O-type foot-and-mouth disease virus strain containing VP1 N133K mutation site and construction method of O-type foot-and-mouth disease virus strain

Abstract

The invention belongs to the field of veterinary biological products, and particularly relates to an O-type foot-and-mouth disease strain containing a VP1 N133K mutation site and a construction method of the O-type foot-and-mouth disease strain. The invention provides an attenuated O-type foot and mouth disease virus mutant strain. Asparagine at the 133rd site of VP1 protein is mutated into lysine. The amino acid sequence of the mutated VP1 protein is as shown in SEQ ID NO: 2. The invention also provides a construction method of the attenuated O-type foot-and-mouth disease virus mutant strain, wherein N133K mutation is introduced by adopting a reverse genetics technology. The FMDV VP1 N133K mutant strain constructed by the method disclosed by the invention is beneficial to research on an FMDV attenuating mechanism. In addition, by reversely introducing VP1 N133K mutation, conservative epitopes can be screened or multivalent vaccines can be designed, and the cross protection capability is improved.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological products, specifically relating to an O-type foot-and-mouth disease virus strain containing the VP1 N133K mutation site and its construction method. Background Technology

[0002] Foot-and-mouth disease (FMD) is a highly contagious and deadly disease caused by the foot-and-mouth disease virus (FMDV), infecting major livestock such as pigs, cattle, and sheep, as well as wild cloven-hoofed animals. The disease spreads rapidly, is highly infectious, and has an extremely high morbidity rate. The World Organisation for Animal Health (OIE) lists it as a reportable animal disease. Outbreaks and epidemics of FMD severely damage livestock productivity and the quality of livestock products, impacting international trade in livestock and their products, and causing enormous economic losses to livestock farming in affected areas. Therefore, effective prevention and control of FMD is of significant strategic importance for the sustainable and healthy development of global livestock farming.

[0003] Currently, the most important means of controlling and preventing FMD is immunization with inactivated vaccines. Inactivated vaccines play a very important role in the prevention and eradication of FMD. However, there are many FMD serotypes, and due to the lack of proofreading function of RNA-dependent RNA polymerase (RDRP), the virus genome is highly variable. Furthermore, immune pressure can also promote the development of FMDV in the direction of vaccine escape. In recent years, mutant strains have been able to evade vaccine immunization.

[0004] The VP1 protein of foot-and-mouth disease virus (FMDV) is a major antigenic target, and its GH loop (residues 141-160) and C-terminal linear epitope (residues 200-213) are the core recognition regions for neutralizing antibodies. Studies have shown that amino acid mutations in VP1 (such as mutations at key sites in the GH loop) may affect antibody binding ability by altering epitope conformation or charge distribution, leading to immune escape. Summary of the Invention

[0005] This invention relates to an O-type foot-and-mouth disease virus (FMDV) mutant strain constructed using reverse genetics technology. The mutant strain has an asparagine (N) mutation at position 133 of the VP1 protein to lysine (K). This mutation significantly reduces the viral titer and pathogenicity, and can be used to study the mechanism of viral attenuation.

[0006] The present invention specifically adopts the following technical solution: This invention provides an attenuated type O foot-and-mouth disease virus mutant strain, which is obtained by mutating the asparagine at position 133 of the VP1 protein of a wild type O foot-and-mouth disease virus strain to lysine. The wild type O foot-and-mouth disease virus strain is FMDV / O / 17002, with the accession number CCTCC NO:V202060. The amino acid sequence of the mutated VP1 protein is shown in SEQ ID NO:2. The nucleotide sequence encoding the mutated VP1 protein is shown in SEQ ID NO:4.

[0007] This invention also provides a method for constructing the aforementioned attenuated type O foot-and-mouth disease virus mutant strain, which involves introducing the N133K mutation using reverse genetics technology. This invention introduces the VP1N133K mutation into the infectious clonal backbone of the FMDV strain FMDV / O / 17002 to construct the mutant strain rVP1-N133K. Specific steps include: Step 1: Primers were designed to introduce the mutation site into a half-length plasmid containing the VP1 gene of the wild-type O foot-and-mouth disease virus strain, resulting in a mutant half-length plasmid. The wild-type O foot-and-mouth disease virus strain is FMDV / O / 17002, and the amino acid sequence of the VP1 protein of this wild-type O foot-and-mouth disease virus strain is shown in SEQ ID NO:1. The half-length plasmid was obtained by ligating the L-VP4-VP3-VP2-VP1 nucleotide sequence of the wild-type O foot-and-mouth disease virus strain to the vector pcDNA3.1. The mutation involved changing the base AAC encoding the 133rd amino acid of the VP1 protein to AAA.

[0008] Step 2: The mutant half-length plasmid obtained in Step 1 and the full-length plasmid containing the full-length gene of the wild-type O foot-and-mouth disease virus strain are ligated after double enzyme digestion to obtain the mutant full-length plasmid. The full-length plasmid is obtained by ligating the nucleotides of the full-length gene of the wild-type O foot-and-mouth disease virus strain to the vector pcDNA3.1. The double enzyme digestion is performed using... Sbf I and Pac Ⅰ Restriction endonucleases.

[0009] Step 3: Transfect the full-length mutant plasmid obtained in Step 2 into BHK-21 cells to rescue the virus and obtain the recombinant virus rVP1-N133K, which is the attenuated type O foot-and-mouth disease virus mutant strain.

[0010] This invention also provides the application of the mutant strain in the study of the attenuation mechanism of foot-and-mouth disease virus.

[0011] In addition, the present invention provides the application of the mutant strain in the preparation of foot-and-mouth disease virus vaccine.

[0012] The beneficial effects of this invention are as follows: 1. The mutant strain constructed in this invention has a reduced viral titer: By comparing the viral titers of rVP1-N133K (recombinant foot-and-mouth disease virus mutant strain) and rWT (foot-and-mouth disease wild strain), it was found that VP1 N133K significantly reduced the viral titer of FMDV.

[0013] 2. This invention found through neonatal mouse challenge experiments that the VP1 N133K mutation reduces the lethality of FMDV in neonatal mice.

[0014] 3. Attenuation of the mutant strain constructed in this invention: The FMDV VP1 N133K mutant strain constructed in this invention is helpful for studying the mechanism of FMDV attenuation.

[0015] 4. Application of the mutant strain constructed in this invention in vaccine optimization: By introducing the VP1 N133K mutation in reverse, conserved epitopes can be screened or multivalent vaccines can be designed to improve cross-protection capabilities. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the FMDV VP1 mutation site of the present invention.

[0017] Figure 2 This is an electrophoresis image of the enzyme digestion products of the recombinant plasmid. M is the DNA marker of DL 15000, 1 is the enzyme digestion band of the original full-length plasmid pSK-HB2017P1, and 2 and 3 are the enzyme digestion bands of the 1st and 2nd mutant plasmids of pSK-HB2017P1-VP1-N133K.

[0018] Figure 3 Comparison of cell morphology 24 h after transfection of the original plasmid pSK-HB2017P1 and the mutant plasmid pSK-HB2017P1-VP1-N133K into BHK-21 cells.

[0019] Figure 4 Comparison of indirect immunofluorescence results between rWT and rVP1-N133K recombinant strains (using FMDV-3D polyclonal antibody as primary antibody). MOCK indicates transfection reagent control.

[0020] Figure 5 Comparison of viral titers for rWT and rVP1-N133K.

[0021] Figure 6 Comparison of plaque phenotypes between rWT and rVP1-N133K recombinant strains.

[0022] Figure 7 Comparison of multi-step growth curves for rWT and rVP1-N133K recombinant strains.

[0023] Figure 8Comparison of pathogenicity of rWT and rVP1-N133K infection in 3-day-old Kunming suckling mice. A represents LD50. 50 The results show that B represents the survival curves of suckling mice infected with rVP1-N133K and rWT, and 8C represents the viral load in organs of suckling mice after infection with rVP1-N133K or rWT.

[0024] Preserved biological material: FMDV / O / 17002; Date of preservation: September 17, 2020; Name of depositary institution: China Center for Type Culture Collection; Accession number: CCTCC NO:V202060; Address of the depositary institution: Wuhan University, Wuhan, China; Classification and naming: Foot-and-mouth disease virus strain O / 17002 (FMDV / O / 17002 strain). Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments of this invention will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0026] CutSmart, as described in the embodiments Sbf I. Pac I. All were purchased from New England Biotechnology (Beijing) Co., Ltd. Three-day-old Kunming suckling mice were purchased from the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0027] Example 1 1. Construction of type O foot-and-mouth disease virus strain rVP1-N133K containing the VP1 N133K mutation site FMDV VP1 mutation sites such as Figure 1 As shown. The amino acid sequences of the original VP1 and the mutant VP1 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0028] 1.1 Construction of recombinant plasmids Using the half-length plasmid pSK-HB2017P1 (pSK-HB207P1 is obtained by ligating the nucleotide sequence of L-VP4-VP3-VP2-VP1 from FMDV / O / 17002 (accession number: CCTCC NO:V202060) to the vector pcDNA3.1) as a template, the VP1 gene fragment containing the N133K mutation was amplified by PCR. The PCR amplification system was as follows: 10 μL of 2×M5-Mutase Mix (Beijing Polymer Biotechnology Co., Ltd.), 0.4 μL of forward mutation primer (10 μM), 0.4 μL of reverse mutation primer (10 μM), 1-10 ng of half-length plasmid pSK-HB2017P1, and ddH2O to a final volume of 20 μL. The PCR program was as follows: 95℃ for 2 min, (94℃ for 25 s, 60℃ for 25 s, 68℃ for 3 min, 25 cycles), 68℃ for 5 min. Primer sequences used: Forward mutation primer: 5'-AACGGGAA A TGCAAATACACCGGGGGGCC-3' (containing AAC→AAA mutation); reverse mutation primer: 5'-TATTTGCA T TTCCCGTTGTAAACGGTGGCC-3'.

[0029] Take 8 μL of the above PCR product, add 1 μL of 10×M5-Remase Buffer and 1 μL of M5 Remase enzyme (Beijing Polymer Biotechnology Co., Ltd.), incubate at 37℃ for 1 h to remove the original plasmid. Transform the PCR product after removing the original plasmid into E. coli DH5α, plate it, pick single colonies, extract the plasmid, and obtain the mutant plasmid pSK-Z123XJLP1-VP1-N133K.

[0030] use Sbf I and Pac I. The recombinant mutant plasmid pSK-HB2017P1-VP1-N133K and the full-length plasmid pOHB2017 (pSK-HB2017 is obtained by ligating the full-length nucleotide sequence of FMDV / O / 17002 into the vector pcDNA3.1) were double-digested with enzymes. The double-digestion system was: CutSmart 5 μL, plasmid 1 μg, Sbf Ⅰ1μL, Pac I. Add 1 μL of ddH2O to a final volume of 50 μL, and react at 37℃ for 20 min. After double digestion, perform agarose gel electrophoresis on the digested products, cut the gel, and recover the 6000 bp target band after digestion with pSK-HB2017P1-VP1-N133K and the 8000 bp target band after digestion with the full-length plasmid pOHB2017. Ligate these bands to obtain the recombinant plasmid pOHB2017-VP1-N133K.

[0031] The recombinant plasmid pOHB2017-VP1-N133K was used Sbf I and Pac I. Double enzyme digestion was performed for identification, and plasmids that were correctly identified were sent to Qingke Biotechnology Co., Ltd. for sequencing identification.

[0032] The results showed that the restriction enzyme digestion bands of the recombinant plasmid pOHB2017-VP1-N133K were consistent with those of the original full-length plasmid pSK-HB2017P1, both yielding two bands of 8000bp and 6000bp, consistent with the expected size (see...). Figure 2 Sequencing results of the recombinant plasmid pOHB2017-VP1-N133K also showed that the VP1 N133K mutation was successfully introduced into the 8000bp target band in the full-length plasmid pOHB2017.

[0033] The nucleotide sequences of the original VP1 and the mutant VP1 are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0034] 1.2 Rescue of Recombinant Viruses BHK-21 monolayers cultured under standard conditions were transferred to T25 cell flasks. Once the cell density reached 70%-80%, the full-length plasmid pOHB2017 and the recombinant plasmid pOHB2017-VP1-N133K were transfected into BHK-21 cells using the transfection reagent jetPRIME (purchased from Polyplus Transfection®) (refer to the instruction manual for specific procedures). Four hours after transfection, the transfection medium was replaced with DMEM containing 2% FBS, and the cells were continuously cultured at 37°C in a 5% CO2 incubator, observing for cytopathic effects. Seventy-two hours after transfection, the cells were harvested, subjected to two freeze-thaw cycles, continuously passaged in BHK-21 cells, and stored at -80°C for future use.

[0035] The results showed that both the original full-length plasmid pOHB2017 and the recombinant plasmid pOHB2017-VP1-N133K exhibited typical cytopathic effects (CPE) in BHK-21 cells 24 hours after transfection. Cells in the transfection control (MOCK) showed a fibrous distribution and were nearly confluent, while the diseased cells became larger, rounder, and grape-like in shape, with a large number dying (see [link to original text]). Figure 3 The genetically engineered viruses rescued after transfecting BHK-21 cells with the original full-length plasmid pOHB2017 and the recombinant plasmid pOHB2017-VP1-N133K were named rWT and rVP1-N133K, respectively.

[0036] 1.3 PCR identification of recombinant virus Viral supernatants from the fourth generation of rWT and rVP1-N133K were collected and viral RNA was extracted using the Omega Viral RNA Kit (purchased from Omega Bio-Tek). RT-PCR amplification was performed using primers VP1-3253-F: 5'-CGCTCGGCAACAGACCAC-3'; VP1-3907-R: 5'-GTTCAAGGACTGTTTCACAGGTG-3' (RT-PCR kit purchased from Takara, RR055A). The PCR amplification products of VP1 were obtained and sent to Qingke Biotechnology Co., Ltd. for sequencing identification to verify the correctness of the recombinant virus. The RT-PCR reaction system was as follows: PrimeScript 1 Step EnzymeMix 2 μL, 2×1 Step Buffer 25 μL, VP1-3253-F (20 μM) 1 μL, VP1-3907-R (20 μM) 1 μL, template RNA 1 μg, and RNase-free dH2O to a final volume of 50 μL.

[0037] The RT-PCR reaction program was as follows: 50℃ for 30 min; 94℃ for 2 min; 94℃ for 30 sec, 60℃ for 30 sec, 72℃ for 90 sec, for 30 cycles.

[0038] Sequencing results showed that the rVP1-N133K recombinant virus contained the VP1 N133K mutation, indicating that the present invention successfully constructed a recombinant FMDV containing VP1 N133K substitution. The sequencing sequences of rWT and rVP1-N133K VP1 are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively. It should be noted that the sequencing sequences are partial sequences of the PCR amplification products of the above-mentioned VP1.

[0039] 1.4 Indirect immunofluorescence identification of recombinant virus When BHK-21 monolayer cells in six-well plates reached a confluence density of 70%-80%, they were inoculated with parental virus rWT and recombinant virus rVP1-N133K, respectively. The expression of specific proteins in the virus-inoculated cells was detected using indirect immunofluorescence. The specific steps were as follows: S1: After inoculating cells with the virus for 12 hours, discard the culture medium, wash three times with PBS, add pre-cooled anhydrous ethanol, and fix at -20℃ for 30 minutes. S2: Wash 3 times with PBS, add 5% BSA and block at room temperature for 1 hour; S3: Wash 3 times with PBS, add 1:500 diluted polyclonal antibody against FDMV nonstructural protein 3D (FMDV-3D polyclonal antibody, which is obtained by expressing and purifying FMDV 3D protein from E. coli, and collecting serum after immunizing 8-week-old New Zealand rabbits twice, which is the rabbit anti-polyclonal antibody against FMDV 3D), and incubate at 37°C for 1 hour; S4: Wash 3 times with PBS, add FITC-labeled goat anti-rabbit IgG secondary antibody diluted 1:1000, and incubate at 37°C for 1 hour; S5: Wash 3 times with PBS, add DAPI diluted 1:1000, incubate at room temperature for 10 min, wash 3 times with PBS to remove excess DAPI, and take pictures under a fluorescence microscope.

[0040] The results showed that BHK-21 cells inoculated with parental virus rWT and recombinant virus rVP1-N133K specifically bound to the FMDV-3D polyclonal antibody and exhibited green fluorescence, while control cells showed no fluorescence (see [link to study]). Figure 4 This indicates that the present invention successfully constructed recombinant FMDV, and the mutation of VP1 N133K did not affect the rescue of infectious FMDV.

[0041] 2. Recombinant virus titer, plaque phenotype, and multi-step growth curve The fourth-generation parental virus rWT and recombinant virus rVP1-N133K were serially diluted 10-fold, and then the different dilutions of virus were inoculated onto 96-well BHK-21 monolayer cells. After 72 hours, the cytopathic effects wells were observed and counted, and the viral titers of parental virus rWT and recombinant virus rVP1-N133K were calculated using the Reed-Muench method.

[0042] The fourth-generation parental virus rWT and recombinant virus rVP1-N133K were serially diluted 10-fold. Then, the different dilutions of virus were seeded into 6-well plates of BHK-21 or PK-15 cells that had formed a confluent monolayer, 200 μL / well. The cells were incubated at 37°C in a 5% CO2 incubator for 1 h, with shaking every 10 min to prevent the cells from drying out. After incubation, the virus solution was discarded, and 2 mL of astragalus gum mixture (MEM and 1.2% astragalus gum mixed 1:1, with 1% FBS added) was added. The cells were incubated at 37°C for 48 h. After incubation, the culture medium was discarded, and the cells were gently washed 3 times with PBS. Pre-cooled anhydrous ethanol was added, and the cells were fixed at -20°C for 30 min. The anhydrous ethanol was discarded, and the cells were gently washed 3 times with PBS. The cells were stained overnight with 1% crystal violet, and washed 5 times with PBS. The viral plaques were observed, and the viral plaque-forming units (PFU / mL) were calculated.

[0043] Fourth-generation parental virus rWT and recombinant virus rVP1-N133K were used to infect 6-well monolayers of BHK-21 or PK-15 cells with an MOI of 0.1. After 1 hour of adsorption, the virus solution was removed, and the cells were washed three times with PBS. DMEM was added, and the cells were incubated at 37°C in a 5% CO2 incubator. The supernatant was collected at 4, 8, 12, 16, 20, and 24 hours after inoculation, and the viral titer (TCID) was measured in 96-well monolayers of BHK-21 (or PK-15) cells. 50 ), and plotted the multi-step growth curve of the virus.

[0044] The results showed that the viral titer of the recombinant virus rVP1-N133K was significantly lower than that of the parental virus rWT (see [link to results]). Figure 5 The plaque phenotypes of the parental virus rWT and the recombinant virus rVP1-N133K are similar (see...). Figure 6 The multi-step growth curves show that VP1 N133K enhances the replication ability of FMDV in the early stage, but weakens it compared to rWT in the later stage of replication (see...). Figure 7 This indicates that the VP1 N133K mutation affects FMDV replication.

[0045] 3. LD50 determination and challenge test in suckling rats The parental virus rWT and recombinant virus rVP1-N133K were serially diluted 10-fold and used to infect 3-day-old Kunming suckling mice (n=8), with 0.2 mL per mouse. The mortality of suckling mice within 72 hours after infection was recorded, and the LD50 of the parental virus rWT and recombinant virus rVP1-N133K was calculated using the Reed-Muench method.

[0046] The parental virus rWT and recombinant virus rVP1-N133K were diluted to 20 LD50 / 0.2 mL and used to infect 3-day-old Kunming suckling mice (n=12). The mortality of suckling mice within 96 hours after infection was recorded and growth curves were plotted.

[0047] The results showed that VP1 N133K significantly reduced the LD50 of FMDV, and at the same dose infection level, survival time and mortality were significantly lower than rWT (see [reference needed]). Figure 8 ). Figure 8 A is LD 50 The measurement results showed that the LD50 of rVP1-N133K was... 50 It is significantly reduced compared to rWT. Figure 8 B represents the survival curves of suckling mice infected with rVP1-N133K and rWT. The survival time and mortality rate of rVP1-N133K were significantly reduced. Figure 8 C represents the viral load in organs of suckling mice infected with rVP1-N133K or rWT. The viral load in the heart, lungs, and muscles of suckling mice infected with rVP1-N133K was significantly reduced.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An attenuated type O foot-and-mouth disease virus mutant strain, characterized in that, It involves mutating asparagine to lysine at position 133 of the VP1 protein of wild-type O foot-and-mouth disease virus strain.

2. The attenuated type O foot-and-mouth disease virus mutant strain according to claim 1, characterized in that, The wild type O foot-and-mouth disease virus strain is FMDV / O / 17002, and its preservation number is CCTCC NO:V202060.

3. The attenuated type O foot-and-mouth disease virus mutant strain according to claim 1, characterized in that, The nucleotide sequence encoding the mutated VP1 protein is shown in SEQ ID NO:

4.

4. The method for constructing an attenuated type O foot-and-mouth disease virus mutant strain as described in claim 1, characterized in that, Mutation sites are introduced using reverse genetics techniques.

5. The construction method according to claim 4, characterized in that, Includes the following steps: Step 1: Design primers to introduce the mutation site into a half-length plasmid containing the VP1 gene of wild-type O foot-and-mouth disease virus strain to obtain the mutant half-length plasmid. Step 2: The mutant half-length plasmid obtained in Step 1 and the full-length plasmid containing the full-length gene of wild type O foot-and-mouth disease virus strain are ligated after double enzyme digestion to obtain the mutant full-length plasmid. Step 3: Transfect the full-length mutant plasmid obtained in Step 2 into BHK-21 cells to rescue the virus and obtain an attenuated type O foot-and-mouth disease virus mutant strain.

6. The construction method according to claim 5, characterized in that, In step 1, the wild type O foot-and-mouth disease virus strain is FMDV / O / 17002 strain, and the preservation number of the wild type O foot-and-mouth disease virus strain is CCTCC NO:V202060; the amino acid sequence of the VP1 protein of the wild type O foot-and-mouth disease virus strain is shown in SEQ ID NO:

1.

7. The construction method according to claim 5, characterized in that, In step 1, the mutation is to change the base AAC encoding the 133rd amino acid of the VP1 protein to AAA.

8. The construction method according to claim 5, characterized in that, In step 1, the half-length plasmid is obtained by ligating the nucleotides of L-VP4-VP3-VP2-VP1 of the wild-type O foot-and-mouth disease virus strain to the vector pcDNA3.1; in step 2, the full-length plasmid is obtained by ligating the nucleotides of the full-length gene of the wild-type O foot-and-mouth disease virus strain to the vector pcDNA3.

1.

9. The application of the mutant strain according to any one of claims 1-3 in the study of the attenuation mechanism of foot-and-mouth disease virus.

10. The use of the mutant strain according to any one of claims 1-3 in the preparation of foot-and-mouth disease virus vaccine.

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