NL2028806B1

NL2028806B1 - Tilv-2 protein or gene encoding tilv-2 protein in preparation of subunit vaccine against tilapia lake virus

Info

Publication number: NL2028806B1
Application number: NL2028806A
Authority: NL
Inventors: Zhu Bin
Original assignee: Univ Northwest A&F
Priority date: 2021-07-22
Filing date: 2021-07-22
Publication date: 2022-06-03
Also published as: NL2028806A

Abstract

The present invention provides use of TILV-2 protein, a gene encoding TILV-2 protein, a recombinant plasmid containing the gene or recombinant bacteria containing the gene in preparation of subunit vaccine against Tilapia Lake virus. The present invention belongs to the technical ﬁeld of vaccine engineering. The present invention uses TILV- 2 protein as the immunogenic protein to prepare the subunit vaccine against Tilapia Lake virus, ﬁlls the gap in the research of subunit vaccine against Tilapia Lake virus, effectively avoids the biological safety risks caused by using whole pathogen to produce vaccines, and facilitates the Tilapia large-scale production of vaccine against Lake virus. It has been verified that a protection rate was up to 72% after immunizing the Tilapia with the subunit vaccine against Tilapia Lake virus prepared by the method of the present invention.

Description

TILV-2 PROTEIN OR GENE ENCODING TILV-2 PROTEIN IN

PREPARATION OF SUBUNIT VACCINE AGAINST TILAPIA LAKE VIRUS TECHNICAL FIELD

[01] The present invention relates to the technical field of vaccine engineering, in particular to the use of TILV-2 protein or a gene encoding TIL V-2 protein in preparation of subunit vaccine against Tilapia Lake virus.

BACKGROUND ART

[02] There are more than 100 species of Tilapia. Because of its advantages in omnivore nature, fast growth, and strong fecundity, 1t has been introduced and cultivated in many countries and has become the world's second important fish after carp. It is the main source of edible protein in many developing countries. It is also an important source of income for many fishermen and farmers. Since 2009, Tilapia Lake virus disease caused by Tilapia Lake virus (Tilapia Lake Virus, TiLV) infection has erupted in many countries, resulting in death of a large number of wild and farmed Tilapia, causing serious economic losses.

[03] Tilapia Lake virus disease is also known as Tilapia syncytical hepatitis (SHT). The pathogen TiLV is a newly discovered virus in recent years, and its classification status has not been confirmed. Based on its biological characteristics, TiLV is currently classified as a new virus in the Orthomyxoviridae family. TiLV infects Tilapia in the stage of early development. Because the immune system of the larval fish has not yet fully developed, the protective effect of vaccine on the larvae is limited. Prevention and control measures, include reducing the spread of pollutants through equipment, vehicles or personnel, breeding disease-resistant or insensitive fish species, developing TiLV- infected fish fry free of specific pathogens, eliminating virus in the body of Tilapia broodstock that have been infected by TiLV, and developing effective TiLV prevention and control vaccines, etc.

[04] Prior art document CN106456737A discloses a vaccine for preventing Tilapia Lake virus disease, specifically a vaccine composition containing an attenuated strain of Tilapia Lake virus (TiLV), which is used to protect Tilapia from (TiLV) infection. However, the vaccine using the attenuated strain of Tilapia Lake virus (TiLV) as the immunogen still retains a certain degree of virulence, and the virulence of the virus may be restored, which posing a greater biological safety risk.

SUMMARY

[05] The objective of the present invention is to provide the use of TILV-2 protein, a gene encoding TILV-2 protein, a recombinant plasmid containing the gene, or recombinant bacteria containing the gene in preparation of subunit vaccine against Tilapia Lake virus, which effectively avoids biosafety risks caused by vaccine production using a whole pathogen.

[06] In order to achieve the above-mentioned objective of the invention, the present invention provides the following technical solutions.

[07] The present invention provides the use of TILV-2 protein, a gene encoding TILV-2 protein, a recombinant plasmid containing the gene or recombinant bacteria containing the gene in preparation of subunit vaccine against Tilapia Lake virus, wherein the amino acid sequence of the TILV-2 protein is set forth in SEQ ID NO. : 2.

[08] Preferably, the nucleotide sequence of the encoding gene is set forth in SEQ ID NO. : 1.

[09] Preferably, the original plasmid of the recombinant plasmid comprises pET32a.

[10] Preferably, the insertion sites of the encoding gene in the recombinant plasmid are BamHI and Xhol. [MI] Preferably, the original bacteria of the recombinant bacteria include Escherichia coll.

[12] Preferably, the recombinant bacteria are constructed by a method comprising the following steps:

[13] extracting total RNA of the Tilapia Lake virus, and reverse-transcripting the total RNA of the Tilapia Lake virus to cDNA;

[14] performing PCR amplification using a primer set T1, with cDNA as a template, to obtain an amplification product; the primer set T1 comprises TIF and TIR; the nucleotide sequence of TIF is set forth in SEQ ID NO. : 3, the nucleotide sequence of TIR is set forth in SEQ ID NO. : 4;

[15] recovering the amplification product and performing a first DNA purification to obtain a purified amplification product;

[16] double digesting the purified amplification product with BamHI and Xho! and ligating the digestion product to a carrier of pMD19T to obtain pMD19T-2-TiLV, and transforming pMD19T-TiLV-2 into £. coli competent cells, screening for a positive strain, extracting the pMD19T-TiLV-2 in the positive strain;

[17] digesting the obtained pMD19T-TiLV-2 present in the positive strain and the pET-32a (+) with BamHI and Xhol, recovering and performing a second DNA purification, respectively, and recombining the product obtained by the second DNA purification to pET32a-TiLV-2 by using T4 DNA ligase;

[18] transforming the pET32a-TiLV-2 into €. coli competent cells to obtain the recombinant bacteria.

[19] Preferably, the PCR amplification in step 2) comprising: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C 1 min, 35 cycles; and extension at 72°C for 10 min.

[20] Preferably, the PCR amplification in step 2) uses an amplification system in which includes, calculated as 25 pL, the following components: 2 x PCR Mix 12.5 pL, TIF and TIR each 1 uL, template 1 uL, ddH20 9.5 pL, with the molar concentration of TIF and TIR being 10 mM, respectively.

[21] Preferably, the TILV-2 protein is prepared by a method comprising the following steps:

[22] SI: mixing a culture of the recombinant bacteria obtained according to the use of any of the above technical solution with isopropyl B-D-thiogalactoside, performing induced expression at a final concentration of isopropyl B-D-thiogalactoside concentration of 0.5-1.0 mmol/L to obtain a bacterial culture of induced expression;

[23] S2: centrifuging the bacterial culture of induced expression, collecting the precipitates, re-suspending precipitates to obtain a re-suspended solution, and sonicating the re-suspended solution to obtain a sonicated product;

[24] S3: centrifuging the sonicated product, collecting precipitates, and washing precipitates dissolving the washed precipitates with a denaturation solution containing 6—10 mol/L of urea to obtain a dissolved solution;

[25] S4: dissolving the dissolved solution in PBS containing 8 mol/L urea, performing a first dialysis to give a first dialysate; centrifuging the first dialysate to give supernatant; performing a second dialysis on the supernatants containing 6.0, 4.0, 2.0 and 1.0 mol/L urea sequentially to give a second dialysate; performing a second dialysis on the second dialysate in 1 * PBS to obtain a crude extract solution;

[26] wherein dialysis bags used for the first dialysis, the second dialysis and the third dialysis each independently has a cut-off molecular of 8000-14000D;

[27] atime for the first dialysis is 6 hours;

[28] atime for the second dialysis in PBS with different concentrations of urea is each 5hours;

[29] and a time of the third dialysis is 9-16 h;

[30] SS: purifying the crude extract solution using a nickel column with a His tag to obtain eluate, wherein a 500 mM imidazole elution buffer is used during the purification, and volume of the eluate is 10 times of column volume; [BI] So: freeze-drying the eluate to give freeze-dried powder containing TILV-2 protein.

[32] The present invention provides the use of TILV-2 protein, a gene encoding TILV-2 protein, a recombinant plasmid containing the gene or recombinant bacteria containing the gene in preparation of subunit vaccine against Tilapia Lake virus. In the present invention, TILV-2 protein is used as immunogenic protein to prepare the subunit vaccine against Tilapia Lake virus, which fills the gap in the research of subunit vaccine against Tilapia Lake virus, effectively avoids the biological safety risks caused by vaccine production by using whole pathogen, and facilitates large-scale production of the vaccine against Tilapia Lake virus. It has been verified that the subunit vaccine against Tilapia Lake virus prepared by the method of the present invention gives a protection rate up to 72% after immunizing Tilapia. The biosafety test shows that the vaccine has very high biosafety.

BRIEFT DESCRIPTION OF THE DRAWINGS

[33] FIG. 1 shows the identification results for double digestion of pET32a-TiLV-2 recombinant vector; M: DNA marker, 1: target band.

[34] FIG. 2 shows the expression results of pET32a-TILV-2 protein; M: protein marker, 1: target band.

[35] FIG. 3 shows the results for Western blot expression of pET32a-TILV-2 protein.

[36] FIG. 4 is the result after the pET32a-TILV-2 protein was purified; M: protein marker, 1: target band;

[37] FIG. 5 shows the results for Western blot expression of purified pET32a-TILV- 2 protein.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[38] The present invention provides the use of TILV-2 protein, a gene encoding TILV-2 protein, a recombinant plasmid containing the gene, or recombinant bacteria containing the gene in preparation of subunit vaccine against Tilapia Lake virus, and the 5 amino acid sequence of the TILV-2 protein is set forth in SEQ ID NO. : 2, specifically:

[39] MSQFGKSFKGRTEVTITEYRSHTVKDVHRSLLTADKSLRKSFCFRNALN

QFLDKDLPLLPIRPKLESRVAVKKSKLRSQLSFRPGLTQEEAIDLYNKGYDGDS VSGALQDRVVNEPVAYSSADNDKFHRGLAALGYTLADRAFDTCESGFVRAIP TTPCGFICCGPGSFKDSLGFVIKISEFWHMYDGFQHFVAVEDAKFLVSKSPSFW LAKRLAKRLNLVPKEDPSVAAAECPCKRVWEASFARAPTALDPFGGRAFCDQ GWVYHRDVGYATANHISQETLFQQALSVKNLGPQGSANVSGSIHTALDRLRA AYSRGTPASRSILQGLANLITPVGENFECDLDKRKLNIKALRSPERYITIEGLVV

NLDDVVRGFYLDKAKVTVLSRSKWMGYEDLPQKPPNGTFYCRKRKAMLLIS CSPGTYAKKRKVAVQEDRFKDMRVENFREVAENMDLNQ (Accession number: QDC17494.1).

[40] In the present invention, there is no special requirement on the source of the TILV-2 protein, as long as the protein can be prepared by conventional chemical synthesis or other protein expression systems in the field.

[41] Inthe present invention, the nucleotide sequence of the encoding gene for TIL V- 2 protein is shown in SEQ ID NO. : 1, specifically:

[42] atgagtcagtttgggaaatcattcaagggcagaactgaagtcacaataaccgaatatcgctcccatactgtcaaag atgtgcacagaagcttacttacggctgacaaatctctaagaaagtcattctgctttaggaacgccctaaaccagttcttggataa agacttgcetcttetgcccattcggccaaaattagaatccagggttgctgtgaaaaagtctaagctgaggagtcagctgtcgtt cagacccggtttaactcaggaggaagcaattgatctttacaacaagggctatgatggtgacagcgtctcaggtgccttacaag atagggtagtcaatgagcctgtagcttactcgagcgcagataatgataaatttcacaggggcttagcggctctagggtacactt tggctgatagagcatttgatacstgcgaatccggcttcstgagagcaatccctaccactccatgcgggttcatatsttgegggc caggitctttcaaagattcacttggatttgtgataaaaatcagcgaattctggcacatgtatgacgggtttcaacacttegtegce gtcgaagatgctaagttcctagtaagtaagtctccttcsttttggttggcaaaacgtcttgcaaagaggctgaatctggttccaa aagaggatccatctgtagcagcagctgagtgcccttgtaaaagagtgtgggaagctagttttgetagggcacctactgcacta gatccatttggaggtagggccttctgegaccagggttggstgtaccacagggacgtaggstatgcaacagctaaccacatat cacaggagacgctttttcaacaagcgctttcagtgaagaaccttgggccgcaaggtagtgcaaatgtctcaggctcaatacat accgccctggacaggctcagagcagcatatagtaggggaacgcccgcctctagatccatactgcaaggscttgcaaatctc atcacacctgtaggtgagaactttgaatgcgatctcgacaagaggaagctcaatataaaggcattacgttctcccgagaggta cattacgatagagggcctggttgtaaacttggacgatgtggtcagagggttctaccttgacaaggcgaaggtcactgtcctct cgaggtcaaagtggatgggttacgaggacctgcctcagaaacctccaaacggtacattttactgcagaaagaggaaggcaa tgettctaatctcatgtagtccaggtacgtacgcaaagaagcgaaaagtggcagtgcaggaggatcgttttaaagatatgagg gttgagaatttccgggaggtagcggaaaatatggatctaaatcagtagccaaacggtacattttaactgcagaaagaggaag gcaatgcttctaatctcatgtagtccaggtacgtacgcaaagaagcgaaaagtggcagtgcaggaggatcgttttaaagatat gagggettgagaatttccgggaggtagcggaaaatatggatctaaatcagtagccaaacggtacattttaactgcagaaagag gaaggcaatgcttctaatctcatgtagtccaggtacgtacgcaaagaagcgaaaagtggcagtgcaggaggatcgttttaaa gatatgagggitgagaatttccgggaggtagcggaaaatatggatctaaatcagtag (Accession number: MK42501 1.1).

[43] In the present invention, the original plasmid of the recombinant plasmid preferably includes pET32a.

[44] In the present invention, the insertion sites of the encoding gene in the recombinant plasmid are preferably BamHI and Xhol.

[45] In the present invention, the original bacteria of the recombinant bacteria preferably include Escherichia coli.

[46] In the present invention, the recombinant bacteria is preferably constructed by a method including the following steps:

[47] extracting total RNA of the Tilapia Lake virus, and reverse-transcripting the total RNA of the Tilapia Lake virus to cDNA;

[48] performing PCR amplification using a primer set T1, with cDNA as a template, to obtain an amplification product; the primer set Tl comprises TIF and TIR; the nucleotide sequence of TIF is set forth in SEQ ID NO. : 3, the nucleotide sequence of TIR is set forth in SEQ ID NO. : 4;

[49] recovering the amplification product and performing a first DNA purification to obtain a purified amplification product;

[50] double digesting the purified amplification product with BamHI, Xhol and ligating the digestion product to a carrier of pMD19T to obtain pMD19T-2-TiLV, and transforming pMD19T-TiLV-2 into £. coli competent cells, screening for a positive strain, extracting the pMD19T-TiLV-2 in the positive strain; [SI] digesting the obtained pMD19T-TiLV-2 present in the positive strain and the pET-32a (+) with BamHI and Xhol, recovering and performing a second DNA purification, respectively, and recombining the product obtained by the second DNA purification to pET32a-TiLV-2 by using T4 DNA ligase;

[52] transforming the pET32a-TiLV-2 into E. coli competent cells to obtain the recombinant bacteria. [S3] Inthe present invention, it is first to extract total RNA of Tilapia Lake virus, then the total RNA of Tilapia Lake virus is reverse transcripted to cDNA.

[54] In the present invention, the Tilapia Lake virus is preferably the Thai VETKU- TVO01 (Accession No. KU751821).

[55] In the present invention, the total RNA of Tilapia Lake virus is preferably extracted using an RNA extraction kit, and the RNA extraction kit is preferably purchased from Beijing CoWin Biosciences Co., Ltd. In the present invention, it is preferable to use a reverse transcription kit to reverse transcript the total RNA of Tilapia Lake virus into cDNA, and the reverse transcription kit is preferably purchased from Takara Company.

[56] In the present invention, after the cDNA is obtained, primer set T1 is subjected to PCR amplification using cDNA as a template to obtain an amplification product. The primer T1 comprises TIF and TIR, and the nucleotide sequence of TIF is shown as SEQ ID NO.: 3, specifically TIF (BamHI). 5'-cgggatccatgtctatcatcagetac-3', and the nucleotide sequence of TIR is shown as SEQ ID NO. : 4, specifically, TIR (Xhol):. 5'- gactcgagttagtcgacaataggtccctc-3'.

[57] In the present invention, the primer set T1 is preferably designed according to the gene sequence of TiLV segment2 (TiLV-2) in GenBank (Accession No. MK425011.1). [S8] In the present invention, the PCR amplification in step 2) preferably comprises: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C 1 min, 35 cycles; and extension at 72°C for 10 min. The PCR amplification in step 2) uses an amplification system in which includes, calculated as 25 uL, the following components: 2 * PCR Mix 12.5 pL, TIF and TIR each 1 pL, template 1 uL, ddH20 9.5 uL, with the molar concentration of TIF and TIR being 10 mM, respectively.

[59] Inthe present invention, after amplification product is obtained, the amplification product is recovered, and a first DNA purification is performed to afford a purified amplification product.

[60] In the present invention, recovery of the amplification product comprises: subjecting the amplification product to agarose gel electrophoresis, cutting and recovering the electrophoresis product, and performing a first DNA purification to afford the purified amplification product. In the present invention, the agarose gel electrophoresis is conducted at a constant voltage 120V for 25 min. In the present invention, the first DNA purification is performed using a DNA purification kit and the DNA purification kit is preferably purchased from Axygen Company.

[61] In the present invention, after obtaining the purified amplification product, the purified amplification product is digested with BamHI and Xho! and then ligated to the pMD19-T vector to obtain pMD19T-TiLV-2, and pMD19T-TiLV-2 is transformed into E. coli competent cells, positive strain is screened out, and pMD19T-2-TiLV in the positive strain is extracted.

[62] In the present invention, the positive strain is preferably obtained by blue-white screening. In the present invention, after the positive strain of pMDI9T-TiLV-2 is extracted, it is preferable to further include performing PCR, double digestion and sequence identification of pMD19T-TiLV-2 in the positive strain.

[63] After obtaining the pMDI19T-TiLV-2 in the positive strain, the obtained pMD19T-TiLV-2 in the positive strain and the pET-32a (+) are subjected to be double digested by BamHI and Xhol recovered, and to second DNA purification, and T4 DNA ligase is used to recombine the second DNA purification product into pET32a-TiLV-2. In the present invention, the second DNA purification is preferably carried out using a DNA purification kit, and the DNA purification kit is preferably purchased from Axygen Company.

[64] After obtaining pET32a-TiLV-2, the pET32a-TiLV-2 is transformed into £. coli competent cells to obtain recombinant bacteria. In the present invention, the £. coli competent cells are preferably £. coli BL21 competent cells or £. coli DE3 competent cells.

[65] In the present invention, the method for preparing the TIL V-2 protein preferably includes the following steps:

[66] SI: mixing a culture of the recombinant bacteria obtained according to the use of the above technical solution with isopropyl B-D-thiogalactoside, performing induced expression at a final concentration of isopropyl B-D-thiogalactoside concentration of

0.5-1.0 mmol/L to obtain a bacterial culture of induced expression;

[67] S2: centrifuging the bacterial culture of induced expression, collecting the precipitates, re-suspending precipitates to obtain a re-suspended solution, and sonicating the re-suspended solution to obtain a sonicated product;

[68] S3: centrifuging the sonicated product, collecting precipitates, and washing precipitates dissolving the washed precipitates with a denaturation solution containing 6 —10 mol/L of urea to obtained a dissolved solution;

[69] S4: dissolving the dissolved solution in PBS containing 8 mol/L urea, performing a first dialysis to give a first dialysate; centrifuging the first dialysate to give supernatant; performing a second dialysis on the supernatants containing 6.0, 4.0, 2.0 and 1.0 mol/L urea sequentially to give a second dialysate; performing a second dialysis on the second dialysate in 1 x PBS to obtain a crude extract solution;

[70] wherein dialysis bags used for the first dialysis, the second dialysis and the third dialysis each independently has a cut-off molecular of 8000-14000D;

[71] atime for the first dialysis is 6 hours;

[72] atime for the second dialysis in PBS with different concentrations of urea is each 5 hours;

[73] and a time of the third dialysis is 9-16 h;

[74] SS: purifying the crude extract solution using a nickel column with a His tag to obtain eluate, wherein a 500 mM imidazole elution buffer is used during the purification, and volume of the eluate is 10 times of column volume;

[75] S6: freeze-drying the eluate to give freeze-dried powder containing TILV-2 protein.

[76] In the present invention, the bacterial culture of the recombinant bacteria constructed by the use of the above technical solution is firstly mixed with isopropyl B- D-thiogalactoside to induce expression.

[77] In the present invention, the final concentration of isopropyl B-D-thiogalactoside in the bacterial culture is preferably 0.5-1.0 mmol/L, more preferably 0.8 mmol/L. In the present invention, OD600 of the bacterial culture is preferably 0.5-0.7, more preferably 0.6. In the present invention, the time for induced expression is preferably 4— 6h, more preferably 5 h, and the temperature for induced expression is preferably 37°C.

[78] After the bacteria are induced to express, the induction expression product is centrifuged, the precipitates are collected and re-suspended to obtain a re-suspended solution. The re-suspended solution is sonicated to obtain sonication product. In the present invention, the rotation speed for the centrifugation is preferably 10000-12000 r/min; the temperature for the centrifugation is preferably 4°C; the time for the centrifugation is preferably 8-12 min, more preferably 10 min. In the present invention, the sonication is preferably conducted in an ice bath, and the time for sonication is preferably 30-50 min, more preferably 40 min. The ultrasonic power is preferably 400 W. In the present invention, sonication in ice bath allows the temperature of the protein to be kept at a low level, preventing the protein from denaturation.

[79] After the sonication product is obtained, the sonication product is centrifuged, the precipitates are collected and washed, and a denaturation solution containing 6-10 mol/L of urea is used to dissolve the washed precipitates to afford a dissolved solution. In the present invention, the rotation speed for the centrifugation is preferably 10000- 12000 r/min; the temperature for the centrifugation is preferably 37°C, and the time for the centrifugation is preferably 18-22 min, more preferably 20 min. In the present invention, the denaturation solution preferably contains 8 mol/L of urea. In the present invention, the precipitates are preferably washed with deionized water.

[80] After dissolution is obtained, the dissolved solution is dissolved in PBS containing 8 mol/L urea, and a first dialysis is performed to give a first dialysate. The first dialysis 1s centrifuged, and the supernatant is collected. The supernatant is subjected to a second dialysis sequentially in PBS containing in 6.0, 4.0, 2.0, 1.0 mol/L of urea to afford a second dialysate. The second dialysate is subjected to a third dialysis in [xPBS to obtain a crude extract solution;

[81] The cut-off molecular weight for the dialysis bags used in the first dialysis, the second dialysis and the third dialysis is independently 8000-14000D, preferably 10000- 12000D:;

[82] the time for the first dialysis is 6 hours;

[83] the time for the second dialysis is 5 hours for each dialysis in PBS with different concentrations of urea; and

[84] the time for the third dialysis is 9-16 h.

[85] In the present invention, the rotation speed for the centrifugation is preferably 10000-12000 r/min, the temperature for the centrifugation is preferably 4°C, and the time for the centrifugation is preferably 8-12 min, more preferably 10 min.

[86] After obtaining the crude extract, a His-tagged nickel column is used to purify the crude extract solution to obtain eluate, wherein a 500 mM imidazole elution buffer 1s used during the purification, and volume of the eluate is 10 times of column volume.

[87] After obtaining the eluate, the eluate is freeze-dried to obtain a freeze-dried powder containing TILV-2 protein.

[88] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all the embodiments. Based on the embodiments of the present invention, all other embodiments made by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

[89] Example 1

[90] Preparation of TILV-2 protein of Tilapia Lake virus

[91] 1. Materials and methods

[92] 1.1 Material

[93] 1.1.1 Virus strains, bacteria strains and vectors

[94] Tilapia Lake virus TiLV is a Thai isolate VETKU-TVO1 (Accession No. KU751821). pET-32a (+) carrier is product from Beijing Solarbio Science & Technology Co., Ltd. Escherichia coli DH5a and BL21 (DE3) strains are products from Beijing CoWin Biosciences Co., Ltd.

[95] 1.1.2 Reagents

[96] Virus RNA extraction kit, DM 2000 DNA Marker, PCR amplification kit, staining reagent DAB, BCA protein assay kit are products from Beijing CoWin Biosciences Co., Ltd. Reverse transcription kit, restriction endonucleases (BamHI, Xho I), and T4 DNA ligase were purchased from Takara Company. DNA purification kit was purchased from Axygen Company. The primers were synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd. Protein marker was purchased from Tiangen BiochTech (Beijing) Co., Ltd. Murine histidine monoclonal antibody and horseradish peroxidase labeled goat anti-mouse IgG were purchased from Abcam Company. Other conventional reagents are domestically produced and analytically pure.

[97] 1.1.3 Test equipment

[98] ALC-1100.2 electronic balance, Beijing Sartorius Instrument System Co., Ltd; HH-4 digital display constant temperature water bath, Shanghai Techeng Machinery Equipment Co., Ltd.; H1650-W desktop high-speed microcentrifuge, Hunan Xiangyi laboratory instrument development Co., Ltd.; 1-15K high-speed refrigerated centrifuge, American Sigma; SY-360 ultrasonic extractor, Shanghai Ningshang Ultrasonic Instrument Co., Ltd; Classic ultrapure water meter, Veolia Water Treatment Technology (Shanghai) Co, Ltd.; ultrasonic cell disrupter, Ningbo biological Technology Co., Chicago; DYCP 31CN-type horizontal agarose electrophoresis, DYCZ-24DN mini dual vertical electrophoresis, and WD-9413A gel image analysis system were purchased from Beijing Liuyi Instrument Factory; Thermo Multiskan MK3 microplate reader, Thermo Fisher Scientific Inc.; BIO-RAD PCR machine, BIO-RAD Corporation.

[99] 1.2 Test method

[100] 1.2.1 PCR amplification to obtain the target gene of G antigen protein

[101] By referring to the instructions of the viral RNA extraction kit, the total RNA of the TiLV virus was extracted, and the reverse transcription kit was used to reverse transcript the RNA into cDNA. According to the gene sequence of TiLV segment2 (TiL V-2) in GenBank (Accession No. MK425011.1), the primer T1 is designed as follows:

[102] TIF, (BamHI). 5-CGGGATCCATGTCTATCATCAGCTAC-3' (as shown in SEQ ID NO. :3);

[103] A TIR (Xhol).. 5' -GACTCGAGTTAGTCGACAATAGGTCCCTC-3' (as shown in SEQ ID NO. : 4).

[104] Reaction conditions for PCR amplification: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 35 cycles, and extension at 72°C for 10 min. The PCR product was subjected to identification by agarose gel electrophoresis under the electrophoresis conditions of 120V constant voltage for 25 min. The electrophoresis product was cut, recovered, and the DNA product was purified by a DNA purification kit. The purified product was digested with BamHI and Xhol and then ligated to pMD19-T vector, and the product was named pMDI19T-TiLV-2. Then pMDI9T-TiLV-2 was transformed into E. coli DH50 competent cells, screened for a positive strain by blue-white screening and the pMDIOT-TILV-2 plasmid was extracted for PCR, double digestion and sequence identification.

[105] 1.2.2 Construction of prokaryotic expression strain of antigen protein

[106] The recombinant pMD19T-TiLV-2 plasmid and pET-32a (+) plasmid were subjected to purification by BamHI and Xhol double digestion, agarose gel electrophoresis, gel cutting, recovery, and purification by DNA purification kit. T4 DNA ligase was recombined into pET32a-TiL V-2. The recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells, and the recombinant plasmid was extracted and identified by PCR, double digestion and sequence identification.

[107] 1.2.3 Induced expression of TILV-2 protein

[108] The recombinant plasmid £. coli BL21 (DE3) containing pET32a-TiLV-2 was spread on a LB plate (containing ampicillinum) and cultured overnight at 37°C. Single colonies were picked and inoculated in LB broth (containing ampicillinum), cultured overnight at 37°C with shaking. Then 1% of culture was taken out for scale-up culturing in a new medium till OD600 was 0.6 when IPTG (isopropyl-D-thiogalactoside beta) with a final concentration of 0.8 mmol/L was added and induced for 4 — 6h. Then the culture was centrifuged, and the supernatant was discarded, and the centrifugation precipitates were washed 2 times with 1 x PBS, and an equal volume of sample buffer was added, mixed, boiled for 5 min, then electrophoresis was performed using 10% polyacrylamide gel. The purified protein lanes were transferred to nitrocellulose membrane, the mouse histidine monoclonal antibody was used as the primary antibody, and the goat anti-mouse IgG was used as the secondary antibody to incubate. After washing, DAB was used for color development and Western Blot analysis was performed.

[109] 1.2.4 Preparation of TILV-2 protein

[110] A large amount of the induced bacterial culture was collected, and centrifuged in a 50 mL centrifuge tube at 4°C for 10 min at 12,000 r/min. The precipitates were collected, and the buffer was disrupted with ultrasound to re-suspend the precipitates. The re-suspend solution was sonicated in an ice bath for 40 min, centrifuged at 12,000 r/min for min, and the resulting precipitates was rinsed with deionized water, then a denaturation solution containing 8 mol/L of urea was added to dissolve the precipitates and was centrifuged at 4°C for 10 min 12,000 r/min. The supernatant was collected and successively subjected to dialysis in PBS containing 6.0, 4.0, 2.0 and 1.0 mol/L urea,

and finally in I “PBS overnight. The protein was purified by a His- tagged nickel column kit. The purification included the following steps: collecting the purified protein liquid, freezing it and evacuate it into freeze-dried powder for preservation. When using, the freeze-dried powder was dissolved in sterile water, and the protein concentration was determined by a BCA protein assay kit.

[111] The purification steps of recombinant protein on nickel column are as follows:

[112] Assembly of the chromatographic column: the Ni-Agarose filler was mixed well and added to the chromatographic column. The column was allowed to stand at room temperature for 10 minutes, and the liquid outlet was opened to allow the liquid to flow out.

[113] Equilibration of the column: the nickel affinity column was washed with 5 volumes of deionized water to the column, and then the column was equilibrated with 8 volumes of Binding buffer to the column.

[114] Sample loading: after the sample was added to the column, the eluate was collected at a flow rate of 10 times of column volume/hour, and then the column was washed with 15 times volume of Binding Buffer to column so as to wash away the impurities.

[115] 10 times of column volume of Elution Buffer (500 mmol/L imidazole elution buffer) was added to the nickel affinity column, and the collected eluate was the target protein sample.

[116] SDS-PAGE electrophoresis was performed on the target protein collected under different elution conditions to analyze the optimal eluted protein concentration. At the same time, 5 column volumes of Binding Buffer and 5 column volumes of deionized water was used to wash the column, and then the column was equilibrated with 3 column volumes of 20% ethanol and the column was stored at 4°C.

[117] The purification condition for determining the recombinant protein was that the protein was eluted with 10 column volumes of S00 mmol/L imidazole elution buffer. All protein samples were purified in batches using Ni-Agarose His Tag Protein Purification Kit to obtain all purified proteins.

[118] 2. Results and analysis

[119] 2.1 Amplification, cloning and identification results of TiLV-2 gene

[120] The TiLV-2 gene was amplified by PCR, and a target band of about 1388 bp was obtained. The target gene was cloned into pET32a (+) vector to obtain a recombinant vector pET32a-2-TiLV, the results for double digestion is shown in FIG. 1.

[121] 2.2 Expression and purification results of TiLV-2 protein

[122] The recombinant expression plasmid pET32a-TiLV-2 was expressed in E. coli BL21. After 4 hours of induction, SDS-PAGE electrophoresis showed that the relative molecular weight of the expressed fusion protein was about 63 KDa (FIG. 2), which was consistent with the expected result. According to BandScan software analysis, the highest expression level of the protein accounted for 28.4% of the total bacterial protein, and results for Western Blot analysis showed that there was a clear identification band at 63 KDa (FIG. 3), indicating that the target protein can be recognized by anti-His monoclonal antibody, confirming that it has good antigen reactivity. The purified target protein has only one obvious identification band at 63 KDa and can be recognized by His monoclonal antibody (FIG. 4 and FIG. 5), indicating that the protein was successfully purified.

[123] Example 2

[124] Determination of the immune effect of TILV-2 protein prepared in Example 2

[125] 1. Materials and methods

[126] 1.1 Materials

[127] 1.1.1 Experimental animals

[128] About 500 Tilapia (average weight: 3.0+0.5 g) were provided by the Xinmin National Domestic Fish Fine Breeding Farm of Shaanxi Provincial Fisheries General Station.

[129] 1.2 Test method

[130] Xinmin National Domestic Fish Fine Breeding Farm of Shaanxi Provincial Fisheries General Station fished about 500 Tilapia. The TILV-2 protein prepared in Example 1 was dissolved and diluted into a suspension with distilled water, and injected at doses of 0.5, 1.0, and 2.0 pg/g Tilapia, respectively, with 100 Tilapia for each dose. After immunization, they were put into 5 m’ breeding ponds, and aeration was carried out. 100 fish were stocked in each breeding pond, and the water temperature was controlled at 27-28°C. 14, 21, 28, 49, 56 and 70 days after the first immunization, 50 fish were randomly collected and tested for antibody titer and challenge test in the laboratory.

[131] 50 Tilapia as collected were divided into 5 groups for parallel duplicate test, with 10 Tilapia in each group, The Tilapia were sacrificed by using a spine cut method, and

2.0 mL 0.6% saline was added. The tissue was homogenized using a 12000 rpm homogenizer. After centrifuging at 3000 rpm for 10 min in a high-speed refrigerated centrifuge, the supernatant was transferred to a new tube and frozen at -80°C. An indirect agglutination method was used for antibody detection. The selected sensitization carrier was the sheep erythrocytes prepared by a one-step glutaraldehyde method.

[132] After 28 days of immunization, 50 fish were randomly selected from the above groups of test fish and placed in a new fish tank for a challenge test. In addition, the water temperature in the fish tank was controlled at 25+0.5°C during the challenge test, and other breeding conditions remained unchanged. The mode of challenge was injection, the injection site was the abdominal cavity of the fish, and each fish was injected with 50 pL of 4.0x107 TCID 50/mL TiLV virus solution. The test fish were observed regularly, and the disease conditions were recorded in time, and the dead Tilapia were stored in time for virus testing. The formula for calculating immune protection is:

[133] RPS = (1- mortality rate of immunization group/mortality rate of control group) x100.

[134] 2. Results and analysis

[135] It can be seen from Table 1 that the titer of the tilapia antibody after immunization increases significantly with time, reaching a peak level on days 21 to 28, and the immunization titer begins to decline on day 49, but it still remains at a very high level.

Onday 70, the immunization titer begins to drop but still remains at a very high antibody level. After immunization by Tilapia Lake virus vaccine, the protection rate can reach up to 72%.

[136] Table 1 Test results for indirect agglutination reaction for antibody titer of Tilapia immunized by TILV-2subunit vaccine Antibody titer Group idd 21 d 28d 49 d 56 d 704d Cogtrol group {187 (254 0.261 0.254 0.155 3.185 pET3Za (+) empty group {152 0.261 2.276 0.263 0.186 0.383

0.5 ue/g 0.248 0.265 0.274 0.269 0.254 0.262

1.0 pgs 8.251 8.239 8.413 0,359 0,325 0.296

2.0 ngs 3.382 0.397 23.425 0.374 0.345 0.313

[137] It can be seen from Table 2 that the protection rate of Tilapia in the control group and the empty protein group after challenge is 0%, while the protection rate of the immunized Tilapia after challenge will increase with the increase of the vaccine dose, After the Tilapia is immunized with 2.0 pg/g of vaccine, the protection rate may be up to 72% after challenge.

[138] Table 2 Challenge results for Tilapia immunized with TIL V-2subunit vaccine Protection Group oo rate (%) Control group 0 PET32a(*)emptygrop 0

0.5 ng/g 46

1.0 ng/g 60

2.0 ng/g 72

[139] After 70 days of cultivation, Tilapia Lake virus disease did not occur in the two ponds. The method of the present invention has broad prospects in the prevention and control of Tilapia Lake virus disease.

[140] The above are only a preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

SEQLTXT-1

SEQUENCE LISTING <110> Northwest A&F University <120> TILV-2 PROTEIN OR GENE ENCODING TILV-2 PROTEIN IN PREPARATION OF SUBUNIT

VACCINE AGAINST TILAPIA LAKE VIRUS <130> GWP202105325 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 1374 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of the encoding gene <400> 1 atgagtcagt ttgggaaatc attcaagggc agaactgaag tcacaataac cgaatatcgc 60 tcccatactg tcaaagatgt gcacagaagc ttacttacgg ctgacaaatc tctaagaaag 120 tcattctgct ttaggaacgc cctaaaccag ttcttggata aagacttgcc tcttctgccc 180 attcggccaa aattagaatc cagggttgct gtgaaaaagt ctaagctgag gagtcagctg 240 tcgttcagac ccggtttaac tcaggaggaa gcaattgatc tttacaacaa gggctatgat 300 ggtgacagcg tctcaggtgc cttacaagat agggtagtca atgagcctgt agcttactcg 360 agcgcagata atgataaatt tcacaggggc ttagcggctc tagggtacac tttggctgat 420 agagcatttg atacgtgcga atccggcttc gtgagagcaa tccctaccac tccatgcggg 480 ttcatatgtt gcgggccagg ttctttcaaa gattcacttg gatttgtgat aaaaatcagc 540 gaattctggc acatgtatga cgggtttcaa cacttcgtcg ccgtcgaaga tgctaagttc 600 ctagtaagta agtctccttc gttttggttg gcaaaacgtc ttgcaaagag gctgaatctg 660 gttccaaaag aggatccatc tgtagcagca gctgagtgcc cttgtaaaag agtgtgggaa 720 gctagttttg ctagggcacc tactgcacta gatccatttg gaggtagggc cttctgcgac 780 cagggttggg tgtaccacag ggacgtaggg tatgcaacag ctaaccacat atcacaggag 840 acgctttttc aacaagcgct ttcagtgaag aaccttgggc cgcaaggtag tgcaaatgtc 900 Pagina 1

SEQLTXT-1 tcaggctcaa tacataccgc cctggacagg ctcagagcag catatagtag gggaacgccc 960 gcctctagat ccatactgca agggcttgca aatctcatca cacctgtagg tgagaacttt 1020 gaatgcgatc tcgacaagag gaagctcaat ataaaggcat tacgttctcc cgagaggtac 1080 attacgatag agggcctggt tgtaaacttg gacgatgtgg tcagagggtt ctaccttgac 1140 aaggcgaagg tcactgtcct ctcgaggtca aagtggatgg gttacgagga cctgcctcag 1200 aaacctccaa acggtacatt ttactgcaga aagaggaagg caatgcttct aatctcatgt 1260 agtccaggta cgtacgcaaa gaagcgaaaa gtggcagtgc aggaggatcg ttttaaagat 1320 atgagggttg agaatttccg ggaggtagcg gaaaatatgg atctaaatca gtag 1374 <210> 2 <211> 457 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the TILV-2 protein <400> 2 Met Ser Gln Phe Gly Lys Ser Phe Lys Gly Arg Thr Glu Val Thr Ile 1 5 10 15 Thr Glu Tyr Arg Ser His Thr Val Lys Asp Val His Arg Ser Leu Leu

Thr Ala Asp Lys Ser Leu Arg Lys Ser Phe Cys Phe Arg Asn Ala Leu 40 45 Asn Gln Phe Leu Asp Lys Asp Leu Pro Leu Leu Pro Ile Arg Pro Lys 50 55 60 Leu Glu Ser Arg Val Ala Val Lys Lys Ser Lys Leu Arg Ser Gln Leu 65 70 75 80 Ser Phe Arg Pro Gly Leu Thr Gln Glu Glu Ala Ile Asp Leu Tyr Asn 85 90 95 Lys Gly Tyr Asp Gly Asp Ser Val Ser Gly Ala Leu Gln Asp Arg Val Pagina 2

SEQLTXT-1 100 105 110 Val Asn Glu Pro Val Ala Tyr Ser Ser Ala Asp Asn Asp Lys Phe His 115 120 125 Arg Gly Leu Ala Ala Leu Gly Tyr Thr Leu Ala Asp Arg Ala Phe Asp 130 135 140 Thr Cys Glu Ser Gly Phe Val Arg Ala Ile Pro Thr Thr Pro Cys Gly 145 150 155 160 Phe Ile Cys Cys Gly Pro Gly Ser Phe Lys Asp Ser Leu Gly Phe Val 165 170 175 Ile Lys Ile Ser Glu Phe Trp His Met Tyr Asp Gly Phe Gln His Phe 180 185 190 Val Ala Val Glu Asp Ala Lys Phe Leu Val Ser Lys Ser Pro Ser Phe 195 200 205 Trp Leu Ala Lys Arg Leu Ala Lys Arg Leu Asn Leu Val Pro Lys Glu 210 215 220 Asp Pro Ser Val Ala Ala Ala Glu Cys Pro Cys Lys Arg Val Trp Glu 225 230 235 240 Ala Ser Phe Ala Arg Ala Pro Thr Ala Leu Asp Pro Phe Gly Gly Arg 245 250 255 Ala Phe Cys Asp Gln Gly Trp Val Tyr His Arg Asp Val Gly Tyr Ala 260 265 270 Thr Ala Asn His Ile Ser Gln Glu Thr Leu Phe Gln Gln Ala Leu Ser 275 280 285 Val Lys Asn Leu Gly Pro Gln Gly Ser Ala Asn Val Ser Gly Ser Ile 290 295 300 His Thr Ala Leu Asp Arg Leu Arg Ala Ala Tyr Ser Arg Gly Thr Pro Pagina 3

SEQLTXT-1 305 310 315 320 Ala Ser Arg Ser Ile Leu Gln Gly Leu Ala Asn Leu Ile Thr Pro Val 325 330 335 Gly Glu Asn Phe Glu Cys Asp Leu Asp Lys Arg Lys Leu Asn Ile Lys 340 345 350 Ala Leu Arg Ser Pro Glu Arg Tyr Ile Thr Ile Glu Gly Leu Val Val 355 360 365 Asn Leu Asp Asp Val Val Arg Gly Phe Tyr Leu Asp Lys Ala Lys Val 370 375 380 Thr Val Leu Ser Arg Ser Lys Trp Met Gly Tyr Glu Asp Leu Pro Gln 385 390 395 400 Lys Pro Pro Asn Gly Thr Phe Tyr Cys Arg Lys Arg Lys Ala Met Leu 405 410 415 Leu Ile Ser Cys Ser Pro Gly Thr Tyr Ala Lys Lys Arg Lys Val Ala 420 425 430 Val Gln Glu Asp Arg Phe Lys Asp Met Arg Val Glu Asn Phe Arg Glu 435 440 445 Val Ala Glu Asn Met Asp Leu Asn Gln 450 455 <2105 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of T1F <400> 3 cgggatccat gtctatcatc agctac 26 <2105 4 Pagina 4

SEQLTXT-1

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence of T1R

<400> 4 gactcgagtt agtcgacaat aggtccctc 29 Pagina 5

Claims

Conclusions I. Use of TILV-2 protein, a gene encoding TILV-2 protein, a recombinant plasmid containing the gene, or recombinant bacteria containing the gene in preparation of a subunit vaccine against Tilapia Lake virus, with characterized in that the amino acid sequence of the TIL V-2 protein is as set forth in SEQ ID NO:2.

Use according to claim 1, characterized in that the nucleotide sequence of the coding gene is as set forth in SEQ ID NO.:1.

Use according to claim 1, characterized in that the original plasmid of the recombinant plasmid comprises pET32a.

Use according to claim 3, characterized in that the insertion sites of the coding gene in the recombinant plasmid are BamHI and XhoI.

Use according to claim 1 or 4, characterized in that the original bacteria of the recombinant bacteria comprise Escherichia coli.

Use according to claim 5, characterized in that the recombinant bacteria are constructed by a method comprising the following steps: 1) extracting total RNA of the Tilapia Lake virus and reverse transcription of the total RNA of the Tilapia Lake virus to cDNA; 2) performing PCR amplification using a primer set TI, with cDNA as template, to obtain an amplification product; wherein the primer set TI comprises TIF and TIR, the nucleotide sequence of TIF is set forth in SEQ ID NO.:3, the nucleotide sequence of TIR is set forth in SEQ ID NO.:4; 3) recovering the amplification product and performing a first DNA purification to obtain purified amplification product; 4) double digesting the purified amplification product with BamHI, XhoI and ligating the digestion product to a support of pMD19T to obtain pMD19T-2-TiLV, and transforming pMD19T-TiL V-2 into competent E.coli cells , screening for a positive strain, extracting pMD19T-TiL V-2 from the positive

-19- strain; 5) digesting the resulting pMD19T-TiLV-2 contained in the positive strain and the pET-32a (+) with BamHI and XhoI, respectively, recovering and performing a second DNA purification and recombining the product obtained by the second DNA purification to pET32a-TiLV-2 using T4 DNA ligase; 6) transforming the pET32a-TiLV-2 into competent F.coli cells to obtain the recombinant bacteria.

Use according to claim 6, characterized in that the PCR amplification in step 2) comprises: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and elongation at 72°C 1 min, 35 cycles; and extension at 72°C for 10 min.

Use according to claim 6 or 7, characterized in that the PCR amplification in step 2) uses an amplification system which, calculated as 25 µL, comprises the following components: 2 x PCR Mix 12.5 µL, TIF and TIR each 1 µl, template 1 µL, ddH 2 O 9.5 µL, where the molar concentration of TIF and TIR is 10 mM, respectively.

Use according to any one of claims 6 to 8, characterized in that the TILV-2 protein is prepared by a method comprising the following steps: S1 : mixing a culture of the recombinant bacteria obtained according to the use of any one of claims 6-8 with isopropyl-B-D-thiogalactoside, performing induced expression at a final isopropyl-p-D-thiogalactoside concentration of 0.5-1.0 mmol/L to obtain a bacterial culture of induced expression; S2: centrifuging the bacterial culture of induced expression, collecting the precipitates, resuspending precipitates to obtain a resuspended solution, and sonicating the resuspended solution to obtain a sonicated product; S3: centrifuging the sonicated product, collecting precipitates and washing precipitates and dissolving the washed precipitates with a denaturation solution comprising 6-10 mol/L urea to make a dissolved solution

-20- get; S4: dissolving the dissolved solution in PBS with 8 mol/L urea, performing a first dialysis to provide a first dialysate; centrifuging the first dialysate to provide supernatant; sequentially performing a second dialysis on the supernatants containing 6.0, 4.0, 2.0 and 1.0 mol/L urea to provide a second dialysate; performing a second dialysis on the second dialysate in 1*PBS to obtain a raw extract solution; wherein dialysis bags are used for the first dialysis, the second dialysis and the third dialysis each independently having a molecular cut-off of 8000-14000D;

a time for the first dialysis is 6 hours; a time for the second dialysis in PBS with different concentrations of urea 1s every 5 hours; and a time for the third dialysis 9 - 4 pm 1s; S5 : purifying the raw extract solution using a His-tagged nickel column to obtain an eluate, wherein a 500 mM imidazole elution buffer is used during the purification, and the volume of the eluate is 10 times the column volume; S6: lyophilization of the eluate to provide lyophilized powder containing TIL V-2 protein.