Method for constructing gluc-labeled live imaging model based on porcine rotavirus nmtl strain and application thereof

Abstract

The application discloses a method for constructing a GLuc marker live imaging model based on a porcine rotavirus NMTL strain and application thereof, and belongs to the technical field of biological medicines. In the application, a P2A self-cleavage peptide and a Gaussian luciferase GLuc coding sequence are inserted into the 3' end of the NSP3 coding sequence of the G9P[23] type porcine rotavirus NMTL strain, a reverse genetic system containing a C3P3-G1 auxiliary plasmid is used, and transfection parameters are optimized to rescue a recombinant virus rNMTL-GLuc. The virus can stably express GLuc in a suckling mouse in vivo, and through in vivo bioluminescence imaging, the replication kinetics of the porcine rotavirus in the suckling mouse in vivo is first revealed, that is, a replication peak is reached on the third day after infection. The application realizes continuous and non-terminal dynamic monitoring of the infection process of the virus in the same individual, and provides a high-sensitivity visual tool for the study of the pathogenic mechanism of the porcine rotavirus and the screening of antiviral drugs.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a method and application for constructing a GLuc-labeled in vivo imaging model based on the porcine rotavirus NMTL strain. Background Technology

[0002] Porcine rotavirus (PoRV) is one of the important pathogens causing diarrhea in suckling piglets, which can cause diarrhea, dehydration, growth retardation and even death. In recent years, the epidemic pattern of PoRV in pig herds in my country has changed, and genotypes such as G9P

[23] have gradually become important epidemic combinations. Existing vaccines or detection systems based on traditional genotypes have limited adaptability to new epidemic strains. Therefore, it is necessary to establish research tools that match the current epidemic genotypes.

[0003] Although existing studies have used reverse genetics techniques to insert reporter genes (such as NLuc and UnaG) into rotaviruses, the existing techniques are mostly limited to high-throughput screening at the cellular level. Current research on porcine rotavirus mainly focuses on other viral backbones or in vitro detection. For the G9P

[23] type porcine rotavirus NMTL strain, although the existing techniques have been disclosed, the use of GLuc for labeling has not been disclosed, and its in vivo imaging model still lacks applicable tools.

[0004] Furthermore, different reporter genes exhibit varying compatibility and expression stability across different viral backbones, necessitating the development of a porcine rotavirus marker technology suitable for real-time in vivo monitoring. Summary of the Invention

[0005] The main purpose of this application is to propose a method and application for constructing a GLuc-labeled in vivo imaging model based on the porcine rotavirus NMTL strain, aiming to solve the technical problems of the lack of research tools that match the current prevalent genotype (G9P

[23] ) and the inability to perform non-terminal, continuous dynamic monitoring of porcine rotavirus infection in vivo. Although the existing technology discloses the NMTL strain, it does not disclose the use of GLuc to label it, nor does it disclose its application in the suckling mouse in vivo imaging model.

[0006] To achieve the above objectives, in a first aspect, this application proposes a method for constructing a GLuc-labeled in vivo imaging model based on the porcine rotavirus NMTL strain, comprising the following steps: (1) Construction of recombinant plasmid: A reporter expression cassette was inserted at the 3' end of the non-structural protein NSP3 coding sequence of G9P

[23] type porcine rotavirus NMTL strain before the stop codon to construct the plasmid pT7-NMTL-NSP3-P2A-GLuc; the reporter expression cassette contains, from 5' to 3', a nucleic acid sequence encoding the P2A self-cleaving peptide and a nucleic acid sequence encoding the Gaussian luciferase GLuc; (2) Reverse genetic rescue: The plasmid pT7-NMTL-NSP3-P2A-GLuc and wild-type infectious clone plasmids of the remaining 10 genomic segments of the NMTL strain were co-transfected with helper plasmid C3P3-G1 into BHK-T7 host cells. After 48 hours of transfection, the cells were co-cultured with MA104 cells to rescue recombinant porcine rotavirus rNMTL-GLuc. (3) Construction of in vivo imaging model: Non-human mammals were infected with the recombinant porcine rotavirus rNMTL-GLuc to obtain an in vivo imaging model for dynamic monitoring of viral replication and spread in vivo.

[0007] In some embodiments, the nucleic acid sequence encoding the P2A self-cleaving peptide is as shown in SEQ ID NO.1; and / or, The nucleic acid sequence encoding Gaussian luciferase GLuc is shown in SEQ ID NO.2.

[0008] In some embodiments, the nucleotide sequence of the plasmid pT7-NMTL-NSP3-P2A-GLuc is shown in SEQ ID NO.3.

[0009] In some embodiments, step (2) further includes rescuing the recombinant porcine rotavirus rNMTL-GLuc and continuously passaged it to P5 in MA104 cells, wherein the reporter expression cassette remains genetically stable and the GLuc expression activity remains stable during passage to P5.

[0010] In some embodiments, in step (3), the non-human mammal is selected from suckling mice; The infection was administered via oral gavage at a dose of 2 × 10⁻⁶. 7 PFU / each.

[0011] In some implementations, step (3) further includes: Intraperitoneal injection of coelenterate was administered to suckling mice infected with the recombinant porcine rotavirus rNMTL-GLuc at a dose of 5 mg / kg, and bioluminescence images were acquired 5 min after administration.

[0012] In some implementations, step (3) further includes: From day 1 to day 7 post-infection of the suckling mice, bioluminescent signals in the abdominal intestinal region of the suckling mice were collected daily at the same time point and using the same exposure time and imaging parameters. Viral dynamics curves were constructed based on the collected signal intensity. These curves were used to indicate the level of viral replication in the body, with the peak signal intensity occurring on day 3 post-infection.

[0013] Secondly, this application also proposes a live imaging model, which is constructed using the method proposed in the first aspect of this application for constructing a GLuc-labeled live imaging model based on the porcine rotavirus NMTL strain.

[0014] Thirdly, this application also proposes an application of the live imaging model proposed in the second aspect of this application in any of the following uses: (a) Dynamic monitoring of the replication, transmission and tissue tropism of porcine rotavirus in non-human mammals; (b) In vivo efficacy evaluation of anti-swine rotavirus drugs; (c) Real-time assessment of the immunogenicity of swine rotavirus vaccine.

[0015] The method proposed in this application for constructing a GLuc-labeled in vivo imaging model based on the porcine rotavirus NMTL strain involves site-specific insertion of a reporter expression cassette (containing a P2A peptide and a reporter gene) after the end of the coding region of the porcine rotavirus NSP3 protein. Post-translational separation of the NSP3 protein and the reporter protein (such as GLuc) is mediated by a self-cleaving peptide (e.g., the P2A peptide). This allows the reporter virus to maintain the normal function of the NSP3 protein while expressing the reporter protein, and also preserves the virus's infectivity and replication capabilities. This design effectively addresses the challenge of reporter gene insertion disrupting viral function, ensuring the infectivity and genetic stability of the reporter virus. When this recombinant porcine rotavirus is used to infect non-human mammals (such as suckling mice), the bioluminescent signal changes with the infection process. This can be used to characterize the dynamic changes in recombinant virus-related signals. By injecting the animal with a corresponding substrate (e.g., coelenterate) and detecting the bioluminescent signal using an in vivo imaging system, non-terminal, continuous, and dynamic monitoring of viral replication and propagation in a living animal can be achieved. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0017] Figure 1A schematic diagram and verification diagram of the construction of the reporter plasmid pT7-NMTL-NSP3-P2A-GLuc provided in the embodiments of this application; a is a schematic diagram of a portion of the plasmid structure; b is a diagram of agarose gel electrophoresis verification; c is a diagram of GLuc activity verification. Figure 2 This is a schematic diagram and verification diagram of the auxiliary plasmid used in the embodiments of this application (Note: The auxiliary plasmid used in this experiment is based on a published article: doi:10.1128 / JVI.01294-20). a) Gel electrophoresis results after fragment amplification; b) Schematic diagram of helper plasmid construction; c) Western blotting (WB) verification of helper plasmid pCAGGS-T7RNAP; d) Western blotting (WB) verification of helper plasmid pCAGGS-NP868R; e) Western blotting (WB) verification of helper plasmid pCAGGS-T7RNAP-P2A-NP868R; f) Western blotting (WB) verification of helper plasmid C3P3-G1; g) Fluorescence microscopy observation of helper plasmid C3P3-G1. Figure 3 A rescue verification diagram of the recombinant report virus rNMTL-GLuc provided for embodiments of this application: a) Graph showing the effect of cytopathic effect; b) Graph showing the quantitative detection of reporter gene activity; Figure 4 This is a diagram showing the detection of viral protein expression in infected cells provided in an embodiment of this application; Figure 5 This is a virus titer detection graph provided in an embodiment of this application; Figure 6 This is a diagram showing the results of a plaque experiment using recombinant virus provided in an embodiment of this application. Figure 7 Genetic stability test diagram of recombinant virus provided in the embodiments of this application: a) Analysis of RT-PCR results for NSP3 fragments from different viral passages; b) Sanger sequencing analysis of RT-PCR products; Figure 8 This is a graph showing the stability test of recombinant virus expression provided in an embodiment of this application. Figure 9 Figure showing the results of the substrate reaction time optimization experiment of the recombinant virus rNMTL-GLuc in neonatal mice provided in the embodiments of this application: a) is an in vivo bioluminescence imaging image of suckling mice at 1 min, 3 min, 5 min and 7 min after intraperitoneal injection of the substrate (coelenterin); b) is a quantitative graph of the corresponding change in luminescence intensity (RLU) over time. Figure 10 This is a flowchart of an in vivo imaging experiment using neonatal mice, provided in an embodiment of this application. Figure 11 Representative bioluminescent imaging images of the abdominal intestinal region of suckling mice infected with recombinant virus rNMTL-GLuc from day 1 to day 7, as provided in the embodiments of this application; Figure 12 A quantitative graph showing the change in relative luminescence intensity (RLU) over time in the region of interest (ROI) on the abdomen of a suckling mouse, as provided in an embodiment of this application. Figure 13 This is a summary diagram of other observation results from animal experiments on suckling mice according to embodiments of this application; a) is the incidence of diarrhea in different groups of suckling mice on days 0-7 after infection with recombinant virus rNMTL-GLuc; b) is the fecal characteristic score of different groups of suckling mice on days 0-7 after infection with recombinant virus rNMTL-GLuc; c) is the growth rate of body weight relative to initial body weight in different groups of suckling mice on days 0-7 after infection with recombinant virus rNMTL-GLuc.

[0018] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0019] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0020] Furthermore, if the embodiments of this application involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the use of "and / or" or "and / or" throughout the text includes three parallel solutions. For example, "A and / or B" includes solution A, solution B, or a solution that simultaneously satisfies A and B. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed in this application.

[0021] Porcine rotavirus (PoRV) is an important pathogen causing diarrhea in piglets. In recent years, genotypes such as G9P

[23] have become important prevalent combinations, and research tools adapted to them are urgently needed. Current research on in vivo infection of porcine rotavirus mainly relies on sacrificing animals at different time points after infection and collecting tissue samples for virus titer detection, immunostaining, or nucleic acid quantitative analysis. These methods are endpoint detection and cannot continuously and dynamically track the replication, spread, and distribution of the virus in the same animal, making it difficult to accurately analyze the virus infection dynamics and causing a large consumption of experimental animals and an increase in time costs.

[0022] To overcome this limitation, reporter virus technology has emerged. By inserting reporter genes such as luciferase into the viral genome, the non-terminal nature of bioluminescent signals allows for continuous and dynamic monitoring of viral activity. However, inserting exogenous genes into rotavirus often presents challenges: inappropriate insertion sites or connection methods can disrupt the integrity and function of the viral genome, leading to decreased viral replication capacity, genetic instability, or even irreversibility. Therefore, how to stably and efficiently integrate reporter genes into the rotavirus genome without significantly affecting the virus's basic infectivity, and make it suitable for in vivo imaging studies, is a key technical problem that urgently needs to be solved in this field.

[0023] Based on the above issues, please refer to Figure 1 In one aspect, embodiments of this application propose a recombinant porcine rotavirus whose genome contains a reporter expression cassette fused to the end of the coding region of the non-structural protein NSP3 gene. The reporter expression cassette includes, from 5' to 3', a nucleic acid sequence encoding a P2A peptide and a nucleic acid sequence encoding a reporter gene. Through ribosome jumping mediated by the P2A peptide, NSP3 and the reporter protein express two products respectively.

[0024] By inserting a reporter expression cassette at the end of the coding region of the NSP3 gene, essential for viral replication, the expression of the reporter gene is correlated with the viral replication process. The introduction of the P2A peptide ensures effective post-translational dissociation of the NSP3 protein and the reporter protein, thereby minimizing interference from exogenous sequences on the normal functions of NSP3 (such as participation in viral mRNA translation and shutdown of host cell protein synthesis) and maintaining the virus's basic replication capacity. Simultaneously, the expression, secretion, or accumulation of reporter proteins (such as secretory luciferase) enables non-terminal, continuous, and dynamic monitoring of viral replication levels in in vitro cell culture supernatants or in vivo animals, effectively addressing the technical challenge of traditional endpoint detection methods failing to continuously track the dynamics of viral infection within the same animal.

[0025] In some embodiments, the viral backbone of the recombinant porcine rotavirus includes the G9P

[23] type porcine rotavirus NMTL strain. The G9P

[23] type porcine rotavirus NMTL strain is a virus isolated from diarrheal piglets, and its genome sequence is known, facilitating reverse genetics operations.

[0026] In some embodiments, the self-cleaving peptide is preferably P2A. The P2A peptide is derived from porcine teschovirus-1. Its working principle involves a "ribosome jump" during ribosomal translation between the C-terminal glycine and proline residues of the P2A peptide, preventing peptide bond formation and thus efficiently dissociating the upstream protein (NSP3) from the downstream protein (reporter protein). This strategy facilitates the relatively synchronous expression of NSP3 and the reporter protein without introducing an additional protease cleavage step, simplifying the design and improving efficiency.

[0027] In some embodiments, the reporter gene includes at least one of the following: Gaussian luciferase GLuc gene, nanoluciferase NLuc gene, and firefly luciferase FLuc gene.

[0028] In some embodiments, the recombinant porcine rotavirus is recombinant porcine rotavirus rHB0923-Gluc with accession number CCTCC NO: V202667, deposited at the China Center for Type Culture Collection (CCTCC); classification name: porcine rotavirus; Latin scientific name: none (or not applicable, subject to the accession certificate); address: Wuhan University, Wuhan, China; postal code: 430072; accession date: June 29, 2026.

[0029] Gaussian luciferase (GLuc) is a secretory luciferase that catalyzes the oxidation of its substrate, coelenterate, to produce luminescence. It boasts advantages such as extremely low background signal, high sensitivity, wide dynamic range, and rapid and convenient detection. As a reporter gene, GLuc is secreted into the culture supernatant after cell infection, facilitating high-throughput in vitro drug efficacy screening. In in vivo imaging applications, after intraperitoneal injection of the substrate, GLuc produces a strong bioluminescent signal in vivo, which can serve as a measurable reporter signal for in vivo detection, aiding in the dynamic monitoring of the infection process.

[0030] This application also proposes a method for constructing a GLuc marker in vivo imaging model based on the porcine rotavirus NMTL strain, using a reverse genetics system, including the following steps: S1. Construction of recombinant plasmid: A reporter expression cassette was inserted at the 3' end of the non-structural protein NSP3 coding sequence of G9P

[23] type porcine rotavirus NMTL strain before the stop codon to construct the plasmid pT7-NMTL-NSP3-P2A-GLuc; the reporter expression cassette contains, from 5' to 3', a nucleic acid sequence encoding the P2A self-cleaving peptide and a nucleic acid sequence encoding the Gaussian luciferase GLuc.

[0031] S2. Reverse genetic rescue: The plasmid pT7-NMTL-NSP3-P2A-GLuc, wild-type infectious clones of the remaining 10 genomic segments of the NMTL strain, and helper plasmid C3P3-G1 were co-transfected into BHK-T7 host cells. After 48 hours of transfection, the cells were co-cultured with MA104 cells to rescue recombinant porcine rotavirus rNMTL-GLuc.

[0032] Among them, the wild-type infectious clonal plasmids of the remaining 10 genomic segments of the NMTL strain are pT7-NMTL-VP1, pT7-NMTL-VP2, pT7-NMTL-VP3, pT7-NMTL-VP4, pT7-NMTL-VP6, pT7-NMTL-VP7, pT7-NMTL-NSP1, pT7-NMTL-NSP2, pT7-NMTL-NSP4, and pT7-NMTL-NSP5.

[0033] "Reverse genetics" refers to a technology that involves cloning the full-length viral genome cDNA into a plasmid, directionally modifying the viral genome at the DNA level, and then transfecting the modified plasmid into host cells to rescue infectious live viral particles. For viruses with segmented genomes, such as rotavirus, reverse genetics requires cloning all viral genome fragments separately into plasmids and co-transfecting them into host cells.

[0034] S3. Construction of in vivo imaging model: Non-human mammals were infected with the recombinant porcine rotavirus rNMTL-GLuc to obtain an in vivo imaging model for dynamic monitoring of viral replication and spread within the body.

[0035] The core of this preparation method lies in precisely manipulating the viral genome through reverse genetics technology, inserting the reporter expression cassette into a predetermined location, and using optimized plasmid combinations to achieve stable virus rescue, ensuring that the resulting recombinant virus has a clear genetic background and that the reporter gene is inserted at an accurate location.

[0036] In some embodiments, the nucleic acid sequence encoding the P2A self-cleaving peptide is shown in SEQ ID NO.1, and the nucleic acid sequence encoding the Gaussian luciferase GLuc is shown in SEQ ID NO.2. In some preferred embodiments, the nucleotide sequence of the plasmid pT7-NMTL-NSP3-P2A-GLuc is shown in SEQ ID NO.3.

[0037] In some embodiments, step S2 further includes rescuing the recombinant porcine rotavirus rNMTL-GLuc and continuously passaged it to P5 in MA104 cells, wherein the reporter expression cassette remains genetically stable and the GLuc expression activity remains stable during passage to P5.

[0038] In some embodiments, in step S3, the non-human mammal is selected from suckling mice; the infection is transmitted via oral gavage at a dose of 2 × 10⁻⁶. 7 PFU / each.

[0039] In some embodiments, step S3 further includes: injecting coelenterate into neonatal mice infected with recombinant porcine rotavirus rNMTL-GLuc via intraperitoneal injection at a dose of 5 mg / kg, and acquiring bioluminescence images 5 min after administration.

[0040] In some embodiments, step S3 further includes: acquiring bioluminescent signals from the abdominal intestinal region of suckling mice at the same time point and using the same exposure time and imaging parameters daily from day 1 to day 7 post-infection. A viral dynamics curve is constructed based on the acquired signal intensity to indicate the viral replication level in vivo, and the peak signal intensity, as experimentally measured, occurs on day 3 post-infection.

[0041] This application also proposes a live imaging model, which is constructed using a method based on the porcine rotavirus NMTL strain to build a GLuc-labeled live imaging model.

[0042] The in vivo imaging model proposed in this application simulates the natural infection route of porcine rotavirus by inoculating suckling mice with rNMTL-GLuc recombinant virus via oral gavage. Standardized intraperitoneal injection of coelenterate and fixed imaging parameters ensure the reproducibility and comparability of the experiment. Compared with traditional in vivo porcine rotavirus infection models (which rely on daily necropsy, tissue homogenization for viral titer detection, or histopathological scoring), this model achieves non-invasive, non-terminal continuous monitoring of the same suckling mouse through in vivo bioluminescence imaging. It can longitudinally track the complete spatiotemporal process of viral infection at the individual level, significantly reducing animal usage, improving data temporal resolution and statistical power, and providing a standardized and quantifiable technical platform for in vivo porcine rotavirus research.

[0043] This application also proposes an application of the above-described live imaging model in any of the following uses: (a) Dynamic monitoring of the replication, transmission and tissue tropism of porcine rotavirus in non-human mammals; (b) In vivo efficacy evaluation of anti-swine rotavirus drugs; (c) Real-time assessment of the immunogenicity of swine rotavirus vaccine.

[0044] The specific luminescent reaction between the GLuc protein expressed by rNMTL-GLuc reporter virus in suckling mice and the coelenterate substrate allows for continuous longitudinal tracking of the same suckling mouse over multiple days without animal sacrifice. This significantly reduces the number of experimental animals used (complying with the 3R principles of animal ethics) and eliminates the interference of inter-individual differences on experimental results. In dynamic monitoring applications, the spatiotemporal distribution of the virus in the intestinal region can be visualized in real time, providing an intuitive tool for studying the in vivo replication kinetics, transmission pathways, and clearance patterns of porcine rotavirus. In antiviral drug evaluation, the difference in bioluminescent signals between the treated group and the control group at the same time point can rapidly determine the in vivo antiviral activity of the drug, shortening the evaluation cycle and reducing experimental costs compared to traditional TCID50 titration or plaque assays. In vaccine efficacy assessment, continuous in vivo imaging after viral challenge in immunized animals allows for real-time evaluation of the onset time, protective strength, and duration of immune protection, providing an efficient, objective, and quantifiable in vivo evaluation method for vaccine development and efficacy assessment.

[0045] The following specific examples provide further details.

[0046] Unless otherwise specified, the reagents, materials and instruments used in the examples are all conventionally selected in the art.

[0047] Example 1: Construction of reporter plasmid pT7-NMTL-NSP3-P2A-GLuc Please see Figure 1 In step a), using an infectious cloning plasmid containing the NSP3 fragment of the PoRV NMTL strain as a backbone, a P2A-GLuc reporter expression cassette was introduced at the end of the NSP3 open reading frame, while preserving the 5' UTR and 3' UTR of the NSP3 fragment. Homologous recombination was used to ligate the insert containing P2A, GLuc, and the vector's homologous arms to the linearized pT7-NMTL-NSP3 vector, yielding pT7-NMTL-NSP3-P2A-GLuc. The constructed plasmid was subjected to agarose gel electrophoresis, as shown in Figure a. Figure 1 As shown in b, from Figure 1 As can be seen from b, the size of the constructed plasmid is comparable to the theoretical value, and further sequencing confirmed the successful construction of plasmid pT7-NMTL-NSP3-P2A-GLuc.

[0048] After construction, the correctness of the insertion direction, junction boundaries, and reading frames was verified by Sanger sequencing. After transfecting the reporter plasmid into BHK-T7 cells, significant GLuc activity was detected in the culture supernatant, while no significant signal was observed in the empty vector or non-reporter controls, indicating that the reporter plasmid possesses expression function (e.g., Figure 1 (As shown in c).

[0049] Example 2: Construction of helper plasmid C3P3-G1 like Figure 2 As shown in Figure a, using BL21(DE3) DNA and ASFV DNA as templates, the target-sized DNA fragment was amplified by PCR, ligated into the pCAGGS vector via homologous recombination, and then verified by Western blotting. Figure 2 c, Figure 2 As shown in d, both methods can enhance protein expression. Figure 2 As shown in Figure b, chimeric plasmids were constructed using P2A and flexible connectors of different lengths. The results are as follows... Figure 2 As shown in Figure e, the chimeric plasmid constructed from the P2A linker peptide did not exhibit good transcriptional enhancement. However, the helper plasmid C3P3-G1, as verified by Western blotting and observed under a fluorescence microscope, showed the following results: Figure 2 f, Figure 2 As shown in Figure g, both methods exhibited good transcriptional enhancement in the BHK-21 control group. The P2A ligation protocol did not achieve the expected results; subsequent virus rescue employed codon optimization and ligated T7 RNA polymerase to NP868R's C3P3-G1 with a (G4S)4 flexible linker peptide.

[0050] Example 3: Rescue of recombinant reporter virus rNMTL-GLuc Ten PoRV NMTL wild-type infectious clones, the pT7-NMTL-NSP3-P2A-GLuc reporter plasmid, and the C3P3-G1 helper plasmid (laboratory-constructed) were co-transfected into BHK-T7 cells (a BHK-21 cell line stably expressing T7 RNA polymerase, preserved in the laboratory), with a control group also included. After transfection, the culture system was changed to conditions suitable for viral replication, and trypsin was added to promote rotavirus infection activation; subsequently, MA104 cells (laboratory-preserved) were added for co-culture to facilitate viral rescue and amplification.

[0051] Cells and culture supernatant were harvested after 72-96 hours of culture, and the virus was released after two freeze-thaw cycles. The virus was then activated with trypsin and inoculated into MA104 cells for passage. The passaged cells were observed under a microscope, and the results are as follows: Figure 3 As shown in Figure a. From Figure 3 As can be seen from Figure a, the rNMTL-GLuc infection group showed a cytopathic effect that was extremely similar to that of the rNMTL group, while the cell morphology of the MOCK control group remained normal.

[0052] To detect the expression of the GLuc reporter gene in recombinant viruses, three commonly used rotavirus cell lines (IPEC-J2, MA104, and Caco-2, stored in the laboratory) were infected with rNMTL-GLuc and control virus rNMTL, respectively, and the luminescence intensity was observed. The results are as follows: Figure 3 As shown in b. From Figure 3 As shown in Figure b, with the extension of infection time (12, 24, 36, 48 h), the luminescence intensity (representing GLuc activity) of the rNMTL-GLuc group increased significantly and showed a good time-dependent growth trend in different cell lines; while the luminescence value of the rNMTL control group (without the GLuc gene) remained at a very low background level. This proves that the GLuc reporter gene has been successfully integrated into the recombinant viral genome and is stably expressed and released during viral replication.

[0053] Example 4: In vitro identification and stability evaluation of rNMTL-GLuc 1. Immunofluorescence analysis (IFA) identification: The expression of viral proteins in the infected cells prepared in Example 3 was detected using anti-PoRV VP6 antibody. The results are as follows: Figure 4 As shown. From Figure 4 As can be seen, green fluorescent signals can be observed in the rNMTL-GLuc infection group, indicating that it can successfully infect and express viral structural proteins.

[0054] 2. Replication kinetics: MA104 cells were infected with an MOI of 0.01, and viral titers were measured at 12 h, 24 h, 36 h, and 48 h. The results are as follows: Figure 5 As shown. From Figure 5 It can be seen that the titer of rNMTL-GLuc is slightly lower than that of rNMTL, but it still has effective infection and replication capabilities.

[0055] 3. Plaque Phenotyping: The infectivity and replication capacity of the recombinant virus rNMTL-GLuc were further analyzed using a plaque formation assay. Specific experimental steps included: The preliminarily rescued and amplified viral supernatant (including the wild-type rNMTL control group and the rNMTL-GLuc experimental group) was activated with an appropriate amount of trypsin and then inoculated into a monolayer of MA104 cells. After adsorption, the inoculation solution was removed, and the cells were covered with low-melting-point agarose medium containing trypsin. The cells were cultured for 3 to 4 days until obvious cytopathic effects were observed. The covering was then removed, and the monolayer of cells was fixed and stained with crystal violet staining solution for observation. The results are shown below. Figure 6 As shown. From Figure 6As can be seen, the wild-type recombinant virus rNMTL formed clear and large plaques on MA104 cells; while the recombinant reporter virus rNMTL-GLuc also produced clear plaques, but the average diameter and area of ​​the plaques were significantly smaller than those of the rNMTL control group, suggesting that the insertion of foreign genes has a certain impact on replication ability but does not eliminate infectivity.

[0056] 4. Genetic Stability: To confirm the genetic stability of the exogenous gene fragment in the recombinant virus rNMTL-GLuc during successive passages, RT-PCR and sequencing were performed on the NSP3 fragment from different passages of P1, P3, and P5 viruses. Specific experimental steps included: extracting total RNA from the wild-type control virus rNMTL and the recombinant virus rNMTL-GLuc at passages P1, P3, and P5; and using specific primers targeting the rotavirus NSP3 gene region and the inserted reporter gene (primer design as follows...). Figure 7 As shown in the diagram below (a), reverse transcription-polymerase chain reaction (RT-PCR) was performed on the NSP3 region (the expected amplification fragment is 358 bp, and the expected amplification fragment containing the reporter gene is 973 bp). The amplification products were subjected to agarose gel electrophoresis, and the PCR products were sequenced by Sanger sequencing to verify the accuracy of the sequences.

[0057] RT-PCR results as follows Figure 7 As shown in Figure a. From Figure 7 As shown in Figure a, the PCR product band of the wild-type virus rNMTL is located at approximately 350 bp (consistent with the expected 358 bp); while the PCR products of the recombinant virus rNMTL-GLuc, after continuous passage to P1, P3, and P5 generations, all exhibited a single, bright, and specific band with consistent molecular weight, located at approximately 1000 bp (consistent with the expected 973 bp). This result demonstrates that during continuous passage culture, the recombinant virus successfully maintained the exogenous GLuc reporter gene fragment, without any loss or large fragment deletion of the reporter gene.

[0058] Sequencing results as follows Figure 7 As shown in b, from Figure 7 As can be seen from Figure b, by analyzing the sequences at the junctions of NSP3-P2A and P2A-GLuc, it can be seen that the recombinant virus maintains complete and accurate connections between the NSP3 and P2A sequences, as well as between the P2A and GLuc sequences, in generations P1, P3, and P5. The sequencing peaks are clear and free of impurities, and no base mutations, deletions, or frameshifts are observed.

[0059] The results of RT-PCR and sequencing show that the constructed recombinant rotavirus rNMTL-GLuc has good genetic stability and can still maintain the complete exogenous reporter gene structure after multiple consecutive passages.

[0060] 5. Expression stability: GLuc activity was detected in different passages of the virus, including P1, P3, and P5. Specific experimental steps included: wild-type control group rNMTL and recombinant virus rNMTL-GLuc from passages P1, P3, and P5 were inoculated into MA104 cells, cultured under identical conditions, and the supernatant was collected; the supernatant was then serially diluted (10⁻⁶ m² / 40⁻¹² ... 0 Up to 10 -4 Subsequently, GLuc activity was measured in culture supernatants of various dilutions using chemiluminescent substrates, and the relative luminescence units (RLUs) were recorded and analyzed. The results are as follows: Figure 8 As shown. From Figure 8 It can be seen that the luminescence signal of wild-type virus rNMTL at all dilutions is at an extremely low background level (approximately 10). 1 Up to 10 2 RLU), does not change with dilution. However, the luminescence intensity trends of the recombinant virus rNMTL-GLuc at three different generations (P1, P3, and P5) are highly consistent: in undiluted (10⁻⁶ ppm), the luminescence intensity at these three generations is significantly higher. 0 All of them showed a high level of luminous signal (approximately 10). 5 Up to 10 6 RLU), as the sample dilution factor increased, its luminescence intensity showed a synchronous logarithmic decrease, and at 10 -2 At higher dilutions, the fluorescence activity gradually approaches background levels. This result indicates that the trends in luminescence activity across different generations of the virus are generally consistent, suggesting that reporter gene expression is relatively stable.

[0061] Example 5: Establishment of an in vivo imaging model of rNMTL-GLuc infection in suckling mice To optimize the imaging conditions of the constructed recombinant rotavirus rNMTL-GLuc in a live animal model, this embodiment used 7-day-old SPF-grade suckling mice as a model to conduct a substrate reaction time experiment. The specific experimental steps included: taking 7-day-old SPF-grade suckling mice and orally inoculating them with rNMTL-GLuc, with a preferred dose of approximately 2 × 10⁻⁶ g / L. 7 PFU / mouse, inoculated at approximately 100 μL. 24 h post-infection, the appropriate GLuc substrate (preferably coelenterate, preferably at a dose of approximately 5 mg / kg) was administered intraperitoneally. Bioluminescence images of the suckling mice were acquired using an in vivo imaging system at 1 min, 3 min, 5 min, and 7 min after substrate administration. The results are as follows: Figure 9 As shown. Figure 9 In Figure a, bioluminescence imaging results in neonatal mice at different reaction time points are shown. Figure 9 Figure b shows further quantitative analysis results, indicating that the relative luminescence intensity (RLU) rose rapidly after substrate administration, approached its peak at 3 min, and remained at a stable high level plateau between 5 and 7 min. Approximately 5 min was ultimately determined to be the optimal imaging acquisition time.

[0062] Furthermore, to verify the ability of the constructed recombinant reporter virus rNMTL-GLuc to track viral infection dynamics in live animals, a dynamic monitoring experiment was conducted for 7 consecutive days using 7-day-old SPF-grade suckling mice as a model. The procedure is as follows: Figure 10 As shown. Specific experimental procedures included: inoculation of rNMTL-GLuc via oral gavage (preferred dose was approximately 2 × 10⁻⁶). 7 PFU / mouse was injected into suckling mice. Imaging was performed daily at the same time point from day 1 to day 7 post-infection. The imaging procedure was as follows: Suckers were first intraperitoneally injected with a GLuc-compatible substrate (preferably coelenterate, preferably at a dose of approximately 5 mg / kg). After approximately 5 minutes of substrate reaction, bioluminescent signals from the abdominal intestinal region of the suckling mice were acquired using an in vivo imaging system. To ensure data comparability, all imaging acquisitions used the same exposure time and parameter settings, and the relative luminescence intensity (RLU) of the ROI was quantitatively analyzed to represent the viral replication level in vivo. The results are as follows: Figure 11 and Figure 12 As shown.

[0063] from Figure 11 As can be seen, weak blue-green luminescence signals can be observed in the abdominal intestinal region of infected suckling mice on the first day; on the second day, the signal rapidly increases and expands, showing green to yellow; by the third day, the luminescence signal reaches its peak, showing a concentrated and bright red high-intensity signal area; then from the fourth day onwards, the signal gradually weakens, and by the seventh day, weak luminescence can still be detected in the abdominal region.

[0064] from Figure 12 As can be seen, focal low-intensity signals are visible in the images on day 1 post-infection, but the ROI quantitative value is lower than that of LOD, and therefore cannot be used as a reliable quantitative result; on day 2, the mean luminescence value jumps to about 2×10 4 RLU; Replication peaks on day 3, with an average value of approximately 3 × 10⁻⁶. 4 RLU (some individuals can reach 5×10) 4 RLU); the signal dropped to approximately 1×10 on the 4th day. 4 RLU, and then the signal remained at a level slightly above LOD from day 5 to day 7.

[0065] Combination Figure 11 and Figure 12The results show that the constructed recombinant reporter virus rNMTL-GLuc can effectively replicate in suckling mice, and the rise, peak and fall of the bioluminescent signal after infection can be continuously and dynamically observed through an in vivo imaging system.

[0066] Example 6: Combined evaluation of diarrhea rate, diarrhea score, and weight change To enhance the reliability of the model evaluation, diarrhea in suckling mice can be recorded simultaneously with in vivo imaging, along with diarrhea scores and changes in mouse weight. Specific experimental steps include: selecting 7-day-old SPF-grade suckling mice and randomly dividing them into an experimental group (rNMTL-GLuc, n=5) and a control group (DMEM, n=5). The experimental group was orally inoculated with recombinant virus rNMTL-GLuc (dose approximately 2 × 10⁻⁶). 7 PFU / mouse), and the control group was inoculated with an equal volume of DMEM medium. From day 1 to day 7 post-inoculation, the following indicators were recorded and evaluated daily for each group of suckling mice: (1) Diarrhea incidence: Observe and record whether each suckling mouse has diarrhea, and calculate the daily diarrhea incidence.

[0067] (2) Diarrhea score: The stool characteristics are quantitatively scored (0 points for normal, 1 point for soft stool, 2 points for loose stool, and 3 points for watery stool), and the daily average score is calculated.

[0068] (3) Weight change: The weight of each suckling mouse was recorded daily, and the weight growth rate relative to the initial weight was calculated.

[0069] The specific results are as follows: Figure 13 As shown. Among them. Figure 13 Figure a shows the incidence of diarrhea in each group of suckling mice. In the experimental group (rNMTL-GLuc), the incidence of diarrhea rapidly increased to 100% from day 1 to day 3 post-infection; it began to subside on day 4, and the diarrhea completely disappeared by day 6 and day 7. In the control group (DMEM), the incidence of diarrhea remained at 0% throughout the entire observation period.

[0070] Figure 13 Figure b shows the stool characteristics scores for each group: The diarrhea score in the experimental group began to rise on day 1, reaching its peak on day 2 to day 3 (average score of approximately 2.5 to 3.0, corresponding to watery stool); the score gradually decreased from day 4, returning to 0 on day 6 and thereafter. The diarrhea score in the control group remained at 0 throughout the entire process.

[0071] Figure 13The figure in Figure 1 shows the weight change trend of each group: the weight of both the experimental group (red line) and the control group (blue line) showed a continuous increasing trend from day 1 to day 7. Although the average weight of the experimental group was slightly lower than that of the control group in the middle and late stages of infection, the difference between the two groups was not statistically significant (ns), indicating that infection with the recombinant virus rNMTL-GLuc did not cause significant growth retardation or weight loss in suckling mice.

[0072] In summary, under the conditions of this experiment, rNMTL-GLuc induced self-limiting diarrhea, and no significant weight loss was observed. By jointly analyzing bioluminescent signal intensity, diarrhea rate, and diarrhea score, a more comprehensive in vivo infection assessment system for PoRV can be constructed.

[0073] The above description is merely an exemplary embodiment of this application and does not limit the patent scope of this application. Any equivalent substitutions made based on the technical concept of this application and the contents of the specification and drawings of this application, or direct / indirect applications in other related technical fields, are included within the patent protection scope of this application.

Claims

1. A method for constructing a GLuc-labeled in vivo imaging model based on porcine rotavirus NMTL strain, characterized in that, Includes the following steps: (1) Construction of recombinant plasmid: A reporter expression cassette was inserted at the 3' end of the non-structural protein NSP3 coding sequence of G9P[23] type porcine rotavirus NMTL strain before the stop codon to construct the plasmid pT7-NMTL-NSP3-P2A-GLuc; the reporter expression cassette contains, from 5' to 3', a nucleic acid sequence encoding the P2A self-cleaving peptide and a nucleic acid sequence encoding the Gaussian luciferase GLuc; (2) Reverse genetic rescue: The plasmid pT7-NMTL-NSP3-P2A-GLuc and wild-type infectious clone plasmids of the remaining 10 genomic segments of the NMTL strain were co-transfected with helper plasmid C3P3-G1 into BHK-T7 host cells. After 48 hours of transfection, the cells were co-cultured with MA104 cells to rescue recombinant porcine rotavirus rNMTL-GLuc. (3) Construction of in vivo imaging model: Non-human mammals were infected with the recombinant porcine rotavirus rNMTL-GLuc to obtain an in vivo imaging model for dynamic monitoring of viral replication and spread in vivo.

2. The method according to claim 1, characterized in that, The nucleic acid sequence encoding the P2A self-cleaving peptide is shown in SEQ ID NO.1; and / or, The nucleic acid sequence encoding the Gaussian luciferase GLuc is shown in SEQ ID NO.

2.

3. The method according to claim 2, characterized in that, The nucleotide sequence of the plasmid pT7-NMTL-NSP3-P2A-GLuc is shown in SEQ ID NO.

3.

4. The method according to claim 1, characterized in that, Step (2) further includes rescuing the recombinant porcine rotavirus rNMTL-GLuc and continuously passaged it to P5 in MA104 cells, wherein the reporter expression cassette remains genetically stable and the GLuc expression activity remains stable when passaged to P5.

5. The method according to claim 1, characterized in that, In step (3), the non-human mammal is selected from suckling mice; The infection was administered via oral gavage at a dose of 2 × 10⁻⁶. 7 PFU / each.

6. The method according to claim 5, characterized in that, Step (3) further includes: Intraperitoneal injection of coelenterate was administered to suckling mice infected with the recombinant porcine rotavirus rNMTL-GLuc at a dose of 5 mg / kg, and bioluminescence images were acquired 5 min after administration.

7. The method according to claim 6, characterized in that, Step (3) further includes: From day 1 to day 7 post-infection of the suckling mice, bioluminescent signals in the abdominal intestinal region of the suckling mice were collected daily at the same time point and using the same exposure time and imaging parameters. Viral dynamics curves were constructed based on the collected signal intensity. These curves were used to indicate the level of viral replication in the body, with the peak signal intensity occurring on day 3 post-infection.

8. A live-body imaging model, characterized in that, It is constructed using the method described in any one of claims 1 to 7.

9. The use of the live imaging model of claim 8 in any of the following applications: (a) Dynamic monitoring of the replication, transmission and tissue tropism of porcine rotavirus in non-human mammals; (b) In vivo efficacy evaluation of anti-swine rotavirus drugs; (c) Real-time assessment of the immunogenicity of swine rotavirus vaccine.

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