Screening and application of high pathogenic porcine reproductive and respiratory syndrome virus subunit vaccine

Abstract

This invention discloses the screening and application of subunit vaccines against highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV). The problem this invention aims to solve is how to screen safe and highly effective subunit vaccines against PRRSV. To address this problem, this invention provides various protein compositions for the prevention of PRRSV, and has screened out the optimal combination. High levels of specific antibodies obtained after immunizing New Zealand White rabbits and piglets with the subunit vaccine prepared using the protein combination method of this invention can effectively neutralize highly pathogenic PRRSV in vitro, providing effective protection for immunized pigs and effectively preventing PRRSV.

Description

Technical Field

[0001] This invention belongs to the field of animal vaccines and veterinary biological products technology, specifically relating to the screening and application of subunit vaccines against highly pathogenic porcine reproductive and respiratory syndrome virus. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a serious transmissible disease of pigs caused by Porcine reproductive and respiratory syndrome virus (PRRSV). The virus was isolated in my country in 1996, confirming the presence of the disease in the country. Pregnant sows infected with PRRSV mainly exhibit reproductive disorders, such as premature birth, abortion, stillbirth, and mummified fetuses in late pregnancy. Weaned piglets infected with PRRSV show respiratory distress, arthritis, diarrhea, and other clinical symptoms. PRRS severely restricts the sustainable development of my country's pig industry and causes incalculable losses to the pig farming sector.

[0003] PRRSV belongs to the Arteritisviridae family and the Arteritisvirus genus. It is an enveloped, single-stranded, positive-sense small RNA virus with a genome length of approximately 15,000 bp and 11 open reading frames. PRRSV pp1a undergoes nine cleavages by proteases, resulting in 10 viral non-structural proteins. NSP1α inhibits IFN-β production through its C-terminal extended region. NSP1β plays a crucial role in PRRSV replication and immune evasion; it not only promotes self-replication through interaction with Ploy(C)-binding protein 2 but also inhibits the production of type I interferon and TNF-α. NSP2 is the largest and most mutable non-structural protein in PRRSV, possessing four regions: a structural domain, a hypervariable region, a transmembrane region, and a C-terminal tail. It exhibits papain-like protease 2 activity and antagonizes type I interferon. NSP3 and NSP5 are two transmembrane proteins involved in membrane rearrangement. NSP4 possesses protease activity and can inhibit innate immune responses. NSP6 has an extremely small molecular weight, and its role in viral infection is currently unclear. NSP7 is cleaved into NSP7α and NSP7β by viral proteases, and NSP7α can interact with NSP9. NSP8 possesses the N-terminal domain of NSP9. The NSP9 sequence is highly conserved, with its C-terminus exhibiting an RNA-dependent RNA polymerase structure, playing a crucial role in viral pathogenicity. It also contains T-cell recognition epitopes, which is beneficial for future vaccine development. NSP10 is the first crystal structure of PRRSV, containing multiple domains: an N-terminal zinc-binding domain, an 1B domain, and a helicase core domain. Its unique C-terminal structure presents a novel open state, playing a key role in helicase activity. NSP11 possesses endonuclease activity and can inhibit the production of type I interferon; the function of NSP12 is also unclear.

[0004] ORF2a encodes GP2a, which plays a crucial role in viral adsorption. ORF3 encodes GP3, which induces neutralizing antibodies during PRRSV infection. ORF4 encodes GP4, with its neutralizing epitope at amino acids 59-67. GP5, encoded by ORF5, possesses excellent antigenicity and is the primary antigen for neutralizing antibody production; therefore, GP5 has been a key protein in vaccine development in recent years. The M protein, encoded by ORF6, can form a GP5-M heterodimer with GP5 in the endoplasmic reticulum, thereby invading viral particles, causing disulfide bond cleavage, participating in PRRSV assembly and budding, and exhibiting good immunogenicity.

[0005] It has been confirmed that inactivated vaccines for PRRS have certain safety and efficacy deficiencies. While live attenuated vaccines can provide better homologous protection, they suffer from numerous safety issues such as virulence reversion, recombinant infections, and high variability. Research on subunit vaccines for PRRS is still in the exploratory stage. Among existing technologies, CN 113563431 B successfully constructed two recombinant baculoviruses, BMP541 and FMP542, that solublely express the PRRSV GP5 and M protein genes, laying the foundation for the development of PRRSV subunit vaccines. CN 109385435 B linked the artificially synthesized GP5 and M tandem sequence GP5M to the pBAC5 plasmid backbone to obtain a recombinant baculovirus Ac-PRRSVGP5M expressing the protein for subunit vaccine preparation, showing promising vaccine prospects. However, due to the complex immunosuppressive mechanism of PRRSV, and the fact that most current PRRS subunit vaccines are based on GP5 and M proteins, they cannot provide complete protection. Therefore, exploring and developing safer and more effective PRRS subunit vaccines has become a key measure for the prevention and control of PRRS. Summary of the Invention

[0006] To address the problems existing in the prior art, the purpose of this invention is to provide a screening and application method for a highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) subunit vaccine, providing an effective immunogen for the development of PRRSV vaccines, enriching the types of PRRSV immunogens, and thus better improving the protection rate against animals infected with porcine reproductive and respiratory syndrome virus.

[0007] To achieve the above objectives, the present invention mainly includes the following:

[0008] In a first aspect, the present invention provides a protein composition for preventing PRRS, the protein composition comprising at least one of tGP2, tGP3, tGP4, tGP5, tM, NSP1αmt, NSP1βmt, NSP2mt, NSP4mt, NSP7α, NSP7β, NSP8, NSP9, NSP10, NSP11mt, and NSP12, the amino acid sequences of which are shown in SEQ ID NO:1-SEQ ID NO:16 respectively.

[0009] The protein composition is a combination of tGP2, tGP3, tGP4, tGP5 and tM with any one or two of NSP1αmt, NSP1βmt, NSP2mt, NSP4mt, NSP7α, NSP7β, NSP8, NSP9, NSP10, NSP11mt and NSP12.

[0010] The protein composition is a combination of tGP2, tGP3, tGP4, tGP5, tM, NSP4mt, and NSP11mt.

[0011] The tGP2, tGP3, tGP4, tGP5, tM, NSP1αmt, NSP1βmt, NSP2mt, NSP4mt, and NSP11mt are obtained by mutations or deletions of proteins GP2, GP3, GP4, GP5, M, NSP1α, NSP1β, NSP2, NSP4, and NSP11, respectively.

[0012] GP2, GP3, GP4, GP5, and M proteins cannot be expressed in their full length in prokaryotes; however, expression can be achieved by truncating them by removing their signal peptides or highly hydrophobic regions. NSP1α, NSP1β, NSP2, NSP4, and NSP11 contain sites that suppress innate immune responses; mutagenesis of these sites can eliminate their suppressive function. NSP7α, NSP7β, NSP8, NSP9, NSP10, NSP11mt, and NSP12 proteins are readily expressed in prokaryotes and do not contain sites that suppress immune responses; therefore, no further mutation or truncation is necessary.

[0013] Specifically, tGP2 (amino acids 56-205) is a truncated protein of the PRRSV structural protein GP2, that is, the GP2 protein with the transmembrane region removed, and its amino acid sequence is shown in SEQ ID NO:1.

[0014] The tGP3 (amino acids 22-255) is a truncated protein of the PRRSV structural protein GP3, that is, the GP3 protein with the transmembrane region removed, and its amino acid sequence is shown in SEQ ID NO:2.

[0015] The tGP4 (amino acids 31-145) is a truncated protein of the PRRSV structural protein GP4, that is, the GP4 protein with the transmembrane region removed, and its amino acid sequence is shown in SEQ ID NO:3.

[0016] The tGP5 (amino acids 127-200) is a truncated protein of the PRRSV structural protein GP5, that is, the GP5 protein with the transmembrane region removed, and its amino acid sequence is shown in SEQ ID NO:4.

[0017] The tM (amino acids 96-175) is a truncated protein of the PRRSV structural protein M, that is, the M protein with the transmembrane region removed, and its amino acid sequence is shown in SEQ ID NO:5.

[0018] The NSP1αmt is the PRRSV non-structural protein NSP1α whose amino acid position 176 is mutated from phenylalanine to alanine, and its amino acid sequence is shown in SEQ ID NO:6.

[0019] The NSP1βmt is a mutant protein of the PRRSV nonstructural protein NSP1β, in which amino acid 124 is mutated from lysine to alanine and amino acid 128 is mutated from arginine to alanine, as shown in SEQ ID NO:7.

[0020] The NSP2mt is the PRRSV non-structural protein NSP2 with the transmembrane region removed and amino acids 54 (cysteine) to 123 (histidine) deleted. Its amino acid sequence is shown in SEQ ID NO:8.

[0021] The NSP4mt is the PRRSV non-structural protein NSP4, in which amino acid 39 is mutated from histidine to alanine, amino acid 64 is mutated from aspartic acid to alanine, and amino acid 118 is mutated from serine to alanine. Its amino acid sequence is shown in SEQ ID NO:9.

[0022] The NSP7α is a PRRSV nonstructural protein, and its amino acid sequence is shown in SEQ ID NO:10.

[0023] The NSP7β is a PRRSV nonstructural protein, and its amino acid sequence is shown in SEQ ID NO:11.

[0024] The NSP8 is a PRRSV nonstructural protein, and its amino acid sequence is shown in SEQ ID NO:12.

[0025] The NSP9 is a PRRSV nonstructural protein, and its amino acid sequence is shown in SEQ ID NO:13.

[0026] The NSP10 is a PRRSV nonstructural protein, and its amino acid sequence is shown in SEQ ID NO:14.

[0027] The NSP11mt is the PRRSV nonstructural protein NSP11, in which amino acids 129 and 144 are mutated from histidine to alanine, and amino acid 173 is mutated from lysine to alanine. Its amino acid sequence is shown in SEQ ID NO:15.

[0028] The NSP12 is a PRRSV nonstructural protein, and its amino acid sequence is shown in SEQ ID NO:16.

[0029] In a second aspect, the present invention provides the use of the above-described protein composition in the preparation of a vaccine for the prevention or treatment of porcine reproductive and respiratory syndrome.

[0030] The vaccine in question is a subunit vaccine.

[0031] In a third aspect, the present invention provides a subunit vaccine for the prevention and treatment of porcine reproductive and respiratory syndrome, wherein the subunit vaccine uses the above-mentioned protein composition as an immunogen or active ingredient.

[0032] The subunit vaccine also includes an adjuvant.

[0033] The adjuvant is one or more of the following: plant adjuvant, bacterial adjuvant, aluminum adjuvant, other inorganic adjuvant, and emulsion adjuvant.

[0034] In a fourth aspect, the present invention provides the use of the above-described protein composition in the preparation of products for epidemiological investigation of porcine reproductive and respiratory syndrome virus.

[0035] The beneficial effects of this invention are:

[0036] This invention provides a protein composition for preventing PRRS, and has screened out the optimal combination from it. Subunit vaccines are developed based on the protein composition, which can overcome the shortcomings of most existing PRRS subunit vaccines, which are based on GP5 and M proteins and cannot provide effective immunogens for the development of PRRSV vaccines. This invention enriches the types of PRRSV immunogens and thus better improves the protection rate against animals infected with highly pathogenic porcine reproductive and respiratory syndrome virus, laying an important foundation for the development of PRRSV subunit vaccines.

[0037] The high levels of specific antibodies obtained after immunizing New Zealand white rabbits and piglets with the subunit vaccine prepared using the protein composition of this invention can effectively neutralize highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) in vitro, thus achieving effective protection against animals infected with highly pathogenic PRRSV. The protein composition for preventing PRRS provided by this invention has good prospects for the development of subunit vaccines. Attached Figure Description

[0038] Figure 1 Figures show the validation of PRRSV viral protein expression. Figures A through P are SDS-PAGE images of NSP1αmt, NSP1βmt, NSP2mt, NSP4mt, NSP7α, NSP7β, NSP8, NSP9, NSP10, NSP11mt, NSP12, tGP2, tGP3, tGP4, tGP5, and tM proteins, respectively. Lanes 1, 3, and 5 represent uninduced bacterial culture; lanes 2, 4, and 6 represent induced bacterial culture.

[0039] Figure 2This is a validation image of PRRSV viral protein purification. Figures A through P show SDS-PAGE images of purified NSP1αmt, NSP1βmt, NSP2mt, NSP4mt, NSP7α, NSP7β, NSP8, NSP9, NSP10, NSP11mt, NSP12, tGP2, tGP3, tGP4, tGP5, and tM proteins, respectively. Lane 1: Bacterial supernatant before column pass-through; Lane 2: Flow-through buffer; Lane 3: Wash buffer; Lane 4: Protein elution buffer 1; Lane 5: Protein elution buffer 2; Lane 6: Protein elution buffer 3.

[0040] Figure 3 The titer of neutralizing antibodies in rabbit hyperimmune serum.

[0041] Figure 4 The purpose of this study was to verify the neutralizing effect of rabbit hyperimmune serum on PRRSV using a fluorescence method. The negative control was the fluorescence detection result of cells not inoculated with the recombinant virus, and the positive control was the fluorescence detection result of cells inoculated with the virus strain without antibody neutralization.

[0042] Figure 5 Body temperature was measured after the viral challenge.

[0043] Figure 6 Post-study viremia level testing. Detailed Implementation

[0044] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0045] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0046] All data analyses in the following examples were performed using Prism 9 statistical software. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used, with P>0.05 (ns) indicating no statistical difference, P<0.05 (*) indicating a statistical difference, P<0.01 (**) indicating a statistically significant difference, and P<0.001 (***) indicating an extremely significant difference.

[0047] The pET-28a-sumo vector in the following examples is a product of Nanjing Genscript Technology Co., Ltd.

[0048] The Freund's complete adjuvant and Freund's incomplete adjuvant in the following examples are products of Beijing Merck Co., Ltd.

[0049] Example 1: Expression and purification of PRRSV viral proteins

[0050] 1. Construction of prokaryotic expression plasmids

[0051] Based on the amino acid sequences of the PRRSV TA-12 (accession number: HQ416720) viral proteins NSP1α, NSP1β, NSP2, NSP4, NSP7α, NSP7β, NSP8, NSP9, NSP10, NSP11, NSP12, GP2, GP3, GP4, GP5, and M, molecular biology techniques were used to delete or mutate the amino acid sequences of NSP1α, NSP1β, NSP2, NSP4, NSP11, GP2, GP3, GP4, GP5, and M proteins. The recombinant plasmids were named pET-28a-sumo-NSP1αmt, pET-28a-sumo-NSP1βmt, and pET-28a-sumo-NSP1βmt, respectively. a-sumo-NSP2mt, pET-28a-sumo-NSP4mt, pET-28a-sumo-NSP7α, pET-28a-sumo-NSP7β, pET-28a-sumo-NSP8, pET-28a-sumo-NSP9, pET-28a-sumo -NSP10, pET-28a-sumo-NSP11mt, pET-28a-sumo-NSP12, pET-28a-tGP2, pET-28a-sumo-tGP3, pET-32a-tGP4, pET-28a-tGP5 and pET-28a-sumo-tM. The primer sequences are shown in Table 1.

[0052] Table 1: Summary Table of Primer Sequences

[0053]

[0054]

[0055] 2. Prokaryotic expression of recombinant viral proteins

[0056] Recombinant plasmids were transformed into Transetta competent cells and incubated overnight at 37°C on LB agar plates with the corresponding antibiotics. The next day, colonies were picked and inoculated into 1.5 mL centrifuge tubes, and 1 mL of LB medium containing 50 μg / mL kanamycin or 100 μg / mL ampicillin was added. The cells were incubated at 37°C and 220 rpm for 10 h. After incubation, the centrifuge tubes were removed, and 6 mL of LB medium containing 50 μg / mL kanamycin or 100 μg / mL ampicillin and 60 μL of the overnight culture were added to 15 mL centrifuge tubes. The cells were then incubated at 37°C and 220 rpm for approximately 3 h using a shaker. The OD value at 600 nm was measured using a spectrophotometer. When the OD value at 600 nm reached 0.4-0.6, the induction conditions were considered met. Aspirate 2 mL of bacterial culture, use 1 mL as a pre-induction control, and 1 mL to preserve the bacterial culture. Add 0.5 mM IPTG inducer to the remaining 4 mL and continue culturing in a shaker at 37°C for 4–5 hours. Harvest the bacterial culture.

[0057] 3. SDS-PAGE identification of recombinant viral proteins expressed in prokaryotes

[0058] After induction, 1 mL of the induced bacterial culture was transferred to a 1.5 mL centrifuge tube and centrifuged with 1 mL of the culture before induction at 3,000 rpm for 5 min. The supernatant was discarded, and 200 μL of PBS was added and vortexed. The mixture was then centrifuged at 3,000 rpm for 5 min. The supernatant was discarded, and the mixture was washed twice with PBS. 20 μL of 5× loading buffer was added, and the mixture was incubated in a 98°C water bath for 10 min. After incubation, the mixture was centrifuged at 12,000 rpm for 15 min. 10 μL of the supernatant was collected and SDS-PAGE was used to verify the induced expression. Figure 1 As shown.

[0059] 4. Large-scale induction of prokaryotic expression of recombinant viral proteins

[0060] Expand the bacterial culture from step 2 to 6 mL. Add the cultured bacterial culture to 400 mL of kanamycin or ampicillin-resistant liquid LB. Incubate at 37°C with shaking. When the OD600 reaches 0.4–0.6, add 0.5 mmol / L IPTG inducer and continue incubating at 37°C with shaking for 4–5 h. After induction, centrifuge at 8,000 rpm for 10 min and discard the supernatant. Add 10 mL of PBS, mix well by pipetting, centrifuge at 3,000 rpm for 5 min and discard the supernatant. Repeat three times.

[0061] 5. Purification of recombinant viral proteins expressed in prokaryotes

[0062] Verification showed that the recombinant viral proteins NSP2mt, NSP4mt, NSP7α, NSP7β, NSP8, and tGP3 were soluble proteins; while NSP1αmt, NSP1βmt, NSP9, NSP10, NSP11mt, NSP12, tGP2, tGP4, tGP5, and tM were insoluble proteins.

[0063] 5.1 Purification of Soluble Proteins

[0064] (1) After induction, the bacterial cells were resuspended in 8 mL of PBS and placed in an ultrasonic cell disruptor for sonication at 200 W for 2 seconds followed by a 4-second interval, for a total of 35 min per tube. After sonication, the bacterial culture was centrifuged at 12,000 rpm for 15 min, and the supernatant was retained.

[0065] (2) Add 1 mL of Ni-NTA purification medium to the protein purification column, let it stand, and wait for the purification medium to completely precipitate on the purification pad and form a layer. Then turn on the control switch and let the liquid flow out at a rate of 4 drops per second.

[0066] (3) Add one column volume of supernatant LE buffer to equilibrate the column, let it stand for 10 minutes, open the control valve, and let the equilibration liquid flow out slowly and evenly.

[0067] (4) Add the supernatant obtained after centrifugation to the pre-equilibrated purification column, mix well, and place in a shaker at 4°C for 30 min. After the binding time is up, drip the liquid from the column at a rate of 6 drops per second and collect the dripping liquid in a centrifuge tube of appropriate size. Repeat this step 3 times to ensure that the purification medium and protein are fully bound.

[0068] (5) Add 5 column volumes of supernatant protein washing buffer to remove impurities. Add washing buffer with imidazole concentrations of 10 mM, 40 mM, 70 mM and 100 mM in sequence, with the drop rate controlled at 4 s / drop.

[0069] (6) Add 2 mL of 250 mM imidazole supernatant protein elution buffer to the protein purification column, place it on a shaker at 4 °C for 30 min to allow the elution buffer to fully bind with the target protein. Open the valve and allow the elution to flow out at a rate of 6 drops per second. Collect the elution buffer in a centrifuge tube and repeat the above steps 3 times.

[0070] 5.2 Purification of Insoluble Proteins

[0071] (1) Resuspend the inclusion body protein cells in 8 mL of PBS, place them in an ultrasonic cell disruptor, and sonicate at 200 W for 2 seconds followed by a 4-second interval, for a total of 35 min per tube. After sonication, centrifuge the bacterial culture at 12,000 rpm for 15 min and discard the supernatant. Add 2 mL of inclusion body LE buffer to the centrifuge tube to resuspend the precipitate, and place the tube in a 4°C freezer to lyse the inclusion bodies overnight. The next day, centrifuge the lysed inclusion bodies at 12,000 rpm for 15 min and retain the supernatant.

[0072] (2) Add 1 mL of Ni-NTA purification medium to the protein purification column, let it stand, and wait for the purification medium to completely precipitate on the purification pad and form a layer. Then turn on the control switch and let the liquid flow out at a rate of 4 drops per second.

[0073] (3) Add an inclusion body LE buffer equal to the column volume to balance the column, let it stand for 10 minutes, open the control valve, and let the balance liquid flow out slowly and evenly.

[0074] (4) Add the supernatant obtained after centrifugation to the pre-equilibrated purification column, mix well, and place in a shaker at 4°C for 30 min. After the binding time is up, drip the liquid from the column at a rate of 6 drops per second and collect the dripping liquid in a centrifuge tube of appropriate size. Repeat this step 3 times to ensure that the purification medium and protein are fully bound.

[0075] (5) Add 5 column volumes of protein washing buffer to remove impurities. Add washing buffer with imidazole concentrations of 10 mM, 50 mM, and 70 mM in sequence, with the drop rate controlled at 4 s / drop.

[0076] (6) Add 2 mL of inclusion body protein elution buffer with an imidazole concentration of 250 mM to the column for protein purification, and place it on a shaker at 4 °C for 30 min to allow the elution buffer to fully bind with the target protein. Open the valve and drop the column at a rate of 6 s / drop, collect the elution buffer in a centrifuge tube, and repeat the above steps 3 times.

[0077] The liquid collected at each step of the protein purification process was validated using SDS-PAGE, such as... Figure 2 As shown, the successfully validated protein was measured at absorbance at A260 and A280 using a spectrophotometer. Protein concentration = 1.45 × A280 - 0.74 × A260.

[0078] Example 2: Preparation of rabbit hyperimmune serum and detection of antibodies in the serum

[0079] 1. Animal experiment grouping and protein dosage

[0080] Twenty-two female 3-month-old New Zealand white rabbits were purchased and randomly divided into 11 groups, with two rabbits in each group. The Ctrl group was injected with 1.2 mg of saline solution, while the other rabbits were injected with a total of 1.2 mg of immune protein each. The grouping is shown in Table 2.

[0081] Table 2 Animal Experiment Groups and Protein Dosage

[0082]

[0083]

[0084] Note: tGPs refers to tGP2, tGP3, tGP4, and tGP5; NSP1α / 1βmt refers to NSP1αmt+NSP1βmt; and NSP7α / 7β refers to NSP7α+NSP7β.

[0085] 2. Protein emulsification and immunity

[0086] (1) Before the first immunization, blood was drawn from the marginal ear vein as a negative control for subsequent tests.

[0087] (2) After the protein concentration is detected by spectrophotometer, the amount of protein used in each group is mixed evenly according to Table 2 and mixed with complete Freund's adjuvant at a volume ratio of 1:1 to emulsify the protein.

[0088] (3) The fully emulsified protein was injected subcutaneously at multiple points on the back of the neck for immunization, and the injection sites were disinfected.

[0089] (4) Before the second and third immunizations, blood is collected from the marginal ear vein and the serum is preserved. The dosage of protein for the second and third immunizations is the same as that for the first immunization, but it needs to be emulsified with incomplete Freund's adjuvant at a 1:1 ratio. The interval between each immunization is 14 days.

[0090] (5) If the antibody level is lower than 1:10 after the third immunization 5 A fourth booster immunization can be administered, following the same method as the second and third immunizations. Seven days after immunization, serum antibody levels should be tested again. If the antibody levels meet the requirements, blood should be drawn from the heart immediately. The collected blood should first be placed in a 37°C incubator for 30 minutes, then placed in a 4°C refrigerator overnight. The next day, the centrifuge tubes should be placed in a centrifuge and centrifuged at 6,000 rpm for 10 minutes. The supernatant should be collected in 1.5 mL centrifuge tubes and stored at -80°C. The collected serum is the hyperimmune serum.

[0091] 3. Detection of antibody levels in rabbit hyperimmune serum

[0092] The serum antibody levels were detected using an indirect ELISA method.

[0093] (1) Antigen coating

[0094] Dilute tGP2, tGP3, tGP4, tGP5, tM, NSP1αmt, NSP1βmt, NSP4mt, NSP11mt, NSP2mt, NSP7α, NSP7β, NSP8, NSP9, NSP10, and NSP12 to 300 ng per well using 1×PBS buffer, and then plate 100 μL of each solution into the wells. Incubate overnight at 4°C.

[0095] (2) Closed

[0096] On the second day, the plate was washed 5 times with PBS'T, patted dry, and then 100 μL of 2.5% skim milk powder was added to each well for sealing. The plate was then incubated at 37°C for 1 hour.

[0097] (3) Incubation of primary antibody

[0098] The serum to be tested was diluted 1:10 with 1×PBS buffer. 2 1:10 3 1:10 4 1:10 5 1:10 6 1:10 7 Proportional dilution. Add 100 μL of diluted serum to each well sequentially, with two replicates for each dilution, and incubate at 37°C for 1 hour.

[0099] After sealing, discard the skim milk powder, wash the plate 5 times with PBS'T, and then pat dry.

[0100] (4) Incubation of secondary antibodies

[0101] After the primary antibody incubation was complete, discard the liquid, wash the plate 5 times with PBS′T, and then blot dry. Dilute HRP goat anti-rabbit IgG (H+L) 1:5,000 with 1×PBS buffer, add 100 μL to each well, and incubate at 37°C for 1 h.

[0102] (5) TMB colorimetric reaction

[0103] After the secondary antibody incubation is complete, discard the liquid, wash the plate 5 times with PBS'T, and then pat dry. Add 100 μL of TMB substrate to each well in a dark environment at room temperature and react for 15 min.

[0104] (6) Termination of reaction

[0105] After adding TMB substrate and reacting for 15 min, add 100 μL of ELISA stop solution to each well to terminate the reaction.

[0106] (7) Read OD value

[0107] After termination, read the absorbance of the ELISA plate on the microplate reader. Set the OD value to the absorbance at 450 nm. The final result is used to determine the serum antibody level according to the formula P / N>2 (P is the average absorbance of the test sample at 450 nm, and N is the average absorbance of the negative sample at OD450). The results are shown in Tables 3-8.

[0108] Table 3 tGP2 antibody levels

[0109]

[0110] Table 4 tGP3 antibody levels

[0111]

[0112]

[0113] Table 5 tGP4 antibody levels

[0114]

[0115]

[0116] Table 6 tGP5 antibody levels

[0117]

[0118] Table 7 tM antibody levels

[0119]

[0120] Table 8. Results of Non-structural Protein Antibody Detection: NSP1αmt Antibody Level

[0121]

[0122] NSP1βmt antibody levels

[0123]

[0124]

[0125] NSP4mt antibody level

[0126]

[0127] NSP11mt antibody level

[0128]

[0129] NSP2mt antibody levels

[0130]

[0131] NSP7α antibody level

[0132]

[0133] NSP7β antibody levels

[0134]

[0135] NSP8 antibody levels

[0136]

[0137] NSP9 antibody levels

[0138]

[0139] NSP10 antibody levels

[0140]

[0141] NSP12 antibody levels

[0142]

[0143] The results of antibody level detection in rabbit hyperimmune serum showed that the antibody levels produced by different groups of rabbits after induction with viral proteins were different. The different antibody levels produced by different groups reflected the different neutralizing antibody production capabilities after induction with different combinations. Surprisingly, induction with multiple viral proteins did not affect the production of individual antibodies in the rabbits, which laid the foundation for subsequent neutralizing antibody detection.

[0144] Example 3: Verification of the neutralizing effect of rabbit hyperimmune serum on the virus

[0145] 1. Virus neutralization experiment

[0146] (1) This experiment used GFP-TA-12 recombinant virus (the GFP exogenous gene was inserted into the existing TA-12 strain, which is the existing conventional recombinant virus), and the amount of virus inoculated into each well was uniformly 0.1 MOI.

[0147] (2) When passaged Marc-145 cells, the cells were diluted to 2.0 × 10⁻⁶. 5 Add 100 μL per well in a 96-well plate.

[0148] (3) The rabbit hyperimmune serum prepared in each treatment group in Example 2 was inactivated in a water bath at 56°C for 30 minutes, and the cells were inverted several times every 8 minutes to ensure uniform heating.

[0149] (4) Dilute the inactivated serum with DMEM in a 2-fold serial dilution, and perform serial dilutions to 2-fold. 1-2 6 The diluted serum was mixed with an equal volume of virus solution and incubated at 37°C for 1 hour.

[0150] (5) Transfer the incubated mixture to a 96-well plate of Marc-145 cells, 100 μL per well, with 4 replicates for each dilution, and incubate at 37°C and 5% CO2 for 1 h.

[0151] (6) After 1 hour, the mixture was aspirated, and DMEM maintenance solution with 2% serum was added. The mixture was then cultured at 37°C and 5% CO2.

[0152] (7) After approximately 2 days, once the cell state has stabilized, calculate the neutralization titer using the Karber method. The neutralizing antibody titer results are as follows: Figure 3 As shown, the detection results indicate that NSP1αmt + NSP1βmt, NSP4mt, and NSP11mt can significantly induce the production of neutralizing antibodies. NSP2mt and NSP12mt inhibit the production of neutralizing antibodies induced by other viral proteins. The groups with the most significant effects in inducing neutralizing antibodies were selected for fluorescence and cytopathic effects observation under a fluorescence microscope. The fluorescence results are shown below. Figure 4 As shown.

[0153] 2. Experimental Results

[0154] Upon testing, when rabbit hyperimmune serum was diluted 64-fold, the tGPs+tM+NSP1α / 1βmt, tGPs+tM+NSP4mt, and tGPs+tM+NSP11mt groups still produced high levels of neutralizing antibodies, with titers of 1:58, 1:59, and 1:58, respectively; the neutralizing titer of the tGPs+tM group was 1:17. Fluorescence results showed that the tGPs+tM+NSP1α / 1βmt, tGPs+tM+NSP4mt, and tGPs+tM+NSP11mt groups, respectively, produced high levels of neutralizing antibodies at 2... -4 2 -5 and 2 -4 The virus can be completely neutralized at dilution ratios. These results indicate that mixing mutants of non-structural proteins NSP1αmt, NSP1βmt, NSP4mt, or NSP11mt into the structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV) can significantly induce the production of neutralizing antibodies, with the addition of NSP4mt or NSP11mt showing the most significant effect.

[0155] Example 4: Neutralizing effect of different protein combinations on viruses in rabbit hyperimmune serum

[0156] Based on the experimental results of Examples 2 and 3, in order to investigate the neutralizing effect of different protein combinations on the virus, the following experiment was further designed, setting up experimental groups and comparative examples 1-3, and rabbits were immunized according to the method in Example 2 to obtain hyperimmune serum, and the virus neutralizing antibody titer in rabbit serum was determined according to the method described in Example 3.

[0157] The specific details of the injected protein and dosage for each of the experimental and control groups (1-3) are as follows:

[0158] Experimental group: injected with tGPs+tM+NSP4mt+NSP11mt, 172ug of each protein.

[0159] Comparative Example 1: tGPs+tM were injected, with 240ug injected for each protein.

[0160] Comparative Example 2: NSP4mt was injected at a dose of 1.2mg.

[0161] Comparative Example 3: NSP11mt was injected at a dose of 1.2 mg.

[0162] The results showed that the neutralizing antibody titer was 1:63 when rabbit serum was injected with the tGPs+tM+NSP4mt+NSP11mt protein combination, 1:17 when tGPs+tM protein combination was injected, 1:23 when NSP4mt protein was injected, and 1:21 when NSP11mt protein was injected. These results indicate that the neutralizing antibody titer obtained by immunizing rabbits in the experimental group was significantly higher than that in the control groups 1-3.

[0163] Example 5: Study on the immunoprotective effect of tGPs+tM+NSP4mt+NSP11mt protein composition against porcine PRRSV.

[0164] 1. Experimental Methods

[0165] The tGPs+tM+NSP4mt+NSP11mt protein composition with the best immune effect in Example 4 was further tested in pigs to detect its immune effect.

[0166] This embodiment included an immunization group and a control group. Six 30-day-old Large White pigs were divided into two groups of three. In the immunization group, each pig received an intramuscular injection of tGPs+tM+NSP4mt+NSP11mt, with a total protein content of 1.0 mg and each protein injected at 0.143 mg. The second immunization was repeated three weeks after the first intramuscular injection. Three weeks after the second immunization, the pigs were challenged with a 5×10 mg intramuscular injection per pig. 5 TCID 50TA-12 toxin / mL. The control group did not receive immunization but were directly challenged with the same treatment as the immunization group.

[0167] 2. Experimental Results

[0168] For 16 consecutive days starting from the day of the challenge, clinical symptoms were observed in both the immunized and control groups. The results showed that one pig in the immunized group had runny nose, while all three pigs in the control group had cough, runny nose, conjunctivitis, and difficulty breathing. One pig died on the 9th and 11th days after the challenge.

[0169] The body temperature of pigs in both the immunized and control groups was measured for 16 consecutive days starting from the day of the viral challenge. The average temperature of each group was recorded, and the results are as follows: Figure 5 As shown.

[0170] Serum from the anterior vena cava of pigs in the immunized and control groups was collected before and at 3, 7, 10, and 13 days after viral challenge. Viremia levels in each group were measured using quantitative real-time fluorescence (dye method), and the average values ​​were calculated. The results are shown below. Figure 6 As shown.

[0171] Based on comprehensive observation of clinical symptoms, body temperature measurement, and quantitative fluorescence detection of viremia in porcine hyperimmune serum, it can be concluded that immunization of pigs with the protein composition provided in this invention (tGPs+tM+NSP4mt+NSP11mt) followed by viral challenge resulted in good resistance to highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV). The development of subunit vaccines using this protein composition also demonstrates good protective effects against porcine herds.

[0172] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A protein composition for preventing PRRS, characterized in that, The protein composition comprises a combination of tGP2, tGP3, tGP4, tGP5, tM, NSP4mt, and NSP11mt. The amino acid sequences of tGP2, tGP3, tGP4, and tGP5 are shown in SEQ ID NO:1-SEQ ID NO:4, respectively; the amino acid sequence of tM is shown in SEQ ID NO:5; the amino acid sequence of NSP4mt is shown in SEQ ID NO:9; and the amino acid sequence of NSP11mt is shown in SEQ ID NO:

15.

2. The use of the protein composition according to claim 1 in the preparation of a vaccine for the prevention or treatment of porcine reproductive and respiratory syndrome, characterized in that, The vaccine in question is a subunit vaccine.

3. A subunit vaccine for the prevention and treatment of porcine reproductive and respiratory syndrome, characterized in that, The subunit vaccine uses the protein composition of claim 1 as an immunogen or active ingredient.

4. The subunit vaccine according to claim 3, characterized in that, The subunit vaccine also includes an adjuvant, which is one or more of plant adjuvants, bacterial adjuvants, aluminum adjuvants, and emulsion adjuvants.

5. The use of the protein composition of claim 1 in the preparation of products for the epidemiological investigation of porcine reproductive and respiratory syndrome virus.

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