CN114181912A - Infectious clone of canine parvovirus low virulent strain and application thereof - Google Patents

Abstract

The invention discloses a canine parvovirus low virulent strain infectious clone and application thereof. The genome DNA of an epidemic strain CPV-2c is cloned into a linearized low-copy plasmid pKQLL to construct a canine parvovirus full-length genome infectious clone plasmid pX-CPV; constructing a strain with multiple strains mutated at the NS1 protein phosphorylation site of the virus by taking the pX-CPV as an operation platform; by analysis and comparison of biological properties, low cytotoxic strains are identified, and the results indicate mutant strains, such as CPV-2^T598A/T601ACan be used as a candidate strain of an attenuated vaccine and has good application prospect.

Description

Infectious clone of canine parvovirus low virulent strain and application thereof

Technical Field

The invention belongs to the field of Canine Parvovirus (CPV) prevention and control, and relates to a preparation method and application of infectious clone of a canine parvovirus attenuated vaccine strain.

Background

Canine parvovirus is a virulent and highly infectious disease caused by infection of canine parvovirus on canidae and other carnivora animals, and typical clinical symptoms comprise significant reduction of leukocyte count, hemorrhagic enteritis, severe vomiting, rapid dehydration, myocarditis and the like, so that the canine parvovirus is an important factor causing morbidity and mortality of puppies, and greatly threatens the health of canine animals. In 1977, the American academy Eugster and Nairn were first isolated from the feces of sick dogs to obtain CPV-2, and the discovery of this virus was reported in New Zealand, Australia, Belgium, Japan, and the like. The first report in 1982 by Begonist et al found hemorrhagic enteritis in CPV-2 infected dogs, Xuhankun et al confirmed the prevalence of the virus the next year. CPV-2 is widely prevalent throughout the world, has a genome replacement rate similar to that of RNA viruses, has continuously changing antigenicity, sequentially presents multiple variant strains such as CPV-2a, CPV-2b and CPV-2c, and gradually expands the susceptible host range.

CPV-2 belongs to the genus parvovirus and the family parvoviridae, is a single-stranded negative-strand non-enveloped DNA virus, the genome length is about 5.0kb, the non-coding regions at the 5 'and 3' ends are hairpin structures formed by inverted repeats, the coding regions mainly code for two Open Reading Frames (ORFs) ORF1 and ORF2, ORF1 codes for two non-structural proteins (NS1 and NS2), and ORF2 codes for two capsid proteins (VP1 and VP 2). NS1 is the most major non-structural protein of CPV-2, and performs multiple functions during the parvovirus infection cycle, including involvement in viral genome replication, trans-regulation of viral and host protein expression, and involvement in inducing autophagy and apoptosis in host cells, and is the major contributor to cytotoxicity caused by viral infection. Phosphorylation of the NS1 protein is a post-translational modification, and is involved in regulating viral replication and affecting cell cycle arrest and cytotoxicity following infection. VP2 is the main component of parvovirus capsid, and is the main antigenic protein of CPV-2, playing an important role in controlling viral host range and tissue tropism. New antigenic variants are continuously generated due to amino acid substitutions of the VP2 protein.

CPV-2 is mainly controlled by attenuated live vaccination measures at present. Although antibody and cell-mediated immune responses can be stimulated by such vaccines, effective protective immunity against CPV-2 is not provided, and one of the main reasons for immune failure is the continuous circulation of different antigenic variants of CPV-2, and the protective efficacy of live attenuated vaccines based on CPV-2 and CPV-2b is reduced since CPV-2c is the main epidemic strain.

The application of reverse genetics in virology research provides an operation platform for directly manipulating genetically modified viruses, and is an effective tool for researching virus pathogenic mechanisms, cross-species transmission and vaccine development. However, no report that effective CPV attenuated vaccine strains are obtained by mutating the NS1 protein of CPV is found at present, and it is still difficult to obtain attenuated CPV infectious clones by mutation.

Disclosure of Invention

The invention aims to provide infectious clone of a canine parvovirus attenuated strain and application thereof.

An infectious clone of a canine parvovirus mutant strain, wherein the mutant site of the mutant strain comprises one or two of 598 th and 601 th amino acid sites (both belong to phosphorylation sites) of wild-type canine parvovirus NS1 protein, and the amino acid at the mutant site is replaced by non-phosphorylatable amino acid after mutation.

Preferably, the mutation site further comprises one or more of other phosphorylation sites (except the 598 th and 601 th amino acid sites, such as the 584 th, 592 th and 617 th amino acid sites) of the wild-type canine parvovirus NS1 protein.

Preferably, the mutation site further comprises one or more of non-phosphorylation sites of wild-type canine parvovirus NS1 protein.

Preferably, the mutation sites are particularly the 598 and 601 amino acid sites of the wild-type canine parvovirus NS1 protein, and the amino acids at the mutation sites are simultaneously replaced after mutation (such as T598A/T601A).

Preferably, the wild-type canine parvovirus is CPV-2, such as CPV-2 c.

Preferably, the nucleotide sequence of the infectious clone is shown in SEQ NO. ID.27.

A mutant canine parvovirus obtained by virus rescue using the above infectious clone.

Preferably, the genomic sequence of the mutant canine parvovirus is shown as SEQ NO. ID.27.

The application of the mutant canine parvovirus in preparing the canine parvovirus attenuated vaccine.

Preferably, the attenuated vaccine is a live attenuated vaccine.

A recombinant plasmid for virus rescue, which comprises the infectious clone.

Preferably, the recombinant plasmid further comprises a vector backbone for linking the infectious clones, wherein the vector backbone employs a shuttle plasmid (e.g., the low copy plasmid pKQLL) to ensure that the recombinant plasmid can be clonally propagated in bacteria before transfecting animal cells (e.g., F81 cells) and to avoid the problem of the hairpin structure of the non-coding region of the viral genome not being able to replicate correctly.

The invention has the beneficial effects that:

the invention discovers effective mutation sites (598 th and 601 th sites) and mutation modes of the obtained low-pathogenicity mutant strains by performing amino acid substitution on phosphorylation sites of NS1 protein of canine parvovirus epidemic strains and combining construction of corresponding mutant virus full-length genome infectious clones, virus rescue and analysis and comparison of biological characteristics, and obtains the obtained mutant strains (such as CPV-2)^T598A/T601A) Can be used as a candidate strain of an attenuated vaccine and has good application prospect.

Drawings

FIG. 1 is a flow chart of the construction of the CPV-2 full-length genomic infectious cloning plasmid pX-CPV (showing the EcoR I genetic marker sites formed by nonsense mutations).

FIG. 2 shows the restriction enzyme identification of pX-CPV; in the figure: lane 1 shows the restriction-free pX-CPV, lane 2 shows the BamH I/Xho I double restriction of pX-CPV, and lane 3 shows the EcoR I single restriction of pX-CPV.

FIG. 3 is the cytopathic effect observed in viral rescue of pX-CPV transfected F81 cells (when the rescued strain X-CPV was blindly passed to passage 5); in the figure: mock is a control group of cells not infected with virus.

FIG. 4 shows the single-restriction identification of EcoR I from the 5 th generation of X-CPV.

FIG. 5 shows the sequencing result of specific genome fragment of X-CPV at generation 5.

FIG. 6 shows the results of detecting the expression of VP2 protein after X-CPV infection of 5 th generation cells; in the figure: mock is a cell control group without virus infection, and beta-actin is an internal reference.

FIG. 7 is a diagram showing the results of single enzyme digestion identification of the stability of the genetic marker carried by X-CPV by EcoR I; in the figure: lane 1 is the parent strain CPV-XY, lane 2 is the 1 st generation X-CPV, lane 3 is the 5 th generation CPV-XY, lane 4 is the 10 th generation X-CPV, lane 5 is the 15 th generation X-CPV.

FIG. 8 shows the result of detecting the DNA copy number of the rescued strain X-CPV and the parent strain CPV-XY; in the figure: ns indicates that there is no significant difference.

FIG. 9 shows the results of flow cytometry to determine the cell death rate of the rescued strain X-CPV and the parent strain CPV-XY at different time points of infection; in the figure: mock is a control group of cells not infected with virus.

FIG. 10 shows the results of the CCK-8 method for detecting cell viability of the strain X-CPV rescued by cell infection and the parent strain CPV-XY at different time points; in the figure: indicates that there was a significant difference, ns indicates that there was no significant difference, and Mock is a control group of cells not infected with virus.

FIG. 11 shows strain CPV-2 with mutation in phosphorylation site of NS1 protein^T584A、CPV-2^S592A、CPV-2^T598A/T601AAnd CPV-2^T617AThe PCR identification result of (1); in the figure: lane 1 is control (water), lane 2 is X-CPV, and lane 3 is CPV-2^T584A Lane 4 is CPV-2^S592A Lane 5 is CPV-2^T598A/T601A Lane 6 is CPV-2^T617A。

FIG. 12 shows the CPV-2c full-length genome infectious clone plasmid pX-CPV with mutation of the phosphorylation site of NS1 protein^T584A、pX-CPV^S592A、pX-CPV^T598A/T601AAnd pX-CPV^T617ACytopathic effects observed in viral rescue by transfection of F81 cells (rescued strain CPV-2)^T584A、CPV-2^S592A、CPV-2^T598A/T601AAnd CPV-2^T617ABlind passage to passage 5).

FIG. 13 shows strain CPV-2 with mutation in phosphorylation site of NS1 protein^T584A、CPV-2^S592A、CPV-2^T598A/T601A、CPV-2^T617AThe detection result of the DNA copy number of the parent strain CPV-XY; in the figure: indicates that there is a significant difference.

FIG. 14 shows that strain CPV-2 with mutation in phosphorylation site of NS1 protein detected by flow cytometry^T584A、CPV-2^S592A、CPV-2^T598A/T601A、CPV-2^T617AResults of cell death rate at different time points of infection from the parent strain CPV-XY; in the figure: mock is a control group of cells not infected with virus.

FIG. 15 shows that strain CPV-2 with mutation of phosphorylation sites of NS1 protein is detected by CCK-8 method^T584A、CPV-2^S592A、CPV-2^T598A/T601A、CPV-2^T617AResults of cell viability at different time points after the parent strain CPV-XY; in the figure: mock is a control group of cells not infected with virus, indicating significant differences.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.

CPV-2 genomic DNA extraction

A CPV-2c epidemic strain CPV-XY (genome reference sequence: MT892649.1) is adopted as a parent strain, and the epidemic strain is separated from Shaanxi province Yanyang city in 3 months in 2021.

Collecting 500 mul parent strain CPV-XY virus suspension, repeatedly freezing and thawing for three times, centrifuging at 6000rpm for 5min, taking 437.5 mul supernatant obtained by centrifugation, adding 50 mul 10% SDS and 12.5 mul proteinase K into the supernatant, incubating at 55 ℃ for 30min, and then extracting virus genome DNA by referring to phenol chloroform method.

Amplification of the terminal non-coding region (containing hairpin structure) and the intermediate coding sequence of the CPV-2 genome

Referring to FIG. 1, the middle coding sequence of the CPV-2 genome was divided into four fragments for amplification (the four fragments were named M1, M2, M3 and M4, respectively), and EcoR I restriction sites were introduced into the CPV-2 genome as genetic markers by nucleotide nonsense mutation (T3566C), and PCR amplification primers for M1, M2, M3 and M4 were as follows.

The upstream primer M1-F:

5'-AAAGACAAACCATAGACCGTTACTG-3'；

the downstream primer M1-R:

5'-TACCGAACAAAGAGTCACCAACCA-3'；

the upstream primer M2-F:

5'-GTGACTCTTTGTTCGGTAAACTT-3'；

the downstream primer M2-R:

(nonsense mutation back base in frame)

The upstream primer M3-F:

(complementary bases of nonsense-mutated bases in frame)

The downstream primer M3-R:

5'-GAGAGGCTCTTAGTTTAGCTTTA-3'；

the upstream primer M4-F:

5'-GCTAAACTAAGAGCCTCTCATACTTGG-3'；

the downstream primer M4-R:

5'-GATACTAATGGTAAGGTTAGTTCACCTTATAGAC-3'。

the extracted CPV-2 genome DNA is used as a template, high fidelity enzyme is applied, and upstream primers M1-F/R amplification fragment M1, M2-F/R amplification fragment M2, M3-F/R amplification fragment M3 and M4-F/R amplification fragment M4 are used respectively, wherein the EcoR I restriction sites are introduced into fragments M2 and M3 through the primers M2-R and M3-F. The PCR amplification products of the fragments M1, M2, M3 and M4 were separated by electrophoresis in 1% agarose gel and recovered by purification. Performing overlap PCR by using the fragments M1 and M2 as a template, using M1-F/M2-R as an upstream primer and a downstream primer to obtain a fragment M (1+2), and performing overlap PCR by using the fragments M3 and M4 as a template and using M3-F/M4-R as an upstream primer and a downstream primer to obtain a fragment M (3+ 4).

The PCR amplification was carried out by optimizing the conventional reaction procedure employed for the above PCR and using the following primers. BamH I and Xho I cleavage sites (see in-frame bases) were introduced into primers T1-F and T6-R.

The upstream primer T1-F:

the downstream primer T1-R:

5'-CGGTCTATGGTTTGTCTTTTATACC-3'；

the upstream primer T6-F:

5'-CCTTACCATTAGTATCAATCTGTCTTTAAGGGGGGG-3'；

the downstream primer T6-R:

the non-coding regions (named T1 and T6) at the 5 'end and the 3' end of CPV-2 were amplified by the downstream primers T1-F/R, T6-F/R, respectively.

Wherein, the optimized PCR reaction conditions are as follows: 5min at 98 ℃; 30 cycles of 98 ℃ for 30s, 64 ℃ for 30s, and 72 ℃ for 30 s; 10min at 72 ℃. The primer concentration was optimized to 0.8. mu. mol/L. The amplification product is separated by 1% agarose gel electrophoresis and then purified and recovered.

Construction of full-Length genome infectious clone of CPV-2

As shown in FIG. 1, the fragments T1 and M (1+2) were subjected to overlap PCR using T1-F/M2-R as upstream and downstream primers to obtain fragment F1; and (3) carrying out overlap PCR on the fragments M (3+4) and T6 by taking M3-F/T6-R as upstream and downstream primers to obtain a fragment F2. Fragments F1 and F2 were cloned sequentially by the seamless Cloning technique using the Cloneexpress II One Step Cloning Kit (vazyme) into Xho I-digested linearized pKQLL vector (Huada gene), and ligated at 37 ℃ for 30 min. The ligation product directly transforms escherichia coli DH5 alpha competence, the obtained product is coated on a kanamycin solid culture medium, after overnight culture, single colony is selected for BamH I and Xho I double enzyme digestion identification and EcoR I single enzyme digestion identification, enzyme digestion products are separated by 1% agarose gel electrophoresis, recombinant plasmids generate specific bands of about 5000bp and 2000bp after BamH I and Xho I double enzyme digestion, and generate specific bands of about 4500bp and 2500bp after EcoR I single enzyme digestion (figure 2). And (3) sequencing and identifying the positive colony to a company, and successfully constructing a CPV-2 full-length genome infectious clone plasmid pX-CPV according to the result, namely obtaining the recombinant plasmid containing the CPV-2 full-length genome infectious clone.

4. Virus rescue of CPV-2 strain X-CPV carrying EcoR I genetic marker

The constructed recombinant plasmid pX-CPV is transfected into F81 cells (Punuisan) after being cloned and propagated, the transfected F81 cells and the supernatant thereof are collected 72 hours after being transfected by Lipo8000 transfection reagent (Biyunyun day), repeated freezing and thawing is carried out for 3 times, centrifugation is carried out for 10min at 12000rpm and 4 ℃, the collected supernatant is inoculated into newly cultured F81 cells and then continuously cultured for 72 hours, F81 cells and the supernatant thereof are collected and repeatedly frozen and thawed for 3 times, virus rescue is carried out by continuous passage according to the method (namely continuously adopting the newly cultured F81 cells), and a parent strain CPV-XY is set as a positive control.

Observing the pathological changes of the inoculated F81 cells by an optical microscope every day, wherein typical cytopathies such as wire drawing, shedding, rounding and the like appear in F81 cells of an infected parent strain CPV-XY and a rescued strain X-CPV (figure 3); collecting F81 cells infected by the 5 th generation X-CPV and the parent strain CPV-XY and supernatant thereof, extracting DNA as a template, and carrying out PCR amplification by using a pair of specific primers E-F and E-R crossing the EcoR I genetic marker:

an upstream primer E-F:

5'-GACAATCTTGCACCAATGAGTGATG-3'；

the downstream primer E-R:

5'-GGTTAAAGTTAATATTTTGAATCCA-3'；

amplifying to generate specific bands (not digested, in an untreated lane in FIG. 4) of about 1300bp in both the X-CPV and CPV-XY infection groups by using an upstream primer E-F/R, performing EcoR I single digestion identification after the PCR products are harvested and purified, and performing enzyme digestion on only the CPV-XY infection group to generate specific bands of about 500bp and 800bp (FIG. 4); meanwhile, the amplified specific band is sequenced, and the virus morphology is observed by combining a sequencing result and an electron microscope, so that CPV-2 strain X-CPV carrying the EcoR I genetic marker is successfully rescued (figure 5).

Adding a proper amount of RIPA lysate (Biyun day) into cell precipitates (respectively collected 5 th generation X-CPV and F81 cells infected by parent strain CPV-XY), performing ice bath lysis for 30min, centrifuging at 12000rpm at 4 ℃ for 10min, collecting protein supernatant, adding protein loading buffer solution, performing SDS polyacrylamide gel electrophoresis separation, performing western blotting by using CPV VP2 antibody to detect the expression condition of VP2 protein of CPV-2, and detecting the expression of VP2 protein in a parent strain CPV-XY infection group and a 5 th generation X-CPV infection group (figure 6).

5. Stability test of rescued X-CPV strain carrying genetic marker

During the continuous passage of the CPV-2 strain X-CPV carrying the EcoR I genetic marker, collecting X-CPV infected F81 cells at 1 st, 5 th, 10 th and 15 th generations, extracting DNA as a template, taking parent strain CPV-XY genome DNA as a reference, utilizing an upstream primer E-F/R PCR to amplify a specific strip, and performing single enzyme digestion on the specific strips amplified from the 1 st, 5 th, 10 th and 15 th generation X-CPVs and the parent strain CPV-XY after glue collection and purification, separating and identifying the enzyme digestion products by 1% agarose gel electrophoresis, wherein the specific strips of about 500bp and 800bp are generated in the 1 st, 5 th, 10 th and 15 th generation X-CPV infection groups, which indicates that the X-CPV can stably carry an EcoR I genetic marker (figure 7).

6. Biological Properties (replication and cytopathic Properties) of rescued X-CPV carrying genetic marker Strain

F81 cells were harvested at 5X 10⁵Cell/well amount cells were inoculated into 6-well cell culture plates for overnight culture, and then culture was continued after inoculating the rescued strain X-CPV and the parent strain CPV-XY, respectively, at the same titer (1MOI), while setting a control group of cells not infected with virus. F81 cells and supernatants thereof at 12h, 24h and 36h after virus infection are collected, DNA is respectively used as a template, the DNA copy number of CPV-2 at different time points after virus infection is detected, and the result shows that the rescued strain X-CPV has the same replication capacity with the parent strain CPV-XY (figure 8); f81 cells at 12h, 24h and 36h after virus infection are collected, and the cell death rate of different time points after virus infection is detected by a 7-AAD flow cytometric staining method; f81 cells in good state were cultured at 1X 10⁵Amount per well cells were seeded into 96 well cell culture plates overnight and the same drops were culturedThe X-CPV with the degree of 1MOI and the parent strain CPV-XY are respectively diluted in a gradient manner and inoculated into F81 cells in a 96-well cell culture plate, and the cell viability of 12h, 24h and 36h after the virus infection is detected by a CCK-8 kit (Biyun day). The results show that X-CPV has the same cytopathogenic ability as the parent strain CPV-XY (FIG. 9, FIG. 10).

Preparation of CPV-2 attenuated vaccine strain

One, two or more sites are randomly selected from the amino acid sites (mainly phosphorylation sites) of the NS1 protein of CPV-2 for amino acid substitution. The experimental procedure is exemplified below.

7.1 primer design

X-CPV or CPV-XY is taken as a wild strain, 4 different amino acid substitutions (T584A, S592A, T598A/T601A and T617A) are respectively introduced into the phosphorylation sites of the C terminal of the NS1 protein (reference sequence: QTF73976.1, corresponding to 270-2276bp of a genome reference sequence MT892649.1), and the following primers are designed according to the corresponding genome reference sequences.

The upstream primer Hind III-F:

5'-CATTAAGTTAGTGTGTAAGCTTCCA-3'；

the upstream primer Hind III-R:

5'-AGATAAGCAGCGTAAGCTTCGT-3'；

upstream primer T584A-F (A1750G, see in frame for bases after mutation):

downstream primer T584A-R:

upstream primer S592A-F (A1774G; G1775C):

the downstream primer S592A-R:

upstream primer T598A/T601A-F (A2061G/A2070G):

the downstream primer T598A/T601A-R:

upstream primer T617A-F (A1849G):

downstream primer T617A-R:

7.2 construction of full-Length genomic infectious clones of mutant strains and Virus rescue of mutant strains

Taking X-CPV genome DNA as a template, respectively carrying out PCR amplification on 4 groups of 8 fragments by using designed corresponding upstream and downstream primers for introducing amino acid substitutions: T584A-F1, T584A-F2, S592A-F1, S592A-F2, T598A/T601A-F1, T598A/T601A-F2, T617A-F1 and T617A-F2.

Respectively taking PCR amplification products of upper and lower arms of T584A-F1, T584A-F2, S592A-F1, S592A-F2, T598A/T601A-F1, T598A/T601A-F2 and T617A-F1 and T617A-F24 groups as templates, and performing overlap PCR amplification to obtain fragments T584A-F (required primers are Hind III-F/R and T584A-F/R), S592A-F (required primers are Hind III-F/R and S592A-F/R), T598A/T601A-F (required primers are Hind III-F/R and T598A/T601A-F/R) and T617A-F (required primers are Hind III-F/R and T617F 45-F617A-F617)/R), performing seamless cloning connection on the 4 fragments purified by the glue harvest and a linearized pKQLL vector purified by the glue harvest after HindIII single enzyme digestion to respectively construct 4 different strains (CPV-2) with NS1 protein phosphorylation site mutation (amino acid substitution)^T584A、CPV-2^S592A、CPV-2^T598A/T601AAnd CPV-2^T617A) Full-length genomic infectious clone plasmid pX-CPV of^T584A、pX-CPV^S592A、pX-CPV^T598A/T601AAnd pX-CPV^T617A。

Respectively transfecting F81 cells with the constructed infectious clone plasmids of 4 mutant strains, collecting the transfected F81 cells and supernatant thereof after 72 hours, repeatedly freezing and thawing for 3 times, centrifuging at 12000rpm and 4 ℃ for 10min, collecting the supernatant, inoculating the supernatant into newly cultured F81 cells, continuing to culture, and continuously passaging for virus rescue according to the method. F81 cells infected with X-CPV and 4 mutant strains and supernatants thereof are collected, DNA is respectively extracted as a template, the full-length sequence of NS1 is amplified by the upstream primer NS1-F/R, and sequencing is carried out after PCR identification:

the upstream primer NS 1-F:

5'-ATGTCTGGCAACCAGTATACTGAGG-3'；

the downstream primer NS 1-R:

5'-ATCCAAGTCGTCTCGAAAATCTT-3'；

the results showed that 2004bp specific bands were amplified (FIG. 11). Continuously passaging to 5 th generation, and respectively infecting mutant strain CPV-2^T584A、CPV-2^S592A、CPV-2^T598A/T601AAnd CPV-2^T617AThe F81 cells all exhibited cytopathic effects (CPE, fig. 12).

7.3 comparison of the biological Properties of the mutant strains with the wild type Strain (X-CPV) and screening of Low-pathogenicity mutant strains

Respectively inoculating the rescued X-CPV and the mutant strain CPV-2 with the same titer (1MOI)^T584A、CPV-2^S592A、CPV-2^T598A/T601AAnd CPV-2^T617AMeanwhile, a cell control group not infected with virus is set. Collecting F81 cells 12h, 24h and 36h after virus infection and supernatant thereof, respectively extracting DNA as a template, detecting the DNA copy number of CPV-2 at different time points of virus infection, and obtaining the resultDisplaying CPV-2^T584A、CPV-2^S592A、CPV-2^T617AAnd X-CPV has the same DNA copy number at different time points after infection, has the same replication capacity (p > 0.05), CPV-2^T598A/T601AThe copy number of (b) was not significantly different from that of X-CPV at 12h after infection (p > 0.05), while it was significantly lower at 24h and 36h after infection than that of X-CPV and the other 3 mutant strains (p < 0.05), indicating that CPV-2^T598A/T601AIs significantly reduced (fig. 13).

F81 cells at 12h, 24h and 36h after virus infection are collected, the cell death rate of different time points of virus infection is detected by a 7-AAD flow cytometric staining method, and the result shows that CPV-2^T598A/T601ACell mortality due to infection was lower than that of X-CPV (FIG. 14) and CPV-2^T584A、CPV-2^S592A、CPV-2^T617A。

Different strains with the same titer (1MOI) are respectively diluted in a gradient way and inoculated into F81 cells in a 96-well cell culture plate, the cell viability of 12h, 24h and 36h after virus infection is detected by a CCK-8 kit (Biyun day), and the result shows that (figure 15) compared with the F81 cells without virus infection, F81 cells are respectively infected with X-CPV and CPV-2^T584A、CPV-2^S592A、CPV-2^T617AThe cell viability was significantly reduced (p < 0.05) at 24h and 36h after infection with CPV-2^T598A/T601The cell viability is not obviously changed after 24h (p is more than 0.05), the cell viability is obviously reduced after 36h after infection (p is less than 0.05), and CPV-2 is infected^T598A/T601AThe viability of the cells of (A) was significantly higher than that of the infected X-CPV and other mutant strains (p < 0.05).

The experimental results of mutant strains according to other experiments show that T598A and T601A are more critical mutation modes for obtaining the CPV-2 attenuated vaccine strain, and in the case of adopting the mutation, the phosphorylation sites of other NS1 proteins (including but not limited to T584A, S592A and T617A) are continuously mutated, so that mutant strains with low cytotoxicity can be obtained through virus rescue.

In summary, the invention takes the genomic DNA of an epidemic strain CPV-2c CPV-XY (MT892649.1) as a template, and divides the full-length viral genome containing Inverted Terminal Repeat (ITR) sequences at two ends (namely, hairpin structure) and a middle coding region into 6 segmentsAnd respectively amplifying, cloning the combined genome fragment into a linearized low-copy plasmid pKQLL by an In-Fusion seamless cloning technology, and constructing a canine parvovirus full-length infectious clone plasmid pX-CPV carrying an EcoR I genetic marker. Using pX-CPV as an operation platform and X-CPV as a wild strain to construct a strain (such as CPV-2) with mutation (such as T584A, S592A, T598A/T601A and T617A at the C terminal) at the phosphorylation site of the NS1 protein^T584A、CPV-2^S592A、CPV-2^T598A/T601A、CPV-2^T617AEtc.). Finally, by analysis and comparison of the biological properties, strains with low cytotoxicity, such as CPV-2, are identified^T598A/T601AThe mutant strain can be used as a candidate strain for preparing the canine parvovirus attenuated vaccine, and has good application prospect.

<110> northwest agriculture and forestry science and technology university

<120> infectious clone of canine parvovirus low virulent strain and application thereof

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<211> 25

<212> DNA

<213> E-F

<400> 13

gacaatcttg caccaatgag tgatg 25

<210> 14

<211> 25

<212> DNA

<213> E-R

<400> 14

ggttaaagtt aatattttga atcca 25

<210> 15

<211> 25

<212> DNA

<213> Hind Ⅲ-F

<400> 15

cattaagtta gtgtgtaagc ttcca 25

<210> 16

<211> 22

<212> DNA

<213> Hind Ⅲ-R

<400> 16

agataagcag cgtaagcttc gt 22

<210> 17

<211> 25

<212> DNA

<213> T584A-F

<400> 17

gcaaagactt agaggcacaa gcggc 25

<210> 18

<211> 25

<212> DNA

<213> T584A-R

<400> 18

gcctctaagt ctttgcaacc tggtg 25

<210> 19

<211> 25

<212> DNA

<213> S592A-F

<400> 19

gcaatcctca ggctcaagac caagt 25

<210> 20

<211> 25

<212> DNA

<213> S592A-R

<400> 20

cttgagcctg aggattgctt gccgc 25

<210> 21

<211> 24

<212> DNA

<213> T598A/T601A-F

<400> 21

ctagctcctc tggctccgga cgta 24

<210> 22

<211> 25

<212> DNA

<213> T598A/T601A-R

<400> 22

ggagccagag gagctagaac ttggt 25

<210> 23

<211> 24

<212> DNA

<213> T617A-F

<400> 23

gagtactcca gatgcgccta ttgc 24

<210> 24

<211> 24

<212> DNA

<213> T617A-R

<400> 24

ggcgcatctg gagtactcca cggt 24

<210> 25

<211> 25

<212> DNA

<213> NS1-F

<400> 25

atgtctggca accagtatac tgagg 25

<210> 26

<211> 23

<212> DNA

<213> NS1-R

<400> 26

atccaagtcg tctcgaaaat ctt 23

<210> 27

<211> 5059

<212> DNA

<213> mutant strain of CPV-2c with or without genetic marker (amino acid substitution: T598A/T601A)

<400> 27

attctttaga accaactgac caagttcacg tacgtatgac gtgatgacgc gcgctgcgcg 60

cgcttcctac ggcagtcaca cgtcatacgt acgctccttg gtcagttggt tctaaagaat 120

gataggcggt ttgtgtgttt aaacttgggc gggaaaaggt ggcgggctaa ttgtgggcgt 180

ggttaaaggt ataaaagaca aaccatagac cgttactgac attcgcttct tgtctttgac 240

agagtgaacc tctcttactt tgactaacca tgtctggcaa ccagtatact gaggaagtta 300

tggagggagt aaattggttg aagaaacatg cagaaaatga agcattttcg tttgttttta 360

aatgtgacaa cgtccaacta aatggaaagg atgttcgctg gaacaactat accaaaccaa 420

ttcaaaatga agagctaaca tctttagtta gaggagcaca aacagcaatg gatcaaaccg 480

aagaagaaga aatggactgg gaatcggaag ttgatagtct cgccaaaaag caagtacaaa 540

cttttgatgc attaattaaa aaatgtcttt ttgaagtctt tgtttctaaa aatatagaac 600

caaatgaatg tgtttggttt attcaacatg aatggggaaa agatcaaggc tggcattgtc 660

atgttttact tcatagtaaa aacttacaac aagcaactgg taaatggcta cgcagacaaa 720

tgaatatgta ttggagtaga tggttggtga ctctttgttc ggtaaactta acaccaactg 780

aaaagattaa gctcagagaa attgcagaag atagtgaatg ggtgactata ttaacataca 840

gacataagca aacaaaaaaa gactatgtta aaatggttca ttttggaaat atgatagcat 900

attacttttt aacaaagaaa aaaattgtcc acatgacaaa agaaagtggc tattttttaa 960

gtactgattc tggttggaaa tttaacttta tgaaatatca agacagacaa attgtcagca 1020

cactttacac tgaacaaatg aaaccagaaa ccgttgaaac cacagtgacg acagcacagg 1080

aaacaaagcg cgggagaatt caaactaaaa aggaagtgtc aatcaaatgt actttgcggg 1140

acttggttag taaaagagta acatcacctg gagactggat gatgttacaa ccagatagtt 1200

atattgaaat gatggcgcaa ccaggaggtg aaaatctttt aaaaaataca cttgaaattt 1260

gtactttgac tttagcaaga acaaaaacag catttgaatt aatacttgaa aaagcagata 1320

atactaagct aactaacttt gatcttgcaa attctagaac atgtcaaatt tttagaatgc 1380

acggatggaa ttggattaaa gtttgtcacg ctatagcatg tgttttaaat agacaaggtg 1440

gtaaaagaaa tacagttctt tttcatggac cagcaagtac aggaaaatct atcattgctc 1500

aagccatagc acaagctgtg ggtaatgttg gttgttataa tgcagcaaat gtaaattttc 1560

catttaatga ctgtaccaat aaaaatttaa tttggattga agaagctggt aactttggtc 1620

aacaagttaa tcaatttaaa gcaatctgtt ctggacaaac aattagaatt gatcaaaaag 1680

gtaaaggaag taagcaaatt gaaccaactc cagtaattat gacaactaat gaaaatataa 1740

caattgtgag aattggatgt gaagaaagac ctgaacatac acaaccaata agagacagaa 1800

tgctgaacat taagttagtg tgtaagcttc caggagactt tggtttggtt gataaagagg 1860

aatggccttt aatatgtgca tggttagtta agcatggttt tgtatcaacc atggctaact 1920

atacacatca ctggggaaaa gtaccagaat gggatgaaaa ctgggcggag cctaaaatac 1980

aagaaggtat aaattcacca ggttgcaaag acttagagac acaagcggca agcaatcctc 2040

agagtcaaga ccaagttcta gctcctctgg ctccggacgt agtggacctt gcactggaac 2100

cgtggagtac tccagatacg cctattgcag aaactgcaaa tcaacaatca aaccaacctg 2160

gcgttactca caaagacgtg caagcgagtc caacgtggtc cgaaatagag gcagacctga 2220

gagccatctt tacttctgaa caattggaag aagattttcg agacgacttg gattaaggta 2280

cgatggcacc tccggcaaag agagccagga gaggtaaggg tgtgttagtg aagtgggggg 2340

aggggaaaga tttaataact taactaagta tgtatttttt tgtaggactt gtgcctccag 2400

gttataaata tcttgggcct gggaacagtc ttgaccaagg agaaccaact aacccttctg 2460

acgccgctgc aaaagaacac gacgaagctt acgctgctta tcttcgctct ggtaaaaacc 2520

catacttata tttctcgcca gcagatcaac gctttataga tcaaactaag gacgctaaag 2580

attggggggg gaaaatagga cattattttt ttagcgctaa aaaggcaatt gctccagtat 2640

taactgatac cccagatcat ccatcaacat caagaccatc aaaaccaact aaaagaagta 2700

aaccaccacc tcatattttc atcaatcttg caaaaaaaaa aaaaaccggt gcaggacaag 2760

taaaaagaga caatcttgca ccaatgagtg atggaggagt tcaaccagac ggtggtcaac 2820

ctgctgtcag aaatgaaaga gctacaggat ctgggaacgg gtctggaggc gggggtggtg 2880

gtggttctgg gggtgtgggg atttctacgg gtacttttaa taatcagacg gaatttaaat 2940

ttttggaaaa cggatgggtg gaaatcacag caaactcaag cagacttgta catttaaata 3000

tgccagaaag tgaaaattat agaagagtgg ttgtaaataa tttggataaa actgcagtta 3060

acggaaacat ggctttagat gatactcatg cacaaattgt aacaccttgg tcattggttg 3120

atgcaaatgc ttggggagtt tggtttaatc caggagattg gcaactaatt gttaatacta 3180

tgagtgagtt gcatttagtt agttttgaac aagaaatttt taatgttgtt ttaaagactg 3240

tttcagaatc tgctactcag ccaccaacta aagtttataa taatgattta actgcatcat 3300

tgatggttgc attagatagt aataatacta tgccatttac tccagcagct atgagatctg 3360

agacattggg tttttatcca tggaaaccaa ccataccaac tccatggaga tattattttc 3420

aatgggatag aacattaata ccatctcata ctggaactag tggcacacca acaaatatat 3480

accatggtac agatccagat gatgttcaat tttacactat tgaaaattct gtgccagtac 3540

acttactaag aacaggtgat gaattygcta caggaacatt ttattttgat tgtaaaccat 3600

gtagactaac acatacatgg caaacaaata gagcattggg cttaccacca tttctaaatt 3660

ctttgcctca agctgaagga ggtactaact ttggttatat aggagttcaa caagataaaa 3720

gacgtggtgt aactcaaatg ggaaacacaa acattattac tgaagctact attatgagac 3780

cagctgaggt tggttatagt gcaccatatt attcttttga ggcgtctaca caagggccat 3840

ttaaaacacc tattgcagca ggacgggggg gagcgcaaac agatgaaaat caagcagcag 3900

atggtgatcc aagatatgca tttggtagac aacatggtca aaaaactacc acaacaggag 3960

aaacacctga gagatttaca tatatagcac atcaagatac aggaagatat ccagaaggag 4020

attggattca aaatattaac tttaaccttc ctgtaacaga agataatgta ttgctaccaa 4080

cagatccaat tggaggtaaa acaggaatta actatactaa tatatttaat acttatggtc 4140

ctttaactgc attaaataat gtaccaccag tttatccaaa tggtcaaatt tgggataaag 4200

aatttgatac tgacttaaaa ccaagactcc atgtaaatgc accatttgtt tgtcaaaata 4260

attgtcctgg tcaattattt gtaaaagttg cacctaattt aacaaatgaa tatgatcctg 4320

atgcatctgc taatatgtca agaattgtaa cttactcaga tttttggtgg aaaggtaaat 4380

tagtatttaa agctaaacta agagcctctc atacttggaa tccaattcaa caaatgagta 4440

tcaatgtaga taaccaattt aactatgtac caagtaatat tggaggtatg aaaattgcat 4500

atgaaaaatc tcaactagca cctagaaaat tatattaaca tacttactat gtttttatgt 4560

ttattacata ttattttaag attaattaaa ttacagcata gaaatattgt acttgtactt 4620

gatataggat ttagaaggtt tgttatatgg tatacaataa ctgtaagaaa tagaagaaca 4680

tttagatcat agttagtagt ttgttttata aaatgtattg taaaccatta atgtatgttg 4740

ttatggtgtg ggtggttggt tggtttgccc ttagaatatg ttaaggacca aaaaaatcaa 4800

taaaagacat ttaaaactaa atggcctcgt atactgtcta taaggtgaac taaccttacc 4860

attagtatca atctgtcttt aagggggggt gggtgggaga tgcacaatat cagtagactg 4920

actggcctgg ttggttgttc tgcttaatca accagaccgt tatgcggtct ggttgattaa 4980

gcgcaaccaa ccaggccagt cagtctactg atgttgtgca tctcccaccc acccccccct 5040

taaagacaga ttgatacta 5059

Claims (10)

1. A mutant canine parvovirus, which is characterized by: the mutant site of the mutant canine parvovirus comprises one or two of 598 and 601 phosphorylation sites of the NS1 protein of the wild canine parvovirus.

2. A mutant canine parvovirus according to claim 1, wherein: the mutation site also includes one or more of the other phosphorylation sites of the wild-type canine parvovirus NS1 protein.

3. A mutant canine parvovirus according to claim 1, wherein: the mutation sites also include one or more of the non-phosphorylation sites of the wild-type canine parvovirus NS1 protein.

4. A mutant canine parvovirus according to claim 1, wherein: the mutation sites are specifically amino acid sites 598 and 601 of wild canine parvovirus NS1 protein.

5. A mutant canine parvovirus according to claim 1, 2, 3 or 4, wherein: and the amino acid at the mutation site is replaced by an amino acid which cannot be phosphorylated after mutation.

6. A mutant canine parvovirus according to claim 1, 2, 3 or 4, wherein: the wild canine parvovirus is CPV-2.

7. A mutant canine parvovirus according to claim 1, 2, 3 or 4, wherein: the genome sequence of the mutant canine parvovirus is shown as SEQ No. ID.27.

8. Use of the mutant canine parvovirus of claim 1 in the preparation of a canine parvovirus attenuated vaccine.

9. An infectious clone of a mutant strain of canine parvovirus, characterized by: the mutant strain has mutation sites including 598 and 601 phosphorylation sites of wild canine parvovirus NS1 protein or both.

10. A recombinant plasmid for virus rescue, characterized by: the recombinant plasmid comprising the infectious clone of claim 9.

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