NADC30, NADC34 and GP5 immune composition and application thereof

Abstract

The invention relates to the technical field of immune compositions, in particular to an NADC30, NADC34 and GP5 immune composition and application thereof. The immune composition comprises an NADC30 pedigree inactivated strain of the PRRSV, an NADC34 pedigree inactivated strain of the PRRSV and a GP5 protein of the PRRSV. The immune composition of NADC30, NADC34 and GP5 is obtained through research, has a remarkably high immune protection effect on pigs, can effectively reduce the morbidity of the porcine reproductive and respiratory syndrome and reduce the cost of the pig raising industry, and has important value in the pig raising industry.

Description

Technical Field

[0001] The present invention relates to the field of immune combination technology, and in particular to a NADC30, NADC34 and GP5 immune combination and application thereof. Background Art

[0002] Porcine Reproductive and Respiratory Syndrome (PRRS) is a disease mainly caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). Its main symptoms include abortion, premature birth, weak piglets or stillbirth in sows, breathing difficulties, coughing, fever in piglets and young pigs, slow growth rate of infected pigs, and reduced feed conversion rate. PRRS has caused serious losses to the pig industry, so it is urgent to develop vaccines against PRRS.

[0003] There are currently a variety of immunization methods for porcine reproductive and respiratory syndrome, such as attenuated vaccines developed using attenuated strains of porcine reproductive and respiratory syndrome virus, inactivated vaccines for porcine reproductive and respiratory syndrome virus, display of porcine reproductive and respiratory syndrome virus GP5 protein on the surface of brewer's yeast, or constructing porcine reproductive and respiratory syndrome virus GP5 protein into expression vectors and then expressing it in eukaryotic cells. However, attenuated vaccines have the risk of reversion to strong virulence, and inactivated vaccines have poor protection. Existing commercially available vaccines are difficult to provide effective cross-protection against multiple prevalent strains. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a NADC30, NADC34 and GP5 immune composition and its application. The immunogenicity to PRRSV is significantly improved by the synergistic cooperation between the PRRSV strains of the NADC30 and NADC34 lineages and the GP5 protein.

[0005] In a first aspect, the present invention provides an immune composition, comprising: an inactivated strain of the NADC30 lineage of PRRSV, an inactivated strain of the NADC34 lineage of PRRSV and the GP5 protein of PRRSV; the inactivated strain of the NADC30 lineage of PRRSV is porcine reproductive and respiratory syndrome virus KQZD30, and the inactivated strain of the NADC34 lineage of PRRSV is porcine reproductive and respiratory syndrome virus KQZD34.

[0006] Porcine reproductive and respiratory syndrome virus KQZD30 and porcine reproductive and respiratory syndrome virus KQZD34 are disclosed in GenBank, and their accession numbers are KQZD34 NSP2 PQ569555 and KQZD30 NSP2 PQ569556 .

[0007] Furthermore, the GP5 protein of PRRSV is derived from the NADC30 lineage virus strain of PRRSV; preferably, the GP5 protein of PRRSV comprises the amino acid sequence shown in SEQ ID NO.1.

[0008] SEQ ID NO.1: MNNSSSHLQLIYNLTICELNGTDWLDKNFDWGGGGSNCMSWRYSCTRYTNFLLDTKGRLYRWRSPVIIEKGGKVDVGGHLIDLKRVVLDGSAATPLTKISAEQWGRPHHHHHHH Furthermore, the volume ratio of the inactivated strain of the NADC30 lineage of PRRSV, the inactivated strain of the NADC34 lineage of PRRSV and the GP5 protein of PRRSV is 1:(0.5-2):(0.5-4); the virus titer of the inactivated strain of the NADC30 lineage of PRRSV is not less than 8 log 10 TCID 50 / ml; the virus titer of the NADC34 lineage inactivated strain of PRRSV is not less than 8log 10 TCID 50 / ml; the concentration of the GP5 protein of PRRSV is not less than 0.1 mg / ml.

[0009] The NADC30 lineage strain of PRRSV and the NADC34 lineage strain of PRRSV in the present invention are obtained by culture and preparation based on Marc145 cells.

[0010] Conventional Marc145 cell culture adopts a two-dimensional single cell layer adherent production method. During industrial scale-up production, it is limited by its adherent performance, and the digestion process also increases the complexity of the process, production time and cost, and the quality is unstable and there are batch differences. Therefore, the present invention domesticates the adherent growth of Marc145 cells into suspended growth.

[0011] The present invention uses the SF1001 culture medium of Wuhan Punosai Life Science Co., Ltd. to directly adapt the adherent Marc145 cells to serum-free suspension. After 90 days of domestication, the Marc145 cells growing in suspension are successfully obtained, but the growth rate is low and cannot meet the needs of large-scale amplification of PRRSV. Therefore, the serum-free culture medium is further optimized, and amino acids, lipids, trace elements, and vitamins are supplemented to better support the growth and maintenance of Marc145 cells.

[0012] The present invention utilizes lentivirus to transduce Marc145, so that Marc145 in a suspended state can stably express receptors CD163-M2 and Rab1a, thereby being susceptible and conducive to the proliferation of NADC30 and NADC34.

[0013] The GP5 protein of PRRSV in the present invention is expressed by an E. coli expression system. The present invention optimizes the GP5 coding sequence, introduces a cspA promoter and a lytic tag, and allows the target protein GP5 to be fully expressed in the supernatant, thereby solving the problem of inclusion bodies and facilitating large-scale preparation.

[0014] In a second aspect, the present invention provides a vaccine comprising the aforementioned immune composition and a vaccine adjuvant.

[0015] Furthermore, the vaccine adjuvant includes: one or more of F38 adjuvant, aluminum salt adjuvant, oil emulsion or particulate adjuvant; the volume ratio of the immune composition to the vaccine adjuvant is (2~6):1.

[0016] In a third aspect, the present invention provides the use of the immune composition in the preparation of a medicament for preventing and treating porcine reproductive and respiratory syndrome.

[0017] The present invention further provides the use of the immune composition in improving the immunogenicity of drugs to porcine reproductive and respiratory syndrome virus.

[0018] The present invention has the following beneficial effects: The present invention has discovered an immunogenic combination, which is obtained by combining an inactivated strain of the NADC30 lineage of PRRSV, an inactivated strain of the NADC34 lineage of PRRSV and the GP5 protein of PRRSV, and has a significantly better protective effect against PRRSV, and there is no antagonism or additional adverse effects between the three. The NADC30, NADC34 and GP5 immune composition provided by the present invention can effectively improve the resistance of pigs to porcine reproductive and respiratory syndrome virus, and has a high economic value in the pig farming industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is the genetic evolution tree of the Nsp2 gene of the PRRSV isolated strain provided in Example 1 of the present invention.

[0021] Figure 2 It is a PRRSV cytopathic effect diagram provided in Example 1 of the present invention; wherein A: Marc145; B: cytopathic effect-NADC30 strain (80-69); B: cytopathic effect-NADC34 strain (72-35).

[0022] Figure 3 This is a statistical graph of virus titer in serum after challenge provided in Example 1 of the present invention.

[0023] Figure 4 This is a diagram of cell density and viability during the suspension adaptation process of Marc145 provided in Example 1 of the present invention.

[0024] Figure 5 This is a graph showing the cell density and survival rate of Marc145 in opti-SF1001 culture medium provided in Example 1 of the present invention.

[0025] Figure 6 The mRNA levels of CD163-M2 and Rab1a in different cells provided in Example 1 of the present invention.

[0026] Figure 7 This is the difference in titer of different Marc145 cells after inoculation provided in Example 1 of the present invention.

[0027] Figure 8 This is a diagram of the test expression results of the recombinant protein GP5 provided in Example 1 of the present invention; wherein M: protein marker; 1: whole bacteria after induction; 2: supernatant after disruption; 3: precipitate after disruption.

[0028] Fig. 9 This is the purification result of the recombinant protein GP5 Ni Focurose 6FF (IMAC) provided in Example 1 of the present invention; wherein M: protein marker; 1: stock solution; 2: flow-through; 3: wash 1; 4: wash 2; 5: elution 1; 6: elution 2.

[0029] Fig.10 This is a graph of KQZD30 viral load in serum samples provided in Example 2 of the present invention.

[0030] Fig.11 This is a graph of KQZD34 viral load in serum samples provided in Example 2 of the present invention.

[0031] Fig.12 This is a graph showing the growth and decline of blue ear ELISA antibodies in piglets after immunization provided in Example 2 of the present invention.

[0032] Fig.13 This is a statistical distribution diagram of the neutralizing antibody titer against KQZD30 provided in Example 2 of the present invention.

[0033] Fig.14 This is a statistical distribution diagram of the neutralizing antibody titer against KQZD34 provided in Example 2 of the present invention.

[0034] Fig.15 This is a graph of KQZD30 viral load in serum samples provided in Example 2 of the present invention.

[0035] Fig.16 This is a graph of KQZD34 viral load in serum samples provided in Example 2 of the present invention.

[0036] Fig.17 This is the KQZD30 viral load in lymph and tonsils 35 days after challenge provided in Example 2 of the present invention.

[0037] Fig.18 This is the KQZD34 viral load in lymph and tonsils 35 days after challenge provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0040] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0041] Example 1 1. Separation of NADC30 and 34 Twenty-six PRRSV-positive samples were randomly selected, 0.5 g of each sample was cut into a 2.0 mL EP tube, and a sterilized steel ball was added. Then 1.5 mL of RPMI 1640 medium (containing 1% penicillin-streptomycin) was added. After balancing, the suspension was ground at 23 Frequency / s for 10 min using a tissue grinder. The suspension was collected and filtered through a 0.45 µm filter and inoculated into the prepared Marc145 cells. After adsorption for 2 h at 37 ℃ and 5% CO2, the supernatant was discarded and 5 mL of maintenance medium (RPMI 1640 medium containing 2% FBS) was added. After 3 days at 37 ℃ and 5% CO2, the supernatant virus liquid was harvested and stored at -80 ℃ for use. After three consecutive passages on Marc145 cells, the virus was successfully isolated by indirect immunofluorescence. At the same time, RNA was extracted from the supernatant of each generation to detect Nsp2 of PRRSV RNA. The purified and recovered Nsp2 fragment was ligated to the PMD19-T vector, and the positive ligation product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing analysis.

[0042] The sequencing results were compared with the reference strains in GenBank using MEGA11.0 to determine their accuracy. Figure 1 ). The genetic evolutionary tree showed that all the isolated strains belonged to the PRRSV-2 type, of which 14 isolated strains belonged to Lineage 1.5 (NADC30); 3 isolated strains belonged to Lineage 5.1 (AY150564); 2 isolated strains belonged to Lineage 8.7 (AY032626); 6 isolated strains belonged to Lineage 1.8 (NADC34); and 1 isolated strain belonged to Lineage 3 (JQ308798).

[0043] 2. NADC30, 34 virus titer determination The 14 NADC30 and 6 NADC34 strains were diluted 10 times and then inoculated into the Marc145 monolayer cells on a 96-well cell culture plate. Each dilution was made into 1 column, 6 wells were inoculated with virus, and 2 wells were set as controls (sterile Hank's solution was used instead of virus solution). Check the cells daily to see if they have cytopathic effects, record the number of wells with cytopathic effects, and stop observing when cytopathic effects stop. Calculate TCID according to the Reed-Muench method 50 The results showed that the virus titers of 80-69, 75-50 and 81-143 in NADC30 strains were relatively high, all exceeding 7.5 log10 TCID 50 / ml, in addition, the virus titers of 72-35 and 44-63 in NADC34 strains exceeded 7.5 log 10 TCID 50 / ml. The specific results are shown in Table 1. Among them, 80-69 and 72-35 cytopathic effects are shown in Figure 2 .

[0044] Table 1 Determination of the toxicity of NADC30 and 34 isolates (log 10 TCID 50 / ml)

[0045] 3. Piglet toxicity test Thirty healthy piglets aged 49 to 56 days were selected, with 5 piglets in each group, for a total of 6 groups, including 5 challenge groups and 1 blank control group. Each piglet in the challenge group was intranasally inoculated with 3.0 ml of 80-69, 75-50, 81-143, 72-35, and 44-63 virus solution (virus content of 1.2×10 5.0 TCID 50 / ml). 28 days after infection, serum was collected and viral load was determined according to “Detection of US, Lelystad, and European-like porcine reproductive and respiratory syndrome viruses and relative quantitation in boar semen and serum samples by real-time PCR (Wasilk et al. 2004)”. The results are shown in Figure 3 As shown in Figure 2, the viral load in the serum of 80-69 strains after challenge was the highest among the NADC30 strains (7.63±0.298 log 10 The viral load in the serum of 72-35 after challenge with NADC34 strain was 7.52±0.148 log 10 Therefore, 80-69 and 72-35 were used as the starting strains for subsequent experiments, 80-69 was named KQZD30, and 72-35 was named KQZD34. The Nsp2 sequences of KQZD30 and KQZD34 RNA have been deposited in the GenBank database, and their accession numbers are PQ569556 and PQ569555, respectively.

[0046] 4. Marc145 suspension adaptation Mar145 was adapted to suspension using SF1001 medium as a serum-free adaptation medium. The adherent cells were digested, centrifuged at 1000 rpm for 5 min, and resuspended in SF1001 medium and inoculated into a 125 ml shake flask with a working volume of 30 ml. The cells were cultured at 130 rpm and 37°C, and samples were taken and counted every 3 days. In case of cell clumping, the cells were allowed to stand at 4°C for 15 min, and the supernatant was aspirated and the precipitated clumped cells were discarded. In case of decreased density, the cell density was increased by resuspending by centrifugation, adding adherent digested cells, or merging cells from parallel culture shake flasks. During the adaptation process, the cell density was always controlled at 1-2×10 6 cells / m range until the cell survival rate increases. The cell density and survival rate of Marc145 during suspension adaptation are shown in Figure 4 The cell density reached its highest value on day 63 (1.87×10 6 The overall trend of cell survival rate was a decrease and then an increase, from 82% when the cells were first transferred to the suspension system, to a minimum of 45%, and then slowly increased to 70%-80%, indicating that the cells were very unadapted to the serum-free suspension culture environment; during the period of 51-90 days, the cell survival rate remained above 80%, and the highest could reach 91%.

[0047] 5. Optimization of Marc145 serum-free medium Suspension culture and stable passage of Marc145 cells were achieved in SF1001 serum-free medium, but the entire adaptation process took a long time, the clumping phenomenon was serious, the cell state recovered slowly, and it was difficult to adapt to the suspension culture environment. The cell expansion rate after adaptation cannot meet the needs of subsequent large-scale amplification of PRRSV, so the serum-free medium needs to be optimized. To support cell growth, 0.5% (w / v) soy protein hydrolysate, zinc sulfate heptahydrate (0.4 mg / L), HyClone LS1000 (lipid additive, 1:1000 dilution), ethanolamine (25 µmol / L), ferric citrate (75 mg / L), vitamin E (0.1 µg / mL), and sodium selenite (100 nmol / L) were added to SF1001 serum-free medium. The medium is opti-SF1001. In order to explore whether opti-SF1001 medium can meet the suspension culture requirements of Marc145 cells, Marc145 was inoculated in opti-SF1001 medium for batch culture. The results are shown in Figure 2. Figure 5 The results showed that Marc145 entered the maintenance phase on day 4, and its maximum cell density was (2.96±0.112)×10 6cells / ml, opti-SF1001 medium has good maintenance ability and can maintain high cell density and survival rate in the later stage of batch culture.

[0048] 6. Marc145 molecular modification CD163 is one of the identified receptors of PRRSV. It was found that its truncated M2 (763bp~2049bp) can promote the infection of non-susceptible cells by PRRSV. In addition, Rab1a, a member of the Ras superfamily of GTPases, enhances PRRSV replication through the autophagy pathway. Therefore, by transducing Marc145 with lentivirus, Marc145 can stably express CD163-M2 and Rab1a, making it susceptible and conducive to the proliferation of NADC30 and NADC34. The specific process is as follows: (1) Cloning of target gene and construction of lentiviral recombinant vector: According to the instructions, total RNA of Marc145 cells was extracted and reverse transcribed to obtain cDNA. Homologous recombination primers were designed based on this as a template. The primer sequences are shown in Table 2. The underlined part is the homologous arm region. The target gene was obtained by PCR. The lentiviral vector pLVX-TRE3G-ZsGreen1 was linearized by double restriction digestion with SmaI and EcoRI. The target gene and lentiviral vector were connected by homologous recombination and transformed into Stbl3 super competent cells. The cells were incubated upside down on a plate containing Ampicillin at 37°C. After 24 hours, a single colony was picked for sequencing. The samples with correct alignment were selected and cultured on a large scale in LB medium containing Ampicillin and plasmids were extracted for subsequent experiments. The obtained recombinant vectors were named pLVX-CD163-M2 and pLVX-Rab1a.

[0049] Table 2 Primers used to amplify target genes CD163-M2 and Rabla

[0050] (2) Packaging, infection and verification of lentivirus: HEK293T cells were inoculated into 6-well plates and cultured in a cell culture incubator until the cell coverage reached 70%-90%. Fresh DMEM+10% FBS medium was replaced. DNA-transfection reagent mixture was prepared: plasmid was added to 125 µl serum-free DMEM medium in the proportion (pLVX (recombinant vector): pCMV-dR8.91: pCMV-VSV-G = 4:3:1) and mixed well. Lipo8000 transfection reagent was added according to the instructions. Supernatant was collected after 72 hours, filtered through a 0.45 µm membrane to remove cell debris, and stored at -80°C. Inoculate 500,000 to 1,000,000 cells into T25 flasks, add 4 ml of lentivirus venom and 8 μg / ml polybrene for infection for 12 h, replace with fresh DMEM+10% FBS medium, continue to culture for 72 h, collect the supernatant to obtain recombinant lentivirus containing LVX-CD163-M2 and LVX-Rab1a.

[0051] (3) Recombinant lentivirus transduction of Marc145: Add 2µg of recombinant lentivirus and 8µl of X-treme GENE HP DNA transfection reagent to 200µl of opti-SF1001 culture medium, mix gently, let stand at room temperature for 25 minutes, then add it to the wells of the plate containing Marc-145 cells adapted to suspension culture, shake the culture plate gently back and forth, place it in a 37℃, 5% CO2 incubator for 6 hours, discard the supernatant, add opti-SF1001 and continue culturing. After 48 hours of culture, add puromycin to a final concentration of 10µg / ml, change the screening medium every 2 days, and continue screening for 2 weeks. Subclone the cells expressing green fluorescence into 96-well plates, and then gradually expand the culture to obtain recombinant cells Marc145-CD163-M2 and Marc145-Rab1a.

[0052] (4) Identification of recombinant cells: In order to identify whether the recombinant cells were successfully constructed, the mRNA level of the target gene in different cells was detected by qPCR. The experimental group was set up as suspension cultured Marc145 cells (Marc145-S), Marc145-CD163-M2 cells and Marc145-Rab1a cells. The total RNA of the sample was extracted according to the FreeZol Reagent (Novozymes) instructions, and then HiScriptIVAll-in-OneUltraRTSuperMixforqPCR (Novozymes) was used for reverse transcription to generate cDNA. The primers required for qPCR were designed using the Primer3plus website, and Taq Pro Universal SYBR qPCR Master Mix (Novozymes) was used. The qPCR primer sequences are shown in Table 3. The system was prepared and the reaction program was set according to the instructions. Finally, according to 2 -ΔΔCt The mRNA levels of related genes were calculated by Figure 6 The mRNA level of CD163-M2 gene in Marc145-CD163-M2 cells increased significantly to 33 times that of Marc145-S group, and the mRNA level of Rab1a gene in Marc145-Rab1a cells increased significantly to 27 times that of Marc145-S group, indicating that both CD163-M2 gene and Rab1a gene were successfully integrated into Marc145 cell chromosomes.

[0053] Table 3 qPCR primers

[0054] 7. Research on the amplification ability of recombinant cell viruses To evaluate the viral amplification ability of recombinant cells Marc145-CD163-M2 and Marc145-Rab1a, cell suspension culture was performed to amplify NADC30 and NADC34. Sterile cell triangular shake flasks were used, the medium Opti-SF1001 was filled with 30%, the MOI was 0.05, and the inoculation density was 2×10 6 cells / ml, and cultured at 130rpm for 24h. The control group suspension cultured Marc145 cells (Marc145-S) were treated in the same way. The results showed that Marc145-CD163-M2 was more conducive to the expansion of KQZD30, and the titer reached 8.61±0.128 log 10 TCID 50 / ml, while Marc145-Rab1a was beneficial to the amplification of KQZD34, with a titer of 8.48±0.045log 10 TCID 50 / ml( Figure 7 ).

[0055] 8. Prokaryotic expression of GP5 (1) Target gene amplification: The GP5 protein coding sequence of NADC30 (the amino acid sequence of GP5 is shown in SEQ ID NO.1) was optimized and cloned into the HindIII / XbaI region of the pUC57 vector (commissioned by Beijing Qingke Biotechnology Co., Ltd.), and then PCR amplified using the primers shown in Table 4 to obtain the target fragment. PCR reaction system (50µL): PrimeSTAR Max Premix (2×) 25µL, primers GP5-F-BamHI and GP5-R-XbaI 1.0µL each, template DNA 2.0µL, double distilled water 21µL. PCR amplification procedure: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 15 seconds, 58℃ annealing for 15 seconds, 72℃ extension for 30~60 sec / kb, 35 cycles; 72℃ thorough extension for 5 minutes, and storage at 2~8℃. PCR products were detected by 1% agarose gel electrophoresis.

[0056] Table 4 Primers used to clone target fragment GP5

[0057] (2) Double digestion of vector: Double digest the pCold NusA vector with BamHI+XbaI at 37°C for 3 hours. The digestion products were electrophoresed on 1% agarose gel and recovered using an agarose gel recovery kit.

[0058] (3) Ligation, transformation and screening of recombinant bacteria The DNA fragments recovered in the above steps and the restriction-cut vector were homologously recombined using the Novizan ClonExpress II One Step Cloning Kit, and the reaction was carried out at 37°C for 30 minutes. The obtained recombinant expression vector was transformed into Escherichia coli DH5α competent cells. Single colonies were picked for PCR identification. After the identification was correct, they were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. After the results were correct, the plasmid was extracted and the extracted plasmid was digested for identification.

[0059] (4) Protein expression test: The extracted plasmid pCold NusA-GP5 was transformed into competent E. coli BL21 (DE3). The obtained recombinant E. coli was cultured in LB liquid medium containing 100µg / mL Ampicillin at 37℃ with shaking until OD600nm=0.6. IPTG was added to the bacterial solution to a final concentration of 1.0 mM, and the expression was induced in a shaking incubator at 16℃ for 20 hours. Take 1mL of bacterial solution and centrifuge at 12000 r / min for 1 minute, discard the supernatant, add an equal volume of PBS to resuspend, and then ultrasonically disrupt. Centrifuge and take the supernatant and precipitate respectively. Add an equal volume of PBS to resuspend the precipitate, then take 100µL of the whole bacteria, supernatant and precipitate resuspension, add 25µL of 5× protein loading buffer, and boil in a boiling water bath for 10 min. SDS-PAGE was used to identify whether the protein was expressed. The results are shown in the table. Figure 8 As shown, the GP5 protein was mainly present in the supernatant with a size of 72 kDa.

[0060] (5) Protein purification: The recombinant protein expressing bacteria were collected and resuspended in Lysis buffer (50 mM Tris-HCl, 50 mM NaCl, 0.4% Triton X-114, pH 7.4). The protein was homogenized three times under high pressure until the liquid was clear (pressure was 600 bar). The supernatant was collected and filtered through a 0.45 µm filter and purified using the AKTA pure 25 protein chromatography purification system. A 40mL Ni Focurose 6FF (IMAC) medium pressure column from Huiyan Bio was used, with a sample volume of 10CV and a flow rate of 5.0mL / min. The column was then washed with 10CV Binding buffer (50mM Tris-HCl, 50mM NaCl, pH 7.4) at a flow rate of 5.0mL / min, then washed with 10CV Washing buffer (50mM Tris-HCl, 50mM NaCl, 60mM imidazole, pH7.4) at a flow rate of 5.0mL / min, and finally eluted with 10CV Elution buffer (50mM Tris-HCl, 50mM NaCl, 300mM imidazole, pH7.4) at a flow rate of 5.0mL / min. The peak protein was collected for further analysis. The purification results are shown in Fig. 9 The concentration of elution 1 was 1.269 mg / ml and that of elution 2 was 0.464 mg / ml as determined by the grayscale method.

[0061] 9. Preparation of viral antigens Virus production: KQZD30 and KQZD34 were amplified according to the study of the amplification ability of recombinant cell viruses. Marc145-CD163-M2 was used to amplify KQZD30, and Marc145-Rab1a was used to amplify KQZD34. The supernatant of the fifth-generation cell culture was filtered through a 0.45µm filter, and the filtered virus solution was concentrated 50 times with a 100 ku ultrafiltration membrane in a Pillicon cassette produced by Millipore for later use.

[0062] Virus inactivation: 2-bromoethylamine was cyclized in 0.175 M sodium hydroxide at 37°C for 1 h to prepare a 0.1 M BEI stock solution. Then, the virus was inactivated by incubation with 1 mM BEI at 37°C for 24 h. Subsequently, BEI was neutralized by incubation with 0.1 mM sodium thiosulfate at 37°C for 2 h.

[0063] Virus purification: The inactivated and concentrated virus solution was subjected to column chromatography on a Cytiva HiPrep 26 / 60 Sephacryl S-500HR prepacked column with a size of 26×600 mm, and the first flow-through peak was collected. The protein concentration of the inactivated and purified virus solution before and after purification was determined by the grayscale method (Table 5).

[0064] Table 5 Protein concentration before and after virus purification

[0065] Example 2 1. Vaccine preparation: (1) NADC30 inactivated vaccine group: Purified KQZD30 and F38 adjuvant were emulsified at a ratio of 4:1 (V / V) to prepare a vaccine (antigen content of 8×10 8 TCID 50 / portion, one portion means 2ml, the same below).

[0066] (2) NADC34 inactivated vaccine group: Purified KQZD34 and F38 adjuvant were emulsified at a ratio of 4:1 (V / V) to prepare a vaccine (antigen content of 8×10 8 TCID 50 / first portion).

[0067] (3) GP5 vaccine group: Purified GP5 and F38 adjuvant were emulsified at a ratio of 4:1 (V / V) to prepare a vaccine (antigen content was 400 µg / dose).

[0068] (4) Three-component inactivated vaccine: The purified KQZD30, KQZD34 and GP5 antigens were mixed at a ratio of 1:1:2 (V / V) and then emulsified with F38 adjuvant at a ratio of 4:1 (V / V) to prepare an inactivated vaccine (the content of KQZD30 and KQZD34 antigens was 2×10 8 TCID 50 / head portion; the antigen content of GP5 protein is 200µg / head portion).

[0069] 2. Attack and protection test: The three single-component vaccines and three-component inactivated vaccines prepared above were inoculated into 20 healthy piglets aged 14 to 21 days, and a blank control group and a challenge control group were set up. The specific groups are shown in Table 6. Needle-free immunization was used, 0.4 ml each time, and the second immunization was carried out 21 days after the first immunization. 35 days after the first immunization, the vaccine immunization group and the challenge control group were challenged with viruses, and each pig was intranasally inoculated with 3.0 ml of KQZD30 or KQZD34 virus solution (the virus content was 1.2×10 5.0 TCID 50 / ml). Blood was collected from all pigs at 0d, 5d, 10d, 15d, 20d, 25d, 30d and 35d after challenge to determine viral load. The results showed that the viral loads of KQZD30 and KQZD34 in the blood were 0.356 and 0.252 log, respectively, 35d after challenge with the three-component inactivated vaccine. 10 copies / ml, which were significantly lower than the other three single-component vaccines ( Fig.10 , Fig.11 ). Therefore, the protective efficacy of the three-component inactivated vaccine is higher than that of the single-component vaccine.

[0070] Table 6 Experimental groups and immunization regimen

[0071] Note: “-” indicates no vaccination.

[0072] 3. Comparative test with similar commercial products (1) Antibody and neutralizing antibody detection: The prepared three-component inactivated vaccine, commercial A inactivated vaccine (produced by Company A), commercial B attenuated live vaccine (produced by Company B) and commercial C inactivated vaccine (produced by Company C) were inoculated into 10 healthy piglets aged 14 to 21 days. The three-component inactivated vaccine was needle-free, and the second immunization was performed according to the recommended immunization, 0.4 ml each time, and the second immunization was performed 21 days after the first immunization; the commercial A inactivated vaccine was immunized once according to the instructions, and 1 dose (2.0 ml) was injected into the neck muscle each time; the commercial B attenuated live vaccine was immunized once according to the instructions, and 1 dose (2.0 ml) was injected into the neck muscle each time; the commercial C inactivated vaccine was immunized once according to the instructions, and 1 ml was injected into the neck muscle each time. In order to ensure that the evaluation date is as consistent as possible, the immunization of commercial vaccines was delayed by one week, and a blank control group was set up. Blood was collected on the 14th, 28th, 35th, 42nd and 60th days after the second immunization, and serum was separated for antibody and neutralizing antibody detection.

[0073] (2) Neutralizing antibody detection steps: 1. Digest the Marc-145 cells in good growth state with trypsin, wash twice with PBS, resuspend in DMEM and dilute to 1×10 5 1. Add 100 μL of cells to each well of a 96-well cell culture plate and place it in an incubator until it adheres to the wall; 2. Inactivate the separated serum in a 60°C water bath for 30 min; 3. Dilute the inactivated serum by 2-fold, with a total of 7 dilutions, and make 4 parallel wells for each dilution; 4. 50 Dilute KQZD30 or KQZD34 to 100 TCID 50 / 50 μL; 5. Mix the diluted serum and virus in equal volumes (250 μL each) and place in a 37 ℃ incubator for incubation for 2 h; 6. Add the incubated virus-serum mixture to the 96-well cell culture plate full of Marc-145 cells, 100 μL per well. Incubate in a 37 ℃ incubator for 2 h, aspirate the mixture, wash twice with PBS, and add 2% FBS-DMEM cell culture medium for culture; 7. Check whether the cells have cytopathic effects on the 3rd to 5th day, and record the number of wells with cytopathic effects; 8. Calculate the neutralizing antibody titer of the serum using the Reed-Muench method; The results showed that the antibody levels of the three-component inactivated vaccine exceeded those of other commercial vaccines throughout the monitoring period, and the ELISA antibody S / P values ​​were all above 0.8. Moreover, 60 days after the second vaccination, the ELISA antibody S / P values ​​of the three-component inactivated vaccine were 1.28 times, 1.24 times, and 1.07 times those of the commercial A inactivated vaccine, the commercial B attenuated live vaccine, and the commercial C inactivated vaccine, respectively. Fig.12The neutralizing antibody titers of the three-component inactivated vaccine against KQZD30 or KQZD34 exceeded those of other commercial vaccines throughout the monitoring period. 42 days after the second vaccination, the neutralizing antibody titers of the three-component inactivated vaccine were 1:35.08 and 1:32.27, respectively, which were higher than 1:30, and could provide 100% protection against PRRSV infection ( Fig.13 , Fig.14 ).

[0074] The prepared three-component inactivated vaccine, commercial A inactivated vaccine, commercial B attenuated live vaccine and commercial C inactivated vaccine were inoculated into 20 healthy piglets aged 14 to 21 days according to (1). A blank control group and a challenge control group were set up. The specific grouping is shown in Table 7. 35 days after the first vaccination, the vaccine immunization group and the challenge control group were challenged with KQZD30 or KQZD34 virus solution 3.0 ml (virus content was 1.2×10 5.0 TCID 50 / ml). Blood was collected from all pigs on 0d, 5d, 10d, 15d, 20d, 25d, 30d and 35d after infection, and lymph and tonsils were collected from all pigs 35d after infection. The viral load was determined with reference to “Detection of US, Lelystad, and European-like porcine reproductiveand respiratory syndrome viruses and relative quantitation in boar semen andserum samples by real-time PCR (Wasilk et al. 2004)”. The results showed that after vaccination with the three-component inactivated vaccine, the viral loads of KQZD30 and KQZD34 in the blood 35d after infection were 0.392 and 0.244 log, respectively. 10 copies / ml( Fig.15 , Fig.16 ), the viral loads of KQZD30 and KQZD34 in lymph were 0.120 and 0.072 log, respectively 10 The viral loads of KQZD30 and KQZD34 in tonsils were 0.180 and 0.148 log respectively. 10 copies / ml( Fig.17 , Fig.18 ), which are significantly lower than other commercial vaccines. Therefore, the three-component inactivated vaccine can provide effective protection for immunized pigs, and its protective efficacy is higher than similar products on the market.

[0075] Table 7 Experimental groups and immunization schemes

[0076] Note: “-” indicates no vaccination.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An immune composition, characterized in that include: The NADC30 lineage inactivated strain of PRRSV, the NADC34 lineage inactivated strain of PRRSV, and the GP5 protein of PRRSV; The NADC30 lineage inactivated strain of PRRSV is porcine reproductive and respiratory syndrome virus KQZD30, and the NADC34 lineage inactivated strain of PRRSV is porcine reproductive and respiratory syndrome virus KQZD34.

2. The immune composition according to claim 1, characterized in that The GP5 protein of PRRSV is derived from the NADC30 lineage virus strain of PRRSV.

3. The immune composition according to claim 1, characterized in that The GP5 protein of PRRSV comprises the amino acid sequence shown in SEQ ID NO.

1.

4. The immune composition according to any one of claims 1 to 3, characterized in that The volume ratio of the inactivated strain of the NADC30 lineage of PRRSV, the inactivated strain of the NADC34 lineage of PRRSV and the GP5 protein of PRRSV is 1: (0.5-2): (0.5-4); the virus titer of the inactivated strain of the NADC30 lineage of PRRSV is not less than 8 log 10 TCID 50 / ml; the virus titer of the NADC34 lineage inactivated strain of PRRSV is not less than 8 log 10 TCID 50 / ml; the concentration of the GP5 protein of PRRSV is not less than 0.1 mg / ml.

5. A vaccine, characterized in that The vaccine comprises the immune composition of claim 1 or 2 and a vaccine adjuvant.

6. The vaccine according to claim 5, characterized in that The vaccine adjuvant includes: one or more of F38 adjuvant, aluminum salt adjuvant, oil emulsion or particulate adjuvant; the volume ratio of the immune composition to the vaccine adjuvant is (2~6):

1.

7. Use of the immune composition according to any one of claims 1 to 4 in the preparation of a medicament for preventing and treating porcine reproductive and respiratory syndrome.

8. Use of the immune composition according to any one of claims 1 to 4 for improving the immunogenicity of a drug against porcine reproductive and respiratory syndrome virus.

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