An engineered extracellular vesicle, its preparation method and application, and porcine reproductive and respiratory syndrome vaccine based on extracellular vesicles

Abstract

The present invention relates to an engineered extracellular vesicle, a preparation method and application thereof, and a porcine reproductive and respiratory syndrome vaccine based on the extracellular vesicle. The main component of the vaccine is an engineered extracellular vesicle loaded with PRRSV antigens. The PRRSV antigens include a fusion protein of the GP2 envelope protein of PRRSV and the N-terminal fragment of the SDCBP protein, a fusion protein of the GP3 envelope protein of PRRSV and the N-terminal fragment of the SDCBP protein, a fusion protein of the GP4 envelope protein of PRRSV and the N-terminal fragment of the SDCBP protein, a fusion protein of the GP5 envelope protein of PRRSV and the N-terminal fragment of the SDCBP protein, and a fusion protein of the M envelope protein of PRRSV and the N-terminal fragment of the SDCBP protein. The engineered extracellular vesicles can effectively deliver multiple PRRSV protein antigens and induce specific immune responses.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology technology, and specifically relates to an engineered extracellular vesicle, a preparation method and application thereof, and a porcine reproductive and respiratory syndrome vaccine based on the extracellular vesicle. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is an acute, highly contagious viral disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). Because some affected pigs develop cyanosis of the ears and extremities of the body, it is called "blue ear disease." Clinically, PRRS is characterized by anorexia, fever, reproductive failure, and respiratory distress. Due to its rapid spread, widespread prevalence, and high mortality rate, it has become a serious threat to the swine industry. Currently, there is no specific treatment for PRRS. The disease is primarily controlled through a combination of antiviral drugs, antibiotics, immunomodulators, and traditional Chinese medicines. Vaccination is particularly important for preventing PRRS.

[0003] Currently, widely used porcine reproductive and respiratory syndrome (PRRS) vaccines primarily include attenuated live (ML) and inactivated (INV) vaccines. MLV vaccines can induce a strong immune response, but carry the risks of reversion (the vaccine strain may regain virulence through mutation or recombination) and viral recombination (the vaccine strain and field strain are prone to recombination, producing new strains). They also offer limited cross-protection (the vaccine offers poor protection against heterologous strains). Inactivated vaccines do not pose the risk of reversion or recombination, but they exhibit weaker immunogenicity and limited protective efficacy. There is an urgent need to develop more effective and safe PRRS vaccines.

[0004] Extracellular vesicle vaccines are an emerging vaccine technology that uses extracellular vesicles to deliver antigens in vivo, inducing an immune response and producing specific neutralizing antibodies. Theoretically, the phospholipid bilayer structure of extracellular vesicles enables more efficient uptake and presentation by immune cells than traditional live attenuated and inactivated vaccines, potentially inducing a more effective immune response. However, there are currently no reports of extracellular vesicle vaccines for PRRS.

[0005] The series of envelope proteins on the surface of PRRSV are the first choice as vaccine antigens. However, these proteins are multi-transmembrane proteins with complex structures and hydrophobicity. They are usually expressed at low levels in recombinant expression cells, which limits the use of recombinant PRRSV envelope proteins as antigens in PRRS vaccines. Due to the high difficulty of expressing PRRSV envelope proteins in recombinant expression cells, it is even more difficult to enrich and load them into extracellular vesicles. In addition, the interaction between different envelope proteins is difficult to predict the impact on their loading efficiency in extracellular vesicles and in vivo immunogenicity, further increasing the difficulty of developing PRRS extracellular vesicle vaccines. Summary of the Invention

[0006] The purpose of the present invention is to provide engineered extracellular vesicles and a novel PRRS extracellular vesicle vaccine that can simultaneously and efficiently load and effectively deliver multiple PRRSV antigens and induce specific immune responses.

[0007] To solve the above problems, the first aspect of the present invention provides an engineered extracellular vesicle, which includes an extracellular vesicle and a PRRSV antigen loaded on the extracellular vesicle, wherein the PRRSV antigen includes a fusion protein of the GP2 envelope protein of PRRSV and the N-terminal fragment of the SDCBP protein, a fusion protein of the GP3 envelope protein of PRRSV and the N-terminal fragment of the SDCBP protein, a fusion protein of the GP4 envelope protein of PRRSV and the N-terminal fragment of the SDCBP protein, a fusion protein of the GP5 envelope protein of PRRSV and the N-terminal fragment of the SDCBP protein, and a fusion protein of the M envelope protein of PRRSV and the N-terminal fragment of the SDCBP protein, wherein the N-terminal fragment of the SDCBP protein includes the first 50 to 70 amino acids from the N-terminus of the SDCBP protein.

[0008] PRRSV (Protein Receptor Virus) contains a series of envelope proteins on its surface, including GP2, GP3, GP4, GP5, and M. These envelope proteins are the preferred protein antigens for PRRSV vaccines. However, due to their complex and hydrophobic structures, these envelope proteins are difficult to express in recombinant expression systems, limiting their application in PRRSV vaccines. Studies have shown that wild-type envelope proteins are difficult to express in mammalian cells (293 cells), manifested by very low expression rates and ultimately little enrichment in extracellular vesicles. The present invention, by adding the N-terminal fragment of SDCBP to these transmembrane proteins, simultaneously improves the expression efficiency at the cellular level and the enrichment and loading efficiency in extracellular vesicles for most envelope proteins. In theory, combining multiple antigens increases the number of antigen-antibody binding sites, helping to enhance the immune response. However, interactions between different envelope protein antigens can form heteropolymers, which may alter their enrichment in extracellular vesicles and their immunogenicity compared to monomers. Studies have shown that when the five envelope proteins are co-expressed in recombinant cells with the addition of the N-terminal fragment of the SDCBP protein, the expression levels of each envelope protein are significantly increased. Furthermore, the enrichment and loading efficiency of the five envelope proteins in extracellular vesicles is significantly improved compared to single antigen expression or pairwise co-expression, demonstrating the effects of coordinated expression and synergistic enrichment and loading. Animal immunization experiments demonstrate that the engineered extracellular vesicles of the present invention can effectively deliver the five envelope proteins and induce specific immune responses.

[0009] The specific amino acid sequences of the GP2, GP3, GP4, GP5, and M envelope proteins in the present invention are designed with reference to the PRRSV genome. There are no particular limitations on the PRRSV genome and, in theory, encompass different genotypes known in the art, and are not limited to genes from specific viral strains. The specific amino acid sequences of the GP2, GP3, GP4, GP5, and M envelope proteins include wild-type amino acid sequences or amino acid sequences optimized according to conventional methods in the art. For example, in some specific embodiments of the present invention, the sequences of GP2 envelope protein, GP3 envelope protein, GP4 envelope protein, GP5 envelope protein, and M envelope protein are designed with reference to the PRRSV genome with accession number EF112445.1 in the GenBank. The amino acid sequence of the GP2 envelope protein of PRRSV is shown in SEQ ID NO: 1; the amino acid sequence of the GP3 envelope protein of PRRSV is shown in SEQ ID NO: 2; the amino acid sequence of the GP4 envelope protein of PRRSV is shown in SEQ ID NO: 3; the amino acid sequence of the GP5 envelope protein of PRRSV is shown in SEQ ID NO: 4; and the amino acid sequence of the M envelope protein of PRRSV is shown in SEQ ID NO: 5.

[0010] According to the present invention, when referring to an amino acid sequence, unless otherwise specified, the amino acid sequence referred to includes not only sequences that are completely identical to the sequence but also sequences that can be easily thought of and expected to have equivalent effects based on the sequence by those skilled in the art (for example, those skilled in the art should know that sequences with high identity (for example, 85% or more, particularly 90% or more, and especially 95% or more) usually have equivalent effects).

[0011] The amino acid sequence of the SDCBP protein (syndecan binding protein) of the present invention includes a wild-type amino acid sequence or an amino acid sequence optimized according to conventional methods in the art. In some embodiments of the present invention, the amino acid sequence of the SDCBP protein is shown in SEQ ID NO: 7.

[0012] In an embodiment of the present invention, the N-terminal fragment of the SDCBP protein preferably includes the first 50, first 51, first 52, first 53, first 54, first 55, first 56, first 57, first 58, first 59, first 60, first 61, first 62, first 63, first 64, first 65, first 66, first 67, first 68, first 69 or first 70 amino acids from the N-terminus of the SDCBP protein. Preferably, it includes the first 55 to 65 amino acids from the N-terminus of the SDCBP protein.

[0013] In some specific embodiments of the present invention, the amino acid sequence of the N-terminal fragment of the SDCBP protein is shown in SEQ ID NO: 6.

[0014] In an embodiment of the present invention, the N-terminal fragment of the SDCBP protein is connected to the C-terminus and / or N-terminus of the envelope protein.

[0015] In an embodiment of the present invention, the N-terminal fragment of the SDCBP protein is linked to the envelope protein directly or via a linker, and whether a linker is needed can be determined based on the protein structure, function, stability, etc. Preferably, a flexible peptide linker is used, such as a peptide linker comprising glycine and / or serine residues.

[0016] In some specific embodiments of the present invention, the N-terminal fragment of the SDCBP protein is connected to the C-terminus of the envelope protein.

[0017] The extracellular vesicles involved in the present invention include exosomes, microvesicles or apoptotic bodies.

[0018] The particle size range of the extracellular vesicles involved in the present invention is preferably 100-200 nm, more preferably 100-180 nm, and even more preferably 120-160 nm.

[0019] The extracellular vesicles involved in the present invention are derived from mammalian cells, including but not limited to cells of common mammals in the art, for example, cells of cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice, including but not limited to cells of non-human primates, for example, monkeys, such as cynomolgus monkeys or rhesus monkeys, chimpanzees, including but not limited to human cells, including but not limited to cells of transgenic mammals. Specifically including but not limited to one or more of fibroblast cell lines, embryonic kidney cell lines, renal cell lines, mesenchymal stem cell lines, megakaryocyte lines, platelet cell lines, and lymphocyte lines. Exemplary mammalian cells include Chinese hamster ovary (CHO cells), human embryonic kidney 293 cells (e.g., HEK293, HEK293E, HEK293T), COS cells, BHK cells, NIH3T3 cells, lymphocyte lines (e.g., NS0 myeloma cells and Sp2 / 0 cells), mammary epithelial cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells or placental mesenchymal stem cells, etc.

[0020] The second aspect of the present invention provides a recombinant cell for preparing the above-mentioned engineered extracellular vesicles, wherein the recombinant cell is a recombinant cell expressing the PRRSV antigen.

[0021] In an embodiment of the present invention, the host cell of the recombinant cell is a mammalian cell, including but not limited to the cell of common mammals in this area, for example, cattle, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat and mouse, including but not limited to the cell of non-human primate, for example, monkey, such as the cell of cynomolgus monkey or rhesus monkey, chimpanzee, including but not limited to the cell of people, including but not limited to the cell of transgenic mammal. Specifically include but not limited to one or more in fibroblast line, embryonic kidney cell line, kidney cell line, mesenchymal stem cell line, megakaryocyte line, platelet cell line, lymphocyte line. Exemplary mammalian cell includes Chinese hamster ovary (CHO cell), human embryonic kidney 293 cell (for example, HEK293, HEK293E, HEK293T), COS cell, BHK cell, NIH3T3 cell, lymphocyte line (for example NS0 myeloma cell and Sp2 / 0 cell), mammary epithelial cell, adipose mesenchymal stem cell, umbilical cord mesenchymal stem cell or placental mesenchymal stem cell etc.

[0022] The third aspect of the present invention provides a method for preparing the above-mentioned engineered extracellular vesicles, comprising the step of isolating the engineered extracellular vesicles from the culture fluid of recombinant cells expressing the PRRSV antigen.

[0023] In an embodiment of the present invention, the method for preparing the engineered extracellular vesicles comprises the following steps:

[0024] (1) constructing and preparing a recombinant expression vector system for expressing the PRRSV antigen;

[0025] (2) transfecting cells with the recombinant expression vector system to obtain recombinant cells;

[0026] (3) culturing the recombinant cells to obtain a recombinant cell culture fluid;

[0027] (4) Isolating and obtaining the engineered extracellular vesicles from the recombinant cell culture medium.

[0028] Specifically, the recombinant expression vector system includes a first recombinant expression plasmid containing the coding sequence of a fusion protein of the GP2 envelope protein of the PRRSV and the N-terminal fragment of the SDCBP protein, a second recombinant expression plasmid containing the coding sequence of a fusion protein of the GP3 envelope protein of the PRRSV and the N-terminal fragment of the SDCBP protein, a third recombinant expression plasmid containing the coding sequence of a fusion protein of the GP4 envelope protein of the PRRSV and the N-terminal fragment of the SDCBP protein, a fourth recombinant expression plasmid containing the coding sequence of a fusion protein of the GP5 envelope protein of the PRRSV and the N-terminal fragment of the SDCBP protein, and a fifth recombinant expression plasmid containing the coding sequence of a fusion protein of the M envelope protein of the PRRSV and the N-terminal fragment of the SDCBP protein.

[0029] In an embodiment of the present invention, the expression vector used in the recombinant expression vector system can be an expression vector conventionally used in the art, including a viral plasmid vector and a eukaryotic expression plasmid vector, and preferably a eukaryotic expression plasmid vector.

[0030] In some specific embodiments of the present invention, the expression vector used in the recombinant expression vector system is a pAAVS1 plasmid, a pIRES plasmid, or a pLENTI plasmid.

[0031] In an embodiment of the present invention, the separation can adopt an extracellular vesicle separation and purification method known in the art, including one or more of ultracentrifugation separation, density gradient centrifugation separation, size exclusion chromatography separation, affinity chromatography separation, adsorption chromatography separation, bonded phase chromatography separation, immunoseparation or sieving separation.

[0032] According to some embodiments of the present invention, the recombinant cell culture fluid is first centrifuged at a centrifugal force of less than 1000g to collect the supernatant, and then the supernatant is centrifuged at a centrifugal force of 100,000g to 200,000g to collect the precipitate, which is the engineered extracellular vesicles.

[0033] Furthermore, the recombinant cell culture medium is first centrifuged at a centrifugal force of 200g~500g to collect the supernatant, and then the supernatant is centrifuged at a centrifugal force of 120000g~150000g to collect the precipitate, which is the extracellular vesicles.

[0034] In an embodiment of the present invention, the culture medium is a serum-free culture medium.

[0035] In an embodiment of the present invention, the recombinant cells are cultured at 34° C. to 38° C. and 5% to 10% CO 2 to obtain the recombinant cell culture fluid.

[0036] In the embodiment of the present invention, the transfection operation is to mix the recombinant expression vector, the host cell, and the transfection reagent in a serum-free culture medium, and incubate the mixture at 20° C. to 30° C.

[0037] In an embodiment of the present invention, the host cell of the recombinant cell is one or more of CHO cells, 293 cells, BHK cells, COS cells, NIH3T3 cells, mammary epithelial cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, induced stem cells, NS0 cells or Sp2 / 0 cells.

[0038] A fourth aspect of the present invention provides the use of the engineered extracellular vesicles described above in the preparation of a medicament for preventing, diagnosing, or treating diseases associated with PRRSV infection. The dosage form of the medicament includes, but is not limited to, tablets, granules, pills, capsules, emulsions, ointments, gels, suspensions, solutions, powders, transdermal patches, sprays, suppositories, or implants.

[0039] The fifth aspect of the present invention provides a porcine reproductive and respiratory syndrome vaccine based on extracellular vesicles, which includes the above-mentioned engineered extracellular vesicles and may optionally include pharmaceutically acceptable excipients.

[0040] In embodiments of the invention, specific excipients may be selected based on the intended mode of administration. The pharmaceutically acceptable excipients include, but are not limited to, pharmaceutically, nutritionally or physiologically acceptable carriers.

[0041] In some specific embodiments of the present invention, the auxiliary material includes PBS buffer with a pH value of 7.2-7.6.

[0042] A sixth aspect of the present invention provides a method for preventing PRRSV, comprising administering the aforementioned porcine reproductive and respiratory syndrome vaccine based on extracellular vesicles to a target. The target includes, but is not limited to, one or more of fattening pigs, sows, gilts, and piglets, and the route of administration includes, but is not limited to, one or more of intramuscular, subcutaneous, intradermal, and oral administration.

[0043] Due to the adoption of the above technical solution, the present invention has the following advantages compared with the prior art:

[0044] The present invention simultaneously links the N-terminal fragment of the SDCBP protein to the GP2, GP3, GP4, GP5, and M envelope proteins of PRRSV, and co-expresses the fusion proteins of the five envelope proteins and the N-terminal fragment of the SDCBP protein in recombinant cells. The recombinant cells then produce engineered extracellular vesicles that are simultaneously loaded with five PRRSV antigens. Existing experimental results have shown that when the five envelope proteins modified with the N-terminal fragment of the SDCBP protein are co-expressed, the expression efficiency of each envelope protein and the enrichment and loading efficiency on the extracellular vesicles are significantly improved compared to when expressed individually. Using the engineered extracellular vesicles of the present invention as a PRRS extracellular vesicle vaccine, it is possible to simultaneously present five PRRSV antigens, effectively activating the host's immune response to produce neutralizing antibodies. Compared with the currently widely used attenuated live vaccines and inactivated vaccines, the engineered extracellular vesicles of the present invention have a stronger ability to activate the host immune response. Under the same immunization method, the host vaccinated with the engineered extracellular vesicles of the present invention produces a higher level of neutralizing antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a graph showing the expression positive rate detection results of GP2, GP2-SDCBP60, and GP2-SDCBP298 at the cellular level;

[0046] Figure 2 The particle size distribution diagrams of the engineered extracellular vesicles loaded with GP2, the engineered extracellular vesicles loaded with GP2-SDCBP60, and the engineered extracellular vesicles loaded with GP2-SDCBP298;

[0047] Figure 3 Transmission electron micrographs of engineered extracellular vesicles loaded with GP2, engineered extracellular vesicles loaded with GP2-SDCBP60, and engineered extracellular vesicles loaded with GP2-SDCBP298;

[0048] Figure 4 The WB electrophoresis results of engineered extracellular vesicles loaded with GP2, engineered extracellular vesicles loaded with GP2-SDCBP60, and engineered extracellular vesicles loaded with GP2-SDCBP298 are shown;

[0049] Figure 5 This is a graph showing the expression positive rate detection results of GP3, GP3-SDCBP60, and GP3-SDCBP298 at the cellular level;

[0050] Figure 6 The particle size distribution diagrams of the engineered extracellular vesicles loaded with GP3, the engineered extracellular vesicles loaded with GP3-SDCBP60, and the engineered extracellular vesicles loaded with GP3-SDCBP298;

[0051] Figure 7 Transmission electron micrographs of engineered extracellular vesicles loaded with GP3, engineered extracellular vesicles loaded with GP3-SDCBP60, and engineered extracellular vesicles loaded with GP3-SDCBP298;

[0052] Figure 8 The WB electrophoresis results of engineered extracellular vesicles loaded with GP3, engineered extracellular vesicles loaded with GP3-SDCBP60, and engineered extracellular vesicles loaded with GP3-SDCBP298 are shown;

[0053] Figure 9 This is a graph showing the expression positive rate detection results of GP4, GP4-SDCBP60, and GP4-SDCBP298 at the cellular level;

[0054] Figure 10 The particle size distribution diagrams are for extracellular vesicles loaded with GP4, engineered extracellular vesicles loaded with GP4-SDCBP60, and engineered extracellular vesicles loaded with GP4-SDCBP298;

[0055] Figure 11 Transmission electron micrographs of GP4-loaded extracellular vesicles, GP4-SDCBP60-loaded engineered extracellular vesicles, and GP4-SDCBP298-loaded engineered extracellular vesicles;

[0056] Figure 12 The WB electrophoresis results of GP4-loaded extracellular vesicles, GP4-SDCBP60-loaded engineered extracellular vesicles, and GP4-SDCBP298-loaded engineered extracellular vesicles are shown;

[0057] Figure 13 This is a graph showing the expression positive rate detection results of GP5, GP5-SDCBP60, and GP5-SDCBP298 at the cellular level;

[0058] Figure 14 The particle size distribution diagrams of the engineered extracellular vesicles loaded with GP5, the engineered extracellular vesicles loaded with GP5-SDCBP60, and the engineered extracellular vesicles loaded with GP5-SDCBP298;

[0059] Figure 15 Transmission electron micrographs of engineered extracellular vesicles loaded with GP5, engineered extracellular vesicles loaded with GP5-SDCBP60, and engineered extracellular vesicles loaded with GP5-SDCBP298;

[0060] Figure 16The WB electrophoresis results of the engineered extracellular vesicles loaded with GP5, the engineered extracellular vesicles loaded with GP5-SDCBP60, and the engineered extracellular vesicles loaded with GP5-SDCBP298 are shown;

[0061] Figure 17 The graph shows the expression positive rate detection results of M, M-SDCBP60, and M-SDCBP298 at the cellular level in 293;

[0062] Figure 18 The particle size distribution diagrams of the engineered extracellular vesicles loaded with M, the engineered extracellular vesicles loaded with M-SDCBP60, and the engineered extracellular vesicles loaded with M-SDCBP298;

[0063] Figure 19 Transmission electron micrographs of engineered extracellular vesicles loaded with M, engineered extracellular vesicles loaded with M-SDCBP60, and engineered extracellular vesicles loaded with M-SDCBP298;

[0064] Figure 20 The WB electrophoresis results of the engineered extracellular vesicles loaded with M, the engineered extracellular vesicles loaded with M-SDCBP60, and the engineered extracellular vesicles loaded with M-SDCBP298 are shown;

[0065] Figure 21 The particle size distribution diagram of the engineered extracellular vesicles loaded with fusion proteins of five PRRSV envelope proteins and the N-terminal fragment of SDCBP and the engineered extracellular vesicles loaded with fusion proteins of two PRRSV envelope proteins and the N-terminal fragment of SDCBP;

[0066] Figure 22 Transmission electron micrographs of engineered extracellular vesicles loaded with fusion proteins of five PRRSV envelope proteins and the N-terminal fragment of SDCBP and engineered extracellular vesicles loaded with fusion proteins of two PRRSV envelope proteins and the N-terminal fragment of SDCBP;

[0067] Figure 23 These are the WB electrophoresis results of engineered extracellular vesicles loaded with fusion proteins of five PRRSV envelope proteins and the N-terminal fragment of SDCBP, and engineered extracellular vesicles loaded with fusion proteins of two PRRSV envelope proteins and the N-terminal fragment of SDCBP, wherein, lane 1: blank control; lane 2: GP5-SDCBP60 + M-SDCBP60; lane 3: GP3-SDCBP60; lane 4: GP2-SDCBP60 + GP4-SDCBP60; lane 5: five antigens. DETAILED DESCRIPTION

[0068] Because extracellular vesicles (EVs) originate from cells and share the same phospholipid bilayer membrane structure as cells, using EVs as carriers for antigen delivery naturally offers improved biocompatibility and safety. A series of envelope proteins on the surface of PRRSV are the preferred protein antigens for PRRS vaccines. However, most PRRSV envelope proteins (such as GP2, GP4, GP5, and M) are difficult to express in recombinant cells, with expression levels being very low. Extracellular vesicles secreted by recombinant cells contain virtually no envelope proteins. The present study unexpectedly discovered that an N-terminal (nitrogen-terminal) fragment extracted from the SDCBP protein can effectively promote the expression of GP2, GP4, GP5, and M envelope proteins in recombinant cells.

[0069] In theory, the combination of multiple antigens can stimulate the host to produce more neutralizing antibodies and achieve better immune effects. However, when multiple antigens are co-expressed, the interaction between different envelope proteins makes it more difficult to load extracellular vesicles, and the in vivo immunogenicity may undergo unpredictable changes compared to monomeric antigens. Further research results showed that when a fusion protein of five envelope proteins and the N-terminal fragment of SDCBP is co-expressed in recombinant cells, the enrichment and loading efficiency of each envelope protein on the engineered extracellular vesicles secreted by the recombinant cells expressing the five fusion proteins is significantly higher than the enrichment and loading efficiency of the corresponding envelope protein on the engineered extracellular vesicles secreted by recombinant cells expressing only one envelope protein, only one fusion protein, or two fusion proteins. The five fusion proteins co-expressed in the recombinant cells interact with each other and jointly promote the enrichment and loading effect of each envelope protein on the engineered extracellular vesicles. Animal immunization experiments showed that the engineered extracellular vesicles loaded with the fusion protein of five envelope proteins and the N-terminal fragment of SDCBP can effectively deliver the loaded fusion protein in vivo as a PRRSV antigen to induce a specific immune response and produce high levels of neutralizing antibodies, and the immune effect is better than that of the JXA1 attenuated vaccine.

[0070] The technical solution of the present invention is further illustrated below with reference to specific embodiments and comparative examples.

[0071] In the present invention, it should be noted that, unless otherwise specified or contradicted by the context, the terms or expressions used herein should be read in conjunction with the entire content of this document and as understood by those of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0072] "Porcine reproductive and respiratory syndrome virus", abbreviated as PRRSV, has a series of envelope proteins on its surface, including GP2 envelope protein (abbreviated as GP2 in this invention), GP3 envelope protein (abbreviated as GP3 in this invention), GP4 envelope protein (abbreviated as GP4 in this invention), GP5 envelope protein (abbreviated as GP5 in this invention) and M envelope protein (abbreviated as M in this invention). These envelope proteins are all multi-transmembrane proteins, each of which has certain immunogenicity and is closely related to the neutralizing antibody response of PRRSV virus. They can serve as important targets for the study of porcine reproductive and respiratory syndrome vaccine (also known as porcine blue ear disease vaccine).

[0073] "Extracellular vesicles" are a group of vesicles with a lipid bilayer membrane structure secreted by living cells. They have a typical disc-shaped vesicle structure and can be divided into exosomes, microvesicles and apoptotic bodies according to their size.

[0074] "Engineered extracellular vesicles" refer to cargo-loaded extracellular vesicles formed by artificially modifying natural extracellular vesicles. In the present invention, artificial customization of extracellular vesicles is achieved through genetic engineering.

[0075] "Loading" refers to connecting cargo to extracellular vesicles. In the present invention, the cargo is a modified or unmodified envelope protein, which is a multi-transmembrane protein, thereby being enriched on the membrane of the extracellular vesicle, including the surface of the membrane and embedded in the membrane.

[0076] A "fusion protein" refers to the expression product of two genes obtained through DNA recombination technology. The present invention relates to a fusion protein of the PRRSV envelope protein and the N-terminal fragment of the SDCBP protein (the expression product of the recombination of the N-terminal fragment sequences of the PRRSV envelope protein gene and the SDCBP protein gene) and a fusion protein of the PRRSV envelope protein and SDCBP protein (the expression product of the recombination of the PRRSV envelope protein gene and the SDCBP protein gene).

[0077] "Vaccine" refers to a composition comprising an antigen used for therapeutic treatment or prophylactic immunization against infectious or non-infectious disease.

[0078] "Immunity" refers to the body's ability to identify and eliminate antigenic foreign substances and maintain its own physiological balance and stability.

[0079] "Antigen" refers to a substance with immunogenicity, and in the present invention mainly refers to protein antigens.

[0080] "Antibodies" refer to substances that specifically bind to antigens and are produced by the immune system when foreign antigens stimulate it.

[0081] "Serum" is the pale yellow transparent liquid separated from plasma after blood coagulation.

[0082] "Serum titer" refers to the concentration or activity of specific antibodies in serum. A higher titer indicates a higher concentration or activity of antibodies in the serum.

[0083] "Promoter" refers to a nucleic acid sequence that controls the transcription of a coding sequence. Promoter sequences include sequences that are sufficient for RNA polymerase to recognize, bind, and initiate transcription. A promoter can affect the transcription of a gene on the same nucleic acid molecule as itself or a gene on a different nucleic acid molecule.

[0084] "Host cells" are also called recipient cells, and exemplary host cells include human embryonic kidney cells HEK293. It is understood that the progeny of a single parental cell may not necessarily be completely identical to the original parent in morphology or in genomic or total DNA complement due to natural, accidental, or deliberate mutations.

[0085] The technical solution of the present invention is further described below in conjunction with specific experiments and examples, but the present invention is not limited to the following specific examples.

[0086] The conditions used in the following experiments and examples can be adjusted according to specific requirements. The conditions not specified are generally those used in routine experiments. All instruments, raw materials, and reagents not otherwise specified can be obtained commercially.

[0087] Preparation and corresponding effect experiments of engineered extracellular vesicles loaded with a PRRSV envelope protein, engineered extracellular vesicles loaded with a fusion protein of a PRRSV envelope protein and the nitrogen-terminal fragment of SDCBP, and engineered extracellular vesicles loaded with a PRRSV envelope protein and SDCBP protein.

[0088] Preparation of engineered extracellular vesicles loaded with GP2:

[0089] 1. Construction of a GP2 expression plasmid: Use the pIRESneo plasmid as the expression vector. Design the expression cassette as follows: use the CMV promoter (sequence shown in SEQ ID NO: 8) as the transcription start point, use the SV40 polyA (sequence shown in SEQ ID NO: 9) as the transcription end point, and place the GP2 protein sequence (sequence shown in SEQ ID NO: 1) within the expression cassette.

[0090] The specific steps are as follows:

[0091] 1.1 PCR cloning: Using conventional methods in the art, upstream and downstream primers were designed based on the above expression cassette. The upstream primer (sequence shown in SEQ ID NO: 10), the downstream primer (sequence shown in SEQ ID NO: 11), and the template DNA (sequence shown in SEQ ID NO: 12) were synthesized. In a 0.2 mL EP tube, 25 μL of DNA polymerase (2× Phanta Max Master Mix, Vazyme, P515-01), 2 μL of the upstream primer, 2 μL of the downstream primer, and 10 g of template DNA were added. The volume was made up to 50 μL with RNase-free dd HO (Vazyme, P071-01-AA). Mix thoroughly and place in a PCR instrument. The amplification program was designed based on the primer annealing temperature.

[0092] 1.2 PCR product purification: Purify the PCR product according to the kit instructions (Vazyme, DC301-01).

[0093] 1.3 Enzyme digestion: Add 5 μL rCutSmart to a 0.2 mL EP tube. TM Buffer (BioLabs, 136004s), 0.5 μL Pac I enzyme (BioLabs, R0547L), 0.5 μL Not I enzyme (BioLabs, R3189L), 2 μg of PCR-purified fragment, and 5 μg of pIRESneo vector plasmid. Add RNase-free ddH2O (Vazyme, P071-01-AA) to 10 μL, mix well, and place in a PCR instrument for enzyme digestion at 37°C for 16 hours.

[0094] 1.4 Ligation and transformation: In a 0.2 mL EP tube, add 4 μL of the digestion solution obtained in step 1.3, 1 μL of T4 DNA ligase (Vazyme, N103-01-AA), and 1 μL of T4 DNA ligase buffer (Vazyme, N103-01-AC). After incubation at room temperature for 10 minutes, 5 μL was added to competent cells (DH5α, Vazyme, C502-03), incubated on ice for 30 minutes, incubated at 42°C for 90 seconds, and incubated on ice for 2 minutes. Then, 1 mL of LB medium (BeyoPure™, 04.05.ST156) was added and the cells were cultured in a shaking incubator for 1 hour. After incubation, the cells were centrifuged at 12,000 g for 1 minute, the supernatant was removed, and 100 μL was retained for resuspending and added to a bacterial culture dish (Biosharp, BS-90-D). The cells were evenly spread with a disposable plastic coating rod (Biosharp, BS-PS-A) and stored in a 37°C constant temperature incubator for later use.

[0095] 2. Transfecting Cells

[0096] 2.1 Cell preparation: Expi293F cells (Thermo Fisher, A14527) were cultured at a density of 0.5 × 10 6 / mL, with a viability of over 95%, was inoculated into a 125mL shake flask with a total volume of 25mL. Cultured overnight in an 8% CO2 incubator shaker, and transfection was initiated the next day to obtain cultured Expi293F cells.

[0097] 2.2 Plasmid transfection into cells

[0098] Using TA-293 reagent (Zhuhai Kairui Biotechnology Co., Ltd., K2001), the transfection procedure for a 25 mL cell suspension is as follows: Prepare two 15 mL centrifuge tubes. Add 1.25 mL of serum-free cell culture medium (Aupuma, CD05) and 25 μg of GP2 expression plasmid to one tube and vortex to mix thoroughly. Add 1.25 mL of serum-free cell culture medium and 125 μL of TA-293 transfection reagent to the other tube and vortex to mix thoroughly. Transfer all liquid from the centrifuge tube containing TA-293 transfection reagent to the centrifuge tube containing the plasmid and vortex to mix thoroughly. Incubate at room temperature for 10 minutes to prepare the plasmid-vector complex. Remove Expi293F cells from the incubator and add the prepared plasmid-vector complex dropwise while shaking. Return the tube to the CO2 incubator for 72 hours.

[0099] 3. Isolation and Purification of Extracellular Vesicles

[0100] After 72 hours of shaking culture, the cell culture medium was first centrifuged at 300g, 4°C for 5 minutes, and the supernatant was collected. The supernatant was then ultracentrifuged at 133900g, 4°C for 1 hour, and the precipitate was collected. The precipitate was resuspended in 100 μL PBS (pH 7.4) to obtain engineered extracellular vesicles loaded with GP2, which were stored at 4°C for later use.

[0101] Preparation of engineered extracellular vesicles loaded with a fusion protein (abbreviated as GP2-SDCBP60) containing the N-terminal fragment sequence of GP2 and SDCBP protein (shown in SEQ ID NO: 6):

[0102] The GP2-SDCBP60 expression plasmid was constructed using the GP2 expression plasmid construction method. The upstream primer (SEQ ID NO: 10), downstream primer (SEQ ID NO: 13), and template DNA (SEQ ID NO: 14) for the synthetic PCR cloning step were redesigned based on the adjusted expression cassette (the N-terminal fragment sequence of the SDCBP protein was added to the C-terminus of the GP2 protein sequence in the GP2 expression plasmid). All other steps were the same as for the GP2 protein expression plasmid. The preparation of engineered extracellular vesicles loaded with GP2 was similar to that for the GP2 protein expression plasmid, except that the GP2 expression plasmid in the cell transfection step was replaced with the GP2-SDCBP60 expression plasmid.

[0103] Preparation of engineered extracellular vesicles loaded with a fusion protein of GP2 and SDCBP protein (shown in SEQ ID NO: 7) (abbreviated as GP2-SDCBP298):

[0104] The GP2 expression plasmid was constructed by referring to the GP2 expression plasmid construction method. The upstream primer (sequence shown in SEQ ID NO: 10), downstream primer (sequence shown in SEQ ID NO: 15), and template DNA (sequence shown in SEQ ID NO: 16) for the synthetic PCR cloning step were redesigned based on the adjusted expression cassette (the SDCBP protein sequence was added to the C-terminus of the GP2 protein sequence in the GP2 expression plasmid expression cassette). All other procedures were the same as for the GP2 protein expression plasmid. The preparation of engineered extracellular vesicles loaded with GP2 was also described, with the exception of replacing the GP2 expression plasmid with the GP2-SDCBP298 expression plasmid during the cell transfection step.

[0105] GP2, GP2-SDCBP60, and GP2-SDCBP298 were overexpressed in 293 cells, and the positive rates were detected by flow cytometry (BD FACS Celesta). The results are shown in the figure. Figure 1 As shown, the positive expression rate of GP2 was 1.25%, the positive expression rate of GP2-SDCBP298 was 1.24%, and the positive expression rate of GP2-SDCBP60 was 11.9%.

[0106] The engineered extracellular vesicles loaded with GP2, engineered extracellular vesicles loaded with GP2-SDCBP60, and engineered extracellular vesicles loaded with GP2-SDCBP298 were subjected to NTA analysis (Nanoparticle Tracking AnaKis) using a ZetaView instrument (Fuliu Bioparticle Metrix, Cat. No. N30E). The particle size distribution is shown in Figure 2 The average particle size of the engineered extracellular vesicles loaded with GP2 was 139.0 nm, and the median particle size was 129.8 nm; the average particle size of the engineered extracellular vesicles loaded with GP2-SDCBP60 was 155.2 nm, and the median particle size was 145.2 nm; the average particle size of the engineered extracellular vesicles loaded with GP2-SDCBP298 was 145.6 nm, and the median particle size was 134.7 nm. The particle sizes of the above-mentioned engineered extracellular vesicles were all between 120 nm and 160 nm.

[0107] Transmission electron microscopy (TEM, Thermo FEI, 120 kV) was used to observe the structures of engineered extracellular vesicles loaded with GP2, engineered extracellular vesicles loaded with GP2-SDCBP60, and engineered extracellular vesicles loaded with GP2-SDCBP298, as shown in Figure 2. Figure 3 As shown, the above-mentioned engineered extracellular vesicles all showed typical vesicle structures.

[0108] The engineered extracellular vesicles loaded with GP2, engineered extracellular vesicles loaded with GP2-SDCBP60, and engineered extracellular vesicles loaded with GP2-SDCBP298 were loaded at the same particle number (2E10 total particles) and subjected to WB analysis (GenScript, surePAGE). The results are shown in Figure 2. Figure 4 As shown, the engineered extracellular vesicles loaded with GP2-SDCBP60 had the strongest ability to enrich GP2 protein, showing a band of ~31kDa-50kDa, while the engineered extracellular vesicles loaded with GP2 and the engineered extracellular vesicles loaded with GP2-SDCBP298 had weaker abilities to enrich GP2 protein.

[0109] Preparation of engineered extracellular vesicles loaded with GP3:

[0110] The GP3 expression plasmid was constructed using the same methods as for the GP2 expression plasmid. The upstream primer (sequence shown in SEQ ID NO: 17), downstream primer (sequence shown in SEQ ID NO: 18), and template DNA (sequence shown in SEQ ID NO: 19) for the synthetic PCR cloning step were redesigned based on the adjusted expression cassette (the GP2 protein sequence in the GP2 expression plasmid expression cassette was replaced with the GP3 protein sequence). All other steps were the same as for the GP2 protein expression plasmid. The preparation of engineered extracellular vesicles loaded with GP2 was also performed using the same methods as for the GP2 protein expression plasmid, except that the GP2 expression plasmid was replaced with the GP3 expression plasmid during the cell transfection step.

[0111] Preparation of engineered extracellular vesicles loaded with a fusion protein (abbreviated as GP3-SDCBP60) containing the N-terminal fragment sequence of GP3 and SDCBP protein (shown in SEQ ID NO: 6):

[0112] The GP3-SDCBP60 expression plasmid was constructed using the same methods as for the GP2 expression plasmid. The upstream primer (SEQ ID NO: 17), downstream primer (SEQ ID NO: 13), and template DNA (SEQ ID NO: 20) for the synthetic PCR cloning step were redesigned based on the adjusted expression cassette (the GP2 protein sequence in the GP2 expression plasmid was replaced with the GP3 protein sequence, and the N-terminal fragment sequence of the SDCBP protein was added to the C-terminus of the GP3 protein sequence). All other steps were the same as for the GP2 protein expression plasmid. The preparation of engineered extracellular vesicles loaded with GP2 was similar to that for engineered extracellular vesicles loaded with GP2, except that the GP2 expression plasmid in the cell transfection step was replaced with the GP3-SDCBP60 expression plasmid.

[0113] Preparation of engineered extracellular vesicles loaded with a fusion protein of GP3 and SDCBP protein (shown in SEQ ID NO: 7) (abbreviated as GP3-SDCBP298):

[0114] The GP3-SDCBP298 expression plasmid was constructed using the same methods as for the GP2 expression plasmid. The upstream primer (SEQ ID NO: 17), downstream primer (SEQ ID NO: 15), and template DNA (SEQ ID NO: 21) for the synthetic PCR cloning step were redesigned based on the adjusted expression cassette (the GP2 protein sequence in the GP2 expression plasmid was replaced with the GP3 protein sequence, and the SDCBP protein sequence was added to the C-terminus of the GP3 protein sequence). All other steps were the same as for the GP2 protein expression plasmid. The preparation of engineered extracellular vesicles loaded with GP2 was similar to that for engineered extracellular vesicles loaded with GP2, except that the GP2 expression plasmid in the cell transfection step was replaced with the GP3-SDCBP298 expression plasmid.

[0115] GP3, GP3-SDCBP60, and GP3-SDCBP298 were overexpressed in 293 cells, and the positive rates were detected by flow cytometry (BD FACS Celesta). The results are shown in the figure. Figure 5 As shown, the positive expression rate of GP3 was 50.9%, the positive expression rate of GP3-SDCBP298 was 37.9%, and the positive expression rate of GP3-SDCBP60 was 29.7%.

[0116] NTA analysis was performed on GP3-loaded engineered extracellular vesicles, GP3-SDCBP60-loaded engineered extracellular vesicles, and GP3-SDCBP298-loaded engineered extracellular vesicles using a ZetaView instrument (Fuliu Bioparticle Metrix, Cat. No. N30E). The particle size distribution is shown in Figure 6 The average particle size of the engineered extracellular vesicles loaded with GP3 was 143.5 nm, and the median particle size was 133.6 nm; the average particle size of the engineered extracellular vesicles loaded with GP3-SDCBP60 was 141.4 nm, and the median particle size was 131.0 nm; the average particle size of the engineered extracellular vesicles loaded with GP3-SDCBP298 was 143.3 nm, and the median particle size was 131.5 nm. The particle sizes of the above-mentioned engineered extracellular vesicles were all between 130 nm and 150 nm.

[0117] Transmission electron microscopy (TEM, Thermo FEI, 120 kV) was used to observe the structures of engineered extracellular vesicles loaded with GP3, engineered extracellular vesicles loaded with GP3-SDCBP60, and engineered extracellular vesicles loaded with GP3-SDCBP298, as shown in Figure 2. Figure 7 As shown in the figure, the above extracellular vesicles all have typical vesicle structures.

[0118] The engineered extracellular vesicles loaded with GP3, engineered extracellular vesicles loaded with GP3-SDCBP60, and engineered extracellular vesicles loaded with GP3-SDCBP298 were loaded at the same particle number (2E10 total particles) and subjected to WB analysis (GenScript, surePAGE). The results are shown in Figure 2. Figure 8 As shown, the engineered extracellular vesicles loaded with GP3 had the strongest ability to enrich GP3, showing a band of ~50kDa-58kDa, while the engineered extracellular vesicles loaded with GP3-SDCBP60 and the engineered extracellular vesicles loaded with GP3-SDCBP298 had relatively weak abilities to enrich GP3.

[0119] Preparation of engineered extracellular vesicles loaded with GP4:

[0120] The GP4 expression plasmid was constructed using the same methods as for the GP2 expression plasmid. The upstream primer (sequence shown in SEQ ID NO: 22), downstream primer (sequence shown in SEQ ID NO: 23), and template DNA (sequence shown in SEQ ID NO: 24) for the synthetic PCR cloning step were redesigned based on the adjusted expression cassette (the GP2 protein sequence in the GP2 expression plasmid expression cassette was replaced with the GP4 protein sequence). All other steps were the same as for the GP2 protein expression plasmid. The preparation of engineered extracellular vesicles loaded with GP2 was also performed using the same methods as for the GP2 protein expression plasmid, except that the GP2 expression plasmid was replaced with the GP4 expression plasmid during the plasmid transfection step.

[0121] Preparation of engineered extracellular vesicles loaded with a fusion protein (abbreviated as GP4-SDCBP60) containing the N-terminal fragment sequence of GP4 and SDCBP protein (shown in SEQ ID NO: 6):

[0122] The GP4-SDCBP60 expression plasmid was constructed using the same methods as for the GP2 expression plasmid. The upstream primer (SEQ ID NO: 22), downstream primer (SEQ ID NO: 13), and template DNA (SEQ ID NO: 25) for the synthetic PCR cloning step were redesigned based on the adjusted expression cassette (the GP2 protein sequence in the GP2 expression plasmid was replaced with the GP4 protein sequence, and the N-terminal fragment sequence of the SDCBP protein was added to the C-terminus of the GP4 protein sequence). All other steps were the same as for the GP2 protein expression plasmid. The preparation of engineered extracellular vesicles loaded with GP2 was similar to that for engineered extracellular vesicles loaded with GP2, except that the GP2 expression plasmid in the cell transfection step was replaced with the GP4-SDCBP60 expression plasmid.

[0123] Preparation of engineered extracellular vesicles loaded with a fusion protein of GP4 and SDCBP protein (shown in SEQ ID NO: 7) (abbreviated as GP4-SDCBP298):

[0124] The GP4-SDCBP298 expression plasmid was constructed using the same methods as for the GP2 expression plasmid. The upstream primer (SEQ ID NO: 22), downstream primer (SEQ ID NO: 15), and template DNA (SEQ ID NO: 26) for the synthetic PCR cloning step were redesigned based on the adjusted expression cassette (the GP2 protein sequence in the GP2 expression plasmid was replaced with the GP4 protein sequence, and the SDCBP protein sequence was added to the C-terminus of the GP4 protein sequence). All other steps were the same as for the GP2 protein expression plasmid. The preparation of engineered extracellular vesicles loaded with GP2 was similar to that for engineered extracellular vesicles loaded with GP2, except that the GP2 expression plasmid in the cell transfection step was replaced with the GP4-SDCBP298 expression plasmid.

[0125] GP4, GP4-SDCBP60, and GP4-SDCBP298 were overexpressed in 293 cells, and the positive rates were detected by flow cytometry (BD FACS Celesta). The results are shown in the figure. Figure 9 As shown, the positive expression rate of GP4 was 38.8%, the positive expression rate of GP4-SDCBP298 was 39.7%, and the positive expression rate of GP4-SDCBP60 was 40.0%.

[0126] NTA analysis was performed on the engineered extracellular vesicles loaded with GP4, engineered extracellular vesicles loaded with GP4-SDCBP60, and engineered extracellular vesicles loaded with GP4-SDCBP298 using a ZetaView instrument (Fuliu Bioparticle Metrix, Cat. No. N30E). The particle size distribution is shown in Figure 10 The average particle size of the extracellular vesicles loaded with GP4 was 150.0 nm, and the median particle size was 138.9 nm; the average particle size of the engineered extracellular vesicles loaded with GP4-SDCBP60 was 156.0 nm, and the median particle size was 143.6 nm; the average particle size of the engineered extracellular vesicles loaded with GP4-SDCBP298 was 150.2 nm, and the median particle size was 140.6 nm. The particle sizes of the above-mentioned engineered extracellular vesicles were all between 150 nm and 160 nm.

[0127] Transmission electron microscopy (TEM, Thermo FEI, 120 kV) was used to observe the structures of engineered extracellular vesicles loaded with GP4, engineered extracellular vesicles loaded with GP4-SDCBP60, and engineered extracellular vesicles loaded with GP4-SDCBP298, as shown in Figure 2. Figure 11 As shown, the above-mentioned engineered extracellular vesicles all showed typical vesicle structures.

[0128] The engineered extracellular vesicles loaded with GP4, engineered extracellular vesicles loaded with GP4-SDCBP60, and engineered extracellular vesicles loaded with GP4-SDCBP298 were loaded at the same particle number (2E10 total particles) and subjected to WB analysis (GenScript, surePAGE). The results are shown in Figure 2. Figure 12 As shown, the engineered extracellular vesicles loaded with GP4-SDCBP60 had the strongest ability to enrich GP4, showing a band of ~31kDa-50kDa, while the engineered extracellular vesicles loaded with GP4 and the engineered extracellular vesicles loaded with GP4-SDCBP298 had relatively weak abilities to enrich GP4.

[0129] Preparation of engineered extracellular vesicles loaded with GP5:

[0130] The GP5 expression plasmid was constructed using the same methods as for the GP2 expression plasmid. The upstream primer (SEQ ID NO: 27), downstream primer (SEQ ID NO: 28), and template DNA (SEQ ID NO: 29) for the synthetic PCR cloning step were redesigned based on the adjusted expression cassette (the GP2 protein sequence in the GP2 expression plasmid was replaced with the GP5 protein sequence). All other steps were the same as for the GP2 protein expression plasmid. The preparation of engineered extracellular vesicles loaded with GP2 was also performed using the same methods as for the GP2 protein expression plasmid, except that the GP2 expression plasmid was replaced with the GP5 expression plasmid during the cell transfection step.

[0131] Preparation of engineered extracellular vesicles loaded with a fusion protein (abbreviated as GP5-SDCBP60) containing the N-terminal fragment sequence of GP5 and SDCBP protein (shown in SEQ ID NO: 6):

[0132] The GP5-SDCBP60 expression plasmid was constructed using the same methods as for the GP2 expression plasmid. The upstream primer (SEQ ID NO: 27), downstream primer (SEQ ID NO: 13), and template DNA (SEQ ID NO: 30) for the synthetic PCR cloning step were redesigned based on the adjusted expression cassette (the GP2 protein sequence in the GP2 expression plasmid was replaced with the GP5 protein sequence, and the N-terminal fragment sequence of the SDCBP protein was added to the C-terminus of the GP5 protein sequence). All other steps were the same as for the GP2 protein expression plasmid. The preparation of engineered extracellular vesicles loaded with GP2 was similar to that for engineered extracellular vesicles loaded with GP2, except that the GP2 expression plasmid in the cell transfection step was replaced with the GP5-SDCBP60 expression plasmid.

[0133] Preparation of engineered extracellular vesicles loaded with a fusion protein of GP5 and SDCBP protein (shown in SEQ ID NO: 7) (abbreviated as GP5-SDCBP298):

[0134] The GP5-SDCBP298 expression plasmid was constructed using the same methods as for the GP2 expression plasmid. The upstream primer (SEQ ID NO: 27), downstream primer (SEQ ID NO: 15), and template DNA (SEQ ID NO: 31) for the synthetic PCR cloning step were redesigned based on the adjusted expression cassette (the GP2 protein sequence in the GP2 expression plasmid was replaced with the GP5 protein sequence, and the SDCBP protein sequence was added to the C-terminus of the GP5 protein sequence). All other steps were the same as for the GP2 protein expression plasmid. The preparation of engineered extracellular vesicles loaded with GP2 was similar to that for engineered extracellular vesicles loaded with GP2, except that the GP2 expression plasmid in the cell transfection step was replaced with the GP5-SDCBP298 expression plasmid.

[0135] GP5, GP5-SDCBP60, and GP5-SDCBP298 were overexpressed in 293 cells, and the positive rates were detected by flow cytometry (BD FACS Celesta). The results are shown in the figure. Figure 13 As shown, the positive expression rate of GP5 was 0.47%, the positive expression rate of GP5-SDCBP298 was 11.3%, and the positive expression rate of GP5-SDCBP60 was 14.6%.

[0136] NTA analysis was performed on the engineered extracellular vesicles loaded with GP5, engineered extracellular vesicles loaded with GP5-SDCBP60, and engineered extracellular vesicles loaded with GP5-SDCBP298 using a ZetaView instrument (Fuliu Bioparticle Metrix, Cat. No. N30E). The particle size distribution is shown in Figure 14 The average particle size of the engineered extracellular vesicles loaded with GP5 was 145.6 nm, and the median particle size was 138.5 nm; the average particle size of the engineered extracellular vesicles loaded with GP5-SDCBP60 was 138.0 nm, and the median particle size was 125.0 nm; the average particle size of the engineered extracellular vesicles loaded with GP5-SDCBP298 was 152.7 nm, and the median particle size was 142.2 nm. The particle sizes of the above extracellular vesicles were all between 130 nm and 160 nm.

[0137] Transmission electron microscopy (TEM, Thermo FEI, 120 kV) was used to observe the structures of engineered extracellular vesicles loaded with GP5, engineered extracellular vesicles loaded with GP5-SDCBP60, and engineered extracellular vesicles loaded with GP5-SDCBP298, as shown in Figure 2. Figure 15 As shown, the above-mentioned engineered extracellular vesicles all showed typical vesicle structures.

[0138] The engineered extracellular vesicles loaded with GP5, engineered extracellular vesicles loaded with GP5-SDCBP60, and engineered extracellular vesicles loaded with GP5-SDCBP298 were loaded at the same particle number (2E10 total particles) and subjected to WB analysis (GenScript, surePAGE). The results are shown in Figure 2. Figure 16 As shown, the engineered extracellular vesicles loaded with GP4-SDCBP60 had the strongest ability to enrich GP5, showing a band of ~50kDa-58kDa, while the engineered extracellular vesicles loaded with GP5 and the engineered extracellular vesicles loaded with GP5-SDCBP298 had a very weak ability to enrich GP5.

[0139] Preparation of engineered extracellular vesicles loaded with M:

[0140] The M expression plasmid was constructed using the same methods as for the GP2 expression plasmid. The upstream primer (SEQ ID NO: 32), downstream primer (SEQ ID NO: 33), and template DNA (SEQ ID NO: 34) for the synthetic PCR cloning step were redesigned based on the adjusted expression cassette (the GP2 protein sequence in the GP2 expression plasmid was replaced with the M protein sequence). All other steps were the same as for the GP2 protein expression plasmid. The preparation of engineered extracellular vesicles loaded with GP2 was also performed using the same methods as for the GP2 protein expression plasmid, except that the GP2 expression plasmid was replaced with the M expression plasmid during the cell transfection step.

[0141] Preparation of engineered extracellular vesicles loaded with a fusion protein (abbreviated as M-SDCBP60) containing the N-terminal fragment sequence of M and SDCBP protein (shown in SEQ ID NO: 6):

[0142] The M-SDCBP60 expression plasmid was constructed using the same methods as for the GP2 expression plasmid. The upstream primer (SEQ ID NO: 32), downstream primer (SEQ ID NO: 13), and template DNA (SEQ ID NO: 35) for the synthetic PCR cloning step were redesigned based on the adjusted expression cassette (the GP2 protein sequence in the GP2 expression plasmid was replaced with the M protein sequence, and the N-terminal fragment sequence of the SDCBP protein was added to the C-terminus of the M protein sequence). All other steps were the same as for the GP2 protein expression plasmid. The preparation of engineered extracellular vesicles loaded with GP2 was similar to that for engineered extracellular vesicles loaded with GP2, except that the GP2 expression plasmid in the cell transfection step was replaced with the M-SDCBP60 expression plasmid.

[0143] Preparation of engineered extracellular vesicles loaded with a fusion protein of M and SDCBP protein (shown in SEQ ID NO: 7) (abbreviated as M-SDCBP298):

[0144] The M-SDCBP298 expression plasmid was constructed using the same methods as for the GP2 expression plasmid. The upstream primer (SEQ ID NO: 32), downstream primer (SEQ ID NO: 15), and template DNA (SEQ ID NO: 36) for the synthetic PCR cloning step were redesigned based on the adjusted expression cassette (the GP2 protein sequence in the GP2 expression plasmid was replaced with the M protein sequence, and the SDCBP protein sequence was added to the C-terminus of the M protein sequence). All other steps were the same as for the GP2 protein expression plasmid. The preparation of engineered extracellular vesicles loaded with GP2 was similar to that for engineered extracellular vesicles loaded with GP2, except that the GP2 expression plasmid in the cell transfection step was replaced with the M-SDCBP298 expression plasmid.

[0145] M, M-SDCBP60, and M-SDCBP298 were overexpressed in 293 cells, and the positive rates were detected by flow cytometry (BD FACS Celesta). The results are shown in Figure 2. Figure 17 As shown in the figure, the positive expression rate of M was 2.63%, the positive expression rate of M-SDCBP298 was 21.6%, and the positive expression rate of M-SDCBP60 was 25.0%.

[0146] NTA analysis was performed on engineered extracellular vesicles loaded with M, engineered extracellular vesicles loaded with M-SDCBP60, and engineered extracellular vesicles loaded with M-SDCBP298 using a ZetaView instrument (Fuliu Bioparticle Metrix, Cat. No. N30E). The particle size distribution is shown in Figure 18 The average particle size of the engineered extracellular vesicles loaded with M was 135.1 nm, and the median particle size was 122.4 nm; the average particle size of the engineered extracellular vesicles loaded with M-SDCBP60 was 143.6 nm, and the median particle size was 128.9 nm; the average particle size of the engineered extracellular vesicles loaded with M-SDCBP298 was 141.7 nm, and the median particle size was 131.1 nm. The particle sizes of the above engineered extracellular vesicles were all between 130 nm and 150 nm.

[0147] Transmission electron microscopy (TEM, Thermo FEI, 120 kV) was used to observe the structures of engineered extracellular vesicles loaded with M, engineered extracellular vesicles loaded with M-SDCBP60, and engineered extracellular vesicles loaded with M-SDCBP298, as shown in Figure 2. Figure 19As shown, the above-mentioned engineered extracellular vesicles all showed typical vesicle structures.

[0148] The engineered extracellular vesicles loaded with M, engineered extracellular vesicles loaded with M-SDCBP60, and engineered extracellular vesicles loaded with M-SDCBP298 were loaded with the same number of particles (2E10 total particles) and subjected to WB analysis (GenScript, surePAGE). The results are shown in Figure 2. Figure 20 As shown, the engineered extracellular vesicles loaded with M-SDCBP60 had the strongest ability to enrich M, showing a band of ~25 kDa, while the engineered extracellular vesicles loaded with M and the engineered extracellular vesicles loaded with M-SDCBP298 had relatively weak abilities to enrich M.

[0149] Preparation of engineered extracellular vesicles loaded with fusion proteins of various PRRSV envelope proteins and the nitrogen-terminal fragment of SDCBP and corresponding effect experiments.

[0150] Preparation of engineered extracellular vesicles loaded with two PRRSV envelope proteins (GP5-SDCBP60 and M-SDCBP60):

[0151] Referring to the preparation method of engineered extracellular vesicles loaded with GP2, except that the 25 μg GP2 expression plasmid in the plasmid transfection cell step was replaced with a mixed plasmid of 5 μg GP5-SDCBP60 expression plasmid and 5 μg M-SDCBP60 expression plasmid, the rest of the operations were the same as the preparation method of engineered extracellular vesicles loaded with GP2.

[0152] Preparation of engineered extracellular vesicles loaded with two PRRSV envelope proteins (GP2-SDCBP60 and GP4-SDCBP60):

[0153] Methods: The above-mentioned method for preparing GP2-loaded extracellular vesicles was referred to, except that the 25 μg GP2 expression plasmid in the plasmid transfection cell step was replaced with a mixed plasmid of 5 μg GP2-SDCBP60 expression plasmid and 5 μg GP4-SDCBP60 expression plasmid. The rest of the operations were the same as the method for preparing GP2-loaded engineered extracellular vesicles.

[0154] Preparation of engineered extracellular vesicles loaded with five PRRSV envelope proteins (GP2-SDCBP60, GP3-SDCBP60, GP4-SDCBP60, GP5-SDCBP60, and M-SDCBP60):

[0155] The preparation method refers to the above-mentioned method for preparing engineered extracellular vesicles loaded with GP2, except that the 25 μg GP2 expression plasmid in the plasmid transfection cell step is replaced with a mixed plasmid of 5 μg GP2-SDCBP60 expression plasmid, 5 μg GP3-SDCBP60 expression plasmid, 5 μg GP4-SDCBP60 expression plasmid, 5 μg GP5-SDCBP60 expression plasmid, and 5 μg M-SDCBP60 expression plasmid. The remaining operations are the same as the preparation method of engineered extracellular vesicles loaded with GP2.

[0156] The engineered extracellular vesicles loaded with the envelope proteins of the five PRRSVs and the envelope proteins of the two PRRSVs were analyzed by NTA using a ZetaView instrument (Fuliu Bioparticle Metrix, Cat. No. N30E). The particle size distribution is shown in Figure 21 The average particle size of the engineered extracellular vesicles loaded with five PRRSV envelope proteins was 135.8 nm, and the median particle size was 123.5 nm; the average particle size of the engineered extracellular vesicles loaded with GP5-SDCBP60 and M-SDCBP60 was 135.2 nm, and the median particle size was 120.4 nm; the average particle size of the engineered extracellular vesicles loaded with GP2-SDCBP60 and GP4-SDCBP60 was 142.7 nm, and the median particle size was 129.3 nm.

[0157] The structures of the engineered extracellular vesicles loaded with the envelope proteins of the five PRRSVs and the engineered extracellular vesicles loaded with the envelope proteins of the two PRRSVs were observed using a transmission electron microscope (TEM, Thermo FEI, 120 kV). Figure 22 As shown, typical vesicle structures can be seen in the engineered extracellular vesicles loaded with five PRRSV envelope proteins and the engineered extracellular vesicles loaded with two PRRSV envelope proteins.

[0158] A blank control (extracellular vesicles without cargo, isolated from the culture medium of Expi293F cells not transfected with the plasmid, referring to the steps of isolating and purifying extracellular vesicles in the preparation method of engineered extracellular vesicles loaded with GP2), the engineered extracellular vesicles loaded with the five PRRSV envelope proteins, and the engineered extracellular vesicles loaded with the two PRRSV envelope proteins were loaded at the same particle number (2E10 total particles) and subjected to Western blotting analysis (surePAGE). The results are shown in Figure 2. Figure 23As shown, engineered EVs loaded with fusion proteins of the five PRRSV envelope proteins and the N-terminal fragment of SDCBP showed the best enrichment of each envelope protein, with distinct bands for each envelope protein. However, engineered EVs loaded with GP5-SDCBP60 and M-SDCBP60 only showed the M band, while engineered EVs loaded with GP2-SDCBP60 and GP4-SDCBP60 showed neither GP2 nor GP4 bands. This suggests that when the five fusion proteins are co-expressed in cells, beneficial aggregates may form, and the five fusion proteins interact with each other to promote the enrichment efficiency of each PRRSV envelope protein in EVs.

[0159] In addition, Figure 23 and Figure 4 、 Figure 8 、 Figure 12 、 Figure 16 and Figure 20 By comparison, Figure 23 The color depth of the band of each envelope protein in the engineered extracellular vesicles loaded with the fusion protein of five PRRSV envelope proteins and the N-terminal fragment of SDCBP was significantly darker than that of the corresponding engineered extracellular vesicles loaded with one PRRSV envelope protein, the engineered extracellular vesicles loaded with the fusion protein of one PRRSV envelope protein and the N-terminal fragment of SDCBP, and the engineered extracellular vesicles loaded with one PRRSV envelope protein and SDCBP protein. The enrichment efficiency of each PRRSV envelope protein in the engineered extracellular vesicles loaded with five PRRSV envelope proteins was significantly higher. When plasmids were co-transfected into cells, the amount of each plasmid used when co-transfecting five plasmids was significantly lower than the amount used when transfecting only one plasmid. It is speculated that GP2-SDCBP60, GP3-SDCBP60, GP4-SDCBP60, GP5-SDCBP60 and M-SDCBP60 may have a synergistic expression effect, and their expression levels can be increased when co-transfected.

[0160] Example: Animals were immunized with extracellular vesicles loaded with PRRSV envelope proteins and the immune effects were tested.

[0161] Six-week-old female New Zealand white rabbits were used as immune hosts and were immunized with two injections intramuscularly, 21 days apart. Three groups were set up, with three rabbits in each group. The first group was equipped with extracellular vesicles loaded with PRRSV envelope proteins (the engineered extracellular vesicles loaded with the fusion protein of the five PRRSV envelope proteins and the N-terminal fragment of SDCBP described above were used. The particle concentration of the engineered extracellular vesicles was adjusted to 1.0×10 13The second group was immunized with a live vaccine (JXA1-R strain) (Kelanin, Wuhan Keqian Biological Co., Ltd.) containing 10 particles / mL of virus. 5 TCID 50 / mL, with each injection dose of 1mL / animal. The third group was not immunized and served as a control. Serum samples were collected before immunization, 21 days after the first immunization, and 7 days after the second immunization, and the neutralizing antibody titer of the serum was tested. The serum samples of the first group were labeled 1-1, 1-2, and 1-3, the serum samples of the second group were labeled 2-1, 2-2, and 2-3, and the serum samples of the third group were labeled 3-1, 3-2, and 3-3.

[0162] The method for detecting the neutralizing antibody titer of serum is as follows:

[0163] (1) Preparation of Marc-145 cells: Resuscitate Marc-145 cells in advance, inoculate the cells into a cell culture dish, and culture them in DMEM medium containing 10% fetal bovine serum and 1% double antibody. When the cells grow to 80%, digest them with trypsin and plate them at 5×10 cells per well. 4 The cells were inoculated into a sterile 96-well plate at a density of 100 cells / well and cultured in a 37°C, 5% CO2 incubator until the cell confluence reached 80%-90%. Then, the plate could be used to detect virus neutralizing antibodies.

[0164] (2) Serum sample processing: Place the serum sample in a 56°C water bath for 30 minutes to inactivate complement.

[0165] (3) Virus preparation: The JXA1-R strain (donated by the Virus Laboratory of the China Veterinary Drug Administration) was taken out of liquid nitrogen, quickly thawed in a 37°C water bath, and diluted to 100 TCID using maintenance medium (DMEM medium containing 2% fetal bovine serum and 1% double antibody). 50 JXA1-R strain virus suspension.

[0166] (4) Neutralization reaction: In a sterile 96-well plate, dilute the treated serum sample in a serial dilution, starting from 1:2, and make 4 replicates for each dilution. Add 50 μL of diluted serum sample to each well. Add 50 μL of 100 TCID 50 Mix the JXA1 strain virus suspension gently and incubate it in a 37°C, 5% CO2 incubator for 1 hour to allow the virus to fully neutralize with the antibodies in the serum.

[0167] (5) Virus infection of cells: Marc-145 cells cultured in a sterile 96-well plate were washed twice with PBS, and 100 μL of the virus and serum mixture after the reaction was added to each well. At the same time, a virus control well was set up (only 100 μL of 100 TCID50 The sterile 96-well plates were incubated in a 37°C, 5% CO2 incubator. Cytopathic effect (CPE) was observed daily and recorded until day 7.

[0168] (6) The pathological changes of cells in each well were recorded. The pathological changes of cells were manifested as rounding and shedding of cells. The neutralizing antibody titer (lgPD50) was calculated according to the Reed-Muench method: lgPD50 = the logarithm of the serum dilution with a protection rate higher than or equal to 50% + the distance ratio × the logarithm of the dilution factor, distance ratio = (percentage of serum with a protection rate higher than 50% - 50%) ÷ (percentage of serum with a protection rate higher than 50% - percentage of serum with a protection rate lower than 50%). The results are shown in Table 1.

[0169]

[0170] Table 1 shows that after the first and second immunizations using the engineered extracellular vesicles of the present invention loaded with fusion proteins of five PRRSV envelope proteins and the nitrogen-terminal fragment of SDCBP, neutralizing antibodies against PRRSV are continuously produced in the host, and the neutralizing antibody titer is higher than the neutralizing antibody level after immunization with the highly pathogenic porcine reproductive and respiratory syndrome heat-resistant protectant live vaccine (JXA1-R strain). This indicates that the engineered extracellular vesicles of the present invention loaded with fusion proteins of five PRRSV envelope proteins and the nitrogen-terminal fragment of SDCBP can be used as a porcine reproductive and respiratory syndrome vaccine to achieve effective delivery of multiple PRRSV protein antigens and stimulate immune response to produce high levels of neutralizing antibodies against PRRSV, and the immune effect is better than that of the highly pathogenic porcine reproductive and respiratory syndrome heat-resistant protectant live vaccine (JXA1-R strain).

[0171] The above detailed description of the present invention is intended to enable those skilled in the art to understand the contents of the present invention and implement them, but it does not limit the scope of protection of the present invention. The present invention is not limited to the above embodiments. Any equivalent changes or modifications made based on the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing engineered extracellular vesicles, characterized in that: The following steps are involved: (1) Construct and prepare a recombinant expression vector system for expressing PRRSV antigens; (2) transfecting cells with the recombinant expression vector system to obtain recombinant cells; (3) culturing the recombinant cells to obtain a recombinant cell culture fluid; (4) isolating the engineered extracellular vesicles from the recombinant cell culture fluid, The PRRSV antigens include a fusion protein of the GP2 envelope protein of PRRSV and the N-terminal fragment of the SDCBP protein, a fusion protein of the GP3 envelope protein of PRRSV and the N-terminal fragment of the SDCBP protein, a fusion protein of the GP4 envelope protein of PRRSV and the N-terminal fragment of the SDCBP protein, a fusion protein of the GP5 envelope protein of PRRSV and the N-terminal fragment of the SDCBP protein, and a fusion protein of the M envelope protein of PRRSV and the N-terminal fragment of the SDCBP protein. The amino acid sequence of the GP2 envelope protein of PRRSV is shown in SEQ ID NO: 1; The amino acid sequence of the GP3 envelope protein of PRRSV is shown in SEQ ID NO: 2; The amino acid sequence of the GP4 envelope protein of PRRSV is shown in SEQ ID NO: 3; The amino acid sequence of the GP5 envelope protein of PRRSV is shown in SEQ ID NO: 4; The amino acid sequence of the M envelope protein of PRRSV is shown in SEQ ID NO: 5; The amino acid sequence of the N-terminal fragment of the SDCBP protein is shown in SEQ ID NO: 6; The N-terminal fragment of the SDCBP protein is connected to the C-terminal end of the envelope protein. The recombinant expression vector system includes a first recombinant expression plasmid containing a coding sequence for a fusion protein of the GP2 envelope protein of the PRRSV and the N-terminal fragment of the SDCBP protein, a second recombinant expression plasmid containing a coding sequence for a fusion protein of the GP3 envelope protein of the PRRSV and the N-terminal fragment of the SDCBP protein, a third recombinant expression plasmid containing a coding sequence for a fusion protein of the GP4 envelope protein of the PRRSV and the N-terminal fragment of the SDCBP protein, a fourth recombinant expression plasmid containing a coding sequence for a fusion protein of the GP5 envelope protein of the PRRSV and the N-terminal fragment of the SDCBP protein, and a fifth recombinant expression plasmid containing a coding sequence for a fusion protein of the M envelope protein of the PRRSV and the N-terminal fragment of the SDCBP protein. The host cell of the recombinant cell is an embryonic kidney cell line.

2. The preparation method according to claim 1, characterized in that The expression vector used in the recombinant expression vector system is a eukaryotic expression plasmid vector or a viral plasmid vector; and / or, the separation comprises ultracentrifugation, density gradient centrifugation, size exclusion chromatography, affinity chromatography, adsorption chromatography, bonded phase chromatography, immunoseparation, or sieving separation; And / or, the culture adopts serum-free medium; And / or, the culturing is carried out at 34° C. to 38° C. and 5% to 10% CO 2 .

3. The preparation method according to claim 1, characterized in that The host cell of the recombinant cell is HEK293 cell.

4. The preparation method according to claim 1, characterized in that The expression vectors used in the recombinant expression vector system include pAAVS1 plasmid, pIRES plasmid, and pLENTI plasmid.

5. The engineered extracellular vesicles prepared by the preparation method according to any one of claims 1 to 4.

6. A recombinant cell for preparing engineered extracellular vesicles, characterized in that: The recombinant cell is the recombinant cell according to claim 1.

7. Use of the engineered extracellular vesicles according to claim 5 in the preparation of a medicament for preventing PRRSV infection.

8. A porcine reproductive and respiratory syndrome vaccine based on extracellular vesicles, characterized in that: It comprises the engineered extracellular vesicles according to claim 5 and may optionally comprise pharmaceutically acceptable excipients.

9. The porcine reproductive and respiratory syndrome vaccine based on extracellular vesicles according to claim 8, characterized in that The auxiliary material includes a PBS buffer solution with a pH value of 7.2 to 7.6.

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