EA042902B1 - A CELL LINE FOR E2 PROTEIN EXPRESSION AND ITS USE, E2 PROTEIN AND ITS USE - Google Patents

Description

The present patent application claims the advantage and priority of PRC Patent Application No. 201910443709.1, filed on May 25, 2019, titled Swine Fever E2 Protein Subunit Vaccine, incorporated herein in its entirety by reference.

The field of technology to which the invention belongs

The invention belongs to the field of veterinary biologicals and specifically relates to a cell line for the expression of the E2 protein and its use, the E2 protein and the corresponding subunit vaccine obtained using the E2 protein of the swine fever virus.

Background Art Information

Swine fever is an acute, rapidly developing and highly contagious infectious disease caused by the classical swine fever virus (CSFV), which causes significant losses in the global pig industry. Although the attenuated CSFV vaccine used in the prior art has good immune efficacy and a high level of safety, it is difficult to distinguish between antibodies produced by the body after stimulation with an attenuated live vaccine from those produced by a pig infected with a wild-type virus, which is not an advantage for control of the epidemic process and elimination of the swine fever virus. Research and development of a subunit swine fever virus vaccine may provide the ability to distinguish between a vaccine-immunized animal and an infected animal, and may also facilitate the elimination of CSFV.

Epidemic strains of Chinese swine fever virus can be divided into 2 gene groups and 4 gene subgroups, namely gene subgroups 2.1, 2.2, 2.3 and 1.1. Studies show that between the strains of swine fever virus prevalent in China and the highly virulent traditional strains of Shi-Men and rabbit-attenuated strains used for vaccine production, there is a large difference in the genes encoding antigens. The traditional Chinese rabbit-attenuated vaccine strain belongs to gene subgroup 1.1, but the wild-type virus in this subgroup is only a small part of it, and it is mainly found in relatively remote free-range areas. Given the rapid development of intensive swine production, the main epidemic strain of CSFV in China is gene group 2. During the epidemiological study of CSFV, it was found that the traditional attenuated vaccine based on CSFV gene subgroup 1.1, which is currently widely used, cannot provide good immune protection against currently widespread swine fever virus gene group 2.

The essence of the invention

The invention provides a cell line for the expression of the E2 protein and its use, as well as the E2 protein and its use. The subunit vaccine based on the E2 protein of the swine fever virus, the production of which is described in this application, can provide good immune protection for pigs.

As a first embodiment, the invention provides a cell line for expressing an E2 protein, which is an E2-CHO cell line comprising a nucleotide fragment encoding an E2 protein; where the E2 protein has the amino acid sequence under SEQ ID NO:3.

The nucleotide fragment has the sequence under SEQ ID NO:4.

As a second embodiment, the invention provides for the use of the E2-CHO cell line in recombinant expression of the E2 protein of swine fever virus.

As a third embodiment, the invention provides the E2 protein, the amino acid sequence of which is indicated under SEQ ID NO:3.

The sequence of the nucleotide fragment encoding the E2 protein is listed under SEQ ID NO:4.

As a fourth embodiment, the invention provides a subunit vaccine based on the E2 protein with the amino acid sequence of SEQ ID NO:3. The sequence of the nucleotide fragment encoding the E2 protein is listed under SEQ ID NO:4.

As a fifth embodiment, the invention provides for the use of the E2 protein in the preparation of a vaccine.

As a sixth embodiment, the invention provides a subunit swine fever virus vaccine containing an antigen and a vaccine adjuvant, and where the antigen is an E2 protein, the preparation of which is described in the patent.

The E2 protein-based subunit vaccine according to at least one embodiment of the present invention is characterized by high antigenic stability, high purity, high specificity, does not generate other unrelated antibodies, and also provides a convenient and accurate detection method, which can provide a reliable bases for the production of a subunit vaccine against the swine fever virus and diagnostic reagents for its detection.

- 1 042902

Detailed description of embodiments

Envelope glycoprotein E2 is the main structural protein of CSFV, it is the main determinant of CSFV virulence and host range, is located on the outer surface of the virion and is the main antigenic protein that induces the production of a virus neutralizing antibody and protects the immune pig from attack by CSFV. Therefore, the present application describes the use of the E2 protein of swine fever virus as an important protein molecule for the development of a new vaccine against swine fever virus. A subunit vaccine developed using eukaryotic cells expressing recombinant E2 protein antigens is used to distinguish between an immune response induced by a natural viral infection and an immune response induced by a subunit vaccine. This labeled vaccine will help eliminate the swine fever virus. In particular, the present application provides for the optimization of the gene sequence of the E2 protein of an isolated epidemic strain of swine fever virus of gene group 2 so that the recombinant E2 protein can be expressed using a CHO (Chinese hamster ovary) cell-based expression system. The genetically engineered subunit vaccine obtained by emulsification with a vaccine adjuvant has a good level of safety and efficacy in immunizing pigs and is a reliable protection against epidemic strains, and can be used as a labeled vaccine to eliminate CSFV.

The invention will be described in detail below by way of illustrative embodiments. However, it should be understood that the implementation of any embodiment may be implemented in other ways or mechanisms that are obvious to those skilled in the art without further enumeration, such as, without limitation, changing experimental parameters, changing reagents for the experiment, and the like. The methods used within the scope of this application may be methods frequently used in the field of vaccine production and are not limited to the specific listing of embodiments in this application. A person skilled in the art can implement the present application through other conventional methods.

Example 1 Screening for swine fever virus E2 protein.

In 2018, on a pig farm, pigs that had already been injected with the previous swine fever virus vaccine (which is a whole-virion live vaccine against gene type 1.1) developed typical symptoms of swine fever. In order to isolate the pathogen, the spleen of a dead pig was collected under aseptic conditions, the suspension was homogenized in sterile saline, centrifuged, and the supernatant was collected. After sterilization with supernatant, ST cells (pig testicle) were inoculated. After 96 hours, the first sampling of the supernatant containing the virus was carried out, and after 72 hours, the second sampling of the supernatant containing the virus was carried out. The collected solution containing the virus was purified, and then a virus strain was obtained. The virus was analyzed and the properties of the virus were determined regarding the level, immunogenicity, specificity and purity of the virus, etc., and the results showed the presence of a specific reaction between the isolated strain of the virus and the serum against swine fever virus, and the absence of bacteria, mycoplasmas and contamination with exogenous viruses . In this embodiment, the properties of the strain of the virus to be screened were determined, and the results showed that the strain belonged to the swine fever virus. The results of the challenge test showed that the virus screened for can cause death in pigs, and the results of post-mortem examination showed the presence of bleeding, necrosis and infarction of the internal organs.

The E2 protein gene (referred to as the E2 gene), which is the structural protein of the screened strain and the identified strain, was sequenced. The E2 gene is 1110 bp long. and codes for 370 amino acids. The amino acid sequence is listed under SEQ ID NO:1 and the nucleotide sequence of the coding gene is listed under SEQ ID NO:2. Gene evolution tree construction and nucleotide sequence homology analysis were carried out for the present E2 gene sequence and the E2 gene sequence registered in GenBank, and found that the degree of amino acid homology of the present E2 and the currently used virulent Shi-Men strain is approximately 87%, and the degree the amino acid homology between present E2 and common genotype 2.1d is approximately 98%. The results showed that the virus had mutated and the 1.1 vaccine was no longer capable of protecting against the epidemic strain of swine fever virus.

Example 2 Construction of a recombinant plasmid for the expression of the E2 gene.

1. Splicing and optimization of the E2 gene.

The Pestivirus_E2 domain of the E2 gene was excised and 23aa from the N-terminus and 32aa from the C-terminus were cut to allow for better tertiary folding of the protein and better exposure of the antigenic site of the E2 protein. At the same time, the codons of the E2 gene were optimized, and the optimized and modified amino acid sequence indicated under SEQ ID NO:3 is able to provide a good level of expression of the antigenic site. The nucleotide sequence encoding the amino acid sequence listed under SEQ ID NO:3 is codon-optimized, and the nucleotide sequence of the optimized gene is listed under SEQ ID NO:4.

The sequence of SEQ ID NO:4 was synthesized by Shanghai Bioengineering Co., Ltd. and inserted into the puc57 vector to obtain puc57-seq4.

2. Construction of recombinant plasmid 14.4-E2.

2.1. enzyme digestion reaction.

(1) Mark the 1.5 ml EP tube to be used, add samples and mix in the 1.5 ml EP tube according to the following table; the volume of the reaction mixture was 50 μl, as shown in the table below.__________________

Added Components Volume (µl) Vector 2 mcg Restriction endonuclease BstI 2.5 Restriction endonuclease Hindll 2.5 Yuh buffer 5 Bi-distilled H2O Bringing up to 50

For each enzyme digestion reaction according to the table, a vector was selected from the vector pEE14.4 (plasmid), puc57-seq4 and puc57-seq1, respectively, where puc57-seq1 could be used as a control group. Accordingly, these three vectors were subjected to an enzyme digestion reaction.

(2) The 1.5 ml EP tube from step (1) was placed in a water bath vessel at a constant temperature of 37° C. for 2-3 hours. Three two-enzyme digestion systems were obtained, respectively.

2.2. Isolation of the product of double cleavage by enzymes using a gel.

The aforementioned two-enzyme digestion systems were removed and agarose gel electrophoresis was performed to extract the DNA fragments. Received fragments of the plasmid and DNA fragments of the optimized gene E2 (containing the target gene), respectively.

(1) Labeled EP sample collection tube, adsorption column and collection tube.

(2) An empty labeled EP tube was weighed and the value recorded.

(3) Carefully excised one strip of target DNA from the agarose gel using a scalpel on a gel cutting knife and placed it in a clean 1.5 ml centrifuge tube.

(4) Add 600 μl of extraction buffer to the 1.5 ml centrifuge tube from step (3) and place in a water bath at 50° C. for approximately 5 minutes. During this, the centrifuge tube was continuously gently rotated up and down to ensure complete dissolution of the gel block to obtain the first solution.

(5) Added the first solution obtained in step (4) to the adsorption column, then centrifuged at 10,000 rpm for 30 seconds; the waste liquid was removed from the collection tube and the adsorption column was placed in the collection tube.

(6) Add 600 µl of extraction buffer to the adsorption column, then centrifuge at 10,000 rpm for 30 seconds; the waste liquid was removed from the collection tube and the adsorption column was placed in the collection tube.

(7) Add 600 µl wash buffer to the adsorption column, stand for 3 min, then centrifuge at 10,000 rpm for 30 s; the waste liquid was removed from the collection tube and the adsorption column was placed in the collection tube.

(8) Step (7) was repeated.

(9) Centrifuge an empty adsorption column at 12,000 rpm for 2 min to remove as much wash buffer as possible; the adsorption column was left at room temperature for 10 min and thoroughly dried.

(10) Place the adsorption column in a collection tube, drop 50 µl of elution buffer (preheated to 65°C) into the center of the adsorption membrane, stand for 3 min, and then centrifuge at 12,000 rpm for 2 min.

(11) Removed the centrifuge tube from step (10) from the centrifuge, removed the center portion from the adsorption column, closed the lid of the centrifuge tube, and left the DNA sample in the centrifuge tube.

(12) The DNA sample from step (11) was kept at 4°C for storage, and an agarose gel electrophoresis system was prepared for identification and gel extraction of DNA fragments. Thus, the linearized vector pEE14.4 and the target gene, respectively, can be obtained for the subsequent ligation reaction.

In the present example, the gel extraction kit was purchased from Hangzhou Bioer Technology Co., LTD.

2.3 Ligation reaction.

(1) Labeled the 0.2 ml centrifuge tube to be used.

(2) Add samples to a labeled 0.2 ml centrifuge tube according to

- 3 042902 with a 20 µl reaction mixture as shown in the table below.

Added Components Volume (µl) Linearized vector pEE14.4 50 ~ 200 ig Insertion DNA fragment 20~200ng Ligaz 1 Yuh buffer 2 Bi-distilled H2O Bringing up to 20

The insert DNA fragment in the table is the target gene obtained in step 2.2 (12).

(3) After adding the samples, the components were mixed by pipetting several times.

(4) Placed a 0.2 ml centrifuge tube at 37° C. and left to react for 30 minutes; after completion of the reaction, the reaction tube was quickly placed in an ice bath for 5 minutes to cool. A solution was obtained in which the ligation reaction occurred, containing the reaction products, and the reaction products were plasmids carrying the target gene.

(5) The reaction products from step (4) can be used directly for transformation or stored at -20° C. until later thawed and transformed as needed.

2.4. Transformation reaction.

(1) Quickly add 10 μl of the ligation reaction solution to 100 μl of competent cells (competent cells of Escherichia coli DH5a were selected in this example) in a sample tube, mix by pipetting, and place in an ice bath for 30 minutes.

(2) After step (1), the sample tube was removed, placed in a water bath at 42° C. for 100 seconds, and then quickly placed in an ice bath for 2 minutes.

(3) After step (2), the sample tube was removed, 600 μl of LB liquid medium was added to the sample tube on an ultra-clean work surface, and the sample tube was placed in a shaker at a constant temperature of 37°C, 220 rpm, and incubated in within 1 hour

(4) Petri dishes were prepared for transformation: Petri dishes were prepared for transformation with LB and the corresponding antibiotic according to the resistance of the plasmid.

(5) Application to a petri dish for transformation: remove the sample tube, centrifuge at room temperature at 8000 rpm for 2 minutes; remove 600 μl of supernatant and resuspend the bacteria at the bottom of the tube with the remaining supernatant, place the resuspended fluid with bacteria in the center of the corresponding transformation Petri dish, and spread the fluid with bacteria evenly in the center of the transformation plate with a spatula.

(6) Place the transformation Petri dishes from step (5) in a biochemical incubator at a constant temperature of 37° C. and incubate for 1 hour, then invert the transformation dishes and incubate for 15 hours; and received monoclones (ie a single colony).

(7) Observed and recorded the result of the transformation.

2.5. Extraction of the recombinant plasmid and identification by PCR.

2.5.1. Extraction of the recombinant plasmid.

(1) A 10 μl microdosing tip was used to select a monoclone (containing a recombinant plasmid) from a Petri dish for transformation into 5 ml LB liquid medium containing the antibiotic ampicillin, shaking the bacteria overnight at 37°C, 220 rpm.

(2) Transfer the bacterial solution to a 1.5 ml EP tube with a pipette, centrifuge at room temperature and 12,000 rpm for 2 minutes, and remove the supernatant.

(3) Added 250 µl of the plasmid extraction reagent buffer P1 to the EP tube from step (2) to completely suspend the bacteria.

(4) Add 250 µl of buffer P2 to the solution from step (3), and mix immediately by gently inverting the centrifuge tube 5-10 times, and stand at room temperature for 2-4 minutes.

(5) Add 350 µl of buffer N3 to the solution from step (4), and mix immediately by gently inverting the centrifuge tube 5-10 times, and stand at room temperature for 2-4 minutes.

(6) Centrifuge the solution from step (5) at room temperature, 14,000 rpm for 10 minutes.

(7) Transfer the supernatant from step (6) to the center of the adsorption column, centrifuge at room temperature, 12,000 rpm for 30 seconds, and remove the liquid from the collection tube.

(8) Add 500 µl of buffer PE to the center of the adsorption column, centrifuge at room temperature, 12,000 rpm for 30 seconds, and remove the liquid from the collection tube. Repeated once.

(9) An empty adsorption column was centrifuged at room temperature, 12,000 rpm in

-4042902 for 2 min.

(10) 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 for 5 min at room temperature, and centrifuge at room temperature, 12,000 rpm for 2 min , and stored the DNA solution in a test tube at 4°C.

At this stage, by expanding the cells in culture and extracting the recombinant plasmid, a relatively pure recombinant plasmid (recombinant plasmid 14.4-E2) was obtained; DNA solution from step (10) for the subsequent transfection process.

In this embodiment, the plasmid extraction kit was purchased from QIAGEN.

2.5.2. Identification by PCR.

(1) Mark the PCR tube to be used, add samples and mix according to the following table. The volume of the reaction mixture was 25 μl.

Added Components Volume (µl) Forward primer (10 µM) 0.5 Reverse primer (10 µM) 0.5 Extracted plasmid template 0.5 Blend 2X 12.5 Bi-distilled H2O Bringing up to 25

The extracted plasmid template in the table is the relatively pure recombinant plasmid 14.4-E2 obtained from the extraction of the recombinant plasmid in paragraph 2.5.1.

(2) PCR amplification procedure.

98°С 2 minutes. 98°С 5 s 55°С 15 s 72°C 80 s 72°C 10 min. 4°C constantly

cycles (3) Sequencing. The recombinant plasmid 14.4-E2, identified as positive by PCR, was sent to a sequencing service company for sequencing. The sequencing results showed that the inserted nucleotides in the recombinant plasmid are located according to SEQ ID NO:4 of the sequence listing, and the resulting amino acids are located according to SEQ ID NO:3 of the sequence listing.

3. Construction of recombinant plasmid 14.4-E2-G418.

The method for constructing the recombinant plasmid 14.4-E2-G418 is similar to that described in step 2 of this example.

In particular, the G418 gene stored in the laboratory and the recombinant plasmid 14.4-E2 identified as positive by PCR were digested with SmaI and EcoRI, respectively. The resulting DNA fragments were then ligated, transformed, and identified as in step 2 of this embodiment, so as to construct the recombinant plasmid 14.4-E2-G418.

Example 3 Transfection of CHO-Κι cells.

(1) Preparation. The microbiological safety box was sterilized with ultraviolet radiation for 30 min; heated the F-12 culture medium to 37°C in a water bath preheated to 37°C.

(2) Added 2.5 μg of recombinant DNA (ie, recombinant plasmid 14.4-E2-G418), P3000 (transfection reagent) to 125 μl of OPTI-MEM culture medium and mixed well. 3.75 µl of Lipofectamine 3000 liposomes was added to 125 µl of OPTI-MEM culture medium and mixed well. Liposomes were mixed with recombinant DNA and allowed to stand at room temperature for 10 min.

(3) The 6-well cell plate (CHO-K1 cells) placed horizontally 24 hours ago at 37°C was removed from the incubator, the supernatant medium was removed, the cells were washed three times with preheated culturing OPTI-MEM and removing the medium for culturing OPTI-MEM.

(4) Added 2 ml of 10% F-12 culture medium supplemented with 1% fetal bovine serum containing two kinds of antibodies to each well.

(5) Carefully add the mixture of recombinant DNA and liposomes to the cells in each well, gently mix and culture in a cell culture incubator at 37°C and 5% CO ₂ .

(6) 72 h after transfection of CHO-K1 cells, the supernatant was taken to extract

- 5 042902 protein phenomena and added to G418 cells to ensure selection pressure.

(7) After 144 hours, monoclonal cells were obtained; tested monoclonal cells and provided for the expansion of positive cells (ie, recombinant CHO cells) that can be used for large-scale protein production.

In this embodiment, transfection reagents were purchased from Thermo Fisher Scientific Co., LTD.

Example 4 Protein purification and detection.

1. Expression and identification of recombinant E2 protein in CHO cells.

The cells selected in Example 3 (i.e. cells with a positive result) were cultured at 37° C. for up to 240 hours, and the protein solution was taken every 48 hours for a total of 5 times, and the protein was analyzed in the protein solution taken 5 times, using SDS-PAGE. The results showed that the E2 gene prior to optimization was not expressed in CHO cells, while the optimized E2 gene was expressed in CHO cells.

2. Chromatographic purification of the recombinant E2 protein.

The supernatant, the E2 protein solution collected in step 1, was subjected to affinity chromatography using Ni Sepharose Excel from GE. For use as a loading solution, the collected supernatant was centrifuged at 5000 rpm for 5 minutes.

2.1. The medium was washed with distilled water, the volume of which was 5 column volumes.

2.2. The medium was washed with equilibration buffer, the volume of which was 5 column volumes.

2.3. Loaded sample, while the column can be loaded at the rate of 4 mg of protein per milliliter.

2.4. The medium was washed with wash buffer, the volume of which was 20 column volumes.

2.5. The protein was eluted with 5 column volumes of elution buffer.

3. Western blot test.

Conducted SDS-PAGE for the whole swine fever virus and the protein obtained in step 2 of this example at the same time, transferred the target protein bands onto a PVDF (polyvinylidene fluoride) membrane in a semi-dry manner at 20 volts, and transferred and blotted for 30 min, blocked the transfer membrane with blocking buffer overnight, washed three times with phosphate buffered saline with Tween (PBST) treated with swine fever virus positive serum diluted 1:500 at 37°C for 1.5 h, washed three times with PBST, treated with horseradish peroxidase (HRP) labeled goat anti-pig antibody http://dict.cnki.net/dict_result.aspx?scw=B and f*&tjTvpe=sentence&style= &t=enzymelabeled+antibody diluted 1:2000 at 37°C for 1.5 h, washed three times with PBST, treated with substrate solution for 5 min, and color developed in the chemiDOC gel-documenting system. The results showed that the band corresponding to the whole virus was very faint, while the band corresponding to the E2 protein expressed by the gene with the optimized nucleotide sequence indicated under SEQ ID NO: 4 in the CHO cell was very bright, indicating that the expressed E2 protein was characterized by a good level of immunogenicity.

4. Indirect immunofluorescence.

After the cells (ie, recombinant CHO cells) were fixed, clarified, and blocked, etc., anti-His tag monoclonal antibodies were added. After incubation at 37°C for 2 h, it was washed three times with PBS, and a second green fluorescent protein antibody was added, and incubated at 37°C for 1 h. An inverted fluorescent microscope was used for observation and photodocumentation. The results showed that CHO cells transfected with the non-optimized E2 gene had no specific fluorescence, while CHO cells transfected with the optimized E2 gene had a pronounced specific fluorescence.

Example 5. Obtaining a vaccine and determining its effectiveness.

1. Getting a vaccine.

(1) Sterilized adjuvant 201 at 116°C for 40 min under high pressure conditions; and maintained the temperature of adjuvant 201 in a water bath at approximately 37°C before use.

(2) Preparation of the aqueous phase: The purified protein solution obtained in Step 2 in Example 4 was mixed with sterile saline at an appropriate ratio such that the protein content of each 0.2 ml of the aqueous phase was at least 10 μg/ml.

(3) Emulsification: Take equal weights of oil phase and water phase, emulsify at 10,000

- 6 042902 rpm for 5 min. After emulsification, 10 ml were taken and centrifuged at 3000 rpm for 15 min, so that the level of the precipitated aqueous phase at the bottom of the tube did not exceed

0.5 ml, thus obtaining a vaccine.

2. Determining the effectiveness of the vaccine.

1) Immunological analysis in rabbits.

Japanese white rabbits, each weighing approximately 2.5 kg, were divided into two groups. Five rabbits in the immunization group received subcutaneous injections of 0.2 ml of the subunit vaccine, while the other five rabbits used as the control group were not immunized. Twenty-one days after immunization, blood was taken from the ear vein, serum was separated and antibodies were detected using ELISA. The results showed that all members of the immunization group were positive for antibodies and the blocking rate was more than 60%, while all members of the control group were negative for antibodies and the blocking rate was less than 30% (see Table 1). ).

Table 1

Detection of antibodies 21 days after immunization of rabbits using a subunit vaccine

Group No. Dose used for immunization (ml/rabbit) Days after first immunization (days) Blocking rate (%) Negative/Positive Positive Rate Immunoposterior group 1 0.2 21 72.3 Positive 5/5 2 0.2 21 79.4 Positive 3 0.2 21 77.7 Positive 4 0.2 21 74.1 Positive 5 0.2 21 76.2 Positive Control group 6 / 21 20.3 Negative 0/5 7 / 21 22.1 Negative 8 / 21 17.5 Negative 9 / 21 20.3 Negative 10 / 21 16.4 Negative

2) Results of an immunogenicity study in pigs.

Fifteen healthy susceptible (negative for both swine fever virus antigen and ELISA antibody) pigs were randomly assigned to 3 groups of 5 pigs per group. The first group was immunized with whole virus; each pig was intramuscularly injected with 1.0 ml of the vaccine; and 21 days after the first immunization, a second immunization was performed using the same vaccine at the same dose. The second group was the immunization group with a previously marketed vaccine; each pig was intramuscularly injected with 1.0 ml of the vaccine; and 21 days after the first immunization, a second immunization was performed using the same vaccine at the same dose. The third group, which was the control group, was not immunized. On day 14 after the second immunization, blood samples were taken to detect antibodies against CSFV by ELISA, and then 1.0 ml (containing at least 105.0 MLD) of the strain isolated in example 1 was intramuscularly injected into the neck of each pig, and observed until day 16. If there were dead pigs during the observation period, necropsy should be performed immediately, and for the remaining pigs, necropsy should be performed at the end of the study (16 days after infection) and the results of the study recorded. The results showed that all 5 piglets from the subunit vaccine group and those from the previous vaccine group were antibody positive and the blocking rate was 80% and 60%, respectively. All members of the control group were negative for antibodies and the blocking rate was less than 30%. After challenge, all members of the control group died and all members of the subunit vaccine group were protected, while 3 protected and 2 diseased animals were observed in the pre-vaccine group (see Table 2).

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Claims (10)

table 2 Detection in pigs of antibodies to the E2 protein 35 days after the first immunization Group No. Dose at immunization (ml/pig) Days after first immunization (days) Blocking rate (%) Negative/Positive Protection for 16 days after infection Protection level Subunit vaccine group 1 1.0+1.0 35 88.6 Positive Healthy 5/5 2 1.0+1.0 35 85.5 Positive Healthy 3 1.0+1.0 35 92.1 Positive Healthy 4 1.0+1.0 35 93.7 Positive Healthy 5 1.0+1.0 35 91.9 Positive Healthy Previous vaccine group 6 1.0+1.0 35 60.2 Positive Sick 3/5 7 1.0+1.0 35 69.4 Positive Healthy 8 1.0+1.0 35 68 Positive Healthy 9 1.0+1.0 35 60.4 Positive Sick 10 1.0+1.0 35 70.1 Positive Healthy Control group eleven / 35 15.7 Negative Fallen 0/5 12 / 35 25.3 Negative Fallen 13 / 35 17.2 Negative Fallen 14 / 35 19.6 Negative Fallen 15 / 35 -ZD Negative Fallen Note: 1.0+1.0 means that the immunization groups were immunized twice, 1.0 ml/pig each time; means that the control group was not immunized. In summary, the E2-based subunit vaccine described in the present invention had the highest level of infection and the best protective effect against the epidemic strain, indicating that the epidemic strain had mutated and the previous vaccine on the market no longer existed. able to protect against epidemic strain. An epidemic strain vaccine is highly needed, and the E2 protein-based subunit vaccine of the present invention is safe, stable, and has a good protective effect and a good market prospect. The described embodiments are only a description of the preferred embodiments of the present invention and are not intended to limit the scope of the present patent. Various modifications and improvements can be made by those skilled in the art with respect to the technical solutions of this invention without departing from the spirit of this patent, which should be considered covered by the scope of legal protection defined by the claims of this patent. CLAIM 1. A cell line for the expression of the E2 protein, where the cell line is an E2CHO cell line containing a nucleotide fragment encoding the E2 protein; and the amino acid sequence of the E2 protein is the sequence of SEQ ID NO:3. 2. The cell line according to claim 1, where the sequence of the nucleotide fragment is the sequence under SEQ ID NO:4. 3. Use of the cell line according to claim 1 for the expression of recombinant swine fever virus E2 protein. 4. Use according to claim 3, wherein the sequence of the nucleotide fragment encoding the E2 protein is the sequence under SEQ ID NO:4. 5. Protein E2, where the amino acid sequence of the protein E2 is the sequence under SEQ ID NO:3. 6. E2 protein according to claim 5, where the sequence of the nucleotide fragment encoding the E2 protein is the sequence under SEQ ID NO:4. 7. The use of the E2 protein according to claim 5 to obtain a subunit vaccine. 8. Use according to claim 7, wherein the sequence of the nucleotide fragment encoding the E2 protein is the sequence under SEQ ID NO:4. 9. A subunit swine fever virus vaccine, wherein the subunit vaccine comprises an antigen and a vaccine adjuvant, and the antigen is an E2 protein according to claim 5. 10. The vaccine according to claim 9, where the sequence of the nucleotide fragment encoding the E2 protein is the sequence under SEQ ID NO:4. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2