effects of the invention are as follows: (1) when the E0 protein of the swine fever virus is prepared by using 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 the eukaryotic CHO cells with complete glycosylation modification function are used for preparing the classical swine fever virus E0 protein, the target protein is not expressed or the expression quantity is extremely low, so that the research and development of CSF subunit vaccine are seriously hindered under the conditions; in the invention, the E0 protein fragment obtained by truncating the E0 protein of the swine fever virus can be secreted and expressed in a large amount in CHO cells, and the protein yield is as high as 0.38mg/ml; the immunogenicity of the E0 protein is not affected, and the method can be used for preparing CSF subunit vaccine; (2) based on the truncated expressed hog cholera virus E0 protein fragment, the invention discovers that after amino acids 160, 162, 165 and 166 of the hog cholera virus E0 truncated protein are mutated into glycine G, the expression of the hog cholera virus E0 truncated protein is further promoted, the protein expression is improved by more than 30%, and the immunogenicity is not influenced.
Drawings
FIG. 1 shows the results of E0 truncated protein expression of classical swine fever virus E0 protein, E0 truncated protein, and amino acid mutation;
FIG. 2 shows the purification result of the truncated E0 protein of classical swine fever virus;
FIG. 3 purification results of E0 truncated protein of classical swine fever virus amino acid mutation;
FIG. 4 results of indirect ELISA on swine fever virus E0 truncated protein immune rabbit serum antibody;
FIG. 5 results of indirect ELISA on E0 truncated protein immune rabbit serum antibodies mutated in amino acids of classical swine fever virus.
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, the national institute of veterinary sciences, the national institutes of agricultural sciences, the laboratory animal ethics committee, the national institutes of veterinary sciences, the biological safety committee, have submitted permissions in steps according to the relevant requirements of the biological safety class 3 laboratory (BSL-3) and the swine fever related biological safety, and have been filed in the agricultural rural area, conforming to the requirements of the national biological safety class.
The CSFV vaccine standard strain (purchased from China veterinary microbiological culture collection center, china veterinary drug administration), eukaryotic expression vector pcDNA3.1 (+), and CSFV positive serum were stored by the present laboratory. CHO suspension cell expression system, unstained protein MW marker (26610), RT-PCR amplification kit and 6 XHis Tag Monoclonal anti-body (HIS.H8), unstained protein MW marker (26616) and BCA protein quantitative kit, alexa488-labeled goat anti-rabbit secondary antibodies were purchased from Thermo Fisher Scientific company. Nickel affinity chromatography 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 E0 truncated protein of classical swine fever Virus and E0 truncated protein with amino acid mutation
Gene fragment amplification of E0 protein
Gene fragments of E0 truncated protein of the hog cholera virus E0 protein, E0 truncated protein and amino acid mutation are synthesized by Beijing Liuhua big gene technology limited company, and are identified to be correct by nucleic acid gel electrophoresis. Wherein the gene fragment for encoding the E0 protein of the swine fever virus 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 swine fever 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 of the swine fever virus E0 truncated protein with the coded amino acid mutation is shown as SEQ ID NO.4, and the amino acid sequence is shown as SEQ ID NO. 3; wherein, the amino acid mutated swine fever virus E0 truncated protein refers to that amino acids at 160 th, 162 th, 165 th and 166 th of the swine fever virus E0 truncated protein are mutated into glycine G.
2. Construction and identification of expression plasmids
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 synthesized E0 protein, E0 truncated protein and gene fragments of the E0 truncated protein with mutation of amino acid are respectively cut by restriction enzymes Xho I and Hind III, recovered and purified, the purified and recovered gene fragments and purified pcDNA3.1 vector fragments are respectively connected 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 respectively named pcDNA3.1-CSFV-E0, pcDNA3.1-CSFV-tE0 and pcDNA3.1-CSFV-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 size of the enzyme cutting products accords with the theoretical value after the constructed recombinant expression plasmids pcDNA3.1-CSFV-E0, pcDNA3.1-CSFV-tE0 (E0 truncated protein) and pcDNA3.1-CSFV-mE0 (E0 truncated mutant protein) are subjected to double enzyme cutting by Xho I and Hind III, so that the amplified gene fragments such as E0 protein, E0 truncated protein and E0 truncated protein with amino acid mutation 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
3 recombinant expression plasmids were separately transfected into CHO suspension cells at 32℃with 5% CO, 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 collected cell culture supernatant was centrifuged, filtered through a 0.22 μm filter, and purified according to the GE company nickel affinity chromatography column. SDS-PAGE identifies the purified target protein and protein concentration is determined using BCA protein quantification kit.
Centrifuging to collect cell culture supernatants of 3 transfected recombinant expression plasmids, and performing SDS-PAGE identification, wherein the result is shown in figure 1, the cell culture supernatant transfected pcDNA3.1-CSFV-E0 plasmid does not observe specific target protein bands at about 30kD, which indicates that E0 protein is not expressed basically; and cell culture supernatants transfected with pcDNA3.1-CSFV-tE0 plasmid and pcDNA3.1-CSFV-mE0 plasmid both observe specific target protein bands around 30kD, indicating that both E0 truncated protein and E0 truncated protein with amino acid mutation can be expressed normally.
The expressed E0 truncated protein and the E0 truncated protein mutated in amino acid were purified by using a nickel affinity chromatography column, and eluted protein samples were identified by SDS-PAGE, and the results are shown in FIG. 2 and FIG. 3: a specific target protein band was observed at a position around 30kD, which was consistent with the identification of cell supernatants. The concentration of the E0 truncated protein after purification was measured using a BCA protein quantification kit, and the amount of the target protein secreted and expressed per unit culture volume was calculated, wherein the E0 truncated protein expression amount was about 0.38mg/mL, and the amino acid mutant E0 truncated protein expression amount was about 0.51mg/mL.
Example 2 immunogenicity detection
The immunogenicity of E0 truncated protein and E0 truncated protein immune rabbit serum mutated by amino acid is analyzed by indirect ELISA. Purified 2 target proteins (50 ng/well) were individually coated in ELISA plates with 50mM carbonate buffer 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.
As shown in FIG. 4, the results of the CSFV-E0 truncated protein indirect ELISA are shown, serum antibodies against E0 protein can be detected 7 days after immunization, the antibody level is remarkably increased after 7 days of secondary immunization, the antibody level is kept high on day 28 after immunization, and the antibody level is slightly higher than that of inactivated CSFV antigen. The results prove that the E0 truncated protein expressed by the CHO cells has good immunogenicity.
The results of the indirect ELISA of the CSFV-E0 truncated protein with the mutation of the amino acid are shown in figure 5, the serum antibody against the E0 protein can be detected 7 days after immunization, the antibody level is obviously increased after 7 days of secondary immunization, and the antibody is kept at a higher level on the 28 th day after immunization. The antibody levels were increased compared to the inactivated CSFV antigen, comparable to the CSFV-E0 truncated protein. The results prove that the E0 truncated protein with the amino acid mutation expressed by CHO cells has good immunogenicity.
Sequence listing
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gccagcgagg ggacaaacta tacgtgctgt aagttacaga gacatgaatg gaacaaacat 240
ggatggtgta actggtacaa tatagaccca tggatacagt tgatgaatag aacccaagca 300
aacttggcag aaggccctcc ggccaaggag tgcgctgtga cttgcaggta cgataaagat 360
gccgacatca acgtggtcac ccaggccaga aacaggccaa caaccctgac cggctgcaag 420
aaaggaaaaa atttttcttt tgcgggtaca attatagagg gcccatgtaa tttcgatgtt 480
tccgtggagg atatcttgta tggggatcat 510
<210> 3
<211> 170
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 3
Ala Glu Asn Ile Thr Gln Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly
1 5 10 15
Ile Gln His Ala Met Tyr Leu Arg Gly Val Ser Arg Ser Leu His Gly
20 25 30
Ile Trp Pro Glu Lys Ile Cys Lys Gly Val Pro Thr Tyr Leu Ala Thr
35 40 45
Asp Thr Glu Leu Lys Glu Ile Gln Gly Met Met Asp Ala Ser Glu Gly
50 55 60
Thr Asn Tyr Thr Cys Cys Lys Leu Gln Arg His Glu Trp Asn Lys His
65 70 75 80
Gly Trp Cys Asn Trp Tyr Asn Ile Asp Pro Trp Ile Gln Leu Met Asn
85 90 95
Arg Thr Gln Ala Asn Leu Ala Glu Gly Pro Pro Ala Lys Glu Cys Ala
100 105 110
Val Thr Cys Arg Tyr Asp Lys Asp Ala Asp Ile Asn Val Val Thr Gln
115 120 125
Ala Arg Asn Arg Pro Thr Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn
130 135 140
Phe Ser Phe Ala Gly Thr Ile Ile Glu Gly Pro Cys Asn Phe Asp Gly
145 150 155 160
Ser Gly Glu Asp Gly Gly Tyr Gly Asp His
165 170
<210> 4
<211> 510
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
gccgaaaata taactcaatg gaacctgagt gataacggca ctaatggtat tcagcatgct 60
atgtacctta gaggggttag cagaagcttg catgggatct ggccggaaaa aatatgcaaa 120
ggagtcccca cctacctggc cacagacacg gaactgaaag aaatacaggg aatgatggat 180
gccagcgagg ggacaaacta tacgtgctgt aagttacaga gacatgaatg gaacaaacat 240
ggatggtgta actggtacaa tatagaccca tggatacagt tgatgaatag aacccaagca 300
aacttggcag aaggccctcc ggccaaggag tgcgctgtga cttgcaggta cgataaagat 360
gccgacatca acgtggtcac ccaggccaga aacaggccaa caaccctgac cggctgcaag 420
aaaggaaaaa atttttcttt tgcgggtaca attatagagg gcccatgtaa tttcgatggc 480
tccggcgagg atggcggcta tggggatcat 510
<210> 5
<211> 233
<212> PRT
<213> classical swine fever virus (Classical swine fever virus)
<400> 5
Ala Glu Asn Ile Thr Gln Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly
1 5 10 15
Ile Gln His Ala Met Tyr Leu Arg Gly Val Ser Arg Ser Leu His Gly
20 25 30
Ile Trp Pro Glu Lys Ile Cys Lys Gly Val Pro Thr Tyr Leu Ala Thr
35 40 45
Asp Thr Glu Leu Lys Glu Ile Gln Gly Met Met Asp Ala Ser Glu Gly
50 55 60
Thr Asn Tyr Thr Cys Cys Lys Leu Gln Arg His Glu Trp Asn Lys His
65 70 75 80
Gly Trp Cys Asn Trp Tyr Asn Ile Asp Pro Trp Ile Gln Leu Met Asn
85 90 95
Arg Thr Gln Ala Asn Leu Ala Glu Gly Pro Pro Ala Lys Glu Cys Ala
100 105 110
Val Thr Cys Arg Tyr Asp Lys Asp Ala Asp Ile Asn Val Val Thr Gln
115 120 125
Ala Arg Asn Arg Pro Thr Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn
130 135 140
Phe Ser Phe Ala Gly Thr Ile Ile Glu Gly Pro Cys Asn Phe Asp Val
145 150 155 160
Ser Val Glu Asp Ile Leu Tyr Gly Asp His Glu Cys Gly Ser Leu Leu
165 170 175
Gln Asp Thr Ala Leu Tyr Leu Val Asp Gly Met Thr Asn Thr Ile Glu
180 185 190
Asn Ala Arg Gln Gly Ala Ala Arg Val Thr Ser Trp Leu Gly Arg Gln
195 200 205
Leu Ser Thr Ala Gly Lys Arg Leu Glu Gly Arg Ser Lys Thr Trp Phe
210 215 220
Gly Ala Tyr Ala Leu Ser Pro Tyr Cys
225 230
<210> 6
<211> 699
<212> DNA
<213> classical swine fever virus (Classical swine fever virus)
<400> 6
gccgaaaata taactcaatg gaacctgagt gataacggca ctaatggtat tcagcatgct 60
atgtacctta gaggggttag cagaagcttg catgggatct ggccggaaaa aatatgcaaa 120
ggagtcccca cctacctggc cacagacacg gaactgaaag aaatacaggg aatgatggat 180
gccagcgagg ggacaaacta tacgtgctgt aagttacaga gacatgaatg gaacaaacat 240
ggatggtgta actggtacaa tatagaccca tggatacagt tgatgaatag aacccaagca 300
aacttggcag aaggccctcc ggccaaggag tgcgctgtga cttgcaggta cgataaagat 360
gccgacatca acgtggtcac ccaggccaga aacaggccaa caaccctgac cggctgcaag 420
aaaggaaaaa atttttcttt tgcgggtaca attatagagg gcccatgtaa tttcgatgtt 480
tccgtggagg atatcttgta tggggatcat gagtgcggca gtttgctcca ggacacggct 540
ctgtacctag tagatggaat gaccaacact atagagaatg ccagacaggg agcagccagg 600
gtaacatctt ggctcgggag gcaactcagc actgccggga agaggttgga gggtagaagc 660
aaaacctggt ttggtgccta tgccctatcg ccttactgt 699
Claims (8)
1. The swine fever virus E0 truncated protein is characterized in that the amino acid sequence of the swine fever virus E0 truncated protein is shown as SEQ ID NO. 3.
2. A gene encoding the swine fever virus E0 truncated protein of claim 1, wherein the sequence of the gene is shown in SEQ ID No. 4.
3. Use of a truncated protein of classical swine fever virus E0 as claimed in claim 1 for the preparation of a classical swine fever vaccine or a classical swine fever diagnostic formulation.
4. The use of claim 3, wherein the vaccine is a subunit vaccine.
5. A method of preparing a truncated protein of classical swine fever virus E0 of claim 1, comprising: cloning a gene for encoding the swine fever virus E0 truncated protein into a eukaryotic expression vector to obtain a recombinant plasmid for expressing the swine fever virus E0 truncated protein; and then transfecting the recombinant plasmid into CHO cells, culturing, screening and purifying to obtain the swine fever virus E0 truncated protein.
6. The method of claim 5, wherein the eukaryotic expression vector is pcDNA3.1 vector.
7. The method of claim 6, wherein the CHO cells are CHO suspension cells.
8. The preparation method according to claim 6, wherein the method comprises:
(1) Amplifying a gene fragment encoding a swine fever virus E0 truncated protein by PCR, wherein the gene fragment is shown as SEQ ID NO. 4;
(2) Double-enzyme cutting is carried out on the vector pcDNA3.1 and the gene fragment of the truncated protein of the classical swine fever virus E0 by using restriction endonucleases Xho I and Hind III respectively, and the enzyme cutting fragment and the gene fragment of the truncated protein of the classical swine fever virus E0 are connected by using DNA ligase to obtain a recombinant plasmid pcDNA3.1-CSFV-tE0 for expressing the truncated protein of the classical swine fever virus E0;
(3) Transfecting CHO suspension cells with recombinant plasmid pcDNA3.1-CSFV-tE0, performing suspension culture, collecting cell culture supernatant, and purifying to obtain swine fever virus E0 truncated protein.
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| CN109182248A (en) * | 2018-09-10 | 2019-01-11 | 青岛易邦生物工程有限公司 | A kind of antibody against swine fever virus detection ELISA diagnostic kit |
| CN111973738A (en) * | 2020-09-02 | 2020-11-24 | 天康生物股份有限公司 | Nucleic acid and recombinant protein co-immune vaccine based on hog cholera virus gene, preparation method and application |
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| CN213933883U (en) * | 2020-11-23 | 2021-08-10 | 浙江洪晟生物科技股份有限公司 | Hog cholera virus E2& E0 antibody detection kit |
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