Porcine rotavirus attenuated strain, construction method and application thereof

Abstract

The application discloses a porcine rotavirus attenuated strain and a construction method and application thereof, and belongs to the technical field of genetic engineering. The porcine rotavirus attenuated strain is obtained by mutating a VP7 protein C207 site of a porcine rotavirus NJ2012 strain into S207, and the amino acid sequence of the mutated VP7 protein is shown as SEQ ID NO. 2. The porcine rotavirus attenuated strain is obtained by a reverse genetic operation system, and the recombinant infectious cloning plasmid carrying the VP7-C207S mutant protein coding gene is transfected into host cells to be rescued. The porcine rotavirus attenuated strain constructed by the application has the characteristics of weakened replication ability, reduced pathogenicity, and high-level induced humoral immune response, and has good potential as a candidate strain of a live attenuated PoRV vaccine.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to an attenuated strain of porcine rotavirus, its construction method, and its application. Background Technology

[0002] Porcine rotavirus (PoRV) mainly affects piglets during the lactation period and around weaning. Infection can cause vomiting, acute watery diarrhea, severe dehydration, and growth retardation in piglets. In my country, the positive detection rate of clinical samples varies from 16.8% to 72.77% in different regions and under different farming models, and the epidemiological characteristics show significant regional differences and seasonal fluctuations.

[0003] Vaccination is the most effective means of controlling PoRV infection and reducing economic losses associated with its resulting piglet diarrhea. Reverse genetics, through in vitro targeted manipulation of the viral genome, enables the artificial rescue of infectious viruses and is one of the core technologies for elucidating the pathogenesis of PoRV and designing and developing novel genetically engineered vaccines. VP7, a glycoprotein, forms the smooth outer surface of its capsid in the form of a homotrimer. Its protein sequence contains three structurally well-defined antigenic epitopes, namely 7-1a, 7-1b, and 7-2, which are the core targets of neutralizing antibodies. VP7 can stimulate the production of IgG and generate a protective immune response, making it an ideal target for vaccine and antiviral therapy development.

[0004] Palmitoylation, a reversible post-translational lipid modification of proteins, refers to the process by which a 16-carbon fatty acid (C16:0) covalently binds to one or more cysteine ​​(Cys) residues via a thioester bond to modify a protein. This modification can regulate the hydrophobicity, stability, intracellular transport, subcellular localization, and membrane binding properties of substrate proteins, thus determining the biological characteristics and functional effects of different intracellular proteins. Existing research shows that palmitoylation modification can guide viral proteins to target the lipid raft region of the cell membrane, providing sites for viral assembly and budding release; the absence of modification leads to abnormal viral particle assembly, resulting in a significant reduction in the infectivity of progeny viruses. The core research and development goal of live attenuated vaccines is to precisely and controllably eliminate the pathogenicity of pathogens while fully preserving their antigenic structure and immunogenicity. Targeted modification of palmitoylation sites perfectly meets this core requirement and represents a novel and rational attenuation technology pathway beyond traditional continuous passage attenuation. For example, the palmitoylated mutant strain NS2A / C221S of the NS2A protein of Japanese encephalitis virus exhibits significantly reduced replication capacity in vitro and cells, a substantial decrease in pathogenicity in mice, while maintaining good genetic stability and showing no risk of reversion. However, as a non-enveloped double-stranded RNA virus, there are no reports in the literature regarding whether PoRV's viral protein undergoes palmitoylation modification. Summary of the Invention

[0005] The purpose of this invention is to provide an attenuated porcine rotavirus strain, its construction method, and its application, in order to solve the problems existing in the prior art. Using the reverse genetic system of the previously established G9 type PoRV strain, a recombinant virus with a VP7-C207 site mutation was constructed. This recombinant virus has characteristics such as weakened replication ability, reduced pathogenicity, and the ability to induce a high level of humoral immune response, and has good potential as a candidate strain for a live attenuated PoRV vaccine.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides an attenuated porcine rotavirus strain, which is obtained by mutating the C207 site of the VP7 protein of the porcine rotavirus NJ2012 strain to S207. The amino acid sequence of the mutated VP7 protein is shown in SEQ ID NO.2.

[0007] The present invention also provides a recombinant infectious cloning plasmid for preparing the attenuated porcine rotavirus strain, wherein the recombinant infectious cloning plasmid contains a gene encoding the VP7-C207S mutant protein of the porcine rotavirus strain NJ2012, and the amino acid sequence of the VP7-C207S mutant protein of the porcine rotavirus strain NJ2012 is shown in SEQ ID NO.2.

[0008] Preferably, the nucleotide sequence of the gene is as shown in SEQ ID NO.1.

[0009] The present invention also provides a method for constructing the attenuated porcine rotavirus strain, comprising obtaining the strain by transfecting the recombinant infectious clonal plasmid into a host cell using a reverse genetics system.

[0010] Preferably, the construction method includes the following steps: (1) Using the cDNA of porcine rotavirus NJ2012 strain as a template, the VP7 gene fragment carrying the VP7-C207S mutation site was amplified by site-directed mutagenesis PCR. (2) The VP7 gene fragment carrying the VP7-C207S mutation site was homologously recombinated with the pT7 vector to construct a recombinant infectious clonal plasmid; (3) The recombinant infectious clonal plasmid and the helper plasmid are co-transfected into the host cell; (4) After transfection, co-culture the cells, collect the supernatant by freeze-thaw and centrifugation to obtain the attenuated porcine rotavirus strain.

[0011] Preferably, the primers for amplification comprise nucleotide sequences as shown in SEQ ID 3-6.

[0012] Preferably, the recombinant plasmid co-transfecting the host cell with the recombinant infectious clonal plasmid and the helper plasmid also includes a recombinant plasmid constructed by homologous recombination of the genes encoding VP1, VP2, VP3, VP4, NSP1, NSP2, NSP3, NSP4 or NSP5 proteins of the porcine rotavirus NJ2012 strain with the pT7 vector.

[0013] The present invention also provides the application of the attenuated porcine rotavirus strain in the preparation of a vaccine for the prevention and control of porcine rotavirus infection.

[0014] The present invention also provides a vaccine for preventing and controlling porcine rotavirus infection, the vaccine containing the aforementioned attenuated porcine rotavirus strain.

[0015] Preferably, the vaccine further includes pharmaceutically acceptable excipients, and the vaccine is a live attenuated vaccine.

[0016] The present invention discloses the following technical effects: This invention constructed an infectious clonal plasmid pT7-NJ2012-VP7-C207S with a VP7-C207S mutation, and rescued the recombinant virus rNJ2012-VP7-C207S using a reverse genetics system. The recombinant virus was identified using sequencing and Western blot detection. Biological characterization experiments showed that the viral replication ability was significantly reduced after the C207 mutation. Five-day-old suckling mice were infected with the same titer of rNJ2012-WT and rNJ2012-VP7-C207S. The pathogenicity of the two viruses was assessed by clinical symptom observation, anatomical identification, and RT-qPCR detection. The results showed that rNJ2012-VP7-C207S had weakened pathogenicity in suckling mice compared to rNJ2012-WT. Immunological experiments showed that the VP7-C207S mutation did not alter the virus's ability to induce humoral immunity. The combination of the characteristics of the recombinant virus with this mutation site, such as weakened replication ability, reduced pathogenicity, and the ability to induce a high level of humoral immune response, confirms that this virus has good potential as a candidate strain for a live attenuated PoRV vaccine. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Sequencing and identification of recombinant viruses with site mutations; Figure 2Cytopathic effect and validation results of MA104 cells infected with site-mutated recombinant virus; A: Morphological characteristics under light microscopy; B: Western blot validation results; Figure 3 Biological characteristics of site-mutated recombinant viruses; A: One-step growth curve on MA104 (** P <0.01); B: dsRNA map, 1-11 are the sequences of various viral gene fragments; C: ImageJ software measurement of plaque diameter and plaque staining results (*** P <0.001); Figure 4 The pathogenicity of the site-mutated virus in suckling mice; A: Schematic diagram of the suckling mouse challenge experiment; B: Clinical symptoms in suckling mice after challenge (*) P <0.05, *** P <0.001); C: Results of intestinal toxin load detection in suckling mice after challenge (*) P <0.05,** P <0.01, *** P <0.001); D: Histopathological lesions of the intestines of suckling mice after viral challenge; E: Staining results of histopathological lesions; Figure 5 A: Schematic diagram of mouse immunization test; B: Detection of serum IgG antibody levels by G9-VP7 and P[7]-VP4 proteins; C: Detection of neutralizing antibodies (***) using NJ2012 and AHFY2022 as target viruses. P <0.001). Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Example 1 1. Experimental Materials 1.1 Cells, Viruses, and Plasmids PoRV virus NJ2012 (G9P[7], GenBank accession number: MT874983.1-MT874993.1), AHFY2022 (G9P

[23] , GenBank accession number: OQ979280.1-OQ979290.1), recombinant virus rNJ2012-WT, and MA104 cells were provided by the Veterinary Research Institute of Jiangsu Academy of Agricultural Sciences. BHK-T7 cells were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee, and a cell line stably expressing T7 RNA polymerase was constructed based on this cell line. Eleven infectious clone plasmids encoding all genes of the NJ2012 strain have been published in the literature "Cheng Xi. Simultaneous expression of three Ggenotypes of VP7 proteins in a recombinant porcine rotavirus confersprotective immunity against multiple rotavirus infections". The pT7-SA11-VP7 plasmid and three helper plasmids (pCAG-D1R, pCAG-D12L, and pCAG-FAST-p10) were purchased from the Addgene platform. The helper plasmid C3P3-G1 was kindly provided by Dr. Siyuan Ding of Washington University in St. Louis. The helper plasmids pCAGGS-HA-NSP2 and pCAGGS-HA-NSP5 have been published in the paper "Chen Shuyu. Guanylate-binding proteins GBP1 and GBP2 inhibit porcine rotavirus replication in vitro".

[0025] 1.2 Experimental Reagents The endotoxin-free plasmid extraction kit and gel recovery kit were products of Omega Bio-tek, Inc.; the 2×Green Taq Mix, 2×Phanta Max Master Mix high-fidelity enzyme, homologous recombinase, RNA extraction kit, and reverse transcription kit were products of Vazyme Biotech Co., Ltd.; trypsin and trypsin were products of Sigma-Aldrich; the TransIT-LT1 transfection reagent was a product of Mirus Biotech; the RIPA protein lysis buffer was a product of Beyotime Biotechnology Co., Ltd.; the 7.5% PAGE gel rapid preparation kit was a product of Yamei Biotech Co., Ltd.; the goat anti-mouse IgG-HRP was a product of Solarbio; and the nucleic acid dye was a product of Yisheng Biotech Co., Ltd. The mouse monoclonal antibody against VP6 protein of rotavirus, the mouse monoclonal antibody against VP7 protein (Youlong Biotech), P[7]-VP4 and G9-VP7 coating proteins were provided by the Veterinary Research Institute of Jiangsu Academy of Agricultural Sciences. Among them, the mouse monoclonal antibody against VP6 protein has been disclosed in the literature "Li Kemang. Preparation and preliminary identification of VP6 monoclonal antibody against porcine group A rotavirus"; the mouse monoclonal antibody against VP7 protein has been disclosed in the literature "Isolation and characterization of a G9P

[23] porcine rotavirus strain AHFY2022 in China"; the P[7]-VP4 and G9-VP7 coating proteins have been disclosed in the literature "Tang Xuechao. Recombinant bivalent subunit vaccine combining truncated VP4 from P[7] and P

[23] induces protective immunity against prevalent porcine rotaviruses" and "Li Sufen. VP4-Specific IgAlevel as a correlate of neutralizing antibody and fecal shedding of porcinerotavirus infection".

[0026] 1.3 Laboratory Animals Fifteen SPF-grade 4-week-old female BALB / c mice and six litters of 5-day-old BALB / c suckling mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and were raised at the animal experimental base of the Veterinary Institute of Jiangsu Academy of Agricultural Sciences.

[0027] 2. Plasmid construction This invention is based on the genome sequence of the virus NJ2012. Primers for site mutation were designed using SnapGene 4.1.8 software, and the primer sequences are shown in Table 1. Using the cDNA product of virus NJ2012 as a template, the VP7-C207S fragment was obtained by PCR amplification, and its nucleotide sequence (SEQ ID NO.1) is as follows: 。

[0028] The mutated VP7-C207S amino acid sequence is shown in SEQ ID NO.2. MYGIEYTTVLTFLISIVLLNYILKSLTSAMDFIIYRFLLLIVIVSPFVKTQNYGINLPITGSMDTAYANSSQQETFLTSTLCLYYPTEASTQIGDTEWKNTLSQLFLTKGWPTGSVYFKEYTDIASFSIDPQLYCDYNVVLMKYDSTLELDMSELADLILNEWL CNPMDITLYYYQQTDEANKWISMGQSCTIKVCPLNTQTLGIGSITTNKATFEEVATNEKLAITDVVDGVDHKLDVTNTCTIRNCKKLGPRENVAIIQVGGSEVLDVTADPTTAPQTERMMRVHWKKWWQVFYTVVDYINQIVQVMSKRSRSLNSAAFYYRV*.

[0029] Using plasmid pT7-NJ2012-VP7 as a template, and primers pT7-VP7-F and C207S-R, the target sequence I was obtained according to the PCR system (pT7-VP7-F 2.5 μL, C207S-R 2.5 μL, cDNA 3 μL, 2×PhantaMaxMaster Mix high-fidelity enzyme 25 μL, sterile water 17 μL). Using plasmid pT7-NJ2012-VP7 as a template, and primers C207S-F and pT7-VP7-R, the target sequence II was obtained according to the PCR system (C207S-F 2.5 μL, pT7-VP7-R 2.5 μL, cDNA 3 μL, 2×PhantaMaxMaster Mix high-fidelity enzyme 25 μL, sterile water 17 μL).

[0030] After both systems were prepared, they were amplified under the following reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ for 15 s, 51℃ for 15 s, 72℃ for 30 s, for 35 cycles; 72℃ for 5 min, and stored at 4℃.

[0031] Using the recovered sequence I and fragment II as templates, and pT7-VP7-F / R as primers, the full-length target sequence III was obtained according to the PCR system (2.5 μL each of pT7-VP7-F / R, 3 μL of cDNA, 25 μL of 2×PhantaMax Master Mix high-fidelity enzyme, and 17 μL of sterile water) and PCR program (95℃ pre-denaturation for 3 min; 95℃ for 15 s, 51℃ for 15 s, 72℃ for 1 min, 35 cycles; 72℃ for 5 min, and storage at 4℃).

[0032] The pT7-SA11-VP7 plasmid was used as the template for PCR amplification of the pT7 vector. The PCR system consisted of 2.5 μL each of p3E5-F / R, 3 μL of cDNA, 25 μL of 2×PhantaMax Master Mix high-fidelity enzyme, and 17 μL of sterile water. The PCR program was as follows: 95℃ pre-denaturation for 3 min; 95℃ for 15 s, 51℃ for 15 s, 72℃ for 2 min, for 35 cycles; 72℃ for 5 min, and storage at 4℃.

[0033] After verification by gel electrophoresis, the target band was excised and purified. Subsequently, homologous recombination was used to ligate the full-length target sequence III to the amplification vector, yielding the recombinant plasmid pT7-NJ2012-VP7-C207S. All constructed plasmids were sequenced and verified for accuracy before extraction using an endotoxin-free plasmid extraction kit.

[0034] Table 1 Primers used in this invention 3. Recombinant virus rescue The construction method of recombinant virus rNJ2012-VP7-C207S is as follows: BHK-T7 cells were seeded in a monolayer in a cell plate. After the cell confluence reached 65%-75%, transfection was prepared. TransIT-LT1 transfection reagent was mixed with recombinant plasmids pT7-NJ2012-VP1, pT7-NJ2012-VP2, pT7-NJ2012-VP3, pT7-NJ2012-VP4, pT7-NJ2012-VP6, pT7-NJ2012-VP7-C207S, and p... T7-NJ2012-NSP1, pT7-NJ2012-NSP2, pT7-NJ2012-NSP3, pT7-NJ2012-NSP4, and pT7-NJ2012-NSP5, along with various helper plasmids C3P3-G1, pCAG-FAST-p10, pCAG-D1R, pCAG-D12L, pCAGGS-HA-NSP2, and pCAGGS-HA-NSP5, were added to serum-depleted medium (Opti-MEM). After mixing and standing at room temperature for 30 min, the mixture was evenly added to each cell well. After 24 h of transfection, the supernatant was discarded, and the cells were washed twice with PBS. The medium was then replaced with 1 mL of DMEM basal medium containing a final concentration of 0.1 μg / mL trypsin. After 24 h of culture, MA104 cells were added to transfected BHK-T7 cells. After 4 h, the trypsin concentration in each well was adjusted to 0.5 µg / mL, and secretin was added at a volume ratio of 1:5000. After co-culturing BHK-T7 and MA104 cells for 3 days, the cells were subjected to two freeze-thaw cycles. Cell debris was discarded by centrifugation at 12000 rpm for 5 min, and the supernatant was recovered to obtain the recombinant virus (named rNJ2012-VP7-C207S), which was then stored at -80℃.

[0035] 4. Identification of recombinant viruses 4.1 PCR identification 200 μL of supernatant was taken from both the parental strain and the recombinant virus. Total RNA was extracted according to the RNA extraction kit instructions, and then reverse transcribed into cDNA using a reverse transcription kit. Using 2×Green DNA... Taq Mix and specific primers rVP7-C207S-F and rVP7-C207S-R (Table 1) were used for PCR detection and sent for comparison.

[0036] 4.2 Western blot identification The parental strain and recombinant virus were inoculated into MA104 cells at a MOI of 0.05. After 12 h of infection, RIPA protein lysis buffer containing PMSF was added, and the cells were lysed at 4°C. The supernatant was collected, and protein loading buffer containing thiol reducing agent was added. After complete denaturation by heating in a metal bath at 100°C for 10 min, SDS-PAGE was performed. Mouse monoclonal antibody against VP6 protein (1:4000 dilution) and mouse monoclonal antibody against VP7 protein (1:4000 dilution) were used as primary antibodies, and goat anti-mouse IgG-HRP (1:10000 dilution) was used as secondary antibodies. The cells were detected using a chemiluminescence assay kit.

[0037] 5. Biological characteristics of recombinant viruses 5.1 One-step growth curve The parental viral strain and recombinant virus were inoculated into MA104 cells at a MOI of 0.01. Virus fluid was collected every 12 h post-infection until 60 h (three replicates at each time point). The median tissue culture infectious dose (TCID) of the virus was calculated using the Spear-Karber method. 50 And plot the virus growth curve.

[0038] 5.2 dsRNA-PAGE Following the instructions of the RNA extraction kit, dsRNA was extracted from the parental viral strain and recombinant virus, and a 7.5% PAGE solution was prepared. 12 μL of viral RNA was mixed with 4 μL of 5× Loading Buffer and loaded onto the sample. 1×TBE electrophoresis buffer was added, and electrophoresis was performed at a constant voltage of 180V for 4 h. After electrophoresis, the sample was stained with physiological saline containing 0.03% nucleic acid dye for 30 min and visualized using a gel imaging system.

[0039] 5.3 Plaque Emission Experiment MA104 cells were evenly seeded in 6-well plates. After a confluent monolayer was formed, activated virus samples diluted 10-fold were added, and the plates were incubated for 1 hour. After incubation, the virus solution was discarded, and the plates were washed twice with sterile PBS. Each well was then covered with 2 mL of plaque buffer (a mixture of equal volumes of 1.1% low-melting-point agarose and phenol red-free 2×MEM, along with 0.5 μg / mL trypsin, 1% penicillin-dextrose antibody, and 0.1% dextran), avoiding air bubbles. After the plaque buffer solidified, the plates were incubated upright for 3 days. Each well was then stained with 1 mL of 450 μg / mL neutral red dye, incubated at 37°C for 1.5 hours, the liquid was aspirated, and the plates were inverted for photographs. Plaque diameters were measured using ImageJ software.

[0040] 6. Pathogenicity experiment in suckling mice Except for the negative control group, all other suckling mice were orally inoculated with 100 μL of either the titer-quantified strain rNJ2012-WT or rNJ2012VP7-C207S. All suckling mice were monitored daily for 5 consecutive days post-inoculation, with general activity recorded and fecal characteristics assessed using a diarrhea scoring system. The severity of diarrhea in the suckling mice was assessed using the fecal scoring system described by Boshuizen. Fecal samples and intestinal tissue were collected every 24 hours post-challenge for RT-qPCR analysis to assess viral shedding and tissue distribution. cDNA was extracted using a commercial RNA extraction kit and reverse transcriptase according to the manufacturer's instructions. The expression level of PoRV-NSP5 was then detected by enzymatic digestion probe method (qNSP5-F: CTGCTTCAAACGATCCACTCAC(SEQ ID NO.11); qNSP5-R: TGATCCATAGACACGCC(SEQ ID NO.12); qNSP5-probe: FAM-TCGAATGCAGTTAAGACAAATGCAGACGCT-BHQ1(SEQ ID NO.13)), and RT-qPCR was performed using these as templates.

[0041] After fixing the duodenal, jejunal, and ileal segments of each group of suckling mice with formalin, histopathological sections were prepared and stained with hematoxylin eosin (HE). The pathological changes were then examined under a microscope.

[0042] 7. Mouse Immunization Experiment Four-week-old female BALB / c mice were randomly divided into three groups of five each. Viral stock solution was processed using Spear-Karber TCID50. 50 The assay method is standardized to 10 6 TCID 50 Mice were inoculated at a dose of 300 μL / mL. The trivalent recombinant virus was injected subcutaneously into mice, while the control group received either wild-type virus or an equal volume of PBS. Mice were immunized twice at 14-day intervals.

[0043] The purified G9-VP7 and P[7]-VP4 recombinant proteins were coated onto the microplate at a working concentration of 1 μg / mL at 4℃ overnight. After blocking with 5% skim milk powder for 2 h, the plates were thoroughly washed. The test serum (negative serum and post-immunization serum) was added to the microplate at a ratio of 1:100. Pre-immunization mouse serum was set as a negative control. Goat anti-mouse IgG-HRP diluted 1:20,000 was used as a secondary antibody for incubation. The OD of each well was detected by microplate reader. 450 And record it.

[0044] Mouse serum was inactivated in a metal bath at 56°C for 30 min, then serially diluted with DMEM basal medium, and then mixed with an equal volume of activated 200 TCID45. 50 Mix 0.1 mL of virus solution thoroughly and incubate for 1 h in a cell culture incubator. Then, inoculate the mixture onto an MA104 cell monolayer. Simultaneously, set up serum toxicity controls: negative serum control, positive serum control, virus control, and normal cell control. Observe the dilution of cell wells showing lesions daily until day 3, and calculate the serum neutralizing titer using the Kaeber method.

[0045] 8. Statistical Analysis All experimental data in this invention were obtained using Graphpad Prism 8.0.2 software Student's t Statistical analysis was performed using the test method and two-way ANOVA. ns indicates no significant difference; * indicates... P <0.05 indicates a significant difference; ** indicates a statistically significant difference. P <0.01 indicates a highly significant difference; *** indicates... P <0.001 indicates a highly significant difference.

[0046] 9. Results 9.1 Rescue and Validation of Site-Mutated Recombinant Viruses rNJ2012-VP7-C207S was successfully rescued using a reverse genetics system. RNA was extracted from the obtained recombinant virus and reverse transcribed, followed by PCR amplification and DNA sequencing. Results are as follows: Figure 1 The results showed that the amino acid at position 207 of VP7 was mutated from TGT(C) to TCT(S), indicating that the rNJ2012-VP7-C207S mutant virus strain was successfully obtained.

[0047] MA104 cells were infected with rNJ2012-WT and rNJ2012-VP7-C207S, and detected using mouse monoclonal antibody against VP6 protein and rabbit polyclonal antibody against VP7 protein, respectively, as primary antibodies. Figure 2 As shown, rNJ2012-VP7-C207S can cause MA104 cells to exhibit lesions such as blurred edges, swelling, and fragmentation, while uninfected cells show an epithelial-like appearance, adhere to the cell wall, and form a dense monolayer of cells. Figure 2 (A); Western blot results showed that VP6 and VP7 protein expression could be detected in all virus-infected groups ( Figure 2 (B). The above results indicate that the recombinant virus rNJ2012-VP7-C207S was successfully rescued and can replicate and proliferate normally in MA104 cells.

[0048] 9.2 Biological characteristics of site-mutated recombinant viruses Further analysis was conducted on the in vitro biological characteristics of the site-mutated recombinant virus. One-step growth curve results showed that the replication ability of NJ2012 in MA104 cells was significantly reduced after the VP7-C207S site mutation. Figure 3 (A). rNJ2012-VP7-C207S was passaged and amplified in MA104 cells, and its dsRNA profile was analyzed by gel electrophoresis. Electrophoresis analysis showed that the dsRNA migration pattern of rNJ2012-VP7-C207S was similar to that of the wild-type strain rNJ2012-WT. Figure 3 (B). Plaque assays showed that rNJ2012-VP7-C207S formed only small and sparse plaques, and its viral spreadability was significantly reduced compared to rNJ2012-WT. Figure 3 (C)

[0049] 9.3 Effect of VP7-C207S site mutation on the pathogenicity of NJ2012 strain To investigate the effect of the VP7-C207S mutation on the virulence of the NJ2012 strain, the virus was administered orally at a dose of 10... 6 TCID 50 Five-day-old BALB / c suckling mice were infected with a dose of [specific dosage], and diarrhea in the suckling mice was monitored over 5 days. Figure 4 (A). The results showed that the suckling mice in the control group maintained a normal clinical condition throughout the observation period. Statistical analysis revealed that the diarrhea rate and viral shedding volume of the suckling mice in the rNJ2012-VP7-C207S infection group were significantly lower than those in the rNJ2012-WT infection group ( Figure 4 (B, C)

[0050] To further investigate the correlation between the attenuation of the site-mutated virus and viral replication, the duodenum, jejunum, and ileum tissues of each group of suckling mice were divided into two parts. One part was used to extract RNA for RT-qPCR analysis to assess the tissue distribution of the virus; the other part was fixed in 10% neutral formalin and used HE staining to prepare pathological sections to observe intestinal pathological changes. The results of the detection of viral load in intestinal tissue are shown in […]. Figure 4 In contrast to the rNJ2012-WT infection group, the viral load in the intestinal tissue of suckling mice infected with rNJ2012-VP7-C207S was significantly lower. Pathological histological observations also confirmed this result; the degree and extent of intestinal tissue damage in the rNJ2012-VP7-C207S infection group were significantly less than those in the rNJ2012-WT group. Figure 4 (E).

[0051] These results show that, compared with rNJ2012-WT, rNJ2012-VP7-C207S has reduced pathogenicity in suckling mice.

[0052] 9.5 Effect of VP7-C207S site mutation on the immunogenicity of NJ2012 strain To assess the immunogenicity of rNJ2012-VP7-C207S, equal amounts of rNJ2012-WT, rNJ2012-VP7-C207S, and PBS were administered to 4-week BALB / c mice, and the levels of specific antibodies at different time points after immunization were detected. Figure 5 (A). The result is as follows: Figure 5 As shown in Figure B, the serum levels of G9-VP7 and P[7]-VP4 specific IgG / IgA antibodies in mice immunized with rNJ2012-WT and rNJ2012-VP7-C207S continued to increase with the duration of immunization, reaching a peak at 35 DPI. There was no significant difference in antibody levels between the two groups, and both were significantly higher than those in the PBS group.

[0053] Neutralization test results as follows Figure 5 As shown in Figure C, both rNJ2012-WT and rNJ2012-VP7-C207S can induce high levels of neutralizing antibodies against G9 type PoRV in mice, meaning there is no significant difference in neutralizing titers against NJ2012 and AHFY2022. This result indicates that the VP7-C207S site mutation does not alter the virus's ability to induce neutralizing antibodies.

[0054] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An attenuated strain of porcine rotavirus, characterized in that, The attenuated porcine rotavirus strain was obtained by mutating the C207 site of the VP7 protein of the porcine rotavirus NJ2012 strain to S207. The amino acid sequence of the mutated VP7 protein is shown in SEQ ID NO.

2.

2. A recombinant infectious clonal plasmid for preparing the attenuated porcine rotavirus strain of claim 1, characterized in that, The recombinant infectious cloning plasmid contains a gene encoding the VP7-C207S mutant protein of the porcine rotavirus NJ2012 strain, the amino acid sequence of which is shown in SEQ ID NO.

2.

3. The recombinant infectious clonal plasmid as described in claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. A method for constructing an attenuated porcine rotavirus strain according to claim 1, characterized in that, This includes obtaining the recombinant infectious clonal plasmid as described in claim 2 or 3 by transfecting a host cell using a reverse genetics operating system.

5. The construction method as described in claim 4, characterized in that, The construction method includes the following steps: (1) Using the cDNA of porcine rotavirus NJ2012 strain as a template, the VP7 gene fragment carrying the VP7-C207S mutation site was amplified by site-directed mutagenesis PCR. (2) The VP7 gene fragment carrying the VP7-C207S mutation site was homologously recombinated with the pT7 vector to construct a recombinant infectious clonal plasmid; (3) The recombinant infectious clonal plasmid and the helper plasmid are co-transfected into the host cell; (4) After transfection, co-culture the cells, collect the supernatant by freeze-thaw and centrifugation to obtain the attenuated porcine rotavirus strain.

6. The construction method as described in claim 5, characterized in that, The primers for the amplification include nucleotide sequences as shown in SEQ ID3-6.

7. The construction method as described in claim 5, characterized in that, The recombinant plasmids that co-transfect host cells with the recombinant infectious clonal plasmids and the helper plasmids also include recombinant plasmids constructed by homologous recombination of genes encoding VP1, VP2, VP3, VP4, NSP1, NSP2, NSP3, NSP4 or NSP5 proteins of porcine rotavirus NJ2012 strain with the pT7 vector.

8. The use of the attenuated porcine rotavirus strain as described in claim 1 in the preparation of a vaccine for the prevention and control of porcine rotavirus infection.

9. A vaccine for preventing swine rotavirus infection, characterized in that, The vaccine contains the attenuated porcine rotavirus strain as described in claim 1.

10. The vaccine as described in claim 9, characterized in that, The vaccine also includes pharmaceutically acceptable excipients, and the vaccine is a live attenuated vaccine.

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