CN113425838A

CN113425838A - Recombinant PRRSV virus-like particle antigen-antibody complex and preparation method thereof

Info

Publication number: CN113425838A
Application number: CN202110663821.3A
Authority: CN
Inventors: 陈瑞; 王晶钰; 南雨辰; 武春燕
Original assignee: Shaanxi Nuweilihua Biotechnology Co ltd; Northwest A&F University
Current assignee: Shaanxi Nuweilihua Biotechnology Co ltd; Northwest A&F University
Priority date: 2021-06-16
Filing date: 2021-06-16
Publication date: 2021-09-24
Anticipated expiration: 2041-06-16
Also published as: CN113425838B

Abstract

According to the invention, the GP5-M protein is modified to obtain the recombinant baculovirus capable of efficiently replicating, the culture titer of the recombinant baculovirus is relatively higher, the prepared PRRSV virus particle-like virus particles are further compounded with IgM to prepare an IgM-RPPSV VLPs immune complex, and the IgM-RPPSV VLPs immune complex can stimulate an organism to generate high-concentration antibodies earlier.

Description

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

The technical field is as follows:

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

Background art:

porcine Reproductive and Respiratory Syndrome (PRRS), also known as blue ear disease, is a pathogen of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), which mainly causes reproductive disorders in pregnant sows and respiratory disorders in piglets, often manifested as cough, dyspnea in piglets, premature birth of sows, abortion and even stillbirth. It was discovered in north america since the end of the 20 th century, and to date, the worldwide prevalence and several outbreaks of the disease have led to a tremendous economic loss in the swine industry around the world.

The PRRSV belongs to the arterivirus family, and simian hemorrhagic fever virus, equine arteritis virus and lactate dehydrogenase-promoting virus belong to the same family. And, it was shown from the latest classification results that the RRSV was classified into 2 species of beta arterivirus (Betaarteri virus) under order Nidovirales (Nidovirales), family Arterividae (artividae), and members of the same genus also included Rat arterivirus (Rat arteri virus, RatAV). With the continuous progress of diagnostic technology, the gene and antigen of PRRSV strain are found to have wide diversity, the PRRSV is generally divided into European type and American type, the homology, antigenicity, toxicity and other aspects of the genome of the two types are obviously different, and the homology difference on the nucleotide level is 30-45%. The PRRSV is divided into different pedigrees by scholars, and the current prevalence in china mainly includes: classical PRRSV-2 (lineage 5), highly pathogenic PRRSV (lineage 8), qyyyz-like PRRSV-2 (lineage 3), and NADC30-like PRRSV-2 (lineage 1). The virus is first isolated in China in 1996, is completely outbreak in 2006 in China, and is mainly characterized by high infectivity, high body temperature, high morbidity and high mortality, and even troubles the pig industry at present. The strain is divided into two subtypes of North American type and European type according to gene characteristics, and currently, the North American type PRRSV such as HP-PRRS strain, NADC30-like strain and the like is mainly taken as a main epidemic strain in China.

The structural gene of PRRSV comprises 8 open reading frames, including ORF2, ORF2a, ORF 3-ORF 5, ORF5a, ORF6 and ORF7, and mainly encodes functional protein of virus. Wherein GP2, GP3, GP4 and GP5 belong to envelope glycoproteins modified by glycosylation, and M protein, N protein, GP2a and GP5a belong to non-glycosylated envelope proteins (not modified by glycosylation). Among them, the ratio of GP5 and M protein in the virion is mainly the envelope protein (Mokhtar et al 2016), GP5 can generate high-level neutralizing antibody and cellular immunity (Popescu et al 2017; Kim et al 2013).

VLPs vaccine is a novel genetic engineering vaccine, which recombines structural protein genes capable of generating immune effect in PRRSV into an expression vector for expression, and can accurately simulate the structure of virus particle antigen. VLPs vaccine has no genetic material, has good immune effect, and is expected to become a novel vaccine for preventing PRRS. The main difficulties in developing such vaccines are selection and optimization of gene sequences, selection of expression systems, improvement of expression levels, large-scale purification of VLPs, reduction of cost, selection and addition ratio of adjuvants, evaluation of vaccine efficacy, and the like. The VLPs vaccine stimulates the production of a B lymphocyte-mediated response, CD4⁺T cell proliferation response, cytotoxic somatic response. VLPs and B lymphocyte receptors are in cross-connection, can efficiently recognize MHC class I classical pathways, and take dendritic cells as target cells to cause efficient cellular and humoral immune responses. GP5 and M protein genes are recombined to an escherichia coli body, and the expressed protein can trigger high-titer neutralizing antibody reaction and Th1 cell-mediated immune reaction. A, B two antagonistic epitopes are arranged on the surface of GP5 protein, A epitope weakens the generation of neutralizing antibody, has a covering effect on B epitope which can cause neutralizing effect, and secondly, A epitope also has an effect before B epitope. And researches show that (HP-PRRSV envelope protein 5 glycosylation site mutation and function initial detection) site-directed mutation on the amino acid of the A epitope has certain influence on virus replication, so that the virus titer is reduced, therefore, how to reduce the antagonism of the A epitope on the B epitope is a technical problem to be solved urgently in recombinant PRRSV virus-like particle vaccines, and the inventors and 2018 prepare recombinant PRRSV virus-like particles (Z) with immunogenicityL201811114020.6), however, the problem of late generation of neutralizing antibody titers still faces 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 the correct ratio. After the antigen-antibody complex is formed, the Fc fragment of the antibody molecule has high affinity with the Fc receptor of an Antigen Presenting Cell (APC), so that the virus combined with the antibody can be more effectively combined with the APC, and after the antigen-antibody complex is phagocytized and internalized by the APC, B lymphocytes and T lymphocytes can be activated, and the B lymphocytes are stimulated to become plasma cells, thereby causing strong humoral immunity and cellular immune response. The immune complex formed in vitro stimulates the humoral immune response of the body 100 times of that of the natural antigen. In addition, Fc receptors are widely distributed on the surface of immune effector cells such as APC, and antibodies can mediate the effects of immune effector cells such as antigen uptake by the cells by binding to Fc receptors, and participate in the regulation of immune responses induced by antigens.

At present, the antigen-antibody compound vaccines of avian diseases and other species diseases are researched more, and mainly comprise chicken infectious bursal disease IC vaccines, newcastle disease IC vaccines, Marek IC vaccines, chicken infectious laryngotracheitis IC vaccines, chicken reovirus IC vaccines, gosling plague IC vaccines, dog parvovirus IC vaccines and the like. Among them, the antigen-antibody complex vaccine of bursa of fabricius and Newcastle disease developed by Roman corporation in the United states has been approved by the United states department of agriculture to come into the market, and is one of the vaccines widely used worldwide at present. However, few reports have been made on antigen-antibody complex vaccines for swine. The antigen-antibody complex vaccine has disadvantages, for example, the traditional method for preparing the vaccine by mixing the serum polyclonal antibody and the pathogen is influenced by the preparation of the serum polyclonal antibody, and has the defects of complex preparation process, instable serum antibody content among different batches, easy introduction of exogenous pathogen pollution to the serum of animal origin and the like, so the antigen-antibody complex vaccine has certain limitations, and the problem does not exist by using the monoclonal antibody. The monoclonal antibody 5D9 which has neutralizing activity to PRRSV-I and PRRSV-II viruses is prepared by professor Nanyuchen (CN 111138535A an immunopotentiator and application thereof in vaccine preparation, application number is CN 202010069365.5) of northwest agriculture and forestry science and technology university, and is used as an immunopotentiator, when the formed immune complex is combined with an adjuvant to immunize a mouse, IFN-gamma secretory T cells are obviously increased, which indicates that in the process of inactivated virus immunization, the PRRSV specific antibody 5D9 and a normal oil-in-water adjuvant are combined to immunize to enhance CTL reaction. Animal experiments show that the immune protection rate of the prepared immune complex can achieve higher protective efficacy compared with the vaccine prepared by only adopting the commercial adjuvant ISA 206 and the commercial attenuated vaccine. On the basis, the invention further combines specific antibodies with PRRSV VLPs to explore the protective efficacy of the antibodies on animals.

The invention content is as follows:

based on the development condition of PRRSV vaccine in China at present, the invention aims to provide a recombinant PRRSV virus-like particle antigen-antibody complex and a preparation method thereof, through researching GP5 and M protein, the invention artificially modifies the genes of GP5 and M protein, and adopts a baculovirus expression system to express to obtain the virus-like particle containing GP5-M recombinant protein, and the virus-like particle can generate neutralizing antibody earlier than the unmodified protein sequence, has better immunogenicity and generates higher titer of the antibody; the invention further combines the RPPSV VLPs obtained by preparation with the monoclonal antibody 5D9 provided by professor south Yu Chen to form an immune complex of IgM-RPPSV VLPs, and the immune response of cells induced by the vaccine is improved by the complex vaccine of IgM-RPPSV VLPs.

In order to solve the technical problems, the invention adopts the following technical scheme.

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.

The Ig M antibody is a monoclonal antibody 5D9 which has neutralizing activity to PRRSV-I and PRRSV-II viruses and is prepared by professor Nanyuchen professor (CN 111138535A an immunopotentiator and application thereof in vaccine preparation, application number is CN 202010069365.5) of northwest agriculture and forestry science and technology university.

The recombinant PRRSV virus-like particle is prepared from recombinant baculovirus expressing PRRSV and M proteins of porcine reproductive and respiratory syndrome virus, and the preparation method of the recombinant baculovirus expressing the PRRSV and M proteins of the porcine reproductive and respiratory syndrome virus is characterized by comprising the following steps:

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

(1) artificially modified PRRSV GP5-M gene, is modified on the basis of the sequence of PRRSV-SD16 strain (Genbank Access No: JX 087437.1), and is designed to obtain a gene sequence shown as 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 GP5-M plasmid DNA with DH10 Bac competent cells, carrying out water bath for 30 minutes in an 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 LB plates with gradient bacterial liquids 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 ℃, observing green fluorescence signals of the recombinant baculovirus by using a fluorescence microscope at 24 hours, 48 hours and 72 hours after transfection, collecting cell supernatant at 72 hours after transfection to obtain recombinant baculovirus, then inoculating the recombinant baculovirus into healthy sf9 cells again for amplification culture, collecting virus liquid as virus seeds, and storing at-70 ℃ for later use.

The invention also claims a preparation method of the recombinant PRRSV virus-like particle antigen-antibody complex, which is characterized by comprising the following steps:

(1) preparation of RPPSV VLPs: inoculating the prepared recombinant baculovirus Ac-PRRSV GP5-M 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 at 12000r/min at 4 ℃ for 20 minutes, collecting supernatant, then precipitating target protein by using an ammonium sulfate precipitation method, inactivating the protein solution by using Binary Ethyleneimine (BEI) after heavy suspension for 36-48 hours, then neutralizing by using equivalent sodium thiosulfate, then concentrating by using a 100kD molecular weight filter membrane (EMD Millipore company) by using a 100kD molecular weight filter membrane for 50 times by using a Labscale TFF cutting filter instrument, and purifying RPPSV VLPs particles by using liquid chromatography to obtain RPPSV VLPs;

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

The invention further provides a vaccine, which is characterized by consisting of IgM-RPPSV VLPs immune complex and an adjuvant.

The invention also claims a preparation method of the vaccine, which comprises the following steps: the compounded immune complex of IgM-RPPSV VLPs (calculated by volume) is mixed and emulsified with Montanide ™ ISA 206 water-in-oil adjuvant at a ratio of 46:54 to obtain the vaccine composition.

Based on the technical scheme, the invention has the following advantages and beneficial effects:

firstly, the inventor further focuses on the research of GP5-M virus-like particles on the basis of the original invention patent, and modifies GP5-M protein through artificial design and modification, particularly, the A epitope and the B epitope on GP5 are connected through rigid linker through the artificial modification of sequences, so as to improve the influence of the A epitope on the B epitope, and the virus-like particles can stimulate organisms to generate neutralizing antibodies earlier, compared with the virus-like particles obtained through the original research of the inventor, the virus-like particles can generate neutralizing antibodies with higher titer, after 6 weeks of first immunization, the neutralizing antibody titer reaches over 1:32, wherein the neutralizing antibody titer of 3 mice reaches 1:64, and 2 mice reaches 1:128, and the recombinant PRRSV virus-like particle vaccine has relatively good immune protection effect from the time and titer level generated by the neutralizing antibodies, it helps animals resist virus and prevent disease progression in early stages of infection;

secondly, the prepared GP5-M virus-like particles and the monoclonal antibody 5D9 are further rechecked to prepare an IgM-RPPSV VLPs immune complex, and the IgM-RPPSV VLPs immune complex is adopted to immunize piglets, so that compared with the method of singly adopting the RPPSV VLPs to immunize, the piglet immune complex can stimulate an organism to generate antibodies with higher titer

In conclusion, the recombinant baculovirus capable of efficiently replicating is obtained by modifying GP5-M protein, the culture titer of the recombinant baculovirus is relatively higher, and the IgM-RPPSV VLPs immune complex is further prepared by compounding the prepared PRRSV virus particle-like particles similar to Ig M, and can stimulate an organism to generate antibodies with high concentration earlier.

Description of the drawings:

FIG. 1: construction and identification of recombinant transfer vectors: (A) adopting a primer provided by a gene synthesis company, and obtaining a GP5-M gene fragment of about 1200bp through colony PCR amplification, wherein M is a marker, a channel 1 is a colony PCR amplification result, and a channel 2 is a blank control; (B) and (3) performing enzyme digestion on the recombinant vector, wherein M is a marker, the channel 1 is the extracted and purified recombinant vector which is not subjected to enzyme digestion, the channel 2 is the recombinant vector subjected to enzyme digestion, and the GP5-M gene fragment of about 1200bp and the vector fragment of about 5500bp are obtained after enzyme digestion.

FIG. 2 expression and characterization of recombinant proteins: a is a blank cell; b is a cell infected by the recombinant baculovirus.

FIG. 3: and (5) observing the result of the recombinant protein by using an electron microscope.

FIG. 4: western blot identification result: m is marker, and channel 1 is PRRSV VLP detection result.

FIG. 5: rule of antibody digestion and expansion for immunized piglets with IgM-RPPSV VLPs immune complexes: the first group was the test group, RPPSV VLPs (10. mu.g) + ISA 206 +5D9 (50. mu.g), and the second group was the control group, RPPSV VLPs (10. mu.g) + ISA 206.

The specific implementation mode is as follows:

the present invention will be more clearly understood from the following examples. The technical steps of the invention are conventional in the art, and are either commercial or published reagent materials, unless otherwise specified.

Example 1: construction of recombinant baculovirus Ac-PRRSV GP5-M

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

based on the sequence of PRRSV-SD16 strain (Genbank Access No: JX 087437.1), after artificial modification, the gene sequence is shown as SEQ ID No: 1, sending the PRRSV GP5-M gene shown in the figure to a gene synthesis company for gene sequence synthesis, and respectively arranging Bam H I and Hind III enzyme cutting sites at the 5 'end and the 3' end to obtain an insert.

(2) Construction and identification of recombinant transfer vectors:

plasmid containing a target gene sequence GP5-M and a vector pBAC-5 (provided by Shaanxi Nuo Willi-Hua Biotech Co., Ltd., a vector is provided with a GFP marker gene and can be used for fluorescence detection, see ZL 201811114020.6) are subjected to BamH I and HindIII double enzyme digestion respectively, after recovery and purification, T4 ligase is used for ligation reaction, the cells are transformed to DH5 alpha competent cells by a chemical method, plasmids are extracted after screening positive clones, and a colony PCR (shown in figure 1-A) and plasmid enzyme digestion identification (shown in figure 1-B) are carried out to obtain a successfully constructed recombinant transfer vector pBAC-5-PRRSV GP 5-M.

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

(3) Construction and identification of recombinant Bacmid

Plasmid DNA of the recombinant transfer vector pBAC-5-PRRSV GP5-M is respectively transformed into DH10 Bac competent cells, and the recombinant bacmid-PRRSV GP5-M containing PRRSV target genes is obtained by homologous recombination of the transfer vector and a skeleton vector in the competent cells.

(4) Preparation of recombinant baculovirus was prepared according to the method described in the inventor's prior patent (ZL 201811114020.6), and the specific procedure was as follows:

4.1 recombinant bacmid transfected insect cells: sf9 cells were plated in six-well plates and when the confluence reached 80%, transfection was performed with Lipofectamine3000 as follows:

4.1.1 Add 2. mu.g recombinant bach-PRRSV P5-M and 2. mu. L p3000 to 250. mu.L of opti-MEM medium without serum and antibiotics, mix gently;

4.1.2 Add 4. mu.l Lipofectamine3000 liposomes to 250. mu.l Opti-MEM medium without serum and antibiotics, mix gently;

4.1.3, uniformly mixing the liquid obtained in the step 4.1.1 and the liquid obtained in the step 4.1.2, and standing at room temperature for 10-15 minutes;

4.1.4 the mixture of step 4.1.3 was dropped into the sf9 cells in a six-well plate, and then the sf9 cells in the six-well plate were incubated at a constant temperature of 27 ℃ and observed continuously.

Harvesting and amplification of recombinant baculovirus

4.2.1 harvesting of recombinant baculovirus, namely carefully observing transfected cells every day, collecting the cells and supernatant when the cells become large and irregular and even begin to float to the liquid level, and the like, extracting sample RNA and carrying out RT-PCR detection to identify whether a target gene GP5-M exists, repeatedly freezing and thawing the collected cells and supernatant when obvious cytopathic effect can be seen in 4-5 days after transfection, centrifuging at 4 ℃ for 10 minutes at 12000r/min, collecting the supernatant and marking as P1 generation recombinant baculovirus;

4.2.2 amplification of recombinant baculovirus, namely inoculating the recombinant baculovirus of P1 generation into sf9 cells according to the ratio of 1:10, culturing for 4-5 days at 27 ℃, and harvesting the recombinant baculovirus of P2 generation when cytopathic effect is obviously generated; recombinant baculoviruses of P3 generation and higher were obtained in the same manner, and the collected recombinant baculoviruses were stored at-70 ℃ for further use.

4.2.3 titer determination of recombinant baculovirus: respectively carrying out 10-fold serial dilution on P2 and P3 generation viruses, then inoculating 96-well plate sf9 cells, inoculating 8 wells for each dilution, placing at the constant temperature of 27 ℃ for culture, observing the cytopathic condition every day, and then calculating the virus TCID according to a Karber method₅₀The values result in that the P2 and P3 generation virus titers of the recombinant baculovirus Ac-PRRSV GP5-M are respectively about 10^5.58TCID₅₀A ratio of 10 to 10^6.62TCID₅₀Ml, the virus titer of the present invention was greatly improved compared to the previous study.

(5) Expression and characterization of recombinant proteins

5.1 immunofluorescence detection of recombinant proteins: infecting the recombinant baculovirus with healthy sf9 cell in 10% proportion, setting blank control hole, culturing at 27 deg.c for 48-72 hr, observing, fixing the cell with pre-cooled 80% acetone for 2 hr when the cytopathic effect reaches over 80%, then PBST washed 3 times, 5% skim milk (using TBST preparation) at 4 degrees C overnight closed, then PBST washed 3 times, added 1:1000 dilution of GP5 monoclonal antibody (Shaanxi Nuo Weili Hua biotechnology limited company provides), 37 degrees C protected from light incubation for 1 hours, then PBST is washed for 3 times, FITC fluorescent labeled goat anti-mouse secondary antibody diluted at the ratio of 1:1000 is added, incubation is carried out for 1 hour at the temperature of 37 ℃, after PBST was washed 3 times, the cells infected with the recombinant baculovirus showed a clear green fluorescence when observed under a fluorescence microscope and judged to be non-fluorescent in the control group, thereby confirming that the gene of interest was expressed in insect cells sf9 (FIG. 2).

5.2 recombinant protein electron microscopy observation: the freshly collected recombinant baculovirus liquid is sent to an electron microscope for observation, and the result shows that virus-like particles with the shape similar to that of PRRSV virus particles are detected, the diameter is about 46-66 nm, and the GP5 and the M protein can form the virus-like particles in vitro (figure 3).

5.3 Western blot identification: preparing 10% polyacrylamide gel, adding the PRRSV VLPs sample prepared by the invention, and performing SDS-PAGE electrophoresis; transferring the protein in the SDS-PAGE gel to a PVDF membrane by a wet transfer method; sealing for 2 h at room temperature by using sealing liquid; using a confining liquid 1: diluting a mouse anti-GP 5 monoclonal antibody (provided by Shaanxi Nuo Weili Rihua biotechnology limited) by 200 times, and respectively incubating a PVDF membrane with the monoclonal antibody at 4 ℃ overnight; PBST cleaning PVDF membrane; using a confining liquid 1: diluting an HRP-labeled goat anti-mouse IgG antibody by 4000 times, and incubating a PVDF membrane with the HRP-labeled goat anti-mouse IgG antibody for 1h at room temperature; PBST cleaning PVDF membrane; and uniformly dripping the Thermo exposure liquid on the PVDF membrane, and exposing. As a result, as shown in FIG. 4, the band of the target protein was detected at around 40kDa, which is consistent with the expected protein size.

In the course of research, the inventors have conducted research and trials on various genetic modification schemes, particularly for the a epitope and the B epitope of GP5 protein, wherein control group 1 only uses one rigid linker (eaaak) between the a and B epitopes to obtain control group 1, the nucleic acid sequence of which is shown in SEQ ID No: 3, and the amino acid sequence is shown as SEQ ID No: 4 is shown in the specification; in the control group 2, three flexible linkers (GGGGS) are used to replace GGGGSSHIQLIYNLGGGGS in the invention to connect the GP5 protein and the M protein after modification, and the nucleic acid sequences are shown as SEQ ID No: 5, the amino acid sequence is shown as SEQ ID No: 6 is shown in the specification; the control group 3 is a recombinant baculovirus constructed in the invention patent ZL 201811114020.6.

For the control group 1 and the control group 2, the corresponding recombinant baculovirus is prepared by the method of example 1, and is expressed in the insect cell sf9 to prepare the corresponding virus-like particle, and is used for preparing the corresponding virus-like particle vaccine. Control 3 was a sample of virus-like particles provided by Shaanxi Novowilli Biotech, Inc.

The P3 generation virus titer of the control group 1 recombinant baculovirus was determined to be about 10 based on the same culture conditions as in example 1^5.72TCID₅₀Per ml; the P3 generation virus titer of the control 2 recombinant baculovirus was about 10^5.76TCID₅₀/ml。

Example 2:

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

inoculating the recombinant baculovirus Ac-PRRSV GP5-M prepared in the example 1 into healthy sf9 cells according to the proportion of 10%, culturing for 4-5 days at 27 ℃, repeatedly freezing and thawing to collect the cells and supernatant, centrifuging for 20 minutes at 12000r/min at 4 ℃, collecting the supernatant, then precipitating target protein by using an ammonium sulfate precipitation method, inactivating the protein solution by using Binary Ethyleneimine (BEI) after resuspension for 36-48 hours, then neutralizing by using equivalent sodium thiosulfate, finally mixing with Seppic ISA 206 adjuvant for emulsification to prepare the vaccine, and placing at 2-8 ℃ for later use.

In the preparation process of the vaccine, the recombinant baculovirus of the invention in example 1, the control group 2 and the control group 3 are all adjusted to have the virus titer of 10 after being cultured⁵TCID₅₀Ml, and then continuing with subsequent operations such as freeze-thawing. During the preparation of the vaccine, the concentration of the protein is controlled to be 100 mug/mL.

(2) Vaccine appearance and safety experiments

The vaccine is tested according to the method described in the prior patent (ZL 201811114020.6) of the inventor, and is subjected to character test, aseptic test, exogenous virus test, mycoplasma test and safety test respectively, and the vaccine safety test is also performed on piglets and pregnant sows respectively, the part of the test is completed by Shaanxi Nuoweili Hua Biotechnology Co., Ltd, and the test result shows that the recombinant PRRSV virus-like particle vaccine meets the relevant regulations of the veterinary pharmacopoeia and is safe to the piglets and the pregnant sows.

(3) Immunization of mice

60 female Balb/C mice of 4-6 weeks old are taken, randomly divided into 6 groups of 10 mice, immunized twice, and boosted at an interval of 3 weeks after the first immunization. Wherein example 1-control group 3 were injected with the corresponding recombinant PRRSV virus-like particle vaccines, respectively; the attenuated vaccine group is inoculated with a commercial attenuated live vaccine of JXA1-R strain; the blank group was not injected with the inoculum.

(4) Neutralizing antibody assay

4.1 separating the serum to be detected, inactivating the serum at 56 ℃ for 30min, and storing the inactivated serum at-20 ℃ for later use;

4.2 using a DMEN basic culture medium to continuously dilute the serum to be detected in a multiple ratio on a 96-well plate, wherein the dilution ratio is from 1:2 to 1:256, and each well is 50 mu L;

4.3 dilution of PRRSV SD-16 Virus broth to 200 TCIDs₅₀In the above wells to which serum had been added, 50. mu.L of virus dilution was added to each well at 37 ℃ with 5% CO₂Infecting for 1h in an incubator;

4.4 Add 100. mu.L of Marc-145 cell suspension (ca. 5X 10) to each well⁵one/mL), gently and uniformly shaken, and then placed in 5% CO at 37 DEG C₂Continuously culturing for 6-8h in the incubator; wherein the positive serum of the pig is provided by Shaanxi Nuoweili Hua Biotech limited company, and the titer of the neutralizing antibody is determined to be 1: 16.

4.5 Observation and recording of cytopathic condition until the results stabilize. The specific results are as follows:

TABLE 1 recombinant PRRSV virus-like particle vaccine induced neutralizing antibody levels

Based on the results in table 1 above, the recombinant PRRSV virus-like particle vaccine of example 1 of the present invention can stimulate the organism to generate neutralizing antibodies earlier than the control group 1-3 and the attenuated vaccine, and 80% of mice generate neutralizing antibodies of 1:8 or more after 3 weeks of first immunization; after 6 weeks of first immunization, the neutralizing antibody titer reaches over 1:32, wherein the neutralizing antibody titer of 3 mice reaches 1:64, and the neutralizing antibody titer of 2 mice reaches 1:128, and the time and titer level of the neutralizing antibody are shown, the recombinant PRRSV virus-like particle vaccine of the embodiment 1 relatively has good immune protection effect, which is beneficial to resisting virus and preventing disease deterioration of animals in early infection.

Example 3: preparation of immune complexes of IgM-RPPSV VLPs

(1) Preparation of RPPSV VLPs:

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

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

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

(3) Preparation of vaccines

The compounded immune complex of IgM-RPPSV VLPs (calculated by volume) is mixed and emulsified with Montanide ™ ISA 206 water-in-oil adjuvant at a ratio of 46:54 to obtain the vaccine composition.

(4) Rule of antibody digestion and expansion for immune piglets by IgM-RPPSV VLPs immune complex

4.1 screening 6 first-month-old healthy piglets with double negative PRRSV antigen-antibody, randomly dividing into 2 groups, and 3 pigs in each group. The first group was the test group, RPPSV VLPs (10. mu.g) + ISA 206 +5D9 (50. mu.g), and the second group was the control group, RPPSV VLPs (10. mu.g) + ISA 206.

And (3) performing boosting immunization 30 days after the first immunization, collecting blood 7 days, 14 days, 21 days, 28 days and 60 days after the first immunization respectively, performing antibody detection by using a commercial PRRSV antibody Elisa kit (BioChek), and calculating the average antibody titer of each group by using the OD value as the antibody titer of the maximum dilution multiple of positive serum. The results of the detection are shown in FIG. 5.

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

Sequence listing

<110> Shaanxi Nuo Weili Hua Biotech Co., Ltd

Northwest A & F University

<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 (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 (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 (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 (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 (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 (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

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 recombinant baculovirus expressing PRRSV and M proteins of porcine reproductive and respiratory syndrome virus, and the method for preparing the recombinant baculovirus expressing PRRSV and M proteins of porcine reproductive and respiratory syndrome virus comprises the steps of:

(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;

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 the recombinant baculovirus into 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 according to claim 1, 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 the cells and supernatant, centrifuging at 12000r/min at 4 ℃ for 20 minutes, collecting the supernatant, then precipitating target protein by using an ammonium sulfate precipitation method, inactivating the protein solution by using Binary Ethyleneimine (BEI) after resuspension for 36-48 hours, then neutralizing by using equivalent sodium thiosulfate, then concentrating by using a 100kD molecular weight filter membrane and 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 immunocomplex of claim 1 and an adjuvant.

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