A method for constructing a porcine respiratory syndrome virus

Abstract

The application relates to the technical field of virus genetic engineering, and particularly relates to a construction method of a porcine respiratory syndrome virus. The construction method is as follows: taking a PRRSV HEB1 strain genome (GenBank EF112447.1) as a template and modifying the same, then cloning the modified genome into a pBAC11 carrier, carrying out in-vitro transcription and transfecting Marc-145 cells, and rescuing a recombinant virus rPRRSV-HA-CMV-mC. The recombinant PRRSV strain shows significant replication delay and titer reduction, forming an attenuated phenotype. Further, the recombinant virus is emulsified with an ISA206 oil adjuvant to prepare a vaccine, which shows high immunogenicity in piglet immunization, can significantly reduce clinical symptoms, viremia and lung pathological damage after PRRSV infection, and has the potential to be developed into a safe and effective vaccine.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of virus genetic engineering, and particularly relates to a construction method of porcine respiratory syndrome virus. BACKGROUND

[0002] Porcine respiratory syndrome is an infectious disease caused by porcine respiratory syndrome virus, which is extremely harmful to the pig industry. Its main characteristics are sow reproductive disorders and respiratory symptoms in piglets and growing pigs. PRRSV belongs to the Arteriviridae family and has high genetic variability, which poses a great challenge to disease prevention and control. Currently, vaccine development, pathogenesis research, and other aspects of PRRSV rely on effective construction and culture of the virus. Traditional virus construction methods have problems such as complex operation, unstable virus activity, low construction efficiency, and are difficult to meet the needs of scientific research and practical application. With the continuous development of genetic engineering technology, new ideas and methods are provided for the construction of PRRSV. For example, through reverse genetics technology to manipulate the viral genome, it can more accurately construct virus strains with specific biological characteristics. However, the existing construction methods based on genetic engineering still need to be improved in terms of virus rescue efficiency and genetic stability. Therefore, it is of great practical significance to develop an efficient and stable construction method for porcine respiratory syndrome virus.

[0003] Traditional research and control methods have certain limitations, so it is necessary to develop more effective virus construction methods to further study the biological characteristics, pathogenesis, and development of new vaccines of the virus. SUMMARY

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

[0005] To achieve the above technical effects, the present application adopts the following technical solutions:

[0006] Firstly, the present application provides a construction method of porcine respiratory syndrome virus, comprising the following steps:

[0007] (a) using PRRS V HEB1 strain genome (GenBank EF112447.1) as a template, inserting an HA tag sequence between the 45-46th nucleotides downstream of the start codon in the ORF5 gene coding region, the HA tag sequence is shown as SEQ ID NO: 1;

[0008] (b) replacing the natural promoter region (14285-14320bp) of the ORF6 gene (corresponding to genomic position 14285-14809bp) with a CMV promoter (SEQ ID NO: 2);

[0009] (c) inserting a fluorescent reporter gene mCherry (SEQ ID NO: 3) upstream of the polyA signal site (15320bp) in the 3'UTR region (15171-15358bp) and linking it to the C-terminus of ORF7 (N protein) via a 2A self-cleaving peptide;

[0010] (d) cloning the modified genome into a pBAC11 vector, in vitro transcribing and transfecting Marc-145 cells to rescue the recombinant virus rPRRSV-HA-CMV-mC.

[0011] Further, the PRRSV HEB1 strain genome sequence is shown as SEQ ID NO: 4.

[0012] Further, the CMV promoter replacement range in step (b) covers the core region of the ORF6 natural promoter.

[0013] Preferably, the amino acid sequence of the 2A self-cleaving peptide in step (c) is shown as SEQ ID NO: 5.

[0014] Further, the pBAC11 vector comprises a T7 promoter and an HDV ribozyme sequence for in vitro transcription to generate virus RNA with precise ends.

[0015] Further, the RNA polymerase used for in vitro transcription is T7 RNA polymerase, and the transcription reaction conditions include: 37℃ reaction for 2 hours, and adding RNA cap analogs.

[0016] Preferably, the method of transfecting Marc-145 cells is liposome-mediated transfection.

[0017] In a second aspect, the present application also provides a recombinant PRRSV strain obtained by the construction method of the porcine respiratory syndrome virus provided in the first aspect.

[0018] Further, the recombinant PRRSV strain provided by the present application comprises a GP5 protein with an HA tag, a CMV promoter-driven M protein expression, and a mCherry-2A-N fusion protein connected to the 3'UTR region.

[0019] Further, the structure of the recombinant PRRSV strain is as follows:

[0020] 5'UTR-ORF1a / b-ORF2-ORF3-ORF4-[HA]-ORF5-CMV-ORF6-2A-mCherry-polyA-3'UTR.

[0021] Further, the strain expresses mCherry fluorescent protein in infected cells, which can be detected by fluorescence microscopy or flow cytometry.

[0022] In a third aspect, the application also provides a vaccine for preventing porcine reproductive and respiratory syndrome, comprising the recombinant PRRSV strain provided in the foregoing second aspect, and a pharmaceutically acceptable carrier.

[0023] Compared with the prior art, the application has the following beneficial effects:

[0024] The application provides a construction method and application of a live attenuated vaccine of porcine reproductive and respiratory syndrome virus (PRRSV). The genome of PRRSV HEB1 strain is amplified by segment PCR, a recombinant plasmid containing an HA tag, a CMV promoter and an mCherry reporter gene is constructed by using a seamless cloning technique, and a recombinant virus rPRRSV-HA-CMV-mC is rescued by enzyme cutting linearization, in vitro transcription and cell transfection. The recombinant PRRSV strain shows significant replication delay and titer reduction, forming an attenuated phenotype. Further, the recombinant virus is emulsified with ISA206 oil adjuvant to prepare a vaccine, which shows high immunogenicity in piglet immunization, can significantly reduce clinical symptoms, viremia and lung pathological damage after PRRSV infection, and has the potential to develop into a safe and effective vaccine. DETAILED DESCRIPTION

[0025] The following examples are only used to more clearly illustrate the technical solutions of the application, and therefore only serve as examples, but cannot be used to limit the protection scope of the application. Any equivalent modifications and substitutions of the examples described below made by those skilled in the art are also within the scope of the application. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the application should be encompassed in the scope of the application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. All reagents or instruments not mentioned by the manufacturer are conventional products that can be purchased on the market.

[0026] In order to better illustrate the application, numerous specific details are given in the specific embodiments below. Those skilled in the art should understand that the application can also be implemented without some specific details. In some embodiments, methods, means, apparatus and steps that are well known to those skilled in the art are not described in detail, in order to highlight the main idea of the application.

[0027] Example 1: Construction of recombinant plasmid

[0028] 1.1 Preparation of experiment

[0029] Before the experiment, prepare the reagents required for PCR reaction, including high-fidelity DNA polymerase, dNTPs mixture, 10x PCR buffer, and sterilized ultrapure water, etc.

[0030] 1.2 Preparation of template

[0031] Extract the viral RNA of PRRSV HEB1 strain (GenBank EF112447.1), and synthesize full-length cDNA by RT-PCR as the PCR template.

[0032] 1.3 Primer design and segment amplification

[0033] Amplify PRRSV HEB1 genome (EF112447.1) by segment PCR and introduce modification elements. Design four segments, see Table 1.

[0034] Table 1. PCR primer design

[0035]

[0036] The PCR reaction program is: 98℃ 30s; 35x (98℃ 10s, 62℃ 15s, 72℃ 1min / kb); 72℃ 10min.

[0037] 1.3 Seamless cloning:

[0038] Transform Stbl3 competent cells and spread on LB plates containing chloramphenicol (12.5 μg / mL). After 1% agarose gel electrophoresis separation and purification, mix the four segments (segments A, B, C, D) at a molar ratio of 1:1:1, add Gibson Assembly Master Mix, incubate at 50℃ for 60 minutes to complete Gibson assembly, and then transform the product into Stbl3 competent cells. Spread on Amp-resistant plates and culture for 16 hours. Screen positive clones and verify by sequencing.

[0039] Example 2: Virus rescue

[0040] 2.1 Linearization of template

[0041] Add 1 μg of linearized DNA to a 20 μL in vitro transcription system containing 2 μL T7 RNA polymerase (NEB), 2 μL 10× transcription buffer, 2 μL 10 mM cap analog m7GpppG (NEB), 2 μL 100 mM DTT, 1 μL RNase inhibitor (40 U / μL), and 2 μL NTP mixture (containing 25 mM ATP, CTP, GTP, and UTP). Incubate the reaction system in a 37°C metal bath for 2 hours, gently mixing every 30 minutes. After transcription, add 1 μL of DNase I (RNase-free) and treat at 37°C for 15 minutes to degrade the DNA template.

[0042] 2.3 RNA Purification: RNA was then purified using LiCl precipitation: 1 / 10 volume of 3M NaOAc (pH 5.2) and 2.5 volumes of pre-cooled anhydrous ethanol were added, and the mixture was 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 then dissolved in 50 μL of RNase-free water. RNA integrity was verified by 1% agarose gel electrophoresis (expected 15.5 kb band), and the concentration was determined using Nanodrop (target concentration ≥2 μg / μL).

[0043] 2.4 Cell transfection:

[0044] Marc-145 cells were used Cells were seeded at a density of 6-well plates in DMEM medium containing 10% fetal bovine serum and incubated at 37°C. Culture for 24 hours until 80% confluence. Replace 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 separately 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 with maintenance medium containing 2% FBS.

[0045] 2.5 Virus Harvest:

[0046] Forty-eight hours post-transfection, mCherry red fluorescence expression (excitation / emission: 587 / 610 nm) was observed under an inverted fluorescence microscope, confirming successful virus rescue. Cell supernatant was collected, and after three cycles of freezing at -80℃ and thawing at 37℃, cell debris was removed by centrifugation at 3000 rpm for 10 minutes at 4℃. The supernatant was aliquoted and stored at -80℃ as a reserve of P0 generation virus.

[0047] 2.5 Viral Replication Dynamics Analysis

[0048] P0 generation virus was inoculated to monolayer Marc-145 cells (T75 culture flask) at MOI = 1, and the virus titers (TCID 50 / mL) at various time points were determined as shown in Table 2:

[0049] Table 2 Virus titers at various time points

[0050]

[0051] p<0.05, p<0.01, p<0.001 (t test); data are mean ± SD (n = 3);

[0052] The above experimental results show that the recombinant virus rPRRSV-HA-CMV-mC exhibits significant replication delay and titer reduction characteristics. Specifically, within the 12 to 36 hour critical replication window period after infection of Marc-145 cells, the titer of the recombinant virus is always significantly lower than that of the wild-type PRRSV HEB1 strain (p<0.01). This difference reaches a peak at 24 hours; by the 48 hour infection point, the wild-type virus reaches a replication peak of 10 8.2 TCID 50 / mL, at which time the titer of the recombinant virus is still significantly lagging (p<0.001). In the plateau maintenance period (60-72 hours), the titer difference between the two viruses continues to exist, with the wild-type being stably at a high level plateau, while the recombinant virus still maintains at a lower level. This persistent titer reduction indicates that the triple modification of the inserted HA tag, CMV promoter replacement, and mCherry reporter system has not completely blocked viral replication, but significantly weakened the replication efficiency of the virus, forming a typical attenuated phenotype.

[0053] Example 3: Vaccine preparation

[0054] 3.1 Adjuvant emulsification and vaccine formulation

[0055] Take 10 7The culture supernatant of PFU virus was mixed with ISA206 oil adjuvant (Seppic) at a ratio of 7:3 (v / v). The high-pressure homogenizer (10,000 rpm) was used for emulsification for 10 minutes to form a uniform water-in-oil (W / O) emulsion. The emulsion stability test was performed: 1 drop of emulsion was dropped into room temperature water, and it was qualified if it remained spherical and did not spread. The final vaccine preparation was divided into sterile glass bottles and stored at 4°C for standby. The emulsion stability test was performed: 1 drop was added to water, and it was qualified if it remained spherical and did not spread. The final vaccine preparation was divided into sterile glass bottles and stored at 4°C for standby.

[0056] 3.2 Immunization procedure:

[0057] Four-week-old PRRSV-negative piglets (ELISA antibody-negative, PCR virus-free) were selected and randomly divided into an immunization group (n=10) and a control group (n=10). The immunization group was injected with 2 mL of vaccine (containing 10 5.0 PFU virus) intramuscularly, and the control group was injected with an equal amount of PBS. Booster immunization was performed 21 days later.

[0058] 3.3 Detection of immune effect

[0059] 3.3.1 Serum was collected before immunization, 2 weeks after the first immunization, and 2 weeks after the booster immunization, and the antibody level was detected.

[0060] The collected serum was subjected to the following experiments:

[0061] ELISA detection: PRRSVN protein monoclonal antibody kit (IDEXX) was used to detect the antibody titer;

[0062] Virus neutralization test: The serum was diluted by 2-fold, and then incubated with 100 TCID 50 of virus, and the neutralization titer was calculated;

[0063] The experimental results are shown in Table 3:

[0064] Table 3 Dynamic change of antibody level

[0065]

[0066] The experimental results show that:

[0067] 2 weeks after the first immunization, the antibody titer was 7.2±0.8 , which was significantly higher than that before immunization (p<0.001). The N protein antibody detected by ELISA is a virus nucleocapsid antibody, reflecting the recognition of humoral immunity to internal antigens of the virus, indicating that the recombinant virus in the vaccine has triggered the initial immune response of the body, and B cells begin to secrete specific IgG antibodies. The neutralizing antibody titer at the same period was 3.5±0.6 , indicating that the vaccine-induced antibodies not only can recognize viral antigens, but also have the ability to neutralize viral particles and block their infection of host cells. The early production of neutralizing antibodies (only 2 weeks) shows that the vaccine has strong immunogenicity and can quickly activate the humoral immune pathway.

[0068] The ELISA antibody titer increased to 10.8 ± 1.2 , about 3.6 times higher than after the first immunization, reflecting the "recall effect" of the secondary immune response - memory B cells rapidly proliferate and differentiate into plasma cells, secreting large amounts of antibodies, forming higher levels of antibody titers. The neutralizing antibody titer also increased to 6.0 ± 0.9 , about 2.5 times higher than after the first immunization, indicating that the booster immunization effectively enhanced the breadth and strength of neutralizing antibodies, possibly by cross-reacting to recognize more epitopes on the surface of the virus, further enhancing the neutralization ability against wild-type virus.

[0069] In summary, the recombinant vaccine can induce a sustained and efficient specific humoral immune response in piglets through the first-boost immunization program. The dynamic growth of ELISA antibodies and neutralizing antibodies is causally related to the protection effect (such as suppression of viremia and reduction of lung lesions) in the challenge protection evaluation in Example 3, verifying the immunogenicity and protective efficacy of the vaccine.

[0070] 3.3.2 Challenge protection evaluation

[0071] On day 28 after the booster immunization, all piglets were inoculated intranasally with 10 5.0 TCID 50 of wild-type PRRSV HEB1 strain, divided into two groups, one group for clinical symptom scoring and viremia monitoring, and the other group for pathological examination:

[0072] (1) Clinical symptom scoring (0-6 points) was monitored for 10 consecutive days after challenge (n=5): body temperature (>40℃, 1 point), respiratory rate (>50 times / min, 1 point), depression (1 point), etc. The experimental results are as follows:

[0073] Immune group: After challenge, most piglets maintained a body temperature of 38.5-39.5℃, with normal respiratory rate and no obvious depression, with overall clinical symptom score ≤1 point, only a few piglets had transient low fever (body temperature 39.8℃, score 1 point).

[0074] Control group: From the second day after challenge, all piglets had a body temperature of >40℃ (score 1 point), respiratory rate of >50 times / min (score 1 point), and obvious depression (score 1 point), with an average clinical symptom score of 3 points, and the symptoms persisted until day 10 after challenge.

[0075] (2) Virus load monitoring for 10 consecutive days after challenge (n=5): blood samples were collected every other day, and virus load was detected by RT-qPCR (primers targeting ORF7 gene). The results are shown in Table 4:

[0076] Table 4. Virus load detection results of piglets after challenge (n=5)

[0077]

[0078] : p<0.01 compared with the control group (two-sample t-test); : virus load was lower than the detection lower limit (LOD=2.0 log 10 copies / mL);

[0079] The above experimental results show that under the attack of the wild strain, the immune group maintains a very low virus replication level throughout, indicating that the vaccine-induced humoral immunity and cellular immunity synergistically act to effectively block the spread of initial infection. At the same time, at the peak of virus replication, the peak of both the immune group and the control group appeared on the 6th day, but the virus load was significantly different, confirming that the vaccine significantly weakens the virus proliferation ability. On the 8th-10th day, the load of the immune group decreased to 2.3±0.2 log 10 on the 8th day and was completely cleared (<2.0 log 10 ) on the 10th day; the control group still had a high load, indicating that the vaccine provided by the present application has the effect of accelerating virus clearance.

[0080] (3) Necropsy analysis (n=5)

[0081] Five pigs / group were euthanized on the 7th day after challenge, and lung lesion scores (0-4 points) were evaluated according to the following criteria:

[0082] 0 points: no lesion; 1 point: <10% lung parenchyma lesion; 2 points: 10-30% lesion; 3 points: 30-50% lesion; 4 points: >50% lesion;

[0083] Immune group: the lung parenchyma lesions of the 5 piglets were all <10%, with an average lesion score of 0.8±0.2 points, mainly showing mild interstitial pneumonia.

[0084] Control group: the lung parenchyma lesions of the 5 piglets necropsied at the same time were all >30%, with an average lesion score of 3.2±0.5 points, showing extensive hemorrhage, consolidation, and widened alveolar septum.

[0085] The above experimental results show that:

[0086] The immunized piglets only showed slight or no clinical symptoms after challenge, while the control group showed typical high fever, dyspnea and depression, indicating that the immune response induced by the vaccine can effectively inhibit the pathogenicity of the wild-type virus. The significant difference in clinical symptom score (≤1 point for the immunized group vs. 3 points for the control group) directly reflects the protective efficacy of the vaccine.

[0087] The viral load of the immunized group was always lower than that of the control group after challenge, and significantly decreased (p<0.01) from the 8th day, and the virus was completely cleared by the 10th day. This indicates that the synergistic effect of neutralizing antibodies and cellular immunity induced by the vaccine effectively controls the replication and spread of the virus in the body. The viral load of the control group remained at a high level (peak 7.1 log 10 copies / mL), which confirms the strong pathogenicity of the wild-type virus.

[0088] The lung lesion score of the immunized group (0.8±0.2) was significantly lower than that of the control group (3.2±0.5), indicating that the vaccine can protect the lungs from viral invasion and reduce typical pathological changes such as interstitial pneumonia. This is consistent with the mechanism of neutralizing antibodies blocking viral adsorption to host cells and reducing lung tissue damage.

[0089] In combination with the reduced replication efficiency of the recombinant virus in Example 2, the vaccine achieves attenuation through triple modification (insertion of HA tag, replacement of CMV promoter, and mCherry reporter system) while retaining immunogenicity, inducing high-titer ELISA antibodies (log210.8) and neutralizing antibodies (log26.0), and ultimately showing good protective effect in the challenge experiment.

[0090] The above examples are only used to illustrate the technical solutions of the present application and not to limit it. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the purpose and scope of the present application, and they should be covered in the scope of the claims of the present application. The technical, shape and structure parts not described in detail in the present application are well-known technologies.

Claims

1. A method for constructing porcine respiratory syndrome virus, characterized in that, Includes the following steps: (a) Using the PRRSV HEB1 strain genome as a template, insert a nucleotide sequence of HA tag between nucleotides 45-46 downstream of the start codon in the coding region of the ORF5 gene, the nucleotide sequence of which is shown in SEQ ID NO:

1. (b) The natural promoter region of the ORF6 gene is replaced with the CMV promoter, the nucleotide sequence of which is shown in SEQ ID NO:2; (c) Insert a fluorescent reporter gene mCherry upstream of the polyA signal site in the 3'UTR region. The nucleotide sequence of the fluorescent reporter gene mCherry is shown in SEQ ID NO:3, and it 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 as described in claim 1, characterized in that: The nucleotide sequence of the PRRSVHEB1 strain genome is shown in SEQ ID NO:

4.

3. The method for constructing a porcine respiratory syndrome virus as described in claim 1, characterized in that: In step (b), the CMV promoter replacement range covers the core region of the ORF6 natural promoter.

4. The method for constructing a porcine respiratory syndrome virus as described in claim 1, characterized in that: 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, characterized in that: The RNA polymerase used for in vitro transcription is T7 RNA polymerase, and the transcription reaction conditions include: reaction at 37°C for 2 hours, and addition of an RNA cap analog.

6. The method according to claim 1, characterized in that: 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 a GP5 protein with an inserted HA tag, an M protein expressed by the CMV promoter, and an mCherry-2A-N fusion protein.

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 the prevention of porcine respiratory and reproductive syndrome, characterized in that, It includes the recombinant PRRSV strain of claim 8 and a pharmaceutically acceptable vector.

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