CN111996202A

CN111996202A - Indocard recombinant virus and recombinant vaccine strain of recombinant O-type foot-and-mouth disease virus VP1 gene, and preparation method and application thereof

Info

Publication number: CN111996202A
Application number: CN202010212908.4A
Authority: CN
Inventors: 郑海学; 杨帆; 朱紫祥; 魏婷; 曹伟军; 刘华南; 郑敏; 张伟; �田宏; 张克山; 刘永杰; 党文; 马旭升; 李丹; 茹毅; 何继军; 郭建宏; 刘湘涛
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2020-03-24
Filing date: 2020-03-24
Publication date: 2020-11-27
Anticipated expiration: 2040-03-24
Also published as: CN111996202B

Abstract

The invention provides a Senecan recombinant virus of a recombinant O-type foot-and-mouth disease virus VP1 gene, a recombinant vaccine strain, a preparation method and application thereof, relating to the technical field of genetic engineering. The SVV/FJ/001 strain is subjected to gene deletion mutation transformation, and meanwhile, VP1 gene of O-type foot-and-mouth disease virus is fused into SVA cDNA to obtain Senecan recombinant virus, wherein the recombinant virus can express the fused exogenous FMDV VP1 gene, and the expression product has good reactogenicity; the prepared vaccine strain has high antigen productivity, obviously reduced pathogenicity and even no pathogenicity to pigs, can not only stimulate the immune response of SVA after an inactivated vaccine immunizes animals, but also generate the immunocompetence aiming at the fusion gene, obviously improves the biological safety of the vaccine strain, and can be used for preventing and controlling the seneca virus and one or more non-seneca viruses.

Description

Indocard recombinant virus and recombinant vaccine strain of recombinant O-type foot-and-mouth disease virus VP1 gene, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a seneca recombinant virus and a recombinant vaccine strain of a recombinant O-type foot-and-mouth disease virus VP1 gene, and a preparation method and application thereof.

Background

Senecavir a (SVA), also known as senecavir (Senecavirus), Seneca Valley Virus (SVV), belongs to the genus Senecavirus (Senecavirus) of the Picornaviridae (Picornaviridae), and is also the only member of this genus. The virus infected pig can cause primary vesicular disease of the pig, and is difficult to distinguish from clinical symptoms caused by foot-and-mouth disease, pig vesicular disease, vesicular stomatitis and the like. After SVA infection, weaned piglets and pigs of all ages which are kept, fattened and bred can be affected with vesicular lesions, and are accompanied with clinical symptoms such as lameness, fever, anorexia, lethargy and the like, and newborn piglets can have symptoms such as continuous diarrhea, dehydration and the like besides the symptoms, and even die suddenly.

Foot and Mouth Disease Virus (FMDV) belongs to the genus of Foot and mouth disease virus (Aphthovius) of the family of Picornaviridae (Picornaviridae), and comprises seven serotypes A, O, C, Asial, SAT1, SAT2 and SAT3, and no cross protection exists among the types. The total length of the viral genome is about 8.5kb, and consists of a 5 'non-coding region, an ORF region and a 3' non-coding region. The coding region encodes a polyprotein, which is gradually degraded by viral self-protease and host cell protease to form various intermediates and mature 4 structural proteins (VP4, VP2, VP3 and VP1) and 8 non-structural proteins (L)^pro2A, 2B, 2C, 3A, 3B, 3C, 3D). Among them, VP1 is the most important capsid protein encoded by structural protein gene, and is the main antigenic protein for determining FMDV antigenicity, inducing organism to produce neutralizing antibody and stimulating protective immune responseIn the family of picornaviridae, VP1 is recognized as the most immunogenic protein, and therefore, as an antigen gene of FMDV, VP1 protein is expressed, and the biological activity and immunogenicity thereof are maintained, thus playing an important role in research of FMDV genetic engineering vaccines.

Disclosure of Invention

In view of the above, the present invention aims to provide a seneca virus recombinant nucleic acid of a recombinant O-type foot-and-mouth disease virus VP1 gene, a seneca recombinant virus comprising the recombinant nucleic acid, a seneca recombinant virus encoded by the recombinant nucleic acid, a seneca recombinant vaccine strain comprising the seneca recombinant virus, and a preparation method and applications thereof. The SVV/FJ/001 strain is subjected to gene deletion mutation transformation, and meanwhile, VP1 gene of O-type foot-and-mouth disease virus is fused into SVAcDNA to obtain Senecan recombinant virus, wherein the recombinant virus can express the fused exogenous FMDV VP1 gene, and the expression product has good reactogenicity; the prepared vaccine strain has high antigen productivity, obviously reduced pathogenicity and even no pathogenicity to pigs; the inactivated vaccine is used for immunizing animals to generate the immunocompetence of SVA and the immunocompetence aiming at the fusion gene. The vaccine strain obviously improves the biological safety and can be used for preventing and controlling the epidotic fever virus and one or more non-epidotic fever viruses.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a seneca virus recombinant nucleic acid, wherein the sequence of the recombinant nucleic acid comprises deletion mutation modification of a 5' UTR gene sequence of a seneca virus strain, and a VP1 gene of O-type foot-and-mouth disease virus is inserted between Sph I and Not I enzyme cutting sites of seneca virus cDNA.

Preferably, the nucleotide sequence of the 5'UTR gene modified by deletion mutation of the 5' UTR gene sequence of the Seneca virus strain is shown as SEQ ID NO. 1; the nucleotide sequence of the inserted VP1 gene of the O-type foot-and-mouth disease virus is shown in SEQ ID NO. 2.

Preferably, the Seneca virus strain comprises the SVV/FJ/001 strain.

The invention also provides application of the recombinant nucleic acid in preparation of the Seneca virus recombinant nucleic acid or Seneca recombinant vaccine strain of the recombinant O-type foot-and-mouth disease virus VP1 gene.

The invention also provides a seneca recombinant virus of the recombinant O-type foot-and-mouth disease virus VP1 gene containing the recombinant nucleic acid.

The invention also provides a seneca recombinant virus of the recombinant O-type foot-and-mouth disease virus VP1 gene coded by the recombinant nucleic acid.

The invention also provides a recombinant vaccine strain containing the recombinant virus.

Preferably, the epicaic recombinant vaccine strain can not only stimulate the immunological activity of animals to the epicaic virus, but also generate the immunological activity to the foot-and-mouth disease virus.

The invention also provides a construction method of the seneca recombinant virus, which comprises the following steps:

(1) using cDNA of O/BY/2010 strain as a template, and amplifying BY using a specific primer pair to obtain VP1 gene of O-type FMDV; the specific primer pair for amplifying the VP1 gene comprises an upstream primer and a downstream primer OS-R, the upstream primer comprises OVP1-F0 and OVP1-F, the nucleotide sequence of the upstream primer OVP1-F0 is shown as SEQ ID No.3, the nucleotide sequence of the upstream primer OVP1-F is shown as SEQ ID No.4, and the nucleotide sequence of the downstream primer OS-R is shown as SEQ ID No. 5;

(2) respectively amplifying to obtain an S1 fragment and an S20 fragment of the SVV/FJ/001 strain by using a specific primer pair by taking the cDNA of the SVV/FJ/001 strain as a template; the specific primer pair for amplifying the S1 fragment comprises an upstream primer and a downstream primer SVA-1R, wherein the upstream primer comprises SVA-1F0 and SVA-1F; the nucleotide sequence of the upstream primer SVA-1F0 is shown as SEQ ID NO.6, and the nucleotide sequence of the upstream primer SVA-1F is shown as SEQ ID NO. 7; the nucleotide sequence of the downstream primer SVA-1R is shown as SEQ ID NO. 8;

the specific primer pair for amplifying the S20 fragment comprises an upstream primer SVA-2F0 and a downstream primer SVA-2R, the nucleotide sequence of the upstream primer SVA-2F0 is shown as SEQ ID NO.9, and the nucleotide sequence of the downstream primer SVA-2R is shown as SEQ ID NO. 10;

(3) fusing the VP1 gene fragment obtained in the step (1) with the S20 gene fragment to obtain an S2-OVP1 fragment;

(4) respectively connecting the S1 fragment and the S2-OVP1 fragment with a pMD 20T vector to obtain subclone plasmids PMD-S1 and PMD-S2-OVP 1;

(5) amplifying by using mutation primers SVA-m5UTRF and SVA-m5 UTRA by using the subcloning plasmid PMD-S1 as a template to obtain a subcloning plasmid PMD-mS1, wherein the nucleotide sequence of the SVA-m5 UTRA is shown as SEQ ID NO.11, and the nucleotide sequence of the SVA-m5 UTRA is shown as SEQ ID NO. 12;

(6) carrying out double enzyme digestion on plasmid PMD-mS1 by PacI and SphI, carrying out double enzyme digestion on plasmid PMD-S2-OVP1 by SphI and NotI, recovering a gene fragment, and inserting the gene fragment into eukaryotic transcription plasmid prO/CHA/99 subjected to double enzyme digestion by PacI and NotI in a connecting and replacing manner to obtain recombinant plasmid prSVV/FJ-M-OVP 1;

(7) transfecting the obtained recombinant plasmid prSVV/FJ-M-OVP1 to the Seneca virus sensitive cells to obtain the Seneca recombinant virus of the recombinant O-type FMDVVP1 gene.

Preferably, in the step (3), fusion PCR is carried out by using primers OVP1-F and SVA-2R to fuse gene segments; the nucleotide sequence of the OVP1-F is shown as SEQ ID NO.4, and the nucleotide sequence of the SVA-2R is shown as SEQ ID NO. 10.

Preferably, the mutated 5' UTR gene segment of step (5) comprises a nucleic acid sequence as shown in SEQ ID No.1, or comprises said recombinant nucleic acid.

Preferably, the Senecavirus sensitive cells of step (7) comprise BHK-21 cells, PK-15 cells, ST cells, SK-RST cells, IBRS-2 cells, H1299 cells or 293T cells.

The invention also provides the use of the seneca recombinant virus of the recombinant O-type FMDVVP1 gene or the seneca recombinant virus prepared by the method in the preparation of the seneca recombinant vaccine.

The invention also provides a Selenecar recombinant vaccine of the recombinant O-type FMDVVP1 gene.

The invention also provides application of the epicaic recombinant vaccine strain or the epicaic recombinant vaccine in preparing a medicament for preventing and/or controlling related diseases of animals caused by epicaic virus and foot-and-mouth disease virus.

Preferably, the animal comprises a pig, a cow or a sheep.

The invention provides a seneca virus recombinant nucleic acid of a recombinant O-type foot-and-mouth disease virus VP1 gene, a seneca recombinant virus containing the recombinant nucleic acid, a seneca recombinant virus coded by the recombinant nucleic acid, a seneca recombinant vaccine strain containing the seneca recombinant virus, a preparation method and application thereof. According to the resources such as epinakavirus molecular epidemiology and early-stage accumulated epidemic strains, an established high-efficiency reverse genetic operation technology platform is utilized, SVV/FJ/001 strain gene analysis is carried out, full-length cDNA for gene deletion mutation modification of the strain is constructed, VP1 gene of O-type foot-and-mouth disease virus is fused in SVAcDNA, virus rescue, recombinant strain biological characteristic determination and the like are carried out, the successfully rescued recombinant virus is shown to have cell infection characteristic and high yield characteristic similar to wild type, meanwhile, the recombinant virus can express fused O-type FMDV VP1 protein and has good reactogenicity, pathogenicity research results show that the pathogenicity of the recombinant virus is obviously reduced or even has no pathogenicity to pigs, the biological safety of the strain is obviously improved, therefore, the preparation method can be utilized, SVA is used as a virus framework inserted with exogenous pathogenic antigen gene to construct chimeric vaccine strains, after the animals are immunized, the immunological activity aiming at the parent framework strain and the inserted exogenous antigen gene can be obtained at the same time.

The antigen gene P1 gene of the SVV/FJ/001 strain of the invention has homology of more than 98.6 percent with the isolated SVA epidemic strain gene reported in China, and has high homology with the epidemic strain in other countries, because the P1 gene is the antigen gene of SVA and can induce organisms to generate neutralizing antibodies, the SVV/FJ/001 strain is used as a template, reverse genetic operation technology is utilized, SVA infectious cDNA clone is constructed through amplification and transformation, and meanwhile, the O-type foot-and-mouth disease virus antigen gene VP1 is inserted into the SVA infectious clone to successfully rescue recombinant viruses, thereby not only realizing antigen matching and immune responsiveness of the SVA strain and ensuring pertinence of the SVA strain, but also effectively expressing the inserted foot-and-mouth disease antigen gene, showing that the SVA can be used as a virus skeleton expressed by exogenous genes by using the construction method, used for chimeric expression of antigen genes of different exogenous pathogens and realizes the research and application of chimeric vaccine based on SVA.

The recombinant virus rSVV/FJ-M-OVP1 has high virus titer, the lesion time after stable passage is about 12-18 h, and the poison value is 10^6.5TCID₅₀/mL～10^10.0TCID₅₀/mL。

The recombinant virus rSVV/FJ-M-OVP1 of the seneca carries out partial deletion and mutation transformation on the 5' UTR of the virus, and simultaneously, an O-type FMDV VP1 gene capable of being efficiently expressed is embedded, compared with the pathogenicity of SVV/FJ/001, the recombinant virus strain obviously reduces the pathogenicity of the virus strain to pigs, even has no pathogenicity to the pigs, and obviously improves the biological safety.

The scheme of the invention realizes a more active and effective construction mode of the SVA recombinant/chimeric vaccine strain, realizes innovation of the SVA virus seed preparation process, and has great application value.

Drawings

FIG. 1 shows the results of electrophoresis of the SVA S1 fragment and the S2-OVP1 fragment amplified in example 2, wherein 1 is the amplification product of the S1 fragment; 2 is an S2-OVP1 fragment amplification product; m is DL 5000 DNA Marker;

FIG. 2 is a schematic diagram showing the construction method of the recombinant plasmid prSVV/FJ-M-OVP1 of Selenkavirus in example 2;

FIG. 3 is the cytopathic effect (CPE) caused by infection of BHK-21 cells with the recombinant virus rSVV/FJ-M-OVP1 strain of example 3, wherein A represents normal BHK-21 cells; b represents BHK-21 cells presenting CPE;

FIG. 4 shows the results of indirect immunofluorescence of rSVV/FJ-M-OVP1 strain inoculated with recombinant virus in example 4, wherein A represents a normal cell control and B represents the results of detection after rSVV/FJ-M-OVP1 strain inoculated with recombinant virus;

FIG. 5 shows the result of ELISA detection of the reactivity of rSVV/FJ-M-OVP1 strain recombinant virus in example 4 with FMDV type O-specific antibody;

FIG. 6 shows the results of Westernblot analysis of rSVV/FJ-M-OVP1 in example 4.

Detailed Description

The Selcarca virus strain of the present invention preferably includes SVV/FJ/001 strain. The SVV/FJ/001 strain is preferably the SVV/FJ/001 strain which is preserved in China center for type culture Collection and has the preservation number of CCTCC NO. V201802, and has been disclosed in China granted patent "Sernica Valley virus vaccine and preparation method and application" ZL201810003888.2 ".

The invention preferably carries out deletion mutation transformation on the 5'UTR gene sequence of the SVV/FJ/001 strain, and the nucleotide sequence of the 5' UTR gene of the SVV/FJ/001 strain after transformation is shown in SEQ ID NO. 1. The nucleotide sequence of the VP1 gene of the inserted type O foot-and-mouth disease virus is preferably shown as SEQ ID NO. 2.

The recombinant vaccine strain of the epinocardia seriolae can not only stimulate the immunocompetence of animals to the epinocardia virus, but also generate the immunocompetence to the foot-and-mouth disease virus.

The invention takes cDNA of O/BY/2010 strain as a template, and obtains VP1 gene of O-type FMDV BY amplifying with a specific primer pair; the specific primer pair for amplifying the VP1 gene comprises an upstream primer and a downstream primer OS-R, the upstream primer comprises OVP1-F0 and OVP1-F, the nucleotide sequence of the upstream primer OVP1-F0 is shown as SEQ ID No.3, the nucleotide sequence of the upstream primer OVP1-F is shown as SEQ ID No.4, and the nucleotide sequence of the downstream primer OS-R is shown as SEQ ID No. 5. The invention preferably designs and synthesizes a primer for amplifying VP1 according to the O/BY/2010 strain gene sequence (Genebank: JN998085) of the O-type foot-and-mouth disease virus:

OVP1-F0：5'-gatgcaatcaggcgacgtcgagaccaaccctggccctatgaccacttcgacgggcgag-3'(SEQ ID NO.3)；

OVP1-F：5'-cccagcatgcttccctttcgcagctacaagcagaagatgctgatgcaatcaggcgacgtc-3' (SEQ ID NO.4), the horizontal line part is the enzyme cutting site of SphI;

OS-R：5'-caggatcgggttgtcagaagcgggcccagggttggactccac-3'(SEQ ID NO.5)。

preferably, the method comprises the steps of extracting total RNA of an O/BY/2010 strain, carrying out reverse transcription to synthesize first strand cDNA, carrying out amplification BY using primers OVP1-F0 and OS-R BY using the reverse transcribed first strand cDNA as a template, and carrying out second-round amplification BY using an amplification product as a template and using primers OVP1-F and OS-R to obtain a gene fragment containing O-type foot-and-mouth disease virus VP1, wherein the gene fragment is shown as SEQ ID NO. 2. The amplification conditions for the PCR of the present invention are preferably: 5min at 94 ℃; 30s at 94 ℃, 30s at 57 ℃, 50s at 72 ℃ after 35 cycles; 10min at 72 ℃.

The method takes cDNA of SVV/FJ/001 strain as a template, and utilizes a specific primer pair to respectively amplify to obtain an S1 fragment and an S20 fragment of the SVV/FJ/001 strain; the specific primer pair for amplifying the S1 fragment comprises an upstream primer and a downstream primer SVA-1R, wherein the upstream primer comprises SVA-1F0 and SVA-1F; the nucleotide sequence of the upstream primer SVA-1F0 is shown as SEQ ID NO.6, and the nucleotide sequence of the upstream primer SVA-1F is shown as SEQ ID NO. 7; the nucleotide sequence of the downstream primer SVA-1R is shown as SEQ ID NO. 8; the specific primer pair for amplifying the S20 fragment comprises an upstream primer SVA-2F0 and a downstream primer SVA-2R, wherein the nucleotide sequence of the upstream primer SVA-2F0 is shown as SEQ ID NO.9, and the nucleotide sequence of the downstream primer SVA-2R is shown as SEQ ID NO. 10.

In the present invention, the source of the cDNA of the SVV/FJ/001 strain is not particularly limited, but is preferably obtained by extracting RNA and then reverse transcription.

The sequences of the primers used for amplifying the S1 fragment and the S20 fragment are shown as follows:

SVA-1F0：5'-gtgaggacgaaactataggaaaggaattcctatagtcttgaaagggggggctgggcc-3'(SEQ ID NO.6)；

SVA-1F：5'-ataggtttaattaatgttaagcgtctgatgagtccgtgaggacgaaactatagga-3' (SEQ ID NO.7), the horizontal line part is PacI enzyme cutting site;

SVA-1R：5'-gggaagcatgctggggcaccaggcac-3' (SEQ ID NO.8), the horizontal line part is the enzyme cutting site of SphI;

SVA-2F0：5'-gtggagtccaaccctgggcccgcttctgacaacccgatcctg-3'(SEQ ID NO.9)；

SVA-2R：5'-ttttctagagcggccgct₃₈-3' (SEQ ID NO.10), with the NotI cleavage site in the horizontal line and t38 representing 38nt of Poly (T).

In the invention, a PacI enzyme digestion site and a hammerhead enzyme core sequence are introduced into an upstream primer SVA-1F, SVA-1F0 for amplifying an S1 fragment to ensure that infectious virus RNA is generated after transcription through shearing modification, and a downstream primer SVA-1R contains a SphI enzyme digestion site; the upstream primer for amplifying the S20 fragment is SVA-2F0, is used for amplification and is fused with an OVP1 fragment, and the downstream primer SVA-2R contains a NotI enzyme cutting site and 38nt poly (T). In the present invention, it is preferable to amplify the S1 fragment by first amplifying the S1 fragment with the primers SVA-1F0 and SVA-1R, using the amplification product as a template, and then performing a second amplification with the primers SVA-1F and SVA-1R to obtain the S1 fragment.

The amplification system and procedure for the S1 fragment and S20 fragment of the present invention are not particularly limited, and may be preferably performed according to the method described in "molecular biology laboratory Manual of Fine Manual" (Main eds., F.M. Osber, R.E. Kingston, J.G. Sadmann, Ma-Shi-Jun, Shujiong-translation, Beijing: scientific Press, 2004).

The VP1 gene fragment obtained in the step (1) is fused with the S20 gene fragment to obtain an S2-OVP1 fragment. The invention preferably utilizes primers OVP1-F and SVA-2R to carry out fusion PCR to fuse gene segments; the nucleotide sequence of the OVP1-F is shown as SEQ ID NO.4, and the nucleotide sequence of the SVA-2R is shown as SEQ ID NO. 10. The amplification conditions of the fusion PCR of the present invention are preferably: 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, 57 ℃ for 30s, and 72 ℃ for 4min for 30 s; 10min at 72 ℃.

The S1 fragment and the S2-OVP1 fragment are respectively connected with a pMD 20T vector to obtain subclone plasmids PMD-S1 and PMD-S2-OVP 1. The present invention preferably collects the S1 fragment and S2-OVP1 fragment from the amplification product by gel recovery. The present invention is not particularly limited to the system and procedure for the connection.

The subclone plasmid PMD-S1 is used as a template, mutation primers SVA-m5UTRF and SVA-m5 UTRA are used for amplification to obtain the subclone plasmid PMD-mS1, the nucleotide sequence of the SVA-m5UTRF is shown as SEQ ID No.11, and the nucleotide sequence of the SVA-m5 UTRA is shown as SEQ ID No. 12. The sequences of the mutation primers are respectively as follows:

SVA-m5UTRF：5’-gttctagcctactcgttttttcccctactcactcattcgtgttgtaactacaggat-3’(SEQ ID NO.11)；

SVA-m5UTRR：5’-atcctgtagttacaacacgaatgagtgagtaggggaaaaaacgagtaggctagaac-3’(SEQ ID NO.12)。

by using the scheme of the invention, the mutated 5' UTR gene segment comprises a nucleic acid sequence preferably shown as SEQ ID NO.1 or comprises the recombinant nucleic acid.

The invention uses PacI and SphI double enzyme digestion of plasmid PMD-mS1 and SphI and NotI double enzyme digestion of plasmid PMD-S2-OVP1 to recover gene segments, and inserts the gene segments into eukaryotic transcription plasmid prO/CHA/99 which is subjected to PacI and NotI double enzyme digestion to obtain recombinant plasmid prSVV/FJ-M-OVP 1. The recombinant plasmid prSVV/FJ-M-OVP1 contains a modified SVV/FJ/001 full-length gene and is simultaneously chimeric with an O-type FMDVVP1 gene.

The recombinant plasmid prSVV/FJ-M-OVP1 is transfected into seneca virus sensitive cells to obtain the seneca recombinant virus of the recombinant O-type FMDVVP1 gene. The Seneca virus sensitive cell comprises BHK-21 cell, PK-15 cell, ST cell, SK-RST cell, IBRS-2 cell, H1299 cell or 293T cell.

The invention also provides a Selenecar recombinant vaccine of the recombinant O-type FMDVVP1 gene. The preparation method of the senecan recombinant vaccine is not particularly limited, and the vaccine can be prepared by using a conventional method in the field.

The invention also provides application of the epicaic recombinant vaccine strain or the epicaic recombinant vaccine in preparing a medicament for simultaneously preventing and/or controlling related diseases of animals caused by epicaic virus and foot-and-mouth disease virus.

The animals according to the present invention preferably include pigs, cattle or sheep.

In the present invention, the seneca recombinant virus/vaccine strain may preferably further comprise one or more antigenic genes of non-seneca valley virus strains, the non-seneca valley virus strains preferably comprising: foot-and-mouth disease virus, porcine circovirus, classical swine fever virus, porcine parvovirus and porcine reproductive and respiratory syndrome virus, so that the corresponding seneca recombinant vaccine prepared from the seneca recombinant virus can prevent and/or control related diseases caused by one or more non-seneca valley viruses besides animal seneca valley viruses.

The present invention provides a recombinant nucleic acid of Seneca virus, a method for producing the same, and applications thereof, which are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

The experimental procedures used in the following examples were carried out under conventional conditions, unless otherwise specified, as described in molecular biology laboratory Manual (ed. F.M. Osber, R.E. Kingston, J.G. Sedman, ed., Mashimi, Shujiong, Beijing: scientific Press, 2004).

Example 1

Acquisition of type O foot-and-mouth disease virus VP1 gene

The O/BY/2010 strain was deposited in the foot-and-mouth disease reference laboratory of the state designated BY the department of veterinary agriculture, and was publicly available through a letter of commission approved BY the department of veterinary agriculture. The primers for amplifying VP1 were designed and synthesized according to the O/BY/2010 strain gene sequence (Genebank: JN 998085):

OVP1-F：5'-cccagcatgcttccctttcgcagctacaagcagaagatgctgatgcaatcaggcgacgtc-3'(SEQ ID NO.4)；

OS-R：5'-caggatcgggttgtcagaagcgggcccagggttggactccac-3'(SEQ ID NO.5)。

in the specific primers, SphI enzyme cutting sites and SVA gene sequences are introduced into an upstream primer OVP1-F, OVP1-F0 used for amplifying OVP1 fragments, and a downstream primer is OS-R used for amplification and fused with the SVA-S20 fragment.

Extracting total RNA of O/BY/2010 BY using RNAeasy Mini Kit (Qiagen), synthesizing first strand cDNA BY Reverse transcription with primer OligodT, preparing 20 muL reaction system BY using PrimeScript Reverse Transcriptase (TaKaRa) Reverse Transcriptase with extremely strong extensibility, reacting at 42 ℃ for 1h for standby, using the Reverse transcribed first strand cDNA as a template, amplifying BY using primers OVP1-F0 and OS-R, using the amplification product as a template, performing second amplification BY using primers OVP1-F and OS-R to obtain a gene fragment containing O type foot-and-mouth disease virus VP1, wherein the amplification is performed BY using LA with excellent performance suitable for long fragment amplification

(TaKaRa Co., Ltd.) DNA polymerase, a 50. mu.L reaction system was prepared according to the product instructions, and the amplification conditions were as follows: 5min at 94 ℃, 30s at 57 ℃, 50s at 72 ℃ after 35 cycles, 10min at 72 ℃, and purifying and recycling the PCR amplification product. The sequencing result shows that the type O foot-and-mouth disease virus VP1 is the nucleotide sequence shown in SEQ ID NO. 2.

Example 2

Construction of infectious clone of Seneca virus of recombinant type O foot-and-mouth disease virus VP1 gene

The SVV/FJ/001 strain used was deposited in the China center for type culture Collection (microbial Collection number: CCTCC NO: V201802), (disclosed in the granted patent "Sernica Valley Virus vaccine and its preparation method and application" ZL201810003888.2, "which is incorporated herein by reference in its entirety), and synthetic amplification primers were designed based on SVA genomic sequences (Genebank: KY 747510):

SVA-1F：5'-ataggtttaattaatgttaagcgtctgatgagtccgtgaggacgaaactatagga-3'(SEQ ID NO.7)；

SVA-1R：5'-gggaagcatgctggggcaccaggcac-3'(SEQ ID NO.8)；

SVA-2F0：5'-gtggagtccaaccctgggcccgcttctgacaacccgatcctg-3'(SEQ ID NO.9)；

SVA-2R：5'-ttttctagagcggccgct₃₈-3'(SEQ ID NO.10)；

SVA-m5UTRF：5'-gttctagcctactcgttttttcccctactcactcattcgtgttgtaactacaggat-3'(SEQ ID NO.11)；

SVA-m5UTRR：5'-atcctgtagttacaacacgaatgagtgagtaggggaaaaaacgagtaggctagaac-3'(SEQ ID NO.12)。

in the specific primers, a PacI enzyme digestion site and a hammerhead enzyme core sequence are introduced into an upstream primer SVA-1F, SVA-1F0 for amplifying an S1 fragment so as to ensure that infectious virus RNA is generated by shearing modification after transcription, and a downstream primer SVA-1R contains a SphI enzyme digestion site; the upstream primer for amplifying the S20 fragment is SVA-2F0, is used for amplification and is fused with an OVP1 fragment, and the downstream primer SVA-2R contains a NotI enzyme cutting site and 38nt poly (T). SVA-m5UTRF and SVA-m5UTRR are primers for deletion and site-directed mutagenesis of the 5' UTR gene on the S1 fragment.

Total RNA of SVV/FJ/001 was extracted with RNAeasy Mini Kit (Qiagen), first strand cDNA was synthesized by Reverse transcription using SVA-2R as a primer, and PrimeScript Reverse Transcriptase (TaKaRa) Reverse Transcriptase having a very high elongation ability was used as a productPreparing a20 mu L reaction system, reacting at 42 ℃ for 1h for later use, using the reverse transcription first strand cDNA as a template, amplifying by using primers SVA-1F0 and SVA-1R, using the amplification product as a template, and performing second amplification by using primers SVA-1F and SVA-1R to obtain a first gene segment S1; and amplifying by using primers SVA-2F0 and SVA-2R by using the reverse transcribed first strand cDNA as a template to obtain a second gene segment S20. The LA for amplification is suitable for long-fragment amplification and has excellent performance

(TaKaRa Co., Ltd.) DNA polymerase, a 50. mu.L reaction system was prepared according to the product instructions, and the amplification conditions were as follows: 5min at 94 ℃, 30s at 57 ℃, 3min at 72 ℃ for 30s, purifying and recovering PCR amplification products after 35 cycles and 10min at 72 ℃.

Performing fusion PCR by using the gene fragment containing the O-type foot-and-mouth disease virus VP1 obtained in example 1 and the amplified S20 gene fragment as templates, adding primers OVP1-F and SVA-2R into the reaction to obtain an SVA S2-OVP1 fragment, wherein the amplification conditions are as follows: 5min at 94 ℃, 30s at 57 ℃, 30 min at 72 ℃ and 30s at 4min, purifying and recovering PCR amplification products after 30 cycles and 10min at 72 ℃. The electrophoresis results of the amplification products are shown in FIG. 1, and the sizes of S1 and S2-OVP1 are 3506bp and 4587bp, respectively, which are consistent with the expected sizes.

The S1 and S2-OVP1 gene fragments are respectively recovered by glue, connected with a pMD 20T vector, transformed into JM109 competent cells, screened, sequenced and identified as positive clones which are respectively named as PMD-S1 and PMD-S2-OVP 1. Amplifying by using plasmid PMD-S1 as template and primer SVA-m5 UTRT and SVA-m5 UTRT, deleting and mutating 5' UTR gene on S1 fragment, amplifying by using high fidelity

HS (TaKaRa company) DNA polymerase, according to the product instruction, preparing 50 μ L reaction system, the amplification conditions are: 5min at 95 ℃; 1min at 95 ℃, 1min at 55 ℃ and 6min at 68 ℃ after 20 cycles; digesting the amplification product by DpnI for 10min at 68 ℃, removing the template plasmid, transforming DH5 alpha competent cells, screening, sequencing and identifying positive clones, namely PMD-mS1, and displaying mS1 containing deleted mS1 by sequencing resultsThe 5' UTR gene modified by mutation is a nucleotide sequence shown as SEQ ID NO. 1.

Plasmid PMD-mS1 is subjected to double enzyme digestion by PacI and SphI, plasmid PMD-S2-OVP1 is subjected to double enzyme digestion by SphI and NotI, target fragments are respectively recovered, then plasmid prO/CHA/99 (disclosed in an authorized patent of 'Asia 1 type foot-and-mouth disease recombinant virus and a preparation method thereof, application of' ZL201310175323.X 'and' A type foot-and-mouth disease recombinant vaccine strain and a preparation method thereof, application of 'ZL 201310175324.4, and the whole content of which is incorporated into the application by reference) containing O type foot-and-mouth disease virus O1 strain is subjected to double enzyme digestion by PacI and NotI, vector fragments are purified and recovered, are connected by T4 ligase and transformed into JM109 competent cells, and are subjected to enzyme digestion, sequencing and positive cloning to obtain SVA recombinant plasmid prSVV/FJ-M-OVP1 containing chimeric 5' UTR modified O type foot-mouth disease virus VP1 gene, and a construction method is shown in figure 2.

Example 3

Rescue of recombinant O-type foot-and-mouth disease virus VP1 gene senocard recombinant virus and culture characteristics of different cells

3.1 rescue of recombinant Selenecar viruses

By using

Plasmid Plus Maxi Kit (QIAGEN Co.) the recombinant Plasmid prSVV/FJ-M-OVP1 from example 2 was prepared, used for transfection when BHK-21 cells grew to 80%, in liposomal Lipofectamine^TM2000(Invitrogen) 4. mu.g of recombinant plasmid was transfected into BHK-21 cells, together with liposome controls and normal cell controls, and placed in a 5% CO-containing chamber₂And (3) in a 37 ℃ incubator, transfecting for 6h, removing the supernatant, adding an MEM (minimum essential medium) culture medium, continuously culturing, observing the cell state and the cytopathic condition, harvesting the virus when about 90% of cells have lesions, repeatedly freezing and thawing for 3 times, and then inoculating the BHK-21 cells again until the virus can stably produce cytopathic effect, and the lesions of cell rounding and falling off, gradually forming plaques and disintegrating into fragments appear. The resulting recombinant virus was named rSVV/FJ-M-OVP1, in FIG. 3, A: a normal control BHK-21 cell picture is obtained; b: infection of BHK-containing virus with rescued recombinant virus rSVV/FJ-M-OVP121。

3.2 culture Properties of recombinant Selenecar viruses in different cells

The recombinant Securium insignis virus rSVV/FJ-M-OVP1 strain obtained by the 3.1 rescue is infected into different cells, and the result shows that the recombinant strain can proliferate in BHK-21 cells, PK-15 cells, ST cells, SK-RST cells, IBRS-2 cells, H1299 cells or 293T cells and other cells and cause typical cytopathic effect, and has similar culture characteristics with wild parent strain SVV/FJ/001 strain on the different cells.

Example 4

Identification of Senecan recombinant virus of recombinant O-type foot-and-mouth disease virus VP1 gene

4.1RT-PCR identification of the stability of recombinant viruses

The supernatant of BHK-21 cells infected by the stably passaged rSVV/FJ-M-OVP1 strain is extracted by RNAeasy Mini Kit (Qiagen), total RNA is extracted, after reverse transcription, S2-OVP1 gene fragment fused with OVP1 gene and S1 gene containing 5'UTR are amplified, purified and recovered, and then sequenced, and the result shows that the obtained fragment inserted with foreign gene and 5' UTR gene are consistent with the theoretical sequence. The recombinant viruses of passage 5, 10, 15 and 20 are amplified by the same method, and sequencing shows that the OVP1 gene inserted in the recombinant viruses during passage and site-directed deletion mutation of 5' UTR can stably exist.

4.2 Indirect immunofluorescence identification of SVA Virus antigens

Repeatedly freezing and thawing the culture of the BHK-21 cells infected by the rSVV/FJ-M-OVP1 strain, inoculating the culture into a six-hole plate (the monolayer cells grow to 60-70%) with BHK-21 cells at the bottom, wherein a glass slide is placed on the six-hole plate, and placing the six-hole plate with the BHK-21 cells growing on the bottom (the monolayer cells grow to 60-70%) in a six-hole plate containing 5% CO₂After 24 hours, indirect immunofluorescence is carried out according to a conventional method in a 37 ℃ incubator, wherein a primary antibody is SVA guinea pig positive serum, a secondary antibody is FITC labeled goat anti-guinea pig IgG (Sigma company), and a normal cell control is arranged. Green-specific fluorescence was seen in cells inoculated with the culture of rSVV/FJ-M-OVP1 strain, whereas no fluorescence was seen in the normal cell control (FIG. 4).

4.3 ELISA detection

Repeatedly freezing and thawing culture of SVV/FJ/001 strain, rSVV/FJ-M-OVP1 strain and O/BY/2010 strain, respectively inactivating, diluting with coating solution, coating an ELISA reaction plate, detecting the reactivity of the coating antigen and the specific antibody of the O-type FMDV BY an indirect ELISA method BY using a normal cell culture as a control, wherein the primary antibody is the specific monoclonal antibody of the O-type FMDV, and the secondary antibody is HRP-labeled goat anti-mouse IgG (Sigma). The rSVV/FJ-M-OVP1 strain can specifically bind to the type O FMDV antibody, and the reactivity of O/BY/2010 strain to the antibody is similar, and neither SVV/FJ/001 strain nor cell culture react with the type O FMDV specific antibody (FIG. 5).

4.4Westernblot analysis

Collecting SVV/FJ/001 strain, rSVV/FJ-M-OVP1 strain, O/BY/2010 strain infected cells and normal control cells, processing cell protein samples according to a Westernblot method, performing SDS-PAGE electrophoresis, membrane transfer, and sealing, wherein the primary antibody is O-type FMDV rabbit polyclonal antibody, and the secondary antibody is HRP-labeled goat anti-rabbit IgG (Sigma). The results show that the rSVV/FJ-M-OVP1 strain infected cell sample can detect a specific reaction band at about 25kD, which is slightly larger than the O/BY/2010 strain sample band, and the reason is presumed to be 2A fusion expression. In contrast, protein bands were not observed in both the SVV/FJ/001 strain samples and the normal cell control (FIG. 6).

Example 5

Pathogenicity test of Seneca recombinant virus of recombinant O-type foot-and-mouth disease virus VP1 gene

5.1 test of the pathogenicity of recombinant Selenecar Virus on cells

Digesting susceptible cells of SVA such as BHK-21 cells, PK-15 cells, ST cells, IBRS-2 cells, etc. in example 3 according to a conventional method, adding DMEM complete medium containing 10% fetal bovine serum, plating the cells in 12-well plates containing 5% CO at 37 deg.C₂The culture is carried out in an incubator until the cell monolayer grows to 80-90 percent for standby. Viral fluids were diluted 10-fold with DMEM, and each dilution (10)^-5.0～10^-10.0) The virus solution was added to a cell plate, 4 wells for each dilution, and the plate was placed at 37 ℃ with 5% CO₂The cells were cultured in the incubator (2), observed for 4 days, and half of the infection amount (TCID) of the virus to different cells such as BHK-21 cells was measured by the Reed-Muench method₅₀). According to this method, the virus titer of rSVV/FJ-M-OVP1 strain on different cells such as BHK-21 was measured, and calculation was performedTCID of rSVV/FJ-M-OVP1 strain₅₀Is 10^-6.5/mL～10^-10.0/mL。

The Reed-Muench calculation Method is prior art in the art and is described in detail in the prior document "Reed, L.J.and Muench, H. (1938)," A Simple Method of Estimating Fine percentage Endpoints ". The American Journal of Hygene 27: 493-497", which is hereby incorporated by reference into the present application.

5.2 pathogenicity test of recombinant Seneca Virus in pigs

9 pigs for the screening test in the non-affected area are detected by a neutralization test, and the SVV neutralizing antibody titer is less than 1: 4. according to the conditions of the established virus attacking model of the SVV/FJ/001 strain to the pig, the prepared rSVV/FJ-M-OVP1 strain is subjected to virus attacking by adopting an injection way, two gradients are set for the virus attacking dosage, and the two gradients are respectively 2 multiplied by 10⁹TCID₅₀，2×10¹⁰TCID₅₀Simultaneously, SVV/FJ/001 strain cytotoxicity is set as a contrast, and the counteracting dosage is 2 multiplied by 10⁹

TCID

₅₀2 mL/head, continuously observing for 13 days, observing and recording the morbidity, judging the morbidity of the animals according to the symptoms of blisters on hooves, rhinoscopes and lips, and scoring (5 points, the judgment standard is made according to the judgment standard of the foot-and-mouth disease, and the higher the point is, the more serious the clinical symptoms are). The challenge results show that typical clinical symptoms appear from the next day after the control of the wild parent strain SVV/FJ/001 strain, blisters appear on hooves, and some blisters appear on a rhinoscope. After the recombinant Seneca virus rSVV/FJ-M-OVP1 strain is challenged, no clinical symptoms appear in both challenged dose groups, and the results are shown in Table 1.

TABLE 1 clinical symptoms of the porcine pathogenicity test with recombinant Seneca Virus

Example 6

Preparation and immunoassay of Sai Nei card recombinant vaccine of recombinant O type foot-and-mouth disease virus VP1 gene

6.1 vaccine preparation

The obtained rSVV/FJ-M-OVP1 strain was cultured according to the method of example 3, the virus was inoculated into the susceptible cells forming a monolayer in an amount of 1% of the culture medium, when the lesion reached 80% or more, the cell culture medium containing the virus was harvested, and after repeated freeze-thawing, it was stored at-70 ℃ for future use. The virus content was determined as described in example 5, and the TCID of the virus was calculated according to the Reed-Muench method₅₀Results were no less than 10 per ml of virus fluid^6.5TCID₅₀. Inactivating with 1.5mmol/L of Binary Ethylene Imine (BEI) (Sigma Co.) at 30 deg.C for 36h, adding blocking agent sodium thiosulfate solution, standing overnight at 4 deg.C, and storing. Inactivated antigen was screened with ISA206 adjuvant (SEPPIC, france) at a ratio of 1: emulsifying at a ratio of 1 to prepare the water-in-oil-in-water vaccine, and subpackaging for later use.

6.2 vaccine immunization test

The obtained seneca recombinant virus inactivated vaccine qualified in security inspection is used for immunizing 5 pigs, and 2 non-immune controls are arranged at the same time to determine the immune efficacy. Experimental pigs were purchased from non-affected areas and tested by neutralization experiments, and the titer of SVA neutralizing antibody was < 1: 4; the O-type antibodies detected by FMD liquid blocking ELISA (LPB-ELISA) produced by national foot-and-mouth disease reference laboratory are all less than 1: 4. the second immunization is carried out at 28d of the first immunization, blood is collected and serum is separated at 7 days, 14 days, 21 days, 28 days and 42 days after the immunization, the SVA is subjected to neutralizing antibody detection and the detection result is counted, and the 42d serum is used for detecting the O-type FMDV antibody, so that the vaccine has good immunogenicity, can effectively stimulate the serum SVA neutralizing antibody (shown in table 2), can generate specific antibodies of FMDV, and has the titer of more than or equal to 64.

TABLE 2 detection results of SVA neutralizing antibody levels after Immunity of Senecan recombinant vaccines

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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Claims

1. The sequence of the recombinant nucleic acid comprises deletion mutation transformation of a 5' UTR gene sequence of a seneca virus strain, and insertion of a VP1 gene of O-type foot-and-mouth disease virus between Sph I and Not I enzyme cutting sites of seneca virus cDNA.

2. The use of the recombinant nucleic acid of claim 1 in the preparation of seneca virus recombinant nucleic acid or seneca recombinant vaccine strain of recombinant type O foot-and-mouth disease virus VP1 gene.

3. A seneca recombinant virus comprising the recombinant foot-and-mouth disease virus type O VP1 gene of the recombinant nucleic acid of claim 1.

4. A seneca recombinant virus of the recombinant O-type foot-and-mouth disease virus VP1 gene encoded by the recombinant nucleic acid of claim 1.

5. A recombinant vaccine strain comprising the recombinant Seika vims of claim 3 or 4.

6. The method for constructing a seneca recombinant virus according to claim 3 or 4, comprising the steps of:

7. Use of the seneca recombinant virus of the recombinant O-type FMDVVP1 gene of claim 3 or 4 or the seneca recombinant virus prepared by the method of claim 6 in the preparation of a seneca recombinant vaccine.

8. A senecan recombinant vaccine of recombinant O-type FMDVVP1 gene.

9. Use of the seneca recombinant vaccine strain of claim 5 or the seneca recombinant vaccine of claim 8 for the preparation of a medicament for the simultaneous prevention and/or control of related diseases caused by seneca virus and foot-and-mouth disease virus in animals.

10. The use of claim 9, wherein the animal comprises a pig, a cow or a sheep.