Echinococcus granulosus mRNA (messenger Ribonucleic Acid) vaccine and preparation method thereof

Abstract

The invention belongs to the field of biology, and discloses an echinococcus granulosus mRNA vaccine and a preparation method thereof. After codon optimization is carried out on an EgG1Y162 protein coding sequence of echinococcus granulosus, a T7 promoter, a 5 '-untranslated region (5' UTR) sequence and a Kozak sequence are added to the front end of the sequence, a 3 '-untranslated region (3' UTR) and a poly A tail (Poly A) are added to the rear end of the sequence, an mRNA vaccine template DNA sequence is formed, and then mRNA is obtained, and the mRNA has the advantages of being good in immunogenicity, high in protection strength and the like when existing in an echinococcus granulosus vaccine form.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a fine-grained Echinococcus larvae mRNA vaccine and its preparation method. Background Technology

[0002] Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by infection with Echinococcus granulosus (Eg). It is endemic in many countries in Asia, South America, the Middle East, and Australia, and has a global distribution. After infection, the larvae form cysts within the host (such as the liver, lungs, and brain). These cysts cause organ dysfunction (such as abnormal liver function, respiratory distress, and neurological symptoms) through mechanical compression, and induce local fibrosis and chronic inflammation. Cyst rupture can release antigens, triggering anaphylactic shock or disseminated infection. Simultaneously, the parasite evades immune clearance through immune escape (Th2 immune shift). Humans, as incidental hosts, may experience long-term wasting diseases (anemia, hypoproteinemia) after infection. Children are more prone to developing severe illness, and treatment relies on surgery (high risk of relapse) and long-term drug control, seriously threatening health and the livestock industry.

[0003] While early vaccines offered strong immune protection due to antigenic diversity, their crude antigenic components were complex, production costs were high, and large-scale production was difficult. Synthetic peptide vaccines, although overcoming the shortcomings of traditional vaccines through artificial design, are susceptible to host factors and the composition of the peptides themselves in terms of immunogenicity, and cannot achieve vertical transmission of immunity. Genetically engineered recombinant vaccines, while highly effective, safe, and providing high protection rates, require multiple doses (2-3 times) to achieve long-lasting immunity. Transgenic plant vaccines, despite their low production costs and ease of administration, have relatively limited protective efficacy, offering only partial protection against protocercariae, and their oral immunization efficiency may be affected by the digestive environment. All types of vaccines still face challenges in terms of stability, protective efficacy, and large-scale application. Therefore, research on vaccines against Echinococcus granulosus must continue, and the emergence of novel mRNA vaccines is needed to address the problems existing in current technologies.

[0004] mRNA vaccines offer significant advantages. An mRNA vaccine is a vaccine that delivers an mRNA sequence encoding an antigen protein into host cells via a vector molecule, where the corresponding antigen protein is expressed, thereby inducing an immune response. Compared to other vaccines such as inactivated vaccines, subunit vaccines, and DNA vaccines, mRNA vaccines can induce both T-cell and B-cell immune responses and do not carry the risks of infection or insertional mutagenesis. mRNA vaccines exhibit significant safety and efficacy, and their short development cycle and ease of large-scale production have made them increasingly popular in recent years. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a fine-grained Echinococcus mRNA vaccine and its preparation method, which addresses the shortcomings of the prior art.

[0006] To solve the above-mentioned technical problems, the present invention discloses the following technical solution:

[0007] In a first aspect, the present invention discloses an mRNA, which is obtained by codon optimization of the EgG1Y162 protein coding sequence of Echinococcus granulosus, adding a T7 promoter, a 5' untranslated region (5'UTR) and a Kozak sequence to the front end of the sequence, and adding a 3' untranslated region (3'UTR) and a polyadenylated tail (Poly A) to the back end, thereby constructing an mRNA vaccine template DNA sequence; when this mRNA exists in the form of Echinococcus granulosus mRNA vaccine, it has advantages such as good immunogenicity and strong protective efficacy.

[0008] The nucleotide sequence of the codon-optimized EgG1Y162 gene is shown in SEQ ID NO.6.

[0009] The nucleotide sequences of the T7 promoter, 5' untranslated region, Kozak sequence, 3' untranslated region and polyadenylate are shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.8, SEQ ID NO.4 and SEQ ID NO.5, respectively.

[0010] Secondly, the present invention also discloses a method for preparing the mRNA described in the first aspect above.

[0011] The method includes the following steps:

[0012] Step 1: Construct a vector containing the nucleotide sequence shown in SEQ ID NO.1 to obtain a plasmid;

[0013] Step 2: Linearize the plasmid to obtain a linearized plasmid;

[0014] Step 3: Transcribe, cap, and purify the obtained linearized plasmid to obtain mRNA.

[0015] Thirdly, the present invention also discloses the use of the mRNA for vaccine preparation described in the first aspect above.

[0016] The vaccine in question is the EgG1Y162-mRNA vaccine for the prevention of Echinococcus granulosus.

[0017] Specifically, the vaccine is to be administered to sheep, cattle, and camels.

[0018] Fourthly, the present invention also discloses a vaccine containing the mRNA described above.

[0019] Specifically, the vaccine comprises liposomes and mRNA encapsulated within the liposomes.

[0020] In the aforementioned vaccine, the liposomes include cationic lipids and distearate phosphatidylcholine (DSPC).

[0021] Cholesterol, dimyristic glycerol-polyethylene glycol 2000 (DMG-PEG2000), wherein the molar ratio of the cationic lipid, DSPC, cholesterol, and DMG-PEG2000 is 45-55:8-12:33.5-43.5:1.2-1.8, preferably 50:10:38.5:1.5.

[0022] The liposomes include the cationic liposome 1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecapoxy)hexyl]amino]octanoate (SM-102).

[0023] In this invention, the average particle size of the liposomes in the vaccine is 90-98 nm.

[0024] In this invention, the nucleotide sequence of the EgG1Y162 gene is shown in GenBank accession number AB462014.

[0025] This invention selects and optimizes the EgG1Y162 antigen coding sequence of *Echinococcus granulosus* to construct a *Echinococcus granulosus* mRNA vaccine. After codon optimization of the *Echinococcus granulosus* EgG1Y162 protein coding sequence, a T7 promoter, a 5' untranslated region (5'UTR), and a Kozak sequence are added to the front end of the sequence, and a 3' untranslated region (3'UTR) and a polyadenylated tail (Poly A) are added to the back end, forming the mRNA vaccine template DNA sequence. This sequence is then ligated into a plasmid vector to construct the recombinant plasmid GS-CMV-EgG1Y162. The recombinant plasmid is linearized, transcribed, capped, and purified to obtain mRNA, which is then packaged using LNP technology to prepare the *Echinococcus granulosus* EgG1Y162-mRNA-LNPs vaccine.

[0026] Beneficial effects:

[0027] 1. The Echinococcus granulosus EgG1Y162-mRNA-LNPs vaccine of the present invention optimizes the EgG1Y162 antigen sequence by codon optimization, and then selects a suitable promoter, 5'UTR, 3'UTR and 3' end PolyA to combine into a complete mRNA nucleic acid encoding Echinococcus granulosus EgG1Y162.

[0028] 2. The Echinococcus granulosus (EgG1Y162) mRNA-LNPs vaccine prepared by this invention has been proven safe, effective, and without toxic side effects through animal safety tests. The mRNA vaccine degrades into nucleotides in vivo and does not integrate into the host genome. The mRNA vaccine can elicit a strong immune response in vivo, including humoral and cellular immunity, effectively preventing Echinococcus granulosus infection of the liver, and has good market application prospects. Attached Figure Description

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0030] Figure 1 The image shows the plasmid map of the recombinant plasmid GS-CMV-EgG1Y162 in Example 1.

[0031] Figure 2 Electrophoretic identification of the linearized enzyme digestion products of the recombinant plasmid in Example 1.

[0032] Figure 3 Immunofluorescence detection of EgG1Y162-mRNA expression in Example 3.

[0033] Figure 4 The particle size and potential diagrams of EgG1Y162-mRNA-LNPs in Example 4 are shown.

[0034] Figure 5 Transmission electron microscopy image of EgG1Y162-mRNA-LNPs in Example 4.

[0035] Figure 6 HE staining of mouse tissue after immunization in Example 5.

[0036] Figure 7 The antibody titer in the serum of mice after immunization in Example 6 is shown.

[0037] Figure 8 The transcriptional levels of IFN-γ, IL-4, TNF-α and IL-10 in the spleen tissue of mice after immunization in Example 6.

[0038] Figure 9 The images show the liver lesion changes in immunized mice after infection with Echinococcus granulosus and the results of HE staining of the tissues.

[0039] Figure 10 The results show the changes in body weight, liver specific gravity, and spleen specific gravity of the immunized mice in Example 7 after infection with Echinococcus granulosus.

[0040] Figure 11The results show the changes in liver vesicle volume in immunized mice in Example 7 after infection with Echinococcus granulosus.

[0041] Figure 12 The results of serum AST / ALT in mice that were immunized in Example 7 after infection with Echinococcus granulosus larvae.

[0042] Figure 13 Flow cytometry of spleen lymphocytes from immunized mice in Example 7 after infection with Echinococcus granulosus. Detailed Implementation

[0043] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0044] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0045] Example 1: Construction of the antigen expression vector for the EgG1Y162-mRNA-LNPs vaccine

[0046] The EgG1Y162-mRNA-LNPs vaccine sequence contains the following elements: a T7 promoter, a 5' untranslated region (5'UTR) and a Kozak sequence, a codon-optimized EgG1Y162 gene, a 3' untranslated region (3'UTR), and a polyadenylated nucleotide (Poly A), with a plasmid-linearized restriction enzyme site (BspQI) linked downstream of the Poly A tail structure (plasmid map shown). Figure 1 (As shown). Among them, the nucleotide sequence of the T7 promoter is shown in SEQ ID NO.2, the nucleotide sequence of the 5'UTR is shown in SEQ ID NO.3, the nucleotide sequence of the Kozak sequence is shown in SEQ ID NO.8, the nucleotide sequence of the 3'UTR is shown in SEQ ID NO.4, and the nucleotide sequence of Poly A is shown in SEQ ID NO.5; the codon-optimized EgG1Y162 gene mRNA sequence is shown in SEQ ID NO.6, and the amino acid sequence is shown in SEQ ID NO.7.

[0047] The mRNA vaccine sequence was synthesized by Nanjing Genscript Biotech Co., Ltd., and cloned into the GS-CMV vector. The nucleotide sequence of the antigen expression of the EgG1Y162-mRNA-LNPs vaccine is shown in SEQ ID NO.1.

[0048] The synthesized DNA sequence was verified by sequencing, and the bands were clear, well separated, and correctly positioned (e.g., Figure 2 As shown in the image, the plasmid was named GS-CMV-EgG1Y162 (plasmid map shown in the image). Figure 1 As shown), the highly efficient expression engineered strain (glycerol cryopreserved strain) constructed and provided by the company was inoculated into LB liquid medium for activation and amplification, and then placed in a constant temperature shaking incubator at 30℃ and 200rpm for 24 hours for primary amplification culture. Plasmids were extracted using the Plasmid Mini Kit, purchased from Nanjing Novizan Biotechnology Co., Ltd. According to the instructions, 5 mL of overnight bacterial culture was centrifuged at 11,500 × g for 1 min, the supernatant was discarded, and the cells were resuspended in Buffer P1 containing RNase A until no clumps remained. Buffer P2 (gently inverted to mix, avoiding vortexing) and Buffer P3 (immediately inverted to neutralize) were added sequentially, and the mixture was centrifuged at 13,400 × g for 10 min, collecting the supernatant. The supernatant was transferred to a DNA adsorption column, centrifuged at 13,400 × g for 30–60 sec, and washed twice sequentially with Buffer PW1 (essential for endA+ host bacteria) and PW2 (ethanol diluted). After centrifugation and drying, the cells were eluted with 20 μL of preheated Elution Buffer (pH 7.0–8.5). The plasmid concentration was measured using an ultra-micro UV-Vis spectrophotometer and stored at -20℃ for later use.

[0049] Example 2: mRNA transcription verification experiment

[0050] 1. Linearization of plasmid GS-CMV-EgG1Y162

[0051] The plasmid obtained in Example 1 was digested with BspQI restriction endonuclease (purchased from Shanghai Aibisin Biotechnology Co., Ltd.), and the reaction system is shown in Table 1 below:

[0052] Table 1. Reaction system of restriction endonuclease BspQI digestion.

[0053] Components volume plasmid ≤1μg BspQ I 1μL 10×BspQ I Buffer 2μL RNase-free ddH2O Up to 20μL

[0054] After gentle mixing, briefly incubate. React at 50°C for 1 hour; the amount of enzyme required to completely break down 1 μg of λDNA is defined as 1 activity unit (U). Heat at 70°C for 20 min to thermally inactivate the endonuclease. The plasmid concentration was measured using an ultra-micro UV-Vis spectrophotometer and stored at -20℃ for later use.

[0055] 2. Capped transcription of linearized plasmids

[0056] The EasyCap T7Co-transcription Kit with CAG Trimer was purchased from Nanjing Novizan Biotechnology Co., Ltd. Following the product instructions, all components except the T7RNA Polymerase Mix were vortexed and mixed thoroughly, briefly centrifuged to collect the residue at the bottom of the tube, and stored on ice for later use. The following components were added sequentially (refer to Table 2 for details), and each component was gently mixed with a pipette, briefly centrifuged, and incubated at 37°C for 4 hours. 2 μL of DNase I was added to the reaction system, and the mixture was incubated at 37°C for 30 minutes to digest the transcribed DNA template.

[0057] Table 2. In vitro transcription system of linearized plasmids

[0058] Components volume <![CDATA[RNase-free ddH2O Up to]]> To 20μl 10×Transcription Buffer 2μI N1-Me-Pseudo UTP (100mM) 1.5μI ATP Solution (100mM) 1.5μI CTP Solution (100mM) 1.5μI GTP Solution (100mM) 1.5μI CAG Trimer 1μI T7RNA Polymerase Mix 2μI Template a (1 μg) xμl

[0059] 3. mRNA purification

[0060] mRNA was purified by precipitation using lithium chloride. 7.5M LiCl (DNase / RNase free) was purchased from Thermo Fisher Scientific (China) Co., Ltd. The specific purification steps are as follows: Add 30 μL of DEPC-treated water to the transcription system, then add 25 μL of 7.5M LiCl (DNase / RNase free) to adjust the LiCl concentration to 2.5M. Incubate at -20℃ for 30 min. Transfer the product to a 1.5 mL EP tube and centrifuge at 13,682 × g for 15 min. Discard the supernatant, add 1 mL of 75% ethanol solution, invert several times to wash the RNA clumps, and centrifuge at 13,682 × g for 15 min. Repeat the washing steps. Depending on the size of the RNA clumps, add an appropriate volume of DEPC-treated water, incubate at room temperature for 5 min to fully dissolve the RNA, then pipette several times, and finally aliquot and use. Concentration was measured using an ultra-micro UV-Vis spectrophotometer and stored at -20℃ for future use.

[0061] Example 3: Validation assay of EgG1Y162-mRNA protein expression

[0062] 1. Cell transfection and sample preparation of mRNA

[0063] The obtained mRNA was transfected into HEK-293T cells using Lipomaster 2000 Transfection Reagent, purchased from Nanjing Novizan Biotechnology Co., Ltd. The specific steps were as follows, following the instructions: cells were passaged 24 hours before transfection, with a seeding density of approximately 2 × 10⁶ cells per well. 5Cells. Overnight culture. Add 25 μL of opti-MEM medium and 1.5 μL of Lipomaster 2000 Transfection Reagent to a 1.5 ml sterile centrifuge tube, and gently mix with a pipette. Add 25 μL of opti-MEM medium and 0.5 μg of mRNA to a 1.5 ml sterile centrifuge tube, and gently mix with a pipette. Add the mRNA / opti-MEM mixture to the Lipomaster 2000 Transfection Reagent / opti-MEM mixture, gently mix with a pipette, incubate at room temperature for 5 min, then add to the medium, gently agitating the culture dish to ensure even distribution. Incubate overnight for 24 h. After 24 h, discard the cell supernatant, wash twice with PBS, pipette the cells, centrifuge at 214 × g for 5 min, and collect the cells.

[0064] 2. Immunofluorescence verification of mRNA after cell transfection

[0065] Transfected HEK-293T cells were washed twice with PBS, pipette tips were used to remove the cells, and centrifuged for 5 min at 214 × g and 4°C. The supernatant was discarded, and the cells were resuspended in PBS. The cell suspension was then placed on an adhesion slide and incubated for 15 min. The supernatant was discarded, and the cells were fixed at room temperature with 4% paraformaldehyde (purchased from Beijing Baishayi Technology Co., Ltd.) for 30 min. The cells were washed three times with PBS for 5 min each time. The cells were then blocked at 37°C for 45 min with 5% BSA (purchased from Aibixin (Shanghai) Biotechnology Co., Ltd.). Anti-EgGY162 polyclonal antibody serum (provided by the laboratory, diluted 1:500) was added, and the cells were incubated at 37°C for 1 h. The cells were washed three times with PBS for 5 min each time. Rabbit anti-mouse secondary antibody (diluted 1:200) (purchased from Wuhan Boster Biological Engineering Co., Ltd.) was added, and the cells were incubated at 37°C in the dark for 45 min. The cells were washed three times with PBS for 5 min each time. DAPI quencher, purchased from Beijing Solarbio Science & Technology Co., Ltd., was used for mounting. After mounting, cells were observed using a laser confocal microscope. Results are as follows: Figure 3 The results showed that the fluorescence signal intensity was high after the mRNA was transfected into the cells, and the target protein exhibited clear and specific staining in the expected cytoplasm.

[0066] Example 4: Preparation of EgG1Y162-mRNA-LNPs vaccine

[0067] The mRNA obtained in Example 2 was packaged using liposome packaging technology to prepare an mRNA liposome nanoparticle vaccine, as detailed below:

[0068] The lipids were dissolved in anhydrous ethanol at a molar ratio of SM102:DSPC:cholesterol:DMG-2000 = 50:10:38.5:1.5, with the concentration of SM102 being 5.68 mg / mL, to obtain the alcohol phase. The purified mRNA was diluted to 120 μg / mL with citrate buffer (pH 4.0) to obtain the aqueous phase. The aqueous and alcohol phases were mixed using a microfluidic mixer at a volume ratio of 3:1 and a total flow rate of 12 ml / min to form lipid nanoparticles. After obtaining the formulation, it was diluted with DPBS and concentrated by ultrafiltration using ultrafiltration tubes purchased from Merck Millipore (Shanghai) Trading Co., Ltd., to obtain the EgG1Y162-mRNA-LNPs vaccine.

[0069] The LNP particle size, zeta potential, and encapsulation efficiency of the vaccine were identified. A particle size analyzer was used for testing, and the results are as follows: Figure 4 The final EgG1Y162-mRNA-LNPs vaccine, as shown, has a particle size of 94.43 nm, a dispersion index (PDI) of 0.197, and potential results as follows. Figure 4 The value was -0.969, and the encapsulation efficiency was approximately 88.83%. This invention utilizes phosphotungstic acid negative staining combined with low-temperature, high-pressure freezing technology to achieve high-resolution imaging of the vaccine's ultrastructure under a transmission electron microscope. The results are as follows: Figure 5 The EgG1Y162-mRNA-LNPs vaccine is a uniform spherical particle (approximately 90-98 nm in diameter) with an electron-dense core and an outer layer of single or multiple lipid structures, consistent with the core-shell model.

[0070] Table 3. LNP particle size, PDI, potential, and encapsulation efficiency for identifying vaccines.

[0071] Note: LNP concentration is the concentration of LNP during microfluidic coating; mRNA concentration is the total concentration of mRNA calculated based on the nitrogen-phosphorus ratio during microfluidic coating.

[0072] Example 5: Safety Trial of EgG1Y162-mRNA-LNPs Vaccine

[0073] Eight SPF mice were randomly divided into two groups: a vaccine group (n=5) and a control group (n=3). Mice were injected intramuscularly into their hind limbs. The vaccine group received 5 μg of vaccine, while the control group received the same volume of PBS. The mice's mental state and appetite were observed, and the injection sites were monitored daily for induration, abscesses, and ulceration. Tissue samples were collected for HE staining. Results are as follows: Figure 6In this experiment, no activity inhibition, abnormal weight fluctuations, or local inflammatory reactions were observed (no redness, swelling, or induration at the injection site), indicating that the vaccine has no acute toxicity. Liver histopathology showed that the liver lobule structure was intact, the hepatocyte nuclei were uniform in morphology, and there was no necrosis, fatty degeneration, or inflammatory cell infiltration, confirming that the vaccine did not cause liver damage and demonstrating that the vaccine has good safety.

[0074] Example 6: Efficacy evaluation of EgG1Y162-mRNA-LNPs vaccine in SPF mice

[0075] 1. Experimental grouping and immunization

[0076] To verify the immunization efficacy of the EgG1Y162-mRNA-LNPs vaccine, an SPF mouse animal experiment was conducted. Eight SPF mice were randomly divided into two groups: an EgG1Y162-mRNA-LNPs vaccine group (n=5) and a blank control group (n=3). The vaccine group was immunized with 5 μg of EgG1Y162-mRNA-LNPs; the blank control group was injected with an equal volume of phosphate-buffered saline (PBS). Two weeks after the initial immunization, both the vaccine group and the blank control group received booster immunizations with the same dose.

[0077] 2. Antibody titer detection after immunization

[0078] Serum was collected 3 weeks after the initial immunization to detect antibody titers. EgG1Y162 protein was diluted to 2 μg / mL with CBS buffer and coated onto ELISA plates (100 μL / well, overnight at 4°C). After washing with PBST, the plates were blocked with 200 μL / well of 5% skim milk at 37°C for 1 hour. After discarding the blocking solution and washing, 100 μL / well of serially diluted (1:200 to 1:25600) polyclonal antibody serum was added and incubated at 37°C for 1 hour, with a blank control included. After washing, 100 μL / well of 1:2000 diluted HRP-labeled secondary antibody was added and reacted at 37°C for 1 hour. After TMB color development (50 μL / well, 37°C in the dark for 10 min) and H2SO4 termination, the OD450 value was measured using an ELISA reader. Results are as follows: Figure 7 The results showed that the OD450 of both the mRNA-LNP group and the blank control group decreased with increasing sample dilution; the OD450 of the mRNA-LNP group was significantly higher than that of the blank control group at all dilutions, suggesting that it can enhance the detection intensity of target biological signals or the expression level of related molecules.

[0079] 3. Extraction of total RNA

[0080] Add 1 mL of Trizol to 50 mg of tissue, bead grinder to disrupt the protein (70 Hz, 30 s / 60 s cycles), and let stand for 15 min. Centrifuge at 13,682 × g for 10 min at 4 °C. Collect the supernatant, add 200 μL of chloroform, vortex vigorously for 15 s, and let stand to separate the layers. Centrifuge at 13,682 × g for 10 min at 4 °C, collect the upper aqueous phase, and add an equal volume of -20 °C pre-cooled isopropanol to precipitate the RNA. After centrifugation, discard the supernatant, wash the precipitate twice with 1 mL of 75% ethanol (v / v), and centrifuge at 13,682 × g for 5 min to remove all liquid. Dry at room temperature and dissolve in DEPC water.

[0081] 4. Reverse transcription of total RNA

[0082] HiScript III RT SuperMix for qPCR (+gDNA wiper) (R323-01), purchased from Nanjing Novizan Biotechnology Co., Ltd., was used for transcription into cDNA. The reverse transcription reaction system is shown in Tables 4 and 5:

[0083] Table 4 Reverse transcription reaction system

[0084] Components volume <![CDATA[RNase-free ddH2O]]> to 16μl 4×gDNA wiper Mix 4μl template RNA Total RNA: 1 pg - 1 μg

[0085] Gently pipette to mix. Incubate at 42°C for 2 minutes.

[0086] Add 5×HiScript III qRT SuperMix directly to the reaction tube in step 1.

[0087] Table 5 Reverse transcription reaction system

[0088] Components volume 5×HiScript III qRT SuperMix 4μl The reaction solution in step 1 16μl

[0089] Gently mix with a pipette. Place in a PCR instrument for reaction.

[0090] The reverse transcription reaction conditions are as follows: 37℃ for 15 min (reverse transcription reaction); 85℃ for 5 s (reverse transcriptase inactivation reaction); stored at -20℃.

[0091] 5. qPCR detection of transcriptional changes of IFN-γ, IL-4, TNF-α and IL-10 in spleen tissue.

[0092] The expression levels of IFN-γ, IL-4, TNF-α, and IL-10 in spleen tissue were detected by qPCR using Taq Pro Universal SYBR qPCR Master Mix (purchased from Nanjing Novizan Biotechnology Co., Ltd.). The qPCR reaction system formulation is shown in Table 6; the qPCR primer sequences are shown in Table 7; and the qPCR reaction system is shown in Table 8.

[0093] Table 6 qPCR reaction system

[0094] Components volume 2×Taq Pro Universal SYBR qPCR Master Mix 10.0μl Primer 1 (10μM) 0.4μl Primer 2 (10μM) 0.4μl Template DNA / cDNA xμl <![CDATA[ddH 2 O]]> To 20.0μl

[0095] Table 7 Primer sequences for qPCR reaction

[0096]

[0097]

[0098] The Bio-Rad Real-Time PCR instrument, purchased from Bio-Rad Biomedical Products (Shanghai) Co., Ltd., was used for qPCR reactions.

[0099] Table 8 qPCR reaction system

[0100]

[0101] The results are as follows Figure 8 As shown, the levels of INF-γ, IL4, IL10, and TNF-α in the spleen tissue of the vaccine group mice were significantly higher than those in the control group. The sample data within the group were concentrated, and the treatment effect showed good reproducibility.

[0102] Example 7: Immunoprotective effect of EgG1Y162-mRNA-LNPs vaccine in mice infected with hepatic echinococcosis.

[0103] 1. Immunization and parasite challenge in mice

[0104] To evaluate the immunogenicity of the mRNA-LNP vaccine, in vivo experiments were conducted in mice. Six-week-old SPF-grade BALB / c mice (n=18) were randomly assigned to three groups (n=6 per group): the vaccine group, the model group, and the protein group. The vaccine group was immunized with 5 μg of EgG1Y162-mRNA-LNPs; the model group was injected with an equal volume of phosphate-buffered saline (PBS); and the protein group was injected with a water-in-oil emulsion containing 200 μg of EgG1Y162 protein emulsified with Complete Freund's Adjuvant (CFA). Two weeks after the initial immunization, the vaccine and model groups received booster immunizations with the same dose. The protein group received a booster immunization using 200 μg of EgG1Y162 protein emulsified with Incomplete Freund's Adjuvant (IFA).

[0105] At week 5 of the experiment, mice in each group were injected via the portal vein with 3000 Echinococcus granulosus larvae per mouse. On day 120 post-infection, the mice were weighed and sacrificed, and liver tissue was obtained and photographed. Liver wet weight was measured, and liver index (liver weight / body weight × 100%) and spleen index (spleen weight / body weight × 100%) were calculated. Results showed that compared to the vaccine group, both the model group and the protein group had significantly higher liver and spleen indices. Liver tissue was placed in 5 mL centrifuge tubes, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). Images showed that the vaccine group exhibited a uniform purplish-blue color and regular morphology, while the model group showed multiple vacuoles in the hepatocyte cytoplasm. The number of liver cysts was significantly higher in the model group. Serum ALT and AST levels obtained from the model group, proteome group, and vaccine group showed that the vaccine group had significantly lower ALT and AST levels than the model group, exhibiting a superior hepatoprotective effect compared to the proteome group. Exogenous proteins had limited effect on improving liver injury. Figure 9 , 10 Results 11 and 12 showed that the vaccine demonstrated significant protective efficacy against hepatic echinococcosis by reducing parasite load, inhibiting the progression of liver fibrosis, and improving liver function.

[0106] 2. Flow cytometry of mouse spleen lymphocytes

[0107] Under aseptic conditions, the spleen was obtained by grinding the homogenate on a glass slide using the ground surface of the slide, as described in Step 1 of Example 7. ≤5 mL of homogenate was collected, filtered through a 200-mesh nylon mesh, and centrifuged at 300×g for 5 min, discarding the supernatant. The precipitate was resuspended in 2 mL of erythrocyte lysis buffer, incubated at 4°C for 15 min, and then centrifuged under the same conditions after adding PBS to the top. If lysis was incomplete, the process was repeated. The cells were resuspended in 10 mL of PBS and filtered twice. After trypan blue staining and counting, 5×10⁻⁶ cells were collected. 6 Live cells were stimulated with 1 μL / mL Leukocyte Activation Cocktail and purchased from BioLegend Biotechnology Co., Ltd. After incubation at 37℃ and 5% CO2 for 4 h, the reaction was stopped on ice, and cells were collected by centrifugation. Cells were incubated in 30 μL of blocking buffer containing CD16 / 32 (10 μg / mL) at 4℃ in the dark for 20 min, followed by incubation with fluorescently labeled anti-CD3 (0.25 μg) and anti-CD4 (0.5 μg) antibodies for 30 min, and then washed with flow cytometry buffer. Cells were then treated with 250 μL of fixation / permeabilization buffer for 20 min, washed with 1× permeabilization buffer, and stained with intracellular antibodies using the same method. Finally, cells were resuspended in 350 μL buffer and analyzed by flow cytometry. Results showed that the spleen of the vaccine group had CD4... + The proportion of T cells was significantly higher than that in the model group and the proteomic group (p<0.05). Figure 13 ).

[0108] Safety tests of the vaccine showed that the Echinococcus granulosus (EgG1Y162-mRNA-LNPs) vaccine of this invention was administered to SPF mice at a dose of 5 μg. After inoculation, the SPF mice exhibited good mental status, normal eating habits, and no induration, abscess, or ulceration occurred at the injection site during daily observation. Autopsy at week 3 post-inoculation revealed complete absorption of the vaccine at the intramuscular injection site in the hind legs, with no abnormalities compared to the control group.

[0109] The mRNA vaccine prepared in this invention evoked strong humoral and effective cellular immunity in mice. Mice were immunized with 5 μg of the mRNA vaccine, and serum was collected 21 days later. The antibody titer in the immunized mice serum was detected by indirect ELISA at an antigen-coating concentration of 5 μg / mL. Results showed that the serum titer in the experimental group reached 1:25600, and the transcriptional levels of IFN-γ, IL-4, TNF-α, and IL-10 in spleen tissue were increased.

[0110] Vaccine efficacy studies demonstrated that the Echinococcus granulosus (EgG1Y162-mRNA-LNPs) vaccine of this invention provides good protection against the corresponding hepatic echinococcosis. We evaluated the immunogenicity of the Echinococcus granulosus (EgG1Y162-mRNA-LNPs) vaccine using SPF mouse animal experiments. Results showed that three doses of 5 μg mRNA vaccine, spaced two weeks apart, significantly inhibited echinococcosis growth after challenge.

[0111] All the above animal test results show that the EgG1Y162-mRNA-LNPs vaccine provided by this invention is safe and effective, and can provide important technical reserves for the prevention and control of hepatic echinococcosis, and can induce cross-protection against echinococcosis larvae. The EgG1Y162-mRNA-LNPs vaccine prepared by this invention provides a new vaccine option for the prevention and control of echinococcosis.

[0112] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. An mRNA, characterized in that, The nucleotide sequence includes the T7 promoter, the 5' untranslated region, the Kozak sequence, the codon-optimized EgG1Y162 gene, the 3' untranslated region, and polyadenylate; the nucleotide sequence of the codon-optimized EgG1Y162 gene is shown in SEQ ID NO.

6.

2. The mRNA according to claim 1, characterized in that, The nucleotide sequences of the T7 promoter, 5' untranslated region, Kozak sequence, 3' untranslated region, and polyadenylate are shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.8, SEQ ID NO.4, and SEQ ID NO.5, respectively.

3. The method for preparing mRNA according to claim 1 or 2, characterized in that, A vector containing the nucleotide sequence shown in SEQ ID NO.1 was constructed to obtain a plasmid; the obtained plasmid was linearized to obtain a linearized plasmid; the obtained linearized plasmid was transcribed and purified by capping to obtain mRNA.

4. Use of the mRNA according to claim 1 or 2 in the preparation of vaccines.

5. A vaccine, characterized in that, It contains the mRNA as described in claim 1 or 2.

6. The use according to claim 4 or the vaccine according to claim 5, characterized in that, The vaccine is an EgG1Y162 mRNA vaccine for the prevention of Echinococcus granulosus infection; preferably, the vaccine is an EgG1Y162 mRNA vaccine for the prevention of Echinococcus granulosus infection in intermediate hosts; the intermediate hosts include cattle, sheep and camels.

7. The use according to claim 4 or the vaccine according to claim 5, characterized in that, The vaccine comprises liposomes and mRNA encapsulated within the liposomes.

8. The use or vaccine according to claim 7, characterized in that, The liposomes comprise cationic lipids, distearylphosphatidylcholine, cholesterol, and dimyristicoglycerol-polyethylene glycol 2000; the molar ratio of the cationic lipids, distearylphosphatidylcholine, cholesterol, and dimyristicoglycerol-polyethylene glycol 2000 is preferably 45-55:8-12:33.5-43.5:1.2-1.8, more preferably 50:10:38.5:1.5; the cationic lipids are preferably 1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate.

9. The use according to claim 3 or the vaccine according to claim 4, characterized in that, The particle size of the vaccine is 90-98 nm.

10. The use of the vaccine according to any one of claims 5-9 in the preparation of a medicament for the prevention of Echinococcus granulosus infection.

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