Construction method of porcine respiratory syndrome virus

Abstract

The invention relates to the technical field of virus gene engineering, in particular to a construction method of a porcine respiratory syndrome virus, which comprises the following steps: taking a PRRSV HEB1 strain genome (GenBankEF112447.1) as a template, modifying the template, cloning the modified genome to a pBAC11 vector, and rescuing a recombinant virus rPRRSV-HA-CMV-mC through in vitro transcription and Marc-145 cell transfection. The recombinant PRRSV strain shows remarkable replication delay and titer reduction, and an attenuated phenotype is formed. The recombinant virus and an ISA206 oil adjuvant are further emulsified to prepare the vaccine, the vaccine shows efficient immunogenicity in piglet immunization, clinical symptoms, viremia and lung pathological injury after PRRSV infection can be remarkably reduced, and the vaccine has the potential of being developed into a safe and effective vaccine.

Description

Technical Field

[0001] The present invention relates to the technical field of viral genetic engineering, in particular to a method for constructing porcine respiratory syndrome virus. Background Art

[0002] Porcine respiratory syndrome (PRRS), caused by the porcine respiratory syndrome virus (PRS), is a highly detrimental infectious disease affecting the swine industry. Its primary characteristics are reproductive failure in sows and respiratory symptoms in piglets and finishing pigs. PRRSV (Porcine Respiratory Syndrome) belongs to the family Arteriviridae and exhibits high genetic variability, posing significant challenges to disease prevention and control. Currently, vaccine development and pathogenicity studies against PRRSV rely on the effective construction and culture of the virus. Traditional viral construction methods suffer from complex procedures, unstable viral activity, and low construction efficiency, making them inadequate for both scientific research and practical applications. The continuous advancement of genetic engineering technology has provided new approaches and methods for constructing PRRSV. For example, reverse genetics techniques, which manipulate the viral genome, enable more precise construction of virus strains with specific biological properties. However, existing genetic engineering-based construction methods still need to be improved in terms of virus rescue efficiency and genetic stability. Therefore, developing efficient and stable methods for constructing PRRSV is of great practical significance.

[0003] Traditional research and prevention methods have certain limitations, so it is necessary to develop more effective virus construction methods to deeply study the biological characteristics and pathogenic mechanisms of the virus and develop new vaccines. Summary of the Invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a method for constructing porcine respiratory syndrome virus. The recombinant PRRSV strain obtained by the porcine respiratory syndrome virus construction method has high immunogenicity, can significantly reduce the clinical symptoms, viremia and lung pathological damage after PRRSV infection, and has the potential to develop a safe and effective vaccine.

[0005] In order to achieve the above technical effects, the present invention adopts the following technical solutions: First, the present invention provides a method for constructing porcine respiratory syndrome virus, comprising the following steps: (a) Using the PRRSV HEB1 strain genome (GenBank EF112447.1) as a template, an HA tag sequence was inserted between nucleotides 45-46 downstream of the start codon of the ORF5 gene coding region, wherein the HA tag sequence is shown in SEQ ID NO: 1; (b) the native promoter region (14285-14320 bp) of the ORF6 gene (corresponding to genomic position 14285-14809 bp) was replaced with the CMV promoter (SEQ ID NO: 2); (c) The fluorescent reporter gene mCherry (SEQ ID NO: 3) was inserted upstream of the polyA signal site (15320 bp) in the 3'UTR region (15171-15358 bp) and linked to the C-terminus of ORF7 (N protein) via a 2A self-cleaving peptide; (d) The modified genome was cloned into the pBAC11 vector, transcribed in vitro, and transfected into Marc-145 cells to rescue the recombinant virus rPRRSV-HA-CMV-mC.

[0006] Furthermore, the genome sequence of the PRRSV HEB1 strain is shown in SEQ ID NO:4.

[0007] Furthermore, the CMV promoter replacement range in step (b) covers the core region of the natural promoter of ORF6.

[0008] Preferably, the amino acid sequence of the 2A self-cleaving peptide in step (c) is as shown in SEQ ID NO:5.

[0009] Furthermore, the pBAC11 vector contains a T7 promoter and an HDV ribozyme sequence, which are used for in vitro transcription to generate viral RNA with precise ends.

[0010] Furthermore, the RNA polymerase used in the in vitro transcription is T7 RNA polymerase, and the transcription reaction conditions include: reacting at 37° C. for 2 hours and adding RNA cap analogs.

[0011] Preferably, the method of transfecting Marc-145 cells is liposome-mediated transfection.

[0012] In a second aspect, the present invention further provides a recombinant PRRSV strain, which is obtained using the construction method of porcine respiratory syndrome virus provided in the first aspect.

[0013] Furthermore, the recombinant PRRSV strain provided by the present invention comprises an HA-tagged GP5 protein, an M protein expression driven by a CMV promoter, and an mCherry-2A-N fusion protein connected to the 3'UTR region.

[0014] Furthermore, the structure of the recombinant PRRSV strain is as follows: 5'UTR-ORF1a / b-ORF2-ORF3-ORF4-[HA]-ORF5-CMV-ORF6-2A-mCherry-polyA-3'UTR.

[0015] Furthermore, the strain expresses mCherry fluorescent protein in infected cells, which can be detected by fluorescence microscopy or flow cytometry.

[0016] In a third aspect, the present invention further provides a vaccine for preventing porcine respiratory and reproductive syndrome, comprising the recombinant PRRSV strain provided in the second aspect, and a pharmaceutically acceptable carrier.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for constructing a live attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine and its application. The PRRSV HEB1 strain genome is amplified by segmented PCR, and a recombinant plasmid containing an HA tag, a CMV promoter, and an mCherry reporter gene is constructed using seamless cloning technology. The recombinant virus, rPRRSV-HA-CMV-mC, is rescued through enzyme linearization, in vitro transcription, and cell transfection. The recombinant PRRSV strain exhibits significant replication delay and reduced titer, resulting in an attenuated phenotype. The recombinant virus is further emulsified with ISA206 oil adjuvant to prepare a vaccine, which exhibits highly effective immunogenicity in piglet immunization and can significantly reduce clinical symptoms, viremia, and lung pathological lesions following PRRSV infection. The vaccine has the potential to be developed into a safe and effective vaccine. DETAILED DESCRIPTION

[0018] The following examples are only used to more clearly illustrate the technical scheme of the present invention and are therefore only used as examples, and cannot be used to limit the scope of the present invention. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, the equalization conversions and modifications made without departing from the spirit and scope of the present invention should all be encompassed within the scope of the present invention. In the examples, if specific conditions are not indicated, they are carried out according to normal conditions or the conditions recommended by the manufacturer. All reagents or instruments that do not indicate the manufacturer are conventional products that can be purchased commercially.

[0019] To better illustrate the present invention, numerous specific details are provided in the following detailed description. Those skilled in the art will appreciate that the present invention can be practiced without certain specific details. In other embodiments, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present invention.

[0020] Example 1: Construction of recombinant plasmid 1.1 Experimental Preparation Before the experiment, prepare the reagents required for the PCR reaction, including high-fidelity DNA polymerase, dNTPs mixture, 10× PCR buffer, and sterile ultrapure water; 1.2 Template preparation Viral RNA of PRRSV HEB1 strain (GenBank EF112447.1) was extracted and full-length cDNA was synthesized by RT-PCR and used as PCR template.

[0021] 1.3 Primer design and segmented amplification The PRRSV HEB1 genome (EF112447.1) was amplified by segmented PCR and modified elements were introduced. Four segments were designed, as shown in Table 1.

[0022] Table 1 PCR primer design

[0023] The PCR reaction program was as follows: 98°C for 30 s; 35× (98°C for 10 s, 62°C for 15 s, 72°C for 1 min / kb); 72°C for 10 min.

[0024] 1.3 Seamless cloning: Stbl3 competent cells were transformed and plated on LB plates containing chloramphenicol (12.5 μg / mL). The four fragments (fragments A, B, C, and D) were separated and purified by 1% agarose gel electrophoresis, mixed at a 1:1:1 molar ratio, and Gibson Assembly Master Mix was added to incubate at 50°C for 60 minutes to complete Gibson assembly. The products were transformed into Stbl3 competent cells, plated on Amp-resistant plates, and cultured for 16 hours. Positive clones were screened and verified by sequencing.

[0025] Example 2: Virus rescue 2.1 Template Linearization Add 1 μg of linearized DNA to 20 μL of in vitro transcription system: 2 μL of T7 RNA polymerase (NEB), 2 μL of 10× transcription buffer, 2 μL of 10 mM cap analog m7GpppG (NEB), 2 μL of 100 mM DTT, 1 μL of RNase inhibitor (40 U / μL), and 2 μL of NTP mix (containing 25 mM ATP, CTP, GTP, and UTP). Incubate the reaction at 37°C in a thermostatted metal bath for 2 hours, gently mixing every 30 minutes. After transcription, add 1 μL of DNase I (RNase-free) and incubate at 37°C for 15 minutes to degrade the DNA template.

[0026] 2.3 RNA Purification: RNA was subsequently purified by LiCl precipitation: 1 / 10 volume of 3M NaOAc (pH 5.2) and 2.5 volumes of pre-chilled anhydrous ethanol were added and precipitated at -20°C for 1 hour. The precipitate was collected by centrifugation at 12,000 rpm for 20 minutes at 4°C, washed twice with 70% ethanol, and dissolved in 50 μL of RNase-free water. RNA integrity was verified by electrophoresis on a 1% agarose gel (expected 15.5 kb band), and the concentration was determined using a Nanodrop (target concentration ≥ 2 μg / μL).

[0027] 2.4 Cell transfection: Marc-145 cells were cultured with The cells were seeded in 6-well plates at a density of 10% fetal bovine serum in DMEM medium at 37°C. Culture for 24 hours until 80% confluency is achieved. Replace the medium with 1 mL of serum-free Opti-MEM® medium before transfection. Prepare the liposome-RNA complex: dilute 5 μg of in vitro transcribed RNA and 10 μL of Lipofectamine 3000 in 125 μL of Opti-MEM®. Incubate at room temperature for 5 minutes, mix, and continue incubation for 20 minutes. Add the complex dropwise to the cells and gently shake to mix. Incubate at 37°C for 6 hours, then replace the medium with 2% FBS.

[0028] 2.5 Virus Harvest: 48 hours after transfection, observe mCherry red fluorescence expression (excitation / emission: 587 / 610 nm) under an inverted fluorescence microscope to confirm successful virus rescue. Collect the cell supernatant and freeze / thaw at -80°C / 37°C three times. Centrifuge at 3000 rpm for 10 minutes at 4°C to remove cell debris. Aliquot the supernatant and store at -80°C as the P0 virus stock.

[0029] 2.5 Analysis of viral replication kinetics The P0 virus was inoculated into a confluent monolayer of Marc-145 cells (T75 culture flask) at an MOI of 1. The virus titer (TCID 50 The results of the determination are shown in Table 2: Table 2 Virus titer determination results at each time point

[0030] p<0.05, p<0.01, p<0.001 (t test); data are mean ± SD (n=3); The above experimental results show that the recombinant virus rPRRSV-HA-CMV-mC exhibits significant replication delay and titer reduction characteristics. Specifically, during the critical replication window of 12 to 36 hours after infection of Marc-145 cells, the titer of the recombinant virus was always significantly lower than that of the wild-type PRRSV HEB1 strain (p < 0.01). This difference reached a peak at 24 hours; by the 48-hour infection node, the wild-type virus reached a replication peak of 10 8.2 TCID 50 / mL, at which point the recombinant virus titer still lagged significantly behind (p<0.001). During the plateau maintenance period (60-72 hours), the titer difference between the two viruses persisted, with the wild-type virus stabilizing at a high plateau, while the recombinant virus remained at a lower level. This sustained decrease in titer indicates that the triple modification of the HA tag insertion, CMV promoter replacement, and mCherry reporter system, while not completely blocking viral replication, significantly weakened the virus's replication efficiency, resulting in a typical attenuated phenotype.

[0031] Example 3: Vaccine preparation 3.1 Adjuvant emulsification and vaccine preparation Take 10 7 The culture supernatant of PFU virus was mixed with ISA206 oil adjuvant (Seppic) at a ratio of 7:3 (v / v). Emulsification was performed using a high-pressure homogenizer (10,000 rpm) for 10 minutes to form a uniform water-in-oil (W / O) emulsion. The emulsion stability test was performed: if one drop of the emulsion remained intact and spherical when added to room temperature water, the emulsion passed. The final vaccine formulation was aliquoted into sterile glass bottles and stored at 4°C until used. The emulsion stability test was performed: if one drop of the emulsion remained spherical when added to water, the emulsion passed. The final vaccine formulation was aliquoted into sterile glass bottles and stored at 4°C until used.

[0032] 3.2 Immunization Procedure: Four-week-old PRRSV-negative piglets (ELISA antibody negative, PCR virus-free) were randomly divided into an immunization group (n=10) and a control group (n=10). The immunization group was injected intramuscularly with 2 mL of vaccine (containing 10 5.0 The control group was injected with an equal amount of PBS. A booster immunization was performed 21 days later.

[0033] 3.3 Immune effect detection 3.3.1 Collect serum before immunization, 2 weeks after the first immunization, and 2 weeks after the booster immunization to test antibody levels; The collected serum was used for the following experiments: ELISA test: Use the PRRSVN protein monoclonal antibody kit (IDEXX) to measure the antibody titer; Virus neutralization test: serum was diluted in multiple ratios and mixed with 100 TCID 50 Virus co-incubation was performed and the neutralization titer was calculated; The experimental results are shown in Table 3: Table 3 Dynamic changes in antibody levels

[0034] The experimental results show that: 7.2±0.8 2 weeks after the first vaccination , which was significantly higher than before immunization (p<0.001). The N protein antibody detected by ELISA is the virus nucleocapsid antibody, which reflects the recognition of the internal antigen of the virus by humoral immunity, indicating that the recombinant virus in the vaccine has stimulated the body's primary immune response and B cells have begun to secrete specific IgG antibodies. The neutralizing antibody titer during the same period was 3.5±0.6 This indicates that vaccine-induced antibodies not only recognize viral antigens but also have the ability to neutralize viral particles and block their ability to infect host cells. The early production of neutralizing antibodies (only 2 weeks) indicates that the vaccine is highly immunogenic and can quickly activate humoral immune pathways.

[0035] Two weeks after booster immunization, the ELISA antibody titer increased to 10.8±1.2 , an increase of about 3.6 times compared to the first vaccination, reflecting the "recall effect" of the secondary immune response - memory B cells rapidly proliferate and differentiate into plasma cells, secrete a large amount of antibodies, and form a higher level of antibody titer. The neutralizing antibody titer simultaneously increased to 6.0±0.9 , an increase of about 2.5 times compared with the first vaccination, indicating that booster immunization effectively enhanced the breadth and strength of neutralizing antibodies, and may recognize more epitopes on the virus surface through cross-reaction, further enhancing the neutralizing ability against wild-type viruses.

[0036] In summary, the recombinant vaccine can induce piglets to produce sustained and efficient specific humoral immune responses through the first-immunization-boosting immunization program. The dynamic growth of ELISA antibodies and neutralizing antibodies forms a causal relationship with the protective effect of the virus attack in Example 3 (such as viremia inhibition and lung lesion reduction), which verifies the immunogenicity and protective efficacy of the vaccine.

[0037] 3.3.2 Evaluation of attack and protection On the 28th day after booster immunization, all piglets were vaccinated intranasally for 10 5.0 TCID 50 The wild-type PRRSV HEB1 strain was divided into two groups. One group underwent clinical symptom scoring and viremia monitoring, and the other group underwent pathological testing. (1) After the infection, the clinical symptom scores (0-6 points) were monitored for 10 consecutive days (n=5): body temperature (>40℃, 1 point), respiratory rate (>50 times / minute, 1 point), mental depression (1 point), etc. The experimental results are as follows: Immunized group: After infection, the body temperature of most piglets remained at 38.5-39.5℃, the respiratory rate was normal, there was no obvious mental depression, and the overall clinical symptom score was ≤1 point. Only a few piglets had a short-term low fever (body temperature 39.8℃, score 1 point).

[0038] Control group: From the second day after the infection, the body temperature of all piglets remained above 40°C (score 1 point), the respiratory rate was above 50 times / min (score 1 point), and the spirit was obviously depressed (score 1 point). The average clinical symptom score was 3 points, and the symptoms lasted until the 10th day after the infection.

[0039] (2) Viremia was monitored for 10 consecutive days after infection (n=5): blood was collected every other day, and viral load was detected by RT-qPCR (primers targeting ORF7 gene). The experimental results are shown in Table 4: Table 4 Viral load test results of piglets after challenge (n=5)

[0040] : p < 0.01 compared with the control group (two-sample t test); :The viral load was below the limit of detection (LOD=2.0 log 10 copies / mL); The above experimental results show that under the attack of wild strains, the immunized group maintained extremely low levels of viral replication throughout the entire process, indicating that the humoral immunity induced by the vaccine and the cellular immunity work synergistically to effectively block the initial spread of the virus infection. At the same time, the peak of viral replication occurred on the 6th day in both the immunized and control groups, but the difference in viral load was obvious, confirming that the vaccine significantly weakened the ability of the virus to proliferate. On days 8-10, the viral load in the immunized group dropped to 2.3±0.2 log on the 8th day. 10 , completely cleared on day 10 (<2.0log 10 ); The control group still maintained a high load, indicating that the vaccine provided by the present invention has the effect of accelerating virus clearance.

[0041] (3) Autopsy analysis (n=5) On the 7th day after challenge, 5 animals / group were euthanized and lung lesion scores were performed (0-4 points) according to the following scoring criteria: 0 points: no lesions; 1 point: <10% lung parenchymal lesions; 2 points: 10-30% lesions; 3 points: 30-50% lesions; 4 points: >50% lesions; In the immunized group, the lung parenchymal lesions of the five piglets were all less than 10%, with an average lesion score of 0.8±0.2 points, mainly manifested as mild interstitial pneumonia.

[0042] Control group: The lung parenchymal lesions of the five piglets autopsied during the same period were all greater than 30%, with an average lesion score of 3.2±0.5 points, showing extensive hemorrhage, consolidation and widening of alveolar septa.

[0043] The above experimental results show that: Piglets in the vaccinated group showed only mild or no clinical symptoms after challenge, while those in the control group developed typical high fever, dyspnea, and depression, indicating that the vaccine-induced immune response effectively suppressed the pathogenicity of the wild-type virus. The significant difference in clinical symptom scores (≤1 in the vaccinated group vs. 3 in the control group) directly reflects the protective efficacy of the vaccine.

[0044] The viral load in the immunized group was always lower than that in the control group after the challenge, and it decreased significantly from the 8th day (p < 0.01), and the virus was completely cleared on the 10th day. This shows that the neutralizing antibodies and cellular immunity induced by the vaccine work together to effectively control the replication and spread of the virus in the body. The viral load in the control group remained high (peak 7.1log 10 copies / mL), confirming the strong pathogenicity of the wild-type virus.

[0045] The lung lesion score in the immunized group (0.8±0.2 points) was significantly lower than that in the control group (3.2±0.5 points), indicating that the vaccine protected the lungs from viral invasion and alleviated typical pathological changes such as interstitial pneumonia. This is consistent with the mechanism by which neutralizing antibodies block viral attachment to host cells and reduce lung tissue damage.

[0046] Combined with the characteristics of reduced replication efficiency of the recombinant virus in Example 2, the vaccine was attenuated through triple transformation (HA tag insertion, CMV promoter replacement, and mCherry reporter system) while retaining immunogenicity, inducing the production of high-titer ELISA antibodies (log210.8) and neutralizing antibodies (log26.0), and ultimately showing good protection in the challenge experiment.

[0047] The above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art will appreciate that the technical solutions of the present invention may be modified or replaced with equivalents without departing from the spirit and scope of the technical solutions of the present invention, and such modifications or equivalents shall be encompassed by the claims of the present invention. Any techniques, shapes, and structures not described in detail herein are well known.

Claims

1. A method for constructing porcine respiratory syndrome virus, characterized in that: The following steps are involved: (a) Using the PRRSV HEB1 strain genome as a template, an HA tag sequence was inserted between nucleotides 45-46 downstream of the start codon of the ORF5 gene coding region, wherein the HA tag sequence is shown in SEQ ID NO: 1; (b) replacing the natural promoter region of the ORF6 gene with a CMV promoter, wherein the CMV promoter sequence is shown in SEQ ID NO: 2; (c) inserting a fluorescent reporter gene mCherry upstream of the polyA signal site in the 3'UTR region, wherein the fluorescent reporter gene mCherry sequence is shown in SEQ ID NO: 3 and is linked to the C-terminus of ORF7 via a 2A self-cleaving peptide; (d) The modified genome was cloned into the pBAC11 vector, transcribed in vitro, and transfected into Marc-145 cells to rescue the recombinant virus rPRRSV-HA-CMV-mC.

2. The method for constructing a porcine respiratory syndrome virus according to claim 1, wherein: The genome sequence of the PRRSV HEB1 strain is shown in SEQ ID NO:

4.

3. The method for constructing a porcine respiratory syndrome virus according to claim 1, wherein: The CMV promoter replacement range in step (b) covers the core region of the natural promoter of ORF6.

4. The method for constructing a porcine respiratory syndrome virus according to claim 1, wherein: The amino acid sequence of the 2A self-cleaving peptide in step (c) is shown in SEQ ID NO:

5.

5. The method according to claim 1, wherein: The RNA polymerase used in the in vitro transcription is T7 RNA polymerase, and the transcription reaction conditions include: reacting at 37° C. for 2 hours and adding RNA cap analogs.

6. The method according to claim 1, wherein: The method for transfecting Marc-145 cells is liposome-mediated transfection.

7. A recombinant PRRSV strain, characterized in that: The recombinant PRRSV strain is constructed by any one of the methods of claims 1-6, and comprises HA-tagged GP5 protein, CMV promoter-driven M protein expression, and mCherry-2A-N fusion protein connected to the 3'UTR region.

8. The recombinant PRRSV strain according to claim 7, characterized in that: The strain expresses mCherry fluorescent protein in infected cells, which is detected by fluorescence microscopy or flow cytometry.

9. A vaccine for preventing porcine respiratory and reproductive syndrome, characterized in that: Comprising the recombinant PRRSV strain according to claim 8, and a pharmaceutically acceptable carrier.

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