CN113425838B - Recombinant PRRSV virus-like particle antigen-antibody complex and preparation method thereof - Google Patents

Abstract

In the present invention, by transforming the GP5-M protein, a recombinant baculovirus capable of efficient replication is obtained, and its culture titer is relatively higher, and IgM is further compounded by using virus-like particles similar to the prepared PRRSV virus particles and IgM to prepare IgM -RPPSV VLPs immune complex, the IgM-RPPSV VLPs immune complex can stimulate the body to produce high concentrations of antibodies earlier.

Description

Recombinant PRRSV virus-like particle antigen-antibody complex and preparation method thereof

Technical field:

The invention belongs to the field of biotechnology, and relates to a recombinant PRRSV virus-like particle antigen-antibody complex and a preparation method thereof.

Background technique:

Porcine reproductive and respiratory syndrome (PRRS), also known as "blue ear disease", is caused by Porcine reproductive and respiratory syndrome virus (PRRSV), which mainly causes the reproduction of pregnant sows. Obstacles and breathing difficulties in piglets, usually manifested as coughing, dyspnea in piglets, preterm birth, miscarriage or even stillbirth in sows. Since its discovery in North America in the late 1980s, the worldwide epidemic and several outbreaks of the disease have caused huge economic losses to the swine industry around the world.

PRRSV belongs to the arteritis virus family, which includes simian hemorrhagic fever virus, equine arteritis virus and lactate-stimulating dehydrogenase virus. According to the latest classification results, RRSV is classified into the genus Betaarteri virus under the order Nidovirales and Arterividae, and members of the same genus also include Rat arteri virus. , RatAV) 2 species. With the continuous advancement of diagnostic technology, it has been found that there is a wide diversity of PRRSV strain genes and antigens. In general, PRRSV is divided into European type and American type, and the two types have obvious genomic homology, antigenicity, and virulence. The differences in homology at the nucleotide level range from 30 to 45%. Some scholars have divided PRRSV into different lineages. The current popular ones in China are: classic PRRSV-2 (lineage 5), highly pathogenic PRRSV (lineage 8), QYYZ-like PRRSV-2 (lineage 3) and NADC30-like PRRSV -2 (Lineage 1). The virus was first isolated in China in 1996 and broke out in China in 2006. It is mainly characterized by high infectivity, high body temperature, high morbidity and high mortality. It has been plaguing the pig industry until now. According to the genetic characteristics, it is divided into two subtypes: North American type and European type. At present, North American type PRRSV such as HP-PRRS strain and NADC30-like strain are the main circulating strains in China.

The structural genes of PRRSV include 8 open reading frames, including ORF2, ORF2a, ORF3~ORF5, ORF5a, ORF6 and ORF7, which mainly encode the functional proteins of the virus. Among them, GP2, GP3, GP4, and GP5 are glycosylated envelope glycoproteins, while M protein, N protein, GP2a, and GP5a are non-glycosylated envelope proteins (not glycosylated). Among them, GP5 and M proteins account for a relatively high proportion in virions and are the main envelope proteins (Mokhtar et al. 2016), and GP5 can produce high levels of neutralizing antibodies and cellular immunity (Popescu et al. 2017; Kim et al. 2013).

VLPs vaccine is a new type of genetically engineered vaccine. The structural protein genes that can produce immune effects in PRRSV are recombined into expression vectors for expression, which can accurately simulate the structure of virus particle antigens. VLPs vaccine has no genetic material and good immune effect, and is expected to become a new type of vaccine for PRRS prevention. The main difficulties in the development of such vaccines lie in the selection and optimization of gene sequences, the selection of expression systems, the improvement of expression levels, the large-scale purification of VLPs, how to reduce costs, the selection and addition ratio of adjuvants, and how to evaluate vaccine efficacy. VLPs vaccines can stimulate B lymphocyte-mediated responses, CD4 ⁺ T cell proliferation responses, and cytotoxic somatic responses. VLPs and B lymphocyte receptors are cross-linked, can efficiently recognize the MHC class I classical pathway, target dendritic cells, and cause efficient cellular and humoral immune responses. GP5 and M protein genes were recombined into E. coli, and the expressed proteins could trigger high-titer neutralizing antibody responses and Th1-type cell-mediated immune responses. There are two antagonistic epitopes, A and B, on the surface of GP5 protein. The A epitope weakens the production of neutralizing antibodies and covers the B epitope that can cause neutralization. effect. And studies have shown (HP-PRRSV envelope protein 5 glycosylation site mutation and functional exploration), site-directed mutation of the amino acid of the A epitope has a certain impact on virus replication, resulting in a decrease in virus titer. Reducing the antagonism of A epitope to B epitope is an urgent technical problem to be solved in recombinant PRRSV virus-like particle vaccines. The inventors and in 2018 prepared recombinant PRRSV virus-like particles with immunogenicity (ZL201811114020.6), However, there is still a problem of late generation of neutralizing antibody titers in actual production, and there is still room for improvement.

Antigen-antibody complex vaccines, also known as immune complex (IC) vaccines, are made by mixing specific antibodies with antigens in appropriate proportions. After the antigen-antibody complex is formed, the Fc fragment of the antibody molecule has a high affinity with the Fc receptor of the antigen presenting cell (APC), making it easier and more effective for the virus bound to the antibody to bind to the APC. After the antigen-antibody complex is phagocytosed and internalized by APC, B lymphocytes and T lymphocytes can be activated, and B lymphocytes can be stimulated to become plasma cells, thereby causing strong humoral and cellular immune responses. The humoral immune response stimulated by immune complexes formed in vitro is 100 times that of natural antigens. In addition, Fc receptors are widely distributed on the surface of immune effector cells such as APC. By binding to Fc receptors, antibodies can mediate the uptake of antigens by immune effector cells, and participate in the regulation of antigen-induced immune responses.

At present, there are many researches on antigen-antibody complex vaccines for poultry diseases and diseases of other species, mainly including chicken infectious bursal IC vaccine, Newcastle disease IC vaccine, Marek IC vaccine, chicken infectious laryngotracheitis IC vaccine, chicken reovirus Virus IC vaccine, gosling plague IC vaccine and canine parvovirus IC vaccine, etc. Among them, the antigen-antibody complex vaccine for chicken bursal bursa and Newcastle disease developed by American Roman Company has been approved by the US Department of Agriculture and is one of the vaccines widely used in the world. However, there are few reports on pig antigen-antibody complex vaccines. Antigen-antibody complex vaccines also have shortcomings. For example, the traditional method of preparing vaccines by mixing serum polyclonal antibodies with pathogens is affected by the preparation of serum polyclonal antibodies. The preparation process is complicated, and the serum antibody content between different batches is unstable. Animal-derived serum is prone to introduce exogenous pathogenic contamination and other disadvantages, so it has certain limitations, while the use of monoclonal antibodies does not have such problems. Professor Nan Yuchen from Northwest Agriculture and Forestry University (CN111138535A, an immune enhancer and its application in vaccine preparation, application number CN202010069365.5) prepared a single compound with neutralizing activity against both PRRSV-I and PRRSV-II viruses. When the antibody 5D9 was cloned and used as an immune enhancer, when the formed immune complex was combined with adjuvant to immunize mice, IFN-γ-secreting T cells were significantly increased, suggesting that in the process of inactivated virus immunization, PRRSV-specific antibody 5D9 Co-immunization with normal oil-in-water adjuvant enhanced CTL responses. Animal experiments show that the immune protection rate of the prepared immune complex can achieve higher protective efficacy than the vaccine prepared only with commercial adjuvant ISA 206 and the commercial attenuated vaccine. On this basis, the present invention further combines specific antibodies with PRRSV VLPs to explore its protective effect on animals.

Invention content:

Based on the current development of domestic PRRSV vaccines, the present invention aims to provide a recombinant PRRSV virus-like particle antigen-antibody complex and a preparation method thereof. By studying GP5 and M proteins, the present invention artificially artificially synthesizes the genes of GP5 and M proteins. Transformation, using a baculovirus expression system for expression to obtain virus-like particles containing GP5-M recombinant protein, the virus-like particles can produce neutralizing antibodies earlier than unmodified protein sequences, and have better immunogenicity, The titer of the produced antibody is higher; the present invention further combines the prepared RPSV VLPs with the monoclonal antibody 5D9 provided by Professor Nan Yuchen to form an "IgM-RPPSV VLPs" immune complex, and the "IgM-RPPSV VLPs" complex Enhancement of vaccine-induced cellular immune responses.

In order to solve the above technical problems, the present invention adopts the following technical solutions.

A recombinant PRRSV virus-like particle antigen-antibody complex, characterized in that the antigen-antibody complex is prepared from a recombinant PRRSV virus-like particle whose amino acid sequence is shown in SEQ ID No: 2 and an IgM antibody.

The IgM antibody is prepared by Professor Nan Yuchen of Northwest Agriculture and Forestry University (CN111138535A, an immune enhancer and its application in vaccine preparation, application number CN202010069365.5), which is resistant to both PRRSV-I and PRRSV-II viruses. Monoclonal antibody 5D9 with neutralizing activity.

The recombinant PRRSV virus-like particles are prepared from recombinant baculovirus expressing porcine reproductive and respiratory syndrome virus PRRSV and M protein, and the preparation method of the recombinant baculovirus expressing porcine reproductive and respiratory syndrome virus PRRSV and M protein , is characterized in that, comprises the steps:

Step 1, the construction of recombinant baculovirus expressing PRRSV GP5 and M protein, through the following steps:

(1) Artificially transform the PRRSV GP5-M gene, based on the sequence of the PRRSV-SD16 strain (Genbank Accession No: JX087437.1), and design the PRRSV GP5-M gene whose gene sequence is shown in SEQ ID No: 1;

(2) Construction of baculovirus transfer vector: Synthesize the PRRSV GP5-M gene whose gene sequence is shown in SEQ ID No: 1, and set Bam H I and Hind III enzyme cleavage sites at the 5' and 3' ends, respectively, The insert fragment was obtained; with the pBAC-5 plasmid as the backbone, the vector and the insert fragment were digested with Bam H I and Hind III enzymes, and then ligated with T4 ligase overnight at 16°C, and positive clones were obtained by transformation and screening, and the recombinant plasmid was extracted and identified by enzyme digestion. , to obtain the baculovirus transfer vector pBAC-5-PRRSVGP5-M;

Step 2, construction of recombinant baculovirus Ac-PRRSV GP5-M:

(1) Acquisition and identification of recombinant bacmid rBac-PRRSV GP5-M: Take the correct baculovirus transfer vector pBAC-5-PRRSVGP5-M plasmid DNA identified by sequencing and mix it with DH10 Bac competent cells, and after 30 minutes of ice bath, Heat shock in a water bath for 45 seconds at 42°C, then ice bath for 5 minutes, add SOC liquid medium, and shake at 37°C for 2 hours. After 10-fold serial dilution, each gradient bacterial solution is coated on LB plates and screened by life company. The kit screened and purified positive colonies, and extracted recombinant bacmid rBac-PRRSVGP5-M.

(2) Obtainment of recombinant baculovirus Ac-PRRSV GP5-M: The recombinant bacmid rBac-PRRSVGP5-M extracted in the previous step was transfected into sf9 cells using the lipofection reagent Lipofectamine 3000 and kept at 28°C Culture observation, the green fluorescent signal of recombinant baculovirus was observed by fluorescence microscope at 24 hours, 48 hours and 72 hours after transfection, and the cell supernatant was collected at 72 hours after transfection to obtain recombinant baculovirus, which was then inoculated again The healthy sf9 cells were expanded and cultured, and the virus liquid was collected as a virus seed and stored at -70°C for future use.

The present invention also claims to protect the preparation method of the recombinant PRRSV virus-like particle antigen-antibody complex, characterized in that the method comprises:

(1) Preparation of RPPSV VLPs: The prepared recombinant baculovirus Ac-PRRSV GP5-M was inoculated into healthy sf9 cells at a proportion of 10%, and after culturing at 27°C for 4-5 days, the cells and supernatant were collected by repeated freezing and thawing, and the cells and supernatants were collected by repeated freezing and thawing. After centrifugation at 12000 r/min for 20 minutes at ℃, the supernatant was collected, and then the target protein was precipitated by ammonium sulfate precipitation. After resuspending, the protein solution was inactivated by diethyleneimine (BEI) for 36-48 hours, and then used, etc. The amount of sodium thiosulfate was neutralized, and then the 100kD molecular weight filter membrane (EMD Millipore company) was used to concentrate 50 times by the LabscaleTFF filter filter, and then the RPPSV VLPs particles were purified by liquid chromatography to obtain the RPPSV VLPs;

(2) Preparation of IgM-RPPSV VLPs immune complex: RPPSV VLPs and monoclonal antibody 5D9 (calculated by mass) were mixed in a ratio of 1:5, and placed at 37 °C for 2 hours to form a complex.

The present invention further claims a vaccine, characterized in that the vaccine is composed of IgM-RPPSV VLPs immune complex and adjuvant.

The present invention also claims a method for preparing the vaccine, comprising the steps of: mixing the compounded "IgM-RPPSVVLPs" immune complex (calculated by volume) with Montanide™ ISA 206 water-in-oil adjuvant at a ratio of 46:54. After emulsification, a vaccine composition is obtained.

Based on the above technical solutions, the present invention has the following advantages and beneficial effects:

First, on the basis of the original invention patent, the inventor further focused on the research of GP5-M virus-like particles, and through artificial design and transformation, the GP5-M protein was transformed, especially through the artificial transformation of the sequence to make the A on GP5. The epitope and the B epitope are connected by a rigid linker, thereby improving the influence of the A epitope on the B epitope, so that the virus-like particle can stimulate the body to produce neutralizing antibodies earlier. The obtained virus-like particles can produce higher titers of neutralizing antibodies. After 6 weeks of the first vaccination, the titers of neutralizing antibodies all reached 1:32 or more, and the neutralizing antibody titers of 3 mice reached 1:64. 2 only reached 1:128. From the point of view of the time and titer level of neutralizing antibody production, the recombinant PRRSV virus-like particle vaccine of the present invention has relatively good immune protection effect, which helps animals to resist the virus and prevent the disease in the early stage of infection. deterioration;

Secondly, the present invention further verifies the prepared GP5-M virus-like particles and monoclonal antibody 5D9 to prepare IgM-RPPSV VLPs immune complexes, and uses the IgM-RPPSV VLPs immune complexes to immunize piglets, compared with using RPSV alone VLPs immunization can stimulate the body to produce higher titers of antibodies earlier

To sum up, the present invention obtains a recombinant baculovirus capable of efficient replication by modifying the GP5-M protein, the culture titer is relatively higher, and the prepared PRRSV virus particles are similar to virus-like particles and Ig. M is further compounded to prepare an IgM-RPPSV VLPs immune complex, and the IgM-RPPSV VLPs immune complex can stimulate the body to produce a high concentration of antibodies earlier.

Description of drawings:

Figure 1: Construction and identification of recombinant transfer vector: (A) Using the primers provided by Gene Synthesis Company, the GP5-M gene fragment of about 1200bp can be amplified by colony PCR, where M is marker and channel 1 is colony PCR amplification As a result, channel 2 is a blank control; (B) the result of enzyme digestion of the recombinant vector, where M is marker, channel 1 is the extracted and purified recombinant vector without enzyme digestion, channel 2 is the recombinant vector after enzyme digestion, and obtained after enzyme digestion The GP5-M gene fragment of about 1200bp and the vector fragment of about 5500bp.

Figure 2 Expression and identification of recombinant protein: A is blank cell; B is recombinant baculovirus-infected cell.

Figure 3: Electron microscopy results of recombinant proteins.

Figure 4: Western blot identification results: M is marker, channel 1 is PRRSV VLP detection result.

Figure 5: Antibody fluctuation rule of IgM-RPPSV VLPs immune complex immunized piglets: the first group was the test group, RPPSV VLPs (10μg) + ISA 206 + 5D9 (50μg), the second group was the control group, RPPSV VLPs (10μg) ) + ISA206.

Detailed ways:

The present invention can be more clearly understood from the following examples. The technical steps described in the present invention, unless otherwise specified, are conventional technical means in the art, or commercialized or disclosed reagent materials.

Example 1: Construction of recombinant baculovirus Ac-PRRSVGP5-M

(1) Synthesis of PRRSV GP5 and M gene sequences:

Based on the sequence of the PRRSV-SD16 strain (Genbank Accession No: JX087437.1), after artificial modification, the PRRSV GP5-M gene with the gene sequence shown in SEQ ID No: 1 was obtained, and sent to a gene synthesis company for gene sequence synthesis , and set Bam H I and Hind III restriction sites at the 5' and 3' ends, respectively, to obtain an insert.

(2) Construction and identification of recombinant transfer vector:

The plasmid containing the target gene sequence GP5-M and the vector pBAC-5 (provided by Shaanxi Novi Lihua Biotechnology Co., Ltd., with a GFP marker gene on the vector, can be used for fluorescence detection, see ZL201811114020.6) for Bam H I and Hind III double-enzyme digestion, after recovery and purification, T4 ligase was used for ligation reaction, chemically transformed into DH5α competent cells, positive clones were screened, and plasmids were extracted, which were identified by colony PCR (Figure 1-A) and plasmid digestion (Figure 1-A). 1-B), the successfully constructed recombinant transfer vector pBAC-5-PRRSV GP5-M was obtained.

Based on the results in Figure 1-A, it can be seen that using the primers provided by the gene synthesis company, the GP5-M gene fragment of about 1200 bp can be amplified by colony PCR; while the results in Figure 1-B show that the GP5-M gene fragment of about 1200 bp obtained after digestion GP5-M gene fragment and a vector fragment of about 5500bp, the above results show that the present invention successfully constructed the recombinant transfer vector pBAC-5-PRRSV GP5-M.

(3) Construction and identification of recombinant Bacmid

The plasmid DNA of the recombinant transfer vector pBAC-5-PRRSV GP5-M was transformed into DH10 Bac competent cells respectively, and the transfer vector and the backbone vector in the competent cells were homologous to obtain a recombinant bacmid rBac- PRRSV GP5-M.

(4) Preparation of recombinant baculovirus, according to the method described in the inventor's previous patent (ZL201811114020.6), to prepare a recombinant baculovirus, the specific steps are as follows:

4.1 Recombinant bacmid transfected insect cells: sf9 cells were plated to six-well plates, and when the cell confluence reached 80%, transfected with Lipofectamine3000, a lipofection reagent, as follows:

4.1.1 Add 2μg recombinant bacmid rBac-PRRSVGP5-M and 2μL p3000 to 250μL opti-MEM medium without serum and antibiotics, mix gently;

4.1.2 Add 4μl Lipofectamine3000 liposome to 250μl opti-MEM medium without serum and antibiotics, and mix gently;

4.1.3 Mix the liquids in step 4.1.1 and step 4.1.2 evenly, and let stand for 10-15 minutes at room temperature;

4.1.4 Add the mixture in step 4.1.3 dropwise to the sf9 cells in the six-well plate, and then incubate the sf9 cells in the six-well plate at a constant temperature of 27°C for continuous observation.

Harvesting and Amplification of Recombinant Baculovirus

4.2.1 Harvest of recombinant baculovirus: Carefully observe the transfected cells every day. When the cells become larger, irregular or even begin to float to the liquid level, collect the cells and supernatant, and extract sample RNA. And carry out RT-PCR detection to identify the existence of the target gene GP5-M. Generally, when obvious cytopathic changes are seen on the 4th to 5th day after transfection, the collected cells and supernatant are repeatedly frozen and thawed, and centrifuged at 12000r/min at 4℃. For 10 minutes, the supernatant was collected and labeled as P1 generation recombinant baculovirus;

4.2.2 Amplification of recombinant baculovirus: Inoculate sf9 cells with P1 generation recombinant baculovirus at 1:10, culture at 27°C for 4-5 days, and harvest P2 generation recombinant baculovirus when cytopathic changes are evident; Recombinant baculoviruses of generation P3 and higher were obtained by the same method, and the collected recombinant baculoviruses were stored at -70°C for future use.

4.2.3 Determination of titer of recombinant baculovirus: 10-fold serial dilution of P2 and P3 generation viruses respectively, then inoculate sf9 cells in 96-well plate, inoculate 8 wells for each dilution, culture at 27°C and observe daily. cytopathic conditions, and then calculated the TCID ₅₀ value of the virus according to the Karber method, and obtained that the P2 and P3 generation virus titers of the recombinant baculovirus Ac-PRRSV GP5-M were about 10 ^5.58 TCID ₅₀ /ml and 10 ^6.62 TCID ₅₀ /ml, respectively , compared with the previous research, the virus titer of the present invention is greatly improved.

(5) Expression and identification of recombinant proteins

5.1 Immunofluorescence detection of recombinant protein: Recombinant baculovirus infects healthy sf9 cells according to the proportion of 10%, and sets blank control wells, incubates at 27 °C for 48-72 hours and continues to observe, when the cytopathic effect reaches more than 80%, use pre-cooling The cells were fixed with 80% acetone for 2 hours, then washed 3 times with PBST, blocked with 5% nonfat milk (prepared with TBST) overnight at 4°C, then washed 3 times with PBST, and added GP5 monoclonal antibody (produced by Shaanxi Nuo) at a 1:1000 dilution. (Provided by Weilihua Biotechnology Co., Ltd.), incubate at 37 °C for 1 hour in the dark, then wash 3 times with PBST, add FITC fluorescently-labeled goat anti-mouse secondary antibody at a dilution of 1:1000, incubate at 37 °C for 1 hour, and wash 3 times with PBST The results were observed and judged under a fluorescence microscope. The cells infected with the recombinant baculovirus showed obvious green fluorescence while the control group had no fluorescence, which confirmed that the target gene could be expressed in insect cells sf9 (Figure 2).

5.2 Electron microscope observation of recombinant protein: The freshly collected recombinant baculovirus liquid was observed by electron microscope, and the results showed that virus-like particles with a shape similar to PRRSV virus particles were detected, with a diameter of about 46-66 nm, which confirmed that GP5 and M protein can be formed in vitro virus-like particles (Figure 3).

5.3 Western blot identification: configure a 10% polyacrylamide gel, add the PRRSVVLPs sample prepared by the present invention, and then perform SDS-PAGE electrophoresis; transfer the protein in the SDS-PAGE gel to PVDF membrane by wet transfer method; use blocking solution to block at room temperature 2 h; use blocking solution to dilute mouse anti-GP5 monoclonal antibody (provided by Shaanxi Novilihua Biotechnology Co., Ltd.) 1:200, and incubate PVDF membrane with monoclonal antibody overnight at 4°C; wash PVDF membrane with PBST; 1: 4000-fold dilution of HRP-labeled goat anti-mouse IgG antibody, and the PVDF membrane was incubated with it for 1 h at room temperature; the PVDF membrane was washed with PBST; Thermo exposure solution was evenly added dropwise to the PVDF membrane and exposed. The results are shown in Figure 4. The target protein band was detected around 40kDa, which was consistent with the expected protein size.

During the research process, the inventors have studied and tried a variety of genetic modification schemes, especially for the A epitope and B epitope of the GP5 protein, in which the control group 1 uses only one rigid linker between the A and B epitopes (EAAAK) to obtain control group 1, whose nucleic acid sequence is shown in SEQ ID No: 3, and the amino acid sequence is shown in SEQ ID No: 4; control group 2 uses three flexible linkers (GGGGS) to replace "GGGGSSHIQLIYNLGGGGS" in the present invention The modified GP5 protein and M protein are connected, the nucleic acid sequence is shown in SEQ ID No: 5, and the amino acid sequence is shown in SEQ ID No: 6; the control group 3 is the recombinant rod-shaped structure constructed by the invention patent ZL201811114020.6 Virus.

For control group 1 and control group 2, the method of Example 1 was used to prepare the corresponding recombinant baculoviruses, and the corresponding virus-like particles were prepared by expressing them in insect cells sf9, which were used for the subsequent preparation of corresponding virus-like particle vaccines . In control group 3, virus-like particle samples were provided by Shaanxi Novilihua Biotechnology Co., Ltd.

It was determined that based on the same culture conditions in Example 1, the P3 generation virus titer of the recombinant baculovirus in the control group 1 was about 10 ^5.72 TCID ₅₀ /ml; the P3 generation virus titer of the recombinant baculovirus in the control group 2 was about 10 ^5.76 _TCID50 /ml.

Example 2:

(1) Preparation of recombinant PRRSV virus-like particle vaccine:

The recombinant baculovirus Ac-PRRSV GP5-M prepared in Example 1 was inoculated into healthy sf9 cells at a proportion of 10%. After culturing at 27°C for 4-5 days, the cells and supernatant were collected by repeated freezing and thawing. After centrifugation for 20 minutes, the supernatant was collected, and then the target protein was precipitated by ammonium sulfate precipitation. After resuspending, the protein solution was inactivated by diethyleneimine (BEI) for 36-48 hours, and then the same amount of sodium thiosulfate was used. Neutralized, and finally mixed with Seppic ISA 206 adjuvant to emulsify the vaccine, and placed at 2-8°C for use.

In the vaccine preparation process, after the recombinant baculoviruses of Example 1, Control Group 1, Control Group 2 and Control Group 3 of the present invention were cultured, they were adjusted to a virus titer of 10 ⁵ TCID ₅₀ /ml, and then continued follow-up Freezing and thawing etc. During vaccine preparation, the control protein concentration was 100 μg/mL.

(2) Vaccine appearance and safety experiments

According to the method described in the inventor's previous patent (ZL201811114020.6), the vaccine was tested, and the trait test, sterility test, exogenous virus test, mycoplasma test, and safety test were carried out respectively, and the vaccine was carried out for piglets and pregnant sows respectively. Safety test, this part of the experiment was completed by Shaanxi Novi Lihua Biotechnology Co., Ltd. The test results show that the recombinant PRRSV virus-like particle vaccine of the present invention complies with the relevant regulations of the veterinary pharmacopoeia, and is safe for piglets and pregnant sows.

(3) Mouse immunization

A total of 60 female Balb/C mice aged 4-6 weeks were randomly divided into 6 groups with 10 mice in each group, and were immunized twice in total. Wherein Example 1-control group 3 were injected with the corresponding recombinant PRRSV virus-like particle vaccine respectively; the attenuated vaccine group was vaccinated with commercially available JXA1-R strain live attenuated vaccine; the blank group was not vaccinated.

(4) Neutralizing antibody assay

4.1 Separate the serum to be tested, inactivate it at 56°C for 30 minutes, and store it at -20°C for later use;

4.2 Use DMEN basal medium to serially dilute the serum to be tested on a 96-well plate, from 1:2 to 1:256, 50 μL per well;

4.3 Dilute the PRRSV SD-16 virus culture solution to 200 TCID ₅₀ , add 50 μL of virus dilution solution to each well of the above-mentioned wells that have been added with serum, and infect them in a 37°C 5% CO ₂ incubator for 1 hour;

4.4 Add 100 μL of Marc-145 cell suspension (about 5×10 ⁵ cells/mL) to each well, shake it evenly, and place it in a 37°C 5% CO ₂ incubator for 6-8 hours; pigs are positive The serum was provided by Shaanxi Novi Lihua Biotechnology Co., Ltd., and the neutralizing antibody titer was determined to be 1:16.

4.5 Observe and record cytopathic conditions until the results are stable. The specific results are as follows:

Table 1 Neutralizing antibody levels induced by recombinant PRRSV virus-like particle vaccine

Based on the results in Table 1 above, it can be seen that the recombinant PRRSV virus-like particle vaccine of Example 1 of the present invention can stimulate the body to produce neutralizing antibodies earlier than the control group 1-3 and the attenuated vaccine. Mice produced neutralizing antibodies of more than 1:8; and after 6 weeks of the first vaccination, the titers of neutralizing antibodies reached more than 1:32, of which 3 mice reached 1:64, and 2 reached 1 : 128, from the time and titer level of neutralizing antibody generation, the recombinant PRRSV virus-like particle vaccine of Example 1 of the present invention has relatively good immune protection effect, which helps animals to resist virus and prevent disease in the early stage of infection deterioration.

Example 3: Preparation of "IgM-RPPSV VLPs" immune complexes

(1) Preparation of RPPSV VLPs:

The recombinant baculovirus Ac-PRRSV GP5-M prepared in Example 1 was inoculated into healthy sf9 cells at a proportion of 10%. After culturing at 27°C for 4-5 days, the cells and supernatant were collected by repeated freezing and thawing. After centrifugation for 20 minutes, the supernatant was collected, and then the target protein was precipitated by ammonium sulfate precipitation. After resuspending, the protein solution was inactivated by diethyleneimine (BEI) for 36-48 hours, and then the same amount of sodium thiosulfate was used. Neutralize, then use 100kD molecular weight filter membrane (EMD Millipore Company) to concentrate 50 times through Labscale TFF filter cutting device, and then purify RPPSV VLPs particles by liquid chromatography to obtain RPPSV VLPs.

(2) Preparation of IgM-RPPSV VLPs immune complexes:

RPPSV VLPs and monoclonal antibody 5D9 (by mass) were mixed at a ratio of 1:5 and placed at 37 °C for 2 hours to form a complex.

(3) Preparation of vaccines

The complexed "IgM-RPPSV VLPs" immune complex (calculated by volume) was mixed with Montanide™ ISA 206 water-in-oil adjuvant at a ratio of 46:54 to obtain a vaccine composition after mixing and emulsification.

(4) Antibody fluctuation rule of piglets immunized with IgM-RPPSV VLPs immune complex

4.1 Screen 6 one-month-old healthy piglets with double-negative PRRSV antigen and antibody, and randomly divide them into 2 groups with 3 pigs in each group. The first group was the experimental group, RPPSV VLPs (10 μg) + ISA 206 + 5D9 (50 μg), and the second group was the control group, RPPSV VLPs (10 μg) + ISA 206.

Booster immunization was performed 30 days after the first immunization. Blood was collected on the 7th, 14th, 21st, 28th, and 60th after the first immunization, and the commercial PRRSV antibody Elisa kit (BioChek) was used for antibody detection. The OD value was The antibody titer at the maximum dilution of the positive serum was calculated, and the average antibody titer of each group was calculated. The detection results are shown in Figure 5.

Based on the results in Figure 5, it can be seen that the use of IgM-RPPSV VLPs immune complex to immunize piglets can stimulate the body to produce higher titers of antibodies earlier than immunization with RPPSV VLPs alone.

sequence listing

<110> Shaanxi Nuoweilihua Biotechnology Co., Ltd.

NWAFU

<120> Recombinant PRRSV virus-like particle antigen-antibody complex and preparation method thereof

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1197

<212> DNA

<213> Artificial Sequence

<400> 1

atgttgggga agtgcttgac cgcgtgctgt tgctcgcgat tgcttttttt gtggtgtatc 60

gtgccgtctt atcttgctgt gctcgtcaac gccgaagctg ccgctaagga agctgccgct 120

aagagctctc atattcagtt gatttataac ttaacgctat gtgagctgaa tggcacagat 180

tggctggcac aaaaatttga ctgggcagtg gagacttttg tcatcttccc cgtgttgact 240

cacattgttt cctatggggc actcaccacc agccatttcc ttgacacagt tggtctggcc 300

actgtgtcca ccgccggata ttatcacggg cggtatgtct tgagtagcat ttacgcagtc 360

tgtgctctgg ctgcgctgat ttgctttgtc attaggcttg cgaagaactg catgtcctgg 420

cgctactctt gtaccagata taccaacttc cttctggaca ctaagggcag actctatcgt 480

tggcggtcgc ccgtcattgt ggagaaaggg ggtaaggttg aggtcgaagg tcacctgatc 540

gacctcaaga gagttgtgct tgatggttcc gcggcaaccc ctttaaccag agtttcagcg 600

gaacaatggg gtcgtctcgg aggtggagga tcatctcata ttcagttgat ttataactta 660

ggaggtggag gatcaatggg gtcgtctcta gacgacttct gcaatgatag cacagctcca 720

cagaaggtgc ttttggcgtt ttccattacc tacacgccag tgatgatata tgctctaaag 780

gtaagtcgcg gccgactgct agggcttctg caccttttga tcttcctgaa ttgtgctttt 840

accttcgggt acatgacatt cgtgcacttt gagagcacaa atagggtcgc gctcactatg 900

ggagcagtag ttgcacttct ttggggagtg tactcagcca tagaaacctg gaaattcatc 960

acctccagat gccgtttgtg cttgctaggc cgcaagtaca ttctggcccc tgcccaccac 1020

gtcgaaagtg ccgcgggctt tcatccgatt gcggcaaatg ataaccacgc atttgtcgtc 1080

cggcgtcccg gctccactac ggtcaacggc acattggtgc ccgggttgaa aagcctcgtg 1140

ttgggtggca gaaaagctgt taagcaggga gtggtaaacc ttgttaaata tgccaaa 1197

<210> 2

<211> 399

<212> PRT

<213> Artificial Sequence

<400> 2

Met Leu Gly Lys Cys Leu Thr Ala Cys Cys Cys Ser Arg Leu Leu Phe

1 5 10 15

Leu Trp Cys Ile Val Pro Ser Tyr Leu Ala Val Leu Val Asn Ala Glu

20 25 30

Ala Ala Ala Lys Glu Ala Ala Ala Lys Ser Ser His Ile Gln Leu Ile

35 40 45

Tyr Asn Leu Thr Leu Cys Glu Leu Asn Gly Thr Asp Trp Leu Ala Gln

50 55 60

Lys Phe Asp Trp Ala Val Glu Thr Phe Val Ile Phe Pro Val Leu Thr

65 70 75 80

His Ile Val Ser Tyr Gly Ala Leu Thr Thr Ser His Phe Leu Asp Thr

85 90 95

Val Gly Leu Ala Thr Val Ser Thr Ala Gly Tyr Tyr His Gly Arg Tyr

100 105 110

Val Leu Ser Ser Ile Tyr Ala Val Cys Ala Leu Ala Ala Leu Ile Cys

115 120 125

Phe Val Ile Arg Leu Ala Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys

130 135 140

Thr Arg Tyr Thr Asn Phe Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg

145 150 155 160

Trp Arg Ser Pro Val Ile Val Glu Lys Gly Gly Lys Val Glu Val Glu

165 170 175

Gly His Leu Ile Asp Leu Lys Arg Val Val Leu Asp Gly Ser Ala Ala

180 185 190

Thr Pro Leu Thr Arg Val Ser Ala Glu Gln Trp Gly Arg Leu Gly Gly

195 200 205

Gly Gly Ser Ser His Ile Gln Leu Ile Tyr Asn Leu Gly Gly Gly Gly

210 215 220

Ser Met Gly Ser Ser Leu Asp Asp Phe Cys Asn Asp Ser Thr Ala Pro

225 230 235 240

Gln Lys Val Leu Leu Ala Phe Ser Ile Thr Tyr Thr Pro Val Met Ile

245 250 255

Tyr Ala Leu Lys Val Ser Arg Gly Arg Leu Leu Gly Leu Leu His Leu

260 265 270

Leu Ile Phe Leu Asn Cys Ala Phe Thr Phe Gly Tyr Met Thr Phe Val

275 280 285

His Phe Glu Ser Thr Asn Arg Val Ala Leu Thr Met Gly Ala Val Val

290 295 300

Ala Leu Leu Trp Gly Val Tyr Ser Ala Ile Glu Thr Trp Lys Phe Ile

305 310 315 320

Thr Ser Arg Cys Arg Leu Cys Leu Leu Gly Arg Lys Tyr Ile Leu Ala

325 330 335

Pro Ala His His Val Glu Ser Ala Ala Gly Phe His Pro Ile Ala Ala

340 345 350

Asn Asp Asn His Ala Phe Val Val Arg Arg Pro Gly Ser Thr Thr Val

355 360 365

Asn Gly Thr Leu Val Pro Gly Leu Lys Ser Leu Val Leu Gly Gly Arg

370 375 380

Lys Ala Val Lys Gln Gly Val Val Asn Leu Val Lys Tyr Ala Lys

385 390 395

<210> 3

<211> 1182

<212> DNA

<213> Artificial Sequence

<400> 3

atgttgggga agtgcttgac cgcgtgctgt tgctcgcgat tgcttttttt gtggtgtatc 60

gtgccgtctt atcttgctgt gctcgtcaac gccgaagctg ccgctaagag ctctcatatt 120

cagttgattt ataacttaac gctatgtgag ctgaatggca cagattggct ggcacaaaaa 180

tttgactggg cagtggagac ttttgtcatc ttccccgtgt tgactcacat tgtttcctat 240

ggggcactca ccaccagcca tttccttgac acagttggtc tggccactgt gtccaccgcc 300

ggatattatc acgggcggta tgtcttgagt agcatttacg cagtctgtgc tctggctgcg 360

ctgatttgct ttgtcattag gcttgcgaag aactgcatgt cctggcgcta ctcttgtacc 420

agatatacca acttccttct ggacactaag ggcagactct atcgttggcg gtcgcccgtc 480

attgtggaga aagggggtaa ggttgaggtc gaaggtcacc tgatcgacct caagagagtt 540

gtgcttgatg gttccgcggc aaccccttta accagagttt cagcggaaca atggggtcgt 600

ctcggaggtg gaggatcatc tcatattcag ttgatttata acttaggagg tggaggatca 660

atggggtcgt ctctagacga cttctgcaat gatagcacag ctccacagaa ggtgcttttg 720

gcgttttcca ttacctacac gccagtgatg atatatgctc taaaggtaag tcgcggccga 780

ctgctagggc ttctgcacct tttgatcttc ctgaattgtg cttttacctt cgggtacatg 840

acattcgtgc actttgagag cacaaatagg gtcgcgctca ctatgggagc agtagttgca 900

cttctttggg gagtgtactc agccatagaa acctggaaat tcatcacctc cagatgccgt 960

ttgtgcttgc taggccgcaa gtacattctg gcccctgccc accacgtcga aagtgccgcg 1020

ggctttcatc cgattgcggc aaatgataac cacgcatttg tcgtccggcg tcccggctcc 1080

actacggtca acggcacatt ggtgcccggg ttgaaaagcc tcgtgttggg tggcagaaaa 1140

gctgttaagc agggagtggt aaaccttgtt aaatatgcca aa 1182

<210> 4

<211> 394

<212> PRT

<213> Artificial Sequence

<400> 4

Met Leu Gly Lys Cys Leu Thr Ala Cys Cys Cys Ser Arg Leu Leu Phe

1 5 10 15

Leu Trp Cys Ile Val Pro Ser Tyr Leu Ala Val Leu Val Asn Ala Glu

20 25 30

Ala Ala Ala Lys Ser Ser His Ile Gln Leu Ile Tyr Asn Leu Thr Leu

35 40 45

Cys Glu Leu Asn Gly Thr Asp Trp Leu Ala Gln Lys Phe Asp Trp Ala

50 55 60

Val Glu Thr Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr

65 70 75 80

Gly Ala Leu Thr Thr Ser His Phe Leu Asp Thr Val Gly Leu Ala Thr

85 90 95

Val Ser Thr Ala Gly Tyr Tyr His Gly Arg Tyr Val Leu Ser Ser Ile

100 105 110

Tyr Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu

115 120 125

Ala Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn

130 135 140

Phe Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val

145 150 155 160

Ile Val Glu Lys Gly Gly Lys Val Glu Val Glu Gly His Leu Ile Asp

165 170 175

Leu Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Leu Thr Arg

180 185 190

Val Ser Ala Glu Gln Trp Gly Arg Leu Gly Gly Gly Gly Ser Ser His

195 200 205

Ile Gln Leu Ile Tyr Asn Leu Gly Gly Gly Gly Ser Met Gly Ser Ser

210 215 220

Leu Asp Asp Phe Cys Asn Asp Ser Thr Ala Pro Gln Lys Val Leu Leu

225 230 235 240

Ala Phe Ser Ile Thr Tyr Thr Pro Val Met Ile Tyr Ala Leu Lys Val

245 250 255

Ser Arg Gly Arg Leu Leu Gly Leu Leu His Leu Leu Ile Phe Leu Asn

260 265 270

Cys Ala Phe Thr Phe Gly Tyr Met Thr Phe Val His Phe Glu Ser Thr

275 280 285

Asn Arg Val Ala Leu Thr Met Gly Ala Val Val Ala Leu Leu Trp Gly

290 295 300

Val Tyr Ser Ala Ile Glu Thr Trp Lys Phe Ile Thr Ser Arg Cys Arg

305 310 315 320

Leu Cys Leu Leu Gly Arg Lys Tyr Ile Leu Ala Pro Ala His His Val

325 330 335

Glu Ser Ala Ala Gly Phe His Pro Ile Ala Ala Asn Asp Asn His Ala

340 345 350

Phe Val Val Arg Arg Pro Gly Ser Thr Thr Val Asn Gly Thr Leu Val

355 360 365

Pro Gly Leu Lys Ser Leu Val Leu Gly Gly Arg Lys Ala Val Lys Gln

370 375 380

Gly Val Val Asn Leu Val Lys Tyr Ala Lys

385 390

<210> 5

<211> 1185

<212> DNA

<213> Artificial Sequence

<400> 5

atgttgggga agtgcttgac cgcgtgctgt tgctcgcgat tgcttttttt gtggtgtatc 60

gtgccgtctt atcttgctgt gctcgtcaac gccgaagctg ccgctaagga agctgccgct 120

aagagctctc atattcagtt gatttataac ttaacgctat gtgagctgaa tggcacagat 180

tggctggcac aaaaatttga ctgggcagtg gagacttttg tcatcttccc cgtgttgact 240

cacattgttt cctatggggc actcaccacc agccatttcc ttgacacagt tggtctggcc 300

actgtgtcca ccgccggata ttatcacggg cggtatgtct tgagtagcat ttacgcagtc 360

tgtgctctgg ctgcgctgat ttgctttgtc attaggcttg cgaagaactg catgtcctgg 420

cgctactctt gtaccagata taccaacttc cttctggaca ctaagggcag actctatcgt 480

tggcggtcgc ccgtcattgt ggagaaaggg ggtaaggttg aggtcgaagg tcacctgatc 540

gacctcaaga gagttgtgct tgatggttcc gcggcaaccc ctttaaccag agtttcagcg 600

gaacaatggg gtcgtctcgg aggtggagga tcaggaggtg gaggatcagg aggtggagga 660

tcaatggggt cgtctctaga cgacttctgc aatgatagca cagctccaca gaaggtgctt 720

ttggcgtttt ccattaccta cacgccagtg atgatatatg ctctaaaggt aagtcgcggc 780

cgactgctag ggcttctgca ccttttgatc ttcctgaatt gtgcttttac cttcgggtac 840

atgacattcg tgcactttga gagcacaaat agggtcgcgc tcactatggg agcagtagtt 900

gcacttcttt ggggagtgta ctcagccata gaaacctgga aattcatcac ctccagatgc 960

cgtttgtgct tgctaggccg caagtacatt ctggcccctg cccaccacgt cgaaagtgcc 1020

gcgggctttc atccgattgc ggcaaatgat aaccacgcat ttgtcgtccg gcgtcccggc 1080

tccactacgg tcaacggcac attggtgccc gggttgaaaa gcctcgtgtt gggtggcaga 1140

aaagctgtta agcagggagt ggtaaacctt gttaaatatg ccaaa 1185

<210> 6

<211> 395

<212> PRT

<213> Artificial Sequence

<400> 6

Met Leu Gly Lys Cys Leu Thr Ala Cys Cys Cys Ser Arg Leu Leu Phe

1 5 10 15

Leu Trp Cys Ile Val Pro Ser Tyr Leu Ala Val Leu Val Asn Ala Glu

20 25 30

Ala Ala Ala Lys Glu Ala Ala Ala Lys Ser Ser His Ile Gln Leu Ile

35 40 45

Tyr Asn Leu Thr Leu Cys Glu Leu Asn Gly Thr Asp Trp Leu Ala Gln

50 55 60

Lys Phe Asp Trp Ala Val Glu Thr Phe Val Ile Phe Pro Val Leu Thr

65 70 75 80

His Ile Val Ser Tyr Gly Ala Leu Thr Thr Ser His Phe Leu Asp Thr

85 90 95

Val Gly Leu Ala Thr Val Ser Thr Ala Gly Tyr Tyr His Gly Arg Tyr

100 105 110

Val Leu Ser Ser Ile Tyr Ala Val Cys Ala Leu Ala Ala Leu Ile Cys

115 120 125

Phe Val Ile Arg Leu Ala Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys

130 135 140

Thr Arg Tyr Thr Asn Phe Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg

145 150 155 160

Trp Arg Ser Pro Val Ile Val Glu Lys Gly Gly Lys Val Glu Val Glu

165 170 175

Gly His Leu Ile Asp Leu Lys Arg Val Val Leu Asp Gly Ser Ala Ala

180 185 190

Thr Pro Leu Thr Arg Val Ser Ala Glu Gln Trp Gly Arg Leu Gly Gly

195 200 205

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Gly Ser

210 215 220

Ser Leu Asp Asp Phe Cys Asn Asp Ser Thr Ala Pro Gln Lys Val Leu

225 230 235 240

Leu Ala Phe Ser Ile Thr Tyr Thr Pro Val Met Ile Tyr Ala Leu Lys

245 250 255

Val Ser Arg Gly Arg Leu Leu Gly Leu Leu His Leu Leu Ile Phe Leu

260 265 270

Asn Cys Ala Phe Thr Phe Gly Tyr Met Thr Phe Val His Phe Glu Ser

275 280 285

Thr Asn Arg Val Ala Leu Thr Met Gly Ala Val Val Ala Leu Leu Trp

290 295 300

Gly Val Tyr Ser Ala Ile Glu Thr Trp Lys Phe Ile Thr Ser Arg Cys

305 310 315 320

Arg Leu Cys Leu Leu Gly Arg Lys Tyr Ile Leu Ala Pro Ala His His

325 330 335

Val Glu Ser Ala Ala Gly Phe His Pro Ile Ala Ala Asn Asp Asn His

340 345 350

Ala Phe Val Val Arg Arg Pro Gly Ser Thr Thr Val Asn Gly Thr Leu

355 360 365

Val Pro Gly Leu Lys Ser Leu Val Leu Gly Gly Arg Lys Ala Val Lys

370 375 380

Gln Gly Val Val Asn Leu Val Lys Tyr Ala Lys

385 390 395

Claims (6)

1. A recombinant PRRSV virus-like particle antigen-antibody complex, characterized in that the antigen-antibody complex is formed by amino acid sequences shown as SEQ ID No: 2 and Ig M antibody.

2. The recombinant PRRSV virus-like particle antigen-antibody complex of claim 1, wherein the Ig M antibody is monoclonal antibody 5D 9.

3. The recombinant PRRSV virus-like particle antigen-antibody complex of claim 1, wherein the recombinant PRRSV virus-like particle is prepared from a recombinant baculovirus expressing the protein of PRRSV GP5 and M of porcine reproductive and respiratory syndrome virus, and the method for preparing the recombinant baculovirus expressing the protein of PRRSV GP5 and M of porcine reproductive and respiratory syndrome virus comprises the steps of:

step one, constructing a recombinant baculovirus expressing PRRSV GP5 and M protein by the following steps:

(1) artificially modified PRRSV GP5-M gene, which is modified based on the sequence of PRRSV-SD16 strain, and the gene sequence is designed to be as shown in SEQ ID No: 1, PRRSV GP5-M gene;

(2) construction of baculovirus transfer vectors: the synthetic gene sequence is shown as SEQ ID No: 1, and respectively arranging BamH I and Hind III enzyme cutting sites at the 5 'end and the 3' end to obtain an insert; taking a pBAC-5 plasmid as a framework, performing enzyme digestion on a vector and an insert fragment through BamH I and Hind III, performing overnight connection at 16 ℃ through T4 ligase, performing transformation screening to obtain a positive clone, extracting a recombinant plasmid, and performing enzyme digestion identification to obtain a baculovirus transfer vector pBAC-5-PRRSV GP 5-M;

step two, constructing the recombinant baculovirus Ac-PRRSV GP 5-M:

(1) obtaining and identifying recombinant bacmid-PRRSV GP 5-M: mixing a baculovirus transfer vector pBAC-5-PRRSV VGP5-M plasmid DNA with DH10 Bac competent cells, carrying out water bath for 30 minutes after ice bath, carrying out water bath heat shock for 45 seconds at 42 ℃, then carrying out ice bath for 5 minutes, adding an SOC liquid culture medium, carrying out oscillation culture for 2 hours at 37 ℃, coating an LB (Luria-Luma-Blume) plate with each gradient bacterial liquid after 10-time serial dilution, screening and purifying positive bacterial colonies by using a screening kit of life company, and extracting recombinant bacmid rBac-PRRSV GP 5-M;

(2) obtaining a recombinant baculovirus Ac-PRRSV GP 5-M: transfecting the recombinant bacmid rBac-PRRSV GP5-M extracted in the last step into sf9 cells by using a liposome transfection reagent Lipofectamine3000, continuously culturing and observing at 28 ℃, collecting cell supernatant 72 hours after transfection to obtain recombinant baculovirus, then inoculating healthy sf9 cells again for amplification culture, collecting virus liquid as a virus seed, and storing at-70 ℃ for later use.

4. The method for preparing the recombinant PRRSV virus-like particle antigen-antibody complex of claim 3, wherein the method comprises:

(1) preparation of RPPSV VLPs: inoculating the prepared recombinant baculovirus Ac-PRRSV GP5-M of claim 3 to healthy sf9 cells according to a proportion of 10%, culturing for 4-5 days at 27 ℃, repeatedly freezing and thawing to collect cells and supernatant, centrifuging for 20 minutes at 12000r/min at 4 ℃, collecting supernatant, then precipitating target protein by using an ammonium sulfate precipitation method, inactivating the protein solution by using binary ethyleneimine after resuspension for 36-48 hours, then neutralizing by using equivalent sodium thiosulfate, then concentrating by using a 100kD molecular weight filter membrane by using a Labscale TFF cutting filter instrument for 50 times, and purifying RPPSV VLPs particles by liquid chromatography to obtain RPPSV VLPs;

(2) preparation of immune complexes of IgM-RPPSV VLPs: RPPSV VLPs were mixed with monoclonal antibody 5D9 at a ratio of 1:5 by mass, and left at 37 ℃ for 2 hours to form a complex.

5. A vaccine comprising the IgM-RPPSV VLPs immunocomplexes produced by the method of claim 4 and an adjuvant.

6. A method for preparing the vaccine of claim 5, comprising the steps of: and mixing and emulsifying the compounded IgM-RPPSV VLPs immune complex and Montanide ™ ISA 206 water-in-oil adjuvant in a ratio of 46:54 by volume calculation to obtain the vaccine.

Images