Recombinant protein composed of PCV2 Cap and PRV gD dominant antigen epitopes, coding gene, vaccine and application

Abstract

The invention discloses a recombinant protein composed of PCV2 Cap and PRV gD dominant antigen epitopes, a coding gene, a vaccine and application, and belongs to the technical field of biological medicine. The amino acid sequence of the recombinant protein is as shown in SEQ ID NO. 2. The nucleotide sequence of the gene for coding the recombinant protein is as shown in SEQ ID NO. 1. According to the invention, dominant epitopes of PCV2 Cap protein and PRV gD protein are connected in series and expressed into recombinant protein by using a CHO-Cap-gD engineering cell fed-batch culture mode, the culture and purification mode is simple, the protein content is high after purification, high-level corresponding antibodies can be generated, and wild virus attack can be resisted. In addition, a porcine circovirus type 2 antibody and a porcine pseudorabies antibody can be detected at the same time by establishing an ELISA method by using a coated elisa plate.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a recombinant protein composed of PCV2 Cap and PRV gD dominant antigen epitopes, a coding gene, a vaccine and uses. Background Art

[0002] Porcine circovirus (PCV) and porcine pseudorabies virus (PRV) are currently widespread in pig farms, causing serious economic losses. PCV is an immunosuppressive virus that reduces the immunity of piglets, so these two viruses are prone to co-infection. PCV can cause multiple diseases such as multisystemic wasting syndrome in post-weaned piglets, porcine dermatitis and nephrotic syndrome, and reproductive disorders in pigs. The PCVs discovered so far include PCV1, PCV2, PCV3, and PCV4, among which PCV2 causes the most serious impact and losses. PCV2 is one of the smallest animal viruses discovered so far, with a virus particle diameter of only about 16~18nm, and its nucleic acid is a single-stranded circular DNA of 1.7kb. Cap protein is its only structural protein and also an immunogenic protein. PRV is a virus that can cause reproductive disorders in sows, persistent high fever and neurological symptoms and even death in piglets, as well as respiratory symptoms in medium and large pigs. Pigs are the only natural host of PRV. PRV expresses a variety of envelope glycoproteins, among which gD, gC and gD proteins are its main immunogenic antigens, especially gD protein, which has a strong ability to induce neutralizing antibodies.

[0003] Since co-infection with PCV2 and PRV is relatively common, there is currently no related bivalent vaccine in use or in clinical trial submission. The patents related to the multi-vaccine that have been published are mainly for PCV2 and PRV whole virus inactivated antigens, or the subunit antigens of PCV2 and PRV need to be expressed separately, which is a relatively complicated process. Therefore, it is of great practical significance to develop a bivalent vaccine for PCV2 and PRV and express its antigens simply and efficiently. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a recombinant protein composed of PCV2 Cap and PRV gD dominant antigenic epitopes, an encoding gene, a vaccine and uses. The present invention utilizes the disclosed dominant epitopes of porcine circovirus type 2 Cap protein and pseudorabies virus gD protein to construct a recombinant protein by tandem expression, thereby solving the problem of the current lack of PCV2 and PRV dual vaccine.

[0005] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is: The object of the present invention is to provide a recombinant protein composed of PCV2 Cap and PRV gD dominant antigenic epitopes, the amino acid sequence of which is shown in SEQ ID NO.2.

[0006] Another object of the present invention is to provide a gene encoding the above recombinant protein, the nucleotide sequence of which is shown as SEQ ID NO.1.

[0007] The present invention constructs a nucleic acid sequence as shown in SEQ ID NO.1 into a vector plasmid, linearizes the plasmid with an endonuclease and then transfects CHO cells to obtain CHO-Cap-gD engineered cells, and culture the engineered cells in a fed-batch culture method to obtain recombinant proteins. A secretion signal peptide and a His tag are added to the N-terminus and the C-terminus of the recombinant protein, respectively, so that the recombinant protein is secreted in the culture medium outside the cells. After centrifugation to remove cell debris, the characteristics of the His tag are used to make the purification method very simple.

[0008] Furthermore, the construction process of CHO-Cap-gD engineered cells is as follows: (1) selecting the dominant antigenic epitopes of PCV2 Cap protein and PRV gD protein, and designing (GGGGS) 3 amino acids after the selected dominant antigenic epitope of PCV2 Cap protein and before the dominant antigenic epitope of PRV gD protein for flexible connection to form the amino acid sequence of the recombinant protein; (2) Optimize the codons according to the amino acid sequence of the designed recombinant protein and the codon preference of CHO cells, and construct the optimized codons into the pCHO1.0 vector plasmid; (3) Transform the constructed pCHO1.0 plasmid into TOP10 competent cells and select positive colonies for PCR and sequencing verification; (4) Verify the correct colony, extract the plasmid and linearize it, transfect it into CHO cells, and perform pressure screening; (5) Verification of target protein expression; (6) Screening of monoclonal CHO-Cap-gD engineered cells that highly express the target recombinant protein.

[0009] Furthermore, the steps of feeding and culturing CHO-Cap-gD engineered cells are as follows: (1) Resuscitate CHO-Cap-gD cells, adjust the density to 500,000 / mL with culture medium, and culture at 37°C, 80% relative humidity, 8% CO2, and 150 rpm; (2) After several days of cell proliferation, the cell density is adjusted to 500,000 cells / mL using culture medium for expanded culture; (3) When the cells continue to proliferate to about 10 million / mL, adjust the culture temperature to 33°C; (4) Add 5% feed medium every 2 days to replenish the glucose concentration to 6 g / L~7 g / L; (5) After 14 days of culture at reduced temperature, the cell culture supernatant was harvested by centrifugation, the target protein was purified by nickel column affinity chromatography, and the protein content was detected by BCA method.

[0010] Another object of the present invention is to provide a recombinant plasmid comprising the above gene.

[0011] Another object of the present invention is to provide a transgenic cell line comprising the above gene or recombinant plasmid.

[0012] Another object of the present invention is to provide an engineered bacterium comprising the above gene or recombinant plasmid.

[0013] Another object of the present invention is to provide the use of the above recombinant protein in the preparation of preparations for diagnosing, preventing, detecting or treating porcine circovirus type 2 and porcine pseudorabies.

[0014] Another object of the present invention is to provide a kit for co-detection of porcine circovirus type 2 and porcine pseudorabies antibody, which comprises the above-mentioned recombinant protein.

[0015] Furthermore, an ELISA detection method can be established based on the recombinant protein, which can be used for the screening of double-negative pigs for porcine circovirus type 2 and pseudorabies, and can also be used for the detection of porcine circovirus type 2 or pseudorabies antibodies.

[0016] Another object of the present invention is to provide a vaccine, which comprises the above-mentioned recombinant protein and a pharmaceutically acceptable adjuvant.

[0017] Furthermore, adjuvants include, but are not limited to, oily adjuvants, aqueous adjuvants, aluminum gel adjuvants, nucleic acid adjuvants, cytokine adjuvants and other adjuvants that have the ability to enhance the immune response to antigens or change the type of immune response.

[0018] Another object of the present invention is to provide a multivalent combination vaccine comprising the above-mentioned recombinant protein.

[0019] When the recombinant protein is diluted to a certain concentration with an appropriate diluent, and then mixed with one or more antigens including but not limited to inactivated antigens of other viruses / bacteria / fungi / mycoplasma / chlamydia / parasites, subunit antigens, virus-like particle antigens, fusion expression antigens, attenuated antigens, chimeric virus antigens, etc., and then mixed and emulsified in appropriate proportions with oily adjuvants, aqueous adjuvants, aluminum gel adjuvants, nucleic acid adjuvants, cytokine adjuvants and other substances that can enhance the immune response ability of antigens or change the type of immune response, to prepare a multivalent combination vaccine, it can prevent diseases caused by infections including porcine circovirus type 2 and pseudorabies virus and the above-mentioned other viruses / bacteria / fungi / mycoplasma / chlamydia / parasites.

[0020] Beneficial effects of the present invention: The present invention utilizes the CHO-Cap-gD engineered cell flow-batch culture method to express the dominant epitopes of PCV2 Cap protein and PRV gD protein in series as a recombinant protein. The culture and purification methods are simple, the protein content is high after purification, and specific immunoblotting reactions can be produced with PCV2 and PRV positive sera. The recombinant protein is prepared into a vaccine, or combined with other antigens to prepare a multi-link multivalent vaccine, which can produce high levels of corresponding antibodies and resist wild virus attacks. The ELISA method established by using the recombinant protein to coat an enzyme-labeled plate can also simultaneously detect porcine circovirus type 2 and porcine pseudorabies antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the PCR verification diagram of the positive colony; among them, band M is the marker; band 1 is the negative control; band 2 is the positive colony; Figure 2 The figure is the result of SDS-PAGE detection of purified Cap-gD recombinant protein; wherein, band M is a marker; band 1 is purified Cap-gD recombinant protein; Figure 3 These are the results of specific immunoblotting detection of Cap-gD recombinant protein with PCV2-positive serum and PRV-positive serum; among them, band M is the marker; band 1 is the PCV2-positive serum; and band 2 is the PRV-positive serum. DETAILED DESCRIPTION

[0022] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0023] Example 1 Construction of CHO-Cap-gD engineered cells 1. Plasmid construction The dominant epitope amino acids and nucleic acid sequences of PCV2 Cap protein and PRV gD protein were selected respectively, and three consecutive GGGGS amino acids were designed for flexible connection. The codons were optimized using the codon preference of CHO cells and synthesized. The synthesized gene sequence is shown in SEQ ID NO.1, and the amino acid sequence of the recombinant protein encoded by it is shown in SEQ ID NO.2, which is then constructed into the pCHO1.0 vector plasmid.

[0024] 2. Transform TOP10 competent cells with the constructed pCHO1.0 plasmid Take 100 μL of TOP10 competent cells and place them on ice to thaw naturally, add 1 μL of plasmid, heat shock at 42°C for 90 seconds after 30 minutes, add 500 μL of LB liquid culture medium, culture at 37°C in a shaker for 60 minutes, take 100 μL of bacterial solution to spread on kanamycin plates, culture at 37°C for 12 hours, pick positive colonies and perform PCR verification with the primers in Table 1, program: 95°C 5min, 95°C 30s, 60°C 30s, 72°C 90s, 30 cycles, 72°C 5min, 15°C 10min, and send the products for sequencing.

[0025] Table 1 PCR validation primers

[0026] The results are as follows Figure 1 As shown, PCR verification of positive colonies showed a band consistent with the expected size, and sequencing confirmed that the constructed sequence was correct.

[0027] 3. Transformation of CHO cells Select the verified positive colonies and expand them with kanamycin-resistant LB liquid medium, then extract the plasmid and linearize the plasmid with NruI endonuclease. Add 50μL of linearized plasmid and 1.45mL of serum-free medium to a tube bottle, add 50μL of transfection reagent and 1.45mL of serum-free medium to another tube bottle, then slowly add the transfection reagent liquid to the plasmid tube, gently mix and let stand at room temperature for 10 minutes, subculture CHO cells with a viability of more than 95% in advance, adjust the density to 1 million / mL with culture medium, take 30mL of inoculated cell shaker, add 3mL of transfection mixture dropwise to the cells, and culture the cells at 37°C, 8% CO2, 150rpm.

[0028] 4. CHO cell pressure screening 48 hours after transformation, CHO cells were cultured statically with low-pressure culture medium (containing 20 μg / mL puromycin + 200 nM methotrexate) at 37°C, 80% relative humidity, and 8% CO2. The cell viability and density decreased and then increased. When the cell viability began to increase, the rotation speed was adjusted to 150 rpm. After 4 days, the cells were passaged with low-pressure culture medium. When the cell density exceeded 1 million and the viability exceeded 85%, the cells were cultured with high-pressure culture medium (containing 50 μg / mL puromycin + 1000 nM methotrexate) under the same conditions, and the cells were passaged every 3 days. The cell density was adjusted to 300,000 / mL until the cell viability exceeded 90%. The cells were centrifuged and frozen with freezing solution.

[0029] 5. Target protein expression verification After culturing the cells after high pressure screening, the supernatant was taken after 5 days, and the target band was detected by SDS-PAGE. Western Blot was performed with PCV2 positive serum and PRV positive serum to verify whether the target protein was expressed. The results are shown in Figure 2 and Figure 3 .

[0030] like Figure 2 As shown, SDS-PAGE detection of the recombinant protein bands showed a 49 kD target band, indicating that the target protein was obtained.

[0031] like Figure 3 As shown, the test results showed that the recombinant protein could react specifically with PCV2 positive serum and PRV positive serum by immunoblotting.

[0032] 6. Single cell cloning with high expression of target protein The cells after high pressure screening were diluted to a theoretical value of 3 cells / mL with cloning medium, and 200 μL / well was inoculated into a 96-well plate. After culturing for 17 days, the supernatant was collected and the OD was detected using a BCA kit. 570 Value, select OD 570 The cells with the highest OD value in 24 wells were inoculated into 24 well plates, then expanded into 6 well plates, and the supernatant was taken again to detect OD value using BCA kit. 570 Value, select OD 570 The cells in the 6 wells with the highest values ​​were frozen, and the remaining cells were inoculated into a shake flask for shaking culture. After 14 days of fed-batch culture, the supernatant was taken and the OD was detected using a BCA kit. 570 Value, OD 570 The cryopreserved cells with the highest value are the CHO-Cap-gD engineered cells that are finally screened for high expression of recombinant proteins.

[0033] Example 2 CHO-Cap-gD cell culture and recombinant protein expression and purification 1. After CHO-Cap-gD cells are revived, they are diluted to 500,000 / mL with culture medium and cultured at 37°C, 80% relative humidity, 8% CO2, and 150 rpm.

[0034] 2. After 3 to 5 days, when the cells have grown to a certain concentration, dilute the cells to 500,000 / mL with culture medium for expanded culture.

[0035] 3. The expanded cultured cells continue to grow to 8-10 million / mL and the culture temperature is adjusted to 33°C.

[0036] 4. Take samples every 2 days to test the glucose concentration in the culture bottle, add 5% feed medium, and replenish the glucose concentration to 6g / L~7g / L.

[0037] 5. After 14 days of cooling and culturing, the cell culture supernatant was harvested by centrifugation and the target protein was purified by nickel column affinity chromatography.

[0038] 6. Use the BCA method to detect the content of the purified target protein.

[0039] 7. Results The CHO-Cap-gD cells grew normally and were cultured for 14 days after adjusting the temperature to 33°C. The target protein content was 1.653 mg / mL by BCA assay.

[0040] Example 3 Preparation and efficacy evaluation of porcine circovirus type 2 and pseudorabies vaccine 1. Preparation of porcine circovirus type 2 and pseudorabies vaccine The recombinant protein after BCA detection was diluted to 50 μg / mL with PBS, mixed with an equal amount of ISA 206 adjuvant, and stirred at 500 rpm for 10 minutes to prepare a porcine circovirus type 2 and pseudorabies bivalent vaccine. The finished vaccine contained 25 μg / mL of recombinant protein.

[0041] 2. Select 30 pigs aged 3-4 weeks that were negative for porcine circovirus type 2 and pseudorabies and divide them into 6 groups, with 5 pigs in each group. In the first and second groups, each pig was injected with 2 mL of porcine circovirus type 2 and pseudorabies bivalent vaccine intramuscularly in the neck. In the third group, each pig was injected with an inactivated porcine circovirus type 2 vaccine (ZJ / C strain) intramuscularly in the neck. In the fourth group, each pig was injected with 1 dose of live pseudorabies vaccine (TP strain) intramuscularly in the neck. The fifth and sixth groups were not vaccinated as the control group. After 14 days, they were vaccinated again with the same dose. 14 days after the second vaccination, blood was collected to measure porcine circovirus type 2 ELISA antibody and pseudorabies gD ELISA antibody. At the same time, 14 days after the second vaccination, the first, third and fifth groups were vaccinated with porcine circovirus type 2 ZJ / C strain (10 7.0 TCID 50 / mL) was used for virus challenge, with 1.0mL / head per nose drop and 2.0mL / head per neck intramuscular injection. On the 4th and 7th days after virus challenge, each pig was inoculated with 2mL of keyhole limpet hemocyanin emulsified with Freund's incomplete adjuvant (KLH / ICFA, 1mg / mL). On the 28th day after virus challenge, the pigs were killed and the inguinal lymph nodes were taken for detection of porcine circovirus type 2 antigen by immunohistochemistry. The results were determined according to the porcine circovirus type 2 morbidity and protection criteria. Fourteen days after the second vaccination, the second, fourth and sixth groups were immunized with porcine pseudorabies virus GD-82 strain (10 6.5 TCID 50 / mL) of poison, 2mL / head of nose drops, and continuous observation for 14 days after the poison challenge, and the diagnosis was made according to the swine pseudorabies incidence and protection standards.

[0042] 3. Protection standard for porcine circovirus type 2: negative result of immunohistochemical test of inguinal lymph nodes. Pathogenesis standard for porcine circovirus type 2: positive result of immunohistochemical test of inguinal lymph nodes.

[0043] 4. Protection standards for pseudorabies in pigs: (1) Body temperature is not higher than 40.5℃ or higher than 40.5℃ but not more than 2 days; (2) No respiratory symptoms such as coughing, sneezing or dyspnea; (3) No neurological symptoms such as paddling, circling, banging against the wall, drooling, muscle tremors. Standards for pseudorabies in pigs: (1) Body temperature is higher than 40.5℃ and lasts for 3 days or more; (2) One of the respiratory symptoms such as coughing, sneezing or dyspnea occurs; (3) One of the neurological symptoms such as paddling, circling, banging against the wall, drooling, muscle tremors or death occurs; any two of them are considered to be onset. Antibody detection and virus challenge results after immunization with the two-combination vaccine are shown in Table 2.

[0044] Table 2 Antibody detection and virus challenge results after immunization with the bivalent vaccine

[0045] According to the test results in Table 2, the levels of porcine circovirus type 2 ELISA antibodies of porcine circovirus type 2 and pseudorabies bivalent vaccine and porcine circovirus type 2 (ZJ / C strain) are equivalent, and both can protect pigs against the attack of wild-type porcine circovirus, with a protection rate of 100%. The levels of pseudorabies neutralizing antibodies of porcine circovirus type 2 and pseudorabies bivalent vaccine and live pseudorabies vaccine (TP strain) are equivalent, and both can protect pigs against the attack of wild-type porcine pseudorabies virus, with a protection rate of 100%.

[0046] Example 4 Preparation and efficacy evaluation of combined vaccines This example is illustrated by the preparation of a triple vaccine for porcine circovirus type 2, pseudorabies, and mycoplasma pneumoniae. However, it should not be understood that the recombinant protein can only be used for the preparation of a triple vaccine for porcine circovirus type 2, pseudorabies, and mycoplasma pneumoniae.

[0047] 1. Preparation of triple vaccine for porcine circovirus type 2, pseudorabies and mycoplasma pneumonia The recombinant protein after BCA detection was diluted to 100 μg / mL with PBS, and the inactivated Mycoplasma hyopneumoniae (HP-G strain) was diluted to 4×10 8.0 CCU / mL, mixed in equal amounts as the aqueous phase, and then mixed with an equal amount of ISA 206 adjuvant, stirred at 500rpm for 10 minutes to prepare a trivalent vaccine of porcine circovirus type 2, pseudorabies, and mycoplasma pneumonia. The finished vaccine contained 25μg / mL of recombinant protein and 1×10 inactivated mycoplasma pneumoniae (HP-G strain). 8.0 CCU / mL.

[0048] 2. Preparation of inactivated vaccine against porcine mycoplasma pneumonia (HP-G strain) The inactivated Mycoplasma hyopneumoniae (HP-G strain) was diluted with PBS to contain 2×10 8.0 CCU / mL, and then mixed with an equal amount of ISA 206 adjuvant and stirred at 500rpm for 10 minutes to prepare the inactivated vaccine of swine mycoplasma pneumonia. The finished vaccine contained 1×10 inactivated swine pneumonia Mycoplasma (HP-G strain) 8.0 CCU / mL.

[0049] 3. A total of 45 pigs aged 3-4 weeks that were negative for porcine circovirus type 2, pseudorabies, and mycoplasma pneumonia were selected and divided into 9 groups, with 5 pigs in each group. Each pig in the first, second, and third groups was injected with 2 mL of a triple vaccine for porcine circovirus type 2, pseudorabies, and mycoplasma pneumonia intramuscularly in the neck. Each pig in the fourth group was injected with an inactivated porcine circovirus type 2 vaccine (ZJ / C strain) intramuscularly in the neck. Each pig in the fifth group was injected with one dose of live pseudorabies vaccine (TP strain) intramuscularly in the neck. Each pig in the sixth group was injected with 2 mL of an inactivated mycoplasma pneumonia vaccine (HP-G strain) intramuscularly in the neck. The seventh, eighth, and ninth groups were not vaccinated as the control group. After 14 days, they were immunized twice with the same dose. 14 days after the second immunization, blood was collected to measure porcine circovirus type 2 ELISA antibody, pseudorabies gD ELISA antibody, and mycoplasma pneumonia IHA antibody. At the same time, porcine circovirus type 2 ZJ / C strain (10 7.0 TCID 50 / mL) was used for virus challenge, with 1.0mL / head per nose drop and 2.0mL / head per neck intramuscular injection. On the 4th and 7th days after virus challenge, each pig was inoculated with 2mL of keyhole limpet hemocyanin emulsified with Freund's incomplete adjuvant (KLH / ICFA, 1mg / mL). On the 28th day after virus challenge, the pigs were killed and the inguinal lymph nodes were taken for porcine circovirus type 2 antigen detection by immunohistochemistry. The porcine circovirus type 2 disease incidence and protection criteria were used for judgment. Fourteen days after the second vaccination, the second, fifth and eighth groups were immunized with pseudorabies virus GD-82 strain (10 6.5 TCID 50 / mL) was used for the challenge, and 2mL / head was dripped into the nose. After the challenge, the pigs were observed for 14 consecutive days and judged according to the incidence and protection standards of pseudorabies in pigs. On the 14th day after the second vaccination, the third, sixth and ninth groups were challenged with HP-G strain of Mycoplasma hyopneumoniae tissue poison, and 5mL / head (including 100MID) was injected into the trachea. The pigs were killed on the 28th day after the challenge, and the lesions of Mycoplasma pneumonia in each test pig were scored according to the Goodwin 55-point scoring method, and the reduction rate of pneumonia lesions was calculated, and the results were judged according to the incidence and protection standards of Mycoplasma pneumonia in pigs.

[0050] 4. Protection and morbidity criteria for porcine circovirus type 2 and pseudorabies: Determine according to Example 3.

[0051] 5. Protection standard for swine mycoplasma pneumonia: The lung lesion score of the challenge control pigs should not be less than 10 points, and the reduction rate of pneumonia lesions in the immunized pigs should not be less than 60%. The morbidity standard for swine mycoplasma pneumonia: The lung lesion score of the challenge control pigs should not be less than 10 points, and the reduction rate of pneumonia lesions in the immunized pigs should be less than 60%. The antibody detection and challenge results after triple vaccine immunization are shown in Table 3.

[0052] Table 3 Antibody detection and virus challenge results after triple vaccine immunization

[0053] According to the test results in Table 3, the levels of porcine circovirus type 2 ELISA antibodies of the trivalent vaccine of porcine circovirus type 2, pseudorabies, and porcine mycoplasma pneumoniae and porcine circovirus type 2 (ZJ / C strain) are comparable, and both can protect pigs against the attack of wild-type porcine circovirus strains, with a protection rate of 100%. The levels of pseudorabies neutralizing antibodies of the trivalent vaccine of porcine circovirus type 2, pseudorabies, and porcine mycoplasma pneumoniae and the live pseudorabies vaccine (TP strain) are comparable, and both can protect pigs against the attack of wild-type pseudorabies virus strains, with a protection rate of 100%. The levels of mycoplasma pneumonia IHA antibodies of the trivalent vaccine of porcine circovirus type 2, pseudorabies, and porcine mycoplasma pneumoniae and the inactivated mycoplasma pneumoniae vaccine (HP-G strain) are comparable, and both can protect pigs against the attack of wild-type Mycoplasma pneumoniae (HP-G strain), with a protection rate of 100%.

[0054] Example 5 Establishment of an ELISA kit for co-detection of porcine circovirus type 2 and pseudorabies antibody 1. ELISA plate coating: dilute the recombinant protein to 1 μg / mL with pH 9.6 carbonate buffer, coat the ELISA plate at 100 μL / well, and place it at 2-8°C for overnight coating.

[0055] 2. Blocking of ELISA plate: discard the coating solution, block the ELISA plate with PBS containing 5% skim milk, 100 μL / well, incubate at 37°C for 60 minutes, discard the blocking solution, wash the plate 3 to 5 times with PBST at 300 μL / well, let the ELISA plate dry naturally to complete the coating. It can be stored in a sealed bag with a moisture-proof bag at 2 to 8°C away from light or used immediately.

[0056] 3. Porcine circovirus type 2 and pseudorabies antibody detection: Take out the ELISA plate and restore it to room temperature, dilute the serum to be tested 100 times, set up positive control (porcine circovirus type 2 and pseudorabies double positive serum) and negative control (porcine circovirus type 2 and pseudorabies double negative serum), add 100 μL / well to the ELISA plate, incubate at 37°C for 60 minutes, wash the plate 3-5 times with PBST at 300 μL / well, add HRP-labeled sheep anti-porcine enzyme-labeled secondary antibody at 100 μL / well, incubate at 37°C for 60 minutes, wash the plate 3-5 times with PBST at 300 μL / well, add TMB substrate at 100 μL / well, incubate at room temperature in the dark for 15 minutes, add 2M sulfuric acid stop solution at 50 μL / well, and detect OD 450 .

[0057] 4. Result determination: Positive control OD 450 Should be > 1.0, negative control OD 450 Should be <0.2, then the test is established; sample S / P value = (sample OD 450 - Negative control OD 450 ) / (positive control OD 450 - Negative control OD 450 ), when the S / P value of the sample is less than 0.3, it is negative, indicating that both porcine circovirus type 2 and pseudorabies antibodies in the sample are negative; when the S / P value of the sample is ≥0.3, it is positive, indicating that at least one of the porcine circovirus type 2 and pseudorabies antibodies in the sample is suspicious or positive.

[0058] 5. Three batches of porcine circovirus type 2 and porcine pseudorabies antibody co-detection ELISA kits were used to detect common swine disease positive sera to determine the specificity of the kits and the stability of the three batches of kits. The results are shown in Table 4.

[0059] Table 4 Detection results of ELISA kits for common swine disease positive sera (S / P value)

[0060] According to the test results in Table 4, the kit was negative for positive sera of common swine diseases other than porcine circovirus type 2 and pseudorabies, indicating that the kit did not cross-react with positive sera of other common swine diseases and had good specificity. The kit was positive for positive sera of porcine circovirus type 2 and pseudorabies, and the coefficient of variation between the three batches of kits was less than 5%, indicating that the kit had good stability.

[0061] 6. The same 20 serum samples were tested using the porcine circovirus type 2 and pseudorabies antibody co-detection ELISA kit, the Guangzhou Yueyang Biological porcine circovirus type 2 antibody ELISA detection kit, and the IDEXX pseudorabies gD antibody ELISA detection kit to determine the correlation between the test results of the porcine circovirus type 2 and pseudorabies antibody co-detection ELISA kit and the Guangzhou Yueyang Biological porcine circovirus type 2 antibody ELISA detection kit or the IDEXX pseudorabies gD antibody ELISA detection kit. The results are shown in Table 5.

[0062] Table 5 Comparison results of co-detection ELISA kits and PCV2 and PRV gD kits

[0063] According to the test results in Table 5, it can be seen that the samples detected as negative by the co-detection ELISA kit for porcine circovirus type 2 and pseudorabies antibody were all negative when tested with the Guangzhou Yueyang Biological Porcine Circovirus Type 2 Antibody ELISA Detection Kit and the IDEXX Porcine Pseudorabies gD Antibody ELISA Detection Kit; the samples detected as positive by the co-detection ELISA kit for porcine circovirus type 2 and pseudorabies antibody were tested with the Guangzhou Yueyang Biological Porcine Circovirus Type 2 Antibody ELISA Detection Kit and the IDEXX Porcine Pseudorabies gD Antibody ELISA Detection Kit, and at least one of them was suspicious or positive. The co-detection ELISA kit cannot distinguish between porcine circovirus type 2 and pseudorabies antibody, but can be used for the screening of double-negative pigs for porcine circovirus type 2 and pseudorabies antibody. Compared with the detection of porcine circovirus type 2 and pseudorabies antibody separately, the co-detection ELISA kit saves more time and cost.

[0064] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A recombinant protein composed of PCV2 Cap and PRV gD dominant antigenic epitopes, characterized in that: The amino acid sequence of the recombinant protein is shown in SEQ ID NO.

2.

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

1.

3. A recombinant plasmid, characterized in that: Comprising the gene according to claim 2.

4. A transgenic cell line, characterized in that Comprising the gene according to claim 2, or the recombinant plasmid according to claim 3.

5. An engineered bacterium, characterized in that: Comprising the gene according to claim 2, or the recombinant plasmid according to claim 3.

6. Use of the recombinant protein according to claim 1 in the preparation of a preparation for diagnosing, preventing, detecting or treating porcine circovirus type 2 and porcine pseudorabies.

7. A kit for co-detection of porcine circovirus type 2 and porcine pseudorabies antibody, characterized in that: Comprising the recombinant protein according to claim 1.

8. A vaccine, characterized in that The invention comprises the recombinant protein according to claim 1, and a pharmaceutically acceptable adjuvant thereof.

9. The vaccine according to claim 8, characterized in that The adjuvant is an oily adjuvant, an aqueous adjuvant, an aluminum gel adjuvant, a nucleic acid adjuvant, a cytokine adjuvant, or an adjuvant that can enhance the antigen immune response capability or change the immune response type.

10. A multivalent combination vaccine, characterized in that: Comprising the recombinant protein according to claim 1.

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