CN113512555B

CN113512555B - Recombinant PRRSV virus-like particle and preparation method thereof

Info

Publication number: CN113512555B
Application number: CN202110663798.8A
Authority: CN
Inventors: 陈瑞; 王晶钰; 南雨辰; 武春燕
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2021-06-16
Filing date: 2021-06-16
Publication date: 2022-05-06
Anticipated expiration: 2041-06-16
Also published as: CN113512555A

Abstract

According to the invention, the GP5-M protein is modified to obtain the recombinant baculovirus capable of being efficiently copied, the culture titer of the recombinant baculovirus is relatively higher, the vaccine is further prepared by utilizing the virus-like particles similar to the PRRSV virus particles, the vaccine can generate a neutralizing antibody with higher titer, and the antibody digestion and expansion regulation of immune piglets shows that the vaccine can stimulate an organism to generate a high-concentration antibody earlier, and can stimulate the organism to generate higher average antibody titer after secondary immunization, so that better immune protection is realized.

Description

Recombinant PRRSV virus-like particle 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 and a preparation method thereof.

The background art comprises the following steps:

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 family of arteriviruses, and the 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).

The PRRSV can infect pigs of various ages (Murtaugh et al.2011), invade the immune system of a pig body to cause persistent infection and secondary infection of other bacteria or viruses, so far, the development of the pig industry in China is still restricted, but no effective treatment measures exist, and the prevention and control are generally carried out in the mode of immune vaccines at present. Because PRRSV has the characteristics of gene diversity, easy variability, persistent infection, poor cross protection among heterologous strains and the like, the characteristics of viral proteins related to the induction of protective immunity are rarely understood at present, and a blank exists in the research on neutralizing antibodies related to the protective effect of vaccines, so that the vaccine general immunization adopted by most Chinese farms is one of the main methods for preventing and controlling PRRS at present. Currently, the following vaccine categories are mainly used: conventional inactivated vaccines, attenuated live vaccines, live vector vaccines, virus-like particles (VLPs) vaccines, nucleic acid vaccines, subunit vaccines, antigen-antibody complex vaccines, and the like. At present, traditional vaccines are commercially applied, and most of genetic engineering vaccines are still in the research and development stage and cannot be commercially applied for a while.

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 gene are recombined to colibacillus body, and the expressed protein can triggerHigh titer neutralizes antibody responses and Th1 type cell-mediated immune responses. 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. Research shows that (glycosylation site mutation and function initial detection of HP-PRRSV envelope protein 5), site-directed mutation of amino acid of an A epitope has certain influence on virus replication, so that virus titer is reduced, and therefore, how to reduce antagonism of the A epitope on the B epitope is a technical problem to be solved in recombinant PRRSV virus-like particle vaccines, and the inventor prepares a recombinant PRRSV virus-like particle (ZL 201811114020.6) with immunogenicity in 2018.

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 and a preparation method thereof, the GP5 and the M protein are researched, the GP5 and the gene of the M protein are artificially modified, a baculovirus expression system is adopted for expression to obtain the virus-like particle containing the GP5-M recombinant protein, the virus-like particle can generate a neutralizing antibody earlier than the unmodified protein sequence, the immunogenicity is better, the titer of the generated antibody is higher, and the animal protection can be better realized.

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

A recombinant PRRSV GP5-M gene sequence of a porcine reproductive and respiratory syndrome virus is characterized in that the PRRSV GP5-M gene sequence is shown as SEQ ID No: 1 is shown.

A recombinant PRRSV GP5-M protein, which is characterized in that the amino acid sequence of the PRRSV-GP5 protein is shown as SEQ ID No: 2, respectively.

A baculovirus transfer vector pBAC-5-PRRSV GP5-M, wherein the baculovirus transfer vector comprises the nucleotide sequence of SEQ ID No: 1, and the PRRSV-GP5 gene shown in figure 1.

A preparation method of recombinant baculovirus expressing PRRSV and M proteins of 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: and mixing plasmid DNA of a baculovirus transfer vector pBAC-5-PRRSV GP5-M which is correctly sequenced and identified with DH10 Bac competent cells, carrying out water bath heat shock for 45 seconds at 42 ℃ after ice bath for 30 minutes, 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 bacteria liquid after 10-time serial dilution, screening and purifying positive 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.

The invention also claims the application of the recombinant baculovirus expressing the PRRSV GP5 and M protein in the preparation of a subunit vaccine, wherein the subunit vaccine comprises a PRRSV virus-like particle and an adjuvant.

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 a rigid linker through the artificial modification of sequences, so that the influence of the A epitope on the B epitope is improved, the virus-like particles can stimulate the organism 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 the 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 of the invention 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, through researching the antibody swelling rule of the twice-immunized piglets, compared with the virus-like particles of a control group 2, the recombinant PRRSV virus-like particle vaccine adopts GGGGSSHIQLIYNLGGGGS to connect at the GP5-M connecting position, compared with the simple GGGGSGGGGSGGGGS connection, the recombinant PRRSV virus-like particle vaccine can stimulate organisms to generate antibodies with higher concentration, and compared with the vaccine researched on line, the vaccine has better protection effect.

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, the vaccine is further prepared by utilizing the virus-like particles similar to the prepared PRRSV virus particles, the vaccine can generate a neutralizing antibody with higher titer, and the antibody digestion and expansion rule of immunized piglets shows that the vaccine can stimulate an organism to generate an antibody with high concentration earlier, and stimulate the organism to generate higher average antibody titer after secondary immunization, so that better immune protection is realized.

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 is a schematic view of: western blot identification result: m is marker, and channel 1 is PRRSV VLP detection result.

FIG. 5: and (4) determining the average antibody titer of the vaccine immunized piglets.

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 target gene sequence GP5-M and vector pBAC-5 (provided by Shaanxi Novrili Biotechnology Co., Ltd., vector with GFP marker gene for fluorescence detection, see ZL 201811114020.6) are subjected to Bam H I and HindIII double enzyme digestion respectively, after recovery and purification, T4 ligase is used for ligation reaction, chemical transformation is carried out to DH5 alpha competent cells, plasmid extraction is carried out after positive cloning is screened, colony PCR (figure 1-A) and plasmid enzyme digestion identification (figure 1-B) are carried out, and the successfully constructed recombinant transfer vector pBAC-5-PRRSV GP5-M is obtained.

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 rBac-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 P1 generation recombinant baculovirus into sf9 cells according to the ratio of 1:10, culturing for 4-5 days at 27 ℃, and harvesting P2 generation recombinant baculovirus 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: serially diluting the P2 and P3 generation viruses by 10 times respectively, then inoculating sf9 cells in a 96-well plate, inoculating 8 wells in each dilution, culturing at a constant temperature of 27 ℃, observing the cytopathic condition every day, and then calculating the TCID of the viruses according to a Karber method₅₀The values show 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.

The method of example 1 is adopted for the control group 1 and the control group 2 respectively to prepare corresponding recombinant baculovirus, and the recombinant baculovirus is expressed in the insect cell sf9 to prepare corresponding virus-like particles and is used for preparing corresponding virus-like particle vaccines. 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 group 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 Marc-145 cell suspension (ca. 5X 10) to each well⁵one/mL), after being gently and uniformly shaken, the mixture is placed in a 5% CO2 incubator at 37 ℃ for continuous culture for 6 to 8 hours; 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 recombinant PRRSV virus-like particle vaccine of the embodiment 1 relatively has good immune protection effect in terms of the time and titer level of the generation of the neutralizing antibody, which is beneficial to resisting viruses and preventing disease deterioration of animals in the early infection stage.

Example 3: rule of antibody digestion and expansion for vaccine immunized piglets

(1) Recombinant PRRSV virus-like particle vaccines and control 2 vaccines were prepared according to the method of example 2, while a control 3 vaccine sample (vaccine prepared in patent ZL 201811114020.6) was provided by noville bio-technologies limited, shanxi.

(2) 10 one-month-old healthy piglets with double negative PRRSV antigen antibodies are screened, wherein 1 is randomly selected as a blank control group, and the remaining 9 are randomly divided into 3 groups, and each group has 3 pigs. The first group is used for immunizing Ac-PRRSV GP5-M vaccine (the amino acid sequence is shown as SEQ ID No. 2), the second group is used for immunizing control group 2 vaccine (the amino acid sequence is shown as SEQ ID No. 6), the third group is used for immunizing control group 3 vaccine (the vaccine prepared by the invention patent ZL201811114020.6 is prepared and provided by Shaanxi Nuo Weili Hua Biotech Co., Ltd.), and the fourth group is used for preparing blank control group. And (3) performing boosting immunization 30 days after the first immunization, collecting blood respectively at 14 days, 28 days, 60 days, 90 days, 120 days and 150 days after the first immunization, 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 positive serum maximum dilution factor, wherein the titer of a blank control group is always less than 1:2 and is not shown on the figure. The results of the detection are shown in FIG. 5.

Based on the detection results shown in fig. 5, the Ac-PRRSV GP5-M vaccine of the present invention can achieve higher average antibody titer than before, which can reach an average antibody titer of 1:2000 or more 28 days after priming, and the average antibody titer reaches the highest value of 1:3200 after 60 days after priming, which is 30 days after secondary priming, whereas the vaccine composition of the control group 3 can also produce higher antibody titer, which reaches the maximum average antibody titer 60 days after priming, whereas the comparison of the control groups 1 to 3 shows that the amino acid sequence of the present invention is SEQ ID No: the Ac-PRRSV GP5-M vaccine of 2 can stimulate an organism to generate high-concentration antibodies earlier, and can stimulate the organism to generate higher average antibody titer after secondary immunization, thereby realizing better immune protection.

Sequence listing

<110> northwest agriculture and forestry science and technology university

<120> recombinant PRRSV virus-like particle 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 GP5-M gene of a porcine reproductive and respiratory syndrome virus is characterized in that the PRRSV GP5-M gene has a sequence shown as SEQ ID No: 1 is shown.

2. A recombinant PRRSV GP5-M protein, which is characterized in that the amino acid sequence of the PRRSV GP5-M protein is shown as SEQ ID No: 2, respectively.

3. A baculovirus transfer vector pBAC-5-PRRSV GP5-M, wherein the baculovirus transfer vector comprises the nucleotide sequence of SEQ ID No: 1, and the PRRSV-GP5-M gene shown in the specification.

4. A preparation method of recombinant baculovirus expressing PRRSV GP5-M protein of porcine reproductive and respiratory syndrome virus is characterized by comprising 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;

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 heat shock at 42 ℃ for 45 seconds after ice bath for 30 minutes, then carrying out ice bath for 5 minutes, adding an SOC liquid culture medium, carrying out oscillation culture at 37 ℃ for 2 hours, carrying out 10-time serial dilution, coating LB (lysogeny broth) plates with gradient bacteria liquid, screening and purifying positive colonies by using a screening kit of life company, and extracting recombinant bacmid rBac-PRRSV GP 5-M;

5. The use of the recombinant baculovirus expressing PRRSV GP5-M protein prepared according to the preparation method of claim 4 in the preparation of subunit vaccines.

6. The use of claim 5, wherein the subunit vaccine comprises PRRSV virus-like particles and an adjuvant, the virus-like particles are obtained by inoculating the recombinant baculovirus Ac-PRRSV GP5-M prepared by the preparation method of claim 4 into healthy sf9 cells according to a proportion of 10%, culturing at 27 ℃ for 4-5 days, repeatedly freezing and thawing to collect the cells and supernatant, centrifuging at 12000r/min at 4 ℃ for 20 minutes to collect the supernatant, then precipitating the target protein by using an ammonium sulfate precipitation method, inactivating the protein solution by using divinyl imine after resuspension for 36-48 hours, and then neutralizing by using equivalent sodium thiosulfate.

7. A porcine reproductive and respiratory syndrome virus vaccine, which is characterized by comprising PRRSV virus-like particles and an adjuvant, wherein the virus-like particles are obtained by inoculating the recombinant baculovirus Ac-PRRSV GP5-M prepared by the preparation method of claim 4 into healthy sf9 cells according to a proportion of 10%, culturing at 27 ℃ for 4-5 days, repeatedly freezing and thawing to collect cells and supernatant, centrifuging at 4 ℃ for 12000r/min for 20 minutes to collect supernatant, then precipitating target protein by using an ammonium sulfate precipitation method, inactivating a protein solution by using binary ethyleneimine after resuspension for 36-48 hours, and then neutralizing by using equivalent sodium thiosulfate.