Chicken tembusu virus antigen protein, subunit vaccine and application thereof

Abstract

The invention provides a chicken tembusu virus antigen protein, a subunit vaccine and application thereof, belongs to the technical field of vaccine preparation, and solves the technical problem that a chicken tembusu virus subunit vaccine special for chicken does not exist in the market at present. According to the chicken tembusu virus antigen protein provided by the invention, the amino acid sequence of the antigen protein is selected from E-GL protein as shown in SEQ ID NO: 3 or E-HA protein as shown in SEQ ID NO: 6. The method can be applied to challenge protection of chicken tembusu virus strains, and has the advantages of high safety, strong effectiveness, high expression quantity and great reduction of production cost.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of vaccine preparation, and particularly relates to a chicken Tembusu virus antigen protein, a subunit vaccine and application thereof. BACKGROUND

[0002] Tembusu virus (TMUV) belongs to the Ntaya virus group of the Flavivirus genus of the Flaviviridae family. TMUV was introduced into China in 2010. The virus mainly affects the reproductive system of ducks, leading to a significant decrease in egg production of laying ducks, and seriously restricts the development of waterfowl breeding industry in China. In recent years, the host spectrum of TMUV infection has expanded to chickens, geese, sparrows and mice, posing a threat to the biosecurity and public health safety of poultry farms.

[0003] At present, the main means to prevent and control the spread of the virus is vaccination. Traditional vaccines use complete pathogens as antigens for vaccine preparation, which has certain safety risks. Moreover, the use of whole viruses will cause selective pressure on viruses and accelerate the mutation of viruses. Compared with traditional vaccines, subunit vaccines have the advantages of high safety, good stability, easy batch production and low production cost. However, there is no chicken Tembusu virus subunit vaccine specifically for chickens on the market at present. Therefore, it is urgent to develop a safe and efficient subunit vaccine to prevent the infection of chicken Tembusu virus. SUMMARY

[0004] The application provides a chicken Tembusu virus antigen protein, a subunit vaccine and application thereof to solve the technical problem that there is no chicken Tembusu virus subunit vaccine specifically for chickens on the market at present. The chicken Tembusu virus subunit vaccine has the advantages of high safety, strong effectiveness and low production cost, and can be effectively used to prevent the infection of chicken Tembusu virus.

[0005] To achieve the above purpose, the technical scheme adopted by the application is as follows: a chicken Tembusu virus antigen protein, wherein the amino acid sequence of the antigen protein is selected from E-GL protein as shown in SEQ ID NO: 3 or E-HA protein as shown in SEQ ID NO: 6.

[0006] In one embodiment, the nucleotide sequence of the codon-optimized E-GL protein is as shown in SEQ ID NO: 5.

[0007] In one embodiment, the nucleotide sequence of the codon-optimized E-HA protein is as shown in SEQ ID NO: 7.

[0008] The application also provides a chicken Tembusu virus subunit vaccine prepared from the E-GL protein.

[0009] The application also provides a chicken Tembusu virus subunit vaccine prepared from the E-HA protein.

[0010] The application also provides a chicken Tembusu virus subunit vaccine prepared by the following steps: constructing a recombinant plasmid for expressing the E gene based on the optimized chicken Tembusu virus E gene; transfecting CHO-K1 cells with the recombinant plasmid, culturing, screening, amplifying and purifying positive monoclonal cells to obtain the corresponding recombinant chicken Tembusu virus E protein; adding an adjuvant to the recombinant chicken Tembusu virus E protein to obtain the chicken Tembusu virus subunit vaccine.

[0011] In one embodiment, the content of the E-GL protein in the chicken Tembusu virus subunit vaccine is 1436 μg / mL.

[0012] In one embodiment, the content of the E-HA protein in the chicken Tembusu virus subunit vaccine is 584 μg / mL.

[0013] The application also provides the use of the chicken Tembusu virus subunit vaccine in the protection against chicken Tembusu virus strain infection.

[0014] Compared with the prior art, the application has the advantages and positive effects that the chicken Tembusu virus subunit vaccine solves the technical problem that there is no chicken Tembusu virus subunit vaccine for chickens on the market, and the production of the subunit vaccine does not involve live virus culture, and the vaccine does not contain whole viruses, so the safety is high; the E protein capable of producing neutralizing antibodies is expressed to prepare the vaccine, and a relatively high antibody can be produced in 14 days, so the effectiveness is strong; the subunit vaccine can be used as a DIVA vaccine, and has important application value for the purification of epidemic diseases and epidemiological detection; the antigen protein is obtained by optimizing the nucleic acid molecular coding of the chicken Tembusu virus E gene, and the expression amount can be as high as 1436 μg / mL, the vaccine is prepared after being diluted by 14 times, the production cost is greatly reduced, the productivity is improved, and the market competitiveness of the vaccine is increased. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1The enzyme cutting picture provided by the embodiment of the present application, wherein M: from top to bottom 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp, 5000bp, 8000bp; Lane 1: pEE14.4 plasmid enzyme cutting; Lane 2: pUC57-E-NOPT recombinant plasmid enzyme cutting; Lane 3: pUC57-E-GL recombinant plasmid enzyme cutting; Lane 4: pUC57-E-HA recombinant plasmid enzyme cutting; Figure 2 The recombinant plasmid identification PCR provided by the embodiment of the present application, wherein M: from top to bottom 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp, 5000bp, 8000bp; Lane 1: pUC57-E-NOPT recombinant plasmid PCR; Lane 2: pUC57-E-GL recombinant plasmid PCR; Lane 3: pUC57-E-HA recombinant plasmid PCR; Lane 4: negative control; Figure 3 The SDS-PAGE analysis diagram provided by the embodiment of the present application, wherein M: from bottom to top 15KDa, 25KDa, 35KDa, 40KDa, 55KDa, 70KDa, 100KDa, 130KDa, 180KDa; Lane 1: negative control supernatant; Lane 2: E-NOPT supernatant; Lane 3: E-GL supernatant; Lane 4: E-HA supernatant; Figure 4 The western blot diagram provided by the embodiment of the present application, wherein M: from bottom to top 15KDa, 25KDa, 35KDa, 40KDa, 55KDa, 70KDa, 100KDa, 130KDa, 180KDa; Lane 1: negative control supernatant; Lane 2: E-GL supernatant; Lane 3: E-HA supernatant. DETAILED DESCRIPTION

[0016] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0017] The embodiment of the application provides a chicken Tembusu virus antigen protein, a subunit vaccine and application thereof, the chicken Tembusu virus subunit vaccine is prepared by using chicken Tembusu virus E protein encoded by an optimized nucleic acid molecule of chicken Tembusu virus E gene, the cell strain of the chicken Tembusu virus subunit vaccine has a high expression of 1436 mu g / mL, and the vaccine is prepared after being diluted by 14 times, so that the production cost is greatly reduced, productivity is improved, and market competitiveness is increased. The TMUV is a single-stranded RNA virus, which is composed of three structural proteins and seven non-structural proteins, wherein the structural proteins include C, Pr M and E proteins, and the non-structural proteins include NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 proteins. The E protein is a main antigen component of the virus, can participate in immune response after virus infection, and induces a protective immune response. In the production process of the chicken Tembusu virus subunit vaccine, live virus culture is not involved, and the vaccine does not contain whole virus, the E protein capable of producing neutralizing antibodies is expressed to prepare the vaccine, the safety is high, the effectiveness is strong, and the chicken Tembusu virus subunit vaccine has important application values for disease purification and epidemiological detection.

[0018] Example 1 Optimization of chicken Tembusu virus E gene The full-length of the chicken Tembusu virus E gene is 1551 bp, the nucleotide sequence is shown as SEQ ID NO:1; and the E gene encodes 516 amino acids, and the amino acid sequence is shown as SEQ ID NO:2.

[0019] The antigen site of the chicken Tembusu virus E gene is analyzed, different signal peptides are added at the N terminal, the transmembrane region and the unnecessary region of the E gene are removed, and different signal peptide combinations and codon optimization are simultaneously performed. The amino acid sequence of the optimized Gaussia luc signal peptide is shown as SEQ ID NO:3, the nucleotide sequence without codon optimization is shown as SEQ ID NO:4, and the nucleotide sequence with codon optimization is shown as SEQ ID NO:5. The amino acid sequence of the HA signal peptide is shown as SEQ ID NO:6, and the corresponding optimized nucleotide sequence is shown as SEQ ID NO:7. The optimized gene is synthesized by Shanghai Shengong Bioengineering Co., Ltd. and is connected into a pUC57 plasmid.

[0020] Example 2 Construction of recombinant plasmid for expressing E gene 2.114.4-E recombinant plasmid construction 2.1.1 Enzyme digestion reaction 2.1.1.1 Label the 1.5 mL EP tube needed, and add samples and mix in the 1.5 mL EP tube according to the following table 1: the reaction system is 50 mu L, and the samples are added as shown in the following table 1: Table 1 Enzyme digestion reaction sample component table

[0021] 2.1.1.2 Place 1.5 mL EP tube in step 2.2.1.1 in a 37℃ constant temperature water bath, and water bath for 2-3 h.

[0022] 2.1.2 Gel recovery of double enzyme digestion product Take out the above double enzyme digestion system, and perform agarose gel electrophoresis to recover the DNA fragments therein. The enzyme digestion picture is shown in Figure 1 .

[0023] (1) Label the sample collection EP tube, adsorption column and collection tube; (2) Weigh the labeled empty EP tube, and record the value; (3) Carefully cut the single target DNA band from the agarose gel with a scalpel and put it into a clean 1.5 mL centrifuge tube; (4) Add 600 μL PC buffer to the 1.5 mL centrifuge tube in step (3) and place it in a 50℃ water bath for about 5 min, and constantly turn the centrifuge tube up and down to ensure that the gel block is fully dissolved; (5) Column equilibration: Put the adsorption column into the collection tube in advance, and then add 500 μL of equilibration solution BL to the adsorption column CB2, centrifuge at 12,000 rpm for 1 min, and then put the adsorption column back into the collection tube after discarding the waste liquid in the collection tube; (6) Add the solution obtained in step (5) to the adsorption column CB2, stand for 2 min, centrifuge at 10,000 rpm for 30 s, and then put the adsorption column CB2 into the collection tube after discarding the waste liquid in the collection tube; (7) Add 600 μL of rinse solution PW buffer to the adsorption column, stand for 3 min, centrifuge at 10,000 rpm for 30 s, and then put the adsorption column CB2 into the collection tube after discarding the waste liquid in the collection tube; (8) Repeat step (7); (9) Centrifuge the empty adsorption column at 12,000 rpm for 2 min to remove as much rinse solution as possible, and then place the adsorption column at room temperature for 10 min to dry completely; (10) Put the adsorption column CB2 into the collection tube, and add 50 μL of Elution buffer preheated at 65℃ to the middle of the adsorption membrane, stand for 3 min, and centrifuge at 12,000 rpm for 2 min; (11) Take out the centrifuge tube in step (10) from the centrifuge, discard the middle adsorption column CB2, cover the centrifuge tube cap, and reserve the DNA sample in the centrifuge tube; (12) Place the DNA sample in step (11) at 4℃ for storage, and prepare for agarose gel electrophoresis to identify the gel-recovered DNA fragments.

[0024] 2.1.3 Ligation reaction (1) Label the 0.2 mL centrifuge tubes needed; (2) Add the 20 μL reaction system according to Table 2 below to the labeled 0.2 mL tube; Table 2 Ligation reaction sample component table

[0025] (3) After completing the sample addition, mix the components gently with a pipette by blowing several times; (4) Place the 0.2 mL centrifuge tube in a 37°C reaction for 30 min, and after the reaction is completed, immediately place the reaction tube in an ice water bath for cooling for 5 min; (5) The reaction product of step (4) can be directly used for transformation experiment, or can be stored at -20°C, and thawed for transformation when needed.

[0026] 2.1.4 Transformation reaction (1) Quickly add 10 μL of the ligation reaction solution to 100 μL of competent cells, and mix well by blowing, and ice bath for 30 min; (2) After step (1) is completed, take out the sample tube and place it in a 42°C water bath for 100 s, and then immediately ice bath for 2 min; (3) After step (2) is completed, take out the sample tube, and in the clean bench, add 600 μL of liquid LB medium to the sample tube, and then place the sample tube in a 37°C constant temperature incubator, 220 rpm, and incubate for 1 h; (4) Prepare the transformation plate, and prepare the LB resistance plate for transformation according to the plasmid resistance; (5) Plate: Take out the sample tube in step (3), centrifuge at room temperature, 8,000 rpm, 2 min, remove 600 μL of supernatant, resuspend the bacteria at the bottom of the remaining supernatant, and place the resuspended bacterial solution in the corresponding transformation plate center, and evenly spread the bacterial solution in the center of the transformation plate with a bacterial spreading rod; (6) Place the plate in step (5) upright in the biochemical constant temperature incubator, and incubate at 37°C for 1 h, and then invert the transformation plate for incubation for 15 h; (7) Observe and record the transformation results.

[0027] 2.1.5 Plasmid extraction and PCR identification 2.1.5.1 Plasmid extraction (1) Use a 10 μL pipette tip to pick a single colony from the transformation plate into 5 mL of LB liquid medium containing ampicillin, and shake at 37°C, 220 rpm overnight; (2) Aspirate the bacterial solution into a 1.5 mL EP tube, centrifuge at room temperature, 12,000 rpm, 2 min, discard the supernatant; (3) Add 250 μL of plasmid extraction reagent P1 buffer to the EP tube in step (2), and suspend the bacteria thoroughly; (4) Add 250 μL of P2 buffer to the solution in step (3), mix immediately by gently inverting the centrifuge tube 5-10 times, and stand at room temperature for 2-4 min; (5) Add 350 μL of P3 buffer to the solution in step (4), mix immediately by gently inverting the centrifuge tube 5-10 times, and stand at room temperature for 2-4 min; (6) Centrifuge the solution in step (5) at room temperature, 14,000 rpm, 10 min; (7) Transfer the supernatant solution in step (6) to the center of the adsorption column, centrifuge at room temperature, 12,000 rpm, 30 s, and discard the liquid in the collection tube; (8) Add 500 μL of Buffer DW1 to the center of the adsorption column, centrifuge at room temperature, 12,000 rpm, 30 s, and discard the liquid in the collection tube; (9) Add 500 μL of wash solution to the center of the adsorption column, centrifuge at room temperature, 12,000 rpm, 30 s, and discard the liquid in the collection tube. Repeat once; (10) Empty the adsorption column, centrifuge at room temperature, 12,000 rpm, 2 min; (11) Place the adsorption column in a clean 1.5 mL centrifuge tube, add 30 μL of Elution buffer to the center of the adsorption membrane, stand at room temperature for 5 min, centrifuge at room temperature, 12,000 rpm, 2 min, and store the DNA solution in the tube at 4°C.

[0028] 2.1.5.2 PCR identification (1) Label the PCR tubes to be used, add samples according to Table 3, and mix, and the reaction system is 25 μL; Table 3 PCR identification sample addition table

[0029] (2) The PCR amplification program is shown in Table 4; Table 4 PCR amplification program table

[0030] (3) The positive plasmid in the PCR identification is sent to a sequencing company for sequencing, and the recombinant plasmid identification PCR reference is shown in the appendix. Figure 2

[0031] Example 3 Transfection of CHO-K1 cells​ (1) Preparation: ultraviolet sterilization in biosafety cabinet for 30 min; F-12 culture solution is placed in a 37°C water bath to preheat to 37°C; (2) 2.5 μg of recombinant DNA, P3000 is added to 125 μL of OPTI-MEM culture solution, mixed well; 3.75 μL of lipotectamine 3000 is added to 125 μL of OPTI-MEM culture solution, mixed well; the liposome is mixed with the recombinant DNA, and is left at room temperature for 10 min; (3) The 6-well plate cells plated 24 hours ago are taken out from the 37°C incubator, the supernatant culture medium is discarded, the cells are washed with pre-warmed OPTI-MEM culture solution three times, and the OPTI-MEM culture solution is discarded; (4) 2 mL of 10%, 1% double-antibody fetal bovine serum F-12 culture solution is added to each cell well; (5) The mixture of recombinant DNA and liposome is gently added to each cell well, mixed gently, and cultured in a 37°C, 5% CO2 cell incubator; (6) 72 hours after transfection, the supernatant is collected for protein detection, and the cells are added with G418 for pressure screening; (7) After 144 hours, the monoclonal cells are detected, and the positive cells are expanded. It can be used for large-scale production of protein.

[0032] Example 4: The cell strains expressing the proteins shown in Table 5 are constructed according to the above example method: Table 5: Information table of protein-expressing cell strains

[0033] Example 5: Protein purification and detection 5.1 Expression and identification of recombinant E protein in CHO cells The cells selected in Example 3 are continuously passaged and domesticated into suspension cells; the suspension cells are cultured at 37°C for 240 hours, and the supernatant is collected for SDS-PAGE protein analysis; it is found that the E-NOPT gene is not expressed in CHO cells, while the optimized E-GL and E-HA genes are expressed in CHO cells.

[0034] 5.2 Protein content detection The supernatant harvested in 6.1 is used to detect the total protein content with the Bradford protein detection kit, the protein purity is detected by SDS-PAGE, the content of the target protein is calculated, and the SDS-PAGE analysis diagram is shown in the attached Figure 3 .

[0035] Table 6: Information table of protein content detection

[0036] 5.3 Chromatography purification of recombinant E protein The collected E-GL and E-HA supernatant was subjected to affinity chromatography with Ni Sepharose excel medium of GE, and the collected supernatant was centrifuged at 5000 rpm for 5 min as the sample loading liquid; 5.3.1 The medium was washed with 5 times column volume of distilled water; 5.3.2 The medium was washed with 5 times column volume of equilibration buffer; 5.3.3 The sample was loaded, and the column could hang 4 mg of protein per milliliter; 5.3.4 The medium was washed with 20 times column volume of washing buffer; 5.3.5 The protein was eluted with 5 times column volume of elution buffer.

[0037] 5.4 Western blot test The protein prepared in 5.3 was subjected to SDS-PAGE, and the target protein band was transferred to a PVDF membrane by semi-dry transfer at 20 volts for 30 min. The transfer membrane was blocked with blocking solution overnight, washed with PBST for 3 times, incubated with 1:500 diluted chicken Tembusu virus positive serum at 37℃ for 1.5 h, washed with PBST for 3 times, incubated with 1:1000 diluted HRP-labeled goat anti-chicken enzyme-labeled antibody at 37℃ for 1.5 h, washed with PBST for 3 times, and incubated with substrate solution for 5 min. Color development was performed on a chemiDOC. As shown in the western blot figure, it was indicated that the expressed E protein had good immunogenicity. Figure 4

[0038] Example 6 Preparation of vaccine and detection of effect 6.1 Preparation of vaccine (1) Preparation of oil phase: 95 parts of veterinary white oil and 1 part of aluminum stearate were placed in an oil phase preparation tank and heated to 80℃, then 5 parts of Span-80 was added, and when the temperature reached 115℃, it was maintained for 30 min, and then cooled for standby; (2) Preparation of water phase: the protein liquid purified in 5.3 was mixed with sterile normal saline in a proper proportion so that the protein content in the water phase was not less than 100 ug / mL; (3) Emulsification: 1.5 parts of oil phase was placed in a high-speed shearing machine, and the motor was started to stir slowly, while 1 part of water phase was added slowly, and emulsified at 10000 r / min for 5 min. After emulsification, 10 mL was centrifuged at 3000 r / min for 15 min, and the water phase separated from the bottom of the tube should not exceed 0.5 ml.

[0039] 6.2 Potency test of vaccine ​SPF chickens aged 21-35 days were randomly divided into 4 groups, 10 chickens in each group. The first group was injected with 0.3 mL of commercial duck Tembusu vaccine, the second group was injected with 0.3 mL of E-GL vaccine, the third group was injected with 0.3 mL of E-HA vaccine, and the fourth group was not immunized as a control. After 14 and 21 days, blood samples were collected from all chickens in the four groups, and antibody detection was performed using a French ID.VET flavivirus antibody detection kit. The kit is commercially available, and the specific detection results are shown in Tables 7 and 8. Referring to Tables 7 and 8, the detection results show that 14 days after immunization, the first group injected with the commercial duck Tembusu vaccine had 4 / 10 positive antibodies, and the average S / N% was 53.19%; the second group and the third group injected with the subunit vaccine of the application both had 10 / 10 positive antibodies, the average S / N% of the second group was 25.69%, and the average S / N% of the third group was 26.37%; the fourth group without injection of vaccine was negative; 21 days after immunization, the first group injected with the commercial duck Tembusu vaccine had 8 / 10 positive antibodies, and the average S / N% was 37.98%; the second group and the third group injected with the subunit vaccine of the application both had 10 / 10 positive antibodies, the average S / N% of the second group was 10.23%, and the average S / N% of the third group was 11.39%; the fourth group without injection of vaccine was negative.

[0040] Table 7: Serum antibody detection results at 14 days

[0041] Note: In Table 7, S / N%≤40% is positive; 40%<S / N%≤50% is suspicious; and S / N%>50% is negative.

[0042] Table 8: Serum antibody detection results at 21 days

[0043] Note: In Table 8, S / N%≤40% is positive; 40%<S / N%≤50% is suspicious; and S / N%>50% is negative.

[0044] In summary, the E subunit vaccine of the present application produces antibodies in 14 days, with a positive rate of 10 / 10, and produces higher titer antibodies in 21 days, with a S / N% of 10.23%; while the commercial duck Tembusu inactivated vaccine produces antibodies in 21 days, with a positive rate of 8 / 10, and a S / N% of 37.98%. From the results of the animal experiment, it can be seen that the antibody levels of the E-GL and E-HA subunit vaccines developed by the inventors are equivalent, and antibodies are produced in 14 days after immunization, but the expression amount of E-GL protein is about 1436 μg / mL, and the E-HA protein content is only 584 μg / mL, which shows that the signal peptide in E-GL can better secrete E protein to the extracellular, and after dilution by 14 times for vaccine production, the production cost can be greatly reduced.

[0045] The above-described embodiments are merely intended to describe the preferred embodiments of the present application, and are not intended to limit the scope of the present application. Various modifications, alterations, evolutions, improvements, and the like of the technical solutions of the present application made by those skilled in the art without departing from the design spirit of the present application shall fall within the protection scope of the present application defined by the claims.

Claims

1. A chicken Tembusu virus antigenic protein, characterized in that, The amino acid sequence of the antigen protein is selected from the E-GL protein as shown in SEQ ID NO: 3 or the E-HA protein as shown in SEQ ID NO:

6.

2. The chicken Tembusu virus subunit vaccine according to claim 1, characterized in that, The codon-optimized nucleotide sequence of the E-GL protein is shown in SEQ ID NO:

5.

3. The chicken Tembusu virus subunit vaccine according to claim 1, characterized in that, The codon-optimized nucleotide sequence of the E-HA protein is shown in SEQ ID NO:

7.

4. A chicken Tembusu virus subunit vaccine, characterized in that, Prepared from the E-GL protein of claim 1.

5. A chicken Tembusu virus subunit vaccine, characterized in that, Prepared from the E-HA protein of claim 1.

6. The chicken Tembusu virus subunit vaccine according to claim 4 or 5, characterized in that, Prepared by the following steps: Constructing a recombinant plasmid expressing the E gene based on the optimized chicken Tembusu virus E gene; Transfecting CHO-K1 cells with the recombinant plasmid, culturing, screening, amplifying and purifying the positive monoclonal cells to obtain the corresponding recombinant chicken Tembusu virus E protein; Adding an adjuvant to the recombinant chicken Tembusu virus E protein to obtain the chicken Tembusu virus subunit vaccine.

7. The chicken Tembusu virus subunit vaccine according to claim 6, characterized in that, The content of the E-GL protein in the chicken Tembusu virus subunit vaccine is 1436 μg / mL.

8. The chicken Tembusu virus subunit vaccine according to claim 6, characterized in that, The content of the E-HA protein in the chicken Tembusu virus subunit vaccine is 584 μg / mL.

9. The chicken Tembusu virus subunit vaccine of any one of claims 4-8 for use in the protection against infection by a chicken Tembusu virus strain.

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