CN116284272B - Broad-spectrum mRNA vaccine for resisting bovine viral diarrhea virus and application thereof - Google Patents
Broad-spectrum mRNA vaccine for resisting bovine viral diarrhea virus and application thereof Download PDFInfo
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Abstract
The invention relates to a broad-spectrum mRNA vaccine for resisting Bovine Viral Diarrhea Virus (BVDV), a preparation method and application thereof. In particular to trivalent BVDV mRNA aiming at BVDV strains with multiple genotypes, which sequentially comprises a bovine IgG1 secretion signal peptide, a bovine IgG1 Fc fragment, BVDV1a E protein domains I-II, BVDV1b E2 protein domains I-II and BVDV 2E 2 protein domains I-II from the N end to the C end. The invention can simultaneously express antigens of BVDV with different genotypes by using one mRNA, has strong immunogenicity, is effectively secreted at a high level in an animal body, thereby inducing and generating a higher level of specific neutralizing antibodies aiming at BVDV1a, BVDV1b and BVDV2, and can realize ideal immune effect aiming at various BVDV genotype strains.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to the field of animal vaccines, and particularly relates to a broad-spectrum mRNA vaccine for resisting bovine viral diarrhea virus and application thereof.
Background
Bovine viral diarrhea virus (Bovine viral diarrhea virus, BVDV, also known as bovine viral diarrhea virus) is the primary pathogen causing bovine viral diarrhea, which is widely found in wild and domestic ruminant bodies. BVDV belongs to the genus pestivirus and the family flaviviridae. Clinically BVDV mainly causes diarrhea, reproductive disorders, immunosuppression, mucosal erosion necrosis, and other symptoms. The virus exists widely worldwide, and brings great loss to the breeding industry. Especially BVDV can exist and spread in cattle widely and continuously, and seriously influence the production capacity of cattle, so that the milk yield is reduced, the dairy quality is reduced, the growth is delayed, the dysplasia is caused, and the like.
BVDV has mainly two genotypes, BVDV1 and BVDV2, respectively. Wherein BVDV1 is a classical strain comprising BVDV1a, BVDV1b, BVDV 1e, BVDV 1g, BVDV 1h and other gene subtypes. BVDV1 mainly causes diarrhea, and the clinical detection rate is significantly higher than BVDV2.BVDV2 can cause diarrhea as well, but BVDV2 has stronger toxicity and can cause acute onset, thrombocytopenia, bleeding syndrome and other symptoms of adult cattle.
At present, the separated strains in China are mainly BVDV1a gene subtype, BVDV1b gene subtype and BVDV2 genotype. Most of the vaccines on the current market aim at BVDV1a and BVDV1b, but the total potency is low and the protection scope is limited, in addition, no vaccine with better effect on BVDV2 genotype is available, and the cross protection of type 1 and type 2 is weak, so that the vaccine cannot resist the infection of virulent viruses. BVDV is a single-stranded positive-strand RNA encoding a total of 4 structural proteins (C, E, E1 and E2, respectively) and 8 non-structural proteins (Npro, P7, NS2, NS3, NS4A, NS4B, NS5A and NS5B, respectively). The E2 protein is an important structural protein of BVDV, contains main antigenic determinants, has strong immunogenicity, can induce the generation of neutralizing antibodies, and can effectively prevent BVDV infection. It was found that the E2 protein has a low homology between BVDV1 and BVDV2, resulting in monovalent E2 vaccines that can only prevent BVDV strains of the same type. The extracellular domain portion of the E2 protein is largely divided into three domains, with domains I-II exposed on the viral surface. Therefore, the development of multivalent vaccines capable of simultaneously preventing BVDV type 1 and BVDV type 2 is particularly important for preventing and controlling bovine viral diarrhea diseases.
Various BVDV vaccines have been disclosed in the prior art. Patent document CN115381935a (publication date 2022, 11, 25 days) discloses a method for preparing an inactivated BVDV vaccine, comprising the steps of: and mixing BVDV strain virus liquid and hydrogen peroxide solution until the concentration of hydrogen peroxide is 1% -3%, inactivating the BVDV strain virus liquid to obtain BVDV strain inactivated virus liquid, mixing the BVDV strain inactivated virus liquid with Freund's adjuvant, and emulsifying to obtain the BVDV inactivated vaccine. Patent document CN110331155B (publication date 2022, 5 and 13 days) discloses a construction method of a high-fertility swine fever attenuated marker vaccine carrying 2 BVDV-Erns genes. The Erns gene of BVDV2-890# strain and E2 gene VR1 region of CSFV epidemic strain QZ14 are substituted for corresponding gene fragment of CSFV-C strain by utilizing molecular cloning and reverse genetic method, and 7 specific mutation sites are introduced to obtain recombinant virus rC-Marker2. The obtained recombinant virus has a molecular marker (BVDV 2 Erns), VR1 of a classical swine fever virus type 2 epidemic strain and a specific mutation site. However, the disadvantages of inactivated vaccines mainly include short duration of the immune effect, relatively long vaccination time and the need for multiple vaccinations, as well as the presence of certain adverse effects. The disadvantage of recombinant gene vaccines is the safety problem, the possibility of mutation caused by insertion of a small number of plasmid DNA into the chromosome cannot be completely excluded, and in addition, the problem of immune tolerance is caused, and the antigen protein is continuously produced in the gene vaccine, so that the immune balance of the organism itself can be broken, and the immune tolerance is caused.
The production cycle of inactivated vaccines and recombinant protein vaccines is long, and the process is complex and cannot meet the large-scale inoculation requirement of sudden pandemic. mRNA is a rapid reaction vaccine development platform for coping with explosive epidemic situations. In recent years, mRNA (messenger ribonucleic acid) vaccines developed by some companies at home and abroad start clinical trials, and early data of the safety and effectiveness of mRNA vaccines are provided. mRNA vaccine is synthesized by using linearized plasmid DNA as a template and performing enzyme transcription reaction in vitro, and the synthesis strategy avoids the problems of safety, complex production process and the like which are required to be considered in a living cell culture production mode. The mRNA vaccine platform has the characteristics of safety, effectiveness, short production period and simple process, and is particularly suitable for coping with explosive epidemic situations. It is known in the art that mRNA is translated rapidly, takes effect rapidly, and has the effect of activating immune response; meanwhile, mRNA medicine is simple to produce, easy to reform, quick to synthesize and low in cost; more importantly, mRNA drugs are not limited to dividing cells, have no risk of integration into the host genome, and are subject to automatic degradation in vivo. Therefore, the development of mRNA vaccines is of great advantage. However, multivalent mRNA vaccines effective for BVDV control were not retrieved. There is a need in the art for a fusion protein multivalent antigen BVDV mRNA vaccine that can express multiple BVDV genotypes simultaneously, has strong immunogenicity, and can prevent or treat infection caused by multiple BVDV genotype strains, thereby effectively preventing and controlling bovine viral diarrhea disease, and reducing losses in agriculture and animal husbandry.
Disclosure of Invention
In order to overcome the defects of BVDV inactivated vaccines, recombinant protein vaccines, recombinant gene vaccines and the like in the prior art, the invention provides a tandem antigen fusion protein for simultaneously expressing E2 protein domains I-II of three BVDV genotypes of BVDV1a, BVDV1b and BVDV2, nucleic acid and mRNA encoding the same, trivalent BVDV vaccines containing the mRNA, related preparation methods, applications and the like. The present invention provides a more effective trivalent BVDV mRNA vaccine against multiple genotypes of BVDV strains, as opposed to strategies that express the E2 protein of one BVDV genotype alone, is an antigen selection strategy that is more beneficial for industrial applications. In addition, the antigen fusion protein of the invention fuses a dissolution-assisting secretion-assisting structural domain IgG1Fc, so that the immunogenicity of the E2 protein is obviously enhanced, and the neutralizing antibody against BVDV generated in a subject to be administered is increased.
One aspect of the present invention provides a BVDV antigen fragment comprising 2 immunoglobulin-like (Ig-like) domains of BVDV E2 protein near the N-terminus, i.e., BVDV E2 protein domains I-II, the spatial structural position of which is schematically shown in FIG. 4, and the referenced BVDV E2 protein structure (PDB: 4 ILD), wherein the 2 immunoglobulin-like (Ig-like) domains correspond to the Dom.I and Dom.II in FIG. 4. The immunoglobulin-like domain has self-stabilizing characteristics, and the formed spatial structure is not affected by upstream and downstream sequences; the immunoglobulin-like domain comprises a β -sheet structure in greenk key topology conformation.
Further, the BVDV antigen fragment comprises one or more of BVDV1a E protein domain I-II, BVDV1b E protein domain I-II, and/or BVDV 2E 2 protein domain I-II.
One aspect of the present invention provides a BVDV antigen fusion fragment comprising an IgG1 Fc fragment fused to one or more of BVDV1a E protein domains I-II, BVDV1b E protein domains I-II and/or BVDV 2E 2 protein domains I-II.
Further, the antigen fusion fragment is characterized in that the IgG1 Fc fragment is a bovine IgG1 Fc fragment.
One aspect of the present invention provides a trivalent BVDV antigen fusion fragment comprising an IgG1 Fc fragment, BVDV1a E protein domains I-II, BVDV1b E protein domains I-II, and BVDV 2E 2 protein domains I-II.
Further, the trivalent BVDV antigen fusion fragment is characterized by comprising a bovine IgG1 Fc fragment, a BVDV1a E protein domain I-II, a BVDV1b E protein domain I-II and a BVDV 2E 2 protein domain I-II from the N-terminal to the C-terminal in sequence.
Further, the trivalent BVDV antigen fusion fragment is characterized by further comprising a secretion signal peptide.
In one aspect, the present invention provides a BVDV antigen fusion protein comprising any one of the following sequences:
(I) An amino acid sequence as shown in SEQ ID NO. 1;
(II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1, and has the same function as the amino acid sequence shown in (I); or,
(III) an amino acid sequence having at least 90% sequence identity to the sequence of (I) or (II), and having the same function as the amino acid sequence of (I).
In one aspect, the invention provides a recombinant nucleic acid comprising a nucleic acid encoding said BVDV antigen fragment, or said BVDV antigen fusion fragment, or said trivalent BVDV antigen fusion fragment, or said BVDV antigen fusion protein.
Further, the recombinant nucleic acid is characterized by comprising any one of the following sequences:
(I) A nucleic acid sequence as shown in SEQ ID NO. 2;
(II) a nucleic acid sequence obtained by substituting, deleting or adding one or more nucleotides to the nucleic acid sequence shown in SEQ ID NO.2, and has the same function as the nucleic acid sequence shown in (I); or,
(III) a nucleic acid sequence having at least 90% sequence identity with the sequence of (I) or (II) and having the same function as the nucleic acid sequence of (I).
One aspect of the present invention provides a recombinant mRNA comprising an mRNA encoding said BVDV antigen fragment, or said BVDV antigen fusion fragment, or said trivalent BVDV antigen fusion fragment, or said BVDV antigen fusion protein.
Further, the recombinant mRNA is characterized in that the mRNA further comprises one or more of 5 '-UTR, 3' -UTR, polyA sequence and IRES sequence.
Further, the recombinant mRNA is characterized in that the sequence of the 5 '-UTR is shown as SEQ ID NO.3, the sequence of the 3' -UTR is shown as SEQ ID NO.4, the sequence of the polyA is shown as SEQ ID NO.5, and the IRES sequence is shown as SEQ ID NO. 8.
One aspect of the present invention provides an expression cassette comprising said recombinant nucleic acid, or said recombinant mRNA.
One aspect of the present invention provides a vector comprising said recombinant nucleic acid, or said recombinant mRNA, or said expression cassette; preferably, the vector comprises a self-replicating vector; more preferably, the self-replicating vector comprises a VEE self-replicating vector; most preferably, the sequence of the vector comprises the sequence shown in SEQ ID No.7, SEQ ID No.9, or SEQ ID No. 11.
In one aspect, the invention provides a cell comprising said recombinant nucleic acid, or said recombinant mRNA, or said expression cassette, or said vector.
In one aspect, the present invention provides a recombinant bacterium comprising the recombinant nucleic acid, or the recombinant mRNA, or the expression cassette, or the vector; preferably, the bacterium is escherichia coli; more preferably, whatThe Escherichia coli isE. coli BL21(DE3)、E. coli Origami B (DE 3) orE. coli One of Rosetta Blue (DE 3); most preferably, the E.coli isE. coli BL21(DE3)。
In one aspect, the invention provides a composition comprising said BVDV antigen fragment, or said BVDV antigen fusion fragment, or said trivalent BVDV antigen fusion fragment, or said BVDV antigen fusion protein, or said recombinant nucleic acid, or said recombinant mRNA, or said expression cassette, or said vector, or said cell, or recombinant bacterium.
In one aspect, the invention provides a kit comprising said BVDV antigen fragment, or said BVDV antigen fusion fragment, or said trivalent BVDV antigen fusion fragment, or said BVDV antigen fusion protein, or said recombinant nucleic acid, or said recombinant mRNA, or said expression cassette, or said vector, or said cell, or said recombinant bacterium, or said composition.
One aspect of the invention provides a trivalent BVDV mRNA vaccine comprising:
(a) The recombinant mRNA; and
(b) A pharmaceutically acceptable carrier; preferably, the carrier comprises a lipid; more preferably, the lipid comprises one or more of cationic lipids, ionizable lipids, helper lipids, cholesterol, DMG-PEG 2000.
One aspect of the present invention provides a method for preparing a capped mRNA recombinant expression vector, a non-capped mRNA recombinant expression vector or a self-replicating RNA recombinant expression vector for expressing a recombinant trivalent BVDV mRNA, comprising the steps of:
step a: synthesizing a gene fragment of the recombinant nucleic acid;
step b: constructing a capped mRNA architecture vector, constructing a non-capped mRNA architecture vector, or constructing a VEE self-replicating vector;
the capping mRNA framework vector comprises 5 '-UTR, 3' -UTR and polyA sequences and a vector for fixing the capping mRNA framework, and the sequences are respectively shown as SEQ ID NO. 3-6; or,
the non-capping mRNA framework vector comprises an IRES sequence, a 3' -UTR and a polyA sequence, and a vector for fixing the non-capping mRNA framework, wherein the sequence of the IRES is shown as SEQ ID No. 8; or,
construction of VEE self-replicating vector: self-replicating RNA sequences obtained from the genome of the alphavirus family; the self-replicating RNA sequence comprises a gene encoding an alphavirus self-replicating component, lacking a gene that produces a structural protein of an infectious alphavirus particle; amplifying a plasmid constructed by the self-replicating RNA sequence as a template, and synthesizing to obtain the VEE self-replicating vector; the sequence of the VEE self-replication vector is shown as SEQ ID NO. 10;
Step c: preparing capped mRNA, uncapped mRNA or self-replicating RNA recombinant expression vector;
c, inserting the gene synthesized in the step a into a capping mRNA framework vector to obtain a capping mRNA recombinant expression vector, wherein the sequence of the capping mRNA recombinant expression vector is shown as SEQ ID NO. 7; or,
inserting the gene synthesized in the step a into a non-capping mRNA framework vector to obtain a non-capping mRNA recombinant expression vector, wherein the sequence of the non-capping mRNA recombinant expression vector is preferably shown as SEQ ID NO. 9; or,
and c, inserting the gene synthesized in the step a into a designated position of the VEE self-replication vector to obtain a self-replication RNA recombinant expression vector, wherein the sequence of the self-replication RNA recombinant expression vector is preferably shown as SEQ ID NO. 11.
One aspect of the present invention provides a method for preparing a trivalent BVDV mRNA vaccine, comprising:
(a) Providing the recombinant mRNA;
(b) The mRNA is mixed with lipid particles and incubated, thereby forming mRNA-encapsulated lipid nanoparticles.
One aspect of the present invention provides a method for preparing a trivalent BVDV capped mRNA structured vector vaccine, a non-capped mRNA structured vector vaccine, or a self-replicating RNA structured vector vaccine, comprising the steps of:
Step a: tangentially cutting the capping mRNA recombinant expression vector, the non-capping mRNA recombinant expression vector or the self-replicating RNA recombinant expression vector by using restriction enzyme;
step b: carrying out in vitro co-transcription capping reaction on the linearized capping mRNA recombinant expression vector, adding a 7-methylated guanylate cap structure to the 5' end of transcribed mRNA, and degrading template DNA; or,
carrying out in-vitro uncapped transcription reaction on the linearized uncapped mRNA recombinant expression vector, and degrading template DNA; or,
carrying out in vitro co-transcription capping reaction on the linearized self-replicating RNA recombinant expression vector, adding a 7-methylated guanylate cap structure to the 5' -end of transcribed RNA, and degrading template DNA;
step c: mixing the capped, uncapped and self-replicating mRNA prepared in the step b with lipid respectively, wrapping the mRNA into LNP to obtain mRNA-LNP complex, concentrating and changing liquid to obtain preparation solution, thus obtaining the vaccine.
One aspect of the invention provides the use of said BVDV antigen fragment, or said BVDV antigen fusion fragment, or said trivalent BVDV antigen fusion fragment, or said BVDV antigen fusion protein, or said recombinant mRNA in any one of the following: (I) Preparing a medicament for treating or preventing diseases caused by BVDV infection; or, (II) preparing a medicament for treating or preventing bovine viral diarrhea; or (III) preparing a medicament that induces a specific immune response against BVDV in the subject.
The broad-spectrum mRNA vaccine for resisting bovine viral diarrhea virus has the following beneficial technical effects:
1. the invention can simultaneously express antigens of BVDV with different genotypes by using one mRNA, has strong immunogenicity, is effectively secreted in animals at high level, thereby inducing and generating higher level specific neutralizing antibodies aiming at BVDV1a, BVDV1b and BVDV2, and can realize ideal immune effect aiming at BVDV genotype strains such as BVDV1a, BVDV1b and BVDV 2. Therefore, the trivalent BVDV mRNA vaccine can effectively block cross infection caused by various BVDV strains, and reduce the loss caused to the breeding industry.
2. The antigen fusion protein of the invention fuses a structure domain IgG1 Fc which is used for assisting dissolution and secretion, thus obviously enhancing the immunogenicity of E2 protein and increasing the neutralizing antibody which is generated in vivo and aims at BVDV. Thus, a protective effect can be achieved with a smaller vaccine dose. Therefore, the dosage of the inoculation is reduced correspondingly, the using amount of the liposome is also reduced, and further, the cytotoxicity generated by the liposome is reduced, so that the immunization effect is ensured, and meanwhile, the toxicity of the vaccine is reduced.
3. The mRNA-based vaccine has strong immune response in a mouse experiment, and mRNA has an adjuvant effect, so that the mRNA-based vaccine can induce obvious immune response.
4. The invention relates to a trivalent BVDV mRNA vaccine, which is synthesized by taking linearized plasmid DNA as a template and performing an enzyme transcription reaction in vitro, wherein the synthesis strategy avoids the problems of safety, complex production process and the like which are not considered in a living cell culture production mode.
5. The trivalent BVDV mRNA vaccine of the invention relates to an mRNA universal vaccine platform, and can select a new antigen sequence according to the annual or seasonal variation of strains, and rapidly develop a new vaccine under the condition of not changing the process flow.
Drawings
FIG. 1 is a schematic diagram of the structure of a BVDV trivalent vaccine of the invention, wherein BVDV antigen fusion protein mRNA is secretion signal peptide+bovine IgG1 Fc+BVDV1a E protein domain I-II+BVDV1b E2 protein domain I-II+BVDV 2E 2 protein domain I-II.
FIGS. 2A-2D are graphs showing the results of Western Blot and Dot Blot detection of mRNA and control BVDV antigen fusion proteins of the invention, wherein FIG. 2A is a graph showing the expression of Dot Blot protein of mRNA of the invention, i.e., secretion signal peptide+bovine IgG1 Fc+BVDV1a domain I-II+BVDV1b domain I-II+BVDV2 domain I-II, after in vitro transfection of HEK293T cells, bovine IgG1 Fc+BVDV1a domain I-II+BVDV1b domain I-II+BVDV2 domain I-II; FIG. 2B is a Western Blot protein expression pattern of bovine IgG1 Fc+BVDV1a domain I-II+BVDV1B domain I-II+BVDV2 domain I-II following in vitro transfection of HEK293T cells with mRNA of the invention; FIG. 2C is a graph of Western Blot protein expression following in vitro transfection of HEK293T cells with mRNA (Fc+) of the present invention and a control (not containing bovine IgG1 Fc, i.e., fc-); FIG. 2D is a graph of Dot blot protein expression following in vitro transfection of HEK293T cells with mRNA (Fc+) of the present invention and a control (not containing bovine IgG1 Fc, i.e., fc-).
FIG. 3 is a schematic diagram showing the immunization and sampling process of mice with BVDV antigen fusion protein mRNA.
FIG. 4 is a schematic representation of the spatial structural position of the BVDV E2 domain I-II of the invention, the BVDV E2 protein structure (PDB: 4 ILD) being referred to.
Detailed Description
Definitions and terms
The term "bovine" refers to bovine animals including, but not limited to, eating cows, bulls, ungenerated heifers, cows and calves. Cattle as used herein refer to pregnant and lactating bovine animals. Preferably, the method of the invention is administered to a non-human mammal; preferably, the lactating or pregnant cow and its foetus or lactating calf.
The term "BVDV" refers to bovine viral diarrhea virus, bovine viral diarrhea virus, also known as bovine viral diarrhea virus, BVDV has mainly two genotypes, BVDV1 and BVDV2, respectively. Wherein BVDV1 is a classical strain, mainly causes diarrhea, and has a clinical detection rate obviously higher than BVDV2.BVDV2 can cause diarrhea similarly, but BVDV2 has stronger virulence and can cause acute onset of adult cattle, thrombocytopenia, and symptoms such as bleeding syndrome. BVDV1 comprises BVDV1a, BVDV1b and other genotypes.
The term "immunogenic" refers to the ability of BVDV to elicit an immune response in an animal against either type 1 or type 2 BVDV or against both type 1 and type 2 BVDV. The immune response can be a cellular immune response mediated primarily by cytotoxic T cells or a humoral immune response mediated primarily by helper T cells, whereby activation of B cells results in antibody production.
The term "administration" or "vaccination" means that the mRNA, vaccine composition of the invention is preferably administered to the cow via an intramuscular or subcutaneous route, although other routes of administration can be used, such as, for example, oral, intranasal (e.g. aerosol or other non-injection administration), intralymphatic, intradermal, intraperitoneal, rectal or vaginal administration, or by a combined route. Intramuscular administration to the neck of the animal is preferred. An acceleration regimen (boosting regimens) can be employed and the dosing regimen can be adjusted to provide optimal immunity.
The term "expression" includes any step involving the production of a polypeptide, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
The term "recombinant nucleic acid molecule" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide may be included in a suitable vector, and the vector may be used for transformation into a suitable host cell. The polynucleotide is then expressed in a recombinant host cell to produce, for example, "recombinant polypeptides," "recombinant proteins," "fusion proteins," and the like.
The term "recombinant expression vector" refers to a DNA structure used to express, for example, a polynucleotide encoding a desired polypeptide. Recombinant expression vectors can include, for example, vectors comprising (1) a collection of genetic elements, such as promoters and enhancers, that regulate gene expression; (2) A structure or coding sequence transcribed into mRNA and translated into protein; and (3) transcriptional subunits of appropriate transcriptional and translational initiation and termination sequences. The recombinant expression vector is constructed in any suitable manner. The nature of the vector is not critical and any vector may be used, including plasmids, viruses, phages and transposons. Possible vectors for use in the present disclosure include, but are not limited to, chromosomal, nonchromosomal and synthetic DNA sequences, such as viral plasmids, bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from combinations of plasmids and phage DNA, DNA from viruses such as lentiviruses, retroviruses, vaccinia, adenoviruses, chicken pox, baculovirus, SV40, and pseudorabies. Including self-replicating vectors and non-self-replicating vectors.
The term "mRNA" refers to Messenger RNA, and the Chinese translation "mRNA," which is a single-stranded ribonucleic acid transcribed from one strand of DNA as a template and carrying genetic information to direct protein synthesis.
The term "BVDV mRNA vaccine" includes mRNA-based vaccines against BVDV of different genotypes, mRNA including regular mRNA or self-replicating mRNA. Existing mRNA vaccine platform technologies have two main routes, one based on regular mRNA and the other on self-replicating mRNA; both routes may be applied to the present invention. The self-replicating mRNA vaccine can replicate the sequence of the self-replicating mRNA vaccine by taking the self-replicating mRNA vaccine as a template after being inoculated into an animal body, so that the self-replicating mRNA vaccine has smaller inoculation amount compared with the conventional mRNA vaccine, and the self-replicating mRNA vaccine can induce stronger immune response due to an adjuvant effect formed by immune response induced during self replication, and further enhance humoral and cellular immune responses.
The term "BVDV mRNA vaccine preparation method" includes mRNA preparation using linearized plasmid DNA as a template and enzyme transcription reaction in vitro. The linearized plasmid DNA was subjected to an in vitro transcription reaction using T7 RNA polymerase, and the template DNA was degraded using DNase, the restriction enzyme being the SapI/BspQI enzyme. The prepared mRNA was rapidly mixed with lipids (composition including cationic/ionizable lipids, helper lipids, cholesterol, and DMG-PEG 2000) by a mixer, resulting in precipitation of the lipids and encapsulation of the mRNA into the LNP under charge. The mRNA-LNP complex was then concentrated and pipetted into the paper solution.
The term "BVDV mRNA vaccine application method" comprises the application method of the broad-spectrum bovine viral diarrhea virus vaccine based on mRNA, wherein the application method adopts the application mode of prime-boost administration of the vaccine, and the prime-boost administration of the vaccine uses mRNA-secretion signal peptide+bovine IgG1 Fc+BVDV1a domain I-II+BVDV1b domain I-II++ BVDV2 domain I-II. The administration mode of the vaccine is first injection and enhanced injection, and the times of the enhanced injection are determined according to the requirement.
The term "5' -UTR" refers to a "5' untranslated region" or "5' UTR" that is a portion of a gene that is transcribed into a primary RNA transcript (pre-mRNA) and is located upstream of the coding sequence. Primary transcripts are initial RNA products, comprising introns and exons, produced by transcription of DNA. Many primary transcripts must undergo RNA processing to form physiologically active RNAs. The processing to form mature mRNA involves modification of the ends, excision of introns, capping and/or cleavage of individual rRNA molecules from the precursor RNA. Thus, the 5' UTR of an mRNA is a portion of the mRNA that is not translated into a protein and is located upstream of the coding sequence. In genomic sequences, the 5' UTR is generally defined as the region between the transcription start point and the start codon. The 5 'untranslated region (5' UTR) of vertebrate mRNA can be tens to hundreds of bases in length.
The term "3'-UTR" refers to a "3' -untranslated region" or "3'UTR" that refers to a region that is located at the 3' end of a gene, downstream of the stop codon of a protein coding region, and that is transcribed but not translated into an amino acid sequence, or to a corresponding region in an RNA molecule. The 3' -untranslated region typically extends from the stop codon of the translation product to a poly (a) sequence that is typically attached after the transcription process. The 3' -untranslated region of mammalian mRNA typically has a homologous region known as the AAUAAA hexanucleotide sequence. The sequence may be a poly (a) attachment signal and is often located 10 to 30 bases upstream of the poly (a) attachment site. The 3' -untranslated region may comprise one or more inverted repeats, which may fold to create a stem-loop structure that acts as a barrier to riboexonucleases or interacts with proteins known to improve RNA stability (e.g., RNA binding proteins).
The term "polyA" refers to a "polyadenylation sequence", "poly (a) sequence" or "poly (a) tail" and refers to a sequence of adenosine residues that is typically located at the 3' end of an RNA molecule. The present invention allows such sequences to be attached via a DNA template during RNA transcription based on repeated thymidylate residues in the strand complementary to the coding strand, whereas the sequences are not normally encoded in DNA, but are attached to the free 3' end of RNA by a template independent RNA polymerase after transcription in the nucleus.
The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. Host cells are any type of cellular system that can be used to produce the mRNA of the present invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E.coli cells. Host cells include cultured cells.
The term "recombinant host cell" encompasses host cells which differ from the parent cell upon introduction of a recombinant nucleic acid molecule, recombinant expression vector, mRNA, in particular by transformation. The host cells of the present disclosure may be prokaryotic or eukaryotic, and are primarily cells into which the recombinant nucleic acid molecules, recombinant expression vectors, mRNA, etc. of the present disclosure can be introduced.
The term "transformation, transfection, transduction" has the meaning commonly understood by those skilled in the art, i.e., the process of introducing exogenous DNA, RNA into a host.
The term "treatment" refers to: indicating the reduction or elimination of viral BVDV types 1 and 2. After suffering from a disease, the subject is contacted (e.g., administered) with an mRNA, composition, etc. of the present invention, thereby alleviating the symptoms of the disease as compared to when not contacted, and does not mean that the symptoms of the disease must be completely inhibited. The suffering from the disease is: the body develops symptoms of the disease.
The term "preventing" refers to: indicating the reduction or elimination of viral BVDV types 1 and 2. By contacting (e.g., administering) a subject with an mRNA, composition, etc. of the invention prior to the onset of a disease, the symptoms after the onset of the disease are reduced compared to when not contacted, which does not mean that complete inhibition of the disease is necessary.
The term "effective amount" refers to an amount or dose of a recombinant nucleic acid molecule, recombinant expression vector, mRNA, vaccine or composition of the invention that, upon administration to a subject in a single or multiple doses, produces a desired effect in a patient in need of treatment or prevention. The effective amount can be readily determined by the skilled artisan by considering a number of factors: species such as mammals; size, age, and health; specific diseases involved; the extent or severity of the disease; response of individual patients; specific antibodies administered; mode of administration; the bioavailability characteristics of the administration formulation; a selected dosing regimen; and the use of any concomitant therapy.
The term "pharmaceutically acceptable carrier" refers to auxiliary materials widely used in the field of pharmaceutical production. The main purpose of the carrier is to provide a pharmaceutical composition that is safe to use, stable in nature and/or has specific functionalities, and to provide a method for obtaining an effective absorption in the body of a subject. The pharmaceutically acceptable carrier may be a filler that is inert or may be an active ingredient that provides some function to the pharmaceutical composition (e.g., stabilizes the overall pH of the composition or prevents degradation of the active ingredient in the composition). Non-limiting examples of pharmaceutically acceptable carriers include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, mucilages, disintegrants, lubricants, anti-adherent agents, glidants, gelling agents, absorption-delaying agents, dissolution inhibitors, enhancing agents, adsorbents, buffers, chelating agents, preservatives, coloring agents, flavoring and sweetening agents and the like.
The term "veterinarily acceptable carrier" includes any and all solvents, dispersants, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents and the like. Diluents may include water, saline, dextrose, ethanol, glycerol, and the like. Wherein the isotonic agent may include sodium chloride, glucose, mannitol, sorbitol and lactose. Wherein the stabilizer comprises albumin, etc. Among them, adjuvants include, but are not limited to, RIBI adjuvant system (RIBI inc.), alum, aluminum hydroxide gel, cholesterol, oil-in-water emulsions, water-in-oil emulsions such as, for example, freund's complete and incomplete adjuvant, block Co-polymer (CytRx, atlantaGA), SAF-M (Chiron, emeryville CA), AMPHIGEN adjuvant, saponin, quil a, QS-21 (Cambridge Biotech inc., cambridge MA), GPI-0100 (Galenica Pharmaceuticals, inc., birmingham, AL) or other saponin fraction, monophosphate a, alfutidine lipid-amine adjuvant (avridinipid-amine adjuvant), escherichia coli heat labile toxin (recombinant or otherwise), cholera toxin or muramyl dipeptide. The immunogenic composition can further include one or more other immunomodulators such as, for example, interleukins, interferons or other cytokines.
Unless defined otherwise or clearly indicated by context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.
The invention discloses a vaccine for BVDV with different genotypes based on mRNA and a preparation method thereof, and a person skilled in the art can properly improve the technological parameters by referring to the content of the invention. It is specifically noted that all similar substitutions and modifications will be apparent to those skilled in the art, and they are considered to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
In the vaccine for bovine viral diarrhea virus based on mRNA and the preparation method thereof, the raw materials and the reagents used in the vaccine can be obtained from the market.
The invention is further illustrated by the following examples. Wherein, as a preferable choice, a conventional capping mRNA framework is used for preparing BVDV vaccine, and the conventional capping mRNA is synthesized by using linearized plasmid DNA as a template and performing enzymatic co-transcription capping reaction in vitro.
Example 1 secretion Signal peptide+bovine IgG1 Fc+BVDV1a Domain I-II+BVDV1b Domain I-II BVDV2 Domain I-II Gene fragment Synthesis and vector construction
Inquiring E2 protein genes of each mutant series virus strain of BVDV1a, 1b and 2 genotypes from NCBI, and respectively comparing sequences of E2 proteins of different genotypes to find an E2 protein sequence with the minimum difference; intercepting E2 structural domain I-II according to the reported BVDVE2 protein structure; secretion signal peptide and Fc fragment of bovine IgG1 were selected. Combining the protein domain sequences to obtain antigen fusion protein (from N end to C end in sequence): bovine IgG1 secretion signal peptide+bovine IgG1 Fc fragment+BVDV 1a E2 protein domain I-II+BVDV1b E2 protein domain I-II+BVDV2 E2 protein domain I-II, C-terminal 6His tag was used for expression detection. Then, the sequence is optimized based on the degeneracy of the codons, and finally, a DNA sequence (the sequence is shown as SEQ ID NO. 2) is directly obtained through synthesis (the synthesis of the delegated gold Style company), and the synthesized target gene is finally inserted into a multiple cloning site of the pUC57 vector, so that the plasmid CS-BVDV-210 is obtained. The amino acid sequence of the BVDV antigen fusion protein polypeptide obtained by encoding is shown as SEQ ID NO. 1. FIG. 1 shows a schematic diagram of the structure of a BVDV trivalent vaccine of the invention, namely a BVDV antigen fusion protein mRNA, namely secretion signal peptide+bovine IgG1 Fc+BVDV1a E protein domain I-II+BVDV1bE2 protein domain I-II+BVDV 2E 2 protein domain I-II.
The following vectors (I) - (III) were prepared from the gene fragments encoding BVDV antigen fusion proteins.
(I) Preparation of vector for conventional capping mRNA
Step a: synthesizing a signal peptide-bovine IgG1 Fc fragment-BVDV 1a E domain I-II-BVDV 1b E domain I-II-BVDV 2E 2 domain I-II gene fragment;
step b: construction of capped mRNA architecture vectors
The capping mRNA framework vector comprises 5 '-UTR, 3' -UTR and polyA sequences and a vector pUC57 for fixing the capping mRNA framework, and the sequences are respectively shown as SEQ ID NO. 3-6;
step c: preparing a recombinant plasmid CS-BVDV-210;
the gene synthesized in the step a is inserted into a capping mRNA architecture vector pUC57 to obtain a plasmid CS-BVDV-210, and the sequence of the plasmid CS-BVDV-210 is shown as SEQ ID NO. 7.
(II) preparation of vector from uncapped mRNA
Step a: synthesizing the target gene as in the step a (I);
step b: constructing a non-capping mRNA framework vector;
the non-capping mRNA framework vector comprises an IRES sequence, a 3' -UTR and a polyA sequence, and a vector for fixing the non-capping mRNA framework, wherein the IRES sequence is shown as SEQ ID NO. 8.
Step c: preparing a recombinant plasmid CS-BVDV-220;
inserting the gene synthesized in the step a into a non-capping mRNA framework vector to obtain a plasmid CS-BVDV-220; the sequence of the plasmid CS-BVDV-220 is shown in SEQ ID NO. 9.
(III) self-replicating RNA architecture vector
Step a: synthesizing the target gene as in the step a (I);
step b: constructing a VEE self-replication vector;
self-replicating RNA sequences obtained from the genome of the alphavirus family; the self-replicating RNA sequence comprises a gene encoding an alphavirus self-replicating component, lacking a gene that produces a structural protein of an infectious alphavirus particle; amplifying a plasmid constructed by the self-replicating RNA sequence as a template, and synthesizing to obtain the VEE self-replicating vector; the sequence of the VEE self-replication vector is shown as SEQ ID NO. 10;
step c: preparing a recombinant plasmid CS-BVDV-230;
c, inserting the gene synthesized in the step a into a designated position of a VEE self-replication vector to obtain a recombinant plasmid CS-BVDV-230; the sequence of the plasmid CS-BVDV-230 is shown in SEQ ID NO. 11.
Control system: the bovine IgG1 Fc sequence was removed based on plasmid CS-BVDV-210, wherein the bovine IgG1 Fc nucleic acid sequence is as shown in SEQ ID No. 12.
Table 1 BVDV antigen fusion protein polypeptides and related sequences
| Name of the name | Amino acid sequence |
| BVDV antigens Fusion proteins | MNPLWTLLFVLSAPRGVLSDPRCKTTCDCCPPPELPGGPSVFIFPPKPKDTLTISGTP EVTCVVVDVGHDDPEVKFSWFVDDVEVNTATTKPREEQFNSTYRVVSALRIQHQDWTG GKEFKCKVHNEGLPAPIVRTISRTKGPAREPQVYVLAPPQEELSKSTVSLTCMVTSFY PDYIAVEWQRNGQPESEDKYGTTPPQLDADGSYFLYSRLRVDRNSWQEGDTYTCVVMH EALHNHYTQKSTSKSAGKGGSGGSGGSGGSGGSGGSGGHLDCKPEFSYAIAKSDRIGP LGAEGLTTTWKDYSHEMKLEDTMVIAWCKDGKFVYLQRCTRETRYLAILHSRALPTSV VFKKLFDGRRQEDTVEMDDNFEFGLCPCDAKPIVRGKFNTTLLNGPAFQMVCPIGWTG TVSCMLANRDTLDTAVVRTYRRSKPFPYRQGCGGSGGGGSGGLPDCKPDFSYAIAKND EIGPLGATGLTTQWYEYSDGMRLQDTEVVVWCKDGEFKYLIRCEREARYLAILHTRAL PTSVVFEKILNGKEQEDVVEMDDNFEFGLCPCDAKPLVRGKFNTTLLNGPAFQMVCPI GWTGTVSCTLANKDTLATTVVRTYKRHRPFPYRQGCGGSGGGGSGGFPECKEGFQYAI SKDKKMGLLGPESLTTTWHLPTKKIVDSMVQVWCEGKDLKILRTCTKEERYLVAVHER ALSTSAEFLQISDGTTGPEVIDMPDDFEFGLCPCDSKPVIKGKFNTSLLNGPAFQMVC PQGWTGTIECILANQDTLDTTVIRTYRRTTPFQRRKWCGSG (SEQ ID NO.1) |
| Coding BVDV Antigen fusion DNA of protein Sequence(s) | ATGAACCCTTTATGGACATTGTTGTTCGTACTCAGCGCCCCGCGGGGGGTGCTGAGCG ACCCACGCTGTAAGACGACGTGTGACTGCTGCCCCCCCCCGGAGCTTCCGGGGGGGCC GTCAGTGTTTATCTTTCCGCCCAAGCCGAAAGATACACTGACGATCTCGGGGACTCCG GAGGTCACGTGTGTTGTCGTCGACGTCGGCCACGACGACCCGGAGGTGAAGTTCTCCT GGTTCGTGGACGACGTCGAGGTCAACACCGCGACCACGAAGCCCCGAGAGGAGCAGTT TAACTCGACTTACAGGGTGGTGTCGGCCCTCCGGATCCAGCACCAGGACTGGACCGGA GGGAAGGAGTTCAAGTGCAAGGTGCATAACGAAGGCCTGCCGGCTCCCATCGTCCGGA CCATTTCGCGGACGAAAGGTCCGGCGAGGGAGCCGCAGGTCTACGTTCTAGCTCCTCC TCAGGAGGAGCTATCCAAGAGTACGGTCTCCCTCACCTGCATGGTGACTAGCTTCTAT CCGGATTATATTGCGGTGGAGTGGCAGCGTAACGGTCAGCCGGAGTCGGAGGACAAGT ACGGTACCACTCCACCGCAGTTAGATGCGGATGGAAGCTATTTTCTCTATAGCAGGTT GAGGGTAGACCGTAACTCTTGGCAGGAGGGCGACACATACACGTGTGTCGTCATGCAC GAGGCACTTCACAACCACTATACTCAGAAGAGTACCTCCAAGTCCGCCGGAAAAGGCG GATCCGGCGGATCCGGTGGATCCGGCGGATCCGGCGGTTCCGGCGGATCTGGAGGTCA TCTGGATTGCAAGCCCGAGTTTTCCTATGCGATCGCGAAAAGCGATCGCATAGGACCG CTCGGTGCTGAAGGTTTGACGACTACCTGGAAGGATTACAGCCACGAGATGAAGTTGG AGGACACCATGGTCATCGCGTGGTGTAAGGACGGGAAGTTCGTCTACCTTCAGCGGTG CACGCGGGAGACGCGGTACCTGGCCATCTTACATTCTCGAGCACTACCGACCTCGGTA GTGTTCAAGAAGTTGTTTGATGGCCGGCGTCAGGAGGACACAGTCGAGATGGACGATA ACTTCGAGTTTGGACTGTGTCCTTGTGACGCCAAGCCCATTGTGCGGGGTAAGTTCAA TACTACCCTGCTCAATGGGCCTGCGTTCCAGATGGTGTGTCCCATAGGGTGGACCGGT ACGGTGAGCTGCATGCTCGCCAACCGGGACACCCTAGACACAGCAGTGGTGCGCACCT ATAGGCGGAGCAAGCCGTTCCCCTATAGGCAGGGGTGCGGGGGGTCAGGGGGAGGTGG TAGTGGGGGACTCCCGGACTGCAAGCCCGACTTCTCCTATGCGATCGCGAAGAACGAT GAAATCGGACCGCTCGGTGCCACTGGGTTAACGACCCAGTGGTACGAGTATTCCGATG GCATGCGACTTCAGGACACTGAAGTCGTGGTGTGGTGCAAGGACGGCGAGTTCAAGTA CCTCATCCGGTGTGAGAGGGAGGCCAGATATCTGGCCATCCTCCACACCCGTGCCCTT CCCACTTCAGTGGTCTTTGAGAAGATCCTGAATGGGAAGGAGCAGGAAGATGTCGTCG AGATGGACGACAACTTCGAGTTCGGGCTATGCCCATGTGACGCGAAGCCGCTCGTTCG GGGGAAGTTTAACACGACTCTCCTGAACGGGCCGGCTTTTCAGATGGTATGCCCGATA GGGTGGACCGGTACGGTGAGCTGTACCTTGGCCAACAAGGACACGCTCGCCACTACCG TTGTCCGCACCTATAAGCGCCACCGCCCGTTCCCGTACCGGCAGGGTTGCGGGGGCTC GGGCGGTGGCGGTTCTGGAGGCTTTCCTGAGTGCAAGGAAGGCTTCCAGTACGCGATC AGCAAGGACAAGAAGATGGGCTTGCTTGGTCCGGAGTCCCTCACTACGACCTGGCACC TGCCCACGAAGAAGATCGTGGACAGCATGGTCCAGGTCTGGTGCGAGGGGAAGGATCT GAAGATCCTTCGCACTTGCACCAAGGAGGAGCGGTACTTGGTGGCAGTGCACGAGCGT GCACTGTCCACCAGTGCCGAGTTCCTCCAGATATCGGACGGGACGACCGGACCCGAAG TCATCGACATGCCCGATGACTTCGAGTTCGGTCTGTGCCCGTGCGATAGCAAGCCCGT TATCAAGGGGAAGTTCAATACTTCCCTCTTGAACGGTCCCGCCTTCCAGATGGTCTGC |
| CCGCAGGGGTGGACCGGGACCATCGAGTGTATCCTGGCCAACCAGGATACACTCGATA CGACGGTGATCCGGACCTACCGCCGCACTACGCCGTTCCAGCGGCGTAAGTGGTGTGG GTCCGGATAATGA (SEQ ID NO.2) | |
| 5`-UTR | aggactcactatttgttttcgcgcccagttgcaaaaagtgtcgccacc(SEQ ID NO.3) |
| 3`-UTR | cccgggctggtactgcatgcacgcaatgctagctgcccctttcccgtcctgggtaccc cgagtctcccccgacctcgggtcccaggtatgctcccacctccacctgccccactcac cacctctgctagttccagacacctcccaagcacgcagcaatgcagctcaaaacgctta gcctagccacacccccacgggaaacagcagtgattaacctttagcaataaacgaaagt ttaactaagctatactaaccccagggttggtcaatttcgtgccagccacacc (SEQ ID NO.4) |
| polyA | aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaa (SEQ ID NO.5) |
| Carrier body pUC57 | ggaagagctcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggt atcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcagga aagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgc tggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaag tcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagc tccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttc tcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggt gtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgc tgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgc cactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctac agagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatc tgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggca aacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcag aaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtgg aacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacct agatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaac ttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtcta tttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagg gcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctcc agatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgca actttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagtt cgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacg ctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttaca tgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtca gaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctct tactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtca ttctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggata ataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggg gcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgt gcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaa caggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatact catactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagc ggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttc cccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataa aaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaac ctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccggga gcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaa ctatgcggcatcagagcagattgtactgagagtgcaccatatg (SEQ ID NO.6) |
| CS-BVDV- 210 | tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggt cacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcg ggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactga gagtgcaccatatgtaatacgactcactataAggactcactatttgttttcgcgccca gttgcaaaaagtgtcgccaccATGAACCCTTTATGGACATTGTTGTTCGTACTCAGCG CCCCGCGGGGGGTGCTGAGCGACCCACGCTGTAAGACGACGTGTGACTGCTGCCCCCC CCCGGAGCTTCCGGGGGGGCCGTCAGTGTTTATCTTTCCGCCCAAGCCGAAAGATACA CTGACGATCTCGGGGACTCCGGAGGTCACGTGTGTTGTCGTCGACGTCGGCCACGACG ACCCGGAGGTGAAGTTCTCCTGGTTCGTGGACGACGTCGAGGTCAACACCGCGACCAC GAAGCCCCGAGAGGAGCAGTTTAACTCGACTTACAGGGTGGTGTCGGCCCTCCGGATC CAGCACCAGGACTGGACCGGAGGGAAGGAGTTCAAGTGCAAGGTGCATAACGAAGGCC TGCCGGCTCCCATCGTCCGGACCATTTCGCGGACGAAAGGTCCGGCGAGGGAGCCGCA GGTCTACGTTCTAGCTCCTCCTCAGGAGGAGCTATCCAAGAGTACGGTCTCCCTCACC TGCATGGTGACTAGCTTCTATCCGGATTATATTGCGGTGGAGTGGCAGCGTAACGGTC AGCCGGAGTCGGAGGACAAGTACGGTACCACTCCACCGCAGTTAGATGCGGATGGAAG CTATTTTCTCTATAGCAGGTTGAGGGTAGACCGTAACTCTTGGCAGGAGGGCGACACA TACACGTGTGTCGTCATGCACGAGGCACTTCACAACCACTATACTCAGAAGAGTACCT CCAAGTCCGCCGGAAAAGGCGGATCCGGCGGATCCGGTGGATCCGGCGGATCCGGCGG TTCCGGCGGATCTGGAGGTCATCTGGATTGCAAGCCCGAGTTTTCCTATGCGATCGCG AAAAGCGATCGCATAGGACCGCTCGGTGCTGAAGGTTTGACGACTACCTGGAAGGATT ACAGCCACGAGATGAAGTTGGAGGACACCATGGTCATCGCGTGGTGTAAGGACGGGAA GTTCGTCTACCTTCAGCGGTGCACGCGGGAGACGCGGTACCTGGCCATCTTACATTCT CGAGCACTACCGACCTCGGTAGTGTTCAAGAAGTTGTTTGATGGCCGGCGTCAGGAGG ACACAGTCGAGATGGACGATAACTTCGAGTTTGGACTGTGTCCTTGTGACGCCAAGCC CATTGTGCGGGGTAAGTTCAATACTACCCTGCTCAATGGGCCTGCGTTCCAGATGGTG TGTCCCATAGGGTGGACCGGTACGGTGAGCTGCATGCTCGCCAACCGGGACACCCTAG ACACAGCAGTGGTGCGCACCTATAGGCGGAGCAAGCCGTTCCCCTATAGGCAGGGGTG CGGGGGGTCAGGGGGAGGTGGTAGTGGGGGACTCCCGGACTGCAAGCCCGACTTCTCC TATGCGATCGCGAAGAACGATGAAATCGGACCGCTCGGTGCCACTGGGTTAACGACCC AGTGGTACGAGTATTCCGATGGCATGCGACTTCAGGACACTGAAGTCGTGGTGTGGTG CAAGGACGGCGAGTTCAAGTACCTCATCCGGTGTGAGAGGGAGGCCAGATATCTGGCC ATCCTCCACACCCGTGCCCTTCCCACTTCAGTGGTCTTTGAGAAGATCCTGAATGGGA AGGAGCAGGAAGATGTCGTCGAGATGGACGACAACTTCGAGTTCGGGCTATGCCCATG TGACGCGAAGCCGCTCGTTCGGGGGAAGTTTAACACGACTCTCCTGAACGGGCCGGCT TTTCAGATGGTATGCCCGATAGGGTGGACCGGTACGGTGAGCTGTACCTTGGCCAACA AGGACACGCTCGCCACTACCGTTGTCCGCACCTATAAGCGCCACCGCCCGTTCCCGTA CCGGCAGGGTTGCGGGGGCTCGGGCGGTGGCGGTTCTGGAGGCTTTCCTGAGTGCAAG GAAGGCTTCCAGTACGCGATCAGCAAGGACAAGAAGATGGGCTTGCTTGGTCCGGAGT CCCTCACTACGACCTGGCACCTGCCCACGAAGAAGATCGTGGACAGCATGGTCCAGGT |
| CTGGTGCGAGGGGAAGGATCTGAAGATCCTTCGCACTTGCACCAAGGAGGAGCGGTAC TTGGTGGCAGTGCACGAGCGTGCACTGTCCACCAGTGCCGAGTTCCTCCAGATATCGG ACGGGACGACCGGACCCGAAGTCATCGACATGCCCGATGACTTCGAGTTCGGTCTGTG CCCGTGCGATAGCAAGCCCGTTATCAAGGGGAAGTTCAATACTTCCCTCTTGAACGGT CCCGCCTTCCAGATGGTCTGCCCGCAGGGGTGGACCGGGACCATCGAGTGTATCCTGG CCAACCAGGATACACTCGATACGACGGTGATCCGGACCTACCGCCGCACTACGCCGTT CCAGCGGCGTAAGTGGTGTGGGTCCGGACATCACCATCACCACCACTAATGAcccggg ctggtactgcatgcacgcaatgctagctgcccctttcccgtcctgggtaccccgagtc tcccccgacctcgggtcccaggtatgctcccacctccacctgccccactcaccacctc tgctagttccagacacctcccaagcacgcagcaatgcagctcaaaacgcttagcctag ccacacccccacgggaaacagcagtgattaacctttagcaataaacgaaagtttaact aagctatactaaccccagggttggtcaatttcgtgccagccacaccaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaggaagagctcctcgctcactgactcgctgcgctcggtcgttcggct gcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggg gataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaa aggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaa tcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgttt ccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacc tgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggta tctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgtt cagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagac acgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgt aggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaaca gtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagct cttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagca gattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtct gacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaa ggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtat atatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctca gcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataacta cgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacg ctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcaga agtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagcta gagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcat cgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatca aggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctc cgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcact gcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtac | |
| tcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgt caatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaa acgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatg taacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctg ggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaa atgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttat tgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttc cgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgac attaacctataaaaataggcgtatcacgaggccctttcgtc(SEQ ID NO.7) | |
| IRES | TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGT ATCACGGTACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGT AACACACACCGATCAACAGTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTC TGTTACCCCGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTTCG TTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTC GCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGA CCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGACTCACTATTTGTTTTCGCGCC CAGTTGCAAAAAGTGTCGCCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCT ATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCA CACACCCTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGAC TACTTTGGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAATTG AGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTAT ATATCCCTTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTC ATTGTTAAGTTGAATACAGCAAA (SEQ ID NO.8) |
| CS-BVDV- 220 | tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggt cacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcg ggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactga gagtgcaccatatgtaatacgactcactataAggTTAAAACAGCCTGTGGGTTGATCC CACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGTACCTTTGTGCGCCTGT TTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGATCAACAGTCAGC GTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAAT AGACTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAA ACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGAT CAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGG CCTGCCCATGGGACTCACTATTTGTTTTCGCGCCCAGTTGCAAAAAGTGTCGCCCATG GGACGCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCG GCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGT GTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCATT TTATTCCTATACTGGCTGCTTATGGTGACAATTGAGAGATCGTTACCATATAGCTATT GGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCCTTTGTTGGGTTTATACC ACTTAGCTTGAAAGAGGTTAAAACATTACAATTCATTGTTAAGTTGAATACAGCAAAA TGAACCCTTTATGGACATTGTTGTTCGTACTCAGCGCCCCGCGGGGGGTGCTGAGCGA CCCACGCTGTAAGACGACGTGTGACTGCTGCCCCCCCCCGGAGCTTCCGGGGGGGCCG TCAGTGTTTATCTTTCCGCCCAAGCCGAAAGATACACTGACGATCTCGGGGACTCCGG AGGTCACGTGTGTTGTCGTCGACGTCGGCCACGACGACCCGGAGGTGAAGTTCTCCTG GTTCGTGGACGACGTCGAGGTCAACACCGCGACCACGAAGCCCCGAGAGGAGCAGTTT AACTCGACTTACAGGGTGGTGTCGGCCCTCCGGATCCAGCACCAGGACTGGACCGGAG GGAAGGAGTTCAAGTGCAAGGTGCATAACGAAGGCCTGCCGGCTCCCATCGTCCGGAC CATTTCGCGGACGAAAGGTCCGGCGAGGGAGCCGCAGGTCTACGTTCTAGCTCCTCCT CAGGAGGAGCTATCCAAGAGTACGGTCTCCCTCACCTGCATGGTGACTAGCTTCTATC CGGATTATATTGCGGTGGAGTGGCAGCGTAACGGTCAGCCGGAGTCGGAGGACAAGTA CGGTACCACTCCACCGCAGTTAGATGCGGATGGAAGCTATTTTCTCTATAGCAGGTTG AGGGTAGACCGTAACTCTTGGCAGGAGGGCGACACATACACGTGTGTCGTCATGCACG AGGCACTTCACAACCACTATACTCAGAAGAGTACCTCCAAGTCCGCCGGAAAAGGCGG ATCCGGCGGATCCGGTGGATCCGGCGGATCCGGCGGTTCCGGCGGATCTGGAGGTCAT CTGGATTGCAAGCCCGAGTTTTCCTATGCGATCGCGAAAAGCGATCGCATAGGACCGC TCGGTGCTGAAGGTTTGACGACTACCTGGAAGGATTACAGCCACGAGATGAAGTTGGA GGACACCATGGTCATCGCGTGGTGTAAGGACGGGAAGTTCGTCTACCTTCAGCGGTGC ACGCGGGAGACGCGGTACCTGGCCATCTTACATTCTCGAGCACTACCGACCTCGGTAG TGTTCAAGAAGTTGTTTGATGGCCGGCGTCAGGAGGACACAGTCGAGATGGACGATAA CTTCGAGTTTGGACTGTGTCCTTGTGACGCCAAGCCCATTGTGCGGGGTAAGTTCAAT ACTACCCTGCTCAATGGGCCTGCGTTCCAGATGGTGTGTCCCATAGGGTGGACCGGTA CGGTGAGCTGCATGCTCGCCAACCGGGACACCCTAGACACAGCAGTGGTGCGCACCTA |
| TAGGCGGAGCAAGCCGTTCCCCTATAGGCAGGGGTGCGGGGGGTCAGGGGGAGGTGGT AGTGGGGGACTCCCGGACTGCAAGCCCGACTTCTCCTATGCGATCGCGAAGAACGATG AAATCGGACCGCTCGGTGCCACTGGGTTAACGACCCAGTGGTACGAGTATTCCGATGG CATGCGACTTCAGGACACTGAAGTCGTGGTGTGGTGCAAGGACGGCGAGTTCAAGTAC CTCATCCGGTGTGAGAGGGAGGCCAGATATCTGGCCATCCTCCACACCCGTGCCCTTC CCACTTCAGTGGTCTTTGAGAAGATCCTGAATGGGAAGGAGCAGGAAGATGTCGTCGA GATGGACGACAACTTCGAGTTCGGGCTATGCCCATGTGACGCGAAGCCGCTCGTTCGG GGGAAGTTTAACACGACTCTCCTGAACGGGCCGGCTTTTCAGATGGTATGCCCGATAG GGTGGACCGGTACGGTGAGCTGTACCTTGGCCAACAAGGACACGCTCGCCACTACCGT TGTCCGCACCTATAAGCGCCACCGCCCGTTCCCGTACCGGCAGGGTTGCGGGGGCTCG GGCGGTGGCGGTTCTGGAGGCTTTCCTGAGTGCAAGGAAGGCTTCCAGTACGCGATCA GCAAGGACAAGAAGATGGGCTTGCTTGGTCCGGAGTCCCTCACTACGACCTGGCACCT GCCCACGAAGAAGATCGTGGACAGCATGGTCCAGGTCTGGTGCGAGGGGAAGGATCTG AAGATCCTTCGCACTTGCACCAAGGAGGAGCGGTACTTGGTGGCAGTGCACGAGCGTG CACTGTCCACCAGTGCCGAGTTCCTCCAGATATCGGACGGGACGACCGGACCCGAAGT CATCGACATGCCCGATGACTTCGAGTTCGGTCTGTGCCCGTGCGATAGCAAGCCCGTT ATCAAGGGGAAGTTCAATACTTCCCTCTTGAACGGTCCCGCCTTCCAGATGGTCTGCC CGCAGGGGTGGACCGGGACCATCGAGTGTATCCTGGCCAACCAGGATACACTCGATAC GACGGTGATCCGGACCTACCGCCGCACTACGCCGTTCCAGCGGCGTAAGTGGTGTGGG TCCGGACATCACCATCACCACCACTAATGAcccgggctggtactgcatgcacgcaatg ctagctgcccctttcccgtcctgggtaccccgagtctcccccgacctcgggtcccagg tatgctcccacctccacctgccccactcaccacctctgctagttccagacacctccca agcacgcagcaatgcagctcaaaacgcttagcctagccacacccccacgggaaacagc agtgattaacctttagcaataaacgaaagtttaactaagctatactaaccccagggtt ggtcaatttcgtgccagccacaccaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaggaagagctc ctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcac tcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgt gagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgttttt ccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtgg cgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgc gctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcggg aagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgtt cgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttat ccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagc agccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttg aagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgc tgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccac cgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaagga | |
| tctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaact cacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatcctttt aaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgac agttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcat ccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatc tggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatca gcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccg cctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaa tagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgttt ggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatccccca tgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagtt ggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatg ccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaat agtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgcc acatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactc tcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaact gatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggca aaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttc ctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatat ttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagt gccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgt atcacgaggccctttcgtc (SEQ ID NO.9) | |
| VEE self-replication Carrier body | atgggcggcgcatgagagaagcccagaccaattacctacccaaaatggagaaagttca cgttgacatcgaggaagacagcccattcctcagagctttgcagcggagcttcccgcag tttgaggtagaagccaagcaggtcactgataatgaccatgctaatgccagagcgtttt cgcatctggcttcaaaactgatcgaaacggaggtggacccatccgacacgatccttga cattggaagtgcgcccgcccgcagaatgtattctaagcacaagtatcattgtatctgt ccgatgagatgtgcggaagatccggacagattgtataagtatgcaactaagctgaaga aaaactgtaaggaaataactgataaggaattggacaagaaaatgaaggagctcgccgc cgtcatgagcgaccctgacctggaaactgagactatgtgcctccacgacgacgagtcg tgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggttgacggaccga caagtctctatcaccaagccaataagggagttagagtcgcctactggataggctttga caccaccccttttatgtttaagaacttggctggagcatatccatcatactctaccaac tgggccgacgaaaccgtgttaacggctcgtaacataggcctatgcagctctgacgtta tggagcggtcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaa caatgttctattctctgttggctcgaccatctaccacgagaagagggacttactgagg agctggcacctgccgtctgtatttcacttacgtggcaagcaaaattacacatgtcggt gtgagactatagttagttgcgacgggtacgtcgttaaaagaatagctatcagtccagg cctgtatgggaagccttcaggctatgctgctacgatgcaccgcgagggattcttgtgc tgcaaagtgacagacacattgaacggggagagggtctcttttcccgtgtgcacgtatg tgccagctacattgtgtgaccaaatgactggcatactggcaacagatgtcagtgcgga cgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaacggtcgcacc cagagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcatttg ctaggtgggcaaaggaatataaggaagatcaagaagatgaaaggccactaggactacg agatagacagttagtcatggggtgttgttgggcttttagaaggcacaagataacatct atttataagcgcccggatacccaaaccatcatcaaagtgaacagcgatttccactcat tcgtgctgcccaggataggcagtaacacattggagatcgggctgagaacaagaatcag gaaaatgttagaggagcacaaggagccgtcacctctcattaccgccgaggacgtacaa gaagctaagtgcgcagccgatgaggctaaggaggtgcgtgaagccgaggagttgcgcg cagctctaccacctttggcagctgatgttgaggagcccactctggaagccgatgtcga cttgatgttacaagaggctggggccggctcagtggagacacctcgtggcttgataaag gttaccagctacgatggcgaggacaagatcggctcttacgctgtgctttctccgcagg ctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaagtcatagt gataacacactctggccgaaaagggcgttatgccgtggaaccataccatggtaaagta gtggtgccagagggacatgcaatacccgtccaggactttcaagctctgagtgaaagtg ccaccattgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgccac acatggaggagcgctgaacactgatgaagaatattacaaaactgtcaagcccagcgag cacgacggcgaatacctgtacgacatcgacaggaaacagtgcgtcaagaaagaactag tcactgggctagggctcacaggcgagctggtggatcctcccttccatgaattcgccta cgagagtctgagaacacgaccagccgctccttaccaagtaccaaccataggggtgtat ggcgtgccaggatcaggcaagtctggcatcattaaaagcgcagtcaccaaaaaagatc |
| tagtggtgagcgccaagaaagaaaactgtgcagaaattataagggacgtcaagaaaat gaaagggctggacgtcaatgccagaactgtggactcagtgctcttgaatggatgcaaa caccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtactctca gagcgctcatagccattataagacctaaaaaggcagtgctctgcggggatcccaaaca gtgcggtttttttaacatgatgtgcctgaaagtgcattttaaccacgagatttgcaca caagtcttccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtct caaccttgttttacgacaaaaaaatgagaacgacgaatccgaaagagactaagattgt gattgacactaccggcagtaccaaacctaagcaggacgatctcattctcacttgtttc agagggtgggtgaagcagttgcaaatagattacaaaggcaacgaaataatgacggcag ctgcctctcaagggctgacccgtaaaggtgtgtatgccgttcggtacaaggtgaatga aaatcctctgtacgcacccacctcagaacatgtgaacgtcctactgacccgcacggag gaccgcatcgtgtggaaaacactagccggcgacccatggataaaaacactgactgcca agtaccctgggaatttcactgccacgatagaggagtggcaagcagagcatgatgccat catgaggcacatcttggagagaccggaccctaccgacgtcttccagaataaggcaaac gtgtgttgggccaaggctttagtgccggtgctgaagaccgctggcatagacatgacca ctgaacaatggaacactgtggattattttgaaacggacaaagctcactcagcagagat agtattgaaccaactatgcgtgaggttctttggactcgatctggactccggtctattt tctgcacccactgttccgttatccattaggaataatcactgggataactccccgtcgc ctaacatgtacgggctgaataaagaagtggtccgtcagctctctcgcaggtacccaca actgcctcgggcagttgccactggaagagtctatgacatgaacactggtacactgcgc aattatgatccgcgcataaacctagtacctgtaaacagaagactgcctcatgctttag tcctccaccataatgaacacccacagagtgacttttcttcattcgtcagcaaattgaa gggcagaactgtcctggtggtcggggaaaagttgtccgtcccaggcaaaatggttgac tggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggcatcccag gtgatgtgcccaaatatgacataatatttgttaatgtgaggaccccatataaatacca tcactatcagcagtgtgaagaccatgccattaagcttagcatgttgaccaagaaagct tgtctgcatctgaatcccggcggaacctgtgtcagcataggttatggttacgctgaca gggccagcgaaagcatcattggtgctatagcgcggcagttcaagttttcccgggtatg caaaccgaaatcctcacttgaagagacggaagttctgtttgtattcattcggtacgat cgcaaggcccgtacgcacaattcttacaagctttcatcaaccttgaccaacatttata caggttccagactccacgaagccggatgtgcaccctcatatcatgtggtgcgagggga tattgccacggccaccgaaggagtgattataaatgctgctaacagcaaaggacaacct ggcggaggggtgtgcggagcgctgtataagaaattcccggaaagcttcgatttacagc cgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacatatcattcatgc cgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttggcagag gcttatgagtccatcgctaagattgtcaacgataacaattacaagtcagtagcgattc cactgttgtccaccggcatcttttccgggaacaaagatcgactaacccaatcattgaa ccatttgctgacagctttagacaccactgatgcagatgtagccatatactgcagggac aagaaatgggaaatgactctcaaggaagcagtggctaggagagaagcagtggaggaga | |
| tatgcatatccgacgactcttcagtgacagaacctgatgcagagctggtgagggtgca tccgaagagttctttggctggaaggaagggctacagcacaagcgatggcaaaactttc tcatatttggaagggaccaagtttcaccaggcggccaaggatatagcagaaattaatg ccatgtggcccgttgcaacggaggccaatgagcaggtatgcatgtatatcctcggaga aagcatgagcagtattaggtcgaaatgccccgtcgaagagtcggaagcctccacacca cctagcacgctgccttgcttgtgcatccatgccatgactccagaaagagtacagcgcc taaaagcctcacgtccagaacaaattactgtgtgctcatcctttccattgccgaagta tagaatcactggtgtgcagaagatccaatgctcccagcctatattgttctcaccgaaa gtgcctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacgaga ctccggagccatcggcagagaaccaatccacagaggggacacctgaacaaccaccact tataaccgaggatgagaccaggactagaacgcctgagccgatcatcatcgaagaggaa gaagaggatagcataagtttgctgtcagatggcccgacccaccaggtgctgcaagtcg aggcagacattcacgggccgccctctgtatctagctcatcctggtccattcctcatgc atccgactttgatgtggacagtttatccatacttgacaccctggagggagctagcgtg accagcggggcaacgtcagccgagactaactcttacttcgcaaagagtatggagtttc tggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctccacatcccgctcc gcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagaaccagcctagtt tccaccccgccaggcgtgaatagggtgatcactagagaggagctcgaggcgcttaccc cgtcacgcactcctagcaggtcggtctcgagaaccagcctggtctccaacccgccagg cgtaaatagggtgattacaagagaggagtttgaggcgttcgtagcacaacaacaatga cggtttgatgcgggtgcatacatcttttcctccgacaccggtcaagggcatttacaac aaaaatcagtaaggcaaacggtgctatccgaagtggtgttggagaggaccgaattgga gatttcgtatgccccgcgcctcgaccaagaaaaagaagaattactacgcaagaaatta cagttaaatcccacacctgctaacagaagcagataccagtccaggaaggtggagaaca tgaaagccataacagctagacgtattctgcaaggcctagggcattatttgaaggcaga aggaaaagtggagtgctaccgaaccctgcatcctgttcctttgtattcatctagtgtg aaccgtgccttttcaagccccaaggtcgcagtggaagcctgtaacgccatgttgaaag agaactttccgactgtggcttcttactgtattattccagagtacgatgcctatttgga catggttgacggagcttcatgctgcttagacactgccagtttttgccctgcaaagctg cgcagctttccaaagaaacactcctatttggaacccacaatacgatcggcagtgcctt cagcgatccagaacacgctccagaacgtcctggcagctgccacaaaaagaaattgcaa tgtcacgcaaatgagagaattgcccgtattggattcggcggcctttaatgtggaatgc ttcaagaaatatgcgtgtaataatgaatattgggaaacgtttaaagaaaaccccatca ggcttactgaagaaaacgtggtaaattacattaccaaattaaaaggaccaaaagctgc tgctctttttgcgaagacacataatttgaatatgttgcaggacataccaatggacagg tttgtaatggacttaaagagagacgtgaaagtgactccaggaacaaaacatactgaag aacggcccaaggtacaggtgatccaggctgccgatccgctagcaacagcgtatctgtg cggaatccaccgagagctggttaggagattaaatgcggtcctgcttccgaacattcat acactgtttgatatgtcggctgaagactttgacgctattatagccgagcacttccagc | |
| ctggggattgtgttctggaaactgacatcgcgtcgtttgataaaagtgaggacgacgc catggctctgaccgcgttaatgattctggaagacttaggtgtggacgcagagctgttg acgctgattgaggcggctttcggcgaaatttcatcaatacatttgcccactaaaacta aatttaaattcggagccatgatgaaatctggaatgttcctcacactgtttgtgaacac agtcattaacattgtaatcgcaagcagagtgttgagagaacggctaaccggatcacca tgtgcagcattcattggagatgacaatatcgtgaaaggagtcaaatcggacaaattaa tggcagacaggtgcgccacctggttgaatatggaagtcaagattatagatgctgtggt gggcgagaaagcgccttatttctgtggagggtttattttgtgtgactccgtgaccggc acagcgtgccgtgtggcagaccccctaaaaaggctgtttaagcttggcaaacctctgg cagcagacgatgaacatgatgatgacaggagaagggcattgcatgaagagtcaacacg ctggaaccgagtgggtattctttcagagctgtgcaaggcagtagaatcaaggtatgaa accgtaggaacttccatcatagttatggccatgactactctagctagcagtgttaaat cattcagctacctgagaggggcccctataactctctacggctaacctgaatggactac gacatagtctagtccgccaagtctagaacaagtttgtacaaaaaagcaggctgccacc acccagctttcttgtacaaagtggtggcgcgcctatgttacgtgcaaaggtgattgtc accccccgaaagaccatattgtgacacaccctcagtatcacgcccaaacatttacagc cgcggtgtcaaaaaccgcgtggacgtggttaacatccctgctgggaggatcagccgta attattataattggcttggtgctggctactattgtggccatgtacgtgctgaccaacc agaaacataattgaatacagcagcaattggcaagctgcttacatagaactcgcggcga ttggcatgccgccttaaaatttttattttatttttcttttcttttccgaatcggattt tgtttttaatatttcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaggaagagctagggataacagg gtaat(SEQ ID NO.10) | |
| CS-BVDV- 230 | atgggcggcgcatgagagaagcccagaccaattacctacccaaaatggagaaagttca cgttgacatcgaggaagacagcccattcctcagagctttgcagcggagcttcccgcag tttgaggtagaagccaagcaggtcactgataatgaccatgctaatgccagagcgtttt cgcatctggcttcaaaactgatcgaaacggaggtggacccatccgacacgatccttga cattggaagtgcgcccgcccgcagaatgtattctaagcacaagtatcattgtatctgt ccgatgagatgtgcggaagatccggacagattgtataagtatgcaactaagctgaaga aaaactgtaaggaaataactgataaggaattggacaagaaaatgaaggagctcgccgc cgtcatgagcgaccctgacctggaaactgagactatgtgcctccacgacgacgagtcg tgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggttgacggaccga caagtctctatcaccaagccaataagggagttagagtcgcctactggataggctttga caccaccccttttatgtttaagaacttggctggagcatatccatcatactctaccaac tgggccgacgaaaccgtgttaacggctcgtaacataggcctatgcagctctgacgtta tggagcggtcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaa caatgttctattctctgttggctcgaccatctaccacgagaagagggacttactgagg agctggcacctgccgtctgtatttcacttacgtggcaagcaaaattacacatgtcggt gtgagactatagttagttgcgacgggtacgtcgttaaaagaatagctatcagtccagg cctgtatgggaagccttcaggctatgctgctacgatgcaccgcgagggattcttgtgc tgcaaagtgacagacacattgaacggggagagggtctcttttcccgtgtgcacgtatg tgccagctacattgtgtgaccaaatgactggcatactggcaacagatgtcagtgcgga cgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaacggtcgcacc cagagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcatttg ctaggtgggcaaaggaatataaggaagatcaagaagatgaaaggccactaggactacg agatagacagttagtcatggggtgttgttgggcttttagaaggcacaagataacatct atttataagcgcccggatacccaaaccatcatcaaagtgaacagcgatttccactcat tcgtgctgcccaggataggcagtaacacattggagatcgggctgagaacaagaatcag gaaaatgttagaggagcacaaggagccgtcacctctcattaccgccgaggacgtacaa gaagctaagtgcgcagccgatgaggctaaggaggtgcgtgaagccgaggagttgcgcg cagctctaccacctttggcagctgatgttgaggagcccactctggaagccgatgtcga cttgatgttacaagaggctggggccggctcagtggagacacctcgtggcttgataaag gttaccagctacgatggcgaggacaagatcggctcttacgctgtgctttctccgcagg ctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaagtcatagt gataacacactctggccgaaaagggcgttatgccgtggaaccataccatggtaaagta gtggtgccagagggacatgcaatacccgtccaggactttcaagctctgagtgaaagtg ccaccattgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgccac acatggaggagcgctgaacactgatgaagaatattacaaaactgtcaagcccagcgag cacgacggcgaatacctgtacgacatcgacaggaaacagtgcgtcaagaaagaactag tcactgggctagggctcacaggcgagctggtggatcctcccttccatgaattcgccta cgagagtctgagaacacgaccagccgctccttaccaagtaccaaccataggggtgtat ggcgtgccaggatcaggcaagtctggcatcattaaaagcgcagtcaccaaaaaagatc |
| tagtggtgagcgccaagaaagaaaactgtgcagaaattataagggacgtcaagaaaat gaaagggctggacgtcaatgccagaactgtggactcagtgctcttgaatggatgcaaa caccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtactctca gagcgctcatagccattataagacctaaaaaggcagtgctctgcggggatcccaaaca gtgcggtttttttaacatgatgtgcctgaaagtgcattttaaccacgagatttgcaca caagtcttccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtct caaccttgttttacgacaaaaaaatgagaacgacgaatccgaaagagactaagattgt gattgacactaccggcagtaccaaacctaagcaggacgatctcattctcacttgtttc agagggtgggtgaagcagttgcaaatagattacaaaggcaacgaaataatgacggcag ctgcctctcaagggctgacccgtaaaggtgtgtatgccgttcggtacaaggtgaatga aaatcctctgtacgcacccacctcagaacatgtgaacgtcctactgacccgcacggag gaccgcatcgtgtggaaaacactagccggcgacccatggataaaaacactgactgcca agtaccctgggaatttcactgccacgatagaggagtggcaagcagagcatgatgccat catgaggcacatcttggagagaccggaccctaccgacgtcttccagaataaggcaaac gtgtgttgggccaaggctttagtgccggtgctgaagaccgctggcatagacatgacca ctgaacaatggaacactgtggattattttgaaacggacaaagctcactcagcagagat agtattgaaccaactatgcgtgaggttctttggactcgatctggactccggtctattt tctgcacccactgttccgttatccattaggaataatcactgggataactccccgtcgc ctaacatgtacgggctgaataaagaagtggtccgtcagctctctcgcaggtacccaca actgcctcgggcagttgccactggaagagtctatgacatgaacactggtacactgcgc aattatgatccgcgcataaacctagtacctgtaaacagaagactgcctcatgctttag tcctccaccataatgaacacccacagagtgacttttcttcattcgtcagcaaattgaa gggcagaactgtcctggtggtcggggaaaagttgtccgtcccaggcaaaatggttgac tggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggcatcccag gtgatgtgcccaaatatgacataatatttgttaatgtgaggaccccatataaatacca tcactatcagcagtgtgaagaccatgccattaagcttagcatgttgaccaagaaagct tgtctgcatctgaatcccggcggaacctgtgtcagcataggttatggttacgctgaca gggccagcgaaagcatcattggtgctatagcgcggcagttcaagttttcccgggtatg caaaccgaaatcctcacttgaagagacggaagttctgtttgtattcattcggtacgat cgcaaggcccgtacgcacaattcttacaagctttcatcaaccttgaccaacatttata caggttccagactccacgaagccggatgtgcaccctcatatcatgtggtgcgagggga tattgccacggccaccgaaggagtgattataaatgctgctaacagcaaaggacaacct ggcggaggggtgtgcggagcgctgtataagaaattcccggaaagcttcgatttacagc cgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacatatcattcatgc cgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttggcagag gcttatgagtccatcgctaagattgtcaacgataacaattacaagtcagtagcgattc cactgttgtccaccggcatcttttccgggaacaaagatcgactaacccaatcattgaa ccatttgctgacagctttagacaccactgatgcagatgtagccatatactgcagggac aagaaatgggaaatgactctcaaggaagcagtggctaggagagaagcagtggaggaga | |
| tatgcatatccgacgactcttcagtgacagaacctgatgcagagctggtgagggtgca tccgaagagttctttggctggaaggaagggctacagcacaagcgatggcaaaactttc tcatatttggaagggaccaagtttcaccaggcggccaaggatatagcagaaattaatg ccatgtggcccgttgcaacggaggccaatgagcaggtatgcatgtatatcctcggaga aagcatgagcagtattaggtcgaaatgccccgtcgaagagtcggaagcctccacacca cctagcacgctgccttgcttgtgcatccatgccatgactccagaaagagtacagcgcc taaaagcctcacgtccagaacaaattactgtgtgctcatcctttccattgccgaagta tagaatcactggtgtgcagaagatccaatgctcccagcctatattgttctcaccgaaa gtgcctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacgaga ctccggagccatcggcagagaaccaatccacagaggggacacctgaacaaccaccact tataaccgaggatgagaccaggactagaacgcctgagccgatcatcatcgaagaggaa gaagaggatagcataagtttgctgtcagatggcccgacccaccaggtgctgcaagtcg aggcagacattcacgggccgccctctgtatctagctcatcctggtccattcctcatgc atccgactttgatgtggacagtttatccatacttgacaccctggagggagctagcgtg accagcggggcaacgtcagccgagactaactcttacttcgcaaagagtatggagtttc tggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctccacatcccgctcc gcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagaaccagcctagtt tccaccccgccaggcgtgaatagggtgatcactagagaggagctcgaggcgcttaccc cgtcacgcactcctagcaggtcggtctcgagaaccagcctggtctccaacccgccagg cgtaaatagggtgattacaagagaggagtttgaggcgttcgtagcacaacaacaatga cggtttgatgcgggtgcatacatcttttcctccgacaccggtcaagggcatttacaac aaaaatcagtaaggcaaacggtgctatccgaagtggtgttggagaggaccgaattgga gatttcgtatgccccgcgcctcgaccaagaaaaagaagaattactacgcaagaaatta cagttaaatcccacacctgctaacagaagcagataccagtccaggaaggtggagaaca tgaaagccataacagctagacgtattctgcaaggcctagggcattatttgaaggcaga aggaaaagtggagtgctaccgaaccctgcatcctgttcctttgtattcatctagtgtg aaccgtgccttttcaagccccaaggtcgcagtggaagcctgtaacgccatgttgaaag agaactttccgactgtggcttcttactgtattattccagagtacgatgcctatttgga catggttgacggagcttcatgctgcttagacactgccagtttttgccctgcaaagctg cgcagctttccaaagaaacactcctatttggaacccacaatacgatcggcagtgcctt cagcgatccagaacacgctccagaacgtcctggcagctgccacaaaaagaaattgcaa tgtcacgcaaatgagagaattgcccgtattggattcggcggcctttaatgtggaatgc ttcaagaaatatgcgtgtaataatgaatattgggaaacgtttaaagaaaaccccatca ggcttactgaagaaaacgtggtaaattacattaccaaattaaaaggaccaaaagctgc tgctctttttgcgaagacacataatttgaatatgttgcaggacataccaatggacagg tttgtaatggacttaaagagagacgtgaaagtgactccaggaacaaaacatactgaag aacggcccaaggtacaggtgatccaggctgccgatccgctagcaacagcgtatctgtg cggaatccaccgagagctggttaggagattaaatgcggtcctgcttccgaacattcat acactgtttgatatgtcggctgaagactttgacgctattatagccgagcacttccagc | |
| ctggggattgtgttctggaaactgacatcgcgtcgtttgataaaagtgaggacgacgc catggctctgaccgcgttaatgattctggaagacttaggtgtggacgcagagctgttg acgctgattgaggcggctttcggcgaaatttcatcaatacatttgcccactaaaacta aatttaaattcggagccatgatgaaatctggaatgttcctcacactgtttgtgaacac agtcattaacattgtaatcgcaagcagagtgttgagagaacggctaaccggatcacca tgtgcagcattcattggagatgacaatatcgtgaaaggagtcaaatcggacaaattaa tggcagacaggtgcgccacctggttgaatatggaagtcaagattatagatgctgtggt gggcgagaaagcgccttatttctgtggagggtttattttgtgtgactccgtgaccggc acagcgtgccgtgtggcagaccccctaaaaaggctgtttaagcttggcaaacctctgg cagcagacgatgaacatgatgatgacaggagaagggcattgcatgaagagtcaacacg ctggaaccgagtgggtattctttcagagctgtgcaaggcagtagaatcaaggtatgaa accgtaggaacttccatcatagttatggccatgactactctagctagcagtgttaaat cattcagctacctgagaggggcccctataactctctacggctaacctgaatggactac gacatagtctagtccgccaagtctagaacaagtttgtacaaaaaagcaggctgccacc ATGAACCCTTTATGGACATTGTTGTTCGTACTCAGCGCCCCGCGGGGGGTGCTGAGCG ACCCACGCTGTAAGACGACGTGTGACTGCTGCCCCCCCCCGGAGCTTCCGGGGGGGCC GTCAGTGTTTATCTTTCCGCCCAAGCCGAAAGATACACTGACGATCTCGGGGACTCCG GAGGTCACGTGTGTTGTCGTCGACGTCGGCCACGACGACCCGGAGGTGAAGTTCTCCT GGTTCGTGGACGACGTCGAGGTCAACACCGCGACCACGAAGCCCCGAGAGGAGCAGTT TAACTCGACTTACAGGGTGGTGTCGGCCCTCCGGATCCAGCACCAGGACTGGACCGGA GGGAAGGAGTTCAAGTGCAAGGTGCATAACGAAGGCCTGCCGGCTCCCATCGTCCGGA CCATTTCGCGGACGAAAGGTCCGGCGAGGGAGCCGCAGGTCTACGTTCTAGCTCCTCC TCAGGAGGAGCTATCCAAGAGTACGGTCTCCCTCACCTGCATGGTGACTAGCTTCTAT CCGGATTATATTGCGGTGGAGTGGCAGCGTAACGGTCAGCCGGAGTCGGAGGACAAGT ACGGTACCACTCCACCGCAGTTAGATGCGGATGGAAGCTATTTTCTCTATAGCAGGTT GAGGGTAGACCGTAACTCTTGGCAGGAGGGCGACACATACACGTGTGTCGTCATGCAC GAGGCACTTCACAACCACTATACTCAGAAGAGTACCTCCAAGTCCGCCGGAAAAGGCG GATCCGGCGGATCCGGTGGATCCGGCGGATCCGGCGGTTCCGGCGGATCTGGAGGTCA TCTGGATTGCAAGCCCGAGTTTTCCTATGCGATCGCGAAAAGCGATCGCATAGGACCG CTCGGTGCTGAAGGTTTGACGACTACCTGGAAGGATTACAGCCACGAGATGAAGTTGG AGGACACCATGGTCATCGCGTGGTGTAAGGACGGGAAGTTCGTCTACCTTCAGCGGTG CACGCGGGAGACGCGGTACCTGGCCATCTTACATTCTCGAGCACTACCGACCTCGGTA GTGTTCAAGAAGTTGTTTGATGGCCGGCGTCAGGAGGACACAGTCGAGATGGACGATA ACTTCGAGTTTGGACTGTGTCCTTGTGACGCCAAGCCCATTGTGCGGGGTAAGTTCAA TACTACCCTGCTCAATGGGCCTGCGTTCCAGATGGTGTGTCCCATAGGGTGGACCGGT ACGGTGAGCTGCATGCTCGCCAACCGGGACACCCTAGACACAGCAGTGGTGCGCACCT ATAGGCGGAGCAAGCCGTTCCCCTATAGGCAGGGGTGCGGGGGGTCAGGGGGAGGTGG TAGTGGGGGACTCCCGGACTGCAAGCCCGACTTCTCCTATGCGATCGCGAAGAACGAT GAAATCGGACCGCTCGGTGCCACTGGGTTAACGACCCAGTGGTACGAGTATTCCGATG | |
| GCATGCGACTTCAGGACACTGAAGTCGTGGTGTGGTGCAAGGACGGCGAGTTCAAGTA CCTCATCCGGTGTGAGAGGGAGGCCAGATATCTGGCCATCCTCCACACCCGTGCCCTT CCCACTTCAGTGGTCTTTGAGAAGATCCTGAATGGGAAGGAGCAGGAAGATGTCGTCG AGATGGACGACAACTTCGAGTTCGGGCTATGCCCATGTGACGCGAAGCCGCTCGTTCG GGGGAAGTTTAACACGACTCTCCTGAACGGGCCGGCTTTTCAGATGGTATGCCCGATA GGGTGGACCGGTACGGTGAGCTGTACCTTGGCCAACAAGGACACGCTCGCCACTACCG TTGTCCGCACCTATAAGCGCCACCGCCCGTTCCCGTACCGGCAGGGTTGCGGGGGCTC GGGCGGTGGCGGTTCTGGAGGCTTTCCTGAGTGCAAGGAAGGCTTCCAGTACGCGATC AGCAAGGACAAGAAGATGGGCTTGCTTGGTCCGGAGTCCCTCACTACGACCTGGCACC TGCCCACGAAGAAGATCGTGGACAGCATGGTCCAGGTCTGGTGCGAGGGGAAGGATCT GAAGATCCTTCGCACTTGCACCAAGGAGGAGCGGTACTTGGTGGCAGTGCACGAGCGT GCACTGTCCACCAGTGCCGAGTTCCTCCAGATATCGGACGGGACGACCGGACCCGAAG TCATCGACATGCCCGATGACTTCGAGTTCGGTCTGTGCCCGTGCGATAGCAAGCCCGT TATCAAGGGGAAGTTCAATACTTCCCTCTTGAACGGTCCCGCCTTCCAGATGGTCTGC CCGCAGGGGTGGACCGGGACCATCGAGTGTATCCTGGCCAACCAGGATACACTCGATA CGACGGTGATCCGGACCTACCGCCGCACTACGCCGTTCCAGCGGCGTAAGTGGTGTGG GTCCGGACATCACCATCACCACCACTAATGAacccagctttcttgtacaaagtggtgg cgcgcctatgttacgtgcaaaggtgattgtcaccccccgaaagaccatattgtgacac accctcagtatcacgcccaaacatttacagccgcggtgtcaaaaaccgcgtggacgtg gttaacatccctgctgggaggatcagccgtaattattataattggcttggtgctggct actattgtggccatgtacgtgctgaccaaccagaaacataattgaatacagcagcaat tggcaagctgcttacatagaactcgcggcgattggcatgccgccttaaaatttttatt ttatttttcttttcttttccgaatcggattttgtttttaatatttcaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaggaagagctagggataacagggtaat (SEQ ID NO.11) | |
| Coded cattle IgG1 Fc DNA sequence | GACCCACGCTGTAAGACGACGTGTGACTGCTGCCCCCCCCCGGAGCTTCCGGGGGGGC CGTCAGTGTTTATCTTTCCGCCCAAGCCGAAAGATACACTGACGATCTCGGGGACTCC GGAGGTCACGTGTGTTGTCGTCGACGTCGGCCACGACGACCCGGAGGTGAAGTTCTCC TGGTTCGTGGACGACGTCGAGGTCAACACCGCGACCACGAAGCCCCGAGAGGAGCAGT TTAACTCGACTTACAGGGTGGTGTCGGCCCTCCGGATCCAGCACCAGGACTGGACCGG AGGGAAGGAGTTCAAGTGCAAGGTGCATAACGAAGGCCTGCCGGCTCCCATCGTCCGG ACCATTTCGCGGACGAAAGGTCCGGCGAGGGAGCCGCAGGTCTACGTTCTAGCTCCTC CTCAGGAGGAGCTATCCAAGAGTACGGTCTCCCTCACCTGCATGGTGACTAGCTTCTA TCCGGATTATATTGCGGTGGAGTGGCAGCGTAACGGTCAGCCGGAGTCGGAGGACAAG TACGGTACCACTCCACCGCAGTTAGATGCGGATGGAAGCTATTTTCTCTATAGCAGGT TGAGGGTAGACCGTAACTCTTGGCAGGAGGGCGACACATACACGTGTGTCGTCATGCA CGAGGCACTTCACAACCACTATACTCAGAAGAGTACCTCCAAGTCCGCCGGAAAA (SEQ ID NO.12) |
Example 2 different types of mRNA vaccine preparation
(I) Preparation of vector vaccine based on capped mRNA architecture
Step a: tangentially cutting plasmid CS-BVDV-210 with restriction enzyme;
step b: carrying out in vitro co-transcription capping reaction on the linearized CS-BVDV-210 plasmid, adding a 7-methylated guanylate cap structure to the 5' -end of transcribed mRNA, and degrading the template DNA;
(II) preparation of vector vaccine based on non-capping mRNA framework
Step a: tangentially cutting plasmid CS-BVDV-220 with restriction enzyme;
step b: carrying out in-vitro uncapped transcription reaction on the linearized CS-BVDV-220 plasmid, and degrading template DNA;
(III) preparation of vector vaccines based on self-replicating RNA structures
Step a: tangential restriction of CS-BVDV-230 plasmid with restriction enzyme;
step b: carrying out in vitro co-transcription capping reaction on the linearized CS-BVDV-230 plasmid, adding a 7-methylated guanylate cap structure to the 5' -end of transcribed RNA, and degrading the template DNA;
mixing the mRNA prepared in (I-III) with lipid respectively, wrapping the mRNA into LNP to obtain mRNA-LNP complex, concentrating and changing the solution to obtain the vaccine.
EXAMPLE 3 linearization and in vitro Co-transcription capping of plasmid CS-BVDV-210 and control System to prepare mRNA
Plasmid CS-BVDV-210 and control System linearization in example 1 was performed using BspQI enzyme tangentially and the linearized CS-BVDV-210 and control was recovered by magnetic bead purification. 1% agarose gel electrophoresis, to determine if plasmid CS-BVDV-210 and control were completely linearized.
The linearized CS-BVDV-210 and the control plasmid start in vitro transcription reaction by using T7 RNA polymerase, and the cap analogue 7-methylated guanylate cap structure is directly added into an in vitro transcription reaction system, so that the 5' -end of the mRNA prepared by in vitro transcription carries the cap analogue. After the in vitro transcription reaction is finished, the DNase enzyme is adopted to degrade the template DNA.
Example 4 mRNA-secretion Signal peptide+bovine IgG1 Fc+BVDV1a Domain I-II+BVDV1b Domain I-II BVDV2 Domain I-II and control System and lipid coating
The mRNA of the present invention and the control system of example 1 were co-mixed with the following lipids, namely, the ionizable lipid DHA-1 (Saunopong, cat. No. 0604000930), the helper lipid DSPC (Saunopong, cat. No. 06030001100), cholesterol (Ai Weita, 57-88-5), DMG-PEG2000 (Saunopong, cat. No. 06020112402), in a molar ratio of DHA-1: DSPC: cholesterol: DMG-PEG2000 was 50:10:38.5:1.5, wherein the molar ratio of mRNA to LNP (sum of four lipids) is 1:5. the components were dissolved in absolute ethanol and mixed rapidly by a mixer, causing precipitation of lipids and encapsulation of mRNA into LNP under charge. The mRNA-LNP complex was then concentrated and pipetted into the formulation solution.
EXAMPLE 5 Western Blot and Dot Blot detection of mRNA expression and secretion in cells
Subculturing HEK293T cells until the number of cells is sufficient, digesting with pancreatin to each well of a cell culture 6-well plate, plating CO at 37deg.C 2 The incubator was cultured overnight. The following day, liposome-encapsulated mRNA was transfected into confluent HEK293T cells, and after 48h of culture, cell lysates and supernatant media were collected.
1) Western Blot detection:
electrophoresis: the concentration of the polyacrylamide separating gel is 12%, the protein loading amount is 40ug, the electrophoresis condition of the concentrated gel is 90V, and the electrophoresis condition of the separating gel is 120V.
Transferring: the membrane was transferred using PVDF membrane at 100v voltage for 1h.
Closing: 5% BSA was prepared as blocking solution using 1xTBST and blocked for 1h.
Incubation resistance: using a blocking liquid will 6His antibodies were diluted 1:2500 and incubated overnight at 4 ℃. />
Secondary antibody incubation: the corresponding secondary antibodies were diluted with blocking solution at a dilution ratio of 1:5000 and incubated for 1h at room temperature.
Developing: developing solution A: liquid b=1:1, development was performed in the image forming system.
2) Dot blot detection:
taking 100ul of recovered supernatant culture medium, and dripping the 100ul of recovered supernatant culture medium on a PVDF membrane; after it has dried, use 15% BSA in TBST was used as blocking solution, and blocked for 1h.
Incubation resistance: using a blocking liquid will 6His antibodies were diluted 1:2500 and incubated overnight at 4 ℃.
Secondary antibody incubation: the corresponding secondary antibodies were diluted with blocking solution at a dilution ratio of 1:5000 and incubated for 1h at room temperature.
Developing: developing solution A: liquid b=1:1, development was performed in the image forming system.
The experimental results are shown in fig. 2A-2D, which are graphs of the detection results of the mRNA of the BVDV antigen fusion protein of the present invention and the Western Blot and Dot Blot of the control, wherein fig. 2A is a graph of the Dot Blot protein expression of the mRNA of the present invention, i.e., secretion signal peptide+bovine igg1fc+bvdv 1a domain I-ii+bvdv1b domain I-ii++ BVDV2 domain I-II, bovine igg1fc+bvdv 1a domain I-ii+bvdv1b domain I-II BVDV2 domain I-II after in vitro transfection of HEK293T cells; FIG. 2B is a Western Blot protein expression pattern of bovine IgG1 Fc+BVDV1a domain I-II+BVDV1B domain I-II++ BVDV2 domain I-II following in vitro transfection of HEK293T cells with mRNA of the invention; FIG. 2C is a graph of Western Blot protein expression following in vitro transfection of HEK293T cells with mRNA (Fc+) of the present invention and a control (not containing bovine IgG1 Fc, i.e., fc-); FIG. 2D is a graph of Dot blot protein expression following in vitro transfection of HEK293T cells with mRNA (Fc+) of the present invention and a control (not containing bovine IgG1 Fc, i.e., fc-).
FIGS. 2A and 2B are graphs showing the expression of bovine IgG1 Fc+BVDV1a domain I-II+BVDV1B domain I-II++ BVDV2 domain I-II protein after in vitro transfection of mRNA into HEK293T cells, and the results of WB (FIG. 2B) and Dot Blot (FIG. 2A) indicate that the corresponding target protein can be expressed and secreted with high efficiency after in vitro transfection of mRNA into HEK293T cells. As can be seen from FIGS. 2A and 2B, the mRNA-bovine IgG1 Fc+BVDV1a domain I-II+BVDV1B domain I-II++ BVDV2 domain I-II is expressed and secreted better after in vitro transfection of HEK293T cells with bovine IgG1 Fc+BVDV1a domain I-II+BVDV1B domain I-II BVDV2 domain I-II.
Furthermore, fig. 2C and 2D show that the control not containing bovine IgG1 Fc has low secretory expression relative to the mRNA of the present invention. The mRNA of the invention is proved, and the added bovine IgG1 Fc structural domain plays a vital role in the expression and secretion of target protein polypeptide BVDV antigen fusion protein.
Example 6 detection of neutralizing antibodies against BVDV induced by immunization of mice with mRNA-bovine IgG1 Fc+BVDV1a Domain I-II+BVDV1b Domain I-II BVDV2 Domain I-II vaccine
After 7 days of adaptive feeding of 6-8 week old BALB/c females, 5 were the mRNA-LNP complex group prepared in example 4 (comprising mRNA-bovine IgG1 Fc+BVDV1a domain I-II+BVDV1b domain I-II++ BVDV2 domain I-II) and 5 were the control group (without any administration, only for blood sampling for comparative analysis). The mode of administration was inguinal subcutaneous injection, 3 times per group of animals were immunized, and the doses were 10ug mRNA-LNP/dose at day 0 (D0), day 14 (D14) and day 28 (D28). FIG. 3 shows a schematic diagram of the immunization and sampling process of mice with BVDV antigen fusion protein mRNA of the invention.
FIG. 3 is a schematic diagram showing the immunization and sampling process of BVDV antigen fusion protein mRNA mice, wherein the time point of blood collection is D7/D14/D21/D28/D35/D42, and more than 100 μl of whole blood is taken from each mouse when blood is taken, so that the requirement of 50 μl of serum is met. 5 mice were fixed as a biological sample group, and serum extracted from 5 mice in each group was mixed as one biological sample (250. Mu.l). Thus 1 sample was obtained after each sampling of each sample group.
The method for detecting the titer of the neutralizing antibody is ELISA, and comprises the following specific operations:
after digestion of MDBK cells with pancreatin, 2x105 cells/mL were adjusted with DMEM medium containing 10% fetal bovine serum; the cells were spread evenly on 96-well cell culture plates, 100 ul/well, and incubated overnight in a CO2 incubator at 37 ℃.
The serum to be tested is extinguished at 56 ℃ for 30min, centrifuged at 10,000g for 10min, and diluted 2-fold from 1:8 with DMEM medium containing 2% fetal bovine serum. BVDV suspensions of different subtypes (BVDV 1a: NADL strain (GenBank ID: AJ 133738.1), BVDV 1b: JSP220 1 strain (GenBank ID: OP 856581), BVDV2: C201602 (GenBank ID: MG 420995)) were diluted with DMEM containing 2% fetal bovine serum at an inoculum size of 100TCID 50/50. Mu.l. Equal volumes of virus solution were mixed with different dilutions of serum to be tested and incubated for 1h at 37 ℃. The mixture after incubation was added to MDBK cells in 96-well cell culture plates at 100ul per well, and 4 replicates were made per sample. Positive and negative serum controls (1:8 dilution), blank controls and toxic cell-receiving controls were set simultaneously. Placing the cells into a cell culture box for further culture for 5 days.
Detection is carried out by indirect immunofluorescence experiments, and the specific operation is as follows:
the culture solution is discarded, cells are fixed for 20min at room temperature by adding an immunostaining fixing solution, PBST is washed 3 times, an immunostaining blocking solution is added for blocking at 37 ℃ for 1h, PBST is washed 3 times, BVDV monoclonal antibodies (AHVLA, diluted 1:100) are added, incubation is performed for 1h at 37 ℃ and PBST is washed 3 times, FITC-labeled goat anti-mouse IgG diluted 1:500 is added, 100 μl/hole is added, incubation is performed for 1h at 37 ℃ and PBST is washed 3 times, and the mixture is observed by a fluorescence microscope. Counting the number of positive and negative cell holes, and calculating the neutralizing antibody titer according to a Reed-Muench method.
Experimental results: neutralizing antibody titers against BVDV1a, 1b and 2 genotype strains in mouse serum. Table 2 shows the results of antibody detection (BVDV-1 a type); table 3 shows the results of antibody detection (BVDV-2 type); table 4 shows the results of antibody detection (BVDV-1 b type).
TABLE 2 antibody detection results (BVDV-1 a type)
TABLE 3 antibody detection results (BVDV-2 type)
TABLE 4 antibody detection results (BVDV-1 b type)
Note that: tables 2 to 4/show that no detection was performed
From the results in tables 2-4, it can be seen that mRNA-bovine IgG1 Fc+BVDV1a domain I-II+BVDV1b domain I-II+BVDV2 domain I-II is capable of inducing high levels of neutralizing antibodies in animals. In this example, the subject selected was a mouse. However, it is suggested that the Fc fragment expressed by the mRNA is derived from bovine IgG1, and that the fusion protein of the Fc can increase the half-life of the fusion protein in the bovine body and further amplify the immune response induced in the bovine body. The experimental result of the embodiment shows that the trivalent BVDV mRNA vaccine obtained by the method can be well expressed in mice and induces and generates more remarkable neutralizing antibody titer. The invention can simultaneously express antigens of a plurality of BVDV genotypes by using one mRNA, has strong immunogenicity and obvious immune effect, and thus has good application prospect.
The above examples of the present disclosure are merely examples for clearly illustrating the present disclosure and are not limiting of the embodiments of the present disclosure. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. Any modifications, equivalent substitutions, improvements, etc. that fall within the spirit and principles of the present disclosure are intended to be included within the scope of the claims of the present disclosure.
Claims (17)
1. A BVDV antigen fusion protein is characterized in that the amino acid sequence of the fusion protein is shown as SEQ ID NO. 1.
2. A recombinant nucleic acid, wherein said recombinant nucleic acid is a nucleic acid encoding a BVDV antigen fusion protein according to claim 1.
3. The recombinant nucleic acid according to claim 2, wherein the nucleic acid sequence of the recombinant nucleic acid is shown in SEQ ID No. 2.
4. A recombinant mRNA encoding the BVDV antigen fusion protein of claim 1.
5. The recombinant mRNA of claim 4, further comprising one or more of a 5 '-UTR, a 3' -UTR, a polyA sequence, and an IRES sequence.
6. The recombinant mRNA according to claim 5, wherein the sequence of the 5 '-UTR is shown in SEQ ID NO.3, the sequence of the 3' -UTR is shown in SEQ ID NO.4, the sequence of the polyA is shown in SEQ ID NO.5, and the IRES sequence is shown in SEQ ID NO. 8.
7. An expression cassette comprising the recombinant nucleic acid of claim 2 or 3, or the recombinant mRNA of any one of claims 4-6.
8. A vector comprising the recombinant nucleic acid of claim 2 or 3, or the recombinant mRNA of any one of claims 4-6, or the expression cassette of claim 7.
9. A cell comprising the recombinant nucleic acid of claim 2 or 3, or the recombinant mRNA of any one of claims 4-6, or the expression cassette of claim 7, or the vector of claim 8.
10. A recombinant bacterium comprising the recombinant nucleic acid of claim 2 or 3, or the recombinant mRNA of any one of claims 4-6, or the expression cassette of claim 7, or the vector of claim 8.
11. A composition comprising a BVDV antigen fusion protein according to claim 1, or a recombinant nucleic acid according to claim 2 or 3, or a recombinant mRNA according to any one of claims 4-6, or an expression cassette according to claim 7, or a vector according to claim 8, or a cell according to claim 9, or a recombinant bacterium according to claim 10.
12. A kit comprising a BVDV antigen fusion protein according to claim 1, or a recombinant nucleic acid according to claim 2 or 3, or a recombinant mRNA according to any one of claims 4-6, or an expression cassette according to claim 7, or a vector according to claim 8, or a cell according to claim 9, or a recombinant bacterium according to claim 10, or a composition according to claim 11.
13. A trivalent BVDV mRNA vaccine comprising:
(a) The recombinant mRNA of any one of claims 4-6; and
(b) A pharmaceutically acceptable carrier.
14. A method for preparing a capped mRNA recombinant expression vector, a non-capped mRNA recombinant expression vector or a self-replicating RNA recombinant expression vector for expressing recombinant trivalent BVDV mRNA, comprising the steps of:
step a: synthesizing a gene fragment of the recombinant nucleic acid of claim 2 or 3;
step b: constructing a capped mRNA architecture vector, constructing a non-capped mRNA architecture vector, or constructing a VEE self-replicating vector;
the capping mRNA framework vector comprises 5 '-UTR, 3' -UTR and polyA sequences and a vector for fixing the capping mRNA framework, and the sequences are respectively shown as SEQ ID NO. 3-6; or,
the non-capping mRNA framework vector comprises an IRES sequence, a 3' -UTR and a polyA sequence, and a vector for fixing the non-capping mRNA framework, wherein the sequence of the IRES is shown as SEQ ID No. 8; or,
Construction of VEE self-replicating vector: self-replicating RNA sequences obtained from the genome of the alphavirus family; the self-replicating RNA sequence comprises a gene encoding an alphavirus self-replicating component, lacking a gene that produces a structural protein of an infectious alphavirus particle; amplifying a plasmid constructed by the self-replicating RNA sequence as a template, and synthesizing to obtain the VEE self-replicating vector; the sequence of the VEE self-replication vector is shown as SEQ ID NO. 10;
step c: preparing capped mRNA, uncapped mRNA or self-replicating RNA recombinant expression vector;
c, inserting the gene synthesized in the step a into a capping mRNA framework vector to obtain a capping mRNA recombinant expression vector; or,
inserting the gene synthesized in the step a into a non-capping mRNA framework vector to obtain a non-capping mRNA recombinant expression vector; or,
and c, inserting the gene synthesized in the step a into a designated position of the VEE self-replication vector to obtain the self-replication RNA recombinant expression vector.
15. A method for preparing a trivalent BVDV mRNA vaccine, comprising:
(a) Providing the recombinant mRNA of any one of claims 4-6;
(b) The mRNA is mixed with lipid particles and incubated, thereby forming mRNA-encapsulated lipid nanoparticles.
16. A method for preparing a trivalent BVDV capped mRNA structured vector vaccine, a non-capped mRNA structured vector vaccine, or a self-replicating RNA structured vector vaccine, comprising the steps of:
step a: tangentially ligating the capped mRNA recombinant expression vector, the non-capped mRNA recombinant expression vector, or the self-replicating RNA recombinant expression vector of claim 14 with a restriction enzyme;
step b: carrying out in vitro co-transcription capping reaction on the linearized capping mRNA recombinant expression vector, adding a 7-methylated guanylate cap structure to the 5' end of transcribed mRNA, and degrading template DNA; or,
carrying out in-vitro uncapped transcription reaction on the linearized uncapped mRNA recombinant expression vector, and degrading template DNA; or,
carrying out in vitro co-transcription capping reaction on the linearized self-replicating RNA recombinant expression vector, adding a 7-methylated guanylate cap structure to the 5' -end of transcribed RNA, and degrading template DNA;
step c: mixing the capped, uncapped and self-replicating mRNA prepared in the step b with lipid respectively, wrapping the mRNA into LNP to obtain mRNA-LNP complex, concentrating and changing liquid to obtain preparation solution, thus obtaining the vaccine.
17. Use of the BVDV antigen fusion protein of claim 1, or the recombinant mRNA of any one of claims 4-6, in any one of the following: (I) Preparing a medicament for treating or preventing diseases caused by BVDV infection; or, (II) preparing a medicament for treating or preventing bovine viral diarrhea; or (III) preparing a medicament that induces a specific immune response against BVDV in the subject.
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| CN115364207A (en) * | 2022-06-17 | 2022-11-22 | 华中农业大学 | Vaccine composition for preventing and treating bovine viral diarrhea and preparation method thereof |
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| CN101360510A (en) * | 2005-11-15 | 2009-02-04 | 贝林格尔.英格海姆维特梅迪卡有限公司 | Combination vaccine comprising an attenuated bovine viral diarrhea virus |
| CN115364207A (en) * | 2022-06-17 | 2022-11-22 | 华中农业大学 | Vaccine composition for preventing and treating bovine viral diarrhea and preparation method thereof |
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