Recombinant protein as well as coding gene and application thereof

Abstract

The invention discloses a recombinant protein as well as a coding gene and application thereof, and belongs to the technical field of bioengineering. The invention designs a recombinant protein which is composed of two subunits with different functions, and design and transformation are carried out on the basis of a conserved sequence of an alpha herpes virus protein with an immune escape function, so that the secretory expression of the recombinant protein is promoted, and the defects of inclusion body formation, overhigh host toxicity and high target protein purification difficulty are overcome. Experimental results show that the recombinant protein provided by the invention has a remarkable effect of inhibiting innate immunity of cells, and can effectively inhibit interferon generated by innate immunity of cells, so that the titer of cultured viruses is improved.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a recombinant protein, its encoding gene, and its applications. Background Technology

[0002] Vaccination is an effective way to prevent viral infection. Currently, vaccines are mainly inactivated vaccines and live vaccines. The titer of the cultured virus directly affects the production cost of the vaccine. Therefore, improving the titer of the cultured virus is of great practical significance.

[0003] However, viral culture depends on viral replication within cells. After viral infection, cells generate a natural immune response to resist the virus, with the production of type I interferon (IFN) being crucial. Most proteins in alpha herpesviruses that suppress cellular innate immunity are encoded by late-stage genes, and their protein products are primarily produced in the late stages of viral infection, dependent on viral genome replication and expression. Once viral nucleic acid enters the cell, it triggers an antiviral response, preventing viral invasion and replication. If exogenous anti-innate immune proteins can be added simultaneously with or before viral infection, the cellular innate immune response can be effectively suppressed. Therefore, using mature protein expression systems such as E. coli expression systems, baculovirus-insect cell expression systems, and CHO cell expression systems to express proteins that suppress innate immunity and then adding them to the culture medium of cultured cells to combat cellular innate immunity is a promising approach. However, most of these proteins are rich in basic amino acids, and exogenous expression of these proteins easily leads to inclusion bodies, reducing protein solubility, causing toxicity to the host, inhibiting host growth, and even causing death. Furthermore, their strong positive charge makes purification very difficult. Summary of the Invention

[0004] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a recombinant protein, its encoding gene, and its applications to improve the titer of virus cultures.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: a recombinant protein is provided, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] The present invention provides a gene encoding the above-mentioned recombinant protein, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0007] This invention provides an application of the above-mentioned recombinant protein in improving virus culture titers.

[0008] Furthermore, the virus is at least one of pseudorabies virus, porcine circovirus type 2, and porcine parvovirus.

[0009] This invention provides a method for increasing virus culture titer by adding the aforementioned recombinant protein to the virus culture system during the virus culture process.

[0010] Furthermore, the concentration of recombinant protein is 20-200 ng / mL.

[0011] This invention provides a reagent for increasing virus culture titers, comprising the aforementioned recombinant protein.

[0012] This invention provides an application of the above-mentioned recombinant protein in the preparation of a viral vaccine, wherein the virus is pseudorabies virus, porcine circovirus type 2, or porcine parvovirus.

[0013] This invention offers the following advantages: It modifies the conserved sequence of an α-herpesvirus protein with immune escape capabilities, thereby promoting the secretory expression of the recombinant protein and overcoming the drawbacks of inclusion body formation, excessive toxicity to the host, and difficulty in purifying the target protein. Experimental results show that the recombinant protein of this invention significantly inhibits cellular innate immunity, effectively suppressing interferon produced by cellular innate immunity, thus increasing the viral titer in culture. Attached Figure Description

[0014] Figure 1 The image shows the results of conserved sequence alignment for subunit 1 and subunit 2. Figure 2 Diagram showing the amplification of the recombinant protein gene; Figure 3 This is an SDS-PAGE image of a recombinant protein containing a vector tag. Figure 4 This is an SDS-PAGE image of the recombinant protein. Detailed Implementation

[0015] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0016] Example 1: Plasmid construction and expression of recombinant proteins containing vector tags (1) Plasmid construction: The conserved sequence of the α-herpesvirus protein with immune escape function was selected and modified to form two subunits of the recombinant protein (subunit 1 and subunit 2, whose nucleotide sequences are shown in SEQ ID NO. 3-4 and amino acid sequences are shown in SEQ ID NO. 5-6, respectively). The nucleotide sequence was optimized according to the codon preference of Escherichia coli. (GGGGS)2 was designed for flexible linkage to protect the function of the two subunits of the recombinant protein. The synthesized nucleotide sequence is shown in SEQ ID NO. 2, and the encoded amino acid sequence is shown in SEQ ID NO. 1 (see the conserved sequence alignment diagram). Figure 1 Then it was constructed into the pET-32a(+) vector plasmid (purchased from Novagen). Eco R Ⅰ and Xho Between site I. The constructed sequence was amplified using PCR, and a specific 1100bp band appeared, as shown. Figure 2 As shown. The amplification primer sequences are as follows: F: 5'-CAAGGCCATGGCTGATATCG-3' (SEQ ID NO.7); R: 5'-CAAGGCCATGGCTGATATCG-3' (SEQ ID NO. 8).

[0017] (2) Plasmid transformation: 100 μL of competent BL21(DE3) cells were thawed on ice, 1 μL of the constructed plasmid was added and gently mixed, and the mixture was kept on ice for 30 minutes. The bacterial culture was then heat-shocked in a 42℃ water bath for 90 seconds, followed immediately by a 2-minute ice bath. 500 μL of antibiotic-free LB medium was added, and the culture was incubated at 37℃ with shaking at 180 rpm for 1 hour. An appropriate amount of the transformed competent cells was spread on an LB plate containing ampicillin and incubated upside down at 37℃ overnight. The next day, a single colony was picked and inoculated into a test tube containing ampicillin and incubated at 37℃ with shaking at 180 rpm until the OD600 reached 1.0. The plasmid was extracted using a plasmid extraction kit and sent for sequencing. The remaining bacterial culture was aliquoted and stored at -80℃ with 20% glycerol.

[0018] (3) Culture and expression of recombinant protein with vector tag: After the bacterial strains that were verified by sequencing were taken out, they were inoculated into LB plates containing ampicillin and incubated overnight at 37°C with the plates upside down. The next day, a single colony was picked and inoculated into a test tube containing ampicillin and cultured at 37°C with shaking at 180 rpm until the OD600 was 1.0. Samples were taken and inoculated into Erlenmeyer flasks containing ampicillin at a ratio of 1% to 2% and cultured at 37°C with shaking at 180 rpm until the OD600 was 0.4 to 0.8. IPTG was added to a final concentration of 0.5 mmol / L, the culture temperature was adjusted to 16°C and the rotation speed was 120 rpm, and expression was induced for 20 hours. All bacterial cultures were centrifuged at 5000 rpm at 4°C for 15 minutes, and the supernatant was the expressed recombinant protein with vector tag.

[0019] Example 2: Purification of recombinant protein (1) Purification of recombinant protein containing the carrier tag: Nickel ion affinity chromatography packing material was loaded into a chromatography column. After filtering out the liquid, equilibration buffer (20 mM Tris + 500 mM NaCl + 10 mM imidazole, pH 7.4) was added and the column was equilibrated for 30-60 minutes. After filtering out the equilibration buffer, the harvested recombinant protein containing the carrier tag was added to the chromatography column, shaken evenly, and slowly rotated at 2-8°C for 2 hours. Then, the liquid was filtered out from the bottom of the chromatography column, and washing buffer (20 mM Tris + 500 mM NaCl + 20 mM imidazole, pH 7.4) was added. After shaking evenly, the washing buffer was filtered out, and the protein was washed twice. Finally, elution was performed with elution buffer (20 mM Tris + 500 mM NaCl + 300 mM imidazole, pH 7.4). The elution volume could be adjusted according to the protein concentration. The recombinant protein containing the Trx and His tags was obtained. The SDS-PAGE results are shown below. Figure 3 As shown.

[0020] (2) Recombinant protein cleavage: The eluted recombinant protein containing the carrier tag was added to a dialysis bag and dialyzed with dialysis buffer (20mM Tris-HCl + 150mM NaCl + 2.5mM CaCl2, pH 8.0). The dialysis buffer was changed 3 times during the process. This operation directly replaced the protein solution with thrombin cleavage buffer, which is beneficial for subsequent operations. The dialyzed protein was harvested and then cleaved with thrombin. According to the ratio, 2~3U of thrombin was added for every 1mg of protein. After adding thrombin, the mixture was mixed evenly and cleaved at 20℃ for 16 hours. The His-tagged part was cleaved from the recombinant protein.

[0021] (3) Purification of recombinant protein: The cleaved liquid was purified again using a nickel column as described above. After equilibration of the column, the equilibration buffer was filtered off. The cleaved protein solution was added to the chromatography column and gently shaken at 2-8°C for 2 hours. The liquid was then filtered from the bottom of the chromatography column. The filtered flow-through was the recombinant protein. The recombinant protein was dialyzed against PBS using the method described above. The SDS-PAGE results of the recombinant protein after dialysis are shown below. Figure 4 As shown.

[0022] Example 3: Recombinant protein inhibits the production of IFN-β in cellular innate immunity. (1) Expression and purification of subunit protein 1 and subunit protein 2: The recombinant protein has two flexible connected subunits. These two subunits were expressed as independent proteins, and their inhibitory effect on innate immunity was compared with that of the recombinant protein. The expression method of subunit protein 1 and subunit protein 2 was performed according to the method in Example 1, and the purification method was performed according to the method in Example 2.

[0023] (2) Comparison of the inhibitory effects of subunit protein 1, subunit protein 2 and recombinant protein on IFN-β production by innate immunity of ST cells: ST cells were digested with trypsin and passaged into multiple flasks, added to culture medium, and labeled as 1-14. They were then incubated at 37℃ and 5% After CO2 culture until the cells reached 80% confluence, subunit protein 1 was added to bottles 1 and 2 at a final concentration of 20 ng / mL, subunit protein 1 to bottles 3 and 4 at a final concentration of 200 ng / mL, subunit protein 2 to bottles 5 and 6 at a final concentration of 20 ng / mL, subunit protein 2 to bottles 7 and 8 at a final concentration of 200 ng / mL, recombinant protein to bottles 9 and 10 at a final concentration of 20 ng / mL, and recombinant protein to bottles 11 and 12 at a final concentration of 200 ng / mL. The cells were cultured for another 8 hours. Then, Toll-like receptor 3 agonist Poly(I:C) was added to bottles 1, 3, 5, 7, 9, 11, and 13 at a final concentration of 10 μg / mL. The cells were cultured for another 16 hours. The culture supernatant was then analyzed using a commercial porcine IFN-β content detection kit. The results are shown in Table 1. The addition of subunit protein 1, subunit protein 2 and recombinant protein reduced the amount of IFN-β in ST cells and effectively reduced the amount of IFN-β produced by Poly(I:C) stimulation. Compared with subunit protein 1 and subunit protein 2, the IFN-β level was lower after the addition of recombinant protein, indicating that recombinant protein superimposed the functions of subunit protein 1 and subunit protein 2.

[0024] Table 1 IFN-β content

[0025] Example 4: Recombinant protein increases virus culture titer (1) Recombinant protein increases the culture titer of virulent pseudorabies virus (PRV) strains: ST cells were digested with trypsin and passaged into multiple flasks. Culture medium was added and labeled as 1-6. The cells were cultured at 37℃ and 5% CO2 until the cells reached 80% confluence. Recombinant protein with a final concentration of 20 ng / mL was added to flask 1, and recombinant protein with a final concentration of 200 ng / mL was added to flask 2. 0.5% virus with a concentration of 10 ng / mL was added to flasks 1-3. 6.0 TCID 50 The fourth vial contained PRV GD-82 strain virus solution at a concentration of / mL, and was used as a blank cell control. After culturing for 20-24 hours, the virus solution was harvested, frozen and thawed at -20℃, and samples were taken. The samples were serially diluted 10-fold with serum-free MEM medium, and 10 μL of each solution was used. -5 10 -6 10 -7 10 -8 10 -9 Dilutes were seeded into 96-well plates, with 8 wells per dilution at 100 μL / well. 100 μL of ST cell suspension was added to each well. Eight wells were included as a control without virus. The plates were incubated at 37°C and 5% CO2 for 72–120 hours. The number of wells showing cytopathic effects was observed, and the median tissue culture infectious dose (TCID) of the virus was calculated using the Reed-Muench method. 50 The results, as shown in Table 2, indicate that the addition of recombinant protein increased the culture titer of the highly virulent PRV GD-82 strain.

[0026] Table 2. Culture titers of the highly virulent strain PRV GD-82

[0027] (2) Recombinant protein increases the culture titer of attenuated PRV strains: ST cells were digested with trypsin and passaged into multiple flasks. Culture medium was added, and the flasks were labeled 1-7. The cells were cultured at 37℃ and 5% CO2 until the cells reached 80% confluence. Recombinant protein with a final concentration of 20 ng / mL was added to flasks 1 and 4, and recombinant protein with a final concentration of 200 ng / mL was added to flasks 2 and 5. 0.5% virus with a concentration of 1×10⁻⁶ was added to flasks 1, 2, and 3. 6 TCID 50 / mL of PRV Bartha K-61 strain virus solution was added to bottles 4, 5, and 6, with a virus concentration of 0.5% (1×10⁶). 6 TCID 50 The PRV ΔgE+ΔgI strain virus solution was diluted / mL. The 7th bottle served as a blank cell control. The cells were cultured for another 20-24 hours. The virus solution was harvested, frozen and thawed at -20℃, and then sampled. The samples were serially diluted 10-fold with serum-free MEM medium. 10 mL of the diluted solution was taken. -5 10 -6 10 -7 10-8 10 -9 The virus-containing dilutions were seeded into 8 wells at a rate of 100 μL / well, with 100 μL of ST cell suspension added to each well. Eight wells were included as a control group without virus. The cells were incubated at 37°C and 5% CO2 for 72–120 hours. The number of wells showing cytopathic effects was observed, and the TCID of the virus was calculated using the Reed-Muench method. 50 The results, shown in Table 3, indicate that the addition of recombinant protein increased the culture titers of the attenuated PRV strain Bartha K-61 and ΔgE+ΔgI.

[0028] Table 3. Culture titers of PRV attenuated strain Bartha K-61 and ΔgE+ΔgI.

[0029] (3) Recombinant protein increases the culture titers of porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV): ST cells were digested with trypsin and passaged into multiple flasks. Culture medium was added and labeled 1-4. Recombinant protein with a final concentration of 20 ng / mL was added to flask 1, and recombinant protein with a final concentration of 200 ng / mL was added to flask 2. 0.5% virus with a content of 1×10⁻⁶ was added to flasks 1, 2, and 3. 6 TCID 50 PCV2 strain 162 virus solution was prepared at / mL, with the fourth bottle serving as a blank cell control. The mixture was incubated at 37℃ and 5% CO2 for 72 hours. The virus solution was harvested, frozen and thawed at -20℃, and then sampled. It was serially diluted 10-fold with serum-free MEM medium. 10 mL of the diluted solution was then used. -4 10 -5 10 -6 10 -7Inoculate 8 wells with each dilution at 100 μL / well, adding 100 μL of ST cell suspension to each well. Include 8 wells as a control cell line without virus. Incubate at 37°C and 5% CO2 for 72 hours. Discard the cell culture medium. Add 100 μL of cold methanol / acetone fixative to each well and fix at -15 to -20°C for 30 to 40 minutes. Discard the fixative and place the cells in a fume hood or allow them to air dry for 30 to 60 minutes. Add PBS containing 5% skim milk to each well. Incubate 100 μL of PBS at 37°C for 30-40 minutes, then discard the blocking solution. Wash each well 3-5 times with 300 μL of PBS. After drying, dilute porcine circovirus type 2 polyclonal antibody 1:1000 with PBS containing 5% skim milk, add 100 μL to each well, incubate at 37°C for 2 hours, wash 3-5 times with PBS, and then incubate at 37°C for 1 hour. Observe the fluorescence results under an inverted fluorescence microscope and calculate the TCID of the virus according to the Reed-Muench method. 50 The results, as shown in Table 4, indicate that the addition of recombinant protein increased the culture titer of PCV2 162 strain.

[0030] Table 4. Culture titers of PCV2 strain 162

[0031] ST cells were digested with trypsin and passaged into multiple flasks. Culture medium was added, and the flasks were labeled 1-4. Recombinant protein was added to flask 1 at a final concentration of 20 ng / mL, and to flask 2 at a final concentration of 200 ng / mL. 0.5% virus at a concentration of 1 × 10⁻⁶ was added to flasks 1, 2, and 3. 6 TCID 50 / mL of PPV SC1 strain virus solution was used, with the fourth bottle serving as a blank cell control. The mixture was incubated at 37℃ and 5% CO2 for 72 hours. The virus solution was harvested, frozen and thawed at -20℃, and then sampled. It was serially diluted 10-fold with serum-free MEM medium. 10 -4 10 -5 10 -6 10 -7 The virus-containing dilutions were seeded into 8 wells at a rate of 100 μL / well, with 100 μL of ST cell suspension added to each well. Eight wells were included as a control group without virus. The cells were incubated at 37°C and 5% CO2 for 72–120 hours. The number of wells showing cytopathic effects was observed, and the TCID of the virus was calculated using the Reed-Muench method. 50 The results, as shown in Table 5, indicate that the addition of recombinant protein increased the culture titer of PPV SC1 strain.

[0032] Table 5. Culture titers of PPV SC1 strain

[0033] The above results indicate that the addition of the recombinant protein of the present invention can significantly improve the culture titers of PCV2 and PPV.

[0034] In summary, the recombinant protein of the present invention can significantly improve the culture titer of various viral strains.

[0035] The nucleotide and amino acid sequences involved in this invention are shown below: (1) Amino acid sequence of the recombinant protein: MGAGSYGSVLVYGSVAVKTLAAGFGHELLMTLLAGECSLSAGADAIIRPVGFSLPLRQLVFPAYDMDMDAYAEALAVLSPAALHALGRVFVDLGRALVFLNSCGLSHLDVKGGNIFVNTCGNMITTAVIGDFSLMTLNGGGGSGGGGSMAIPAD LCAGIRCHNRFYEALACDVSAYGAQLFGAPEIFKSVVSVSLAFEVNLQSRRPDCVCLLRLGAGPHLCLLIELKTCRFSANMNTPSKMDQRHGGLCQLRDSASLVAALAPPGPDPVVLAPLLVFVAQRGMRVLKVTRLPAQQTASNASALEAILAGLAEYVPFARA (SEQ ID NO.1); (2)Nucleotide sequence of the recombinant protein: ATGGGTGCTGGTTCTTACGGTTCTGTCCTGGTATATGGTAGCGTTGCGGTTAAAACTCTGGCAGCAGGTTTCGGCCACGAACTGCTGATGACCCTGCTGGCCGGCGAATGTAGCCTGTCTGCCGGTGCCGACGCGATCATTCGTCCGGTTGGTTTCAGCCTGCCGCTGCGTCAGCTGGTTTTTCCGGCGTACGACATGGATATGGATGCCTATGCCGAGGCGCTGGCCGTACTGAGCCCGGCGGCGCTGCACGCTCTGGGCCGTGTGTTTGTGGACCTGGGTCGTGCCCTGGTATTCCTGAATAGCTGTGGCCTGTCCCACCTGGATGTGAAGGGTGGTAACATCTTCGTCAATACTTGTGGTAACATGATCACCACCGCAGTCATCGGTGACTTCTCTCTGATGACCCTGAATGGTGGTGGCGGCTCTGGCGGTGGCGGTAGCATGGCGATTCCGGCTGATCTGTGTGCGGGTATTCGTTGCCATAACCGCTTCTACGAAGCTCTGGCGTGTGACGTAAGCGCATATGGCGCGCAGCTGTTCGGCGCTCCGGAAATCTTCAAAAGCGTTGTTTCCGTGTCCCTGGCGTTCGAAGTTAACCTGCAGTCTCGCCGTCCGGACTGCGTTTGCCTGCTGCGTCTGGGTGCCGGTCCGCACCTGTGCCTGCTGATCGAACTGAAAACCTGCCGTTTCAGCGCAAACATGAACACGCCGAGCAAAATGGATCAGCGTCACGGTGGTCTGTGCCAGCTGCGTGATTCCGCATCCCTGGTCGCGGCTCTGGCTCCTCCGGGTCCGGATCCGGTAGTTCTGGCGCCGCTGCTGGTTTTCGTAGCACAGCGTGGCATGCGCGTGCTGAAAGTGACTCGCCTGCCTGCTCAGCAGACCGCTTCTAACGCGTCCGCGCTGGAAGCGATCCTGGCGGGCCTGGCGGAATATGTACCGTTCGCCCGTGCTTGA (SEQ ID NO.2); (3)Nucleotide sequence of subunit 1: ATGGGTGCTGGTTCTTACGGTTCTGTCCTGGTATATGGTAGCGTTGCGGTTAAAACTCTGGCAGCAGGTTTCGGCCACGAACTGCTGATGACCCTGCTGGCCGGCGAATGTAGCCTGTCTGCCGGTGCCGACGCGATCATTCGTCCGGTTGGTTTCAGCCTGCCGCTGCGTCAGCTGGTTTTTCCGGCGTACGACATGGATATGGATGCCTATGCCGAGGCGCTGGCCGTACTGAGCCCGGCGGCGCTGCACGCTCTGGGCCGTGTGTTTGTGGACCTGGGTCGTGCCCTGGTATTCCTGAATAGCTGTGGCCTGTCCCACCTGGATGTGAAGGGTGGTAACATCTTCGTCAATACTTGTGGTAACATGATCACCACCGCAGTCATCGGTGACTTCTCTCTGATGACCCTGAATTGA (SEQ ID NO.3); (4)Nucleotide sequence of subunit 2: ATGGCGATTCCGGCTGATCTGTGTGCGGGTATTCGTTGCCATAACCGCTTCTACGAAGCTCTGGCGTGTGACGTAAGCGCATATGGCGCGCAGCTGTTCGGCGCTCCGGAAATCTTCAAAAGCGTTGTTTCCGTGTCCCTGGCGTTCGAAGTTAACCTGCAGTCTCGCCGTCCGGACTGCGTTTGCCTGCTGCGTCTGGGTGCCGGTCCGCACCTGTGCCTGCTGATCGAACTGAAAACCTGCCGTTTCAGCGCAAACATGAACACGCCGAGCAAAATGGATCAGCGTCACGGTGGTCTGTGCCAGCTGCGTGATTCCGCATCCCTGGTCGCGGCTCTGGCTCCTCCGGGTCCGGATCCGGTAGTTCTGGCGCCGCTGCTGGTTTTCGTAGCACAGCGTGGCATGCGCGTGCTGAAAGTGACTCGCCTGCCTGCTCAGCAGACCGCTTCTAACGCGTCCGCGCTGGAAGCGATCCTGGCGGGCCTGGCGGAATATGTACCGTTCGCCCGTGCTTGA (SEQ ID NO.4); (5)Amino acid sequence of subunit 1: MGAGSYGSVLVYGSVAVKTLAAGFGHELLMTLLAGECSLSAGADAIIRPVGFSLPLRQLVFPAYDMDMDAYAEALAVLSPAALHALGRVFVDLGRALVFLNSCGLSHLDVKGGNIFVNTCGNMITTAVIGDFSLMTLN (SEQ ID NO.5); (6)Amino acid sequence of subunit 2: MAIPADLCAGIRCHNRFYEALACDVSAYGAQLFGAPEIFKSVVSVSLAFEVNLQSRRPDCVCLLRLGAGPHLCLLIELKTCRFSANMNTPSKMDQRHGGLCQLRDSASLVAALAPPGPDPVVLAPLLVFVAQRGMRVLKVTRLPAQQTASNASALEAILAGLAEYVPFARA (SEQ ID NO.6); (7)Original nucleotide sequence of subunit 1: ATGGGGGCCGGCTCGTACGGCAGCGTGCTCGTGTACGGCTCGGTGGCCGTGAAGACGCTCCGCGCCGGCTTCGGCCACGAGGCCGTCATGACGCTGCTGGCCGCGGAGGCGACGCGCTCCGCCGGCGTCCGCGGCGTGGTGCGCCTGGTGGGGCTCTCGGCGCCGCTGCGCCAGCTCGTGTTCCCGGCCTACGAGATGGACATGGACGCGTACCGCCGCTCGCTCACGGCGCGCCCCGGGCACGTGGTGCACGCCCTGGGGCGCGTCTTCGCCGAGCTCGGGCGCGCGCTCGTGTTCCTCAACAGCCGCGGGCTCAGCCACCTGGACGTCAAGGGCGGCAACATCTTTGTGCGCACGTGCGGCAACATGGTCGTGACGGCCGTCATCGGGGACTTTAGCCTCATGGCCCTCAACTGA (SEQ IDNO.9); (8) Original nucleotide sequence of subunit 2: ATGCGCATCCCGGCGCGCTTCCGGGCGGGGATCCGCTGCCACAACCGCTTCTACGAACGCCTGCGACGCGACCCGTCCGCGTACGGCGAGAGTCTCTTCGGGCTTCCCCGAGAGACGC TTAAGAAGGCGTCCTTGACCCTGGCCTTTGAAGTCAACCTCGGGGTGCGCCGCCCCGACTGCGTGTGCGTGGTGCGGCTGGGGGCGGGCCCGCACCTGTGCTTCCTCATCGAGCTCAAGACGTGCCGCTTCCCC CGGAACCTCAACACGCCCAGCAAGCGCGGGCAGCGGCACGAGGGGCTGTGCCAGCTGCGCGACTCGGCGCGCCTCCTCGCCGCCGCCGTGCCCCCGGGCGGCGAGGAGATCACGCTCGTGCCCCTGCTGGTGTT TGTGGCCCAGCGGAGCATGCGCGTGCTCGACGTGACGCGCCTGCCCTGCACCCAGACGCGCGGCAACGCGAGCGCGATGGCCGCGACCGTGCGCGGCCTCGCCGAGTACGTCGCCGGGCCGCGGCGGTGA (SEQ ID NO.10).

[0036] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant protein, characterized in that, The amino acid sequence of the recombinant protein is shown in SEQ ID NO.

1.

2. A gene encoding the recombinant protein of claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. The application of the recombinant protein according to claim 1 in improving virus culture titers.

4. The application according to claim 3, characterized in that, The virus is at least one of pseudorabies virus, porcine circovirus type 2, and porcine parvovirus.

5. A method for increasing virus culture titer, characterized in that, The viral culture titer is increased by adding the recombinant protein of claim 1 to the viral culture system during the viral culture process.

6. The method according to claim 5, characterized in that, The concentration of the recombinant protein is 20-200 ng / mL.

7. A reagent for increasing virus culture titers, characterized in that, The reagent includes the recombinant protein as described in claim 1.

8. The use of the recombinant protein according to claim 1 in the preparation of a viral vaccine, characterized in that, The virus in question is pseudorabies virus, porcine circovirus type 2, or porcine parvovirus.

Images