Construction and application of recombinant PRRSV (porcine reproductive and respiratory syndrome virus) strain for expressing green fluorescent EGFP (green fluorescent green fluorescent protein)

Abstract

The invention discloses construction and application of a recombinant PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) strain for expressing green fluorescent EGFP (Green Green Fluorescent Protein) protein. Through a reverse genetic manipulation technology, the recombinant PRRSV capable of expressing the green fluorescent EGFP protein is successfully constructed and saved. The modified virus is inoculated to a Marc-145 cell for subculture, and the result shows that the modified virus can trigger cytopathy and stably express EGFP (Green Fluorescent Protein). The obtained recombinant strain shows a stable passage capability in a Marc-145 cell. The method is suitable for tracking virus infection and replication conditions in experiments and screening drugs, the virus detection and quantitative analysis process can be simplified, and the efficiency and accuracy of the experiments are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary biological products, and in particular relates to the construction and application of a recombinant PRRSV strain expressing green fluorescent protein (EGFP). Background Art

[0002] Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most important infectious diseases in the global swine industry, causing significant economic losses annually. PRRS is caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a member of the genus Arterivirus in the family Arteriviridae. Because PRRSV, an RNA virus, lacks specific mutation repair mechanisms and is highly susceptible to recombination mutations, the prevention and control of PRRS has always been a key focus in my country.

[0003] Initially, live attenuated vaccines provided effective protection against PRRSV in my country's swine industry. However, over time, increasing research has suggested that the use of live attenuated vaccines may have promoted recombination of PRRSV, and that they have also encountered issues such as re-enforcement during use, leading to a gradual decline in their use in my country. Drug control, as another prevention and control measure, has also been plagued by inefficiencies in laboratory screening.

[0004] Green fluorescent protein (GFP), a natural, non-toxic fluorescent marker, is widely used in biological research because it emits bright green fluorescence in living cells without the need for an external substrate. Introducing the GFP gene into viral genomes not only enables real-time, dynamic monitoring of viral infection but also provides intuitive visualization for studying interactions between viruses and host cells. By observing GFP expression, researchers can gain insights into key aspects of the viral replication cycle, transmission pathways, and host cell response mechanisms, providing important clues and evidence for uncovering viral pathogenicity. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions: A recombinant PRRSV strain capable of expressing green fluorescent protein (EGFP) is named FX1701-EGFP strain. The main feature of the recombinant PRRSV strain is that it contains EGFP green fluorescent protein.

[0006] The present invention further discloses a method for constructing a recombinant PRRSV strain expressing green fluorescent protein (EGFP), the specific steps of which are as follows: (1) Extract the viral fluid of PRRSV 1501 strain, obtain the full-length viral cDNA by reverse transcription, and use specific primers for PCR amplification to obtain the gene fragment of 1501 strain.

[0007] (2) Treat the gene fragment plasmid with PacⅠ, BstBIⅠ, AfIⅡ, AscⅠ, and MluⅠ endonucleases, connect the gene fragments into a full-length genomic fragment through enzyme ligation and homologous recombination technology, and insert it into the PSKⅡ vector; (3) In a water bath, use EcoRI single enzyme digestion to verify the plasmid and verify the full length of the plasmid to ensure the correct length of the plasmid; (4) The EGFP protein was amplified using specific primers and inserted between ORF1a and ORF2b of the PRRSV reverse genetic vector by homologous recombination to obtain a recombinant plasmid, and the correctness of the inserted sequence was verified by enzyme digestion.

[0008] The primer sequences used in steps (1) to (4) are as follows:

[0009] The present invention further reveals the application of a recombinant PRRSV strain expressing the green fluorescent protein (EGFP) for tracking viral infection and replication in experiments, as well as its broad prospects in drug screening. Experimental results show that detection results of this strain during drug screening are consistent with those of viral detection and quantitative analysis.

[0010] Compared with the prior art, the recombinant PRRSV strain expressing green fluorescent EGFP protein and the construction method thereof disclosed in the present invention have the following significant advantages: (1) Ability to track viral infection and replication more accurately; (2) Achieve more efficient drug screening; (3) Simplify the virus detection and quantitative analysis process, greatly improving experimental efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 A schematic diagram of the plasmid map of the PRRSV reverse genetic vector platform constructed in Example 1 is shown; Figure 2 Presents a gel electrophoresis diagram of double enzyme digestion verification of the PRRSV reverse genetic vector plasmid constructed in Example 1; Figure 3 The pathological changes induced by the reverse genetic vector in marc-145 cells in implementation 1 were recorded; Figure 4 The growth curves of the reverse genetic vector and the original virus in Example 1 were compared; Figure 5 The plasmid map of the PRRSV recombinant EGFP green fluorescent protein reverse genetic vector is implemented in 2; Figure 6 A schematic diagram of gel electrophoresis of the amplified EGFP protein in Example 2 is depicted; Figure 7 The fluorescence signal generated by the recombinant virus in Example 2 in MARC-145 cells is shown; Figure 8 Provides WB images for verification of EGFP protein expression in Example 2; Figure 9 The figure shows the qPCR results of the four drug screenings. DETAILED DESCRIPTION

[0012] The present invention is described in detail below by specific embodiments. Unless otherwise specified, the technical means adopted in the present invention are methods well known to those skilled in the art. In addition, the embodiments should be understood to be illustrative rather than limiting the scope of the present invention, and the spirit and scope of the present invention are limited only by the claims. For those skilled in the art, various adjustments or modifications of the material components and dosages in these embodiments, without departing from the spirit and scope of the present invention, also fall within the scope of protection of the present invention.

[0013] The general experimental methods in the following examples are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition (Beijing: Science Press, 2002). Instrument use is described in the corresponding instrument operating instructions. In the examples of the present invention, viruses were rescued from the FX1701 strain (known) and the FX1701-EGFP strain (the protective strain obtained in the present invention). Cells used included the BHK-21 cell line, the Marc-145 cell line, and primary alveolar macrophages (PAM) (commercially available).

[0014] In the examples of the present invention, DH5α competent cells were purchased from Sangon Biotech (Shanghai) Co., Ltd. Other reagents used in the examples of the present invention include: RNase-Free H2O and trypsin cell digestion solution (phenol red) were purchased from Solarbio; TRIpure Reagent total RNA extraction reagent was purchased from Adlai Biotechnology; 2× PrimeSTAR MAX DNA Polymerase, T4 ligase, and T cloning kit were all purchased from TAKARA; One Step Clone Kit homologous recombination reagent was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; DMEM medium was purchased from HyClone Biochemical Products Co., Ltd.; fetal bovine serum was purchased from Sigma; DNA restriction enzymes and Lipofectamine™ 3000 Transfection Reagent were purchased from Thermo Fisher Scientific. Example 1

[0015] Based on the TJ1501 strain, the reverse genetic vector strain FX1701 adapted to the in vitro passage of Marc-145 cells was successfully obtained.

[0016] 1.1 Schematic diagram of the construction of FX1701 reverse genetic vector using Snapgene software. Figure 1 shown.

[0017] 1.2 Primer design for construction of FX1701 reverse genetic vector Using Snapgene software, the TJ1501 strain was divided into four parts for amplification according to the simulated plasmid schematic diagram, and the corresponding primers were designed (see Table 1).

[0018]

[0019] 1.3 Construction of reverse genetic vectors First, fragments 1501-A, 1501-B, 1501-C, and 1501-D were amplified by PCR to obtain the full-length sequence of the original virus. Subsequently, these fragments were ligated into the PSKⅡ vector using homologous recombination technology.

[0020] 1.3.1 PCR amplification method and system for target fragments In the present invention, amplification of target fragments was performed using PCR technology. The specific PCR amplification system and procedure are detailed in Tables 2 and 3. The commercial kit used was 2× PrimeSTAR MAX DNA Polymerase, purchased from TAKARA.

[0021]

[0022] Using the above method, we can obtain Figure 1 The fragments of the original strain full-length sequence were spliced ​​together as shown in . Separate the fragments by 1% large-pore gel electrophoresis for 35 minutes. The enzyme-cleaved bands were separated by agarose gel electrophoresis, and the target band was cut and purified by gel recovery. The amplified target fragment was stored at -20°C.

[0023] 1.3.2 Vector Enzyme Digestion and Homologous Recombination Methods and Systems In the present invention, all vector enzyme digestion and homologous recombination methods and systems are carried out according to the following steps: First, the PSKⅡ vector is double-digested with PacⅠ and MluⅠ to prepare a linearized vector. The specific system is detailed in Table 4.

[0024]

[0025] According to the above system, enzyme digestion was performed at 37°C for 1 hour. Subsequently, the enzyme-digested bands were separated by agarose gel electrophoresis, and the target band was cut out and purified by gel recovery. The PSKⅡ linearized vector was successfully obtained, and its concentration was measured to be 100 ng / μl and stored at -20°C. The linearized fragments 1701-A, 1701-B, 1701-C, and 1701-D were homologously recombined with the linearized vector PSKⅡ (using the One Step Clone Kit). The specific operation system is detailed in Table 5.

[0026] After the system reacts at 50°C for 30 minutes, immediately place on ice. Transform the system into DH5α competent cells, and pick independent colonies for pure culture. Perform PCR on the bacterial solution using universal primers. Select PCR-positive colonies for overnight propagation, extract the plasmid, and verify it by double enzyme digestion with AscⅠ and BSTbⅠ. Subsequently, perform electrophoresis on a 1% agarose gel to confirm the correct enzyme digestion bands. Store the correct plasmid at -20°C.

[0027] 1.4 Rescue of MARC-145 cells with reverse genetic vectors First, BHK-21 cells were seeded at a density of 6 × 10^5 cells / well in a 6-well cell culture plate using DMEM medium supplemented with 10% FBS and cultured in a 37°C, 5% CO2 incubator until the cell density reached approximately 80%. Cell transfection was performed according to the instructions for Lipofectamine™ 3000 Transfection Reagent.

[0028] The transfection method is as follows: First, mix 2 μg of plasmid, 4 μL of P3000 plasmid, and 50 μL of Opti-MEM reagent to form Solution A. Then, mix 3 μL of Lip3000 and 50 μL of DMEM to form Solution B. Next, mix Solution A and Solution B and incubate at 25°C for 15 minutes. Finally, add the mixture to BHK-21 cells. See Table 6 for specific transfection systems.

[0029] 48 hours after cell transfection, the 6-well plate was sealed and frozen at -80°C. After repeated freezing and thawing twice, all the cell suspensions were collected and centrifuged at 12,000 × g for 5 minutes to collect the transfection supernatant.

[0030]

[0031] Pre-seed MARC-145 cells at a density of 6 × 10^5 cells / well in 2 mL of DMEM medium containing 10% FBS in a 6-well cell culture plate and culture in a 37°C, 5% CO2 incubator. After the MARC-145 cells adhere, overlay 500 μL of the transfection supernatant on the MARC-145 cells. After incubation for 1 hour, discard the supernatant and add DMEM medium containing 2% FBS to continue culturing.

[0032] The rescue results of MARC-145 cells are shown in Figure 3 , observation of lesions proves successful rescue.

[0033] 1.5 Determination of growth characteristics of strains adapted to MARC-145 cell culture First, the TCID50 titer of the fifth-generation reverse genetic vector was determined. The TCID50 assay was performed as follows: MARC-145 cells were seeded at a density of 5 × 10^4 cells / well in DMEM medium supplemented with 10% FBS in a 96-well cell culture plate. The cells were cultured in a 37°C, 5% CO2 incubator. When the cell density reached approximately 80%, the medium was switched to DMEM supplemented with 2% FBS. The viral stock solution was diluted 10-fold, 100-fold, 1000-fold, 10,000-fold, 100,000-fold, and 1,000,000-fold, respectively. Three replicates of each dilution series were plated in a 96-well plate. After adsorption for 1 hour, the plate was replaced with 200 μL of fresh DMEM medium supplemented with 2% FBS. After 72 hours, the number of wells with lesions was counted, and the TCID50 viral titer of the reverse genetic vector was calculated.

[0034] The growth of the reverse genetic vector on MARC-145 cells was evaluated by drawing a multi-step growth curve. The specific method is as follows: each virus liquid was inoculated onto MARC-145 cells in a 6-well plate at an infection dose of 0.01 MOI. The supernatant was collected at 5 time points, namely 12 hours, 24 hours, 36 hours, 48 ​​hours and 72 hours, and frozen in a -80°C refrigerator. After all time points were collected, the TCID50 titer of the supernatant at different time points was determined and a standard curve was drawn. The results of the multi-step growth curve determination are shown in Figure 2. Figure 4 As shown, both the reverse genetic vector and the wild-type strain can be propagated on MARC-145 cells. Example

[0035] Based on the FX1701 reverse genetic vector, the recombinant vaccine candidate strain FX1701-EGFP was successfully obtained by in vitro passage of Marc-145 cells.

[0036] 2.1 Schematic diagram of the construction of recombinant plasmids using Snapgene software. Figure 5 .

[0037] 2.2 Primer design for constructing recombinant plasmids Using Snapgene software, according to the schematic diagram of the simulated plasmid, the recombinant protein was divided into three parts for amplification, and the corresponding primers were designed (see Table 7).

[0038]

[0039] 2.3 Methods for constructing recombinant plasmids First, PF, EGFP, and PR fragments were amplified by PCR to obtain the full-length protein sequences. Subsequently, these fragments were ligated into the FX1701 vector using homologous recombination.

[0040] 2.3.1 Target fragment PCR amplification method and system In the present invention, amplification of target fragments was performed using PCR technology. The specific system and procedure of PCR amplification are detailed in Tables 8 and 9. The commercial kit used was 2× PrimeSTAR MAX DNA polymerase (purchased from TAKARA).

[0041]

[0042] Using the above method, we can obtain Figure 5 Each fragment of the EGFP protein sequence was amplified. Electrophoresis was performed on a 1% large-pore gel for 35 minutes. The cleaved 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°C.

[0043] 2.3.2 Vector Enzyme Digestion and Homologous Recombination Methods and Systems In the present invention, all vector enzyme digestion and homologous recombination methods and systems are carried out according to the following steps. First, the reverse genetics vector is double-digested with ASCII and ECORV to prepare a linearized vector. The specific double-digestion system is detailed in Table 10.

[0044]

[0045] Following the above system, enzyme digestion was performed at 37°C for 1 hour. The resulting bands were then separated by agarose gel electrophoresis, and the target band was excised and purified by gel extraction. The linearized reverse genetic vector was obtained at a concentration of 200 ng / μl and stored at -20°C.

[0046] The linearized fragments PF, EGFP, and PR were homologously recombined with the linearized vector using the One Step Clone Kit. The specific operating system is detailed in Table 11.

[0047]

[0048] After incubating the system at 50°C for 30 minutes, immediately place on ice. Transform the system into DH5α and isolate individual colonies for pure culture. Perform PCR testing using universal primers. Select PCR-positive colonies, propagate the culture overnight, extract the plasmid, and verify by double enzyme digestion. Then, perform electrophoresis on a 1% agarose gel. Plasmids with correct enzyme digestion bands should be stored at -20°C.

[0049] 2.4 Rescue of MARC-145 cells with reverse genetic vectors First, BHK-21 cells were seeded at a density of 6 × 10^5 cells / well in a 6-well cell culture plate using DMEM medium supplemented with 10% FBS and cultured in a 37°C, 5% CO2 incubator until the cell density reached approximately 80%. Cell transfection was performed according to the instructions for Lipofectamine™ 3000 Transfection Reagent.

[0050] The transfection method is as follows: Mix 2 μg of plasmid, 4 μL of P3000 plasmid, and 50 μL of Opti-MEM reagent to form Solution A. Then, mix 3 μL of Lip3000 and 50 μL of DMEM to form Solution B. Then, mix Solution A and Solution B and incubate at 25°C for 15 minutes. Finally, add the mixture to BHK-21 cells. See Table 12 for specific transfection systems.

[0051] 48 hours after cell transfection, the 6-well plate was sealed and frozen at -80°C. After repeated freezing and thawing twice, all cell suspensions were collected and centrifuged at 12,000 × g for 5 minutes to collect the transfection supernatant.

[0052]

[0053] MARC-145 cells were seeded into a 6-well cell culture plate at a density of 6 × 10^5 cells / well using 2 mL of DMEM medium containing 10% FBS and cultured at 37°C in 5% CO2. After the MARC-145 cells attached, 500 μL of the transfection supernatant was overlaid on the MARC-145 cells. After incubation for 1 hour, the supernatant was discarded and DMEM medium containing 2% FBS was added to continue the culture.

[0054] The recombinant virus was serially passaged, and the pathological changes were shown in Figure 7. After stable inheritance, WB verification was performed using EGFP protein monoclonal antibody, and the WB band pattern was shown in Figure 7. Figure 8 . Example 2

[0055] 3.1 Application of recombinant PRRSV strains expressing green fluorescent protein (EGFP) to track viral infection and replication in experiments First, MARC-145 cells were seeded at a density of 6 × 10^5 cells / well in a 6-well cell culture plate using DMEM medium supplemented with 10% FBS. The cells were cultured in a 37°C, 5% CO2 incubator until the cell density reached approximately 80%. Inoculation was then performed. 200 μL of recombinant PRRSV virus expressing the green fluorescent protein (EGFP) was added to the 6-well plate.

[0056] The cells were observed under an inverted fluorescence microscope at 6, 12, 24, 36, 48, and 72 hours post-inoculation (hpi). By comparing the intensity and distribution of the fluorescence signal at different time points, the virus replication and infection status in the cells were successfully observed, and the key time points for virus entry, intracellular replication, and secretion to infect neighboring cells were determined.

[0057] By tracking the entire viral life cycle in cells, we obtained more accurate timelines. We found that the first generation of virions appeared at 16 hours, complete virion release began at 20 hours, peaked at 24 hours, and then entered a phase of cell death. This laid the foundation for subsequent research into the replication mechanism of PRRSV. Example 3

[0058] Applications for implementing drug screening Stable expression of recombinant PRRSV strain expressing green fluorescent protein (EGFP) in MARC-145 cells MARC-145 cells were seeded at a density of 6 × 10^5 cells / well in a 6-well cell culture plate using DMEM medium supplemented with 10% FBS. The cells were cultured in a 37°C, 5% CO2 incubator until the cell density reached approximately 80%. Inoculation was then performed. 200 μl of recombinant PRRSV expressing the green fluorescent protein (EGFP) was added to the 6-well plate. FX1701 was also added as a control.

[0059] Drug screening 4.2.1 Observe MARC-145 cells under a microscope. When pathological changes appear in the field of view, add candidate drugs for screening. Based on the maximum safe concentration determined, add the drugs to six-well plates. At 72 hours, collect the supernatant from the positive control group and the green fluorescence signal from the FX1701-CAP group. Based on the intensity of the fluorescence signal in each drug-treated group, screen for the optimal drug for PRRSV inhibition.

[0060] 4.2.2 The cells in the positive control group were repeatedly frozen and thawed in a -80°C refrigerator and centrifuged at 12,000 rpm for 5 minutes to obtain the cell supernatant. According to the nucleic acid extraction and reverse transcription method in Example 1, the viral load in different experimental groups was detected by qPCR. The experimental results are shown in Figure 2. Figure 9 shown.

[0061] After viral load detection by qPCR method, the results were compared with the fluorescence signal group, and it was found that the two groups of results were consistent, proving that FX1701-CAP can provide accurate, fast and lower-cost results as an antiviral drug screening strain.

Claims

1. A recombinant PRRSV strain capable of expressing green fluorescent protein (EGFP), named FX1701-EGFP strain. Its main characteristics are: The recombinant PRRSV strain contains EGFP green fluorescent protein.

2. The method for constructing a recombinant PRRSV strain expressing green fluorescent protein (EGFP) according to claim 1, comprising the following steps: (1) Extract the PRRSV 1501 strain virus liquid, obtain the full-length viral cDNA by reverse transcription, and use specific primers for PCR amplification to obtain the gene fragment of the 1501 strain; (2) Treat the gene fragments with PacⅠ, BstBI, AfⅡ, AscⅠ, and MluⅠ endonucleases, connect the gene fragments into full-length genomic fragments through enzyme ligation and homologous recombination technology, and insert them into the PSKⅡ vector; (3) Use EcoRI single enzyme digestion to verify the plasmid and verify the full length of the plasmid to ensure the correct length of the plasmid; (4) The EGFP protein was amplified using specific primers and inserted between ORF1a and ORF2b of the PRRSV reverse genetic vector by homologous recombination to obtain a recombinant plasmid, and the correctness of the inserted sequence was verified by enzyme digestion.

3. The method for constructing a recombinant PRRSV strain expressing green fluorescent protein (EGFP) according to claim 2, wherein the primer sequences used in steps (1) to (4) are as follows: 。 4. The recombinant PRRSV strain capable of expressing green fluorescent EGFP protein according to claim 3 is used in experiments to track viral infection and replication, and is applied to drug screening, showing significant application value.

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