CN114949193A - Tick-borne encephalitis virus-like particle vaccine and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of biological products, in particular to a tick-borne encephalitis virus-like particle vaccine and a preparation method thereof. The immunogenic component of the tick-borne encephalitis virus-like particle vaccine is tick-borne encephalitis virus prM-E protein; the tick-borne encephalitis virus prM-E protein comprises an amino acid sequence shown as SEQ ID NO. 1. The invention adopts an insect cell-baculovirus expression system to express and purify a prM-E protein which is a specific structural protein of tick-borne encephalitis virus, so as to obtain tick-borne encephalitis virus-like particles. The tick-borne encephalitis virus-like particle provided by the invention has good immunogenicity, can induce a mouse to generate specific immune response, and has a good prevention effect on tick-borne encephalitis.

Description

Tick-borne encephalitis virus-like particle vaccine and preparation method thereof

Technical Field

The invention relates to the technical field of biological products, in particular to a tick-borne encephalitis virus-like particle vaccine and a preparation method thereof.

Background

Tick-borne encephalitis (TBE), also known as forest encephalitis, is a natural epidemic disease mainly caused by nervous system diseases caused by Tick-borne encephalitis virus (TBEV), can cause fatal encephalitis in humans and animals with long-term sequelae, and seriously threatens human health. With global climate change, the number and activity range of ticks is expanding, and tick-borne disease prevalence and spread become important public health concerns. As one of important tick-borne pathogens, the research on vaccines aiming at tick-borne encephalitis viruses has never been stopped, and the development of novel genetic engineering vaccines with higher safety and better immune effect is a hotspot and a main direction of research in the field.

Tick-borne encephalitis virus is a single-stranded positive-strand RNA virus of the flavivirus family, approximately 11kb in length, containing an open reading frame. A single open reading frame encodes three structural proteins (C, prM and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS 5). Flaviviruses are enveloped viruses with an average diameter of 40-60nm, and the surface of mature virions consists of 90 dimers of the E and M proteins. The double-layer lipid envelope encloses a nucleocapsid (composed of protein C) that encloses the single-stranded positive-stranded RNA genome. The flavivirus C protein can form a nucleocapsid together with a virus genome, can also interact with a liposome to participate in cell membrane remodeling, but does not induce an organism to generate a neutralizing antibody; the transmembrane domain of the C protein can serve as an additional anchor and signal sequence for the prM protein. The prM protein is cleaved by the host furin, producing the pr fragment and the M protein. The M protein is immobilized on the lipid envelope by two transmembrane domains, interacting with the transmembrane domain of the E protein in the mature granule. The E protein is an envelope protein, contains neutralizing antibody epitopes, and plays an important role in immunoprophylaxis.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a tick-borne encephalitis virus-like particle vaccine and a preparation method thereof.

In a first aspect, the invention provides a tick-borne encephalitis virus like particle vaccine, wherein the immunogenic component of the tick-borne encephalitis virus like particle vaccine is tick-borne encephalitis virus prM protein; the tick-borne encephalitis virus prM-E protein comprises an amino acid sequence shown as SEQ ID No. 1.

Furthermore, the tick-borne encephalitis virus prM-E protein is coded by a nucleotide sequence shown in SEQ ID NO. 2.

The tick-borne encephalitis virus prM-E protein is used as an immunogenic component of the virus-like particle vaccine, and the tick-borne encephalitis virus prM-E vaccine has high safety and high immunogenicity under the condition of no virus genetic material, and can cause effective immune response in a body.

When the insect cell-baculovirus expression system expresses foreign proteins, signal peptide assistance is required. In the research process, the invention discovers that when the baculovirus signal peptide is used, the recombinant plasmid can not be successfully constructed, so that only the signal peptide of the flavivirus per se, namely the transmembrane domain of the flavivirus C protein, can be selected. Studies show that the JEV signal peptide of the TBEV same genus is more beneficial to expression and secretion of flavivirus protein, and the research respectively uses the JEV signal peptide and the TBEV signal peptide to construct recombinant protein, and finds that when the TBEV signal peptide is used, the amount of M-E protein obtained after the recombinant baculovirus infects suspension cells is more. Therefore, the transmembrane domain of the TBEV C protein is used as a signal peptide, namely the amino acid sequence at the first 16 positions of SEQ ID NO.1 provided by the invention.

Further, an adjuvant is also included; the adjuvant comprises one or more of Montanide ISA201VG adjuvant, Poly (I: C), Montanide Gel 02PR, Montanide IMS 1313VG NST, polysaccharide, Alum, Freund or Prodavx.

Further, the adjuvant is a Poly (I: C) and Montanide ISA201VG composite adjuvant, and the volume ratio of the immunogenic component to the adjuvant is (5-9): (11-15).

Further, in the Poly (I: C) and Montanide ISA201VG composite adjuvant, the volume ratio of the Poly (I: C) to the Montanide ISA201VG adjuvant is (1-3): (9-13).

In a second aspect, the present invention provides a method for preparing the tick-borne encephalitis virus like particle vaccine, comprising:

the nucleotide sequence shown in SEQ ID NO.2 is constructed on an expression vector, expressed by an expression system and purified to obtain the tick-borne encephalitis virus-like particle vaccine.

Further, the expression vector is a pFBD vector;

the expression system is one or more of an insect cell-baculovirus expression system, a mammalian expression system, a prokaryotic expression system or a yeast expression system.

Further, the purifying comprises:

treating an expression product of the insect cell-baculovirus expression system at 0-4 ℃ for 25-35 minutes;

centrifuging for 10-20 min at the temperature of 0-4 ℃ and the speed of 14000-16000 g/min;

taking supernatant, and performing density gradient centrifugation through potassium tartrate-glycerol.

In a third aspect, the present invention provides a nucleic acid comprising the nucleotide sequence shown as SEQ ID NO. 2.

The invention further provides a biological material comprising the nucleic acid, wherein the biological material is an expression cassette, a vector or a transgenic cell.

The invention has the following beneficial effects:

the invention adopts an insect cell-baculovirus expression system to express and purify a prM-E protein which is a specific structural protein of tick-borne encephalitis virus, so as to obtain tick-borne encephalitis virus-like particles. The tick-borne encephalitis virus-like particle provided by the invention has higher immunogenicity, can induce a mouse to generate specific immune response, obviously improves the quantity and activation level of mouse spleen B cells, T cells and memory T cells, and can improve the level of Th1 and Th2 cytokines, thereby having important significance in the field of tick-borne encephalitis prevention and treatment.

Drawings

FIG. 1 is a schematic diagram of recombinant plasmid pFBD-prM-E-prM-E provided in example 1 of the present invention.

FIG. 2 shows the PCR amplification result of the TBEV prM-E gene provided in example 1 of the present invention; wherein, A: TBEV prM-E (PH) gene PCR amplification result; b: the PCR amplification result of TBEV prM-E (P10) gene.

FIG. 3 is the identification of recombinant plasmid pFBD-prM-E-prM-E provided in example 1 of the present invention; wherein, A: a pFBD-prM-E double enzyme digestion identification result; b: and (3) carrying out double enzyme digestion identification on the pFBD-prM-E-prM-E.

FIG. 4 is a PCR result of recombinant bacmid-prM-E-prM-E provided in example 1 of the present invention; wherein, M: marker; 1: the result of the identification of the rBacmid-prM-E-prM-E PH terminal; 2: rBacmid-prM-E-prM-E P10 terminal identification result

FIG. 5 is a schematic diagram showing the morphological changes of Sf9 cells after infection with recombinant baculovirus according to example 1 of the present invention; wherein, A: recombinant baculovirus rBV-prM-E-prM-E infected Sf9 cells; b: normal Sf9 cells.

FIG. 6 is a schematic diagram showing the result of identifying recombinant baculovirus rBV-prM-E-prM-E provided in example 1 of the present invention; wherein, A-a: indirect immunofluorescence identification results of normal Sf9 cells; a-b: indirect immunofluorescence identification results of recombinant baculovirus rBV-prM-E-prM-E infected Sf9 cells; western Blot identification result (1: rBV-prM-E-prM-E cell suspension, 2: rBV-prM-E culture supernatant and 3: rBV-prM-E-prM-E cell sediment suspension).

FIG. 7 is a schematic diagram of the identification results of TBEV VLPs provided in example 2 of the present invention; wherein, A: western Blot identification result (1: normal Sf9 cells; 2: before purification; 3: after purification); b-a: observing results by an electron microscope; B-B: and (5) observing the result by using an immunoelectron microscope.

FIG. 8 is a diagram showing the detection results of the mouse serum TBEV IgG antibody provided in example 3 of the present invention.

FIG. 9 is a schematic diagram showing the results of flow cytometry analysis of immune cells in inguinal lymph nodes after first-pass provided in example 3 of the present invention; wherein, A-a: mouse lymph node CD11c ⁺ CD80 ⁺ The proportion of double positive cells; a-b: mouse lymph node CD11c ⁺ MHCⅠ ⁺ The proportion of double positive cells; a-c: mouse lymph node CD11c ⁺ MHCⅡ ⁺ The proportion of double positive cells; b-a: mouse lymph node CD19 ⁺ CD40 ⁺ The proportion of double positive cells; B-B: mouse lymph node CD19 ⁺ CD69 ⁺ Double positive cell ratio.

FIG. 10 shows the results of ELISpot assay of spleen cells IFN-. gamma.and IL-4 provided in example 3 of the present invention; wherein, A: the ELISpot detection result of the spleen cell IFN-gamma; b: the results of ELISpot detection of splenocyte IL-4.

FIG. 11 shows the results of the splenocyte proliferation assay provided in example 3 of the present invention.

FIG. 12 shows the results of cytokine detection provided in example 3 of the present invention; wherein, A: th1 type cytokines; b: th2 type cytokine.

FIG. 13 is a schematic diagram showing the results of flow cytometry analysis of mouse splenocytes after three immunizations, provided in example 3 of the present invention; wherein, A: mouse spleen CD19 ⁺ CD69 ⁺ The proportion of double positive cells; b: the proportion of mouse spleen CD4+ CD69+ double positive cells; c-a: mouse spleen CD4 ⁺ /CD44 ⁺ CD62L ⁺ The proportion of double positive cells; c-b: mouseSpleen CD8 ⁺ /CD44 ⁺ CD62L ⁺ Double positive cell ratio.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Example 1 construction and identification of recombinant baculovirus rBV-prM-E-prM-E1, primer design and Synthesis

Specific primers (primer sequences are shown in Table 1) were designed with reference to TBEV far east subtype strain WH2012 (GenBank: KJ755186) prM-E gene. The insect cell-baculovirus expression vector pFastBac Dual has two promoters of PH and P10, and can construct a vector containing two target gene segments, so that two pairs of amplification primers (TBEV-prM-E-PHF + TBEV-prM-E-PHR, TBEV-prM-E-P10F + TBEV-prM-E-P10R) and two pairs of identification primers (PHF + M13R, M13+ P10R) are designed.

TABLE 1 primer sequence information

Note: restriction endonuclease recognition sites are underlined.

2. Construction and identification of recombinant plasmid pFBD-prM-E-prM-E

(1) Amplification of the Gene of interest TBEV prM-E (PH)

TBEV prM-E (PH) gene was amplified using TBEV-prM-E-PHF and TBEV-prM-E-PHR primers (Table 1) using a plasmid containing the objective gene TBEV prM-E as a template, and the reaction conditions are shown in Table 2. The target fragment was separated and recovered by electrophoresis on a 1% agarose gel.

The amplification result shows that the product is consistent with the size of the target fragment (A in FIG. 2), which indicates that the TBEV prM-E (PH) gene is successfully amplified.

TABLE 2 PCR reaction conditions

(2) Identification of recombinant plasmid pFBD-prM-E

After the recovered TBEV prM-E (PH) gene and the pFastBac Dual vector are subjected to double enzyme digestion by NotI and HindIII, the two genes are connected and transformed into Stellar competent cells, the Stellar competent cells are coated on an ampicillin-resistant solid LB plate, inverted culture is carried out for 12h at 37 ℃, a monoclonal strain is picked up and put into ampicillin-resistant liquid LB for shake culture for 12h, plasmids are extracted and subjected to double enzyme digestion identification by NotI and HindIII (A in figure 3), correct plasmids are identified for sequence determination and analysis, the result shows that the sequence homology of the plasmids and the synthesized TBEV prM-E gene sequence is 100 percent, the recombinant plasmid containing the TBEVprM-E gene is successfully constructed, and the plasmid is named as pFBD-prM-E.

(3) Amplification of the Gene of interest TBEV prM-E (P10)

TBEV prM-E (P10) is amplified by taking the recombinant plasmid pFBD-prM-E as a template and TBEV-prM-E-P10F and TBEV-prM-E-P10R as primers. The amplified product was matched in size with the target gene fragment by agarose gel electrophoresis analysis (B in FIG. 2), and the TBEV prM-E (P10) gene was successfully amplified.

(4) Identification of recombinant plasmid pFBD-prM-E-prM-E

The recovered TBEV prM-E (P10) gene and recombinant plasmid pFBD-prM-E are subjected to double enzyme digestion by SmaI and Nhe I, then are connected and transformed into Stellar competent cells, a monoclonal strain is picked, plasmids are extracted and are subjected to double enzyme digestion identification by SmaI and Nhe I (B in figure 3), the size of enzyme digestion products is consistent with that of the TBEV prM-E gene, and a recombinant plasmid containing the TBEV prM-E double copy gene is successfully constructed and named as pFBD-prM-E-prM-E.

3. Preparation and identification of recombinant bacmid-prM-E-prM-E

The recombinant plasmid pFBD-prM-E-prM-E was transformed into DH10Bac competent cells and plated on triple-resistant solid LB plates containing tetracycline (10. mu.g/mL), kanamycin sulfate (50. mu.g/mL), gentamicin (7. mu.g/mL), IPTG and X-Gal, and inverted cultured at 37 ℃ for 40 h. Picking white spots, shaking and culturing at 37 ℃ and 200rpm for 16h, and extracting bacmids for PCR identification. The MCS of the PH promoter can be identified by taking the bacmid as a template and taking PHF and M13R as upstream and downstream primers; M13F and P10R are used as upstream and downstream primers to identify MCS at the P10 promoter.

The results showed that the amplified product corresponded to the size of the desired fragment (FIG. 4), indicating the successful preparation of recombinant bacmid-prM-E-prM-E.

TABLE 3 PCR reaction conditions

4. Rescue of recombinant baculovirus rBV-prM-E-prM-E

Inoculating Sf9 cells into a six-hole plate, culturing for 12h at 27 ℃, changing to a double-non Grace's culture medium when the cells grow to more than 80%, respectively diluting positive recombinant bacmid-E-Linker-PA3(4 mu g) and Cellffectin II Reagent (8 mu L) by using the double-non Grace's culture medium, slightly mixing uniformly, standing for 20min, uniformly adding into the 6-hole plate, and standing and culturing for 5h at 27 ℃, wherein the liquid is changed to Grace's complete culture medium. The cells are statically cultured for 5 days at 27 ℃, the cell state is observed under a microscope every day, and compared with the normal adherent Sf9 cells, the adherent Sf9 cells transfected by the recombinant bacmid-prM-E-prM-E have obvious cytopathic phenomena such as expansion, rounding, large shedding and the like (as shown in figure 5). When 60% of cells have lesions, collecting the supernatant as a first generation recombinant baculovirus named rBV-prM-E-prM-E, and continuing to pass through 3 generations blindly.

5. Identification of recombinant baculovirus rBV-prM-E-prM-E

(1) Indirect immunofluorescence assay

Sf9 cells with good growth state are uniformly paved into a six-hole plate, when the cells grow to 70%, recombinant baculovirus is inoculated, 10 mu L of recombinant baculovirus is inoculated in each hole, the culture medium is discarded after the culture is carried out for 48 hours at the temperature of 27 ℃. Fixing with 70% ethanol solution for 30min, 1:100 dilutions of anti-TBEV E-DIII mouse serum (1% BSA dilution) were used as primary antibody, incubated for 1h at 37 ℃, 1: FITC-labeled goat anti-mouse IgG was diluted at 200 as a secondary antibody, incubated for 1h at 37 ℃ in the absence of light, and washed 3 times with PBS at the end of each operation. Cells were observed under a fluorescent microscope. The results show that adherent Sf9 cells infected with recombinant baculovirus fluoresced significantly in green under the fluorescent microscope (A-a and A-b in FIG. 6) compared to the normal cell control, indicating that TBEV prM-E protein was successfully expressed after infection of the cells with recombinant baculovirus.

(2) Western Blot identification

Infecting Sf9 cells cultured in suspension by recombinant baculovirus, culturing at 27 ℃ and 120rpm for 96h, then collecting infected cell suspension, culture supernatant and cell sediment by low-speed centrifugation, preparing a sample to be loaded, and mixing the sample with the culture solution according to the weight ratio of 1: anti-TBEV E-DIII mouse serum at 300 dilution was primary, 1:20000 diluted HRP-labeled goat anti-mouse IgG was used as a secondary antibody for Western Blot identification. The results show that: a specific protein of interest band (B in FIG. 6) appeared at both 74 and 54kD, and almost no specific protein could be detected in the culture supernatant.

Predicted according to software Editseq: the size of TBEV prM-E protein is about 74kD, the size of TBEVM-E protein is about 54kD, and the surface of mature TBEV granules is mainly composed of M protein and E protein. In conclusion, the TBEV prM-E protein is successfully expressed after the recombinant baculovirus infects the cells, and the expression form is mainly intracellular expression.

Example 2 preparation and characterization of TBEV VLPs

1. Purification of TBEV VLPs

Infecting suspension Sf9 cells with recombinant baculovirus rBV-prM-E-prM-E according to the proportion of 1%, culturing at 27 ℃ for 4d at 120rpm, harvesting cell culture, centrifuging at 3000g/min for 15min to remove cell debris, washing the harvested precipitate with PBS, adding cell lysate (1% PMSF before use) in the proportion of original solution 1/5 into the precipitate, re-suspending the precipitate with a shaker, shaking for 15s after ice bath for 20min, ice bath for 10min, centrifuging at 4 ℃ for 15min at 15000g/min, and taking supernatant. Purifying TBEV virus-like particles by potassium tartrate-glycerol density gradient centrifugation by using an ultracentrifuge, measuring the concentration of the virus-like particles by using a BCA method, subpackaging, and storing at-80 ℃ for later use.

2. Western Blot identification

Protein samples were prepared from TBEV VLPs with different purities, and Western Blot identification was performed with anti-TBEV E-DIII murine serum diluted at 1:300 as the primary antibody and HRP-labeled goat anti-murine IgG diluted at 1:20000 as the secondary antibody. The results showed that a specific protein band (A in FIG. 7) appeared at 54kD, indicating successful purification of TBEV VLPs.

3. Transmission electron microscopy inspection

Diluting the purified TBEV VLPs protein solution by 2 times, taking 20 mu L of the diluted protein solution, dropwise adding the diluted protein solution onto a copper net, absorbing redundant liquid after dyeing, and observing under a transmission electron microscope to identify the form and the size of the TBEV VLPs. As a result, it was found that the protein solution had a large number of spherical, envelope-like particles on the surface and virus-like particles (B-a in FIG. 7) of about 40-60nm, which were similar to the morphological structure of flavivirus.

4. Immuno-electron microscopy detection

Diluting TBEV VLPs twice, taking a sealing film, putting a sample of 30 mu L, putting a copper net on the liquid drop with the front side facing downwards, sticking the sealing film on a light shaking table, and incubating for 15-20min at room temperature. The liquid was blotted with absorbent paper and after the copper mesh was air dried, washed 3 times with 30 μ L PBS for 5 min/time. Blocking was performed with 30 μ L of 3% skimmed milk powder at room temperature for 1 h. anti-TBEV E-DIII murine sera were diluted 50-fold with PBS as primary antibody and incubated for 1h at room temperature. 30 μ L PBS 3 times, 5 min/time. The anti-mouse IgG antibody was diluted 20-fold with PBS as a secondary antibody and incubated at room temperature for 1 h. 30 μ L PBS 3 times, 5 min/time. After the copper mesh was stained, it was observed under an electron microscope (B-B in FIG. 7) that gold particles were attached to the surface of the virus-like particles, indicating that purified TBEV VLPs were successfully obtained in the present invention.

Example 3 evaluation of immune Effect of TBEV VLPs

1. Immunization of mice

To evaluate the immune efficacy of TBEV VLPs, 27 healthy female BALB/C mice, 16-18g, were randomly divided into 3 groups, and experimental mice were adjuvanted with purified TBEV VLPs as antigen, supplemented with Poly (I: C) & Montanide ISA201VG complex adjuvant. According to the antigen: poly (I: C): montanide ISA201VG 35: 10: 55, mixing the antigen with Poly (I: C), emulsifying with Montanide ISA201VG (preheated at 31 ℃), shaking for 10min on a vortex oscillator, and standing for 1h at 21 ℃ after complete emulsification. Each mouse was injected intramuscularly with 100 μ L of immunogen, the grouping protocol was as follows:

TABLE 4 BALB/c mouse immunization and grouping protocol

2. Detection of murine serum TBEV IgG antibodies

The serum of mice in PBS group is used as a negative control, and the serum IgG antibody level of the mice at 3 (2 weeks after first immunization), 4 (1 week after second immunization), 7 (1 week after third immunization), 10, 12 and 18 weeks after TBEV VLPs immunization is detected by using a TBEV antibody indirect ELISA detection method. The method comprises the following steps:

the purified recombinant protein TBEV E-DIII expressed in pronucleus with the final concentration of 1 mug/mL is used as a coating antigen and coated overnight at 4 ℃. 5% of skimmed milk powder is used as sealing liquid; from 1: diluting the serum of the mouse to be detected as a primary antibody at a ratio of 2 times at the beginning of 100; mixing the raw materials in a ratio of 1: a 40000-fold dilution of HRP-labeled goat anti-mouse IgG antibody was used as the secondary antibody. Developing with TMB for 3min, and applying 0.5M H ₂ SO ₄ The color development was terminated. Reading OD value of each hole at the wavelength of 450nm of an enzyme labeling instrument, and determining the OD of the serum to be detected ₄₅₀ Negative serum OD ₄₅₀ The highest serum dilution at > 2.1 was taken as the ELISA antibody titer.

The results are shown in FIG. 8: TBEV-specific IgG antibodies (mean 1:10) can be detected in mouse sera 1 week after the second immunization; the serum IgG antibody level of the mice after the three-immunization is obviously improved and can reach 1:10 at most ^5.5 (average value 1:10) ^4.6 ) (ii) a The level of TBEV IgG antibody in the serum of the mouse slowly decreases at 18 weeks and can still be maintained at 1:10 in 3 months after the three-time immunization ^4.3 。

3. Flow cytometry analysis of immune cells in the mouse inguinal lymph node

In 1 week after the first immunization, 3 mice in TBEV VLPs, Adjuvant and PBS groups are randomly selected, inguinal lymph nodes of the mice are respectively taken to prepare lymphocyte suspensions, cells are stained, and fluorescent signals are detected on a flow cytometry.

(1) Activation of mouse lymph node DC cells

Mouse inguinal lymph node cell line CD80 ⁺ 、MHCⅠ ⁺ 、MHCⅡ ⁺ And CD11c ⁺ Detection of CD11c after fluorescent antibody staining ⁺ CD80 ⁺ 、CD11c ⁺ MHCⅠ ⁺ And CD11c ⁺ MHCⅡ ⁺ Double positive cell ratio.

The results show that: immune group mouse CD11c ⁺ CD80 ⁺ The proportion of double positive cells is obviously higher than that of a PBS control group and an Adjuvant control group (P is less than 0.01, A-a in figure 9); immune group mouse CD11c ⁺ MHCⅠ ⁺ The proportion of double positive cells is obviously higher than that of a PBS control group and an Adjuvant control group (P is less than 0.01, A-b in figure 9); immune group mouse CD11c ⁺ MHCⅡ ⁺ The proportion of double positive cells is significantly higher than that of the PBS control group and the Adjuvant control group (P < 0.01, A-c in figure 9). Taken together, it can be shown that the first immunization of TBEV VLPs in mice promotes DC cell activation.

(2) Recruitment and activation of B cells from mouse lymph nodes

Mouse inguinal lymph node cells, CD19 ⁺ 、CD40 ⁺ And CD69 ⁺ Detection of CD19 following fluorescent antibody staining ⁺ CD40 ⁺ And CD19 ⁺ CD69 ⁺ Double positive cell ratio. The results show that: immunization group mouse CD19 ⁺ CD40 ⁺ The proportion of double positive cells was significantly higher than that of the PBS control group (P < 0.05) (B-a in FIG. 9); immunization group mouse CD19 ⁺ CD69 ⁺ The proportion of double positive cells was significantly higher than that of the PBS control group (P < 0.01) and the Adjuvant control group (P < 0.05) (B-B in FIG. 9). Taken together, it can be shown that the recruitment and/or activation of B cells in lymph nodes is enhanced after the first immunization of TBEV VLPs in mice.

4. Spleen cell activation assay

Separating mouse spleen cell, adjusting the concentration of spleen cell to 2.5 × 10 ⁶ one/mL for use.

(1) ELISpot assay

Detection was performed according to the instructions of the mouse IFN-. gamma.and IL-4ELISpot detection kit, and the steps were as follows: adding 5X 10 of each hole ⁵ Adding TBEVE-DIII as stimulator with final concentration of 10 μ g/mL into each splenocyte, wrapping ELISpot plate with tinfoil paper, and culturing at 37 deg.C with 5% CO ₂ Culturing for 38h in a cell incubator, and using biotin-labeled detection antibody (BVD6-24G2-biotin) diluted by 1:1000 as a primary antibody; 1:1000 dilution of Streptavidin-HRP as a Secondary antibody, ChamberAfter incubation at warm temperature, adding TMB color development solution, performing color development in dark until clear spots appear, and washing the ELISpot plate with tap water to stop color development. And naturally air-drying the ELISpot plate in a dark place, performing full-automatic Spot image acquisition and counting by using an AID enzyme-linked Spot image automatic analyzer, and storing the number of Spot-Forming Cells (SFCs) and Spot images in each hole.

The number of the cell spots of the immune group and the control group in the presence of the stimulant is plotted, and the result shows that the number of the cell spots secreting IFN-gamma in spleen cells of mice in the immune group is remarkably higher than that of an Adjuvant control group (P is less than 0.05, A in figure 10) and the number of the cell spots secreting IL-4 is remarkably higher than that of a PBS control group and that of the Adjuvant control group (P is less than 0.0001, B in figure 10) after TBEV specific antigen stimulation. IFN-gamma and IL-4 are representative cytokines of Th1 type cell immunity and Th2 type humoral immunity respectively, and after a mouse is immunized by TBEV VLPs, an organism can be induced to secrete Th1 type and Th2 type cytokines, and the Th2 type cytokines are mainly used.

(2) Splenic lymphocyte proliferation assay

Taking the separated spleen lymphocytes, and adding the cells at a ratio of 2.5X 10 ⁵ One well was seeded in 96-well cell culture plates and splenic lymphocytes were stimulated with the recombinant protein TBEV E-DIII (final concentration 10. mu.g/mL) at 37 ℃ with 5% CO ₂ Culturing in a cell culture box for 44h, and detecting the proliferation capacity of the splenic lymphocytes in vitro by using a CCK-8 method. Adding 10 μ L of CCK-8 solution into each well, culturing in cell culture box for 4 hr, and detecting OD on microplate reader ₄₅₀ Numerical values.

The result shows that the splenic lymphocyte proliferation index of the mice in the immune group is obviously higher than that of the PBS control group and the Adjuvant control group (P < 0.01, figure 11). In conclusion, after triple immunization with TBEV VLPs, splenic lymphocytes of mice can significantly proliferate under the action of specific antigens.

(3) Cytokine detection

Taking separated spleen lymphocytes at 5 × 10 ⁵ Each well was inoculated in a U-shaped 96-well plate and splenic lymphocytes were stimulated with the recombinant protein TBEV E-DIII (final concentration 10. mu.g/mL). At 37 ℃ 5% CO ₂ The cell culture box is used for culturing for 48 hours. Centrifuging at 4 deg.C in dark at 2500rpm for 10min, collecting 50 μ L supernatant, storing in dry ice, and delivering to Shanghai youNingwei company, using the MSD detection technique, detected Th1 and Th2 cytokines in splenocytes under the effect of TBEV-specific stimuli: IFN-gamma, IL-12p70, TNF-alpha, IL-2, KC/GRO, IL-4, IL-5, IL-6, IL-10, IL-1 beta secretion.

The results show that the Th2 type cytokines IL-5 and IL-6 in the splenocyte culture solution of the immunized mice are significantly higher than those in the Adjuvant control group (P < 0.05, FIG. 12) after the splenocytes are stimulated by TBEV specific stimulators.

(4) Spleen lymphocyte assay

Collecting the spleen lymphocytes after the centrifugation in the previous step, adding a fluorescent antibody premix, and detecting fluorescent signals of the lymphocytes by using a flow cytometer.

i) Recruitment and activation of murine splenic B cells

Mouse spleen cell line CD19 ⁺ 、CD40 ⁺ And CD69 ⁺ Staining with fluorescent antibody, detecting CD19 ⁺ CD69 ⁺ Double positive cell ratio. The results show that: immunization group mouse CD19 ⁺ CD69 ⁺ The proportion of double positive cells was significantly higher than that of the PBS control group (P < 0.05, A in FIG. 13). Taken together, it can be shown that splenic B cells recruited and activated under specific antigen stimulation following TBEV VLPs triaimmunization, and following TBEV VLPs triaimmunization.

ii) recruitment and/or activation of mouse spleen T cells

Mouse spleen cell line CD4 ⁺ And CD69 ⁺ Staining with fluorescent antibody, detecting CD4 ⁺ CD69 ⁺ And CD8 ⁺ CD69 ⁺ Double positive cell ratio. The results show that: immunization group mouse CD4 ⁺ CD69 ⁺ The proportion of double positive cells is significantly higher than that of the Adjuvant control group (P < 0.05, B in figure 13); taken together, spleen CD4 stimulated by specific antigen after triple immunization with TBEV VLPs ⁺ Enhanced T lymphocyte recruitment and activation.

3) Production of mouse spleen memory T cells

Mouse spleen cell line CD4 ⁺ 、CD8 ⁺ 、CD44 ⁺ And CD62L ⁺ Staining with fluorescent antibody, detecting CD4 ⁺ And CD8 ⁺ CD44 in Positive cells ⁺ CD62L ⁺ Double positive cell ratioFor example. The results show that: immunization group mouse CD4 ⁺ /CD44 ⁺ CD62L ⁺ The proportion of double positive cells is significantly higher than that of the PBS control group and the Adjuvant control group (P < 0.01, as C-a in FIG. 13); immune group mouse CD8 ⁺ /CD44 ⁺ CD62L ⁺ The cell ratio was significantly higher than that of the PBS control group and the Adjuvant control group (P < 0.01, C-b in FIG. 13). Taken together, CD4 was produced in the spleen of mice after triple immunization with TBEV VLPs ⁺ And CD8 ⁺ Central memory T cells.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Jilin university

<120> tick-borne encephalitis virus-like particle vaccine and preparation method thereof

<130> KHP221114921.3

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 680

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Ser Ala Ala Asp Trp Thr Ser Trp Leu Leu Val Leu Val Leu Val Gly

1 5 10 15

Val Thr Leu Ala Ala Thr Val Arg Lys Glu Arg Asp Gly Thr Thr Val

20 25 30

Ile Arg Ala Glu Gly Lys Asp Ala Ala Thr Gln Val Arg Val Glu Asn

35 40 45

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

50 55 60

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

65 70 75 80

Asp Val Asp Cys Phe Cys Arg Asn Val Asp Gly Val Tyr Leu Glu Tyr

85 90 95

Gly Arg Cys Gly Lys Gln Glu Gly Ser Arg Thr Arg Arg Ser Val Leu

100 105 110

Ile Pro Ser His Ala Gln Gly Asp Leu Thr Gly Arg Gly His Lys Trp

115 120 125

Leu Glu Gly Asp Ser Leu Arg Thr His Leu Thr Arg Val Glu Gly Trp

130 135 140

Val Trp Lys Asn Lys Val Leu Thr Leu Ala Val Ile Ala Ile Val Trp

145 150 155 160

Leu Thr Val Glu Ser Val Val Thr Arg Val Ala Val Val Val Val Leu

165 170 175

Leu Cys Leu Ala Pro Val Tyr Ala Ser Arg Cys Thr His Leu Glu Asn

180 185 190

Arg Asp Phe Val Thr Gly Thr Gln Gly Thr Thr Arg Val Thr Leu Val

195 200 205

Leu Glu Leu Gly Gly Cys Val Thr Ile Thr Ala Glu Gly Lys Pro Ser

210 215 220

Met Asp Val Trp Leu Asp Ser Ile Tyr Gln Glu Asn Pro Ala Lys Thr

225 230 235 240

Arg Glu Tyr Cys Leu His Ala Lys Leu Ser Asp Thr Lys Val Ala Ala

245 250 255

Arg Cys Pro Thr Met Gly Pro Ala Thr Leu Ala Glu Glu His Gln Ser

260 265 270

Gly Thr Val Cys Lys Arg Asp Gln Ser Asp Arg Gly Trp Gly Asn His

275 280 285

Cys Gly Leu Phe Gly Lys Gly Ser Ile Val Thr Cys Val Lys Ala Ser

290 295 300

Cys Gly Ala Lys Lys Lys Ala Thr Gly His Val Tyr Asp Ala Asn Lys

305 310 315 320

Ile Val Tyr Thr Val Lys Val Glu Pro His Thr Gly Asp Tyr Val Ala

325 330 335

Ala Asn Glu Thr His Ser Gly Arg Lys Thr Ala Ser Phe Thr Val Ser

340 345 350

Ser Glu Lys Thr Ile Leu Thr Met Gly Asp Tyr Gly Asp Val Ser Leu

355 360 365

Leu Cys Arg Val Ala Ser Gly Val Asp Leu Ala Gln Thr Val Ile Leu

370 375 380

Glu Leu Asp Lys Thr Ser Glu His Leu Pro Thr Ala Trp Gln Val His

385 390 395 400

Arg Asp Trp Phe Asn Asp Leu Ala Leu Pro Trp Lys His Glu Gly Ala

405 410 415

Gln Asn Trp Asn Asn Ala Glu Arg Leu Val Glu Phe Gly Ala Pro His

420 425 430

Ala Val Lys Met Asp Val Tyr Asn Leu Gly Asp Gln Thr Gly Val Leu

435 440 445

Leu Lys Ser Leu Ala Gly Val Pro Val Ala His Ile Asp Gly Thr Lys

450 455 460

Tyr His Leu Lys Ser Gly His Val Thr Cys Glu Val Gly Leu Glu Lys

465 470 475 480

Leu Lys Met Lys Gly Leu Thr Tyr Thr Met Cys Asp Lys Thr Lys Phe

485 490 495

Thr Trp Lys Arg Ile Pro Thr Asp Ser Gly His Asp Thr Val Val Met

500 505 510

Glu Val Ala Phe Ser Gly Thr Lys Pro Cys Arg Ile Pro Val Arg Ala

515 520 525

Val Ala His Gly Ser Pro Asp Val Asn Val Ala Met Leu Ile Thr Pro

530 535 540

Asn Pro Thr Ile Glu Thr Asn Gly Gly Gly Phe Ile Glu Met Gln Leu

545 550 555 560

Pro Pro Gly Asp Asn Ile Ile Tyr Val Gly Glu Leu Ser His Gln Trp

565 570 575

Phe Gln Lys Gly Ser Ser Ile Gly Arg Val Phe Gln Lys Thr Arg Lys

580 585 590

Gly Ile Glu Arg Leu Thr Val Ile Gly Glu His Ala Trp Asp Phe Gly

595 600 605

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

610 615 620

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

625 630 635 640

Lys Ile Leu Val Gly Val Val Leu Ala Trp Leu Gly Leu Asn Met Arg

645 650 655

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

660 665 670

Ala Met Thr Leu Gly Val Gly Ala

675 680

<210> 2

<211> 2040

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tcagcagcag actggacaag ttggttgctg gttctggttt tggtgggggt gactctcgct 60

gctacagttc gtaaggaacg tgacggtact actgtgatcc gcgctgaagg caaggacgct 120

gccacccagg tgcgtgttga gaacggcacc tgtgtgatcc tggccacaga catgggctcc 180

tggtgcgacg actccctgac atacgagtgc gtgaccatcg accaaggcga agaacctgtg 240

gacgtcgatt gcttctgccg caacgtcgat ggcgtgtacc tggagtacgg tcgttgtgga 300

aagcaggaag gttccaggac tcgccgctct gtgctgatcc cttcccacgc tcaaggcgac 360

ctcaccggac gcggccacaa gtggctcgaa ggcgactccc tgcgtactca cctgacccgc 420

gtggaaggtt gggtgtggaa gaacaaggtc ctgaccctgg ccgtgatcgc tatcgtctgg 480

ctcacagtgg aaagcgtcgt gacccgtgtc gccgtcgtgg tggtgctgct gtgtctggcc 540

cctgtgtacg ccagccgttg cacacacctc gaaaaccgcg acttcgtgac cggcacacaa 600

ggaactacac gcgtcactct cgtcctggag ttgggaggtt gcgtgaccat caccgctgag 660

ggcaagccaa gcatggacgt gtggctggac tccatctacc aggagaaccc cgctaagaca 720

cgtgaatact gcctccacgc caagctcagc gacacaaagg ttgccgctcg ctgtcctaca 780

atgggcccag ccaccctggc tgaggagcac cagagcggta ccgtctgtaa gcgcgaccag 840

agcgaccgcg gttggggcaa tcactgcggc ctcttcggca agggtagcat cgtgacctgt 900

gtgaaggcca gctgcggagc caagaagaag gccactggcc acgtgtacga cgctaacaag 960

atcgtgtaca ccgtgaaggt ggagcctcac accggtgact acgtggctgc taacgagacc 1020

cacagcggac gtaagaccgc cagcttcacc gtcagctccg aaaagactat cctcactatg 1080

ggcgactacg gcgacgtgag cctgttgtgt cgtgttgcca gcggcgtgga cctcgcccag 1140

acagtgatcc tggaactgga taagacatcc gagcacctgc ccaccgcttg gcaggtgcac 1200

cgtgactggt tcaacgacct cgctctcccc tggaagcacg aaggtgctca gaactggaac 1260

aacgctgagc gtctggtgga gttcggtgcc cctcacgctg tgaagatgga cgtttacaac 1320

ttgggtgacc agaccggtgt tctgctgaag tccctcgccg gtgtgcccgt cgctcacatt 1380

gacggcacta agtaccacct gaaaagcggc cacgtgactt gtgaggttgg actggagaag 1440

ctgaagatga agggactgac ctacacaatg tgtgacaaga ctaagttcac ctggaagcgt 1500

atccctacag actccggaca cgacactgtc gtgatggagg tggccttcag cggtactaag 1560

ccttgtcgta tcccagtgcg cgctgtggct cacggctccc ctgacgtgaa cgtggctatg 1620

ctgatcaccc ctaaccctac tatcgaaacc aacggtggcg gcttcatcga gatgcagctg 1680

ccacccggtg acaacatcat ctacgttggt gaactgagcc accaatggtt ccagaagggt 1740

tctagcatcg gccgcgtgtt ccagaaaaca cgcaagggca tcgagcgtct gaccgtgatc 1800

ggcgagcacg cctgggactt cggctccacc ggcggcttcc tgacctctgt gggtaaagct 1860

ctgcacacag tgctgggtgg tgctttcaac agcctcttcg gtggagtggg cttcctccct 1920

aagatcctgg tgggcgtggt gctggcttgg ctgggcctga acatgcgcaa cccaactatg 1980

agcatgagct tcctgctggc tggtggcctg gtgctggcta tgaccctggg tgtcggcgcc 2040

<210> 3

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tatgcggccg catgtcagca gcagactgga caagtt 36

<210> 4

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tataagcttt taggcgccga cacccagggt 30

<210> 5

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tatcccggga tgtcagcagc agactggaca agttg 35

<210> 6

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tatgctagct taggcgccga cacccagggt 30

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cccagtcacg acgttgtaaa acg 23

<210> 8

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cggaccttta attcaaccc 19

<210> 9

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ttcataccgt cccaccat 18

<210> 10

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

agcggataac aatttcacac agg 23

Claims (10)

1. A tick-borne encephalitis virus-like particle vaccine, characterized in that the immunogenic component of the tick-borne encephalitis virus-like particle vaccine is tick-borne encephalitis virus prM-E protein; the tick-borne encephalitis virus prM-E protein comprises an amino acid sequence shown as SEQ ID No. 1.

2. The tick-borne encephalitis virus like particle vaccine of claim 1, characterised in that the tick-borne encephalitis virus prM protein is encoded by the nucleotide sequence shown in SEQ ID No. 2.

3. The tick-borne encephalitis virus like particle vaccine of claim 1 or 2, further comprising an adjuvant; the adjuvant comprises one or more of Montanide ISA201VG adjuvant, Poly (I: C), Montanide Gel 02PR, Montanide IMS 1313VG NST, polysaccharide, Alum, Freund or Prodavx.

4. The tick-borne encephalitis virus-like particle vaccine according to claim 3, characterised in that the adjuvant is a Poly (I: C) and Montanide ISA201VG composite adjuvant, the volume ratio of the immunogenic component to the adjuvant is (5-9): (11-15).

5. The tick-borne encephalitis virus-like particle vaccine according to claim 4, characterised in that in the Poly (I: C) and Montanide ISA201VG composite adjuvant, the volume ratio of Poly (I: C) and Montanide ISA201VG adjuvant is (1-3): (9-13).

6. The method of making a tick-borne encephalitis virus like particle vaccine of any of claims 1-5, comprising:

the nucleotide sequence shown in SEQ ID NO.2 is constructed on an expression vector, expressed by an expression system and purified to obtain the tick-borne encephalitis virus-like particle vaccine.

7. The method according to claim 6, wherein the expression vector is a pFBD vector; and/or, the expression system is one or more of an insect cell-baculovirus expression system, a mammalian expression system, a prokaryotic expression system, or a yeast expression system.

8. The method of claim 6 or 7, wherein the purifying comprises:

treating an expression product of the insect cell-baculovirus expression system at 0-4 ℃ for 25-35 minutes;

centrifuging for 10-20 min at the temperature of 0-4 ℃ and the speed of 14000-16000 g/min;

taking supernatant, and performing density gradient centrifugation through potassium tartrate-glycerol.

9. A nucleic acid comprising the nucleotide sequence set forth as SEQ ID No. 2.

10. A biological material comprising the nucleic acid of claim 9, wherein the biological material is an expression cassette, a vector, or a transgenic cell.

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