CN111394389A - Infectious clone of Seneca virus based on single plasmid rescue system, construction method and application - Google Patents
Infectious clone of Seneca virus based on single plasmid rescue system, construction method and application Download PDFInfo
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Abstract
The invention provides a single plasmid rescue system aiming at a positive strand RNA virus, infectious clone of a Seneca virus based on the system, a construction method and application, and relates to the technical field of genetic engineering. The invention provides a single plasmid rescue system aiming at a positive strand RNA virus, and application of the rescue system in the research of virus gene function, virus variation, pathogenic mechanism and the like, wherein the rescue system can be used for directionally designing SVA reverse recombinant vaccines and constructing SVA directed mutant strains. The single-plasmid senecard virus rescue system constructed by using the polymerase I and polymerase II promoters, terminators, ribozymes and other elements realizes the editing of virus genomes on the plasmid level, and provides a technical platform for the directional design, construction and rescue of viruses.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an infectious clone of Seneca virus based on a single plasmid rescue system, a construction 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.
The Seneca virus causes epidemic situations in a plurality of countries such as America, Canada, Brazil, Columbia, China, Thailand and the like, the epidemic situation is introduced into China in 2015, the epidemic situation appears in Guangdong for the first time and is rapidly diffused, a large amount of pig farm morbidity is caused in Fujian, Guangxi, Hubei, Henan, Hebei, Shandong, Liaoning and the like, the transmission speed and the pathogenicity are all enhanced compared with those reported before, and researches show that the prior national SVA epidemic strains are complex, and the prevention and control situation is still severe. Because clinical morbidity symptoms caused by the Seneca virus are similar to foot-and-mouth disease, and mixed infection can be formed with the Seneca virus, and severe interference is formed on the prevention and control of the foot-and-mouth disease, a technical platform is urgently needed to develop the research of the Seneca virus basic theory and the creation of prevention and control products. The establishment of SVA infectious clone by utilizing a reverse genetic operation technology platform, the research of the variation and pathogenic mechanism of the Seneca virus, the development of high-efficiency vaccines and other works are very important and urgent.
Disclosure of Invention
In view of the above, the present invention aims to provide a single plasmid high-efficiency rescue system for positive-strand RNA viruses, which includes a method for constructing SVA infectious clone by using the single plasmid rescue system, and application studies for constructing and rescuing SVA by using the rescue system in a targeted manner for basic research and targeted design for preparing reverse recombinant vaccines, such as viral gene function, viral variation and pathogenic mechanism.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a single plasmid rescue system aiming at a positive-strand RNA virus, which is a eukaryotic transcription plasmid containing a nucleic acid sequence of the positive-strand RNA virus, wherein the eukaryotic transcription plasmid contains a human cytomegalovirus RNA polymerase II promoter, a murine RNA polymerase I promoter and a ribozyme sequence at the upstream of the 5' end of the nucleic acid sequence of the virus; the downstream of the 3' end of the virus nucleic acid sequence contains ribozyme, murine polymerase terminator I and polymerase terminator II sequences;
the positive strand RNA viruses include Securidacae.
Preferably, the backbone vector of the eukaryotic transcription plasmid comprises pcDNA3.1.
Preferably, when the positive strand RNA virus is Seneca virus, the nucleotide sequence of Seneca virus is inserted between the PacI and NotI cleavage sites of pcDNA3.1.
Preferably, the nucleotide sequence of the inserted seneca virus is shown as SEQ ID NO. 10.
The invention provides a construction method of full-length infectious clone of Seneca virus, which comprises the following steps: (1) amplifying the A gene of the SVV/FJ/001 strain by using a specific primer pair by using the cDNA of the SVV/FJ/001 strain as a template; the specific primer pair comprises an upstream primer and a downstream primer SVA-AR; the upstream primer comprises SVA-1F0 and SVA-1F, the nucleotide sequence of the upstream primer SVA-1F0 is shown as SEQ ID NO.1, the nucleotide sequence of the upstream primer SVA-1F is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer SVA-AR is shown as SEQ ID NO. 3;
(2) amplifying a B gene of the SVV/FJ/001 strain by using a specific primer pair by using a cDNA of the SVV/FJ/001 strain as a template, wherein the specific primer pair comprises an upstream primer SVA-BF and a downstream primer SVA-2R, the nucleotide sequence of the upstream primer SVA-BF is shown as SEQ ID No.4, and the nucleotide sequence of the downstream primer SVA-2R is shown as SEQ ID No. 5;
(3) respectively connecting the A gene and the B gene obtained by amplification in the steps (1) and (2) with a pMD20T vector, and transforming JM109 competent cells to obtain plasmids PMD-A and PMD-B;
(4) carrying out double enzyme digestion on plasmid PMD-A by PacI and NheI and plasmid PMD-B by NheI and NotI, recovering a target fragment, connecting, replacing and inserting the target fragment into the eukaryotic plasmid subjected to double enzyme digestion by PacI and NotI, and transforming the eukaryotic plasmid into JM109 competent cells to obtain eukaryotic transcription plasmid prSVV/FJ;
the step (1) and the step (2) do not have a temporal restriction relationship.
Preferably, the step (1) of amplifying the A gene comprises: taking cDNA of the SVV/FJ/001 strain as a template, and amplifying by using an upstream primer SVA-1F0 and a downstream primer SVA-AR to obtain a first PCR product; and (3) taking the first PCR product as a template, and amplifying by using an upstream primer SVA-1F and a downstream primer SVA-AR to obtain an A gene.
Preferably, the amplification in step (1) and the amplification in step (2) are performed by the following procedures: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 3min for 30s, 35 cycles; extension at 72 ℃ for 10 min.
The invention also provides application of the full-length infectious clone of the seneca virus constructed by the construction method in rescuing the seneca virus.
The invention provides a method for rescuing an endo-virus, which comprises the following steps: transfecting the eukaryotic transcription plasmid prSVV/FJ into the Seneca virus sensitive cells, and placing the Seneca virus sensitive cells in a medium containing 5% CO2Culturing in an incubator at 37 ℃, harvesting the virus when 80-90% of cells have pathological changes, repeatedly freezing and thawing for 3 times, and then inoculating the seneca virus sensitive cells again until the virus can stably generate the pathological changes of the cells to obtain the recombinant virus rSVV/FJ.
Preferably, the method of transfection comprises lipofection or non-lipofection.
Preferably, the Seneca virus-sensitive cell comprises a BHK-21 cell, a PK-15 cell, an ST cell, an SK-RST cell, an IBRS-2 cell, an H1299 cell or a 293T cell.
Preferably, the cytopathic effect comprises rounding off of the cells, shedding, gradual formation of plaques, and disintegration into fragments.
The invention provides a recombinant virus rSVV/FJ of a seneca, which can proliferate in BHK-21 cells, PK-15 cells, ST cells, SK-RST cells, IBRS-2 cells, H1299 cells or 293T cells and cause cytopathic effect.
Preferably, the gene of the seneca recombinant virus rSVV/FJ contains a NheI molecular marker with silent mutation, and the genetic marker can be identified with a parental wild strain.
The invention also provides the use of the seneca recombinant virus obtained by the method or the recombinant virus in the preparation of SVA recombinant vaccines.
The invention also provides application of the single plasmid rescue system in construction of SVA directed mutant strains.
The invention also provides application of the single particle rescue system in directional design of SVA reverse recombinant vaccines.
The invention provides a single plasmid rescue system aiming at a positive strand RNA virus, infectious cloning and construction method of Seneca virus based on the system, and application of directionally designing SVA reverse recombinant vaccine and constructing SVA directionally mutant strain in research of virus gene function, virus variation, pathogenesis and the like by using the rescue system. The invention constructs a single-particle Sakavirus rescue system by using elements such as polymerase I and polymerase II promoters, terminators, ribozymes and the like, realizes the editing of virus genomes on the plasmid level, and provides a technical platform for the directional design, construction and rescue of viruses.
The SVA infectious clone rescue virus is constructed based on the single plasmid rescue system, so that the directional design of SVA genes can be realized, and the technical problem of rapidly preparing vaccine seed viruses can be solved; and the technology platform can be used for developing the basic research related to SVA virus gene function, virus variation, pathogenic mechanism and the like.
The conventional in vitro transcription system is fussy and unstable in virus rescue operation and expensive in reagent, while the single plasmid rescue system is operated at the plasmid level, is simple and convenient to operate, and has high rescue efficiency and stability. The epikavirus rescued by the system has high consistency with parent virus.
The technology of the invention realizes a more active and effective SVA construction mode and has important application value.
Drawings
FIG. 1 shows the results of electrophoresis of the A fragment and the B fragment of SVA amplified in example 1, wherein 1 is the amplification product of the A fragment, 2 is the amplification product of the B fragment, M is D L5000 DNAmarker;
FIG. 2 is a schematic diagram showing the construction of infectious clones of the full-length cDNA of Seneca virus in example 2;
FIG. 3 is the cytopathic effect (CPE) caused by infection of BHK-21 cells with the recombinant virus rSVV/FJ strain of example 3, wherein A represents normal BHK-21 cells; b represents BHK-21 cells presenting CPE;
FIG. 4 is the cytopathic effect (CPE) induced by the recombinant virus rSVV/FJ strain of example 3 after infection of PK-15 cells and IBRS-2 cells, wherein A represents PK-15 cells presenting CPE; b represents CPE-exhibiting IBRS-2 cells;
FIG. 5 shows the results of indirect immunofluorescence of the rSVV/FJ strain and SVV/FJ/001 strain inoculated in example 4, wherein A represents a normal cell control, B represents the detection result after the rSVV/FJ strain was inoculated, and C represents the detection result after the SVV/FJ/001 strain was inoculated.
Detailed Description
The invention provides a single plasmid rescue system aiming at a positive-strand RNA virus, which is a eukaryotic transcription plasmid containing a nucleic acid sequence of the positive-strand RNA virus, wherein the eukaryotic transcription plasmid contains a human cytomegalovirus RNA polymerase II promoter, a murine RNA polymerase I promoter and a ribozyme sequence at the upstream of the 5' end of the nucleic acid sequence of the virus; the downstream of the 3' end of the virus nucleic acid sequence contains ribozyme, murine polymerase terminator I and polymerase terminator II sequences; the positive strand RNA viruses include Securidacae.
The skeleton vector of the eukaryotic transcription plasmid comprises pcDNA3.1. In the present invention, when the positive-strand RNA virus is Seneca virus, the nucleotide sequence of Seneca virus is inserted between PacI and NotI cleavage sites of pcDNA3.1; the nucleotide sequence of the inserted seneca virus is shown as SEQ ID NO. 10. According to the invention, a hammerhead enzyme core sequence (Hamrz) is introduced at the 5 'end of an SVA full-length cDNA genome, the core sequence of the hammerhead enzyme is 58nt, and the 3' end C of the hammerhead enzyme core sequence is provided with a self-shearing modification site; a core sequence of hepatitis delta enzyme (Hdvrz) having 88nt and having a self-cleavage modification site at the 5 'terminal G was introduced at the 3' end of the SVA full-length cDNA genome. When the infectious clone containing SVA genome is transfected into receptor cells, virus RNA precursors are respectively transcribed and packaged by an RNA polymerase II promoter and an RNA polymerase I promoter regulating element, and then the virus RNA precursors are cut and modified by hammerhead enzyme and hepatitis delta enzyme to generate infectious virus RNA.
The invention provides a construction method of full-length infectious clone of Seneca virus, which comprises the following steps: (1) amplifying the A gene of the SVV/FJ/001 strain by using a specific primer pair by using the cDNA of the SVV/FJ/001 strain as a template; the specific primer pair comprises an upstream primer and a downstream primer SVA-AR; the upstream primer comprises SVA-1F0 and SVA-1F, the nucleotide sequence of the upstream primer SVA-1F0 is shown as SEQ ID NO.1, the nucleotide sequence of the upstream primer SVA-1F is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer SVA-AR is shown as SEQ ID NO. 3;
(2) amplifying a B gene of the SVV/FJ/001 strain by using a specific primer pair by using a cDNA of the SVV/FJ/001 strain as a template, wherein the specific primer pair comprises an upstream primer SVA-BF and a downstream primer SVA-2R, the nucleotide sequence of the upstream primer SVA-BF is shown as SEQ ID No.4, and the nucleotide sequence of the downstream primer SVA-2R is shown as SEQ ID No. 5;
(3) respectively connecting the A gene and the B gene obtained by amplification in the steps (1) and (2) with a pMD20T vector, and transforming JM109 competent cells to obtain plasmids PMD-A and PMD-B;
(4) carrying out double enzyme digestion on plasmid PMD-A by PacI and NheI and plasmid PMD-B by NheI and NotI, recovering a target fragment, connecting, replacing and inserting the target fragment into the eukaryotic plasmid subjected to double enzyme digestion by PacI and NotI, and transforming the eukaryotic plasmid into JM109 competent cells to obtain eukaryotic transcription plasmid prSVV/FJ;
the step (1) and the step (2) do not have a temporal restriction relationship.
The invention takes cDNA of SVV/FJ/001 strain as a template, and uses a specific primer pair to amplify A gene of SVV/FJ/001 strain, wherein the specific primer pair comprises an upstream primer and a downstream primer SVA-AR, the upstream primer comprises SVA-1F0 and SVA-1F, the nucleotide sequence of the upstream primer SVA-1F0 is shown as SEQ ID NO.1, the nucleotide sequence of the upstream primer SVA-1F is shown as SEQ ID NO.2, the nucleotide sequence of the downstream primer SVA-AR is shown as SEQ ID NO.3, the Seneca SVV/FJ/001 strain is preferably preserved in China center of type culture, the SVV/FJ/001 with the preservation number of CCTCC NO. 201802, and has been amplified in China granted patent 'Seneca valley virus vaccine and preparation method thereof and application' Z L201810003888.2, when amplifying the gene, the invention preferably comprises that the first primer is used to amplify the upstream primer SVV/FJ/001, the first primer is used as upstream primer and PCR is used as upstream primer 0:
SVA-1F0：5'-gtgaggacgaaactataggaaaggaattcctatagtcttgaaagggggggctgggcc-3'(SEQ ID NO.1)；
SVA-1F：5'-ataggtttaattaatgttaagcgtctgatgagtccgtgaggacgaaactatagga-3'(SEQ ID NO.2)；
SVA-AR：5'-gttgtcgctagcagggccagggttggtctc-3'(SEQ ID NO.3)。
the program of the invention in the A gene amplification process is preferably pre-denatured at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 3min for 30s, 35 cycles; extension at 72 ℃ for 10 min. The upstream primer SVA-1F, SVA-1F0 used for amplifying the A gene is introduced with PacI enzyme cutting site and hammerhead enzyme core sequence to ensure that infectious virus RNA is generated after transcription through shearing modification, the downstream primer SVA-AR contains NheI enzyme cutting site, TCT of SVA3512-3514 coding serine is synonymously mutated into AGC, and NheI enzyme cutting site is introduced, and the site mutation can be used for constructing full-length cDNA and can be used as molecular marker/genetic marker to be distinguished from parent virus.
The method takes cDNA of an SVV/FJ/001 strain as a template, and uses a specific primer pair to amplify B gene of the SVV/FJ/001 strain, wherein the specific primer pair comprises an upstream primer SVA-BF and a downstream primer SVA-2R, the nucleotide sequence of the upstream primer SVA-BF is shown as SEQ ID No.4, and the nucleotide sequence of the downstream primer SVA-2R is shown as SEQ ID No. 5. The primer sequences used for amplifying the B gene are shown as follows:
SVA-BF：5'-ccccagctagcgacaacccgatcctg-3'(SEQ ID NO.4)；
SVA-2R：5'-ttttctagagcggccgct38-3' (SEQ ID NO. 5). NheI enzyme cutting sites are also introduced into the upstream primer SVA-BF of the amplification gene B, and the downstream primer SVA-2R of the amplification gene B contains NotI enzyme cutting sites and 38nt poly (T). The program of the invention in the B gene amplification process is preferably pre-denatured at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 3min for 30s, 35 cycles; extension at 72 ℃ for 10 min.
In the present invention, although the source of the cDNA of SVV/FJ/001 strain is not particularly limited, it is preferable to extract the total RNA of SVV/FJ/001 using RNAeasy MiniKit (Qiagen) and synthesize the first strand cDNA by reverse transcription using the primer SVA-2R.
The invention connects the A gene and the B gene obtained by the amplification in the steps (1) and (2) with a pMD20T vector respectively, and transforms JM109 competent cells to obtain plasmids PMD-A and PMD-B. The method of the present invention for the ligation and transformation is not particularly limited, and may be a method that is generally used in the art.
The invention uses PacI and NheI double enzyme digestion of plasmid PMD-A and NheI double enzyme digestion of plasmid PMD-B, recovers the target segment, connects, replaces and inserts into the eukaryotic plasmid which is introduced with polymerase promoter gene at the 5 'end, hepatitis D enzyme core sequence at the 3' end and polymerase terminator sequence after PacI and NotI double enzyme digestion, and transforms into JM109 competent cell, and obtains eukaryotic transcription plasmid prSVV/FJ. The construction process of the eukaryotic transcription plasmid prSVV/FJ is preferably as shown in figure 2, and the eukaryotic transcription plasmid prSVV/FJ containing the modified SVV/FJ/001 full-length gene can be obtained by using the method.
The invention also provides application of the full-length infectious clone of the seneca virus constructed by the construction method in rescuing the seneca virus.
The invention also provides a method for rescuing the epikayavirus, which comprises the following steps: transfecting the eukaryotic transcription plasmid prSVV/FJ into the Seneca virus sensitive cells, and placing the Seneca virus sensitive cells in a medium containing 5% CO2Culturing in a 37 ℃ incubator, harvesting virus when 80-90% of cells have pathological changes, repeatedly freezing and thawing for 3 times, inoculating the Seneca virus sensitive cells again,recombinant virus rSVV/FJ. the transfection method of the present invention preferably comprises lipofection or non-lipofection until the virus can stably produce cytopathic effect, when eukaryotic transcription plasmid prSVV/FJ is transfected into senescent-virus-sensitive cells under the mediation of liposome, 80% -90% of the cells are cytopathic, the virus is harvested, the senescent-virus-sensitive cells are inoculated again after repeated freezing and thawing for 3 times until the virus can stably produce cytopathic effect, recombinant virus rSVV/FJ. the senescent-virus-sensitive cells of the present invention preferably comprise BHK-21 cells, PK-15 cells, ST cells, SK-RST cells, IBRS-2 cells, H1299 cells or 293T cellsTM2000(Invitrogen), under which the recombinant plasmid was transfected into BHK-21 cells. The transfection culture according to the invention is preferably carried out in an incubator, preferably containing 5% CO2The temperature of the incubator is preferably 37 ℃.
The cytopathic effect of the present invention preferably comprises rounding off, shedding, gradual formation of plaque, and disintegration into fragments.
The invention also provides a recombinant virus rSVV/FJ of the Seneca, which can proliferate in BHK-21 cells, PK-15 cells, ST cells, SK-RST cells, IBRS-2 cells, H1299 cells or 293T cells and cause cytopathic effect. The gene of the seneca recombinant virus rSVV/FJ contains a silent mutation NheI molecular marker, and the genetic marker can be identified with a parental wild strain.
The invention uses the seneca recombinant virus obtained by the method or the application of the recombinant virus in preparing SVA recombinant vaccine. The seneca recombinant virus obtained by the method has biological characteristics similar to those of a wild parent virus and contains a unique genetic marker, so that the seneca recombinant virus can be used for preparing SVA recombinant vaccines.
The invention utilizes the application of the single particle rescue system in construction of SVA directed mutant strains. The single particle rescue system is used as a technical platform, and virus strains such as SVA gene site-directed mutation, deletion and the like are constructed in a targeted manner, so that a platform and technical support are provided for basic researches such as virus gene functions, virus variation, pathogenic mechanisms and the like.
The invention utilizes the application of the single particle rescue system in the directional design of the SVA reverse recombinant vaccine. The single particle rescue system is used as a technical platform, SVA basic theory research is used as guidance, SVA key genes are mutated and modified, various performances of the seed virus for preparing the vaccine are improved, such as productivity improvement, antigenicity improvement, immunosuppression and pathogenicity reduction, and the like, and a platform and a support are provided for the design and construction of the SVA reverse recombinant vaccine seed virus.
The infectious cloning, construction method and application of seneca virus based on single plasmid rescue system provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Construction of infectious clones of Seneca recombinant viruses
The SVV/FJ/001 strain used was deposited at 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 use" Z L201810003888.2, which is incorporated herein by reference in its entirety), and amplification primers that introduce the rescue elements and cover the SVA whole genome were designed by comparative analysis based on SVA genome sequence (Genebank: KY 747510):
SVA-1F0：5'-gtgaggacgaaactataggaaaggaattcctatagtcttgaaagggggggctgggcc-3'(SEQ ID NO.1)；
SVA-1F：5'-ataggtttaattaatgttaagcgtctgatgagtccgtgaggacgaaactatagga-3'(SEQ ID NO.2)；
SVA-AR：5'-gttgtcgctagcagggccagggttggtctc-3'(SEQ ID NO.3)；
SVA-BF：5'-ccccagctagcgacaacccgatcctg-3'(SEQ ID NO.4)；
SVA-2R：5'-ttttctagagcggccgct38-3'(SEQ ID NO.5)；
in the specific primers, a PacI enzyme cutting site and a hammerhead enzyme core sequence are introduced into an upstream primer SVA-1F, SVA-1F0 for amplifying an A fragment to ensure that infectious virus RNA is generated after transcription through shearing modification, a downstream primer SVA-AR contains a NheI enzyme cutting site, the enzyme cutting site is obtained by analyzing a gene sequence, TCT of coding serine at SVA3512-3514 is subjected to synonymous mutation to AGC, and the NheI enzyme cutting site is introduced, and the site mutation can be used for constructing full-length cDNA and can be distinguished from parent virus as a molecular marker; the upstream primer SVA-BF for amplifying the B fragment also introduces NheI enzyme cutting site, and the downstream primer SVA-2R thereof contains NotI enzyme cutting site and 38nt poly (T).
Extracting total RNA of SVV/FJ/001 with RNAeasy Mini Kit (Qiagen), Reverse transcribing with primer SVA-2R to synthesize first strand cDNA, Reverse transcribing with PrimeScript Reverse Transcriptase (TaKaRa) with strong extending ability, preparing 20 μ L reaction system according to product instruction, reacting at 42 deg.C for 1h, using Reverse transcribed first strand cDNA as template, amplifying with primers SVA-1F0 and SVA-AR, using the amplified product as template, performing second round amplification with primers SVA-1F and SVA-AR to obtain first gene fragment A, using Reverse transcribed first strand cDNA as template, amplifying with primers SVA-BF and SVA-2R to obtain second gene fragment B, and amplifying with L A fragment A with proper length and excellent amplifying performance
(TaKaRa Co., Ltd.) DNA polymerase, preparing a 50 mu L reaction system according to the product specification, wherein the amplification conditions are 94 ℃ for 5min, 94 ℃ for 30s, 57 ℃ for 30s, 72 ℃ for 3min for 30s, and after 35 cycles and 72 ℃ for 10min, purifying and recovering PCR amplification products, the electrophoresis results of the amplification products are shown in FIG. 1, and the sizes of A and B are 3580bp and 3820bp respectively, which are consistent with the expected sizes.
And respectively carrying out gel recovery on the A gene fragment and the B gene fragment, connecting the A gene fragment and the B gene fragment with a pMD20T vector, transforming JM109 competent cells, screening, sequencing and identifying positive clones which are respectively named as PMD-A and PMD-B.
Example 2
Construction of infectious clones of full-Length cDNA of Seneca Virus
The schematic diagram of the construction method is shown in FIG. 2: and carrying out double enzyme digestion on plasmid PMD-A by PacI and NheI, carrying out double enzyme digestion on plasmid PMD-B by NheI and NotI, respectively recovering target fragments, carrying out double enzyme digestion on eukaryotic plasmids containing polymerase promoter genes, polymerase terminators and ribozyme sequences by PacI and NotI, purifying and recovering vector fragments, connecting by T4 ligase, transforming into JM109 competent cells, carrying out enzyme digestion, sequencing and positive cloning identification, and obtaining the eukaryotic transcription plasmid prSVV/FJ containing the modified SVV/FJ/001 full-length gene.
Example 3
Rescue of seneca virus and culture characteristics of different cells
3.1 rescue of Selenecar Virus
By using
Preparation of eukaryotic transcriptional Plasmid prSVV/FJ. from example 2 BHK-21 cells were seeded in T12.5 cell flasks using the Plasmid Plus Maxi Kit (QIAGEN Co.), used for transfection when the cells grew to 80%, in liposomes L ipofectamineTM2000(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 80-90% of cells have pathological changes, repeatedly freezing and thawing for 3 times, and then inoculating the BHK-21 cells again until the virus can stably generate cytopathic changes, and the cells become round and fall off, gradually form plaques and disintegrate into fragments. The resulting recombinant virus was named rSVV/FJ, and in FIG. 3, A: a normal control BHK-21 cell picture is obtained; b: the rescued recombinant virus rSVV/FJ-M infected BHK-21.
3.2 culture Properties of Selenecar Virus on different cells
The recombinant Securium insignis virus rSVV/FJ 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, 293T cells and other cells and cause typical cytopathic effect, and the culture characteristics of the recombinant Securium insignis virus rSVV/FJ/001 strain on the different cells are similar to those of a wild parent strain SVV/FJ/001 strain. The lesions caused by the rSVV/FJ strain on PK-15 cells and IBRS-2 cells are shown in FIG. 4.
Example 4
Identification of Seneca Virus
4.1 RT-PCR identification
Extracting total RNA from supernatant of BHK-21 cells infected by the stably passaged rSVV/FJ strain by using RNAeasy Mini Kit (Qiagen), performing reverse transcription, and amplifying a P1 gene and a P2 gene by using primers for amplifying a P1 gene as follows:
P1F：5'-ggtaatgtccagacaacctc-3'(SEQ ID NO.6)；
P1R：5'-ttgcatcagcatcttctgc-3'(SEQ ID NO.7)；
the primers for amplifying the P2 gene are as follows:
P2F：5'-tcaggcgacgtcgagaccaac-3'(SEQ ID NO.8)；
P2R：5'-ctgtagaaccagagtctgc-3'(SEQ ID NO.9)
the amplified fragment is purified and recovered and then sequenced, and the result shows that the obtained P1 gene is consistent with the reference sequence of SVV/FJ/001 strain, and the molecular marker sequence which is introduced into the P2 gene and is distinguished from parent virus can stably exist.
4.2 Indirect immunofluorescence assay
Repeatedly freezing and thawing the culture in which the rSVV/FJ strain and the SVV/FJ/001 respectively infect the BHK-21 cells, 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 in a medium containing 5% CO2In a 37 ℃ incubator, indirect immunofluorescence is carried out according to a conventional method, 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 both the rSVV/FJ strain and the SVV/FJ/001 cultures, whereas no fluorescence was seen in the normal cell controls (FIG. 5).
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
<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences
<120> infectious clone of Seneca virus based on single plasmid rescue system, construction method and application
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ccccagctag cgacaacccg atcctg 26
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ttttctagag cggccgcttt tttttttttt tttttttttt tttttttttt ttttt 55
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tcaggcgacg tcgagaccaa c 21
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ctgtagaacc agagtctgc 19
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ttgaaagggg gggctgggcc ctcatgccca gtccttcctt tccccttccg gggggtaaac 60
cggctgtgtt tgctagaggc acagaggagc aacatccaac ctgcttttgt ggggaacagt 120
gcggctccaa ttcctgcgtc gccaaaggtg ttagcgcacc caaacggcgc atctaccaat 180
gctattggtg tggtctgcga gttctagcct actcgtttct cccctactca ctcatttaca 240
cacaaaaact gtgttgtaac tacaggattt ggccctcgca cgggatgtgc gataaccgca 300
agattgactc aagcgcggaa agcgttgtaa ccacatgctg ttagtccctt tatggctgtg 360
agatggctat ccacctcgga tcactgaact ggagctcgac cctccttagt aagggaaccg 420
agaggccttc ctgcaacaag ctccgacaca gagtccacgt gattgctacc accatgagta 480
catggttctc ccctctcgac ccaggacttc tttttgaata tccacggctc gatccagagg 540
gtggggcatg atccccctag catagcgagc tacagcggga actgtagcta ggccttagcg 600
tgccttggat actgcctgat agggcgacgg cctagtcgtg tcggttctat aggtagcaca 660
tacaaatatg cagaactctc atttttcttt cgatacagcc tctggcacct ttgaagacgt 720
aaccggaaca aaagtcaaga tcgttgaata ccccagatcg gtgaacaatg gtgtttacga 780
ttcgtccact catttagaga tactgaacct acagggtgaa attgaaattt taaagtcttt 840
caacgaatac caaattcgcg ccgctaaaca acaacttgga ctggacatcg tatacgaact 900
acagggtaat gtccagacaa cctcaaagaa cgattttgat tcccgcggca ataatggtaa 960
catgaccttc aattactacg caaacactta ccagaattca gtagacttct cgacctcctc 1020
gtcggcgtca ggcgccggac ccgggaactc ccggggcgga ttagcgggtc tcctcacaaa 1080
tttcagtgga atcttgaacc ctcttggcta cctcaaagat cacaataccg aagaaatgga 1140
aaactctgct gatcgagtca taacgcaaac ggcgggcaac actgccataa acacgcaatc 1200
atcactgggt gtgttgtgtg cctacgttga agacccgacc aaatctgacc ctccgtccag 1260
cagcacagat caacccacca ccacttttac tgccatcgac aggtggtaca ctggacggct 1320
caattcttgg acaaaagctg taaaaacctt ctcttttcag gccgtcccgc tccctggagc 1380
cttcctgtct aggcagggag gcctcaacgg aggggccttc acggctaccc tacatagaca 1440
tttcttaatg aagtgcgggt ggcaagtgca ggtccaatgc aatttgacac aattccacca 1500
aggcgctctt cttgttgcca tggtccccga aaccaccctt gatgtcaaac ctgacggcaa 1560
ggcaaagagc ttacaagagc tgaatgaaga gcagtgggtg gagatgtctg acgattaccg 1620
gaccgggaaa aacatgcctt ttcagtctct tggcacttac tatcggcccc ctaactggac 1680
ttggggcccc aatttcatca acccctatca agtaacagtc ttcccacacc aaattctgaa 1740
cgcgagaacc tctacctcgg tagacataag tgtcccatac atcggggaga ctcctacgca 1800
atcctcagag acacagaact cctggaccct cctcgttatg gtgcttgtcc ccctggacta 1860
caaggaggga gccacaactg acccagaaat tacattttct gtaaggccta caagtcctta 1920
cttcaacggg cttcgtaacc gtttcacgac cgggacggac gaggagcagg ggcccattcc 1980
cacagcaccc agagaaaatt cgcttatgtt tctctcaacc atccctgacg acactgttcc 2040
tgcttacggg aatgtgcgta cccctcccgt caattacctc cctggtgaaa taaccgacct 2100
cttacaactg gcccgtatac ccactctcat ggcgtttggg cgggtgtctg aacccgagcc 2160
tgcctcagac gcatatgtgc cttacgttgc cgttcctgcc cagttcgacg acaagcctct 2220
catctccttc ccgatcaccc tttcagatcc tgtctaccag aacaccctgg tgggcgccat 2280
cagttcgaac ttcgccaact accgggggtg tatccaaatc actctgacat tttgtggacc 2340
catggtggca agagggaaat tcctgctctc gtattctccc ccaaatggag cacaaccaca 2400
gaccctttct gaagctatgc agtgcacata ctctatttgg gatataggct tgaactctag 2460
ttggaccttt gtcatcccct atatctcgcc cagtgattac cgtgaaactc gggctattac 2520
caactcagtt tattctgctg atggttggtt tagcttgcac aagctgacca aaattactct 2580
accacctgac tgcccacaga gtccctgtat tctctttttc gcctctgctg gtgaggatta 2640
caccctccgc ctccctgttg attgtaatcc ttcctacgtg ttccactcca ccgacaacgc 2700
cgagactggg gttattgagg caggtaacac tgacaccgat ttctctggtg aactggcggc 2760
tcctggctct aaccatacta atgtcaaatt cctgtttgac cgatctcggc tactgaatgt 2820
aattaaggta ctggagaagg acgccgtctt cccccgtcct ttccccacag caacaggtgc 2880
acagcaggac gatggttact tttgtcttct aacaccccgc ccaacagtcg cttcccgacc 2940
cgccactcgt ttcggcctgt acgtcaaccc gtctgacaat ggcgttctcg ctaacacttc 3000
actggatttc aatttttaca gtttggcctg tttcacttac tttagatcag accttgaagt 3060
cacggtggtc tcactggagc cagatctgga attcgccgtg gggtggttcc cctctggcag 3120
tgagtaccag gcttctagct ttgtctacga ccaactgcat gtaccctacc actttactgg 3180
gcgcactccc cgcgctttca ccagcaaggg tggaaaggta tccttcgtgc tcccttggaa 3240
ctctgtctct tccgtgcttc ccgtgcgctg ggggggcgcc tccaagcttt cttctgccac 3300
gcggggtctg ccggctcatg ctgactgggg gaccatttac gcctttatcc cccgtcctaa 3360
cgagaagaaa ggcaccgctg taaagcacgt ggcggtgtac gttcggtaca agaacgcgcg 3420
tgcctggtgc cccagcatgc ttccctttcg cagctacaag cagaagatgc tgatgcaatc 3480
aggcgacgtc gagaccaacc ctggccctgc tagcgacaac ccgatcctgg agtttcttga 3540
agcggaaaac gatctagtca ctctggcctc tctctggaag atggtacact ctgttcaaca 3600
gacctggaga aagtacgtga agaacgacaa tttttggccc aacttgctca gtgagctagt 3660
gggggaaggc tccatcgcct tggccgccac gctatctaac caagcttcag tgaaagctct 3720
cttgggcctg cattttctct ctcgagggct caattacaca gatttttact ctttactgat 3780
agagaaatgc tctagtttct ttactgtaga accgcctcct ccaccagctg aaaatctgat 3840
gaccaagccc tccgtgaagt cgaaattccg aaagctgttt aagatgcaag gacccatgga 3900
cacagtcaaa gactggaacc aaatagccgc cggcttgaag aatttccaat ttgttcgtga 3960
cctagtcaag gaggtggtcg actggctcca ggcctggatc aacaaagaga aagccagccc 4020
tgtcctccag taccagctgg agatgaagaa gctcgggccc gtggctttgg ctcatgatgc 4080
cttcatggcc ggttccgggc cccctcttgg tgacgaccag attgaatacc tccagaacct 4140
caaatctctt gccctaacac tgggaaagac taatttggcc caaagcctca ccactatgat 4200
caatgccaag cagagctccg cccaacgagt cgaacccgtt gtggtggtcc tcagaggcaa 4260
gccgggatgc ggcaaaagct tggcctccac gttgattgcc caggctgtgt ccaagcgtct 4320
ctacggctcg caaagtgtgt attctcttcc tccggaccca gacttcttcg acggatataa 4380
aggacagttt gtaaccttga tggacgatct gggacaaaac ccggatgggc aagatttctc 4440
caccttttgt cagatggtgt cgaccgccca atttcttccc aatatggcgg accttgcaga 4500
gaagggacgt cccttcacct ccaatcttat cattgcgact acaaacctcc ctcactttag 4560
ccctgtcacc attgctgatc cttctgcagt ctctcggcgt atcaactacg acctgactct 4620
agaagtatct gaggcttaca agaagcacac acggctgaat ttcgacctgg ctttcagacg 4680
cactgacgcc ccccccattt acccttttgc tgcccacgtg cccttcgtgg acgtggctgt 4740
gcgcttcaaa aatggtcatc aaagcttcaa tctcctagag ttggtcgact ccatttgtgc 4800
agacattcgg gccaagcaac aaggtgctcg aaatatgcag actctggttc tacagagccc 4860
taacgagaac gacgacaccc ccgtcgacga ggcgttgggt agagttctca cccccgctgc 4920
ggtcgacgag gcgcttgtcg acctcgctcc agatgccgac ccggttggcc gcctggctat 4980
tctcgccaag ctaggtcttg ccctagctgc ggtcacccct ggtttgataa tcttggcagt 5040
gggactctac aagtacttct ctggctctga cacagaccaa gaagaaacag aaactgagga 5100
gcctgctaaa gcgcctagga gcgagaatgc ttatgacggc ccgaagaaaa actccaagcc 5160
ccctggagcg ctctctctta tggaaatgca acagcccaac gtggacatgg gctttgaggc 5220
tgcagttgct aagaaagtgg tcgtccccat taccttcatg gttcccaaca gaccttctgg 5280
acttacacag tccgctcttc ttgtggccgg ccggaccttc ctaatcaatg agcatacatg 5340
gtccaacccc tcctggacca gcttcacaat ccgtggtgag gtgcacactc gtgatgagcc 5400
tttccaaacg gttcatttta ctcaccatgg tcttcccaca gatctgatga tggtacgtct 5460
cggaccgggc aactctttcc ctaacaatct agacaagttt ggacttgacc agatgccggc 5520
acgtaactcc cgtgtggttg gcgtttcggc tagttacggt aacttcttct tctctgggaa 5580
cttcctcggg tttgttgact ccattacctc tgaccaagga acctatgcga gacttttcag 5640
gtacagggtg acgacttaca agggatggtg cggttcggcc ctggtctgtg aggccggtgg 5700
tgtccgacgc ataattggta tgcattctgc tggtgccgct ggtatcggcg ccgggactta 5760
cgtctcaaaa ttaggactga tcaaggccct taaacacctc ggtgagcctc tggctacaat 5820
gcaaggactg atgactgagc tagagcctgg agtcaccgta catgtacccc gaaaatctaa 5880
attgagaaag acgaccgcac acgcggtgta caaaccggag tttgaacctg ctgtgttgtc 5940
aaaatttgat cccagactga acaaggatgt tgacctagat gaggtaattt ggtctaaaca 6000
caccgccaac gtcccttatc aacctccttt gttctacaca tacatgtcag agtacgctca 6060
tcgggttttc tcctttttgg gaaaagacaa tgacgttctg accgtcaaag aagcaatcct 6120
gggcatccct ggactagacc ctatggatcc ccacacagct ccgggtttgc cctacgccat 6180
tagcggtctt cgacgtactg atctcgtcga ttttgcgaac ggcacggtag acccggcact 6240
ggccatgcag atccagaaat tcttagacgg tgactactct gaccatgtct tccaaacttt 6300
tctgaaagat gaaatcagac cctcagagaa ggtccgggcg ggaaaaaccc gcattgtcga 6360
tgtgccctcc ctggcgcact gcattgtggg cagaatgctg cttgggcgct ttgccgccaa 6420
gtttcaatcc catcctggct ttctccttgg ctccgctatc gggtctgacc ccgatgtctt 6480
ctggaccgtc ataggggctc agctcgaggg aagaaagaac acgtatgacg tggactacag 6540
tgcctttgac tcttcacacg gcactggctc cttcgaggct ctcatctatc actttttcac 6600
cgtggacaat ggtttcagcc ctgcgctggg accgtatctc agatccctgg ctgtctcggt 6660
gcacgcttac ggcgagcgtc gcatcaagat taccggaggc ctcccctctg gttgtgccgc 6720
gaccagcctg ctgaacacag tgctcaacaa tgtgatcatc aggactgctc tggcattgac 6780
ctacaaggaa tttgaatatg acatggttga tatcatcgcc tacggtgacg accttttggt 6840
tggtgcggat tacgatctgg acttcaatga ggtggcgcgg cgcgctgcca aactggggta 6900
taagatgact cccgccaaca agggttctgt cttccctccg acttcctctc tctccgatgc 6960
tgtttttcta aaacgcaaat tcgtccaaaa caatgacggc ttatataaac cagttatgga 7020
tttaaagaat ttggaagcca tgctctccta cttcaaacca ggaacactac tcgagaagct 7080
gcaatctgtt tctatgttgg ctcaacattc tggaaaagaa gaatatgata gattgatgca 7140
ccccttcgct gactacggtg ccgtaccgag tcacgagtac ctgcaggcaa gatggagggc 7200
cttgttcgac tgacctggat agcccaacgc gcttcggtgc tgccggcgat tctgggagaa 7260
ctcagtcgga acagaaaagg gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 7319
Claims (9)
1. A single plasmid rescue system aiming at a positive-strand RNA virus is characterized in that the single plasmid rescue system is a eukaryotic transcription plasmid containing a nucleic acid sequence of the positive-strand RNA virus, and the eukaryotic transcription plasmid contains a human cytomegalovirus RNA polymerase II promoter, a murine RNA polymerase I promoter and a ribozyme sequence at the upstream of the 5' end of the nucleic acid sequence of the virus; the downstream of the 3' end of the virus nucleic acid sequence contains ribozyme, murine polymerase terminator I and polymerase terminator II sequences;
the positive strand RNA viruses include Securidacae.
2. A construction method of full-length infectious clone of Selenecar virus is characterized by comprising the following steps: (1) amplifying the A gene of the SVV/FJ/001 strain by using a specific primer pair by using the cDNA of the SVV/FJ/001 strain as a template; the specific primer pair comprises an upstream primer and a downstream primer SVA-AR; the upstream primer comprises SVA-1F0 and SVA-1F, the nucleotide sequence of the upstream primer SVA-1F0 is shown as SEQ ID NO.1, the nucleotide sequence of the upstream primer SVA-1F is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer SVA-AR is shown as SEQ ID NO. 3;
(2) amplifying a B gene of the SVV/FJ/001 strain by using a specific primer pair by using a cDNA of the SVV/FJ/001 strain as a template, wherein the specific primer pair comprises an upstream primer SVA-BF and a downstream primer SVA-2R, the nucleotide sequence of the upstream primer SVA-BF is shown as SEQ ID No.4, and the nucleotide sequence of the downstream primer SVA-2R is shown as SEQ ID No. 5;
(3) respectively connecting the A gene and the B gene obtained by amplification in the steps (1) and (2) with a pMD20T vector, and transforming JM109 competent cells to obtain plasmids PMD-A and PMD-B;
(4) carrying out double enzyme digestion on plasmid PMD-A by PacI and NheI, carrying out double enzyme digestion on plasmid PMD-B by NheI and NotI, recovering a target fragment, connecting, replacing and inserting the target fragment into the eukaryotic plasmid of claim 1 or 2 subjected to double enzyme digestion by PacI and NotI, and transforming the eukaryotic plasmid into JM109 competent cells to obtain eukaryotic transcription plasmid prSVV/FJ;
the step (1) and the step (2) do not have a temporal restriction relationship.
3. The application of the full-length infectious clone of the Seneca virus constructed by the construction method of claim 2 in rescuing the Seneca virus.
4. A method of rescuing an epikavirus comprising the steps of: transfecting the eukaryotic transcription plasmid prSVV/FJ into the Seneca virus sensitive cells, and placing the Seneca virus sensitive cells in a medium containing 5% CO2Culturing in an incubator at 37 ℃, harvesting the virus when 80-90% of cells have pathological changes, repeatedly freezing and thawing for 3 times, and then inoculating the seneca virus sensitive cells again until the virus can stably generate the pathological changes of the cells to obtain the recombinant virus rSVV/FJ.
5. A recombinant Sesamecarin virus rSVV/FJ, which is capable of proliferating and causing cytopathy in BHK-21 cells, PK-15 cells, ST cells, SK-RST cells, IBRS-2 cells, H1299 cells or 293T cells.
6. The seneca recombinant virus rSVV/FJ of claim 5, wherein the gene of the seneca recombinant virus rSVV/FJ comprises a silent mutation NheI molecular marker, and the genetic marker can be identified with a parental wild-type strain.
7. Use of the seneca recombinant virus obtained by the method of claim 4 or the recombinant virus of claim 5 or 6 for the preparation of a recombinant vaccine for SVA.
8. Use of the single plasmid rescue system of claim 1 in construction of SVA directed mutant strains.
9. Use of the single-plasmid rescue system of claim 1 for targeted design of SVA reverse recombinant vaccines.
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| CN113308480A (en) * | 2021-06-17 | 2021-08-27 | 河北农业大学 | A-type seneca virus SVA/HeB full-length infectious cDNA clone and preparation method and application thereof |
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