A linearized mutant plasmid of a tambussu virus sl ii, recombinant virus and use thereof

Abstract

The application discloses a tamsui virus SLII linearization mutant plasmid, a recombinant virus and application, and belongs to the technical field of molecular biology. The application takes pACNR CQW1-Intron infectious cloning as a skeleton, linearizes SLII stem loop structure in the 3'UTR Domain-I region, constructs SLII linearization mutant infectious cloning pACNR-CQW1-SLIILiner, and obtains the recombinant virus CQW1-SLIILiner through rescue. The replication ability of the recombinant virus is limited at the in-vitro cell level, and the recombinant virus shows a significantly attenuated phenotype in an animal model; the recombinant virus can induce specific neutralizing antibodies and cellular immune responses in the body, and has a significant protective effect on a virulent strain attack; therefore, the recombinant virus has the advantages of clear molecular genetic markers, high safety, good immunogenicity and strong genetic stability, and can be applied to preparation of a live attenuated vaccine or related biological products for preventing TMUV infection.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a Tembusu virus SLII linearized mutant plasmid, recombinant virus, and its applications. Background Technology

[0002] Currently, the control of Tembusu virus (TMUV) mainly relies on vaccines, which primarily include inactivated vaccines and traditional live attenuated vaccines. Inactivated vaccines have relatively high safety, but their immunogenicity is limited, often requiring multiple booster immunizations to maintain protective levels, increasing breeding costs and operational complexity.

[0003] Traditional live attenuated vaccines are typically obtained through continuous passage, and their virulence declines due to the accumulation of random mutations. While these vaccines can induce strong humoral and cellular immune responses, their attenuation mechanisms are complex, their molecular basis is unclear, and they lack stable, traceable molecular genetic markers. In the context of ongoing viral circulation and genetic mutation, the cross-protective ability of these vaccines against different circulating strains is uncertain, and the potential risk of reversion to virulence is difficult to assess precisely at the molecular level.

[0004] Therefore, it is necessary to develop a rational attenuated vaccine strategy with a clear molecular genetic basis, controllable virulence, high genetic stability, and good protective effect. Summary of the Invention

[0005] The purpose of this invention is to solve the above-mentioned problems existing in the prior art and to provide a Tembusu virus SLII linearized mutant plasmid, recombinant virus, and its application.

[0006] The technical solution adopted in this invention is as follows:

[0007] A method for preparing a linearized mutant plasmid of Tembusu virus SLII includes the following steps:

[0008] S1. The pACNR-CQW1-Intron plasmid was double-digested using restriction enzyme sites SbfI and NruI to obtain a linearized vector.

[0009] S2. Using pACNR-CQW1-Intron plasmid as a template, the first amplified fragment was obtained by PCR amplification; using the artificially synthesized plasmid pUC57-SLIIliner containing the SLIIliner sequence as a template, the second amplified fragment was obtained by PCR amplification.

[0010] S3. The first amplified fragment and the second amplified fragment are fused by PCR to obtain a recombinant amplified fragment containing the TMUV 3'UTR Domain-I modified sequence. The recombinant amplified fragment is ligated into the linearized pACNR-CQW1-Intron vector in S1. The Tembusu virus SLII linearized mutant plasmid pACNR-CQW1-SLIIliner is constructed through identification and screening.

[0011] The SLIIliner sequence is shown in SEQ ID No. 1, and the first amplified fragment and the second amplified fragment are shown in SEQ ID No. 7 and SEQ ID No. 8, respectively.

[0012] Preferably, the primer sequences for PCR amplification are shown in SEQ ID No. 2 to SEQ ID No. 5.

[0013] Preferably, the method for preparing the artificially synthesized plasmid pUC57-SLIILiner containing the SLIIliner sequence is as follows: replacing the complete Domain-I region in the 3'UTR of pACNR-CQW1-Intron with the SLIIliner sequence to obtain a modified full-length 3'UTR sequence, artificially synthesizing a linearized modified 3'UTR fragment, and then ligating it into the pUC57 vector; the modified full-length 3'UTR sequence is shown in SEQ ID No. 6.

[0014] The present invention also provides a linearized mutant plasmid of Tembusu virus SLII prepared by the above preparation method.

[0015] The present invention also provides a recombinant virus, obtained by transfecting BHK-21 cells with the above-mentioned Tembusu virus SLII linear mutant plasmid for 72 hours.

[0016] The present invention also provides the application of the above-mentioned recombinant virus in the preparation of Tembusu live attenuated vaccine.

[0017] The Domain I region of the TMUV 3'UTR contains four stem-loop structures and is a key region determining the synthesis and type of flavivirus sfRNA. Figure 1 A). This invention utilizes a subgenomic replicon system to perform stepwise deletion mutation screening on key RNA structures in Domain I, aiming to obtain the optimal construct that maximizes the disruption of Domain I structure while preserving RNA replication capability. Results show that as the Domain I region is successively deleted ( Figure 1In B / C, the replication ability of replicons gradually decreases, and replication activity is almost lost upon complete deletion. However, when only a single stem loop is retained, all individual stem loops can support viral replication, with the replication level of stem loop SLII being closest to that of the wild type. Figure 1 (D / E). The above results indicate that 3'UTR-Domain I has a certain tolerance for deletion mutations, and SLII is the core stem-loop structure in this region that maintains TMUV replication capability. To prevent the virus from restoring its RNA functional structure through RNA recombination (such as replicating existing RNA elements) during passage, this invention designs an irreversible structural modification scheme based on the strategy of separately preserving SLII: linearizing SLII to disrupt its hairpin structure. This invention aims to enhance the genetic stability and biosafety of the virus while achieving viral attenuation.

[0018] Therefore, this invention modifies the key SLII stem-loop structure within Domain-I using reverse genetics: linearization is achieved by disrupting complementary pairing of the stem-loop structure, as illustrated in the schematic diagram below. Figure 2 As shown in A, this modification does not alter the coding region sequence, thereby achieving precise regulation of viral replication capacity and virulence expression while maintaining the integrity of the structural protein antigen.

[0019] The beneficial effects of this invention are as follows: Based on the infectious clone pACNR-CQW1 as a backbone, this invention uses reverse genetics to linearize the SLII stem-loop structure within the 3′UTR Domain-I region, constructing the SLII linearized mutant infectious clone pACNR-CQW1-SLIILiner, and rescuing the recombinant virus CQW1-SLIILiner. This modification, by disrupting the key stem-loop structure of the 3′UTR, regulates viral replication and significantly reduces virulence while maintaining good genetic stability. The resulting recombinant virus exhibits limited replication capacity at the in vitro cellular level, displays a significantly attenuated phenotype in animal models, and can induce specific neutralizing antibodies and cellular immune responses, providing significant protection against virulent strains. The attenuated vaccine strain constructed in this invention has advantages such as clear molecular genetic markers, high safety, good immunogenicity, and strong genetic stability, and can be used to prepare live attenuated vaccines or related biological products for the prevention of TMUV infection. Attached Figure Description

[0020] Figure 1 The deletion mutations in the key RNA structure of TMUV 3′UTR-Domain I; (A) Schematic diagram of RNA secondary structure; (B) Schematic diagram of RNA secondary structure deletion mutations; (C) Replicon experiment; (D) Schematic diagram of RNA secondary structure deletion mutations; (E) Replicon experiment.

[0021] Figure 2For the rescue and identification of TMUV recombinant viruses; (A) Design diagram; (B) Plaque; (C) IFA.

[0022] Figure 3 The in vitro characteristics and genetic stability of TMUV recombinant virus; (A) F1 generation virus DEF growth curve; (B) F1 generation virus BHK-21 growth curve; (C) F1 generation virus duck embryo virulence experiment; (D) passage stability; (E) F10 generation virus BHK-21 growth curve; (F) F10 generation virus DEF growth curve; (G) F10 generation virus plaque morphology; (H) F10 generation virus duck embryo virulence experiment.

[0023] Figure 4 The pathogenicity of TMUV recombinant virus; (A) weight changes; (B) clinical symptoms; (C) viremia; (D) tissue load; (E) mortality.

[0024] Figure 5 To assess the protective effect of TMUV recombinant virus against challenge; (A) weight change; (B) clinical symptoms; (C) viremia; (D) tissue load; (E) mortality rate; (F) neutralizing antibody level 14 days after immunization; (G) neutralizing antibody level 14 days after challenge.

[0025] Figure 6 The image shows the pACNR-CQW1-SLIIliner plasmid.

[0026] The full-length infectious cDNA cloning plasmid pACNR-CQW1-Intron of the Tembusu virus (TMUV) CQW1 strain in this invention was constructed and provided by the Avian Disease Prevention and Control Research Center of the College of Veterinary Medicine, Sichuan Agricultural University. Reference: GUO J, HEY, WANG X, et al. Stabilization of a full-length infectious cDNA clone for duck Tembusu virus by insertion of an intron [J]. J Virol Methods, 2020, 283:113922;

[0027] The CQW1 strain of WT virus was rescued from an infectious clone of pACNR CQW1-Intron;

[0028] pUC57-SLIILiner (a plasmid containing the SLIIliner sequence) was synthesized by a biotechnology company;

[0029] The Anti-TMUV mouse polyclonal antibody was prepared in-house by the Poultry Disease Prevention and Control Research Center of the College of Veterinary Medicine, Sichuan Agricultural University. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0031] The reagents used in the following examples are as follows: HiScript II RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit was purchased from Nanjing Novizan Biotechnology. 2×Taq SYBRGreen qPCR Premix was purchased from Changsha Novogene Biotechnology. Nano-Glo luciferase assay system Glo lysis buffer was purchased from Shanghai Promega. TransIntro EL transfection reagent was purchased from Beijing TransGen Biotech. Lipofectamine Messenger MAX reagen RNA transfection reagent was purchased from Shanghai Thermo Scientific. Viral DNA / RNA co-extraction kit was purchased from Beijing Tiangen Biotech. Plasmid miniprep kit and endotoxin-free plasmid extraction kit were both purchased from Guangzhou Meiji Biotechnology. DMEM was purchased from Shanghai Gibco; FBS was purchased from Shanghai Sijiqing.

[0032] Example 1: Preparation of plasmid for linearized SLII mutant of Tembusu virus

[0033] 1. The RNA secondary structure of Domain I in the 3'UTR of TMUV CQW1 strain was predicted using software such as Mfold. This prediction was then combined with previously reported 3'UTR structure data from DENV2 and ZIKV to determine a predictive model for the secondary structure of the TMUV 3'UTR. While maintaining the same base composition and number, bioinformatics tools were used to randomly rearrange the nucleotide sequence of Domain I SLII in the 3'UTR of TMUV CQW1 strain, resulting in a randomly scrambled mutant. Based on the above analysis, the complete Domain-I region in the 3'UTR of pACNR-CQW1-Intron was replaced with the SLIIliner sequence (as shown in SEQ ID No. 1), resulting in the modified full-length 3'UTR sequence (as shown in SEQ ID No. 6). The modified full-length 3'UTR sequence was commissioned to a biotechnology company for whole-genome synthesis, resulting in a linearized modified 3'UTR fragment, which was then ligated into the pUC57 vector to prepare the plasmid pUC57-SLIILiner containing the SLIIliner sequence.

[0034] 2. The pACNR-CQW1-Intron plasmid was double-digested using two restriction enzyme sites, SbfI and NruI, to obtain a linearized vector.

[0035] 3. Using pACNR-CQW1-Intron plasmid as a template, PCR amplification was performed using CQW1 SbfIF (as shown in SEQ ID No. 2) and CQW1 NS5 R (as shown in SEQ ID No. 3) to obtain the first amplified fragment (as shown in SEQ ID No. 7). Simultaneously, using a synthetically produced plasmid containing the SLIIliner sequence (pUC57-SLIIliner) as a template, PCR amplification was performed using primers CQW1NS5-F (as shown in SEQ ID No. 4) and CQW1 P3 R (as shown in SEQ ID No. 5) to obtain the second amplified fragment (as shown in SEQ ID No. 8). The first and second amplified fragments were fused by PCR to obtain a recombinant amplified fragment containing the TMUV 3'UTR Domain-I modified sequence. The recombinant amplified fragment was ligated into the linearized pACNR-CQW1-Intron vector. Through identification and screening, the Tembusu virus SLII linearized mutant plasmid pACNR-CQW1-SLIIliner was constructed, as shown in SEQ ID No. 7. Figure 6 As shown.

[0036] The primer names and sequences are shown in Table 1, the PCR system is shown in Table 2, and the PCR reaction procedure is shown in Table 3.

[0037] Table 1 Primers for constructing the full-length infectious clone pACNR-CQW1-SLIIliner

[0038]

[0039] Table 2 PCR amplification system

[0040]

[0041] Table 3 PCR amplification program

[0042]

[0043] 4. The results of the Mfold RNA secondary structure prediction software showed that the SLIIliner sequence in the mutant plasmid could not form the stable stem-loop structure of the original SLII, and the SLII structure was successfully linearized.

[0044] Example 2 Preparation of recombinant virus

[0045] Two μg of the recombinant plasmid pACNR-CQW1-SLIIliner prepared in Example 1 was transfected into BHK-21 cells with a growth density of approximately 80% using Lipofectamine 3000 and cultured at 37 °C and 5% CO2. After 72 hours of culture following transfection, the cell supernatant was harvested, and immunofluorescence assay confirmed successful virus rescue. Figure 2 (B / C), as the F0 generation virus. The purified recombinant virus was obtained through plaque purification technology and named CQW1-SLIILiner.

[0046] Mutation verification: RNA was extracted from the virus rescued, the 3'UTR region was amplified by RT-PCR and sequenced to confirm the introduced mutation.

[0047] Example 3: Study on the biological properties of recombinant viruses

[0048] The recombinant virus CQW1-SLIILiner in this embodiment was prepared in Example 2.

[0049] 1. Experimental Methods

[0050] 1.1 Growth curve determination

[0051] The experimental virus was the successfully rescued recombinant virus CQW1-SLIILiner, with wild-type CQW1-WT as a control. Virus growth curve determination: Cells were inoculated with a multiplicity of infection (MOI) of 0.001, and supernatant was collected at different time points (24, 36, 48, 72 h) post-infection. Viral titer was determined by plaque assay, and a one-step growth curve was plotted. Genetic stability analysis: The recombinant virus was continuously passaged blindly for 10 generations in BHK-21 cells. Genomic RNA was extracted from each generation of virus, and the full-length 3'UTR was amplified by RT-PCR and sequenced to analyze sequence stability during passage. Duck embryo virulence assay: The virus working solution titer was 1 × 10⁻⁶. 4 TCID 50 / mL, add 100 μL (according to 10³ TCID) 50 Inoculate the allantoic cavity of 9-day-old SPF duck embryos, 8 in each group, and observe the mortality of duck embryos within 7 days.

[0052] 1.2 Toxicity determination

[0053] Experimental animals: 5-day-old SPF ducklings were randomly divided into 5 groups (n=8): DMEM simulated control group, CQW1-WT infection group, CQW1-SLIILiner (F10) immunization group, and commercially available inactivated vaccine (FX2010-180P) immunization group. All animal experiments were conducted in accordance with protocols approved by the animal ethics committee. Immunization and challenge: The immunization group was injected intramuscularly into the leg with 200 μL of virus solution (103.5 TCID50). 50 (Or commercially available vaccines.) A control group was injected with an equal volume of DMEM. Clinical observation and sample collection: Animal mental status, neurological symptoms (such as paralysis), mortality rate, and weight changes were observed and recorded daily. On days 2, 5, and 7 post-infection, three ducks were randomly selected from each group, and blood (for viremia detection) and major organs (brain, spleen, liver, kidneys, etc., for viral load detection and histopathological analysis) were collected. Detection indicators: 1) Viremia: Quantitative detection of viral genome copy number in plasma using qPCR; 2) Tissue viral load: Tissue homogenate was weighed, and viral copy number was detected using qPCR.

[0054] 1.3 Evaluation of the effectiveness of virus attack protection

[0055] On day 14 after the initial immunization, all surviving animals were challenged with the virulent strain CQW1 (10⁵ TCID⁻¹) via intramuscular injection. 50 Following the challenge, observation continued for 14 days. Sample collection and testing: Blood samples were collected on day 1 post-challenge to detect viremia. Serum samples were collected one day before and 14 days post-challenge for serological testing: 1) Neutralizing antibodies: A virus-diluted serum method was used to perform a virus neutralization test on BHK-21 cells, and the serum neutralizing antibody titer was calculated. Efficacy assessment: Morbidity, mortality, and clinical symptoms were recorded after challenge. The efficacy of the vaccine was comprehensively evaluated by combining the neutralizing antibody level and clinical protection results.

[0056] 2. Experimental Results

[0057] 2.1 Growth curves of CQW1-SLIILiner were determined in BHK-21 and DEF cells. The results showed that the recombinant virus CQW1-SLIILiner exhibited significantly reduced proliferation ability in both cell types compared to the wild-type virus. Duck embryo virulence assays indicated that CQW1-SLIILiner only delayed duck embryo death time. Figure 3 A / B / C). After 10 consecutive passages of the recombinant virus in BHK-21 cells, no long-term insertions or deletions due to RNA recombination in the 3'UTR region were detected, indicating that the designed virus has good genetic stability. Figure 3D). Further kinetic analysis of the 10th generation virus revealed that CQW1-SLIILiner was still significantly lower than the wild type; the plaque size formed by the recombinant virus was significantly smaller than that of the wild type (D). Figure 3 E / F / G). Duck embryo virulence tests showed that the virulence of the 10th generation virus was further weakened compared to the 1st generation virus. Figure 3 H).

[0058] 2.2 Starting from day 3 post-inoculation, animals in the wild-type virus infection group experienced significant weight loss and successively developed clinical symptoms such as depression, reduced activity, unsteady gait, and hind limb paralysis, accompanied by obvious viremia. Figure 4 A / B / C). In contrast, the weight changes in the CQW1-SLIILiner group and the commercial vaccine group were not significantly different from those in the simulated control group. No obvious clinical symptoms were observed during the experiment, and the viremia level was much lower than that in the wild-type group (A / B / C). Figure 4 A / B / C). On day 5 post-infection, the viral load in the tissues of animals in each group was measured. The CQW1-SLIILiner group was significantly lower than the wild-type group (A / B / C). Figure 4 D). Ultimately, only 25% (2 / 8) of the wild-type group survived, while no deaths occurred in the remaining vaccine groups. Figure 4 E), indicating that the toxicity of CQW1-SLIILiner to ducklings was significantly reduced.

[0059] 2.3 On the third day after challenge with the virus, the animals in the simulated control group began to show significant weight loss and gradually exhibited typical clinical symptoms of TMUV infection, including depression, reduced activity, unsteady gait, and hind limb paralysis, with significantly elevated viremia levels. In contrast, the animals in the CQW1-SLIILiner group and the commercially available vaccine group showed no significant weight changes, no obvious clinical symptoms during the experiment, and their viremia levels were much lower than those in the simulated control group. Figure 5 AC). Tissue viral load assays showed high levels of viral copies in the simulated control group's heart tissue only on day 5 post-challenge. During the trial, only the simulated control group experienced a 3 / 8 mortality rate; no deaths occurred in the other vaccine groups. Figure 5 D / E). On day 14 post-immunization, the level of neutralizing antibodies in the serum of the CQW1-SLIILiner vaccine group was significantly higher than that of the commercially available vaccine group, approximately 100 times higher. Even on day 14 post-challenge, the level of neutralizing antibodies remained high, indicating that it can provide effective immune protection for ducklings. Figure 5 F / G).

[0060] Based on the combined results of immune protection rate, viremia suppression, tissue viral load control and neutralizing antibody levels, the recombinant virus based on the linearization of the SLII structure of the 3'UTR is superior to existing commercial vaccines in terms of immunogenicity and protective effect.

[0061] In summary, this invention provides a TMUV recombinant virus strain with high safety, stable immunization effect, and strong protective efficacy, which can be applied to the preparation of live attenuated vaccines and has good application prospects.

[0062] The specification and drawings are intended to be illustrative rather than restrictive. Based on the present invention, those skilled in the art can make substitutions and modifications to some of the technical features without creative effort, and all such modifications are within the scope of protection of the present invention.

Claims

1. A method for preparing a linearized mutant plasmid of Tembusu virus SLII, characterized in that, Includes the following steps: S1. The pACNR-CQW1-Intron plasmid was double-digested with restriction endonucleases SbfI and NruI to obtain a linearized vector. S2. Using pACNR-CQW1-Intron plasmid as a template, the first amplified fragment was obtained by PCR amplification; using the artificially synthesized plasmid pUC57-SLIIliner containing the SLIIliner sequence as a template, the second amplified fragment was obtained by PCR amplification. S3. The first amplified fragment and the second amplified fragment are fused by PCR to obtain a recombinant amplified fragment containing the TMUV 3'UTR Domain-I modified sequence. The recombinant amplified fragment is ligated into the linearized pACNR-CQW1-Intron vector in S1. The Tembusu virus SLII linearized mutant plasmid pACNR-CQW1-SLIIliner is constructed through identification and screening. The SLIIliner sequence is shown in SEQ ID No. 1, and the first amplified fragment and the second amplified fragment are shown in SEQ ID No. 7 and SEQ ID No. 8, respectively.

2. The preparation method according to claim 1, characterized in that, The primer sequences for PCR amplification are shown in SEQ ID No. 2 to SEQ ID No.

5.

3. The preparation method according to claim 1, characterized in that, The method for preparing the artificially synthesized plasmid pUC57-SLIILiner containing the SLIIliner sequence is as follows: the complete Domain-I region in the 3'UTR of pACNR-CQW1-Intron is replaced with the SLIIliner sequence to obtain the modified full-length 3'UTR sequence, the linearized modified 3'UTR fragment is artificially synthesized, and then ligated into the pUC57 vector; the modified full-length 3'UTR sequence is shown in SEQ ID No.

6.

4. The Tembusu virus SLII linearized mutant plasmid prepared by the preparation method according to any one of claims 1-3.

5. A recombinant virus, characterized in that, It was obtained by transfecting BHK-21 cells with the Tembusu virus SLII linear mutant plasmid as described in claim 4 for 72 hours.

6. The use of the recombinant virus as described in claim 5 in the preparation of the Tembusu attenuated vaccine.

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