CN111454989A

CN111454989A - Chimeric gene type I encephalitis B virus-like particle vaccine and preparation method and application thereof

Info

Publication number: CN111454989A
Application number: CN202010261589.6A
Authority: CN
Inventors: 田占成; 周亚红; 于瑞明; 高闪电; 独军政; 殷宏; 罗建勋; 刘光远; 关贵全; 刘志杰
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2020-04-04
Filing date: 2020-04-04
Publication date: 2020-07-28
Anticipated expiration: 2040-04-04
Also published as: CN111454989B

Abstract

The invention discloses a chimeric gene type I Japanese encephalitis virus-like particle vaccine as well as a preparation method and application thereof, belonging to the technical field of biology. The chimeric virus-like particle is a recombinant swine fever virus neutralizing epitope chimeric antigen protein based on a genotype I type Japanese encephalitis virus-like particle, a nucleotide sequence of a DNA fragment for encoding the virus-like particle is shown as SEQ # ID # NO.1, and an amino acid sequence of the DNA fragment is shown as SEQ # ID # NO. 2; cloning a DNA fragment shown in SEQ # ID # NO.1 into a eukaryotic expression vector pVAX1 to construct a recombinant expression plasmid; transfecting the recombinant expression plasmid into a BHK-21 cell, collecting cell culture supernatant of the transfected cell, concentrating and purifying to obtain the chimeric gene type I encephalitis B virus-like particles presenting the neutralizing epitope of classical swine fever virus. The virus-like particle can induce an organism to generate specific body fluid and cellular immune response, can be used for immunoprophylaxis of swine fever virus and epidemic encephalitis B virus infection in a pig farm, and can be used for preparing swine fever virus and epidemic encephalitis B virus vaccines.

Description

Chimeric gene type I encephalitis B virus-like particle vaccine and preparation method and application thereof

Technical Field

The invention relates to the technical field of bioengineering and virus vaccines, in particular to a chimeric gene type I Japanese encephalitis virus-like particle presenting a neutralizing epitope of classical swine fever virus, a preparation method and a vaccine thereof.

Background

Epidemic encephalitis B (Japanese encephalitis for short) is a central nervous system infectious disease which is mainly diseased by brain parenchyma after being infected by Japanese Encephalitis Virus (JEV), and can infect animals such as human, horse, pig, bird and the like. Culex tritaeniorhynchus is a transmission medium of the disease, and pigs are an infection source of the disease. The key point of reducing Japanese encephalitis virus infection of human is to reduce Japanese encephalitis epidemic rate in pig farms by adopting a method of immunizing Japanese encephalitis virus vaccine. The Japanese encephalitis virus has five genotypes, in recent years, the Japanese encephalitis virus with the genotype I gradually replaces the Japanese encephalitis virus with the genotype III to become a main epidemic strain, and the Japanese encephalitis virus attenuated vaccine SA14-14-2 for human or livestock at present is mainly a Japanese encephalitis virus strain with the genotype III, but the attenuated vaccine has the defects of strong toxicity, weak cross protection on the Japanese encephalitis virus strain with the genotype I which is partially epidemic and the like. The problem of high cost of the vaccine prevents the pig farm from widely using the attenuated Japanese encephalitis virus vaccine to prevent and control the epidemic Japanese encephalitis in the pig farm. Therefore, the development of a novel gene type I Japanese encephalitis virus vaccine which is low in cost, efficient and safe for preventing and controlling the epidemic type B encephalitis in a pig farm is urgently needed.

The Hog cholera is also called as Hog cholera, is a highly contagious disease of pigs caused by Hog cholera virus (CSFV), takes the pigs as an infection source, has the characteristics of high morbidity and high mortality, and is one of important infectious diseases seriously threatening the healthy development of the pig breeding industry, the comprehensive immunization of Hog cholera lapinized virus vaccine (HC L V) ensures that China controls the pandemic of the Hog cholera, but the variation of the Hog cholera virus, the difficulty in distinguishing the immunized pigs from the serological method detection of the Hog cholera vaccine, the non-standard production and use of the Hog cholera vaccine and other factors cause great difficulty in purifying the Hog cholera virus in China, so the development of a safer novel Hog cholera marker vaccine becomes an inevitable trend.

The Chinese patent application 2018114801802 provides a swine fever virus chimeric virus-like particle, a preparation method and application thereof, and a vaccine, belonging to the technical field of bioengineering and virus vaccines. The swine fever virus chimeric virus-like particle adopts Gag precursor protein of retrovirus as matrix protein, and takes swine fever virus envelope proteins E0 and E2 as surface membrane proteins, so that an ideal and functional virus-like particle is self-assembled, the defects of subunit protein vaccines are overcome, and the immunogenicity, safety and stability of the vaccine are effectively improved; the mosaic virus-like particle can be prepared in a large scale by constructing a baculovirus expression vector to transfect insect cells; the swine fever virus chimeric virus-like particle can be applied to preparation of vaccines for preventing swine fever diseases, preparation of swine fever virus antibody detection preparations or swine fever virus epidemic disease monitoring preparations, and has good practical application value. However, it is clear that this technique has no immunoprophylactic effect on swine epidemic encephalitis B infection.

Disclosure of Invention

The invention provides a chimeric gene type I Japanese encephalitis virus-like particle presenting a neutralizing epitope of classical swine fever virus, and also provides a preparation method of the virus-like particle and a vaccine thereof.

The preparation method of the chimeric gene type-I encephalitis B virus-like particle presenting the hog cholera virus neutralizing epitope comprises the steps of inserting a coding sequence of a hog cholera virus E2 protein neutralizing epitope (CTAVSPTT L RTEVVK) into three L oop sites of a gene type-I encephalitis B virus-like particle coding gene expression cassette by an in vitro gene synthesis method, mutating 108 th phenylalanine (F) of the coding gene type-I encephalitis B virus E protein into alanine (A), obtaining a fusion gene, then using the fusion gene as a template, carrying out amplification by PCR, carrying out DNA gel recovery after carrying out double enzyme digestion on a PCR product and an eukaryotic expression vector pVAX1 by EcoR I and Xba I, connecting the PCR product with the eukaryotic expression vector, then transforming into a trans5 α competent state, extracting a correctly identified positive recombinant bacterium by using a medium-quality-improving plasmid kit, obtaining a high-purity recombinant pVAX 2 plasmid containing a target gene, transfecting the recombinant pVAX 3875 to a BHK cell, and carrying out transfection on a BHK cell culture to obtain a chimeric gene expressing the viral epitope of the chimeric gene, and concentrating the chimeric gene expressing the porcine encephalitis B virus, and purifying the viral particle presenting the porcine virus.

Preferably, the Japanese encephalitis virus belongs to genotype I in the preparation method of the chimeric genotype I Japanese encephalitis virus-like particle presenting the hog cholera virus neutralizing epitope.

Preferably, the PCR amplification primers used in the preparation method of the chimeric gene type I Japanese encephalitis virus-like particle presenting the hog cholera virus neutralizing epitope are SEQ ID No.3 and SEQ ID No. 4.

Preferably, the preparation method of the chimeric gene type I Japanese encephalitis virus-like particle for presenting the hog cholera virus neutralizing epitope (CTAVSPTT L RTEVVK) inserts the coding sequence of the hog cholera virus E2 protein neutralizing epitope (CTAVSPTTT L RTEVVK) into three L oop insertion sites of the gene expression cassette of the gene type I Japanese encephalitis virus-like particle coding gene by an in vitro gene synthesis methodP respectively located in Japanese encephalitis virus E protein₂₂₈-S₂₂₉，T₂₄₆-A₂₄₇And S₂₉₀-S₂₉₁Between amino acid positions.

More preferably, the invention provides a method for preparing chimeric type gene type I Japanese encephalitis virus-like particles for presenting neutralizing epitope of hog cholera virus, which comprises mutating 108 th phenylalanine (F) of encoding gene type I Japanese encephalitis virus E protein in the obtained fusion gene into alanine (A).

Preferably, the preparation method of the chimeric gene type I Japanese encephalitis virus-like particle presenting the hog cholera virus neutralizing epitope is characterized in that a recombinant eukaryotic expression vector of a gene type I Japanese encephalitis virus-like particle coding gene expression cassette containing a hog cholera virus E2 protein neutralizing epitope (CTAVSPTTT L RTEVVK) coding sequence is transiently transfected into cells, transfected cell culture supernatant is collected, and the virus-like particle is obtained after concentration and purification.

Preferably, the cell is a BHK-21 cell and the recombinant vector is a pVAX1 recombinant vector.

The nucleotide sequence and the amino acid sequence of the encoding gene of the chimeric type gene type I encephalitis B virus-like particle presenting the neutralizing epitope of classical swine fever virus are SEQ ID No.1 and SEQ ID No.2 respectively.

The chimeric gene type I encephalitis B virus-like particle presenting the hog cholera virus neutralizing epitope prepared by the method can be used for preparing vaccines for preventing infection of hog cholera virus and epidemic encephalitis B virus in a pig farm.

The principle of the technical scheme of the invention is as follows:

1. the generation principle of the chimeric gene type I Japanese encephalitis virus-like particles is as follows:

the gene I type Japanese encephalitis virus-like particles are used as a transfer release carrier, a linear neutralizing epitope (CTAVSPTT L RTEVVK) of a main immunogenic protein E2 of the classical swine fever virus is displayed on the surface of the gene I type Japanese encephalitis virus-like particles and is constructed on a eukaryotic expression carrier pVAX1, a recombinant pVAX1 plasmid obtained by transient transfection is transferred to a BHK-21 cell, and the protein expressed in the cell is assembled into chimeric type Japanese encephalitis virus-like particles without any Japanese encephalitis virus genetic material and is released into a cell culture solution.

2. The generation principle of the chimeric gene type I Japanese encephalitis virus-like particle expression level optimization is as follows:

the Japanese encephalitis virus-like particle has the same or similar spatial conformation as the natural virus particle, although the Japanese encephalitis virus-like particle does not contain the genetic material of the natural virus particle, the Japanese encephalitis virus-like particle released into a cell culture solution still has the capacity of infecting host cells and reducing cell viability, and is not beneficial to the expression of virus-like particle constituent protein. The key amino acid of structural protein E closely related to cell membrane fusion when Japanese encephalitis virus invades host cells is mutated, for example, 108 th phenylalanine (F) of gene I type Japanese encephalitis virus-like particle constitutive protein E is mutated into alanine (A), so that the secondary infection capacity of the virus-like particles released into a cell culture solution on transfected cells is reduced, the transfected cells have better cell activity, and the expression of virus-like particle constitutive protein is facilitated.

3. The immune principle of the chimeric gene type I Japanese encephalitis virus-like particle is as follows:

envelope glycoprotein E2 is a main immunogenicity protective antigen of the classical swine fever virus, and highly conserved E2 protein linear neutralizing epitope (CTAVSPTT L RTEVVK) in different classical swine fever virus epidemic strains can induce an organism to generate protective immune antibodies against the classical swine fever virus, a plurality of linear neutralizing epitopes are connected in series to generate better immune protective effect than single neutralizing epitope immunity, because chimeric gene I type encephalitis B virus-like particles have the same or similar spatial conformation as natural virus particles but do not have genetic materials of the virus, the virus-like particle vaccine does not need chemical inactivation, the spatial conformation is not destroyed, after the organism is immunized, the infection way of wild type virus can be simulated, the organism natural immune system better recognizes, and induces strong immune response reaction, the virus-like particles are used as a transfer and release carrier to present exogenous antigens, the exogenous antigens can be more effectively presented to antigen presentation cells, the exogenous antigens can be recognized by the organism natural immune system, the immune response is induced, the immune enhancement effect of the virus-like particles can also be obviously improved, the multiple immune antigen presentation of the virus-like particles can be more effectively presented to the antigen presentation of the classical swine fever virus, and the multiple antigen presentation of the chimeric gene antigen of the classical swine fever virus, the multiple antigen-like virus can be effectively constructed, thus the multiple antigen-like virus, the multiple antigen-like particle vaccine can be effectively displayed in the swine fever virus, and the immune virus-like antigen of the swine fever virus vaccine.

Compared with the existing vaccine, the vaccine prepared by the virus-like particle of the invention has the following advantages:

1. the vaccine overcomes the defects of attenuated vaccines of classical swine fever virus and Japanese encephalitis virus, and simultaneously can avoid the defect of poor cross protection of the existing attenuated vaccine of gene type III Japanese encephalitis virus on partial gene type I Japanese encephalitis virus strains. Because the virus-like particles have the space conformation of natural virus particles and the epitope for inducing neutralizing antibodies, and do not have genetic materials of viruses, the virus-like particle vaccine does not need chemical inactivation, and the space conformation of surface glycoprotein is not damaged and is consistent with that of wild viruses, so the virus-like particles have better antigenicity, safety and stability; the obtained virus-like particles can better present the neutralizing epitope of the classical swine fever virus to antigen presenting cells, and are recognized by the natural immune system of an organism to induce strong immune response reaction; recovering, concentrating and purifying the supernatant of the cell culture of the transient transfection to obtain virus-like particles, and obtaining a large amount of antigens which keep natural activity and immunogenicity in a short time, and has the characteristics of safety, high efficiency and low cost.

2. The vaccine can be used for immunizing and preventing infection of swine fever and epidemic encephalitis B in a pig farm, can generate cross immune protection on swine fever viruses of different epidemic strains, can distinguish swine fever attenuated vaccines immune pigs from wild virus infected pigs by a serological method, and can be used as swine fever marker vaccines.

In a word, the invention is very important for preventing the infection of the swine fever virus and the Japanese encephalitis virus in the pig farm, and provides a solid foundation for the immune prevention of the swine fever virus and the Japanese encephalitis in the pig farm.

Drawings

FIG. 1: the composition schematic diagram of a chimeric gene type I Japanese encephalitis virus-like particle coding gene expression box containing a hog cholera virus neutralizing epitope coding sequence.

FIG. 2: a three-dimensional homologous modeling schematic diagram of a chimeric gene type I Japanese encephalitis virus-like particle protein presenting a swine fever virus neutralizing epitope.

FIG. 3: the expression identification result of the virus-like particle constituent protein is shown in a schematic diagram.

FIG. 4: electron microscopy of virus-like particles.

FIG. 5: the results of monitoring the levels of specific antibodies induced after immunization of mice are shown schematically.

FIG. 6: the result of the measurement of the neutralizing antibody titer of the Japanese encephalitis virus and the classical swine fever virus induced after the mice are immunized is shown in a schematic diagram.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of chimeric Gene type I Japanese encephalitis Virus-like particles presenting neutralizing epitope of hog cholera Virus and Immunity Effect test

1 preparation of chimeric gene type I Japanese encephalitis virus-like particles presenting hog cholera virus neutralizing epitope and immune effect test

The invention utilizes virus-like particles of gene I type Japanese encephalitis virus strain JEV/GS/2016/China which are separated and identified in a laboratory to form a protein coding gene sequence to synthesize a gene I type Japanese encephalitis virus-like particle coding gene expression cassette containing a swine fever virus E2 protein neutralizing epitope coding sequence, meanwhile, 108 th phenylalanine (F) of the encoding gene I type encephalitis B virus E protein is mutated into alanine (A), EcoR I and XbaI restriction enzyme sites are added to the two ends of the obtained fusion gene, the fusion gene is cloned to a pVAX1 expression vector, the recombinant pVAX1 plasmid is transiently transfected to BHK-21 cells for culture, transfected cell culture supernatant is collected, concentrated and purified, and the chimeric gene I type encephalitis B virus-like particles for displaying the neutralizing epitope of the hog cholera virus E2 protein are obtained through identification. Respectively immunizing mice with the prepared DNA vaccine and the virus-like particles, analyzing the levels of the swine fever virus neutralizing epitope synthetic peptide and the Japanese encephalitis virus specific antibody, and the titer of the swine fever virus and Japanese encephalitis virus specific neutralizing antibody, and evaluating the immune effect of the virus-like particles. The method comprises the following specific steps:

1 materials and methods

1.1 materials

1.1.1 strains and vectors: the eukaryotic expression vector pVAX1 was stored in the laboratory, and the JEV GS strain of Gene type I was isolated and identified in the inventors' laboratory.

1.1.2 cells and serum: cell line BHK-21 was purchased from ATCC, Inc., USA. BHK-21 cell growth and maintenance solution was DMEM (purchased from Gibco) containing 10% Fetal Bovine Serum (FBS).

1.1.3 polyclonal antibodies and antigens: japanese encephalitis virus prM protein and E protein rabbit-derived specific polyclonal antibody are purchased from Beijing Xinshengke science and technology ltd, and mouse anti-classical swine fever virus E2 protein composite linear neutralizing epitope antigen polyclonal antibody and gene I type JEV EIII recombinant protein are prepared by the inventor.

1.1.4 Experimental animals: female Balb/c mice at 4 weeks of age were purchased from laboratory animal bases of Lanzhou veterinary research institute, national academy of agricultural sciences.

1.2 construction of recombinant vectors

In order to obtain a gene expression cassette for coding a virus-like particle of a gene I type Japanese encephalitis virus containing a sequence of a neutralizing epitope (CTAVSPTT L RTEVVK) of a hog cholera virus E2 protein, a sequence of the neutralizing epitope (CTAVSPTTT L RTEVVK) of the hog cholera virus E2 protein is inserted into a gene expression cassette prME of the virus-like particle of the gene I type Japanese encephalitis virus by an in vitro gene synthesis method, wherein the insertion sites are located at three L oop sites of a GS strain E protein of the gene I type Japanese encephalitis virus and respectively located at an amino acid P₂₂₈-S₂₂₉，T₂₄₆-A₂₄₇And S₂₉₀-S₂₉₁The neutralizing epitope of classical swine fever virus E2 protein (CTAVSPTT L) is inserted between sitesRTEVVK) coding gene sequence to obtain fusion gene E5-6-8; meanwhile, phenylalanine (F) at the 108 th site of the E protein of the gene type I encephalitis B virus GS strain is mutated into alanine (A), and a fusion gene E5-6-8 is obtained_F108A(see figure 1 and figure 2.) the above synthesized gene is used as template, EcoR I restriction site is introduced at the 5 'end of open reading frame by PCR primer (see table 1), Xba I restriction site is introduced at the 3' end, PCR product and eukaryotic expression vector pVAX1 which are recovered by glue are subjected to double enzyme digestion by EcoR I and Xba I, DNA gel recovery is carried out, glue recovery product after enzyme digestion is subjected to ligation and transformation trans-ns 5 α competence, single clone is selected for PCR, enzyme digestion and sequencing identification, plasmid is extracted from recombinant bacteria which are identified correctly by adopting medium quality plasmid kit, and recombinant plasmids containing target gene are obtained and named as pVAX1-E5-6-8 and pVAX1-E5-6-8 respectively_F108A. Fusion gene E5-6-8_F108AThe nucleotide sequence of (A) is shown in SEQ ID No.1 of the sequence table.

Amplification of fusion gene E5-6-8 and mutant gene E5-6-8_F108ASee table 1 for PCR primers.

TABLE one amplification of the wild type fusion gene E5-6-8 and the mutant fusion gene E5-6-8_F108APCR primer of (1)

Note: boxed letters indicate the protective bases of the restriction enzyme sites; underlined letters indicate restriction enzyme site sequences.

1.3 obtaining Virus-like particles

BHK-21 cells are cultured in a 6-well plate, when the cells grow to 90%, cells are transfected according to the proportion of 2 mu g of recombinant plasmid to liposome to 9 mu l (liposome and plasmid compound are incubated for 20min), 2m L MEM culture solution containing 5% FBS and 1% double antibody is added into each well after 6h, cell culture supernatant is collected after 48h, the collected supernatant is centrifuged for 30min at 4 ℃ and 10000rpm in a small low-speed centrifuge, cell debris is discarded, PEG8000 precipitates and a 100KDa ultrafiltration tube are concentrated, chimeric Japanese encephalitis virus sample particles are centrifuged and purified for 3h at 4 ℃ and 35000rpm in a low-temperature ultracentrifuge at 10-50% sucrose density gradient, and virus sample particles are primarily obtained.

1.4 identification of Virus-like particles

1.4.1 recombinant plasmids pVAX1-E5-6-8 and pVAX1-E5-6-8_F108ATransient transfection of BHK-21 cells is carried out respectively, cell culture supernatant is collected after 48h, the cell culture supernatant is centrifuged at 35000rpm for 3h at 4 ℃, and precipitates are collected for appropriate dilution and then subjected to immunoblotting analysis. Taking a rabbit-derived anti-Japanese encephalitis virus E protein antibody and a prM protein antibody (diluted 1: 1000) as primary antibodies, taking goat anti-rabbit IgG marked by HRP as a secondary antibody (diluted 1: 5000), adding a chemiluminescent reagent, developing for 3-10 min in a dark place, taking a picture, and storing the picture. Immunoblotting results show that the obtained virus-like particle as an antigen has a specific band at a corresponding position after reacting with rabbit polyclonal antibodies against Japanese encephalitis virus prM protein and E protein, and the expression levels of virus-like particle constituent protein E and PrM are obviously improved after phenylalanine (F) at the 108 th position of Japanese encephalitis virus E protein is mutated into alanine (A). (see FIG. 3A). E5-6-8_F108AThe amino acid sequence of (1) is shown in a sequence table SEQ ID No. 2.

FIG. 3A shows pVAX1-E5-6-8 and pVAX1-E5-6-8_F108ACell precipitation of transfected BHK21 cells and Western blotting identification of cell culture supernatant. In FIG. 3A, M is protein marker; 1 is pVAX1-E5-6-8 transfection BHK21 cell sediment; 2 is pVAX1-E5-6-8 transfection BHK21 cell culture supernatant; 3 is pVAX1-E5-6-8_F108ACell culture supernatants of transfected BHK21 cells.

1.4.2 empty vector pVAX1 and recombinant plasmids (pVAX1-E5-6-8 and pVAX 1-E5-6-8)_F108A) BHK-21 cells are transfected, after 24 hours of transfection, a commercial rabbit polyclonal antibody (1: 1000) of encephalitis B virus E protein and a self-made polyclonal antibody (1: 500) of swine fever virus E2 protein tandem repeat neutralization epitope (CTAVSPTT L RTEVVK) antigen mouse polyclonal antibody (1: 1000) are used as primary antibodies, goat anti-rabbit IgG (1: 1000) marked by 568 and donkey anti-mouse IgG (1: 1000) marked by 488 are used as secondary antibodies for immunofluorescence co-localization analysis, and fluorescence of collected images is observed by a laser confocal microscopeThe result shows that the rabbit anti-Japanese encephalitis virus E protein polyclonal antibody and the mouse anti-classical swine fever virus E2 protein tandem neutralizing epitope (CTAVSPTTT L RTEVVK) polyclonal antibody can specifically recognize JEV E protein expressed by the gene I type Japanese encephalitis virus-like particles and presented classical swine fever virus E2 protein neutralizing epitope (CTAVSPTTT L RTEVVK). the classical swine fever virus E2 protein neutralizing epitope can be well displayed on the surface of the gene I type Japanese encephalitis virus-like particles, and the classical swine fever virus E2 protein neutralizing epitope does not influence the correct expression and immunoreactivity of the gene I type Japanese encephalitis virus-like particle component proteins at the insertion site of the gene I type Japanese encephalitis virus E protein (see FIG. 3B)

FIG. 3B shows pVAX1-E5-6-8 and pVAX1-E5-6-8_F108AA is an indirect immunofluorescence schematic drawing of a Japanese encephalitis virus E protein rabbit-derived polyclonal antibody as a primary antibody, the indirect immunofluorescence schematic drawing shows specific red fluorescence, B is an indirect immunofluorescence schematic drawing of a home-made classical swine fever virus E2 protein tandem repeat neutralizing epitope (CTAVSPTTT L RTEVVK) antigen mouse-derived polyclonal antibody as a primary antibody, the indirect immunofluorescence schematic drawing shows specific green fluorescence, and C is a position combination drawing of an A drawing and a B drawing, and the position combination drawing shows specific yellow fluorescence.

1.4.3 transfection of recombinant plasmid into BHK-21 cells, centrifugation at 10000rpm for 30min after transfection of cell culture supernatant collected after 48h, discarding cell debris, precipitation by PEG8000 and concentration by 100KDa ultrafiltration tube, centrifugation at 4 ℃, 35000rpm for 3h in a low temperature ultracentrifuge, centrifugation at 10-50% sucrose density gradient to purify virus-like particles, taking purified virus-like particles to appropriately dilute and then adsorbing on a copper mesh for about 2min, staining by phosphotungstic acid and observing the morphology of the virus-like particles by electron microscopy to observe the assembly and formation of virus-like particles of target proteins, observing the virus-like particles assembled in vitro by a transmission electron microscope, staining by phosphotungstic acid, a large number of spherical particles with regular and uniform morphology of 30-50 nm can be seen, see FIG. 4. combining indirect immunofluorescence and immunoblotting test results, it is shown that the neutralizing epitope of classical swine fever virus E2 protein in three encephalitis virus oop sites of encephalitis B virus coding E protein and the 108 th mutant phenylalanine (F) of encephalitis B virus E protein are inserted into the chimeric encephalitis B virus-like particles which are not affected by the chimeric encephalitis B virus (encephalitis B) and the encephalitis B virus.

1.5 evaluation of the Effect of DNA vaccine and Virus-like particles on immunization of Balb/c mice

Dividing 32 female BA L B/c mice with age of 4-6 weeks into 4 groups according to 8 mice per group (pVAX1-E5-6-8 )_F108A，VLP-E5-6-8_F108AAnd PBS), animal immunoassays are performed. Each group of mice was bled from the tail tip before priming and serum was isolated to prepare negative serum. The DNA vaccine immunization group adopts a mode of injecting recombinant plasmids into hind leg muscles for immunization, the immunization is carried out once every 2 weeks, the immunization is carried out three times in total, and the immunization dose is 100 mu g. The virus-like particle immunization groups were immunized once each with the antigen containing the immune adjuvant by cervical subcutaneous injection on days 0 and 14, respectively. The first time adopts complete Freund's adjuvant, the second time adopts incomplete Freund's adjuvant, and the third time adopts the mode of intraperitoneal injection of antigen, and the immunization dose is 2 mug. The immunization dose in PBS group was 200. mu.l. At 14 th, 28 th, 35 th, 42 th and 63d of the mice before and after immunization, serum is prepared by separation through a tail tip blood collection method and is stored at the temperature of 80 ℃ below zero for later use. Sera from 4 mice were randomly taken from each immunization group at 63d post immunization for virus neutralization antibody titration experiments.

1.5.1 evaluation of humoral immune levels in immunized mice by enzyme-linked immunosorbent assay (E L ISA)

Recombinant Japanese encephalitis virus EIII protein 0.5 μ g/well, adding 2-fold dilution of each immune group mouse serum from 1:100 as primary antibody for incubation, adding 1:1000 dilution of each immune group mouse serum as primary antibody for incubation, using HRP-labeled goat anti-mouse IgG (1: 2500) as secondary antibody for E III protein and swine fever virus neutralizing epitope (CTAVSPTTT L RTEVVK) synthetic peptide specific antibody level monitoring, and measuring OD by microplate reader₄₅₀The value is the maximum dilution multiple of serum with P/N > 2.1 as the specific antibody titer. pVAX1-E5-6-8 and pVAX1-E5-6-8 in addition to PBS immune control group_F108ADNA vaccine immunization group and V L P-E5-6-8_F108AThe virus-like particle immune group can induce immune mice to generate eggs aiming at classical swine fever virus E2Synthetic peptide of white neutralizing epitope (CTAVSPTT L RTEVVK) (see figure 5A) and specific E L ISA antibody of gene I type B encephalitis virus EIII protein (see figure 5B). compared with wild type DNA vaccine pVAX1-E5-6-8 immunization group, mutant DNA vaccine pVAX1-E5-6-8_F108AThe level of specific antibody of Japanese encephalitis virus EIII protein induced by immune group is slightly improved (P is less than 0.05), and reaches about 1:1 ten thousand on day 42, and virus-like particle V L P-E5-6-8_F108AThe level of specific antibody of Japanese encephalitis virus EIII protein induced by vaccine immunization group is obviously higher than that of mutant DNA vaccine immunization group (P is less than 0.01). V L P-E5-6-8_F108AThe level of the antibody specific to the synthetic peptide of the neutralizing epitope (CTAVSPTT L RTEVVK) of the classical swine fever virus induced by the vaccine immunization group is obviously higher than that of the wild type DNA vaccine pVAX1-E5-6-8 immunization group and the mutant DNA vaccine pVAX1-E5-6-8_F108AImmunization group (P < 0.01).

1.5.2 immunization of mice induces levels of specific neutralizing antibodies against Japanese encephalitis virus and classical swine fever virus

1.5.2.1 plaque reduction neutralization assay for determination of neutralizing antibody titers against encephalitis b virus in sera of mice from each immunization group: inactivated mouse sera from each immunization group collected on day 63 were removed from 1:10, 2-fold serial dilutions were started and the diluted sera were mixed in equal volumes with virus solution containing 100PFU, and the highest dilution of sera that reduced the number of plaques in 50% of the PBS immune sera-treated virus groups was the neutralizing antibody titer of the sera. The DNA vaccine immunization group and the virus-like particle immunization group can induce immune mice to generate specific neutralizing antibodies against the Japanese encephalitis virus. The results in fig. 6A show that the wild type DNA vaccine pVAX1-E5-6-8 immunization group had an average neutralizing antibody titer against japanese encephalitis virus of 1: 90, respectively; mutant DNA vaccine pVAX1-E5-6-8_F108AThe average neutralizing antibody titer of the immune group against the Japanese encephalitis virus is 1:180, and the virus-like particle vaccine V L P-E5-6-8_F108AThe average neutralizing antibody titer of the anti-Japanese encephalitis virus in the immunized group is 1: 280.

1.5.2.2 determination of hog cholera virus neutralizing antibody titres: inactivated day 63 sera from mice in each immunization group were removed from the mice with serum from 1:2 starting with serial 2-fold dilutions to 1:256, diluted sera with 200TCID₅₀VirusAnd (3) mixing and incubating the liquid in equal volume for 2 hours, then inoculating the virus serum mixture on PK15 cells, repeating the steps for 8 times for each sample, and after 48 hours, judging the titer of the average neutralizing antibody of the serum of each immune group of mice against the classical swine fever virus by adopting a classical swine fever virus E2 protein monoclonal antibody as a primary antibody and an indirect immunofluorescence method. The determination of the titer of the neutralizing antibody of the classical swine fever virus is assisted by Shanghai veterinary research institute of Chinese academy of agricultural sciences. The results in FIG. 6B show that the wild-type DNA vaccine pVAX1-E5-6-8 immunization group induced an average neutralizing antibody titer against classical swine fever virus SM strain of 1: 20; mutant DNA vaccine pVAX1-E5-6-8_F108AThe average neutralizing antibody titer of the anti-CSFV SM strain in the immune group is 1:70, and the virus-like particle vaccine V L P-E5-6-8_F108AThe average neutralizing antibody titer of the anti-CSFV SM strains in the immune group is 1:180, thus showing the neutralizing epitope of CSFV, the chimeric gene type I type Japanese encephalitis virus-like particle V L P-E5-6-8_F108ACan be used as a potential ideal vaccine for preventing the swine fever virus infection.

The analysis of the related test results of the invention shows that: compared with the wild type DNA vaccine pVAX1-E5-6-8, the mutant DNA vaccine pVAX1-E5-6-8_F108ACan obviously improve the immunogenicity of DNA vaccine, which is directly related to the improvement of protein expression level after the 108 th amino acid mutation of JEV E protein, and adopts virus-like particles V L P-E5-6-8_F108AThe vaccine preparation form is used for immunizing mice to induce and generate hog cholera virus neutralizing epitope (CTAVSPTT L RTEVVK) synthetic peptide and Japanese encephalitis virus specific antibody level and the neutralizing antibody level aiming at the hog cholera virus and the Japanese encephalitis virus are obviously higher than that of the DNA vaccine preparation form, so that the chimeric gene I type Japanese encephalitis virus-like particles V L P-E5-6-8 presenting the hog cholera virus neutralizing epitope_F108AIs a potential ideal vaccine for preventing swine fever and epidemic encephalitis B in a pig farm.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences

<120> virus-like particles for swine fever and epidemic encephalitis B, preparation method and application

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>2214

<212>DNA

<213> genotype I Japanese encephalitis virus-like particle nucleotide sequence of hog cholera virus E2 protein neutralizing epitope (E5-6-8F108A)

<400>1

atgagaggag ggaatgagag ctcgattatg tggcttgcca gctcggcaat cgtaatagcc 60

tgcgccggag ccatgaagct atcaaacttt caaggaaaac ttctgatgac catcaacaac 120

acggacattg cggacgtcat cgtgatcccc acctcaaaag gtgaaaacag atgttgggtc 180

cgagcaatcg acgttggtta catgtgtgaa gacaccatca cgtatgaatg tccgaagctt 240

gccgtgggca acgatccgga agacgtggac tgctggtgcg acaatcagga agtctacgtg 300

cagtatggtc gctgcacaca gaccaggcat tccaaacgaa gcagaagatc cgtttcggtc 360

caaacgcatg gggaaagctc actagtgaac aaaaaagagg cttggctgga ttcaacgaag 420

gccacgcgat acctcatgaa aacggagaac tggatcataa ggaaccctgg ttatgctttc 480

ctggcggcgg cacttggatg gatgcttggc agcaacagtg gccaacgtgt ggtgttcact 540

attctcttgc tattggtcgc cccggcttac agttttaact gtcttggaat gggaaatcgg 600

gatttcatag aaggagccag tggagccact tgggtagatc tggtgttgga aggagatagc 660

tgtttgacaa tcatggcaaa cgacaaacca acactagatg tccgcatgat caacattgaa 720

gctagccaac ttgctgaagt caggagttac tgctatcgcg cttcagtcac tgacatttca 780

acggtggctc gatgccccac gactggagaa gcccacaacg aaaaacgtgc cgacagcagc 840

tacgtgtgca aacaaggctt tactgatcgc ggatggggaa atggatgtgg acttttcggg 900

aaaggaagca ttgacacatg cgcaaaattt tcttgtacca ataaggccat tggaagaatg 960

atccaaccag aaaacatcaa gtacgaggtt ggcatattcg tgcacggaac taccacctcg 1020

gaaaaccatg ggaattactc agcgcaagta ggagcgtctc aagcagcaaa gtttactgta 1080

actccaaatg ctccttcaat aacccttaag cttggtgatt atggagaagt cacactggat 1140

tgtgaaccaa ggagtggact gaacactgaa gcgttctatg tcatgaccgt gggttcgaag 1200

tcattcttag tccataggga atggttccat gacctttctc ttccctggac gtccccctgc 1260

acggcagtaa gccccacaac cttgagaaca gaagtggtga agtcaagcac gtgcacggca 1320

gtaagcccca caaccttgag aacagaagtg gtgaaggcat ggagaaacag agaactcctc 1380

atggaatttg aagaggcaca tgccacaaaa caacctgtcg tagctcttgg gtcacaggag 1440

ggaggccccc atcaagcgct ggcaggtgcc attgtggtgg agtactcgtg cacggcagta 1500

agccccacaa ccttgagaac agaagtggtg aagagctcag tgaagttgac atcaggccac 1560

ctgaaatgta ggctaaaaat ggacaaactg gctctgaagg gcacgactta tggcatgtgt 1620

acagaaaaat tctcgttcgc gaaaaatcca gcggacacag gccatggaac agttgtcatt 1680

gagctaacat attctggaag tgatggtccc tgcaaaattc cgattgtctc agttgcgagc 1740

ttaaacgaca tgacccctgt ggggaggttg gtaacagtaa accccttcgt cgcaacatct 1800

agctccaact caaaggtgct ggttgagatg gaacctccct tcggagactc ttatatcgtg 1860

gttggaagag gggacaagca gattaaccat cactggcaca aagctggaag cacgctgggc 1920

aaggctttct caacaacttt gaaaggggct cagagactag cagcgctagg tgacacagcc 1980

tgggacttcg gctccattgg aggggtattc aactccatag ggaaagctgt tcaccaagta 2040

tttggcggtg cattcagaac gctctttggg ggaatgtctt ggatcacaca aggactaatg 2100

ggagccttac tcctttggat gggtgtcaac gcacgagacc ggtcaattgc cctggctttt 2160

ctggccacgg gaggtgtgct cgtgttttta gcgaccaatg tgcatgccga ctaa 2214

<210>2

<211>737

<212>PRT

<213> type Japanese encephalitis virus-like particle amino acid sequence of swine fever virus E2 protein neutralizing epitope gene I (E5-6-8F108A)

<400>2

Met Arg Gly Gly Asn Glu Ser Ser Ile Met Trp Leu Ala Ser Ser Ala

1 5 10 15

Ile Val Ile Ala Cys Ala Gly Ala Met Lys Leu Ser Asn Phe Gln Gly

20 25 30

Lys Leu Leu Met Thr Ile Asn Asn Thr Asp Ile Ala Asp Val Ile Val

35 40 45

Ile Pro Thr Ser Lys Gly Glu Asn Arg Cys Trp Val Arg Ala Ile Asp

50 55 60

Val Gly Tyr Met Cys Glu Asp Thr Ile Thr Tyr Glu Cys Pro Lys Leu

65 70 75 80

Ala Val Gly Asn Asp Pro Glu Asp Val Asp Cys Trp Cys Asp Asn Gln

85 90 95

Glu Val Tyr Val Gln Tyr Gly Arg Cys Thr Gln Thr Arg His Ser Lys

100 105 110

Arg Ser Arg Arg Ser Val Ser Val Gln Thr His Gly Glu Ser Ser Leu

115 120 125

Val Asn Lys Lys Glu Ala Trp Leu Asp Ser Thr Lys Ala Thr Arg Tyr

130 135 140

Leu Met Lys Thr Glu Asn Trp Ile Ile Arg Asn Pro Gly Tyr Ala Phe

145 150 155 160

Leu Ala Ala Ala Leu Gly Trp Met Leu Gly Ser Asn Ser Gly Gln Arg

165 170 175

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

180 185 190

Asn Cys Leu Gly Met Gly Asn Arg Asp Phe Ile Glu Gly Ala Ser Gly

195 200 205

Ala Thr Trp Val Asp Leu Val Leu Glu Gly Asp Ser Cys Leu Thr Ile

210 215 220

Met Ala Asn Asp Lys Pro Thr Leu Asp Val Arg Met Ile Asn Ile Glu

225 230 235 240

Ala Ser Gln Leu Ala Glu Val Arg Ser Tyr Cys Tyr Arg Ala Ser Val

245 250 255

Thr Asp Ile Ser Thr Val Ala Arg Cys Pro Thr Thr Gly Glu Ala His

260 265 270

Asn Glu Lys Arg Ala Asp Ser Ser Tyr Val Cys Lys Gln Gly Phe Thr

275 280 285

Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser Ile

290 295 300

Asp Thr Cys Ala Lys Phe Ser Cys Thr Asn Lys Ala Ile Gly Arg Met

305 310 315 320

Ile Gln Pro Glu Asn Ile Lys Tyr Glu Val Gly Ile Phe Val His Gly

325 330 335

Thr Thr Thr Ser Glu Asn His Gly Asn Tyr Ser Ala Gln Val Gly Ala

340 345 350

Ser Gln Ala Ala Lys Phe Thr Val Thr Pro Asn Ala Pro Ser Ile Thr

355 360 365

Leu Lys Leu Gly Asp Tyr Gly Glu Val Thr Leu Asp Cys Glu Pro Arg

370 375 380

Ser Gly Leu Asn Thr Glu Ala Phe Tyr Val Met Thr Val Gly Ser Lys

385 390 395 400

Ser Phe Leu Val His Arg Glu Trp Phe His Asp Leu Ser Leu Pro Trp

405 410 415

Thr Ser Pro Cys Thr Ala Val Ser Pro Thr Thr Leu Arg Thr Glu Val

420 425 430

Val Lys Ser Ser Thr Cys Thr Ala Val Ser Pro Thr Thr Leu Arg Thr

435 440 445

Glu Val Val Lys Ala Trp Arg Asn Arg Glu Leu Leu Met Glu Phe Glu

450 455 460

Glu Ala His Ala Thr Lys Gln Pro Val Val Ala Leu Gly Ser Gln Glu

465 470 475 480

Gly Gly Pro His Gln Ala Leu Ala Gly Ala Ile Val Val Glu Tyr Ser

485 490 495

Cys Thr Ala Val Ser Pro Thr Thr Leu Arg Thr Glu Val Val Lys Ser

500 505 510

Ser ValLys Leu Thr Ser Gly His Leu Lys Cys Arg Leu Lys Met Asp

515 520 525

Lys Leu Ala Leu Lys Gly Thr Thr Tyr Gly Met Cys Thr Glu Lys Phe

530 535 540

Ser Phe Ala Lys Asn Pro Ala Asp Thr Gly His Gly Thr Val Val Ile

545 550 555 560

Glu Leu Thr Tyr Ser Gly Ser Asp Gly Pro Cys Lys Ile Pro Ile Val

565 570 575

Ser Val Ala Ser Leu Asn Asp Met Thr Pro Val Gly Arg Leu Val Thr

580 585 590

Val Asn Pro Phe Val Ala Thr Ser Ser Ser Asn Ser Lys Val Leu Val

595 600 605

Glu Met Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val Val Gly Arg Gly

610 615 620

Asp Lys Gln Ile Asn His His Trp His Lys Ala Gly Ser Thr Leu Gly

625 630 635 640

Lys Ala Phe Ser Thr Thr Leu Lys Gly Ala Gln Arg Leu Ala Ala Leu

645 650 655

Gly Asp Thr Ala Trp Asp Phe Gly Ser Ile Gly Gly Val Phe Asn Ser

660 665 670

Ile Gly Lys AlaVal His Gln Val Phe Gly Gly Ala Phe Arg Thr Leu

675 680 685

Phe Gly Gly Met Ser Trp Ile Thr Gln Gly Leu Met Gly Ala Leu Leu

690 695 700

Leu Trp Met Gly Val Asn Ala Arg Asp Arg Ser Ile Ala Leu Ala Phe

705 710 715 720

Leu Ala Thr Gly Gly Val Leu Val Phe Leu Ala Thr Asn Val His Ala

725 730 735

Asp

<210>3

<211>34

<212>DNA

<213> Artificial sequence (upstream amplification primer JEV-V L PF)

<400>3

gcggaattca tgagaggagg gaatgagagc tcga 34

<210>1

<211>36

<212>DNA

<213> Artificial sequence (downstream amplification primer JEV-V L PF)

<400>1

gcgtctagat tagtcggcat gcacattagt cgctaa 36

Claims

1. A preparation method of chimeric gene type-I encephalitis B virus-like particles presenting swine fever virus neutralizing epitope is characterized by synthesizing a gene type-I encephalitis B virus-like particle coding gene expression cassette containing a swine fever virus E2 protein neutralizing epitope (CTAVSPTT L RTEVVK) coding sequence, mutating 108 th phenylalanine (F) of coding gene type-I encephalitis B virus E protein into alanine (A), obtaining a synthesized fusion gene, then using the fusion gene as a template, carrying out amplification through PCR, carrying out DNA gel recovery on a PCR product obtained by gel recovery and an eukaryotic expression vector pVAX1 after double enzyme digestion by EcoR I and Xba I, connecting the PCR product with the gel recovery product of the eukaryotic expression vector, then transforming a trans5 α competence, extracting correctly identified positive recombinant bacteria by using a medium-quality-improving plasmid kit, obtaining a recombinant pVAX1 plasmid containing a target gene with high purity, transiently transfecting the recombinant pVAX1 plasmid to a cell, culturing BHK-21 cell, then carrying out transfection on the recombinant bacteria, and carrying out linear culture on the recombinant bacteria to obtain the encephalitis B virus vector expressing the linear encephalitis B virus epitope and purifying the swine fever virus epitope in the collected linear encephalitis B virus particle 2.

2. The method for preparing the chimeric type gene type I japanese encephalitis virus like particle presenting neutralizing epitope of hog cholera virus according to claim 1, wherein said type I japanese encephalitis virus is of type I.

3. The method for preparing the chimeric genotype I type Japanese encephalitis virus like particle presenting neutralizing epitope of hog cholera virus according to claim 2, wherein the nucleotide sequence of PCR amplification primer is SEQ ID No.3 and SEQ ID No. 4.

4. The method for preparing the chimeric gene type I Japanese encephalitis virus-like particle presenting the neutralizing epitope of classical swine fever virus according to claim 3, wherein the sequence encoding the neutralizing epitope of classical swine fever virus E2 protein (CTAVSPTT L RTEVVK) is inserted into the gene type I Japanese encephalitis virus-like particle by in vitro gene synthesisThree L oop insertion sites of the coding gene expression cassette, wherein the insertion sites are respectively positioned at P of Japanese encephalitis virus E protein₂₂₈-S₂₂₉，T₂₄₆-A₂₄₇And S₂₉₀-S₂₉₁Between amino acid positions.

5. The method for preparing the chimeric gene type-I Japanese encephalitis virus like particle presenting neutralizing epitope of hog cholera virus according to any of claims 1 to 4, wherein phenylalanine (F) at position 108 of gene type-I Japanese encephalitis virus E protein in the obtained fusion gene is mutated to alanine (A).

6. The method for preparing the chimeric gene type I Japanese encephalitis virus like particle presenting the neutralizing epitope of hog cholera virus according to claim 5, wherein the nucleotide sequence and amino acid sequence of the coding gene are SEQ ID No.1 and SEQ ID No.2, respectively.

7. Chimeric genotypic type b encephalitis virus like particle presenting a neutralizing epitope of classical swine fever virus produced by any of the methods of claims 1 to 6.

8. Use of the chimeric genotype I japanese encephalitis virus like particles presenting a neutralizing epitope of classical swine fever virus according to claim 7, for the preparation of a vaccine formulation for simultaneous immunization against classical swine fever virus and epidemic encephalitis b virus infection.