Construction and application of rHP-PRRSV-NADC30 Nsp230+131 recombinant strain

Abstract

The invention discloses a method for preparing an rHP-PRRSV-NADC30 Nsp230 + 131 recombinant strain and an application of the rHP-PRRSV-NADC30 Nsp230 + 131 recombinant strain. According to the present invention, by using the reverse genetic manipulation technology, the virus strain is successfully constructed and saved, and the inherent 30 deletion of HP-PRRSV (line 8.7) and the 131 deletion of NADC30-like (line 1.8) are simultaneously retained on the PRRSV Nsp2 protein of the virus strain; the modified virus is inoculated to Marc-145 cells for subculture, and experimental results show that the modified virus can induce cytopathy. The obtained recombinant strain shows a stable passage capability in a Marc-145 cell. In addition, clinical animal experiments further verify the safety and effectiveness of the recombinant strain. No obvious pathological reaction and abnormal clinical manifestation are observed in an experimental animal inoculated with the recombinant strain, which indicates that the recombinant strain has good biological safety. Meanwhile, experimental animals show that the recombinant strain can stimulate effective immune response, an effective immune protection effect is provided for multilineage PRRSV infection, and potential vaccine development prospects are shown.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of veterinary biological products, and specifically relates to a strain taking HP-PRRSV (lineage 8.7) as an infectious cloning skeleton, using reverse genetics technology to recombine the characteristic fragment of Nsp2 gene of NADC30-like strain (lineage 1.8) into the skeleton, and finally obtaining a recombinant virus which retains the inherent △30 deletion of lineage 8.7 and the △131 deletion of lineage 1.8 on the Nsp2 protein at the same time, exhibits new biological characteristics and potential application value. In the field of veterinary biological products, the construction of this recombinant strain provides a new idea and tool for the prevention and control of PRRS, and is expected to play an important role in actual production and improve the resistance to multiple lineage strains. BACKGROUND

[0002] Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most important infectious diseases in the global pig breeding industry, which brings huge economic losses to the global pig industry every year. PRRSV is mainly divided into PRRSV-1 type and PRRSV-2 type, and PRRSV-2 type is mainly in China, among which Lineage 1, 3, 5 and 8 are the main subtypes. Since PRRSV was first discovered in China in 1996, the virus has undergone multiple mutations, especially the HP-PRRSV that appeared in 2006, which led to a large-scale outbreak of the disease in the Chinese breeding industry, causing serious economic losses. In 2014, PRRSV NADC30-like subtype was first isolated in China. At present, the PRRSV strains prevalent in China mainly include Lineage 8.7 and Lineage 1.8, among which the representative strain of Lineage 8.7 is the derivative strain of HP-PRRSV, and the representative strain of Lineage 1.8 is NADC30-like strain.

[0003] In the face of the ever-changing and complex epidemic situation of PRRSV, the immunization effect of traditional vaccines is greatly challenged, and it is difficult to effectively resist the infection of multiple spectrum strains. Different subtypes of PRRSV have significant differences in antigenicity, pathogenicity and other aspects, which makes it difficult for a single strain vaccine to provide broad protection. Therefore, in order to cope with the challenge of multiple spectrum strains in the actual production, it is particularly important to develop a vaccine or biological product that can resist different spectrum strains at the same time, which is of great significance to guarantee the healthy development of China's pig breeding industry and reduce economic losses. Based on this background, the present application successfully constructs a recombinant strain with new biological characteristics through reverse genetics technology, providing a new strategy for the prevention and control of PRRSV. SUMMARY

[0004] Based on this, the present application proposes a construction and application of a rHP-PRRSV-NADC30 Nsp2△30+△131 recombinant strain.

[0005] To solve the above technical problems, the present application adopts the following technical solutions: A rHP-PRRSV-NADC30 Nsp2△30+△131 recombinant strain, named FX-2025-1.8 strain, is characterized in that a recombinant strain containing HP-PRRSV (deletion of 30 amino acids) and NADC30-Like PRRSV (deletion of 131 amino acids) is modified by reverse genetics technology The application of reverse genetics technology to the construction of recombinant PRRSV is as follows: (1) Extract the HP-PRRSV strain virus liquid, obtain the full length by using a nucleic acid extraction kit, and use specific primers for PCR amplification to obtain the linage 8.7 strain full genome fragment; (2) The gene fragment plasmid is treated with Pac I, BstB I, AfI II, Asc I and Mlu I endonuclease, and the gene fragment is connected into a full-length fragment by enzyme digestion and homologous recombination technology, and then inserted into the PSK II vector; (3) Under water bath conditions, the plasmid is verified by EcoR I single enzyme digestion, and the full-length verification of the plasmid is carried out to ensure the correct length of the plasmid; the primer sequences used in steps (1) to (4) are as follows:

[0006] The FX-2025 strain obtained is modified, the partial Nsp2 of the lineage 1.8 strain is replaced with the FX-2025 to synthesize a recombinant strain with characteristics of both lineage 1.8 and lineage 8.7 strains.

[0007] The application further discloses application of the rHP-PRRSV-NADC30 Nsp2△30+△131 recombinant strain in cross-protection effect in a clinical animal experiment. The experimental results show that immunization of the recombinant strain can provide 100% survival protection for the lineage 1.8 and the lineage 8.7 (the survival rate of the lineage 8.7 challenge control group is 20%, and the survival rate of the lineage 1.8 challenge control group is 80%). In addition, after challenge, the indicators such as the body temperature and the clinical symptoms of the piglets in the immunization group are significantly better than those in the non-immunization group, which shows that the recombinant strain has a significant cross-protection effect. At the same time, the experimental animals immunized with the recombinant strain have stable weight gain, and no obvious weight loss phenomenon occurs, which further verifies the reliability of the immunoprotective effect, and provides solid data support for subsequent clinical application.

[0008] The rHP-PRRSV-NADC30 Nsp2△30+△131 recombinant strain disclosed in the application has the following positive effects compared with the prior art: (1) Better resistance to possible multi-lineage strains in real production conditions.

[0009] (2) Multi-lineage protection ability and good immunoprotective ability.

[0010] (3) Reduction of economic demand in real production, making one-shot full-immunization possible in the future. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 A schematic diagram of a plasmid map of the FX-2025 reverse genetic vector platform in Embodiment 1 is shown in the figure. Figure 2 A gel electrophoresis map for verification of double enzyme digestion of the PRRSV reverse genetic vector plasmid constructed in Embodiment 1 is shown in the figure. Figure 3 Appearance of lesions of the reverse genetic vector in Embodiment 1 in marc-145 cells (left side: blank control group, right side: experimental group) Figure 4 A schematic diagram of a plasmid map of the FX-2025-1.8 recombinant strain in Embodiment 2 is shown in the figure. Figure 5 A base deletion alignment map of the lineagel.8 and the lineage 8 amplified in Embodiment 2 is shown in the figure. Figure 6 A comparison chart of the body temperature of the immunization group and the control group in the clinical protection experiment in Embodiment 3 is shown in the figure. Figure 7 A comparison chart of the average daily weight gain of the immunization group and the control group in the clinical protection experiment in Embodiment 3 is shown in the figure. Figure 8Figure 3. Survival rate comparison between the immunized group and the control group in the 3 clinical protection experiments. DETAILED DESCRIPTION

[0012] The present application is described below through specific embodiments. Unless otherwise specified, the technical means used in the present application are methods known to those skilled in the art. In addition, the embodiments are understood to be illustrative rather than limiting the scope of the present application, and the essence and scope of the present application are limited only by the claims. For those skilled in the art, various changes or modifications to the material components and amounts in these embodiments without departing from the essence and scope of the present application also fall within the protection scope of the present application.

[0013] In the following examples, the general experimental methods refer to the "Molecular Cloning: A Laboratory Manual" 3rd edition (Beijing: Science Press, 2002) edited by Sambrook et al., and the use of instruments refers to the instrument operation manual.

[0014] In the embodiments of the present application, virus rescue obtained FX-2025 strain (obtained strain of the present application) and FX-2025-1.8 (protective strain obtained by the present application). The cells are BHK-21 cell line, Marc-145 cell line and primary alveolar macrophage PAM (commercially available).

[0015] In the embodiments of the present application, the DH5α competent cells were purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0016] In the embodiments of the present application, other reagents used: RNase Free H2O, trypsin cell digestion solution (phenol red) were purchased from Solarbio Company; TRIpure Reagent total RNA extraction reagent was purchased from Aidley Biological Company; 2xPrimeSTARMAX DNA Polymearse, T4 ligase, T cloning kit were all purchased from TAKARA Company; One Step Clone Kit homologous recombination reagent was purchased from Beijing Quanshi Gold Biological Co., Ltd.; DMEM culture medium was purchased from HyClone Biological Chemicals Co., Ltd.; Fetal bovine serum was purchased from Sigma Company; DNA restriction endonuclease, LipofectamineTM3000 Transfection Reagent were purchased from Thermo Fisher Company; EMBODIMENT

[0017] Based on the TJ1701 strain (known), the Marc-145 cell in vitro passage adapted reverse genetic vector strain FX2025 strain was obtained.

[0018] 1.1 The results of the schematic diagram of the application of Snapgene software to simulate the construction of FX2025 reverse genetic vector are shown inFigure 1 .

[0019] 1.2 Primer Design for Constructing the FX2025 Reverse Genetic Vector Using the snapgene software, the TJ1701 strain was divided into four parts based on the simulated plasmid diagram for amplification primer design. Specific information on primer construction is shown in Table 1.

[0020]

[0021] 1.3 Construction methods for reverse genetics vectors First, the full-length provirus was obtained by PCR amplification of the FX-2025-A, FX-2025-B, FX-2025-C, and FX-2025-D fragments, and then ligated into the PSKII vector through homologous recombination.

[0022] 1.3.1 PCR amplification method and system for the target fragment In this invention, all target fragments were amplified using PCR. The PCR amplification system and procedure are shown in Tables 2 and 3. The commercially available kit was the 2×PrimeSTAR MAX DNA Polymearse (purchased from TAKARA).

[0023]

[0024] Using the above method, we can obtain Figure 1 The images show fragments of the original strain's full-length sequence assembled. Electrophoresis was performed on a 1% large-well gel for 35 min. Enzyme-digested bands were separated by agarose gel electrophoresis. The target band was cut and purified by gel extraction. The amplified target fragment was stored at -20℃.

[0025] 1.3.2 Vector enzyme digestion and homologous recombination methods and systems All vector digestion and homologous recombination methods and systems in this invention are performed as follows: First, the PSKⅡ vector is prepared by double digestion with PacⅠ and MluⅠ to prepare a linearized vector; the specific double digestion system is shown in Table 4.

[0026]

[0027] Following the above system, enzyme digestion was performed at 37℃ for 1 hour. The digested bands were separated by agarose gel electrophoresis, and the target band was excised and purified by gel recovery. The linearized PSKII vector was obtained, with a measured concentration of 100 ng / μl, and stored at -20℃.

[0028] Linearized fragments FX-2025-A, FX-2025-B, FX-2025-C and FX-2025-D were subjected to homologous recombination with linearized vector PSK II (One Step Clone Kit). The specific system is shown in Table 5.

[0029]

[0030] After the system was placed at 50°C for 30 min, it was immediately placed on ice. Independent colonies were picked and purified using universal primers for bacterial liquid PCR detection. PCR-positive bacteria were selected for overnight expansion of the bacterial liquid, and plasmids were extracted and subjected to double enzyme digestion using Asc I and BSTb I. The digested bands were then subjected to 1% agarose gel electrophoresis. The correct plasmids were reserved at -20°C 1.4 Rescue of reverse genetic vectors on MARC-145 cells BHK-21 cells were first seeded in a 6-well cell culture plate at a density of 6 x 10 5 cells / well in DMEM medium containing 10% FBS and incubated at 37°C in a 5% CO2 incubator until the cell density reached about 80%. Cell transfection was performed according to the LipofectamineTM3000 Transfection Reagent instructions.

[0031] Transfection method: first, 2 μg of plasmid, 4 μL of P3000 plasmid and 50 μL of OPTI-MEM reagent were mixed to form A liquid, and then 3 μL of Lip3000 and 50 μL of DMEM were mixed to form B liquid. Subsequently, A liquid and B liquid were mixed and incubated at 25°C for 15 min. Finally, they were added to BHK-21 cells. The transfection system is shown in Table 6.

[0032] After 48 h of cell transfection, the 6-well plate was sealed and frozen at -80°C. After repeated freezing and thawing twice, the entire cell suspension was obtained, centrifuged at 12000 x g for 5 min, and the transfection supernatant was collected.

[0033]

[0034] MARC-145 cells were first seeded in a 6-well cell culture plate at a density of 6 x 105 cells / well in DMEM medium containing 10% FBS and incubated at 37°C in a 5% CO2 incubator. After the MARC-145 cells adhered, 500 μL of transfection supernatant was added to the MARC-145 cells, incubated for 1 h, and then the supernatant was discarded. DMEM medium containing 2% FBS was added for continued culture.

[0035] The rescue results of Marc-145 cells are shown in Table 7. Figure 3Observing the lesions proves that the rescue was successful.

[0036] 1.6 Determination of growth characteristics of Marc-145 cell culture strains First, the TCID50 titer of the fifth-generation reverse genetics vector was determined. 50 The detection method is as follows: Marc-145 cells were pre-cultured in DMEM medium containing 10% FBS at a concentration of 5 × 10⁶ cells / year. 4 Cells were seeded at a density of approximately 80% per well in 96-well cell culture plates and incubated at 37°C in a 5% CO2 incubator. The medium was then replaced with DMEM containing 2% FBS. The viral stock solution was diluted 10, 100, 1000, 10000, 100000, and 1000000 times. Three replicates of each dilution were seeded into 96 wells. After 1 hour of adsorption, the medium was replaced with 200 μL of fresh DMEM containing 2% FBS. After 72 hours, the wells showing cytopathic effects were counted, and the TCID50 viral titer of the reverse genetic vector was calculated. Example

[0037] The recombinant vaccine candidate strain FX-2025-1.8 was obtained by in vitro passage in Marc-145 cells using the FX-2025 reverse genetic vector. 2.1 A schematic diagram of the construction of recombinant plasmids using Snapgene software is shown below. Figure 4 ; 2.2 Primer Design for Constructing Recombinant Plasmids Primers were designed to amplify the recombinant portion using the snapgene software based on the simulated plasmid diagram. Detailed information on primer construction is shown in Table 7.

[0038] 2.3 Construction methods for recombinant plasmids First, fragment 1.8 was amplified by PCR, and then ligated into the FX-2025 vector via homologous recombination.

[0039] 2.3.1 PCR amplification method and system for the target fragment In this invention, all target fragments were amplified using PCR. The PCR amplification system and procedure are shown in Tables 8 and 9. The commercially available kit used was the 2×PrimeSTAR MAX DNA Polymearse (purchased from TAKARA).

[0040] The 1.8 fragment can be obtained by the above method. The gel electrophoresis is performed for 35 min by using a macroporous 1% gel. The enzyme cutting band is separated by agarose gel electrophoresis. The target band is cut and purified by gel recovery. The target fragment obtained by amplification is stored at -20°C.

[0041] 2.3.2 Vector enzyme cutting and homologous recombination method and system thereof All the vector enzyme cutting and homologous recombination method and system thereof in the present application are performed in the following manner. First, the reverse genetic vector is double-enzyme cut by Pac I and Bstb I to prepare a linearized vector. The specific system of double enzyme cutting is shown in Table 10.

[0042]

[0043] According to the above system, the enzyme cutting is performed at 37°C for 1 h. The enzyme cutting band is separated by agarose gel electrophoresis. The target band is cut and purified by gel recovery. The linearized reverse genetic vector is obtained. The concentration is measured to be 200 ng / μl. The linearized vector is stored at -20°C.

[0044] The linearized fragments PF, EGFP and PR are subjected to homologous recombination (One Step CloneKit) with the linearized vector. The specific system is shown in Table 11.

[0045]

[0046] After the system is placed at 50°C for 30 min, it is immediately placed on ice. The transformation is performed into DH5α. The independent colonies are picked and cultured. The universal primer is used for bacterial liquid PCR detection. The PCR positive bacteria are selected for overnight expansion of the bacterial liquid. The plasmid is extracted. The double enzyme cutting verification is performed. Then, the enzyme cutting band is correctly reserved on the 1% agarose gel electrophoresis. The correct plasmid is stored at -20°C. 2.4 Rescue of reverse genetic vector on MARC-145 cells First, the BHK-21 cells are inoculated in a 6-well cell culture plate at a density of 6×105 cells / well by using DMEM medium containing 10% FBS. The plate is placed in a 37°C, 5% CO2 incubator for culture until the cell density reaches about 80%. The cell transfection is performed according to the LipofectamineTM3000 Transfection Reagent instruction.

[0047] Transfection method: first, 2 μg of plasmid, 4 μL of P3000 plasmid and 50 μL of OPTI-MEM reagent are mixed to form A liquid. Then, 3 μL of Lip3000 and 50 μL of DMEM are mixed to form B liquid. Subsequently, A liquid and B liquid are mixed. After 25°C for 15 min, the BHK-21 cells are added. The transfection system is shown in Table 12.

[0048] 48 hours after cell transfection, the 6-well plate was sealed and frozen at -80°C. After two freeze-thaw cycles, all cell suspension was collected, centrifuged at 12000×g for 5 min, and the transfection supernatant was collected.

[0049]

[0050] MARC-145 cells were seeded at a rate of 6 × 10⁵ cells / well in 2 mL of DMEM medium containing 10% FBS and incubated at 37°C with 5% CO₂. After the MARC-145 cells adhered, 500 μL of transfection supernatant was transferred onto the MARC-145 cells and incubated for 1 h. The supernatant was then discarded, and DMEM medium containing 2% FBS was added for further culture.

[0051] After the recombinant virus was stably passaged and inherited, its recombinant fraction was amplified by PCR and compared. Figure 5 . Example

[0052] Immunoprotective efficacy of recombinant strains 3.1 Detection of the median infectivity of the recombinant FX-2025-1.8 strain First, the TCID50 titer of the 5th generation FX-2025-1.8 was determined. TCID 50 The detection method is as follows: Marc-145 cells were pre-cultured in DMEM medium containing 10% FBS at a concentration of 5 × 10⁶ cells / year. 4 Cells were seeded at a density of approximately 80% per well in 96-well cell culture plates and incubated at 37°C in a 5% CO2 incubator. The medium was then replaced with DMEM containing 2% FBS. The viral stock solution was diluted 10, 100, 1000, 10000, 100000, and 1000000 times. Three replicates of each dilution were seeded into 96 wells. After 1 hour of adsorption, the medium was replaced with 200 μL of fresh DMEM containing 2% FBS. After 72 hours, the wells showing cytopathic effects were counted, and the TCID50 viral titer was calculated.

[0053] 3.2 Immunoprotection Experiment 25 piglets were randomly divided into five groups, one group was blank control group, two groups were immune challenge groups, two groups were challenge control groups. After the piglets were settled in the cage, they were recorded as day 0. Two groups were immunized with FX-2025-1.8 virus liquid, and the temperature was measured and the oral and nasal swabs were collected every day for 28 days. The piglets were immunized twice at 14 days to ensure the effectiveness of the immunization. After 28 days, the challenge groups and the immune challenge groups were inoculated with lineage 1.8 and lineage 8, and the observation and comparison were made to determine whether the recombinant strain had cross-immunity effect. The body temperature, average daily gain and mortality (survival curve) are shown in Figures 6-7 -8.

Claims

1. A rHP-PRRSV-NADC30 Nsp2△30+△131 recombinant strain, which is mainly characterized in that the recombinant strain retains the △30 deletion inherent to lineage 8.7 and the △131 deletion of lineage 1.8 simultaneously on the Nsp2 protein.

2. The method of constructing the rHP-PRRSV-NADC30 Nsp2Δ30+Δ131 recombinant strain of claim 1, characterized in that The rHP-PRRSV-NADC30 Nsp2△30+△131 is constructed by reverse genetic technology, and the specific steps are as follows: (1) Extract the virus liquid of HP-PRRSV (lineage 8.7) strain, obtain the full length by nucleic acid extraction kit, and use specific primers for PCR amplification to obtain the full genome fragment of lineage 8.7 strain; (2) The gene fragment plasmid is treated with Pac I, BstB I, AfI II, Asc I and Mlu I endonuclease, and the gene fragment is connected into the full-length fragment by enzyme digestion and ligation and homologous recombination technology, and then inserted into the PSK II vector; (3) Under water bath conditions, the plasmid is verified by EcoR I single enzyme digestion, and the full-length verification of the plasmid is carried out to ensure the correct length of the plasmid. 3.The construction method of the rHP-PRRSV-NADC30 Nsp2△30+△131 recombinant strain of claim 2, characterized in that the primer sequences used in steps (1) to (4) are as follows: 。 4. The method of constructing the rHP-PRRSV-NADC30 Nsp2Δ30+Δ131 recombinant strain of claim 3, characterized in that The obtained FX-2025 strain is modified, the partial Nsp2 of lineage 1.8 strain is replaced with FX-2025 to synthesize a recombinant strain with characteristics of both lineage 1.8 and lineage 8.7 strains. 5.The application of the rHP-PRRSV-NADC30 Nsp2△30+△131 recombinant strain of claim 1 in clinical animal experiment cross protection effect.

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