A plasmid system based on insect virus FHV RNA1 replicon and its construction and application

Abstract

The present invention discloses an application of an insect virus FHV RNA1 replicon in exogenous gene amplification, wherein the application amplifies the exogenous gene at the mRNA level by utilizing the autonomous replication ability of the insect virus FHV RNA1 replicon; the present invention discloses a plasmid system based on the insect virus FHV RNA1 replicon and a method for constructing the plasmid system; the present invention also discloses a method and application of the plasmid system for expressing proteins in cells, wherein the plasmid system is used to obtain a cloning vector after double enzyme digestion, and after homologous recombination with an exogenous gene, the animal or plant protein is expressed in the cell.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a plasmid system based on an insect virus FHV RNA1 replicon and a construction and application thereof. Background Art

[0002] The functions and regulatory mechanisms of exogenous genes require expression in host cells for further study. Host cells that express exogenous genes are called expression systems and are divided into prokaryotic and eukaryotic systems. Eukaryotic systems include yeast cells, insect cells, mammalian cells, and plant cells. To express exogenous genes in host cells, they must be inserted into expression vectors containing the various elements required for gene expression. Different expression vectors are required for different expression systems. Eukaryotic expression systems can recognize and excise introns in exogenous genes. They also have better regulation of translation levels and can be glycosylated, which helps maintain immunogenicity.

[0003] However, the eukaryotic expression system has the problem of low efficiency in introducing exogenous genes into eukaryotic cells. Eukaryotic expression vectors cannot replicate continuously to achieve high copy numbers. In addition, current eukaryotic expression vectors have boundary barriers and are not universally compatible in different eukaryotic cells. In addition, although some eukaryotic expression vectors can efficiently express exogenous genes, they will also cause physiological interference to host cells or cause host death. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, the first object of the present invention is to provide an application of an insect virus FHV RNA1 replicon in exogenous gene amplification, which can solve the problem of weak exogenous mRNA amplification ability.

[0005] The second object of the present invention is to provide a plasmid system based on the insect virus FHV RNA1 replicon, which can solve the problem of low transcription activity of the vector.

[0006] The third object of the present invention is to provide a method for constructing a plasmid system based on the insect virus FHV RNA1 replicon, which can solve the problem of constructing a eukaryotic expression vector with high replication ability.

[0007] The fourth object of the present invention is to provide a method for expressing proteins in cells using a plasmid system based on the insect virus FHV RNA1 replicon, which can solve the problems of eukaryotic expression vectors being unable to replicate continuously, having species barriers, and interfering with host cells.

[0008] The fifth object of the present invention is to provide an application of a plasmid system based on the insect virus FHV RNA1 replicon, which can solve the problem that eukaryotic expression vectors cannot be shuttled for expression in eukaryotic cells.

[0009] The first object of the present invention is achieved by the following technical solutions:

[0010] The invention discloses an application of an insect virus FHV RNA1 replicon in exogenous gene amplification. The nucleotide sequence of the replicon is shown in SEQ NO.1.

[0011] Furthermore, the exogenous gene is inserted into the open reading frame between the 3' end of the insect virus FHV RNA1 replicon and the B2 coding sequence to amplify the exogenous gene. The B2 coding sequence includes the nucleotide sequence shown in SEQ NO.2.

[0012] The second object of the present invention is achieved by the following technical solutions:

[0013] A plasmid system based on the insect virus FHV RNA1 replicon, wherein the plasmid system uses the insect virus FHV RNA1 replicon; the plasmid system uses a pUC vector as a backbone vector; the nucleotide sequence of the plasmid system sequentially includes a vector promoter, an insect virus FHV RNA1 replicon, a linker sequence, a P2A sequence, a B2 coding sequence, an HDV ribozyme sequence, and a vector terminator.

[0014] Furthermore, there is an enzyme cleavage site between the P2A sequence and the B2 coding sequence; the enzyme cleavage sites are PstI, EcoRI, XhoI, XbaI and HindIII in sequence; and the vector promoter is one of a CMV promoter and a T7 promoter.

[0015] The third object of the present invention is achieved by the following technical solutions:

[0016] A method for constructing a plasmid system based on the insect virus FHV RNA1 replicon, characterized by comprising the following steps:

[0017] Step 1: amplify multiple nucleotide sequence fragments of the plasmid system using primers and templates; Step 2: connect the nucleotide sequence fragments of the multiple plasmid systems to obtain a connection product; Step 3: transfer the connection product to Escherichia coli competent cells for culture; Step 4: culture the Escherichia coli competent cells described in step 3, extract the recombinant plasmid after screening, and complete the construction of a plasmid system based on the insect virus FHV RNA1 replicon.

[0018] Furthermore, in step 1, when amplifying the nucleotide sequence fragments of the plasmid system, the green fluorescent protein expression cassette is used as a template to amplify fragment A; the pUC-T7-FHVRNA1 plasmid is used as a template to amplify fragments B and C, respectively; and the CMV promoter is used as a template to amplify fragment D; the nucleotide sequence of the pUC-T7-FHVRNA1 plasmid is shown in SEQ NO.3; the nucleotide sequence of the green fluorescent protein expression cassette is shown in SEQ NO.4; and the nucleotide sequence of the CMV promoter is shown in SEQ NO.5.

[0019] Furthermore, in the step 1, the green fluorescent protein expression cassette was amplified using the upstream primer [pUC / PstI]-hibit-fwd and the downstream primer msfGFP-[pUC / XhoI]-rev to obtain fragment A, the upstream primer [pUC / PstI]-hibit-fwd nucleotide sequence is shown in SEQ NO.6, and the downstream primer msfGFP-[pUC / XhoI]-rev nucleotide sequence is shown in SEQ NO.7; the upstream primer FHV RNA1 / 1st-fwd and the downstream primer [P2A / PstI]-rev were used to amplify the pUC-T7-FHVRNA1 plasmid as a template to obtain fragment B, the upstream primer FHV RNA1 / 1st-fwd nucleotide sequence is shown in SEQ NO.8, and the downstream primer [P2A / PstI]-rev nucleotide sequence is shown in SEQ NO.9; the upstream primer [XhoI / B2]-fwd and the downstream primer pUC / last Base-rev was amplified using the pUC-T7-FHVRNA1 plasmid as a template to obtain fragment C. The nucleotide sequence of the upstream primer [XhoI / B2]-fwd is shown in SEQ NO.10, and the nucleotide sequence of the downstream primer pUC / last base-rev is shown in SEQ NO.11. The CMV promoter was amplified using the upstream primer pUC / CMV-fwd and the downstream primer CMV / FHV RNA1-ev to obtain fragment D. The nucleotide sequence of the upstream primer pUC / CMV-fwd is shown in SEQ NO.12, and the nucleotide sequence of the downstream primer CMV / FHV RNA1-rev is shown in SEQ NO.13.

[0020] The fourth object of the present invention is achieved by the following technical solutions:

[0021] A method for expressing proteins in cells using a plasmid system based on the insect virus FHV RNA1 replicon, comprising the following steps: performing double enzyme digestion on the plasmid system based on the insect virus FHV RNA1 replicon to obtain a cloning vector; performing homologous recombination between the cloning vector and an exogenous gene to obtain a homologous recombination product; transfecting the homologous recombination product into host cells, culturing the cells, collecting cell samples, and detecting the cells to complete protein expression.

[0022] Furthermore, the double enzyme digestion sites are PstI and XhoI.

[0023] The fifth object of the present invention is achieved by the following technical solutions:

[0024] Application of a plasmid system based on the insect virus FHV RNA1 replicon in protein expression.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention provides a plasmid system for gene expression that exhibits excellent autonomous replication and high replication efficiency without causing any physiological disturbance to host cells. Furthermore, the plasmid system for gene expression constructed in the present invention is capable of bridging multiple barriers, with its genome being able to replicate in insects, mammals, plants, and even yeast cells, encompassing a wide range of eukaryotic expression systems.

[0027] 2. The present invention provides a simple and convenient method for constructing a plasmid system for gene expression. The present invention utilizes a plasmid system for gene expression, which, when expressing exogenous genes in host cells, exhibits high transcriptional activity, exhibits no cytopathic effects, is not a human pathogen, and exhibits high biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Plasmid map based on insect virus FHV RNA1 replicon constructed for the present invention

[0029] Figure 2 The results of the plasmid system of the present invention expressing the exogenous protein msfGFP at different temperatures are shown in FIG. Figure 3 The time course results of the expression of the exogenous protein msfGFP by the plasmid system of the present invention and the results of real-time fluorescence quantitative PCR are shown.

[0030] Figure 4 This is the immunofluorescence result of the exogenous protein msfGFP expressed by the plasmid system of the present invention Figure 5-9 Comparison of protein expression results of the same exogenous gene by the plasmid system of the present invention and the pXJ40 vector DETAILED DESCRIPTION

[0031] The technical solutions of the present invention will be described clearly and completely below with reference to specific embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0032] The functions and regulatory mechanisms of exogenous genes require expression in host cells for further investigation. To express exogenous genes in host cells, they must be inserted into an expression vector containing the necessary components for gene expression. Eukaryotic expression systems can recognize and excise introns within exogenous genes, eliminating the need for cDNA preparation required by prokaryotic expression systems. Eukaryotic expression systems also provide better regulation of translation, and exogenous genes can be glycosylated, which helps maintain immunogenicity.

[0033] However, eukaryotic expression systems suffer from low efficiency in introducing exogenous genes into eukaryotic cells. Eukaryotic expression vectors cannot replicate continuously to achieve high copy numbers. Current eukaryotic expression vectors also have limitations and are not universally compatible with different eukaryotic cells. Furthermore, while some eukaryotic expression vectors can efficiently express exogenous genes, they can also cause physiological disruptions in host cells or even cause host death. Therefore, a method for amplifying exogenous genes based on the insect virus FHV RNA1 replicon is provided to address this issue of weak exogenous mRNA amplification.

[0034] The invention discloses an application of an insect virus FHV RNA1 replicon in exogenous gene amplification. The nucleotide sequence of the replicon is shown in SEQ NO.1.

[0035] Furthermore, the exogenous gene is inserted into the open reading frame between the 3' end of the insect virus FHV RNA1 replicon and the B2 coding sequence to amplify the exogenous gene. The B2 coding sequence includes the nucleotide sequence shown in SEQ NO.2.

[0036] The insect virus FHV belongs to the Nodaviridae family and the genus Alpha-tuberculovirus. Its structure consists of a capsid packaged into a non-enveloped icosahedral virus particle. The FHV genome consists of two single-stranded positive-sense RNAs. Genomic RNA1 encodes RNA polymerase (RdRp), which enables autonomous replication. Genomic RNA2 is primarily responsible for encoding the viral capsid protein precursor. During FHV replication, subgenomic RNA3 encoding B1 and B2 proteins is synthesized. The B2 protein is responsible for inhibiting RNA silencing and can effectively control genome replication. The insect virus FHV has a relatively small genome, so as a vector, it can accommodate exogenous genes with larger genomes.

[0037] On the other hand, the insect virus FHV possesses a unique shuttling ability, enabling genome replication in vertebrates, plants, and even yeast cells, in addition to insects. Furthermore, FHV is non-pathogenic, exhibiting no cytopathic effect in mammalian cells and exhibiting excellent biosafety. Therefore, the present invention provides a plasmid system based on the insect virus FHV RNA1 replicon.

[0038] A plasmid system based on the insect virus FHV RNA1 replicon, wherein the plasmid system uses the insect virus FHV RNA1 replicon; the plasmid system uses a pUC vector as a backbone vector; the nucleotide sequence of the plasmid system sequentially includes a vector promoter, an insect virus FHV RNA1 replicon, a linker sequence, a P2A sequence, a B2 coding sequence, an HDV ribozyme sequence, and a vector terminator.

[0039] Furthermore, there is an enzyme cleavage site between the P2A sequence and the B2 coding sequence; the enzyme cleavage sites are PstI, EcoRI, XhoI, XbaI and HindIII in sequence; and the vector promoter is one of a CMV promoter and a T7 promoter.

[0040] The presence of the multiple single enzyme cleavage sites enables the vector system to accommodate exogenous genes with different genome lengths, and to express more diverse exogenous proteins.

[0041] The present invention provides a method for constructing a plasmid system based on the insect virus FHV RNA1 replicon, which can solve the problem of constructing a eukaryotic expression vector with high replication ability.

[0042] A method for constructing a plasmid system based on the insect virus FHV RNA1 replicon, characterized by comprising the following steps:

[0043] Step 1, amplifying multiple nucleotide sequence fragments of the plasmid system using primers and templates;

[0044] Step 2, connecting the nucleotide sequence fragments of the multiple plasmid systems to obtain a connection product;

[0045] Step 3, transferring the ligation product to Escherichia coli competent cells for culture;

[0046] Step 4: Cultivate the competent E. coli cells described in step 3, extract the recombinant plasmid after screening, and complete the construction of a plasmid system based on the insect virus FHV RNA1 replicon.

[0047] Furthermore, in step 1, when amplifying the nucleotide sequence fragments of the plasmid system, the green fluorescent protein expression cassette is used as a template to amplify fragment A; the pUC-T7-FHVRNA1 plasmid is used as a template to amplify fragments B and C, respectively; and the CMV promoter is used as a template to amplify fragment D; the nucleotide sequence of the pUC-T7-FHVRNA1 plasmid is shown in SEQ NO.3; the nucleotide sequence of the green fluorescent protein expression cassette is shown in SEQ NO.4; and the nucleotide sequence of the CMV promoter is shown in SEQ NO.5.

[0048] Furthermore, in step 1, the reporter gene expression cassette was amplified using the upstream primer [pUC / PstI]-hibit-fwd and the downstream primer msfGFP-[pUC / XhoI]-rev to obtain fragment A, the upstream primer [pUC / PstI]-hibit-fwd sequence is shown in SEQ NO.6, and the downstream primer msfGFP-[pUC / XhoI]-rev is shown in SEQ NO.7; the pUC-T7-FHVRNA1 plasmid was amplified using the upstream primer FHV RNA1 / 1st-fwd and the downstream primer [P2A / PstI]-rev to obtain fragment B, the upstream primer FHV RNA1 / 1st-fwd sequence is shown in SEQ NO.8, and the downstream primer [P2A / PstI]-rev sequence is shown in SEQ NO.9; the upstream primer [XhoI / B2]-fwd and the downstream primer pUC / last The pUC-T7-FHVRNA1 plasmid was amplified using base-rev to obtain fragment C. The upstream primer [XhoI / B2]-fwd sequence is shown in SEQ NO.10, and the downstream primer pUC / last base-rev sequence is shown in SEQ NO.11. The CMV promoter was amplified using the upstream primer pUC / CMV-fwd and the downstream primer CMV / FHV RNA1-ev to obtain fragment D. The upstream primer pUC / CMV-fwd sequence is shown in SEQ NO.12, and the downstream primer CMV / FHVRNA1-rev is shown in SEQ NO.13.

[0049] The present invention also provides a method for expressing proteins in cells using a plasmid system based on the insect virus FHV RNA1 replicon, comprising the following steps: performing double enzyme digestion on the plasmid system based on the insect virus FHV RNA1 replicon to obtain a cloning vector; performing homologous recombination between the cloning vector and an exogenous gene to obtain a homologous recombination product; transfecting the homologous recombination product into host cells, culturing the cells, collecting cell samples, and detecting the cells to complete protein expression.

[0050] Furthermore, the double enzyme digestion sites are PstI and XhoI.

[0051] The present invention also provides an application of a plasmid system based on the insect virus FHV RNA1 replicon in protein expression, which can solve the problem that eukaryotic expression vectors cannot be shuttled for expression in eukaryotic cells.

[0052] Below, with reference to the accompanying drawings and specific embodiments, the present invention is further described: Example 1 Construction of plasmid system

[0053] 1. Construction of FHV RNA1 Plasmid System

[0054] Step 1: Amplify the target fragments according to the template. The target fragments include fragment A, fragment B, fragment C, and fragment D. The target fragments and corresponding primers are shown in Table 1:

[0055] Table 1 Plasmid system homologous recombination primers and templates

[0056]

[0057] Step 2: Digest the original plasmid with DpnI to eliminate template interference during subsequent recombinant plasmid construction. Homologous recombination ligates the target fragments A, B, C, and D to produce a ligation product. The reaction system and conditions are shown in Table 2. The volume of the target fragment is determined by the amount of pUC-T7-FHVRNA1 plasmid, and the molar ratio of pUC-T7-FHVRNA1 plasmid to target fragment is 3:1.

[0058] Table 2 Reaction system and conditions for homologous recombination

[0059]

[0060]

[0061] Step 3: Transform the ligation product into E. coli DH5α competent cells using a heat shock transformation method. An appropriate amount of the transformation product was plated onto LB solid medium containing 100 μg / ml ampicillin. After 12 to 16 hours of incubation, several single colonies of uniform morphology and size were randomly selected and verified by PCR to confirm successful cloning. Two pairs of primers were used for colony PCR. The primer names and sequences are shown in Table 3:

[0062] Table 3 Primer information for colony PCR

[0063]

[0064] After amplification, the corresponding monoclonal colonies were selected for plasmid extraction. The extracted plasmid was double-digested with BglII and XbaI to obtain two fragments of 2444 bp and 5042 bp as the target plasmid. The positive plasmid obtained by sequencing verification was the recombinant plasmid pUC-CMV-FHVRNA1-HIBIT-msfGFP. The plasmid map is shown below. Figure 1 As shown, the construction of a plasmid system for foreign gene expression is completed.

[0065] 2. Screening of the optimal temperature for plasmid systems

[0066] The optimal temperature of FHV RNA1 plasmid system in BHK cells was screened by transfection experiment and protein immunoblotting experiment. The reagents of the transfection experiment were from Nanjing Novozyme Biotechnology Co., Ltd. Transfection Reagent.

[0067] The plasmid pUC-CMV-FHVRNA1-HIBIT-msfGFP constructed in Example 1 was mixed with the transfection reagent and allowed to stand for 15 minutes. The plasmid mixture was then added dropwise to the hamster kidney cell BHK-21 cell line plated in a 12-well plate the day before. The cell lines were cultured at 28°C, 30°C, 32°C, and 37°C for 48 hours. After observing the green fluorescence after incubation at the corresponding temperature, the corresponding protein samples were collected for Western Blot wet transfer detection. The specific steps of Western Blot wet transfer detection are as follows:

[0068] 1. Discard the culture medium in the 12-well plate, wash once with PBS, and add 200 μL of 2×LD and 20 μL of DTT to each well;

[0069] 2. Scrape the cells and place them in a 1.5ml centrifuge tube. Denature the protein sample in a 95°C dry bath for 15 minutes. The sample should be a viscous liquid before denaturation and a fluid state after denaturation. Vortex the protein sample appropriately during this period to check the degree of lysis.

[0070] 3. After denaturing the protein, gently centrifuge it and run gel electrophoresis at 60V for 30 minutes. When the band reaches the dividing line between the stacking gel and the separating gel, switch to 80V for 1.5 hours. After the gel run, transfer the protein to the membrane at 80V for 2 hours. This time can be extended depending on the size of the protein band.

[0071] 4. After the transfer is completed, remove the NC membrane and place it in a clean container. Add PBST to wash; after washing, block it on a shaker at room temperature for 2 hours; recover the blocking solution and add PBST to wash again; after washing, pour the primary antibody until the NC membrane is covered and incubate it at 4°C overnight.

[0072] 5. Recover the primary antibody and wash with PBST five times, 5 minutes each time. Add the secondary antibody and incubate at room temperature in the dark for 1 hour. Since the secondary antibody used is fluorescent, it needs to be recovered in the dark. After recovery, wash the secondary antibody five times with PBST.

[0073] Use a scanner to view the protein bands. Figure 2 It can be seen that at 30°C, the green fluorescence signal is the strongest and the protein expression level is the highest.

[0074] Example 2 Time course detection of plasmid system

[0075] The protein expression levels of the plasmid pUC-CMV-FHVRNA1-HIBIT-msfGFP constructed in Example 1 at five time points at 30°C were verified by transfection experiments and Western blotting. The transfection and Western blotting experiments were performed using the same methods as in Example 1. Green fluorescence was observed at 24 h, 48 h, 72 h, 96 h, and 120 h of culture, and the corresponding cells were sampled for protein and analyzed by Western blotting.

[0076] In addition, the cells at the five time points were respectively subjected to RNA extraction and specific reverse transcription to obtain negative strand cDNA, using a reverse transcription kit from Tiangen Biochemical Technology (Beijing) Co., Ltd. The negative strand cDNA sample was diluted with DEPC water, and the cDNA formed by reverse transcription of each μg RNA was diluted 20 times, followed by real-time fluorescence quantitative PCR detection to verify the replication of the plasmid system of the present invention. The reaction system of each well of the real-time fluorescence quantitative PCR detection was 4.6 μL of cDNA diluent, 0.2 μL of upstream primer, 0.2 μL of downstream primer, and 5 μL of 2×ChamQ Unversal SYBR QPCR Master Mix. The reaction conditions for real-time fluorescence quantitative PCR detection are shown in Table 4, and the primer sequences for real-time fluorescence quantitative PCR detection are shown in Table 5.

[0077] Table 4 Reaction conditions for real-time fluorescence quantitative PCR detection

[0078]

[0079]

[0080] Table 5 Real-time fluorescence quantitative PCR detection primer sequence list

[0081]

[0082] The results of Example 2 are as follows Figure 3As shown in the figure, GFP expression increases over time from 24 to 96 hours, reaching a peak at 96 hours. After 120 hours, GFP expression decreases as cells die. qPCR results show that negative RNA increases over time from 24 to 96 hours, reaching a peak at 96 hours. Combined with these results, it can be seen that as the plasmid system replicates, the exogenous protein is continuously translated and expressed.

[0083] Example 3 Verification of the autonomous replication ability of the plasmid system

[0084] The recombinant plasmid constructed in Example 1 was transfected into BHK cells cultured in a 48-well plate using the same transfection method as in Example 1. The cells were cultured at 30°C for 48 h, 72 h, and 96 h, and immunofluorescence experiments were performed on the cells at the three time points. The specific steps were as follows:

[0085] 1. Discard the culture medium in the well plate and add 200 μL PBS to each well plate for washing.

[0086] 2. Fix the cells by adding 300 μL of ice-cold 100% methanol to each well and incubate at -20°C for 15 minutes. Discard the fixative and add 300 μL of PBS to each well. Wash the plate on a shaker at room temperature for 5 minutes. Repeat three times.

[0087] 3. Add 300 μL of 5% calf serum (Triton is added to enhance cell permeability) to each well and block at room temperature for 1 hour. After blocking, discard the blocking solution, add 300 μL of PBS to each well, and wash the plate on a shaker at room temperature for 5 minutes. Repeat 3 times.

[0088] 4. Dilute the primary antibody (mouse double-stranded RNA antibody) with 1% calf serum at a dilution factor of 500. Add 80 μL of the diluted primary antibody to each well and incubate overnight at 4°C. After incubation, recover the primary antibody and add 300 μL of PBS to each well. Wash the plate on a shaker at room temperature for 5 minutes. Repeat three times.

[0089] 5. Dilute the secondary antibody (mouse antibody) with PBS at a dilution factor of 500; add 80 μL of diluted secondary antibody to each well and incubate at room temperature in the dark for 1 hour; discard the secondary antibody after incubation, add 300 μL PBS to each well, place the well plate on a shaker and wash for 5 minutes at room temperature, and repeat 3 times.

[0090] 6. Dilute DAPI (nuclear staining reagent) with PBS to a dilution factor of 1000; add 100 μL of diluted DAPI to each well and stain at 37°C in the dark for 10 minutes. After staining, discard the DAPI and add 300 μL of PBS to each well. Wash the plate on a shaker at room temperature for 5 minutes. Repeat three times.

[0091] 7. Observe the cells using a fluorescence microscope.

[0092] The results of Example 3 are as follows Figure 4 As shown, it can be seen that green fluorescent protein only appears in cells that produce double-stranded RNA. Therefore, the replication and expression of exogenous genes are inevitably related to the plasmid system of the present application.

[0093] Example 4 Integration of plasmid system and exogenous gene of plant and animal protein

[0094] The plasmid system of the present invention comprises the following steps of expressing foreign genes in cells:

[0095] 1. The recombinant plasmid constructed by homologous recombination in Example 1

[0096] The cloning vector pUC-CMV-FHVRNA1 was obtained by double enzyme digestion of pUC-CMV-FHVRNA1-HIBIT-msfGFP. The double enzyme digestion sites were PstI and XhoI.

[0097] 2. Using cDNAs of four plant virus G proteins, nine sugarcane Ustilago farfara proteins, and six animal virus proteins as templates, primers with homology arms were designed for PCR amplification to obtain different animal and plant protein fragments. The primer sequences for the exogenous gene PCR are shown in Table 6. The exogenous gene PCR reaction system consisted of 1 μL of upstream primer, 1 μL of downstream primer, 10-20 ng of plasmid DNA template, 25 μL of 2×EasyPfu PCR SuperMix, and the system was made up to 50 μL with deionized water. The exogenous gene PCR reaction conditions were pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing for 30 s (annealing temperature was determined based on the Tm values ​​of the upstream and downstream primers), extension at 72°C (0.5 kb / min), and final extension at 72°C for 5 min, for a total of 35 cycles.

[0098] 3. The exogenous gene PCR product was digested with DpnI enzyme to avoid template interference caused by subsequent construction of the recombinant plasmid; the cloning vector pUC-CMV-FHVRNA1 was homologously recombined with the exogenous gene PCR product, and the homologous recombination method was the same as in Example 1.

[0099] 4. The product of homologous recombination of the exogenous gene and the cloning vector is also transferred into Escherichia coli DH5α competent cells. An appropriate amount of the transformation product is spread and cultured in LB solid culture medium containing 100 μg / ml ampicillin. After culturing for 12 to 16 hours, several single clones are selected from each culture medium for plasmid extraction. At the same time, NheI and XbaI double enzyme digestion is performed to verify whether the exogenous gene is successfully constructed. Finally, the positive clones in the above plasmids are selected and sent to the company for sequencing.

[0100] Table 6 Primer sequences of exogenous genes

[0101]

[0102]

[0103]

[0104] Example 5 Analysis of the efficiency of expressing foreign genes in plasmid systems

[0105] The plasmid containing the animal and plant proteins successfully integrated in Example 4 was transfected into the BHK-21 cell line using the same transfection method as in Example 1. A plasmid expressing the same protein in the pXJ40 vector was used as a comparison. The cells were cultured at 28°C, 30°C, and 37°C. After 48 hours, protein samples were collected and Western Blot analysis was performed using the same method as in Example 1. The results are shown in FIG. Figure 5-9 It can be seen that the cloning vector constructed by the plasmid system of the present invention has a more significant WB detection result than the protein expressed by the pXJ40 vector, indicating that the plasmid system of the present invention has a stronger expression efficiency.

[0106] Those skilled in the art can make various other corresponding changes and deformations based on the technical solutions and concepts described above, and all of these changes and deformations should fall within the scope of protection of the claims of the present invention.

Claims

1. A plasmid system based on the insect virus FHV RNA1 replicon, characterized in that: The plasmid system uses the pUC vector as a backbone vector; the nucleotide sequence of the plasmid system includes a vector promoter, an insect virus FHV RNA1 replicon, a linker sequence, a P2A sequence, a B2 coding sequence, an HDV ribozyme sequence, and a vector terminator in sequence; The nucleotide sequence of the insect virus FHV RNA1 replicon is shown in SEQ ID NO.1; The open reading frame between the 3' end of the insect virus FHV RNA1 replicon and the B2 coding sequence is used to insert a foreign gene for amplification of the foreign gene; the B2 coding sequence is the nucleotide sequence shown in SEQ ID NO.2; There are restriction enzyme cutting sites between the P2A sequence and the B2 coding sequence; the restriction enzyme cutting sites are PstI, EcoRI, XhoI, XbaI and HindIII in sequence; and the vector promoter is a CMV promoter.

2. The method for constructing a plasmid system based on the insect virus FHV RNA1 replicon according to claim 1, characterized in that: The following steps are involved: Step 1, amplifying multiple nucleotide sequence fragments of the plasmid system using primers and templates; Step 2, connecting the nucleotide sequence fragments of the multiple plasmid systems to obtain a connection product; Step 3, transferring the ligation product to Escherichia coli competent cells for culture; Step 4: Cultivate the competent E. coli cells described in step 3, extract the recombinant plasmid after screening, and complete the construction of a plasmid system based on the insect virus FHV RNA1 replicon.

3. A method for constructing a plasmid system based on insect virus FHV RNA1 replicon according to claim 2, characterized in that, In step 1, when amplifying the nucleotide sequence fragments of the plasmid system, the green fluorescent protein expression cassette is used as a template to amplify fragment A; the pUC-T7-FHVRNA1 plasmid is used as a template to amplify fragments B and C, respectively; and the CMV promoter is used as a template to amplify fragment D; the nucleotide sequence of the pUC-T7-FHVRNA1 plasmid is shown in SEQ ID NO.3; the nucleotide sequence of the green fluorescent protein expression cassette is shown in SEQ ID NO.4; and the nucleotide sequence of the CMV promoter is shown in SEQ ID NO.

5.

4. A method for constructing a plasmid system based on the insect virus FHV RNA1 replicon according to claim 3, characterized in that: In step 1, upstream primers are used The green fluorescent protein expression cassette was amplified using the upstream primer [pUC / PstI]-hibit-fwd and the downstream primer msfGFP-[pUC / XhoI]-rev to obtain fragment A. The nucleotide sequence of the upstream primer [pUC / PstI]-hibit-fwd is shown in SEQ ID NO.6, and the nucleotide sequence of the downstream primer msfGFP-[pUC / XhoI]-rev is shown in SEQ ID NO.

7. The upstream primer FHV RNA1 / 1st-fwd and the downstream primer [P2A / PstI]-rev were used to amplify the pUC-T7-FHVRNA1 plasmid as a template to obtain fragment B. The nucleotide sequence of the upstream primer FHV RNA1 / 1st-fwd is shown in SEQ ID NO.8, and the nucleotide sequence of the downstream primer [P2A / PstI]-rev is shown in SEQ ID NO.

9. The upstream primer [XhoI / B2]-fwd and the downstream primer pUC / last Base-rev was amplified using the pUC-T7-FHVRNA1 plasmid as a template to obtain fragment C. The nucleotide sequence of the upstream primer [XhoI / B2]-fwd is shown in SEQ ID NO.10, and the nucleotide sequence of the downstream primer pUC / last base-rev is shown in SEQ ID NO.

11. The CMV promoter was amplified using the upstream primer pUC / CMV-fwd and the downstream primer CMV / FHV RNA1-ev to obtain fragment D. The nucleotide sequence of the upstream primer pUC / CMV-fwd is shown in SEQ ID NO.12, and the nucleotide sequence of the downstream primer CMV / FHV RNA1-rev is shown in SEQ ID NO.

13.

5. The method for expressing proteins in cells using a plasmid system based on the insect virus FHV RNA1 replicon according to claim 1, characterized in that: The following steps are involved: The plasmid system based on the insect virus FHV RNA1 replicon is subjected to double enzyme digestion to obtain a cloning vector; the cloning vector is subjected to homologous recombination with the exogenous gene to obtain a homologous recombination product; After the homologous recombination product is transfected into host cells and cultured, cell samples are collected and tested to complete protein expression.

6. The method for expressing proteins in cells using a plasmid system based on the insect virus FHV RNA1 replicon according to claim 5, characterized in that: The double enzyme cutting sites are PstI and XhoI.

7. Use of the plasmid system based on the insect virus FHV RNA1 replicon according to claim 1 in protein expression.

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