Preparation and application of a recombinant bivalent vaccine for porcine reproductive and respiratory syndrome

Abstract

This invention relates to the preparation and application of a recombinant bivalent vaccine for porcine reproductive and respiratory syndrome (PRRS). The recombinant bivalent PRRS vaccine is prepared using gene recombination technology. The genes encoding the fusion proteins of the major structural proteins GP2, GP3, GP4, GP5, and M protein B-cell epitopes of PRRS-like strains NADC30 and NADC34, as well as their recombinant strains, are cloned into a vector, transformed into host bacteria, and prepared through processes such as fermentation, purification, mixing, and emulsification. Animal experiments show that the recombinant bivalent PRRS vaccine can effectively improve the humoral and cellular immune responses in target animals.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and genetic engineering, and mainly relates to the preparation and application of a recombinant bivalent vaccine for porcine reproductive and respiratory syndrome (PRRS). Specifically, using gene recombination technology, the gene encoding the fusion protein of the main structural proteins GP2, GP3, GP4, GP5, and M protein B-cell epitopes of PRRS-like strains NADC30 and NADC34, as well as their recombinant strains, is cloned into a vector. The vector is then transformed into host bacteria, and through processes such as fermentation, purification, mixing, and emulsification, a recombinant bivalent PRRS vaccine is obtained, along with its application in the prevention of PRRS. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRSV) is a highly contagious disease of pigs caused by Porcine Reproductive and Respiration Syndrome (PRRSV), characterized by reproductive disorders in sows and severe respiratory diseases in growing piglets and finishing pigs.

[0003] Pigs infected with PRRSV develop a systemic humoral immune response, producing specific antibodies within a short period. Studies by Nelson et al. showed that the first detected antibodies were against the N protein, followed by the M protein, and then the GP5 antibody. The highest concentration of specific IgG antibodies in the serum peaks approximately 21–49 days after infection. In the early stages of infection, the antibodies produced do not neutralize the virus and therefore cannot prevent PRRSV infection. The production of neutralizing antibodies is characterized by a long duration and low titers, failing to neutralize the PRRS virus in the body. This allows PRRSV to cause persistent infection, potentially leading to viremia in severe cases and significant losses to the pig farming industry.

[0004] In the process of PRRSV infection in pigs, T cells play a crucial role in the development and regulation of antigen-specific immune responses and are an essential component of the body's resistance to PRRSV infection. One week after PRRSV infection, the number of CD8+ T cells in peripheral blood mononuclear cells (PBMCs) in pigs significantly exceeds normal levels, and the growth trend of IFN-γ-secreting cells is positively correlated with the viral load clearance capacity. Specifically, the increased percentage of cytotoxic T lymphocytes in the bronchoalveolar lavage fluid of PRRSV-infected pigs is positively correlated with the decrease in PRRSV in the alveoli. A large number of CD8+ T cells are produced 4–5 weeks after PRRSV infection. T cell subsets are mainly characterized by the differentiation of CD3, CD4, and CD8 and the secretion of IFN-γ. CD4 / CD8 double positivity plays an important role in regulating protective immunity by being responsible for IFN-γ secretion and localizing it to sites of inflammation. In addition, pigs that recovered from PRRSV infection had a strong ability to proliferate lymphocytes and could produce IFN-γ and IL-2 4–12 weeks after infection. These were mainly secreted by Th1 cells. Piglets passively infected by pregnant sows infected with PRRSV showed a decrease in the number of CD4+ T cells at birth and one week after birth, which returned to normal by 14 days, while the number of CD8+ T cells continued to increase.

[0005] An antigenic epitope is a region where an antigen can bind to an antibody. This indicates that antigen-antibody recognition only requires the antibody to bind to the corresponding antigenic epitope, rather than the antibody binding to the entire antigenic group. Based on the characteristics of TCR and BCR, epitopes can be divided into two types: B-cell epitopes and T-cell epitopes.

[0006] Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded positive-sense RNA virus belonging to the order Heliovirales, genus Arteriviridae, and family Arteriviridae, with a diameter of approximately 50–65 nm. PRRSV has been confirmed to have 10 open reading frames (ORFs): ORF1a, ORF1b, ORF2a, ORF2b, ORF3, ORF4, ORF5, ORF5a, ORF6, and ORF7. ORF2a, ORF3, and ORF4 encode three N-glycosylated small envelope proteins (GP2a, GP3, and GP4), respectively. ORF2b is completely embedded within ORF2a and primarily encodes the non-glycosylated minor protein E. ORF5 is one of the largest variable regions in the viral genome, encoding the GP5 glycosylated envelope protein, and exists as a heterodimer with the non-glycosylated membrane protein M encoded by ORF6. ORF7 encodes the virus's only nucleocapsid protein, N.

[0007] The North American type GP2a protein consists of 256 amino acid residues with a molecular weight of approximately 29–30 kDa. GP2a is a major component of the viral envelope protrusion, and studies by Overend et al. have shown that GP2a has an inhibitory effect on apoptosis. GP2a can bind to CD163, an important receptor for PRRSV. PRRSV lacking GP2a cannot grow in Marc-145 cells. However, the deletion of 10 amino acids at the C-terminus of GP2a does not affect its growth in Marc-145 cells, nor does it alter the neutralizing antigenic epitopes of the recombinant virus. Therefore, GP2a may play an important role in PRRSV infection and the regulation of host immunity. B-cell epitopes of the North American type GP2a, identified using peptide scanning technology, are 41–55 amino acids and 121–135 amino acids.

[0008] The GP3 protein is a highly glycosylated membrane protein composed of 265 amino acids. Although antibodies against the PRRSV GP3 protein can be detected in pigs infected with PRRSV, the levels of these antibodies are relatively low. Many researchers speculate that the GP3 protein may participate in the viral neutralization response, along with the PRRSV M protein, to neutralize viral pathogenicity. HP-PRRSV GP3 contains one conserved B-cell epitope of 87–94 amino acids, while low-virulence PRRSV contains one non-conserved B-cell epitope of 59–68 amino acids; neither of these epitopes is a neutralizing epitope.

[0009] GP4 protein is a membrane protein encoded by ORF4. GP4 is an important glycoprotein for PRRSV infection and the production of neutralizing antibodies, and its extracellular domain may be a relevant site for neutralizing antibody production. Linear antigenic epitopes 53aa–67aa, 84aa–95aa, 112aa–123aa, 107aa–115aa, and 59aa–67aa contain neutralizing epitopes and are not conserved. Studies have shown that the extracellular region of the GP4 protein can serve as a neutralizing epitope for American PRRSV. Immunization of mice with recombinant GP4 protein expressed in insect cells yielded anti-American PRRSV mAbs. ELISA and Western blot analysis showed that the mAb prepared in this experiment recognized a conformation-dependent epitope, but the epitope was not precisely localized.

[0010] The GP5 protein is encoded by ORF5. The ORF5 of the American PRRSV consists of 46–51 amino acid residues and has a transmembrane region. This protein is immunogenic, and it is speculated that its R and R / Q rich regions may be related to the regulation of mRNA splicing and transport. A series of reactions in PRRSV, including invasion, adsorption, and proliferation, are all related to GP5. GP5 has six antigenic epitopes, one of which is a linear neutralizing epitope with serotype specificity. Neutralization activity is significantly correlated with anti-GP5 antibody titers. Numerous studies have investigated the application of the GP5 protein in vaccines, finding that immunized pigs can induce the production of neutralizing antibodies against PRRSV. Furthermore, antibodies produced in immunized pigs after challenge with highly pathogenic strains provide some protection. Currently, some GP5 protein neutralizing epitopes (37–45 aa, 117–131 aa, and 149–163 aa) in its extracellular region have been identified. Between 27 and 31 aa at the N-terminus of GP5, there is an inducible epitope similar to human immunodeficiency virus type I, which can interfere with the immune response to the neutralizing epitope, thus delaying the production of neutralizing antibodies.

[0011] The M protein is a membrane protein encoded by ORF6. The American PRRSV M protein consists of 174 amino acids and has a molecular weight of approximately 18 kDa. The M protein is the most conserved and structurally stable structural protein. Neutralizing epitopes may exist on the M protein. The smallest cellular epitope of the M protein that can be recognized by antibodies is located at 151–174 aa, with its core region at 161–165 aa. The T-cell epitopes of the M protein are 9–23 aa, 33–47 aa, 57–71 aa, and 93–107 aa. Summary of the Invention Invention Overview Porcine reproductive and respiratory syndrome virus (PRRSV) contains abundant high-quality antigens on its GP2, GP3, GP4, GP5, and M proteins. Based on the current prevalence of PRRSV in China, we analyzed the sequence characteristics of the major structural proteins GP2, GP3, GP4, GP5, and M proteins of NADC30-like strains, NADC34-like strains, and their recombinant strains using software such as BIMAS, NetCTL1.2, SYFPEITHI, TEPITOPE, and IEDB. Finally, we selected the B-cell epitopes, CTL, and Th-cell epitopes of NADC30-like strains, NADC34-like strains, and their recombinant strains as an antigenic framework, constructed it into the pRSETA vector, optimized the design to create a vaccine antigen fusion protein encoding gene sequence, expressed it in E. coli, and then, through protein extraction, purification, and formulation processes, obtained a composition with ideal immunogenicity: a recombinant bivalent PRRSV vaccine. Immunization of target animals with the recombinant bivalent PRRSV vaccine can induce high levels of humoral and cellular immune responses.

[0013] One objective of this invention is to provide a recombinant bivalent porcine reproductive and respiratory syndrome (PRRS) vaccine immunization composition capable of stimulating humoral and cellular immunity; a second objective of this invention is to provide a method for designing, constructing, and obtaining the encoding nucleotides of the recombinant PRRS vaccine antigen; a third objective of this invention is to provide a genetically engineered bacterial strain capable of expressing the recombinant PRRS vaccine antigen; a fourth objective of this invention is to provide a method for preparing the recombinant PRRS vaccine; and a fifth objective of this invention is to provide the use of the recombinant PRRS vaccine in vaccine formulation and animal immunization.

[0014] In a first aspect, the present invention provides a recombinant bivalent vaccine polypeptide for porcine reproductive and respiratory syndrome (PRRS) and its composition. It contains fusion proteins or polypeptides of the major structural proteins GP2, GP3, GP4, GP5, and M protein B-cell epitopes tandemly with T-cell epitopes of PRRS-like strains NADC30 and NADC34, and their recombinant strains, as shown in Table 1.

[0015] Table 1. Major structural protein epitopes of NADC30 and NADC34 strains and their recombinant strains

[0016] Tandem synthesis can be achieved through genetic engineering or artificial synthesis. In addition to containing B-cell and T-cell epitopes for porcine reproductive and respiratory syndrome (PRRS), the recombinant bivalent PRRSNADC30 / 34 vaccine also contains non-immunogenic substances. These non-immunogenic substances are the linking portions of the polypeptides, lacking immunogenicity and adjuvant activity, and mainly include linker peptides, chemically modified portions, N-terminal signal peptides, and C-terminal polyadenylates. Pharmaceutically acceptable salts refer to salts that are non-toxic, non-irritating, and non-allergenic, and suitable for human or animal tissues. Non-active substances and pharmaceutically acceptable salts are well known to those skilled in the art. Therefore, the amino acid sequence of the recombinant PRRSNADC30 / 34 polypeptide of the recombinant PRRSNADC30 / 34 vaccine is as follows:

[0017] VRALPFTLSSYRRSYGGRTDAPSWGTKHGSGYTAQYHPEIFGIGNGGHQFICAEHDGQNATLPGGRRSPASHVSVRVFQTSGSGFKVIFGNVSGSGGTDWLNDKFGGVIFPALTHI VSYGDIPGCKKVSTLIDELVSRRMYRTMENAGQAAWKQVVSEATLSRIGSGFAIFPTPDSRPKLHDFQQWLCAAYFELLVNYTVCPPCLTRQAATQIYEPNRSLWCRIGNDRCEEDD HDELGFVIPPGLSNEGHCAAYCLRHSNPSPAAFRKVPQCRTAIGTPVYITVTANVTDQNYGSGTSYVQHVKEFTQRSLVIDHVRLLHFMTPETMRWATVLCAAYMSWRYSCTRYTNF LLDTKGKLYRWRSPVIIEKGGKVDVGGHLIDLKRVVLDGSAATPITKISAEQCAAYILAPAHHVESVAGFHPITANDNHAFVVRRPGSTTVNGTLVPGLKSLVLGGRRAVKRGVVNL

[0018] In a second aspect, the present invention provides a nucleotide molecule encoding the recombinant porcine reproductive and respiratory syndrome (PRRS) bivalent vaccine polypeptide described in the first aspect of the present invention. In this invention, the nucleotide can be in RNA or DNA form, synthesized as a tandem sequence through artificial synthesis, then ligated using genetic engineering techniques, cloned into a vector, transformed into *E. coli*, and screened, fermented, and purified to obtain the recombinant PRSRS bivalent vaccine polypeptide. In this invention, the nucleic acid can be subjected to conventional molecular biology operations, such as PCR, restriction endonuclease digestion, and ligation. Restriction sites are added to both the 5' and 3' ends of the nucleic acid design. Preferably, the PRRSNADC30 / 34 encoding nucleotide sequence in this invention is as follows:

[0019] GTT CGT GCA CTG CCG TTT ACC CTG TCC AGC TAC CGT CGC TCT TAC GGT GGCCGT ACT GAC GCT CCG TCC TGG GGC ACT AAG CAC GGT AGC GGT TAT ACG GCA CAG TATCAC CCG GAA ATC TTC GGC ATC GGT AAT GGT GGC CAT CAG TTC ATC TGC GCA GAA CATGAC GGT CAG AAC GCA ACC CTG CCG GGT GGT CGT CGT TCC CCG GCG TCT CAC GTG TCCGTT CGC GTC TTC CAG ACG TCT GGT AGC GGT TTC AAA GTC ATC TTC GGC AAC GTT AGCGGT TCC GGT GGC ACC GAT TGG CTG AAC GAC AAA TTT GGT GGT GTT ATT TTT CCG GCGCTG ACG CAC ATC GTG AGC TAT GGC GAC ATC CCA GGT TGC AAA AAA GTG TCT ACG CTGATC GAT GAG CTG GTT TCT CGC CGT ATG TAC CGT ACC ATG GAA AAT GCT GGC CAG GCTGCC TGG AAG CAG GTT GTG TCT GAG GCT ACC CTG TCC CGT ATC GGC TCC GGT TTC GCGATC TTC CCG ACC CCG GAC TCC CGT CCG AAG CTG CAC GAT TTC CAG CAG TGG CTG TGCGCC GCT TAC TTC GAA CTG CTG GTA AAC TAC ACT GTG TGT CCG CCG TGT CTG ACC CGTCAG GCA GCG ACC CAG ATC TAC GAA CCG AAC CGT AGC CTG TGG TGT CGC ATT GGT AACGAT CGT TGC GAA GAG GAT GAC CAC GAT GAA CTG GGC TTC GTT ATC CCA CCG GGC CTGAGC AAC GAA GGT CAC TGC GCC GCATAC TGT CTG CGT CAC AGC AAC CCG TCC CCG GCAGCC TTC CGC AAA GTT CCT CAA TGT CGT ACC GCA ATC GGT ACC CCG GTA TAC ATT ACCGTG ACT GCT AAC GTT ACC GAT CAG AAC TAC GGT TCT GGC ACT TCT TAC GTC CAG CACGTT AAA GAA TTC ACC CAG CGT TCC CTG GTT ATC GAT CAC GTA CGT CTG CTG CAC TTTATG ACC CCG GAG ACT ATG CGT TGG GCT ACT GTA CTG TGC GCA GCT TAT ATG TCT TGGCGT TAC AGC TGC ACC CGT TAT ACC AAT TTC CTG CTG GAC ACC AAA GGT AAA CTG TACCGT TGG CGT TCT CCG GTG ATC ATC GAG AAG GGT GGC AAG GTC GAT GTT GGT GGT CATCTG ATT GAC CTG AAG CGT GTT GTT CTG GAT GGC TCT GCG GCT ACC CCA ATT ACC AAGATC TCT GCA GAA CAG TGC GCG GCG TAC ATT CTG GCG CCG GCA CAC CAC GTG GAA TCCGTA GCA GGC TTC CAT CCG ATC ACC GCA AAC GAC AAC CAT GCG TTC GTT GTA CGT CGTCCG GGT AGC ACC ACC GTG AAC GGC ACC CTG GTC CCG GGC CTG AAA AGC CTG GTT CTGGGC GGT CGT CGT GCC GTA AAA CGT GGT GTT GTA AAC CTG

[0020] In a third aspect, the present invention provides a vector that, in addition to containing the nucleotide molecule encoding the recombinant porcine reproductive and respiratory syndrome bivalent vaccine as described in the second aspect of the invention, also contains expression control elements operably linked to the nucleotide sequence for expression (transcription and translation) in prokaryotic cells. The most basic expression control elements include promoters, transcription terminators, enhancers, selectivity markers, etc., and these regulatory elements are well known in the art.

[0021] In a fourth aspect, the present invention provides a host cell containing the vector described in the third aspect of the invention. After transformation or transfection with a gene sequence containing the protein encoding the protein described in the present invention, and subsequently testing to demonstrate good genetic and expression stability, the host cell can be used for fermentation expression to produce the recombinant porcine reproductive and respiratory syndrome (PRRS) bivalent vaccine antigen required for this purpose. In this invention, *Escherichia coli* BL21(DE3, Plys) is preferred as the host bacterium for expressing the target protein.

[0022] In a fifth aspect, the present invention provides a method for preparing a recombinant bivalent porcine reproductive and respiratory syndrome (PRRS) vaccine, comprising the following steps: fermentation expression of the PRRSNADC30 / 34 fusion protein by engineered bacteria, followed by crude purification, fine purification processes, and subsequent mixing and formulation processes to obtain the desired recombinant PRSRS bivalent vaccine. The methods involved include, but are not limited to, cell disruption, inclusion body washing, denaturation, affinity chromatography, hydrophobic chromatography, anion exchange chromatography, reversed-phase chromatography, and renaturation.

[0023] In a sixth aspect, the present invention provides a recombinant bivalent vaccine for porcine reproductive and respiratory syndrome, comprising the polypeptide composition described in the first aspect of the present invention and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier described in the present invention is an oil adjuvant, an aqueous adjuvant, etc.

[0024] In a seventh aspect, the present invention provides the application of the recombinant bivalent porcine reproductive and respiratory syndrome (PRRS) vaccine described in the sixth aspect. The recombinant bivalent PRRS vaccine, administered to animals via intramuscular injection, intradermal or subcutaneous injection, or aerosol inoculation at a certain effective dose, can generate sufficiently effective humoral and cellular immune responses (see Examples 5, 6, 7, 8, 9, 10, and 11), stimulating the production of high IgG levels, inducing peripheral blood T lymphocyte proliferation, enhancing cytokine transcription, and increasing cytokine production. Furthermore, in embodiments of the present invention, laboratory safety tests on the vaccine demonstrate that the recombinant bivalent PRRS vaccine of the present invention is safe (see Example 4).

[0025] Furthermore, it should be noted that, based on the disclosure in the context of this application, other substantial aspects of the invention will be apparent to those skilled in the art. Attached Figure Description

[0027] The following figures are used to illustrate specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims. Figure 1 Diagram showing the construction of the expression plasmid pRSETA-PRRSNADC30 / 34 for the recombinant porcine reproductive and respiratory syndrome bivalent vaccine; Figure 2 The vector plasmid restriction enzyme digestion diagram shows that lane 1 is the DNA marker, lane 2 is the blank control, lane 3 is the intact plasmid pRSETA-PRRSNADC30 / 34 sample, and lanes 4 and 5 are pRSETA-PRRSNADC30 / 34 restriction enzyme digestion samples. Figure 3 This is an SDS-PAGE assay image, where lane 1 is the protein marker, lane 2 is the uninduced control sample of pRSETA-PRRSNADC30 / 34 / BL21 (DE3, Plys), and lane 3 is the induced sample of pRSETA-PRRSNADC30 / 34 / BL21 (DE3, Plys). The arrows indicate the expressed target protein. Figure 4 The results of Western blot analysis of PRRSNADC30 / 34 antigen protein from the recombinant porcine reproductive and respiratory syndrome bivalent vaccine are shown. Lane 1 is the pre-stained marker, lane 2 is the negative control, and lane 3 is the purified PRRSNADC30 / 34 antigen protein. Figure 5 The results of SDS-PAGE analysis of the PRRSNADC30 / 34 antigen protein of the recombinant porcine reproductive and respiratory syndrome bivalent vaccine are shown. Lane 1 is the protein marker, and lane 2 is the purified PRRSNADC30 / 34 antigen protein sample. Figure 6 The production index is used to test the safety of pregnant sows. Figure 7 For detecting antibody titers after immunization of piglets; Figure 8 For the detection of IFNγ concentration in serum; Figure 9 To detect the concentration of the cytokine TNF in peripheral blood; Figure 10 For the detection of IL-4 cytokine concentration in peripheral blood; Figure 11 For the detection of IL-6 cytokine concentration in peripheral blood; Figure 12 To detect the concentration of the cytokine IL-10 in peripheral blood; Figure 13 To detect the production frequency of IFNγ-SC in peripheral blood (NADC30); Figure 14 Detection of IFNγ-SC generation frequency in peripheral blood (NADC34); Figure 15 To detect the transcriptional level of the cytokine IL-17 mRNA in peripheral blood; Figure 16 To detect the transcriptional level of the cytokine TGF-β mRNA in peripheral blood; Figure 17The change in the proportion of CD3+ subsets of T lymphocytes in peripheral blood lymphocytes; Figure 18 The change in the proportion of CD3+CD4+ subsets of T lymphocytes in peripheral blood lymphocytes; Figure 19 This represents the change in the proportion of CD3+CD8+ subsets of T lymphocytes in peripheral blood lymphocytes. Detailed Implementation

[0029] The specific test methods described in the embodiments are merely exemplary descriptions used to illustrate the present invention in detail, but do not constitute a limitation on the scope of the present invention.

[0030] Example 1: Construction of Escherichia coli expression vector and expression strain

[0031] 1. The designed polypeptide-encoding nucleotides were sent to GenScript for synthesis. Nucleotides were designed at both ends of the nucleotide fragments. BamH I (5' end) and HindIII (3' end) Restriction enzyme sites were added, and the fragments were cloned into the pMD18T vector after synthesis. Sequencing confirmed that the inserted gene fragment was consistent with the designed sequence. The recombinant plasmid was named pMD18T-PRRSNADC30 / 34. The plasmid was digested with the appropriate restriction endonuclease. The pRSETA plasmid from Invitrogen was used as the E. coli expression vector, and the same restriction endonuclease was used. The digestion conditions were as follows: 10 μl reaction system, 2 μl plasmid, 5 activity units of restriction endonuclease, 1 μl 10× buffer, and deionized water were added to make up the difference. Digestion was carried out at 37℃ for 1.5 hours. After digestion, 1 μl 200mM EDTA was added to stop the reaction. Electrophoresis was performed on a 1% agarose gel for 30 minutes. The 2.6Kbp RSETA plasmid and the 1.41Kb PRRSNADC30 / 34 fragment were excised under UV light and recovered from the gel according to the Solarbio gel extraction kit instructions. The multi-epitope nucleotide fragments were mixed separately with the expression vector at a ratio of 1:2-3 (vector:fragment 1:2-3). The mixture was 15 μl in volume and ligated using T4 DNA ligase overnight at 16°C. The resulting recombinant plasmids were named pRSETA-PRRSNADC30 / 34 (see [link to relevant documentation]). Figure 1 ), transformed competent Escherichia coli BL21(DE3)pLysS.

[0032] 2. Transformation: Thaw pRSETA-PRRSNADC30 / 34 on ice, add 2 μl of ligation reaction solution to each, mix well again, incubate in an ice-water bath for 30 minutes, then at 42°C for 30 seconds, and then quickly return to the ice bath for 1.5 minutes. Add 1 mL of LB medium, incubate at 37°C for 1 hour, centrifuge at 4000g for 10 seconds and discard the supernatant. Resuspend the bacterial cells in 200 μl of LB medium. Spread the bacterial culture evenly on an LB agar plate containing 100 μg / mL ampicillin, and incubate upside down in a 37°C incubator for 12–16 hours until clones are formed.

[0033] 3. Identification: Single clones from the plate were picked and transferred to LB medium, cultured at 37°C with shaking at 200 rpm for 12 hours, plasmids were extracted, and restriction enzymes were used to identify them. BamH I and HindIII Double digestion yielded a 1.41 kb clone of the corresponding PRRSNADC30 / 34 fragment, which can be preliminarily identified as a positive clone (see...). Figure 2 Positive clones were subjected to DNA sequencing to further verify their correctness.

[0034] 4. Induction of expression. Positive clones were cultured overnight, and transferred at a 1:100 ratio the following morning. After culturing for 4 hours, 0.2 mM IPTG was added, and the cells were cultured for another 4 hours to prepare samples. The expression of the target protein was detected by routine SDS-PAGE (see...). Figure 3 The presence of a specific band at 55.4 kDa indicates a correct clone, and its expression accuracy was further confirmed using standard Western blot (see [link to article]). Figure 4 ); Take the correct clone, scale up the culture, and use the selected positive clone as the engineered bacteria to establish the original seed bank. The strain is named pRSETA-PRRSNADC30 / 34 / BL21(DE3, Plys).

[0035] Example 3: Fermentation, purification, and formulation of engineered bacteria

[0036] 1. Fermentation: Inoculate the production strain pRSETA-PRRSNADC30 / 34 / BL21 (DE3, Plys) into 5 mL LB liquid medium (containing 100 μg / mL ampicillin) and incubate at 37°C with shaking at 200 rpm for 10 hours to activate the strain. Then, inoculate the strain into a shake flask at a 1:100 ratio and incubate at 37°C with shaking until OD600=3. The culture medium can then be inoculated into the fermenter at a ratio of 10%. The fermentation medium is a semi-synthetic medium prepared with distilled water. Calibrate the dissolved oxygen and pH electrodes, turn on the tank agitator at 300 rpm, sterilize the tank online, and when the temperature of the culture medium in the tank drops to 37.0°C, calibrate the pH and dissolved oxygen (OD) zero point. The fermentation temperature was 37.0±0.1℃, dissolved oxygen was controlled at around 30-40%, and pH was controlled at around 7.0. IPTG (final concentration of 0.3mM) was added 5 hours after fermentation to induce expression, and fermentation ended after continuous induction for 3 hours.

[0037] 2. Purification: 100g of collected bacterial cells were suspended in 1000ml of Tris-HCl and lysed three times at 4℃ using a low-temperature high-pressure homogenizer. Inclusion bodies were collected by centrifugation at 12000 rpm for 30 min at 4℃. The inclusion body precipitate was mixed with 6M guanidine hydrochloride, 0.3% β-ME, and 20mM Tris-Cl (pH=8.00), stirred at room temperature for 4 hours, and centrifuged at 8000 rpm for 30 min. The supernatant was collected and the precipitate was discarded. The inclusion body supernatant was equilibrated onto an affinity chromatography column with 20mM phosphate buffer (pH=8.0), 0.5M sodium chloride, and 20mM imidazole, and eluted with 20mM phosphate buffer (pH=8.0), 0.5M sodium chloride, and 0.5M imidazole. The recombinant PRRSNADC30 / 34 protein stock solution was obtained. The semi-finished stock solution was analyzed by SDS-PAGE (see...). Figure 5 ).

[0038] 3. Formulation: The purified PRRSNADC30 / 34 fusion protein was sterilized by filtration through a sterile 0.22 μm Millipore filter membrane and then diluted with sterile PBS to 260 μg / mL. The purified PRRSNADC30 / 34 was emulsified with ICTYOLANE 17 adjuvant to prepare a recombinant porcine reproductive and respiratory syndrome (PRRS) bivalent vaccine. Three batches of vaccine were prepared using the same method for later use.

[0039] Example 4: Safety Trial of Recombinant Porcine Reproductive and Respiratory Syndrome Bivalent Vaccine

[0040] 1. Materials

[0041] 1.1 Vaccine: The emulsion of the recombinant porcine reproductive and respiratory syndrome bivalent vaccine was provided by Qingdao Baoruihui R&D Center, with batch numbers 202321, 202322, and 202323.

[0042] 1.2 Experimental animals: 20 Balb / C mice (18-22g), 20 healthy piglets (3-4 weeks old, negative for PRV, ASFV, PRRSV, CSFV, and PCV2 antibodies by ELISA and negative for PRV, ASFV, PRRSV, CSFV, and PCV2 antigens by PCR), and 20 pregnant sows (2-3 months gestation).

[0043] 2 Methods

[0044] 2.1 Safety of the vaccine in mice: 18-22g Balb / C mice were immunized with 5 mice per batch of vaccine, for a total of three batches, immunizing 15 mice in total. The immunization method was: 0.5ml was injected subcutaneously into each mouse. At the same time, 5 blank control mice were set up. The health status of the mice was observed for 10 consecutive days.

[0045] 2.2 Safety of the Vaccine in Piglets: Healthy piglets aged 3-4 weeks were selected and injected with the recombinant bivalent porcine reproductive and respiratory syndrome vaccine. Five piglets per batch were immunized, for a total of 15 piglets. The immunization method was: 4 ml injected intramuscularly behind the ear into each piglet. A control group of 5 piglets was also established. Clinical observation was conducted for 14 days. [Further details regarding immunization procedures and the end of the experiment are omitted as they are not relevant to the main text.]

[0046] 2.3 Safety of the Vaccine in Pregnant Sows: Twenty pregnant sows were randomly divided into four groups of five each. One group was vaccinated with each of the three batches of vaccine. Each sow received a 2ml intramuscular injection of recombinant porcine reproductive and respiratory syndrome (PRRS) bivalent vaccine behind the ear. The remaining group served as a blank control. All experimental groups were penned and fed and observed by designated personnel, with body temperature measured daily until day 7. Feeding and observation continued until farrowing, with detailed records kept of sow abortions, the health status of piglets born, and the number of stillbirths, weak piglets, and mummified fetuses.

[0047] 3 Results

[0048] 3.1 Safety test of the vaccine in mice After immunization, all mice showed no abnormalities in appetite, mental state and health status, consistent with the blank control group, and no deaths occurred. It can be seen that the recombinant porcine reproductive and respiratory syndrome bivalent vaccine is safe for mice, as shown in Table 2.

[0049] Table 2. Results of safety tests of the vaccine in mice.

[0050] 3.2 Safety Trial of Vaccine in Piglets During the entire 14-day observation period, all immunized piglets exhibited normal body temperature, mental state, and appetite. Vaccine absorption was good, and no inflammatory reaction was observed at the injection site. At the end of the trial, all 15 piglets survived without any clinical abnormalities. The results indicate that the recombinant bivalent porcine reproductive and respiratory syndrome vaccine is safe for piglets (see Table 3).

[0051] Table 3. Results of safety trials of the vaccine on piglets

[0052] 3.3 Safety trials of the vaccine in pregnant sows

[0053] 3.3.1 No abnormal clinical reactions were observed in either the immunized or control group sows. Their body temperature, mental state, and feed intake were normal, the vaccine was well absorbed, and there was no inflammatory reaction at the injection site (see Table 4).

[0054] Table 4. Results of safety trials of the vaccine in pregnant sows.

[0055] 3.3.2 Pregnant sows delivered normally, and all piglets were in good health. No abortions, stillbirths, or mummified fetuses were observed. There were no significant differences between groups (P > 0.05). See [link to relevant documentation]. Figure 6 .

[0056] Example 5: Animal grouping, immunization, and evaluation methods for the recombinant porcine reproductive and respiratory syndrome bivalent vaccine.

[0057] 1. Experimental animals: 20 healthy piglets aged 3-4 weeks (negative for PRV, ASFV, PRRSV, CSFV, and PCV2 antibodies detected by ELISA and negative for PRV, ASFV, PRRSV, CSFV, and PCV2 antigens detected by PCR). The experimental pigs were allowed to acclimatize to the environment for one week before the start of the experiment.

[0058] 2. The recombinant bivalent porcine reproductive and respiratory syndrome vaccine was provided by the R&D center, with batch numbers 202321, 202322, and 202323.

[0059] 3 Experimental Design

[0060] 3.1 Grouping and Immunization: Healthy piglets were housed in a BSL-2 isolation room. Animal immunization and sampling were performed. Animals were randomly divided into four groups: three vaccine-immunized groups and a non-immunized control group, with five piglets per group. 2 ml of vaccine was injected intramuscularly behind the ear per piglet. A booster immunization was administered using the same method 14 days after initial immunization.

[0061] 3.2 PBMC and Serum Separation: Blood was collected from the anterior vena cava of all experimental pigs before immunization (day 0), and on days 7, 14, 21, 28, 35, and 42 after immunization, with 8 ml collected from each pig. 3 ml of this blood was not anticoagulated and was injected into numbered sterile centrifuge tubes for serum separation and detection of PRRSV-specific antibodies. The remaining 5 ml of anterior vena cava blood was rapidly injected into corresponding numbered heparin sodium anticoagulation tubes, and the tubes were inverted several times to ensure sufficient contact between the blood and the heparin sodium in the tubes to prevent clotting; this was used for peripheral blood lymphocyte separation.

[0062] 3.3 Evaluation Methods: The titer of immune IgG antibodies in the isolated serum was detected by ELISA. PBMCs were used for T cell typing, cytokine transcriptional change assay, in vitro lymphocyte proliferation assay, and cytokine concentration assay to evaluate the humoral and cellular immunity levels of the recombinant porcine reproductive and respiratory syndrome bivalent vaccine.

[0063] Example 6: ELISA detection of IgG antibody titer

[0064] 1. Methods: A commercially available indirect ELISA antibody detection kit for porcine reproductive and respiratory syndrome was used to detect antibody titers in serum samples. The serum samples to be tested were serially diluted using the 1:200 dilution specified in the kit as the baseline.

[0065] 1.1 Bring the reagent to room temperature and mix by gently shaking or inverting.

[0066] 1.2 Record the positions of the control sample and the test sample on the 12×8 ELISA plate, with 2 wells each for the positive control and negative control, and 1 well for each test sample.

[0067] 1.3 Perform serial dilutions of 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, and 1:25600 on the samples to be tested, and set them aside for later use.

[0068] 1.4 Remove the sealing film from the ELISA plate and add 50 µl of positive control sample, negative control sample, and serially diluted test sample to the corresponding wells. Cover with the sealing film and incubate at 37°C for 60 minutes.

[0069] 1.5 Remove the sealing film, add 300µl of washing solution to each well, rinse 3 times, and blot dry on absorbent paper (turn the plate over and tap it lightly on the paper).

[0070] 1.6 Add 50 µl of MAb anti-porcine IgGs / HRPO conjugate solution to each well. Cover with membrane and incubate at 37 °C for 60 minutes.

[0071] 1.7 Remove the sealing film and repeat step 1.1.5.

[0072] 1.8 Add 50 µl of substrate solution to each well and gently shake the plate for 2 seconds.

[0073] 1.9 Place the plate at room temperature (20-25℃) in the dark for 10 minutes, and then carry out the color development reaction.

[0074] 1.10 Add 50µl of stop solution to each well and gently shake the plate to mix.

[0075] 1.11 Judgment

[0076] 1.11.1 Read the plate at 450 nm using a microplate reader. If the OD of the positive control is... 450 Mean > 0.6, and positive control OD 450 Mean / Negative Control OD 450 If the mean is greater than 6.0, the test is valid.

[0077] 1.11.2 Calculate the IRPC value of the sample. When the IRPC value is >20.0, the sample is judged as positive; when the IRPC value is ≤20.0, the sample is judged as negative.

[0078] 2 Results

[0079] like Figure 7 As shown, compared to the control group, the recombinant bivalent porcine reproductive and respiratory syndrome (PRRS) vaccine immunization group elicited a higher level of antibody immune response. One week after the first immunization, the antibody titer of some samples reached 1:200. Two weeks after the first immunization, the increase in antibody titer in the recombinant bivalent PRRS vaccine group was significantly higher than that in the control group, and the antibody titer continued to rise thereafter. By the end of the experiment, the antibody titer of the immunized pigs was not lower than 1:1600, while that of the control group was lower than 1:200.

[0080] Example 7: ELISA detection of serum IFNγ concentration

[0081] Serum was collected from the experimental animals using standard methods, and the IFNγ concentration was measured according to the instructions of the goat anti-porcine IFNγ ELISA kit. Results showed that after vaccination, the IFNγ concentration continued to rise with increasing immunization time, remaining at a high level for four weeks post-immunization. At the end of the experiment, the IFNγ concentration in the recombinant porcine reproductive and respiratory syndrome (PRRS) bivalent vaccine-immunized group was significantly higher than that in the control group (P < 0.01) (see...). Figure 8 ).

[0082] Example 8: Detection of peripheral blood cytokines

[0083] Serum levels of IL-4, IL-6, IL-10, and TNF-α were measured at 0, 7, 14, 21, 28, 35, and 42 days post-primary immunization according to the instructions of the commercially available kit. Cytokine concentrations for each sample are expressed as the mean of five samples, while cytokine mass at specified time points in each group is calculated as the mean ± standard deviation (SD) of five piglets. Compared with the control group, the immunized group showed significant differences in PBMC TNF and IL-10 at 7 days post-immunization (P<0.05), and at 14 days post-immunization, the immunized group showed highly significant differences in PBMC TNF, IL-4, IL-6, and IL-10 compared with the control group (P<0.01) (see [link to kit]). Figures 9-12This indicates that the recombinant bivalent vaccine for porcine reproductive and respiratory syndrome can elicit a good cellular immune response.

[0084] Example 9: Specific Lymphocyte Proliferation Detection

[0085] 1. Separation of PBMC

[0086] Porcine PBMCs were isolated from fresh venous blood and anticoagulated with 5 mM heparin. The leukocyte layer was collected by centrifugation at 1500 r / min, 37℃ for 30 min. PBMCs were washed twice with 15 ml Hanks balanced saline, resuspended in RPMI medium, and supplemented with 5% fetal bovine serum (Life Technologies, Gaithersburg, MD), 100 U / ml penicillin (Sigma), 100 mg / ml streptomycin (Sigma), 100 U / ml gentamicin (Sigma), 4 mM L-glutamate (Sigma), 1 mM sodium pyruvate (Life Technologies), 10 mM MEM non-essential amino acids (Life Technologies), and 250 mM 2-mercaptoethanol (Sigma).

[0087] 2. ELISPOT Determines IFNγ-SC Frequency Detection in PBMC

[0088] 2.1 The degree of antigen-specific cellular immune response in animals of different immunization groups and control groups was quantified using IFNγ ELISPOT. Peripheral blood mononuclear cells were diluted to a concentration of 5 × 10⁻⁶ cells / mL with medium containing 10% FCS. 6 Cells / mL, 100 μl of cell suspension was seeded into the wells of an ELISPOT plate. Wells containing NADC30-like antigen, NADC34-like inactivated antigen, and ConA stimulation were added as positive control wells, negative control wells, and background control wells. All samples were included in triplicate.

[0089] 2.2 The treated cells were cultured in a 37℃, 50% CO2 incubator for 24 h (12-48 h) (Note: the culture time may be extended to 30 h as appropriate; to ensure the accuracy of the data, the incubator and the ELISA plate must be kept completely still throughout the cell culture process, and the plate should not be moved or shaken. At the same time, measures should be taken to prevent evaporation, such as wrapping the ELISA plate with aluminum foil).

[0090] 2.3 PBMCs were exposed to the antigen at 37℃ in a CO2 environment for 2 hours. Cells were removed after washing 6 times with PBST (0.05% Tween 20). An appropriate amount of biotin-labeled detection antibody (P2C11-biotin) was diluted with PBS containing 0.5% FCS to a concentration of 0.5 μg / mL. 100 μl was added to each well and incubated at room temperature for 2 hours. The liquid in the wells was discarded after incubation, and the plate was washed 5 times with PBS for 10 min each time. An appropriate amount of HRP-labeled streptavidin (HRP Phosphase) was diluted 1000-fold with PBS-0.5% FCS. 100 μl was added to each well and incubated at room temperature for 1 hour. The liquid in the wells was discarded after incubation, and the plate was washed 5 times with PBS for 10 min each time. 100 μl of ready-to-use TMB substrate solution was added to each well and incubated at room temperature in the dark for 15–45 min until spots formed at the bottom of the wells. The colorimetric reaction was terminated by rinsing the wells with double-distilled water. The plates were then dried at room temperature. The spot-forming cell count (SFC) per well was analyzed using an ELISPOT analyzer or under a dissecting microscope. Results were recorded in spot-forming units: IFNγ SFC / well, where each spot represents one IFNγ-secreting cell.

[0091] 2.4 Positive Criteria: Stimulation Index = IFNγ-SFC per well after sample stimulation / IFNγ-SFC per well after blank stimulation ≥ 2.1. Specific IFNγ-producing cells were determined by counting the number of blue dots.

[0092] 3 Results

[0093] To evaluate the development of specific regulatory immune responses induced by recombinant porcine reproductive and respiratory syndrome (PRRS) bivalent vaccine immunization, IFNγ secretion was measured in PBMCs isolated from experimental animals before and after immunization. In this assay, the presence of IFNγ-SCs in unstimulated cultures represented spontaneous background products of nonspecific cytokines. The higher frequency of IFNγ-SCs induced by PBMC cultures stimulated with memory antigens compared to background products indicated the production of virus-specific IFNγ-SCs. The average frequency of specific (memory antigen-stimulated) IFNγ-SCs in each group throughout the experimental period (0–42 days) is shown in the table below. Figure 13 , Figure 14 Forty-two days post-immunization, negligible IFNγ-producing cells were detected in the control group, and the presence of memory antigens in cell cultures was ignored. Twenty-eight days post-immunization, all vaccine groups produced high levels of spontaneously secreted IFNγ (range: 20–50 IFNγ-SC / 10). 6 The results showed a highly significant difference from the unstimulated group and the control group (P < 0.01), indicating that the recombinant bivalent porcine reproductive and respiratory syndrome vaccine can stimulate a specific regulatory immune response.

[0094] Example 10: Changes in the transcription of IL-17 and TGF-β genes in peripheral blood lymphocytes

[0095] Anticoagulated blood was collected at 0, 7, 14, 21, 28, 35, and 42 days post-immunization. Peripheral blood lymphocytes were isolated, and total RNA was extracted from the lymphocytes. The mRNA transcription levels of IL-17 and TGF-β in peripheral blood were detected by real-time quantitative RT-PCR. The reaction system (20L system) was as follows: 1.0 μL cDNA, 0.5 μL each of forward and reverse primers, 10.0 μL 2XSYBR® Premix Ex Taq™, and 8.0 μL RNase-free HO. The reaction program was: 94℃ for 3 min, 94℃ for 20 s, annealing at 20 s, 72℃ for 20 s, for 40 cycles.

[0096] The IL-17 mRNA transcription level peaked in the immunized group 14 days post-immunization, stimulating higher levels of the immune regulatory factor IL-17 in the immunized group compared to the control group (P<0.05). Subsequently, all groups gradually returned to normal levels. The TGF-β mRNA transcription level significantly increased in the immunized group except for a significant increase at 7 days post-immunization, showing a significant difference from the control group (P<0.05). Throughout the experiment, there was no statistically significant difference in TGF-β mRNA transcription levels among the immunized groups. At 42 days post-immunization, there were no significant differences in PBMC cytokine IL-17 and TGF-β transcription levels between all immunized groups and the control group (P>0.05). These results indicate that the recombinant bivalent porcine reproductive and respiratory syndrome vaccine can induce a relatively stronger cellular and non-specific immune response in a short period of time. Figures 15-16 ).

[0097] Example 11: T-cell typing detection after immunization in piglets

[0098] The number of CD3+, CD3+CD4+, and CD3+CD8+ positive cells and the CD4+ / CD8+ ratio were detected in 10,000 isolated PBMCs using flow cytometry.

[0099] 1. Reagent Preparation

[0100] Fluorescent washing buffer: PBS containing 5% fetal bovine serum. Introduce the fetal bovine serum into 450 ml of PBS, mix well, and bring the volume to 500 ml. Filter sterilize through a 0.22 μm filter membrane and store at -20°C for later use. Fluorescent preservation buffer: 100 ml of 0.15 M PBS pH 7.4, 2% glucose, 1% formaldehyde, 0.1% NaN3. Filter sterilize through a 0.22 μm filter membrane and store at -20°C for later use.

[0101] 2. Fractal Operation

[0102] Take 1×10 6Each porcine lymphocyte was transferred into a numbered 1.5ml centrifuge tube, washed with 1ml of fluorescent wash buffer, centrifuged at 1500rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in 100μl of fluorescent wash buffer. 1–5μl (the exact volume should be determined based on pre-experiment results or the manufacturer's recommended dosage) of APC-CD3 (approximately 0.5μg), AlexaFlupr-CD8 (approximately 0.125μg), and PE-CD4 (0.25μg) fluorescent antibodies were added (Note: it is best to protect the fluorescently labeled monoclonal antibodies from light when adding them). The mixture was thoroughly mixed and incubated at 4°C in the dark for 30 minutes. Four blank control tubes were prepared, each containing 2μl of PBS, APC-CD3, and AlexaFlupr-CD8 antibodies. Flupr-CD8 and PE-CD4 were washed twice with fluorescent washing buffer, 1 ml each time, and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded. The cells at the bottom of the tube were resuspended in 200 μl (200-500 μl) of fluorescent preservation buffer and analyzed by flow cytometry to detect the number of CD3+, CD3+CD8+, and CD3+CD4 positive cells in 10,000 cells (the cell count in each sample must reach 10). 6 (The sample should be stored for a short time and tested as soon as possible to prevent the green fluorescence signal from weakening).

[0103] 3 Results

[0104] The results of the cellular immune level test indicate that a certain cellular immune response can be induced 7 days after the first immunization, and the cellular immune response is enhanced after the second immunization.

[0105] 3.1 Changes in the proportion of CD3+ subsets in peripheral blood lymphocytes

[0106] From 14 to 35 days post-immunization, the proportion of CD3+ subsets in peripheral blood lymphocytes of some piglets in the immunized group was significantly higher than that in the control group (P<0.05). Before immunization and 42 days post-immunization, there was no significant difference in the proportion of CD3+ subsets in peripheral blood lymphocytes between the immunized group and the control group (P>0.05). Figure 17 ).

[0107] 3.2 Changes in the proportion of CD3+CD4+ subsets in peripheral blood lymphocytes

[0108] Before immunization, there was no significant difference in the proportion of CD3+CD4+ subsets in peripheral blood lymphocytes between the immunized group and the control group (P>0.05). After immunization, the proportion of CD3+CD4+ subsets in peripheral blood lymphocytes of the vast majority of piglets in the immunized group was significantly different from that in the control group (P<0.05). Figure 18 ).

[0109] 3.3 Changes in the proportion of CD3+CD8+ subsets in peripheral blood lymphocytes

[0110] Before immunization, there was no significant difference in the proportion of CD3+CD8+ subsets in peripheral blood lymphocytes between the immunized group and the control group (P>0.05). After immunization, the proportion of CD3+CD8+ subsets in peripheral blood lymphocytes of most piglets in the immunized group was significantly higher than that in the control group (P<0.05). Figure 19 ).

Claims

1. A recombinant porcine reproductive and respiratory syndrome (PRRS) bivalent vaccine fusion protein, characterized in that, Its amino acid sequence is shown in SEQ ID No. 2 in the sequence listing.

2. A nucleic acid molecule encoding the fusion protein of claim 1, characterized in that, Its gene sequence is shown in SEQ ID No. 1 in the sequence listing.

3. A carrier containing the nucleic acid molecule of claim 2.

4. A host cell comprising the vector of claim 3.

5. A recombinant bivalent vaccine for porcine reproductive and respiratory syndrome, characterized in that, It contains the fusion protein as described in claim 1.

6. A preparation method, characterized in that, The preparation process for the recombinant porcine reproductive and respiratory syndrome bivalent vaccine as described in claim 5 includes, but is not limited to, the construction of prokaryotic expression and eukaryotic expression fermentation process systems, the establishment of purification processes such as affinity, reverse, hydrophobic, and ion exchange, and the establishment of emulsification processes.

7. The use of the recombinant bivalent porcine reproductive and respiratory syndrome vaccine according to claim 5 in the prevention of porcine reproductive and respiratory syndrome.

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