CN116785420B

CN116785420B - mRNA vaccine of bovine viral diarrhea virus and application thereof

Info

Publication number: CN116785420B
Application number: CN202311047976.XA
Authority: CN
Inventors: 尹鑫; 太万博; 姜志刚; 黄鹤; 常继涛
Original assignee: Beijing Hemu Biotechnology Co ltd; Harbin Veterinary Research Institute of CAAS
Current assignee: Beijing Hemu Biotechnology Co ltd; Harbin Veterinary Research Institute of CAAS
Priority date: 2023-08-21
Filing date: 2023-08-21
Publication date: 2023-11-14
Anticipated expiration: 2043-08-21
Also published as: CN116785420A

Abstract

The invention discloses an mRNA vaccine of bovine viral diarrhea virus and application thereof, belonging to the technical field of nucleic acid vaccine. In order to improve the immunoprotection of the bovine viral diarrhea virus mRNA vaccine. The present invention provides an mRNA vaccine of bovine viral diarrhea virus (Bovine viral diarrhea Virus) encoding E2 protein of bovine viral diarrhea virus or truncated E2 protein of bovine viral diarrhea virus. The immunity effect of the bovine viral diarrhea virus is improved.

Description

mRNA vaccine of bovine viral diarrhea virus and application thereof

Technical Field

The invention belongs to the technical field of virus vaccines, and particularly relates to an mRNA vaccine of bovine viral diarrhea virus and application thereof.

Background

Bovine viral diarrhea virus (Bovine Viral Diarrhea Virus, BVDV) can infect cattle causing viral diarrhea—acute, febrile, contagious disease. BVDV is distributed worldwide, the infection condition is complex, and the recessive infection rate is extremely high. It is estimated that the loss of production caused by this amounted to $ 687.8/head, resulting in a significant loss to the global cattle industry. BVDV can cause various symptoms such as slow growth, reproductive disturbance, reduced productivity and the like of cattle. After 50-150 days of gestation, the virus can be vertically transmitted to the fetus through the placenta, at the moment, the immune system of the fetus is not yet developed completely, BVDV is not recognized to cause immune tolerance, the BVDV continuously infects cattle after birth, and the BVDV is provided with the virus for life, and is continuously discharged to the outside through nasal discharge, saliva, semen and the like, so that the BVDV becomes an important infectious source of herds. The disease causes economic loss and biosafety problems for the global breeding industry, and the hazard is huge.

The BVDV vaccines used abroad at present mainly comprise modified attenuated live vaccines, inactivated vaccines and E2 subunit vaccines. In the United states, BVDV vaccines are mainly used in combination with traditional multiple vaccines composed of infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, and the like. The commercial BVDV vaccine in China is only an inactivated vaccine, and the type is single; the BVDV single-combined inactivated vaccine and BVDV and bovine infectious rhinotracheitis combined inactivated vaccine can be divided.

Subunit vaccines and inactivated vaccines have the common disadvantages of requiring an adjuvant and multiple immunizations, often an immune blank susceptible period exists between a single dose and the peak of an antibody level, and the subunit vaccines have the disadvantage of being incapable of maintaining a natural immunogen conformation.

Disclosure of Invention

The invention aims to improve the immunoprotection of an mRNA vaccine of bovine viral diarrhea virus.

The present invention provides an mRNA vaccine of bovine viral diarrhea virus (Bovine viral diarrhea Virus) encoding E2 protein of bovine viral diarrhea virus or truncated E2 protein of bovine viral diarrhea virus.

Further defined, the sequence of the E2 protein of the bovine viral diarrhea virus is shown as SEQ ID NO. 9; the sequence of truncated E2 protein of bovine viral diarrhea virus is shown as SEQ ID NO. 10.

Further defined, the mRNA vaccine consists of the following components: a 5 'untranslated region, a signal peptide sequence, an encoding RNA gene of an E2 protein of bovine viral diarrhea virus or an encoding RNA gene of a truncated E2 protein of bovine viral diarrhea virus, a 3' untranslated region and polyadenylation.

Further defined, the nucleotide sequence of the 5' -terminal untranslated region is shown as SEQ ID NO. 1; the nucleotide sequence of the 3' -end untranslated region is shown as SEQ ID NO. 2; the nucleotide sequence of the poly A is shown as SEQ ID NO. 3; the nucleotide sequence of the signal peptide is shown as SEQ ID NO. 5; the coding RNA gene sequence of the E2 protein of the bovine viral diarrhea virus is shown as SEQ ID NO. 15; the coding RNA gene sequence of the truncated E2 protein of the bovine viral diarrhea virus is shown as SEQ ID NO. 16.

The invention provides an application of a sequence of E2 protein of bovine viral diarrhea virus or a sequence of truncated E2 protein of bovine viral diarrhea virus in preparing an mRNA vaccine for preventing or treating bovine respiratory disease syndrome, wherein the amino acid sequence of E2 protein of bovine viral diarrhea virus is shown as SEQ ID NO. 9; the amino acid sequence of the truncated E2 protein of the bovine viral diarrhea virus is shown as SEQ ID NO. 10.

The invention provides an application of a recombinant plasmid or recombinant microorganism cell containing a DNA molecule for encoding the mRNA vaccine in preparing the mRNA vaccine for preventing or treating bovine respiratory disease syndrome.

The invention provides an application of lipid nano particles containing the mRNA vaccine in preparing the mRNA vaccine for preventing or treating bovine respiratory disease syndrome.

Further defined, the virus that causes the bovine respiratory disease syndrome is bovine viral diarrhea virus.

The invention provides an application of the mRNA vaccine in preparing an antibody of bovine viral diarrhea virus.

The beneficial effects are that:

1. on vaccine design

The research is based on the gene sequence of the E2 protein of the BVDV epidemic subtype 1a gene subtype strain in China, and improves the translation efficiency through codon optimization. The method comprises the steps of adding a signal peptide in front of an RNA sequence for encoding E2 protein to improve the secretion amount of the protein, adding a T7 promoter, a 5'UTR and a cap 1 structure at the front end of the signal peptide sequence, adding elements such as a 3' UTR and a poly A tail (poly A) at the rear end of the RNA sequence for encoding the E2 protein to improve the translation efficiency and stability of an mRNA vaccine, and constructing an E2 target antigen expressed candidate mRNA vaccine. And constructing a candidate mRNA vaccine with high secretion level by expressing the truncated E2 protein.

The expression and extracellular secretion expression of the constructed candidate mRNA vaccine in HEK-293T cells are verified by in vitro cell transfection experiments and Western Blot experiments, and the dimer form of E2 protein is verified, and BVDV tE2 candidate mRNA vaccine expressing truncated E2 protein expresses more secreted E2 monomeric protein and E2 dimer protein.

2. Immune effect

mRNA was prepared by in vitro transcription from plasmids that were confirmed to be expressed. And (3) preparing BVDV E2 mRNA vaccine by mass analysis such as lipid nanoparticle packaging and electron microscope detection. Immunization of mice and calves with the constructed candidate mRNA induces the production of high-level IgG antibodies and neutralizing antibodies against BVDV, and is cross-protective against BVDV-1 subtype strains of various genes. Safety experiments show that the prepared mRNA candidate vaccine has safety. The virus-attacking experiment of the cattle shows that the BVDV E2 mRNA vaccine has good protection effect on the domestic epidemic gene subtype BVDV-1b JL strain, and shows that the mRNA vaccine has cross protection effect on the heterologous epidemic strain in situ.

Drawings

FIG. 1 is a plasmid map of an antigen expression vector;

FIG. 2 is a Western Blot detection result in an antigen expression vector plasmid expression validation experiment;

FIG. 3 is a graph showing agarose gel electrophoresis results of in vitro transcribed mRNA;

FIG. 4 shows the results of Western Blot detection in an in vitro transcribed mRNA protein expression validation experiment;

FIG. 5 is a graph showing the results of electron microscopy analysis after LNP-mRNA packaging; FIG. 5 (A) is after packaging LNP-mRNA of E2 protein of bovine viral diarrhea virus, and FIG. 5 (B) is after packaging LNP-mRNA of truncated E2 protein of bovine viral diarrhea virus;

FIG. 6 is a graph of E2-specific IgG antibody versus time for BVDV mRNA vaccine versus inactivated vaccine immunized mice;

FIG. 7 is a graph of NADL-specific neutralizing antibody versus time for BVDV mRNA vaccine versus inactivated vaccine immunized mice;

FIG. 8 shows the results of E2-specific IgG antibodies and NADL-specific neutralizing antibodies two weeks after immunization of mice 2 with BVDV mRNA vaccine and inactivated vaccine;

FIG. 9 is the results of a cross-neutralization assay of serum from BVDV mRNA vaccine immunized mice;

FIG. 10 shows the NADL-specific neutralizing antibody results of BVDV mRNA vaccine-immunized cattle;

fig. 11 is the NADL specific neutralizing antibody results of BVDV mRNA vaccine and BVDV inactivated vaccine immunized cattle.

Detailed Description

Example 1 construction of antigen expression vector of bovine viral diarrhea Virus mRNA vaccine

1. The construction method of the antigen expression vector plasmid is as follows:

(1) The following gene sequences were synthesized by the golden srey biosystems: t7 promoter, 5' UTR (SEQ ID NO. 1), kozak sequence, tPA signal peptide sequence (SEQ ID NO. 4) and E2 sequence (SEQ ID NO. 6) are respectively arranged from 5' to 3' end, constructed on a pCAGGS vector, and the synthetic plasmid is named pCAGGS-1a-E2;

(2) Using New England Biolabs CoXhoI andKpni restriction enzyme is subjected to double enzyme digestion, and the bag is recovered by agarose gel electrophoresisThe DNA fragment containing E2 gene and pCAGGS vector fragment.

( 3) The primers were synthesized in the Harbin Rui Boxing family company (F: ATATAAGAGCCACCGCTAGCCTCGAGGCCACCATGGACGCCATGAA; R-tE2: GAGGCTCCAGCCTATTATCAGATATCGGTACCTCAAAAGTAGTCCCGGTGG; )

A truncated E2 protein (tE 2) gene sequence (SEQ ID NO. 7) was obtained by performing a PCR amplification reaction using KOD enzyme from TOYOBO company with the recovered DNA fragment containing the E2 gene as a template.

The EGFP gene sequence (SEQ ID NO. 5) was obtained by PCR amplification with KOD enzyme from TOYOBO company using EGFP plasmid stored in the laboratory as a template, as a primer (F: ATATAAGAGCCACCGCTAGCCTCGAGGCCACCATGGACGCCATGAA; R-EGFP: GAGGCTCCAGCCTATTATCAGATATCGGTACCCTACTTGTACAGCTCGTC) synthesized by the Harbin Rui Boxing family company.

(4) The PCR product was subjected to 1% agarose gel electrophoresis, and the PCR fragment DNA was recovered using the Axygen gel recovery kit.

(5) Carrying out homologous recombination by Takara company infusion enzyme, carrying out homologous recombination reaction on the pCAGGS carrier fragment recovered in (2) and the truncated tE2 DNA fragment recovered in (4) and the EGFP DNA fragment respectively, reacting at 50 ℃ for 15min, taking 2.5uL of the product, adding the product into competent DH5 alpha competent cells of the plant family of the Fabricius company, carrying out ice bath for 30min, carrying out heat shock at 42 ℃ for 90s, carrying out ice bath for 2min, adding 1mL of LB culture solution, shaking at 37 ℃ for 30min at 200rpm, centrifuging at 2000rpm for 10min, re-suspending the thalli by 100uL of LB, coating the thalli on a solid agar culture plate with the resistance to the Carna, and picking colonies the next day.

(6) 2mL of plasmids in bacterial solutions were extracted by a plasmid DNA miniprep kit, respectively, and the plasmids were named pCAGGS-1a-tE2 (pCAGGs vector with tE2 DNA fragment attached thereto); pCAGGS-EGFP (pCAGGs vector linked to EGFP). Using New England Biolabs CoXhoI andKpni restriction enzyme is subjected to double enzyme digestion identification, and recombinant plasmid sequence is identified by sequencing through Harborborui Boxing family companyThe columns are correct. The recombinant plasmids pCAGGS-EGFP, pCAGGS-1a-E2 and pCAGGS-1a-tE2 are shown in FIG. 1.

Example 2 mRNA transcription verification test

The plasmids obtained in example 1 (pCAGGS-EGFP; pCAGGS-1a-E2; pCAGGS-1a-tE 2) were each prepared by using New England Biolabs companyBsaI restriction endonuclease was cut to linearize the plasmid. Next, purification of linearized DNA was performed, 1/10 volume of 3M sodium acetate (pH=5.2) and 3 volume of absolute ethanol were added, the system was placed at-20℃for 1 hour, centrifuged at 15000rpm for 30 minutes at 4℃by the centrifuge, the supernatant was discarded, and the DNA was resuspended in DEPC water to determine the DNA concentration.

In vitro transcription was performed using the mMESSAGE mMACHINE ™ T7 Transcription Kit product of Thermofisher company. The reaction was carried out in a PCR apparatus at 37℃for 2 hours. Adding TURBO DNase 1uL into the kit, mixing, and reacting at 37deg.C for 15min in a PCR instrument. Purification was performed using Qiagen's RNA purification kit, RNA on the adsorption column was eluted using DEPC water, the concentration of RNA was measured by a Nanodrop instrument, and RNA was stored at-80 ℃.

1% agarose gel was prepared and the quality of in vitro transcribed RNA was verified by agarose gel electrophoresis. The results are shown in FIG. 3, where the bands of EGFP mRNA, 1a-E2 mRNA and 1a-tE2 mRNA transcribed in vitro are clearly single. The EGFP mRNA sequence transcribed in vitro is shown as SEQ ID NO.20, the 1a-E2 mRNA sequence is shown as SEQ ID NO.18, and the 1a-tE2 mRNA sequence is shown as SEQ ID NO. 19.

The DNA sequence of the 5 'untranslated region (5' UTR) is shown in SEQ ID NO. 1: GAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCGCTAGCCTCGAG.

The DNA sequence of the 3 'untranslated region (3' UTR) is shown as GATATCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTG in SEQ ID NO. 2.

The poly A has a sequence shown in SEQ ID NO.3 and contains 104 bases A: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA.

DNA sequence of T7 promoter: TAATACGACTCACTATAGG;

the DNA sequence of the signal peptide tPA sequence is shown in SEQ ID NO. 4: ATGGACGCCATGAAGAGGGGGCTGTGCTGCGTGCTGCTGCTGTGCGGAGCCGTGTTCGTGAGCGCCTCC.

EGFP nucleotide coding gene sequence is shown in SEQ ID NO. 5: ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAG.

The E2 antigen DNA sequence after codon optimization is shown in SEQ ID NO. 6: ATGCACCTGGACTGTAAGCCTGAGTTCAGCTACGCCATCGCCAAAGACGAGCGGATCGGCCAGCTGGGAGCTGAAGGCCTCACCACCACATGGAAGGAATACAGCCCCGGAATGAAACTGGAAGATACCATGGTCATCGCCTGGTGCGAGGATGGCAAGCTTATGTACCTGCAGCGGTGCACCAGAGAAACAAGATACCTGGCCATCCTGCACACCCGGGCTCTGCCTACCAGCGTGGTTTTCAAGAAGCTGTTCGATGGAAGAAAGCAGGAGGACGTGGTGGAAATGAACGACAATTTTGAGTTCGGCCTGTGCCCTTGTGACGCCAAGCCAATAGTGCGGGGCAAGTTCAACACCACGCTGCTGAACGGCCCCGCCTTCCAGATGGTGTGCCCTATCGGCTGGACCGGCACCGTGTCTTGCACCAGCTTCAACATGGATACACTGGCAACCACAGTGGTTAGAACCTACCGGAGAAGCAAGCCCTTCCCTCACAGACAGGGCTGCATCACACAGAAGAACCTGGGCGAGGACCTGCACAACTGCATCCTGGGCGGCAATTGGACCTGTGTGCCTGGCGACCAGCTGCTGTATAAGGGCGGCAGCATCGAGAGCTGCAAGTGGTGCGGCTACCAATTTAAAGAAAGCGAAGGCCTGCCCCACTACCCCATTGGAAAATGCAAGCTGGAAAACGAGACAGGCTACAGACTGGTGGACAGCACCAGCTGTAATAGAGAAGGTGTGGCCATCGTGCCACAGGGCACCCTGAAGTGCAAGATCGGCAAAACAACAGTGCAGGTGATCGCCATGGACACAAAGCTGGGCCCAATGCCTTGCAGACCCTACGAGATCATCAGCAGCGAGGGCCCTGTGGAAAAGACAGCCTGCACTTTTAACTACACCAAAACCCTGAAGAATAAGTACTTCGAGCCTAGAGACTCCTACTTCCAGCAGTACATGCTGAAGGGCGAGTATCAGTACTGGTTTGACCTGGAAGTGACAGACCACCACCGGGACTACTTTGCCGAGAGCATCCTGGTGGTGGTGGTCGCCCTGCTCGGAGGCAGATACGTGCTGTGGCTGCTCGTGACCTACATGGTGCTCAGCGAGCAGAAAGCCCTGGGC.

The truncated E2 protein tE2 antigen DNA sequence after codon optimization is shown in SEQ ID NO. 7: ATGCACCTGGACTGTAAGCCTGAGTTCAGCTACGCCATCGCCAAAGACGAGCGGATCGGCCAGCTGGGAGCTGAAGGCCTCACCACCACATGGAAGGAATACAGCCCCGGAATGAAACTGGAAGATACCATGGTCATCGCCTGGTGCGAGGATGGCAAGCTTATGTACCTGCAGCGGTGCACCAGAGAAACAAGATACCTGGCCATCCTGCACACCCGGGCTCTGCCTACCAGCGTGGTTTTCAAGAAGCTGTTCGATGGAAGAAAGCAGGAGGACGTGGTGGAAATGAACGACAATTTTGAGTTCGGCCTGTGCCCTTGTGACGCCAAGCCAATAGTGCGGGGCAAGTTCAACACCACGCTGCTGAACGGCCCCGCCTTCCAGATGGTGTGCCCTATCGGCTGGACCGGCACCGTGTCTTGCACCAGCTTCAACATGGATACACTGGCAACCACAGTGGTTAGAACCTACCGGAGAAGCAAGCCCTTCCCTCACAGACAGGGCTGCATCACACAGAAGAACCTGGGCGAGGACCTGCACAACTGCATCCTGGGCGGCAATTGGACCTGTGTGCCTGGCGACCAGCTGCTGTATAAGGGCGGCAGCATCGAGAGCTGCAAGTGGTGCGGCTACCAATTTAAAGAAAGCGAAGGCCTGCCCCACTACCCCATTGGAAAATGCAAGCTGGAAAACGAGACAGGCTACAGACTGGTGGACAGCACCAGCTGTAATAGAGAAGGTGTGGCCATCGTGCCACAGGGCACCCTGAAGTGCAAGATCGGCAAAACAACAGTGCAGGTGATCGCCATGGACACAAAGCTGGGCCCAATGCCTTGCAGACCCTACGAGATCATCAGCAGCGAGGGCCCTGTGGAAAAGACAGCCTGCACTTTTAACTACACCAAAACCCTGAAGAATAAGTACTTCGAGCCTAGAGACTCCTACTTCCAGCAGTACATGCTGAAGGGCGAGTATCAGTACTGGTTTGACCTGGAAGTGACAGACCACCACCGGGACTACTTT.

The amino acid sequence of the E2 antigen after codon optimization is shown as SEQ ID NO. 9:

MHLDCKPEFSYAIAKDERIGQLGAEGLTTTWKEYSPGMKLEDTMVIAWCEDGKLMYLQRCTRETRYLAILHTRALPTSVVFKKLFDGRKQEDVVEMNDNFEFGLCPCDAKPIVRGKFNTTLLNGPAFQMVCPIGWTGTVSCTSFNMDTLATTVVRTYRRSKPFPHRQGCITQKNLGEDLHNCILGGNWTCVPGDQLLYKGGSIESCKWCGYQFKESEGLPHYPIGKCKLENETGYRLVDSTSCNREGVAIVPQGTLKCKIGKTTVQVIAMDTKLGPMPCRPYEIISSEGPVEKTACTFNYTKTLKNKYFEPRDSYFQQYMLKGEYQYWFDLEVTDHHRDYFAESILVVVVALLGGRYVLWLLVTYMVLSEQKALG。

the amino acid sequence of the truncated E2 protein tE2 antigen after codon optimization is shown in SEQ ID NO. 10:

MHLDCKPEFSYAIAKDERIGQLGAEGLTTTWKEYSPGMKLEDTMVIAWCEDGKLMYLQRCTRETRYLAILHTRALPTSVVFKKLFDGRKQEDVVEMNDNFEFGLCPCDAKPIVRGKFNTTLLNGPAFQMVCPIGWTGTVSCTSFNMDTLATTVVRTYRRSKPFPHRQGCITQKNLGEDLHNCILGGNWTCVPGDQLLYKGGSIESCKWCGYQFKESEGLPHYPIGKCKLENETGYRLVDSTSCNREGVAIVPQGTLKCKIGKTTVQVIAMDTKLGPMPCRPYEIISSEGPVEKTACTFNYTKTLKNKYFEPRDSYFQQYMLKGEYQYWFDLEVTDHHRDYF。

the RNA sequence of the 5 'untranslated region (5' UTR) is shown as SEQ ID NO.11

GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAG。

The RNA sequence of the 3 '-terminal untranslated region (3' UTR) is shown in SEQ ID NO. 12: GAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUG.

The RNA sequence of poly A is shown in SEQ ID NO. 13:

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。

the RNA sequence of tPA signal peptide is shown in SEQ ID NO. 14:

AUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCC。

the sequence of the E2 antigen RNA after codon optimization is shown as SEQ ID NO. 15:

AUGCACCUGGACUGUAAGCCUGAGUUCAGCUACGCCAUCGCCAAAGACGAGCGGAUCGGCCAGCUGGGAGCUGAAGGCCUCACCACCACAUGGAAGGAAUACAGCCCCGGAAUGAAACUGGAAGAUACCAUGGUCAUCGCCUGGUGCGAGGAUGGCAAGCUUAUGUACCUGCAGCGGUGCACCAGAGAAACAAGAUACCUGGCCAUCCUGCACACCCGGGCUCUGCCUACCAGCGUGGUUUUCAAGAAGCUGUUCGAUGGAAGAAAGCAGGAGGACGUGGUGGAAAUGAACGACAAUUUUGAGUUCGGCCUGUGCCCUUGUGACGCCAAGCCAAUAGUGCGGGGCAAGUUCAACACCACGCUGCUGAACGGCCCCGCCUUCCAGAUGGUGUGCCCUAUCGGCUGGACCGGCACCGUGUCUUGCACCAGCUUCAACAUGGAUACACUGGCAACCACAGUGGUUAGAACCUACCGGAGAAGCAAGCCCUUCCCUCACAGACAGGGCUGCAUCACACAGAAGAACCUGGGCGAGGACCUGCACAACUGCAUCCUGGGCGGCAAUUGGACCUGUGUGCCUGGCGACCAGCUGCUGUAUAAGGGCGGCAGCAUCGAGAGCUGCAAGUGGUGCGGCUACCAAUUUAAAGAAAGCGAAGGCCUGCCCCACUACCCCAUUGGAAAAUGCAAGCUGGAAAACGAGACAGGCUACAGACUGGUGGACAGCACCAGCUGUAAUAGAGAAGGUGUGGCCAUCGUGCCACAGGGCACCCUGAAGUGCAAGAUCGGCAAAACAACAGUGCAGGUGAUCGCCAUGGACACAAAGCUGGGCCCAAUGCCUUGCAGACCCUACGAGAUCAUCAGCAGCGAGGGCCCUGUGGAAAAGACAGCCUGCACUUUUAACUACACCAAAACCCUGAAGAAUAAGUACUUCGAGCCUAGAGACUCCUACUUCCAGCAGUACAUGCUGAAGGGCGAGUAUCAGUACUGGUUUGACCUGGAAGUGACAGACCACCACCGGGACUACUUUGCCGAGAGCAUCCUGGUGGUGGUGGUCGCCCUGCUCGGAGGCAGAUACGUGCUGUGGCUGCUCGUGACCUACAUGGUGCUCAGCGAGCAGAAAGCCCUGGGC。

the truncated E2 protein tE2 antigen RNA sequence is shown in SEQ ID NO. 16:

AUGCACCUGGACUGUAAGCCUGAGUUCAGCUACGCCAUCGCCAAAGACGAGCGGAUCGGCCAGCUGGGAGCUGAAGGCCUCACCACCACAUGGAAGGAAUACAGCCCCGGAAUGAAACUGGAAGAUACCAUGGUCAUCGCCUGGUGCGAGGAUGGCAAGCUUAUGUACCUGCAGCGGUGCACCAGAGAAACAAGAUACCUGGCCAUCCUGCACACCCGGGCUCUGCCUACCAGCGUGGUUUUCAAGAAGCUGUUCGAUGGAAGAAAGCAGGAGGACGUGGUGGAAAUGAACGACAAUUUUGAGUUCGGCCUGUGCCCUUGUGACGCCAAGCCAAUAGUGCGGGGCAAGUUCAACACCACGCUGCUGAACGGCCCCGCCUUCCAGAUGGUGUGCCCUAUCGGCUGGACCGGCACCGUGUCUUGCACCAGCUUCAACAUGGAUACACUGGCAACCACAGUGGUUAGAACCUACCGGAGAAGCAAGCCCUUCCCUCACAGACAGGGCUGCAUCACACAGAAGAACCUGGGCGAGGACCUGCACAACUGCAUCCUGGGCGGCAAUUGGACCUGUGUGCCUGGCGACCAGCUGCUGUAUAAGGGCGGCAGCAUCGAGAGCUGCAAGUGGUGCGGCUACCAAUUUAAAGAAAGCGAAGGCCUGCCCCACUACCCCAUUGGAAAAUGCAAGCUGGAAAACGAGACAGGCUACAGACUGGUGGACAGCACCAGCUGUAAUAGAGAAGGUGUGGCCAUCGUGCCACAGGGCACCCUGAAGUGCAAGAUCGGCAAAACAACAGUGCAGGUGAUCGCCAUGGACACAAAGCUGGGCCCAAUGCCUUGCAGACCCUACGAGAUCAUCAGCAGCGAGGGCCCUGUGGAAAAGACAGCCUGCACUUUUAACUACACCAAAACCCUGAAGAAUAAGUACUUCGAGCCUAGAGACUCCUACUUCCAGCAGUACAUGCUGAAGGGCGAGUAUCAGUACUGGUUUGACCUGGAAGUGACAGACCACCACCGGGACUACUUU。

EGFP protein RNA sequence is shown in SEQ ID NO. 17:

AUGGUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGUGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAG。

the sequence of the 1a-E2 mRNA molecule encoding the E2 antigen in the mRNA vaccine is shown in SEQ ID NO. 18:

5’cap-GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGCACCUGGACUGUAAGCCUGAGUUCAGCUACGCCAUCGCCAAAGACGAGCGGAUCGGCCAGCUGGGAGCUGAAGGCCUCACCACCACAUGGAAGGAAUACAGCCCCGGAAUGAAACUGGAAGAUACCAUGGUCAUCGCCUGGUGCGAGGAUGGCAAGCUUAUGUACCUGCAGCGGUGCACCAGAGAAACAAGAUACCUGGCCAUCCUGCACACCCGGGCUCUGCCUACCAGCGUGGUUUUCAAGAAGCUGUUCGAUGGAAGAAAGCAGGAGGACGUGGUGGAAAUGAACGACAAUUUUGAGUUCGGCCUGUGCCCUUGUGACGCCAAGCCAAUAGUGCGGGGCAAGUUCAACACCACGCUGCUGAACGGCCCCGCCUUCCAGAUGGUGUGCCCUAUCGGCUGGACCGGCACCGUGUCUUGCACCAGCUUCAACAUGGAUACACUGGCAACCACAGUGGUUAGAACCUACCGGAGAAGCAAGCCCUUCCCUCACAGACAGGGCUGCAUCACACAGAAGAACCUGGGCGAGGACCUGCACAACUGCAUCCUGGGCGGCAAUUGGACCUGUGUGCCUGGCGACCAGCUGCUGUAUAAGGGCGGCAGCAUCGAGAGCUGCAAGUGGUGCGGCUACCAAUUUAAAGAAAGCGAAGGCCUGCCCCACUACCCCAUUGGAAAAUGCAAGCUGGAAAACGAGACAGGCUACAGACUGGUGGACAGCACCAGCUGUAAUAGAGAAGGUGUGGCCAUCGUGCCACAGGGCACCCUGAAGUGCAAGAUCGGCAAAACAACAGUGCAGGUGAUCGCCAUGGACACAAAGCUGGGCCCAAUGCCUUGCAGACCCUACGAGAUCAUCAGCAGCGAGGGCCCUGUGGAAAAGACAGCCUGCACUUUUAACUACACCAAAACCCUGAAGAAUAAGUACUUCGAGCCUAGAGACUCCUACUUCCAGCAGUACAUGCUGAAGGGCGAGUAUCAGUACUGGUUUGACCUGGAAGUGACAGACCACCACCGGGACUACUUUGCCGAGAGCAUCCUGGUGGUGGUGGUCGCCCUGCUCGGAGGCAGAUACGUGCUGUGGCUGCUCGUGACCUACAUGGUGCUCAGCGAGCAGAAAGCCCUGGGCUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。

the sequence of the 1a-tE2 mRNA molecule of the truncated E2 protein in the mRNA vaccine is shown in SEQ ID NO. 19:

5’cap-GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGCACCUGGACUGUAAGCCUGAGUUCAGCUACGCCAUCGCCAAAGACGAGCGGAUCGGCCAGCUGGGAGCUGAAGGCCUCACCACCACAUGGAAGGAAUACAGCCCCGGAAUGAAACUGGAAGAUACCAUGGUCAUCGCCUGGUGCGAGGAUGGCAAGCUUAUGUACCUGCAGCGGUGCACCAGAGAAACAAGAUACCUGGCCAUCCUGCACACCCGGGCUCUGCCUACCAGCGUGGUUUUCAAGAAGCUGUUCGAUGGAAGAAAGCAGGAGGACGUGGUGGAAAUGAACGACAAUUUUGAGUUCGGCCUGUGCCCUUGUGACGCCAAGCCAAUAGUGCGGGGCAAGUUCAACACCACGCUGCUGAACGGCCCCGCCUUCCAGAUGGUGUGCCCUAUCGGCUGGACCGGCACCGUGUCUUGCACCAGCUUCAACAUGGAUACACUGGCAACCACAGUGGUUAGAACCUACCGGAGAAGCAAGCCCUUCCCUCACAGACAGGGCUGCAUCACACAGAAGAACCUGGGCGAGGACCUGCACAACUGCAUCCUGGGCGGCAAUUGGACCUGUGUGCCUGGCGACCAGCUGCUGUAUAAGGGCGGCAGCAUCGAGAGCUGCAAGUGGUGCGGCUACCAAUUUAAAGAAAGCGAAGGCCUGCCCCACUACCCCAUUGGAAAAUGCAAGCUGGAAAACGAGACAGGCUACAGACUGGUGGACAGCACCAGCUGUAAUAGAGAAGGUGUGGCCAUCGUGCCACAGGGCACCCUGAAGUGCAAGAUCGGCAAAACAACAGUGCAGGUGAUCGCCAUGGACACAAAGCUGGGCCCAAUGCCUUGCAGACCCUACGAGAUCAUCAGCAGCGAGGGCCCUGUGGAAAAGACAGCCUGCACUUUUAACUACACCAAAACCCUGAAGAAUAAGUACUUCGAGCCUAGAGACUCCUACUUCCAGCAGUACAUGCUGAAGGGCGAGUAUCAGUACUGGUUUGACCUGGAAGUGACAGACCACCACCGGGACUACUUUUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。

the RNA sequence of the in vitro transcribed complete EGFP mRNA is shown as SEQ ID NO. 20:

5’cap-GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGGUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGUGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAGGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。

example 3E 2 protein expression validation test

(1) The purified in vitro transcribed RNA was subjected to verification expression by transfecting HEK-293T cells with Mirus TransIT ™ -mRNA Transfection Kits reagent. 293T cells were plated uniformly in six well plates and RNA transfected using the Mirus company TransIT ™ -mRNA Transfection Kits kit with 2. Mu.g mRNA per well. After 48 h is transfected, collecting cell supernatant, concentrating the supernatant by using 10kDa concentration tube of Millipore company, respectively adding 5×loading buffer containing DDT and 5×loading buffer without DTT for sample treatment, and obtaining Western Blot result by reducing SDS-PAGE and non-reducing SDS-PAGE after electrophoresis by using anti-His tag antibody of protein company;

(2) As a result, as shown in FIG. 4, protein bands corresponding to the expected sizes appeared in the test groups transfected with 1a-E2 mRNA and 1a-tE2 mRNA, as compared with the vector control group pCAGGs-tPA-EGFP mRNA. And the protein content in the supernatant of the 1a-tE2 mRNA test group is far higher than that of the pCAGGs-tPA-E2 mRNA test group, which shows that the tE2a target antigen sequence expression protein carrying tPA signal peptide has high-efficiency secretion characteristics.

EXAMPLE 4 preparation of mRNA vaccine

By passing throughBsaI restriction endonuclease the plasmid pCAGGS-1a-E2 successfully constructed in example 1; the pCAGGS-1a-tE2 was subjected to single cleavage to linearize the circular DNA. The DNA molecule was purified by 3M sodium acetate (ph=5.2), absolute ethanol, 0.5M EDTA: adding in1/10 volume of 3M sodium acetate (PH=5.2), 3 volume of absolute ethanol, 1/20 volume of 0.5M EDTA, mixing, placing at-20 ℃, centrifuging at 15000rpm to obtain precipitated DNA molecules. The next purification was performed using proteinase K to remove contaminating proteins. Linearized DNA molecules were extracted by phenol/chloroform method and the DNA was dissolved in nuclease free water for in vitro transcription.

The linearized DNA molecule was transcribed in vitro by the product T7 High Yield RNA Transcription Kit (N1-Me-pseudoUTP) from Norpran, which gave rise to a large number of mRNAs with N1-Me-pseudoUTP modifications aimed at reducing the natural immune response induced by the mRNA vaccine. The in vitro transcribed mRNA was then subjected to Capping modifications, as per the instructions of the Cap 1 Capping System from offshore Co. Extracting in vitro transcribed mRNA molecules containing cap structures by a phenol-chloroform method, and packaging the in vitro transcribed mRNA molecules to prepare the LNP-mRNA vaccine.

The specific experimental steps are as follows: first, the preparation of the alcohol phase, the lipid was prepared as a cationic lipid (SM 102): distearoyl phosphatidylcholine (DSPC): cholesterol: DMG-2000 (available from saino bond company) =50:10:38.5:1.5 (mass ratio) in absolute ethanol; next, an aqueous phase was prepared, and a citric acid buffer (50 mM) at ph4.0 was used as a solute for dissolving mRNA; packaging of mRNA was performed by microfluidic apparatus with an alcoholic phase to aqueous phase (volume ratio 1:3). Then diluted with RNase-free PBS buffer, concentrated and changed using a 30 kDa ultrafiltration tube. Adding PBS solution with equal volume of sucrose concentration of 20%, adjusting mRNA concentration to 60 μg/ml, sucrose concentration to 10%, and filtering with 0.22 μm filter membrane to obtain 1a-E2 and 1a-tE2LNP-mRNA vaccine, packaging, and storing at-20deg.C.

The plastid nanoparticle comprises at least one of cationic lipid, distearoyl phosphatidylcholine (DSPC), cholesterol, and DMG-2000.

The prepared 1a-E2 and 1a-tE2LNP-mRNA vaccine particles are verified to be uniform and consistent through an electron microscope detection result (figure 5).

Example 5 mouse immune evaluation experiment

The 1a-E2 mRNA vaccine prepared in example 4 was used to evaluate the immunization effect of BALB/c mice immunized with 1a-tE2 mRNA vaccine. 20 mice purchased from Liaoning long-life biotechnology Co., ltd at 8 weeks of age SPF BALB/c were randomly divided into 44 groups, 5 mice/group, and 20ug (100 uL) 1a-E2 mRNA vaccine, 20ug (100 uL) 1a-tE2 mRNA vaccine, 60uL BVDV (1 a-NM01 strain) inactivated vaccine, respectively; 100uL PBS. After 3 weeks of immunization, booster immunization was performed with the same dose volume of vaccine as the first immunization, after which mice were collected blood every two weeks and serum was isolated for antibody detection.

(1) Detection of anti-E2 IgG antibodies and neutralizing antibodies

The immune serum was assayed for specific anti-E2 antibody titers by an indirect ELISA method based on E2 protein as coating antigen. Coating ELISA plate with purified E2 protein (33 ng/hole), diluting the serum separated at different time points after boosting, adding into corresponding ELISA plate, sequentially adding HRP-anti-mouse IgG secondary antibody, TMB, and after color development is terminated A ₄₅₀ OD values were measured and antibody titers were determined. The results are shown in fig. 6, which shows that the mice with the highest serum-specific anti-E2 antibody titers two weeks after the mRNA vaccine was subjected to secondary immunization, the mRNA vaccine induced high-level specific anti-E2 IgG antibodies compared with the BVDV inactivated vaccine, and effectively induced humoral immune responses in the body (fig. 6, table 1).

Neutralizing antibody titers in immune sera were analyzed by neutralization assay. Equal volumes of NADL-mcherry report virus and equal volumes of mouse serum at different dilutions were neutralized at 37℃for 1h, then added to MDBK cells, and after infection for 2h, replaced with DMEM medium containing 2% horse serum. After 3 days the NADL-mcherry infection range and cell number were analyzed by a zeiss high throughput living cell analyzer and IC50 was calculated, and neutralizing antibody titer was defined as the serum dilution at which 50% of NADL-mcherry infection was neutralized. The results are shown in FIG. 7 and Table 2, where the titers of anti-NADL nAb in mice two weeks after the mRNA vaccine was double-immunized were highest, and the mRNA vaccine induced high levels of specific anti-NADL nAb compared to the BVDV inactivated vaccine.

Further comparing antibodies two weeks after peak immunity boost (fig. 8), BVDV mRNA vaccines induced significantly elevated E2-specific IgG antibodies, NADL-specific neutralizing antibodies compared to BVDV inactivated vaccines.

TABLE 1 E2-specific IgG antibody titres induced by BVDV E2 mRNA vaccine and inactivated vaccine immunization mice

TABLE 2 neutralizing antibody titre specific for NADL induced by BVDV E2 mRNA vaccine and inactivated vaccine immunized mice

(2) Cross-protective detection

Cross-neutralizing antibody titers in serum of immunized BVDV E2 mRNA vaccine mice were analyzed by neutralization assays with other BVDV strains of different genotypes. Equal volumes of different gene subtype BVDV strains VEDEVAC, 3877 and the like and equal volumes of mouse serum with final dilution of 200 are respectively added into MDBK cells after neutralization for 1h at 37 ℃, and replaced by DMEM culture solution containing 2% horse serum after infection for 2h. BVDV infected cells were observed by indirect immunofluorescence experiments using BVDV polyclonal antiserum from VMRD and 488-labeled anti-coat IgG antibody from invitrogen after 3 days, BVDV infection range and cell number were analyzed by Zeiss high throughput living cell analyzer, and inhibition ratios of BVDV E2 mRNA vaccine immunized mouse serum at 200 dilution factors to BVDV strains of different genotypes were calculated. The results are shown in FIG. 9, where mRNA vaccine immunized mice serum has up to 90% inhibition of BVDV-1 strains of various different genotypes and cross-protection of BVDV strains of different genotypes, which induces cross-neutralizing antibodies in mice.

(3) Detection of cellular immune response

Antigen-specific T cells were tested by cytokine staining (ICCS) experiments to assess cellular immunity, and murine lymphocytes were inoculated into culture plates after vaccine immunization, stimulated with the E2 protein peptide pool. Cells were incubated with anti-CD 28 antibody at 37℃and 5% CO ₂ Co-stimulation under conditions. DMSO was used as negative control. PMA/ionomycin was used as positive control. Subsequently, experiments were performed using the immobilization/permeabilization kit, using the following cytokine antibodies: anti-IFN-gamma, anti-TNF-alpha, anti-IL-2 and anti-IL-4 interact with spleen cells and finally cytokine specific lymphocyte numbers are analyzed by flow cytometry.

Results several weeks after boost vaccination, experimental results indicate that BVDV E2 mRNA vaccine vaccines induce antigen-specific, multifunctional CD8 expressing IFN-gamma, IL-2 and TNF-a ⁺ T cells; BVDV E2 mRNA vaccine induced higher CD4 than BVDV inactivated vaccine ⁺ And CD8 ⁺ Memory T cells.

EXAMPLE 6 safety test of mRNA vaccine

Immunization was performed on 2 month old calves. 10 calves, 5 calves each, one group of muscles inoculated with high dose mRNA vaccine, 600 μg/head; another group was injected with PBS as a control. The same dose and route was followed for the second inoculation 3 weeks after the first inoculation. And (5) observing the body health condition of the calves. All cattle in the PBS group and the mRNA vaccine group calf have good health status and normal body temperature, which indicates that the vaccine has good safety.

EXAMPLE 7 mRNA vaccine immunization cattle experiments

8 healthy susceptible cows of 3-4 months of age are used, 4 cows in each group. One group of neck intramuscular injection 1a-E2 mRNA vaccine 200ug, the other group of neck intramuscular injection 10 ⁷ TCID ₅₀ BVDV inactivated virus vaccine. BVDV inactivated virus vaccines are prepared in the laboratory: BVDV virus was cultured in MDBK cells, after four days the cells were harvested and freeze-thawed 3 times at-80℃and BVDV was inactivated by sigma company Binary Ethyleneimine (BEI), the virus and MONTANIDE were inactivated as BVDV ^TM The volume ratio of the ISA 15A VG adjuvant is 85 percent to 15 percent, and the BVDV inactivated vaccine is prepared. The second immunization is carried out after 21 days, and the inoculation dosage is the same as that of the first immunization. BVDV inactivated virus vaccine the immunized bovine group is respectively immunized for the third time and the fourth time 4 weeks after the last immunization, and the inoculation dose is the same as that of the first immunization.

1a-E2 mRNA immunogroups were subjected to blood collection and serum separation respectively 3 weeks before immunization, 3 weeks after the first immunization, and 1 week after the booster immunization. BVDV inactivated vaccine immunization groups were blood collected and serum isolated 1 week after the fourth immunization.

Neutralizing antibody titers in immunized bovine serum were analyzed by neutralization assay. Equal volumes of reporter virus NADL-mcherry and equal volumes of different dilutions of bovine serum were neutralized at 37℃for 1h, added to MDBK cells, and after infection for 2h, replaced with DMEM medium containing 2% horse serum. After 3 days the NADL-mcherry infection range and cell number were analyzed by a zeiss high throughput living cell analyzer and IC50 was calculated, and neutralizing antibody titer was defined as the serum dilution at which 50% of NADL-mcherry infection was neutralized. The results are shown in FIG. 10, where mRNA vaccines induced high levels of anti-NADL nAb titers following booster immunization. The mRNA vaccine induced about 2-fold higher levels of anti-NADL nAb compared to BVDV inactivated vaccine (fig. 11, table 3).

TABLE 3 neutralizing antibody titre specific for NADL induced by BVDV E2 mRNA vaccine and BVDV inactivated vaccine immunization of cattle

Example 8 mRNA vaccine challenge experiments

4 healthy susceptible cows of 3-4 months of age are used, 2 cows in each group. One group was injected with 200ug of 1a-E2 mRNA vaccine intramuscularly in the neck and the other group was injected with PBS as a control. The second immunization is carried out after 21 days, and the inoculation dosage is the same as that of the first immunization. Each cow is inoculated with 1b-JL strain (virus content is not less than 10) by intranasal spraying 21 days after the second immunization ^6.5 FAID ₅₀ /ml) 6ml. Before the toxin is attacked, observing that the cow has no clinical symptoms such as cough, fever and the like, judging that the cow is only BVDV negative through PCR detection, and measuring the rectum temperature of the cow before the toxin is attacked as a cow basic body temperature value; bovine blood was taken before challenge and the number of leukocytes was examined as the basal number of leukocytes in bovine subjects. After the virus is challenged, the body temperature of the cattle is measured every day, blood is collected every other day, the white blood cell number of the blood is measured, and BVDV virus separation is carried out.

The experiment result of the virus attack of the cattle shows that the control cattle injected with PBS has fever after the virus attack, and the cattle immunized with the 1a-E2 mRNA vaccine only has normal body temperature; the white blood cell count results showed that the white blood cell count of the PBS control group was significantly higher than that of the control group, with the 1a-E2 mRNA vaccinated group, due to the decrease in white blood cell count after challenge.

Experimental results show that BVDV E2 mRNA vaccine has good protection effect on domestic epidemic gene subtype BVDV-1b JL strains, and shows that the mRNA vaccine has cross protection effect on heterologous local epidemic strains.

Example 9 method for preparing antibodies by mRNA vaccine

And (3) preparing the monoclonal antibody from the prepared 1a-E2 mRNA vaccine and the BALB/c mice immunized with the 1a-tE2 mRNA vaccine. 10 8 week old SPF BALB/c mice purchased from Liaoning long biotechnology Co., ltd were randomly divided into 2 groups, 5 mice/group, and 20ug1a-E2 mRNA vaccine and 20ug 1a-tE2 mRNA vaccine were immunized respectively. After 3 weeks of immunization, booster immunization was performed with the same dose volume of vaccine as the first immunization, followed by blood collection from mice and determination of serum antibody titers. The immunized mouse spleen cells were fused with SP2/0 cells. Positive cell clones are screened through ELISA method, indirect immunofluorescence and the like to obtain hybridoma cell strains which stably secrete specific monoclonal antibodies, and the prepared monoclonal antibodies can be used for establishing a diagnosis method for detecting BVDV pathogens and antibodies.

Claims

1. An mRNA vaccine of bovine viral diarrhea virus (Bovine viral diarrhea Virus), comprising the following components: a 5 'untranslated region, a kozak sequence, a signal peptide sequence, a coding RNA sequence of a truncated E2 protein of bovine viral diarrhea virus, a 3' untranslated region and polyadenylation; the amino acid sequence of the truncated E2 protein of the bovine viral diarrhea virus is shown as SEQ ID NO. 10; the coding RNA sequence of the truncated E2 protein of the bovine viral diarrhea virus is shown as SEQ ID NO. 16; the nucleotide sequence of the signal peptide is shown as SEQ ID NO. 4.

2. The mRNA vaccine of claim 1, wherein the 5' untranslated region has the nucleotide sequence set forth in SEQ ID No. 1; the nucleotide sequence of the 3' -end untranslated region is shown as SEQ ID NO. 2; the nucleotide sequence of the poly A is shown as SEQ ID NO. 3.

3. The application of RNA for encoding truncated E2 protein of bovine viral diarrhea virus in preparing mRNA vaccine for preventing or treating bovine respiratory disease syndrome caused by bovine viral diarrhea virus is characterized in that the amino acid sequence of truncated E2 protein of bovine viral diarrhea virus is shown as SEQ ID NO. 10; the sequence of the RNA for encoding the truncated E2 protein of the bovine viral diarrhea virus is shown as SEQ ID NO. 16.

4. Use of a recombinant plasmid or recombinant microbial cell comprising a DNA molecule encoding the mRNA vaccine of claim 1 for the preparation of an mRNA vaccine for preventing or treating bovine respiratory disease syndrome caused by bovine viral diarrhea virus.

5. Use of lipid nanoparticles comprising the mRNA vaccine of claim 1 for the preparation of an mRNA vaccine for preventing or treating bovine respiratory disease syndrome caused by bovine viral diarrhea virus.

6. Use of the mRNA vaccine of claim 1 for the preparation of antibodies to bovine viral diarrhea virus.