CN111944770A - Foot-and-mouth disease virus attenuated mutant strain and preparation method and application thereof - Google Patents
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Abstract
The invention provides a foot-and-mouth disease virus attenuated mutant strain and a preparation method and application thereof, belonging to the technical field of biotechnology and biological products. The attenuated foot-and-mouth disease virus mutant strain is obtained by mutating Lb AUG in a parent foot-and-mouth disease virus leader protein gene into ATC or AAA, wherein the replication capacity of the mutant virus in BHK-21 cells is similar to that of a parent strain, but the replication level on PK-15 cells is reduced, the pathogenicity to suckling mice is obviously weakened, and the mutation type can be stably inherited. The invention can obviously weaken O-type foot-and-mouth disease virus after mutating 2 nd initiation codon AUG in the leader protein gene to ATC or AAA on the basis of the OZK/93-08 vaccine strain genome structure, is a method for developing genetic engineering attenuated vaccine strains, and provides a safer platform for the production of foot-and-mouth disease O-type inactivated vaccine antigens.
Description
Technical Field
The invention belongs to the technical field of biotechnology and biological products, and particularly relates to a foot-and-mouth disease virus attenuated mutant strain and a preparation method and application thereof.
Background
Foot-and-mouth disease (FMD) is an acute, hot, highly contagious animal epidemic disease caused by FMDV (foot-and-mouth disease virus) and mainly infects cloven-hoof animals, including cattle, pigs, sheep, goats, and the like. The disease is one of animal infectious diseases reported by the world animal health Organization (OIE) legal practice, and is listed as a first animal epidemic disease in China. Currently, the most effective control strategy for foot-and-mouth disease is achieved mainly by vaccination with inactivated vaccines and slaughter of infected and exposed animals. Some countries in European Union and south America immunize and inoculate foot-and-mouth disease virus inactivated vaccines for decades, have effectively controlled the foot-and-mouth disease, and are considered as benchmarks for preventing and controlling the foot-and-mouth disease by the inactivated vaccines. In the scheme for preventing and treating the middle-long term animal epidemic diseases in China (2012-2020), which is released in 5 months in 2012, foot-and-mouth disease is definitely regarded as the epidemic disease which is preferentially prevented and controlled, and vaccine immunization is taken as a core technical means for prevention, control and purification.
The production of inactivated vaccines requires expensive facilities in which virulent strains of foot-and-mouth disease virus are propagated in large quantities, with the risk of the virus escaping from the production facility. In order to reduce the hidden danger of virus dispersion in the vaccine manufacturing process, one method is to develop a foot-and-mouth disease virus strain with improved biological safety. The replication capacity of the engineering strain in BHK-21 cells (a seed cell line for producing the foot-and-mouth disease vaccine) should not be reduced, but the replication capacity in other interferon totipotent cells is weakened or the pathogenicity to sensitive animals is weakened, namely, an attenuated strain. Therefore, the foot-and-mouth disease vaccine virulent strain is transformed from the source of 'seed virus' to obtain the attenuated virus strain, and the method is of great importance for improving the biological safety in the vaccine production process.
The leader (L) protein is a papain-like protease, is a virulence factor encoded by the foot-and-mouth disease virus, and plays an important role in the pathogenesis of the foot-and-mouth disease virus. Therefore, manipulation of the leader protein gene may result in an engineered, viable strain of foot-and-mouth disease virus. Translation of the leader protein begins at two different start codons (Lab AUG and Lb AUG) separated by 84 nucleotides (La region), which results in the leader protein existing in two forms, called the Lab protein and the Lb protein. The selection and utilization of initiation codon is one of the core steps influencing the initiation of viral protein translation, and determines the translation efficiency of viral proteins, so that two initiation AUG in L gene play an important role in foot-and-mouth disease virus replication. Previous studies have analyzed the effect of two initial AUG mutations on virus replication, and the results indicate that the Lab AUG mutation to UGG does not prevent the rescue of foot and mouth disease virus, but the Lb AUG mutation to UUU does not rescue viable foot and mouth disease virus. Another study showed that the insertion of a 57nt transposon in the La region results in a decrease in the availability of Lb AUG, which results in a reduction in the virulence of the virus in cattle. Furthermore, insertion of an epitope tag (Flag) in the La region does not affect the survival of the virus, but eliminates or greatly reduces the translation activity of the virus using the Lab AUG initiation protein. These previous results indicate that the selective use of Lab AUG and Lb AUG is critical to the replication and virulence of foot and mouth disease virus. So far, no Lb AUG modified foot-and-mouth disease virus is available at home and abroad, and no attenuated strain caused by Lb AUG modified foot-and-mouth disease virus is reported.
Disclosure of Invention
In view of the above, the present invention aims to provide a attenuated mutant strain of foot-and-mouth disease virus, wherein the mutant strain has attenuated phenotype and has application potential for improving biological safety in vaccine production.
The invention provides a foot-and-mouth disease virus attenuated mutant strain, which mutates LbAUG in a foot-and-mouth disease virus leader protein gene into ATC or AAA.
Preferably, the nucleotide sequence of the leader protein mutated to ATC is shown in SEQ ID No. 2.
Preferably, the nucleotide sequence of the leader protein mutated to AAA is shown in SEQ ID No. 4.
Preferably, the nucleotide sequence of the leader protein is shown as SEQ ID No. 5.
Preferably, the foot and mouth disease virus comprises O, A, C, SAT1, SAT2, SAT3 and Asia17 serotype foot and mouth disease viruses.
Preferably, the attenuated strain of foot-and-mouth disease type O is obtained by mutating LbAUG of the leader protein to ATC or AAA based on the strain OZK/93-08.
The preparation method of the foot-and-mouth disease virus attenuated mutant strain comprises the following steps:
and (3) carrying out point mutation on the cDNA plasmid containing the foot-and-mouth disease virus full-length genome to obtain a mutated recombinant plasmid, and carrying out virus rescue on the mutated recombinant plasmid transfected cells to obtain the foot-and-mouth disease virus mutant strain.
Preferably, the primers for point mutation are ATC-F/ATC-R primer pairs and AAA-F/AAA-R primer pairs;
the nucleotide sequence of the ATC-F is shown as SEQ ID No. 8;
the nucleotide sequence of the ATC-R is shown as SEQ ID No. 9;
the nucleotide sequence of the AAA-F is shown as SEQ ID No. 12;
the nucleotide sequence of the AAA-R is shown as SEQ ID No. 13.
Preferably, the PCR amplification procedure used for the point mutation is pre-denaturation at 94 ℃ for 5min, followed by 20min at 98 ℃ and 1min/1kb at 68 ℃ for 35 cycles, followed by 10min at 72 ℃.
The invention provides application of the attenuated foot-and-mouth disease virus mutant strain in preparation of a foot-and-mouth disease vaccine.
The invention provides a foot-and-mouth disease virus attenuated mutationAnd (3) mutating LbAUG in the foot-and-mouth disease virus leader protein gene into ATC or AAA. And (3) rescuing the foot-and-mouth disease virus mutant strain by using the recombinant plasmid with correct sequence determination. The indirect immunofluorescence test result shows that 2 strains of LbAUG modified O type foot-and-mouth disease virus (rV-O/ATC and rV-O/AAA) are successfully obtained. 2 strains of mutant viruses are continuously passaged on cells, and sequencing results show that 2 base mutations respectively introduced on the leader protein gene can stably exist, which indicates that the mutant strain has high genetic stability. The determination results of the plaque phenotype and the one-step growth curve show that the replication capacity of the rV-O/ATC and the rV-O/AAA on BHK-21 cells is similar to that of a parent strain, but the titer of the rV-O/ATC and the rV-O/AAA virus on PK-15 cells is obviously lower than that of the parent virus; while the control LbAUG was mutated to UGG or CUG, the rescued viruses rV-O/TGG and rV-O/CTG were also stably inherited, but their replication capacity was similar to the parental strain. The results of the pathogenicity measurement of the suckling mice show that the LD of rV-O/ATC and rV-O/AAA is compared with the parent strain50Obviously reduced, has obviously weakened pathogenicity on suckling mice, and LD of rV-O/TGG and rV-O/CTG50Slightly lower than the parent strain. The above results demonstrate that rV-O/ATC and rV-O/AAA have attenuated phenotypes. This shows that the attenuated virus strain provided by the invention is applied to organisms or spread to the environment, the proliferation of the virus strain is greatly limited, and the biological safety of the attenuated virus strain as a vaccine strain in production is greatly improved. Therefore, the foot-and-mouth disease virus O-type mutant strain prepared by the method can be used as a seed virus of the O-type foot-and-mouth disease vaccine with improved biological safety.
Drawings
FIG. 1 is a schematic diagram of the full-length genome structure of a LbAUG modified foot-and-mouth disease virus mutant;
FIG. 2 is an indirect immunofluorescence identification of rescued viruses, with scales of 10 μm for each plot;
FIG. 3 is a replication competent assay of rescued viruses, wherein A: rescue the plaque phenotype of the virus; b: one step growth curve for rescue of virus.
Detailed Description
The invention provides a foot-and-mouth disease virus attenuated mutant strain, which mutates LbAUG in a foot-and-mouth disease virus leader protein gene into ATC or AAA (see figure 1).
In the present invention, the nucleotide sequence of the leader protein mutated to ATC is preferably as shown in SEQ ID No. 2. The nucleotide sequence of the leader protein mutated to AAA is preferably shown in SEQ ID No. 4. The nucleotide sequence of the leader protein is preferably shown as SEQ ID No. 5.
In the invention, as LbAUG in the foot-and-mouth disease virus leader protein gene is a conserved codon, the two mutation schemes provided by the invention are suitable for all serotypes of foot-and-mouth disease viruses. The foot and mouth disease virus includes O, A, C, SAT1, SAT2, SAT3 and Asia17 serotypes. To illustrate that both mutation schemes can be used to prepare attenuated foot and mouth disease virus mutants, the present example specifically tests foot and mouth disease virus type O as parent virus, but this should not be construed as limiting the scope of the present invention. When the O type foot-and-mouth disease virus is used for mutation, the foot-and-mouth disease virus attenuated mutant strain is constructed by taking the genome of the OZK/93-08 virus strain as a framework and mutating LbAUG in the leader protein gene into ATC or AAA.
In the invention, the preparation method of the foot-and-mouth disease virus attenuated mutant strain preferably comprises the following steps of mutating LbAUG to obtain 2 kinds of recombinant plasmids with different mutations on the basis of full-length infectious clone plasmids of the foot-and-mouth disease virus strain OZK/93-08, and carrying out virus rescue by using the screened positive recombinant plasmids to obtain the foot-and-mouth disease virus mutant strain.
In the present invention, the mutation is preferably performed using a site-directed mutagenesis technique. Primers used for point mutations are shown in Table 1. Meanwhile, in order to verify whether mutation of the LbAUG site into any codon can influence the replication capacity of the virus, the invention also provides 2 contrast mutation schemes, LbAUG is mutated into TGG and CTG (shown in figure 1), the mutation method is the same as the method, and primers for point mutation are shown in table 1.
TABLE 1 Point mutation primer information
The amplification procedure of the primers is as follows: after pre-denaturation at 94 ℃ for 5min, 20min at 98 ℃ and 1min/1kb at 68 ℃ for 35 cycles, 10min at 72 ℃.
In the present invention, the selected positive recombinant plasmid is subjected to sequencing. The method for rescuing virus is preferably to linearize the correctly sequenced mutant recombinant plasmid with Not I in LipofectamineTM2000(Invitrogen), transferred into BSR/T7 cells for foot and mouth disease virus rescue, and the transfected cells were incubated at 37 ℃ for 48 hours, and after freezing, the supernatant was collected to obtain the rescued virus strain. After the virus strain is obtained, it is preferably verified. The verification result is that BHK-21 cells infected with the rescued virus can react with the foot-and-mouth disease virus MAb 3A24 antibody to generate red fluorescence, while normal BHK-21 cells do not generate fluorescence, which indicates that the synthesis of the foot-and-mouth disease virus 3A protein can be detected in the proliferation process of the rescued virus, and indicates that the rescued virus is indeed the foot-and-mouth disease virus.
In the present invention, the site of viral mutation is often subjected to various forms of back mutation and changes in viral replication ability due to gene mutation. Therefore, the invention carries out genetic stability detection and plaque test and growth curve determination on 2 rescued viruses respectively.
The genetic stability test result shows that the introduced mutant base can keep stable heredity in the passage of the foot-and-mouth disease virus mutant, and other base mutations do not occur in the passage of the rescued virus.
The plaque phenotype and the one-step growth curve are consistent to show that the replication capacity of the rescued viruses rV-O/ATC and rV-O/AAA on BHK-21 cells (cell lines for foot-and-mouth disease vaccine production) is similar to that of a parent strain, but the replication level in PK-15 cells (interferon totipotent cell lines) is reduced, which shows that 2 rescued viruses are likely to have reduced pathogenicity in animals sensitive to the foot-and-mouth disease virus.
In the present invention, since the leader protein is the most important viral protein determining the pathogenic mechanism of foot-and-mouth disease virus, it isFurther verifying the influence of LbAUG mutation on the pathogenicity of foot-and-mouth disease virus, and carrying out a virus-rescuing neonatal rat pathogenicity test. The results show that the LD of rV-O/ATC and rV-O/AAA compared to the parental strains50Obviously reduces the pathogenicity of the milk mouse, which shows that the strategy of utilizing the 2 nd initiation codon in the mutant leader protein gene can generate the weakened foot-and-mouth disease virus. Based on the above, the invention provides an application of the attenuated foot-and-mouth disease virus mutant strain in the production of inactivated foot-and-mouth disease vaccines.
The method for preparing the vaccine is not particularly limited in the present invention, and a method for preparing the vaccine known in the art may be used.
In the case of a virus escape aftosa vaccine manufacturing factory, such aftosa virus will be attenuated in proliferation in other cells than BHK-21 cells. Therefore, the method for modifying the initiation codon in the leader protein gene to accurately engineer the foot-and-mouth disease virus provides a safe platform for producing inactivated vaccine antigens.
The preparation and use of a attenuated foot-and-mouth disease virus mutant strain of the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Description of the sources of the materials
BHK-21 cells were purchased from Chinese veterinary institute under product catalog number BHK21F5620071213 and used to prepare viruses and calculate virus titers. BHK-21 cells are cells used for mass propagation of viruses in the production of foot and mouth disease vaccines, and this continuous cell line is an interferon-deficient cell line with limited ability to antagonize viral replication.
PK-15 was obtained by a conventional commercial purchase route. The PK-15 cell line was able to produce and respond to interferon, and its ability to antagonize viral replication was stronger than that of the BHK-21 cell line.
BSR/T7 cells were purchased from Germany, and are disclosed in the references "Generation of bone resorcinogenic viruses (BRSV) from cDNA: BRSVNS2 is a not-addressed instruction for video reproduction in the future, and the human RSV lead region as a functional BRSV gene promoter ", Journal of virology, 1999, 73 (1): 251-259, the cell is used for the rescue of foot-and-mouth disease virus.
A construction method of a full-length infectious cDNA clone plasmid pOZKF-Z1234 of the foot-and-mouth disease virus OZK/93-08 strain is disclosed (see patent CN102614507A), and the sequence of the L gene is shown as SEQ ID No. 5.
Example 1
1. Construction of mutant full-Length plasmids
In order to obtain the LbAUG mutant foot-and-mouth disease virus, LbAUG is point-mutated into AUC and AAA by a site-directed mutagenesis technology on the basis of the full-length infectious cDNA clone plasmid pOZKF-Z1234 of the foot-and-mouth disease virus OZK/93-08 strain, and plasmids modified by different LbAUG mutations are sequentially constructed. The primers used for point mutation are shown in Table 1, the PCR amplification system is shown in Table 2, and the amplification procedure is as follows: after pre-denaturation at 94 ℃ for 5min, 20min at 98 ℃ and 1min/1kb at 68 ℃ for 35 cycles, 10min at 72 ℃. Whether the resulting recombinant plasmid introduced the expected mutation was verified by sequencing.
TABLE 1 primers for Lb AUG Point mutation
TABLE 2 PCR reaction System
2. Rescue of mutant viruses
The correctly sequenced mutant plasmid was linearized with Not I and amplified in LipofectamineTM2000(Invitrogen), into BSR/T7 cells for foot and mouth disease virus rescue. Transfected cells were incubated at 37 ℃ for 48 hours and the supernatant collected after freezing.
The construction of the mutant full-length cDNA clone and the sequence determination result show that the full-length cDNA clone containing the expected mutation is obtained.
The mutation full-length cDNA clone plasmid and the Wild Type (WT) plasmid which are correctly sequenced are transfected into BSR/T7 cells, the cells are obviously changed by foot and mouth disease viruses after being transfected for 48 hours, the cell morphology of a control group is regular and has no change, the foot and mouth disease viruses are saved, and the saved viruses are respectively named rV-O/ATC and rV-O/AAA.
Comparative example 1
Full-length plasmids and rescued mutant viruses were constructed according to the method of example 1, except that the mutation scheme was varied, LbAUG was point-mutated to UGG (SEQ ID No.1), and primers for point mutation are shown in table 1. The mutation full-length cDNA clone plasmid with correct sequencing is transfected into BSR/T7 cells, the cells have obvious foot and mouth disease virus lesions after being transfected for 48 hours, the cell morphology of a control group is regular and has no lesions, the foot and mouth disease virus is saved, and the saved virus is named as rV-O/TGG.
Comparative example 2
Full-length plasmids and rescued mutant viruses were constructed as in example 1, except that the mutation scheme was varied, and LbAUG was point-mutated to CUG (SEQ ID No.3) and primers for point mutation are shown in table 1. The mutation full-length cDNA clone plasmid with correct sequencing is transfected into BSR/T7 cells, the cells have obvious foot and mouth disease virus lesions after being transfected for 48 hours, the cell morphology of a control group is regular and has no lesions, the foot and mouth disease virus is saved, and the saved virus is named as rV-O/CTG.
Example 2
Indirect immunofluorescence identification of rescued viruses
BHK-21 monolayer cells grow to about 70%, rescued parent virus (WT) and 4 foot-and-mouth disease virus mutants (rV-O/TGG, rV-O/ATC, rV-O/CTG and rV-O/AAA) are respectively inoculated with the infection dose of 10MOI, the cells infected by the virus are fixed in 4% paraformaldehyde after incubation for 4 hours at 37 ℃, rinsing with PBS buffer solution for 3 times, permeating with 0.5% Triton X-100(Sigma-Aldrich) for 15min, adding 1% BSA, placing in a 37 ℃ incubator, sealing for 1h, dripping 3A monoclonal antibody 3A24 of foot-and-mouth disease virus, incubating at 37 ℃ for 1h, rinsing with PBS for 3 times, adding FITC-labeled goat anti-mouse IgG antibody (Sigma), incubating at 37 ℃ for 1h, rinsing with PBS for 3 times, adding DAPI (Beyotime), incubating for 15min, rinsing with PBS for 3 times, and observing fluorescence in virus-infected cells by using a Laser Scanning Confocal Microscope (LSCM).
The identification result of the rescued virus is shown in figure 2, the BHK-21 cells infected with the rescued virus can react with the foot-and-mouth disease virus MAb 3A24 antibody to generate red fluorescence, and the normal BHK-21 cells do not generate fluorescence, which indicates that the synthesis of the foot-and-mouth disease virus 3A protein can be detected in the proliferation process of the rescued virus, and indicates that the rescued virus is indeed the foot-and-mouth disease virus.
Example 3
Genetic stability analysis of rescued viruses
The collected supernatant was serially passaged on BHK-21 cells at an inoculation dose of 0.1 times in volume, and RNA was extracted from the 3 rd, 6 th and 10 th virus generations, respectively, and then residual DNA in the RNA was removed by digestion with RNase-free DNase I (Qiagen), followed by RT-PCR amplification to rescue the leader protein gene of the virus, and the resulting fragment was sequenced using primer (LCF)5'-AGGTAACACGAGACACTC-3' (SEQ ID No.14) to identify the genetic stability of the introduced mutant base.
And (3) analyzing the genetic stability of the leader protein gene of the 3 rd, 6 th and 10 th generation viruses for rescuing the viruses, amplifying the leader protein gene by using the rescued mutant virus genome as a template and carrying out sequence determination, wherein the sequencing result shows that the introduced mutant base can keep stable inheritance in the passage of the foot-and-mouth disease virus mutant, and other base mutations are not generated in the passage of the rescued viruses.
Example 4
Plaque phenotype and one-step growth curve assay for rescued viruses
BHK-21 or PK-15 cells were plated in 6-well plates for 24h, washed with PBS, 200. mu.L of 10 Xvirus dilution was added to each well, and incubated at 37 ℃ in an incubator for 1h, during which time 1 gentle shaking was performed every 10min to prevent drying of the cells. Adding 2mL of Overlay culture solution into each hole, continuously culturing for 48h, sucking the culture solution, rinsing cells with PBS, adding 2mL of fixing solution (methanol: acetone ═ 1:1), fixing at 20 ℃ for 30min, removing the fixing solution, adding crystal violet staining solution, staining for 30min, and observing the plaque form of the rescued virus after rinsing the staining solution with clear water. Inoculating 1MOI virus-saving solution to BHK-21 or PK-15 cells, adsorbing for 1 hr, removing virus solution, adding fresh culture medium, respectively collecting samples at3, 6, 9 and 20 hr, and measuring TCID50To determine viral titers in samples at different time points.
The influence of LbAUG mutations on the replication capacity of foot-and-mouth disease virus was analyzed using plaque phenotype and one-step growth curves. As shown in FIG. 3A, in BHK-21 cells, the 4 mutant foot-and-mouth disease viruses did not show a great difference in plaque phenotype and plaque size compared to the parent virus. In PK-15 cells, the plaques of rV-O/TGG and rV-O/CTG were slightly reduced as compared with WT virus, but the plaques formed by rV-O/ATC and rV-O/AAA were significantly reduced. The growth curves of the mutant foot-and-mouth disease viruses showed (see fig. 3B) that there was no significant change in the titers of the mutant foot-and-mouth disease viruses on BHK-21 cells compared to the parental viruses, which is consistent with the results for the plaque phenotype. In PK-15 cells, the viral titers of rV-O/ATC and rV-O/AAA were significantly lower than those of the parental viruses, while those of rV-O/TGG and rV-O/CTG were similar to those of the parental viruses.
Example 5
Virus-rescued suckling mouse pathogenicity detection
10 of 4 mutant foot-and-mouth disease viruses-4～10-8The diluted solution was subcutaneously inoculated into 1-day-old suckling mice (200. mu.l/mouse, 5 mice/dilution) and the morbidity of the suckling mice was observed until day 7, and the death count of the suckling mice was counted and the LD was calculated according to the Reed-Muench method50。
The pathogenic results of suckling mice show that the LD of parent toxin50Is 10-7.3LD of rV-O/ATC and rV-O/AAA50Is 10-6.1And 10-6.3/. Compared with parent poison, the pathogenicity of rV-O/ATC and rV-O/AAA to suckling mouse is obviously weakened. And LD of rV-O/TGG and rV-O/CTG50Are respectively 10-6.8And 10-6.9rV-O/TGG and rV-O/CTG are slightly less virulent than the parental strain.
Taken together, these results indicate that the rescued mutant viruses rV-O/ATC and rV-O/AAA were attenuated.
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.
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<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences
<120> foot-and-mouth disease virus attenuated mutant strain and preparation method and application thereof
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 603
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgaacacga ctgactgttt tatcgctctg ttacacgttc tcagggagat taaagcactg 60
tttctgtcac gaacacaagg gaaatgggaa ttcacacttc acaacggtga aaagaaggtc 120
ttctacgcca gacccaacaa ccacgacaat tgctggttga acgccatcct ccaactgttc 180
aggtacgtcg acgaaccctt cttcgactgg gtctacgact cacctgagaa ccttactctt 240
gaggcgatca ggcgactcga agaaattact ggtcttgagc tacacgaggg tggaccaccc 300
gcccttgtcg tctggaacat taagcacttg ctctgcaccg gaatcggcac cgcttcgcgg 360
cctagcgagg tgtgtatggt ggacggtaca gacatgtgct tggccgactt ccacgctggt 420
atctttctga agggacaaga ccacgccgta ttcgcctgtg tcacctccga cgggtggtac 480
gcgattgacg acgaggattt ttacccgtgg acaccagacc cggctgacgt tttggttttt 540
gttccgtacg atcaagaacc acttaatgga gaatggaaag caaaggtcca gaagcggctt 600
aag 603
<210> 2
<211> 603
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgaacacga ctgactgttt tatcgctctg ttacacgttc tcagggagat taaagcactg 60
tttctgtcac gaacacaagg gaaaatcgaa ttcacacttc acaacggtga aaagaaggtc 120
ttctacgcca gacccaacaa ccacgacaat tgctggttga acgccatcct ccaactgttc 180
aggtacgtcg acgaaccctt cttcgactgg gtctacgact cacctgagaa ccttactctt 240
gaggcgatca ggcgactcga agaaattact ggtcttgagc tacacgaggg tggaccaccc 300
gcccttgtcg tctggaacat taagcacttg ctctgcaccg gaatcggcac cgcttcgcgg 360
cctagcgagg tgtgtatggt ggacggtaca gacatgtgct tggccgactt ccacgctggt 420
atctttctga agggacaaga ccacgccgta ttcgcctgtg tcacctccga cgggtggtac 480
gcgattgacg acgaggattt ttacccgtgg acaccagacc cggctgacgt tttggttttt 540
gttccgtacg atcaagaacc acttaatgga gaatggaaag caaaggtcca gaagcggctt 600
aag 603
<210> 3
<211> 603
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgaacacga ctgactgttt tatcgctctg ttacacgttc tcagggagat taaagcactg 60
tttctgtcac gaacacaagg gaaactggaa ttcacacttc acaacggtga aaagaaggtc 120
ttctacgcca gacccaacaa ccacgacaat tgctggttga acgccatcct ccaactgttc 180
aggtacgtcg acgaaccctt cttcgactgg gtctacgact cacctgagaa ccttactctt 240
gaggcgatca ggcgactcga agaaattact ggtcttgagc tacacgaggg tggaccaccc 300
gcccttgtcg tctggaacat taagcacttg ctctgcaccg gaatcggcac cgcttcgcgg 360
cctagcgagg tgtgtatggt ggacggtaca gacatgtgct tggccgactt ccacgctggt 420
atctttctga agggacaaga ccacgccgta ttcgcctgtg tcacctccga cgggtggtac 480
gcgattgacg acgaggattt ttacccgtgg acaccagacc cggctgacgt tttggttttt 540
gttccgtacg atcaagaacc acttaatgga gaatggaaag caaaggtcca gaagcggctt 600
aag 603
<210> 4
<211> 603
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atgaacacga ctgactgttt tatcgctctg ttacacgttc tcagggagat taaagcactg 60
tttctgtcac gaacacaagg gaaaaaagaa ttcacacttc acaacggtga aaagaaggtc 120
ttctacgcca gacccaacaa ccacgacaat tgctggttga acgccatcct ccaactgttc 180
aggtacgtcg acgaaccctt cttcgactgg gtctacgact cacctgagaa ccttactctt 240
gaggcgatca ggcgactcga agaaattact ggtcttgagc tacacgaggg tggaccaccc 300
gcccttgtcg tctggaacat taagcacttg ctctgcaccg gaatcggcac cgcttcgcgg 360
cctagcgagg tgtgtatggt ggacggtaca gacatgtgct tggccgactt ccacgctggt 420
atctttctga agggacaaga ccacgccgta ttcgcctgtg tcacctccga cgggtggtac 480
gcgattgacg acgaggattt ttacccgtgg acaccagacc cggctgacgt tttggttttt 540
gttccgtacg atcaagaacc acttaatgga gaatggaaag caaaggtcca gaagcggctt 600
aag 603
<210> 5
<211> 603
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atgaacacga ctgactgttt tatcgctctg ttacacgttc tcagggagat taaagcactg 60
tttctgtcac gaacacaagg gaaaatggaa ttcacacttc acaacggtga aaagaaggtc 120
ttctacgcca gacccaacaa ccacgacaat tgctggttga acgccatcct ccaactgttc 180
aggtacgtcg acgaaccctt cttcgactgg gtctacgact cacctgagaa ccttactctt 240
gaggcgatca ggcgactcga agaaattact ggtcttgagc tacacgaggg tggaccaccc 300
gcccttgtcg tctggaacat taagcacttg ctctgcaccg gaatcggcac cgcttcgcgg 360
cctagcgagg tgtgtatggt ggacggtaca gacatgtgct tggccgactt ccacgctggt 420
atctttctga agggacaaga ccacgccgta ttcgcctgtg tcacctccga cgggtggtac 480
gcgattgacg acgaggattt ttacccgtgg acaccagacc cggctgacgt tttggttttt 540
gttccgtacg atcaagaacc acttaatgga gaatggaaag caaaggtcca gaagcggctt 600
aag 603
<210> 6
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tcacgaacac aagggaaatg ggaattcaca cttcacaac 39
<210> 7
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gttgtgaagt gtgaattccc atttcccttg tgttcgtga 39
<210> 8
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tcacgaacac aagggaaaat cgaattcaca cttcacaac 39
<210> 9
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gttgtgaagt gtgaattcga ttttcccttg tgttcgtga 39
<210> 10
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
tcacgaacac aagggaaact ggaattcaca cttcacaac 39
<210> 11
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gttgtgaagt gtgaattcca gtttcccttg tgttcgtga 39
<210> 12
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tcacgaacac aagggaaaaa agaattcaca cttcacaac 39
<210> 13
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gttgtgaagt gtgaattctt ttttcccttg tgttcgtga 39
<210> 14
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
aggtaacacg agacactc 18
Claims (10)
1. The attenuated foot-and-mouth disease virus mutant is characterized in that LbAUG is mutated into ATC or AAA on the basis of a leader protein of foot-and-mouth disease virus.
2. The attenuated foot-and-mouth disease virus mutant strain of claim 1, wherein the nucleotide sequence of the leader protein mutated to ATC is shown in SEQ ID No. 2.
3. The attenuated foot-and-mouth disease virus mutant strain of claim 1, wherein the nucleotide sequence of the leader protein mutated to AAA is shown in SEQ ID No. 4.
4. The attenuated foot-and-mouth disease virus mutant strain of claim 1, wherein the nucleotide sequence of the leader protein is shown as SEQ ID No. 5.
5. The attenuated mutant strain of foot-and-mouth disease virus of any one of claims 1 to 4, wherein the foot-and-mouth disease virus comprises O, A, C, SAT1, SAT2, SAT3 and Asia17 serotypes of foot-and-mouth disease virus.
6. The attenuated foot and mouth disease virus mutant of claim 5, wherein the attenuated foot and mouth disease virus strain O is obtained by mutating LbAUG of leader protein to ATC or AAA based on OZK/93-08 virus strain.
7. The method for preparing the attenuated mutant strain of foot-and-mouth disease virus of any one of claims 1 to 6, comprising the steps of:
point mutation is carried out on the recombinant plasmid containing the full-length cDNA of the foot-and-mouth disease virus to obtain a mutated recombinant plasmid, and the mutated recombinant plasmid is infected with cells to be rescued to obtain a rescued virus strain.
8. The method of claim 7, wherein the primers for point mutation are ATC-F/ATC-R primer pair and AAA-F/AAA-R primer pair;
the nucleotide sequence of the ATC-F is shown as SEQ ID No. 8;
the nucleotide sequence of the ATC-R is shown as SEQ ID No. 9;
the nucleotide sequence of the AAA-F is shown as SEQ ID No. 12;
the nucleotide sequence of the AAA-R is shown as SEQ ID No. 13.
9. The method of claim 7, wherein the point mutation is performed by PCR amplification at 94 ℃ for 5min, 98 ℃ for 20min, 68 ℃ for 1min/1kb, and 35 cycles at 72 ℃ for 10 min.
10. The use of the attenuated foot-and-mouth disease virus mutant strain of any one of claims 1 to 6 in the preparation of a foot-and-mouth disease vaccine.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117210501A (en) * | 2023-09-13 | 2023-12-12 | 中国农业科学院兰州兽医研究所 | Construction of recombinant foot-and-mouth disease virus strain with foot-and-mouth disease virus 2B protein immunosuppression site mutation |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117210501A (en) * | 2023-09-13 | 2023-12-12 | 中国农业科学院兰州兽医研究所 | Construction of recombinant foot-and-mouth disease virus strain with foot-and-mouth disease virus 2B protein immunosuppression site mutation |
| CN117210501B (en) * | 2023-09-13 | 2024-06-07 | 中国农业科学院兰州兽医研究所 | Construction of recombinant foot-and-mouth disease virus strain with foot-and-mouth disease virus 2B protein immunosuppression site mutation |
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