CN117427154A - Sequence combination for preparing mRNA vaccine for porcine reproductive and respiratory syndrome and application thereof - Google Patents
Sequence combination for preparing mRNA vaccine for porcine reproductive and respiratory syndrome and application thereof Download PDFInfo
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Abstract
The invention discloses a sequence combination for preparing an mRNA vaccine for porcine reproductive and respiratory syndrome and application thereof. Belongs to the technical field of vaccines. The invention aims to improve the effect that the porcine reproductive and respiratory syndrome virus vaccine stimulates the organism to generate stronger humoral immune response. The invention provides a recombinant protein of porcine reproductive and respiratory syndrome virus, which is obtained by mixing recombinant protein 1 and recombinant protein 2 according to a mass ratio of 1:1; the recombinant protein 1 is obtained by fusing GP2 protein and GP4 protein by using a Linker; the recombinant protein 2 is obtained by fusing GP3 protein and Nsp9 protein. The developed PRRSV multi-antigen fusion mRNA vaccine has a certain protection effect on PRRSV, and lays a foundation for developing a safe and efficient ideal PRRSV vaccine.
Description
Technical Field
The invention belongs to the technical field of vaccines, and particularly relates to a sequence combination for preparing an mRNA vaccine for porcine reproductive and respiratory syndrome and application thereof.
Background
Porcine reproductive and respiratory syndrome virus (Porcine Reproductive and Respiratory Syndrome Virus, PRRSV) is one of the most important pathogens affecting pigs worldwide, which causes sow reproductive disorders and respiratory diseases in pigs of all ages, and which brings about a significant economic loss to the global pig industry. At present, PRRSV vaccines in the market mainly comprise live vaccines and inactivated vaccines, the inactivated vaccines have poor immune effects, and attenuated vaccines have the risk of recombination with epidemic strains although the attenuated vaccines have good immune effects. Thus, there is a need to develop novel vaccines that are safe and efficient against PRRSV. The ability to eliminate PRRSV in a humoral immune manner by only a single antigen is limited, so that the ability of PRRSV vaccine to stimulate the body to generate humoral immunity is particularly important.
Disclosure of Invention
The invention aims to improve the effect that the porcine reproductive and respiratory syndrome virus vaccine stimulates the organism to generate stronger humoral immune response.
The invention provides a sequence combination for preparing an mRNA vaccine for porcine reproductive and respiratory syndrome, which is obtained by mixing lipid nanoparticles of mRNA encoding recombinant protein 1 and lipid nanoparticles of mRNA encoding recombinant protein 2 according to a mass ratio of 1:1; the recombinant protein 1 is obtained by fusing GP2 protein and GP4 protein by using a Linker; the recombinant protein 2 is obtained by fusing GP3 protein and Nsp9 protein.
Further defined, the Linker has an amino acid sequence of (GGGGS) 3.
Further defined, the N-terminus of the recombinant protein is linked to a tPA secretory peptide.
Further defined, the C-terminal of the recombinant protein is added with a 3 xflag tag.
Further defined, the amino acid sequence of the tPA secretory peptide is MDAMKRGLCCVLLLCGAVFVSAR.
Further defined, the amino acid sequence of the recombinant protein 1 is shown in SEQ ID NO. 12.
Further defined, the amino acid sequence of the recombinant protein 2 is shown in SEQ ID NO. 14.
Further defined, the method of preparing the lipid nanoparticle of mRNA is as follows:
step 1: plasmid linearization: carrying out single enzyme digestion on the plasmid to form a linearization plasmid;
step 2: in vitro transcription and capping: mixing linearized plasmid, nucleic acid material, co-transcribed capping analogue, HEPES, DTT, T RNA polymerase, rnase inhibitor and spermidine material, incubating for 2-6h at 37 ℃;
step 3: and (3) liposome packaging: the lipid nanoparticle of mRNA is prepared by mixing the mixture of the ionizable cationic liposome, neutral phospholipid, sterol lipid and PEGylated phospholipid with the aqueous solution of mRNA through a microfluidic mixing device.
Further defined, the sequence of mRNA encoding recombinant protein 1 is shown as SEQ ID NO. 18; the sequence of mRNA encoding recombinant protein 2 is shown in SEQ ID NO. 19.
The invention provides an application of the sequence combination of the mRNA vaccine in preparing a medicament for treating or preventing porcine reproductive and respiratory syndrome virus.
The beneficial effects are that: the research develops an mRNA vaccine aiming at PRRSV, recombining a plurality of antigen proteins including GP2-GP4 and GP3-Nsp9, optimizing mRNA-LNP, detecting the in vitro expression level of the mRNA-LNP of different optimization schemes through Western blot, and the result shows that the GP2-GP4 and the GP3-Nsp9 can be effectively expressed. The mixed immunization group of GP2-GP4 and GP3-NSp9 can effectively reduce the titer of N protein antibodies in pigs and the viral load in blood and lungs. The result shows that the PRRSV multi-antigen fusion mRNA vaccine developed by the invention has a certain protection effect on PRRSV, and lays a foundation for developing a safe and efficient ideal PRRSV vaccine.
Drawings
FIG. 1 is a schematic diagram of PRRSV HuN4 mRNA design;
FIG. 2 is a graph of mRNA-LNP Western blot results; (A): western blot results of cell samples; (B): western blot results of supernatant samples; m: protein molecular weight standard; G2G4 (C/G/T): GP2-GP4 (LNP different optimization schemes); G3N9 (C/G/T): GP3-Nsp9 (LNP different optimization schemes); G5M (C/G/T): GP5-M (LNP different optimization protocol); +: transfecting an LNP control; -: cell blank control;
FIG. 3 is a graph showing the results of detecting the body temperature and the N protein antibody level after the PRRSV mRNA-LNP immune pig is detoxified; (a) a PRRSV mRNA-LNP immune pig post-challenge body temperature profile; (B) N protein antibody level changes after PRRSV mRNA-LNP immune pigs are detoxified;
FIG. 4 is a graph showing the results of detecting the copy number of virus replication after virus challenge in PRRSV mRNA-LNP immunized pigs; (a) viral load in serum; (B) viral load in the lung;
FIG. 5 is a diagram showing the appearance of lung after challenge in an mRNA-LNP immunized pig.
Detailed Description
Experimental materials: the highly pathogenic porcine reproductive and respiratory syndrome live vaccine (HuN-F112 strain) (veterinary drug raw word 080011074) is a product of Harbin Vicat biotechnology Co., ltd;
PRRSV HuN4 strain is described in literature pottery, wang Gang, liu Yonggang, et al, research of the thymus injury of piglets by different generations of PRRSV HuN4 strain [ J ]. Chinese Protect veterinarian journal, 2014, 36 (5): 4.DOI: 10.3969/j.issn.1008-0589.2014.05.02.
PCR high fidelity enzyme KOD FX Neo was purchased from Toyobo Biotechnology Co., ltd; DMEM basic is purchased from Thermo Fisher Scientific; australian fetal bovine serum FBS was purchased from alpha bio technologies Co., ltd; anti-Flag mouse monoclonal antibodies were purchased from merck biosystems, germany; dyight 800, goat Anti-Mouse IgG, available from KPL Biotechnology; RIPA high-efficiency lysate is purchased from Beijing Soy Bao technology Co., ltd; primeScript ™ II 1st Strand cDNA Synthesis Kit, one Step TB Green PrimeScript ™ RT-PCR Kit (Perfect Real Time) were purchased from Takara doctor materials technology (Beijing); viral RNA extraction kits were purchased from tenna biotechnology limited; PRRSV N protein ELISA detection kit was purchased from ideexx biotechnology limited.
Experimental animals: PRRSV negative pigs at 4 weeks of age were purchased from the dormitory new farm in harbine.
EXAMPLE 1 construction of recombinant proteins of porcine reproductive and respiratory syndrome Virus
Referring to PRRSV HuN4 genome sequence (GenBank: EF 635006) on NCBI, TMHMM is utilized to predict GP2, GP3, GP4 and GP5 signal peptides and transmembrane regions, GP2, GP3, GP4 and GP5 signal peptides and transmembrane regions are removed, netNGlyc predicts GP5 glycosylation site, GP5 glycosylation site is modified, and the decoy epitope of GP5 is replaced with a second neutralization epitope, the glycosylation site of the second neutralization epitope is mutated, thereby making it easier to generate neutralizing antibodies, and M and Nsp9 sequences both use full-length sequences, (-)X: a signal peptide;X: a transmembrane region;X: glycosylation site) sequences are as follows:
amino acid sequence before GP2 protein modification: (SEQ ID NO. 1)
MKWGLCKASLTKLANFLWMLSRNFWCPLLISSYFWPFCLASQSPVGWWSYASDWFAPRYSVRALPFTLSNYRRSYEAFLSQCQVDIPTWGVKHPLGVLWHHKVSTLIDEMVSRRMYRIMEKAGQAAWKQVVSEATLSRISGLDVVAHFQHLAAIEAETCKYLASRLPMLHNLRLTGSNVTIVYNSTLDQVFAIFPTPGSRPKLHDFQQWLIAVHSSIFSS VAASCTLFVVLWLRIPMLRSVFGFRWLGATFLLNSW；
Amino acid sequence after GP2 protein modification: (SEQ ID NO. 2)
PFCLASQSPVGWWSYASDWFAPRYSVRALPFTLSNYRRSYEAFLSQCQVDIPTWGVKHPLGVLWHHKVSTLIDEMVSRRMYRIMEKAGQAAWKQVVSEATLSRISGLDVVAHFQHLAAIEAETCKYLASRLPMLHNLRLTGSNVTIVYNSTLDQVFAIFPTPGSRPKLHDFQQRIPMLRSVFGFRWLGATFLLNSW；
Amino acid sequence before GP3 protein modification: (SEQ ID NO. 3)
MANSCTFLHIFLRCSFLYSFCCAVVANSNATFCFWFPLVRGNFSFELMVNYTVCPLCPTRQAAAEILEPGKSLWCRIGHDRCSENDHDELGFMVPPGLSSEGHLTSVYAWLAFLSFSYTAQFHPEIFGIGNVSQVYVDIKHQFICAVHDGDNATLPRHDNISAVFQTYYQHQVDGGNWFHLEWLRPFFSSWLVLNVSWFLRRSPANHVSVRVFRTSKPTP PQHQTSLSSRTSAALGMATRPLRRFAKVLSAARR；
Amino acid sequence after GP3 protein modification: (SEQ ID NO. 4)
TFCFWFPLVRGNFSFELMVNYTVCPLCPTRQAAAEILEPGKSLWCRIGHDRCSENDHDELGFMVPPGLSSEGHLTSVYAWLAFLSFSYTAQFHPEIFGIGNVSQVYVDIKHQFICAVHDGDNATLPRHDNISAVFQTYYQHQVDGGN；
Amino acid sequence before GP4 protein modification: (SEQ ID NO. 5)
MAASFLFLLVGFKCFVVSQAFACKPCFSSSLSDIKTNTTAASDFVVLQDISCLRHGDSSSPTIRKSSQCRTAIGTPVYITITANVTDENYLHSSDLLMLSSCLFYASEMSEKGFKVVFGNVSGIVAVCVNFTSYVQHVKEFTQRSLVVDHVRLLHFMTPETMRWATVLACLFAILLAI；
Amino acid sequence after GP4 protein modification: (SEQ ID NO. 6)
CKPCFSSSLSDIKTNTTAASDFVVLQDISCLRHGDSSSPTIRKSSQCRTAIGTPVYITITANVTDENYLHSSDLLMLSSCLFYASEMSEKGFKVVFGNVSGIVAVCVNFTSYVQHVKEFTQRSLVVDHVRLLHFMTPE；
Amino acid sequence before GP5 protein modification: (SEQ ID NO. 7)
MLGKCLTACCCSRLLFLWCIVPFYLAVLVNASNNNSSHIQLIYNLTLCELNGTDWLAQKFDWAVETFV IFPVLTHIVSYGALTTSHFLDTVGLATVSTAGYYHGRYVLSSIYAVCALAALICFVIRLAKNCMSWRYSCTRYTNFLLDTKGRLYRWRSPVIVEKGGKVEVKGHLIDLKRVVLDGSAATPLTRVSAEQWGRL；
Amino acid sequence after GP5 protein modification: (SEQ ID NO. 8)
SHLQLIYTLAVLVTAKYSSSSHIQLIYTLTLCELRGTDWLAQKFDWAVEAKNCMSWRYSCTRYTNFLLDTKGRLYRWRSPVIVEKGGKVEVEGHLIDLKRVVLDGSAATPLTRVSAEQWGRL；
M protein amino acid sequence: (SEQ ID NO. 9)
MGSSLDDFCNDSTAPQKVLLAFSITYTPVMIYALKVSRGRLLGLLHLLIFLNCAFTFGYMTFVHFESTNRVALTMGAVVALLWGVYSAIETWKFITSRCRLCLLGRKYILAPAHHVESAAGFHPIAANDNHAFVVRRPGSTTVNGTLVPGLKSLVLGGRKAVKQGVVNLVKYAK；
Nsp9 protein amino acid sequence: (SEQ ID NO. 10)
FKLLAASGLTRCGRGGLVVTETAVKIVKFHNRTFTLGPVNLKVASEVELKDAVEHNQYPVARPVDGGVVLLRSAVPSLIDVLISGADASPKLLARHGPGNTGIDGTLWDFEAEATKEEVALSAQIIQACDIRRGDAPEIGLPYKLYPVRGNPERVKGVLQNTRFGDIPYKTPSDTGSPVHAAACLTPNATPVTDGRSVLATTMPSGFELYVPTIPASVLDYLDSRPDCPKQLTEHGCEDAALRDLSKYDLSTQGFVLPGVLRLVRKYLFAHVGKCPPVHRPSTYPAKNSMAGINGNRFPTKDIQSVPEIDVLCAQAVRENWQTVTPCTLKKQYCGKKKTRTILGTNNFIALAHRAALSGVTQGFMKKAFNSPIALGKNKFKELQAPVLGRCLEADLASCDRSTPAIVRWFAANLLYELACAEEHLPSYVLNCCHDLLVTQSGAVTKRGGLSSGDPITSVSNTIYSLVIYAQHMVLSYFKSGHPHGLLFLQDQLKFEDMLKVQPLIVYSDDLVLYAESPSMPNYHWWVEHLNLMLGFQTDPKKTTITDSPSFLGCRIINGRQLVPNRDRILAALAYHMKASNVSEYYASAAAILMDSCACLEYDPEWFEELVVGIAQCARKDGYSFPGPPFFLSMWEKLRSNHE；
tPA secretory peptide amino acid sequence (SEQ ID No. 11): MDAMKRGLCCVLLLCGAVFVSPS;
fusion of GP2 and GP4 to obtain fusion peptides 1, GP3 and Nsp9 to obtain fusion peptides 2, GP5 and M to obtain fusion peptide 3, each of fusion peptide 1, fusion peptide 2 and fusion peptide 3 was linked by GS-linker for maintaining conformational integrity between proteins, and tPA secretory peptide was added to the N-terminus of fusion peptide 1, fusion peptide 2 and fusion peptide 3, 3 xflag tag was added to the C-terminus, the structure was as shown in fig. 1, and the amino acid sequence of GS-linker was (GGGGS) 3.
GP2-GP4 fusion protein: the connection sequence from the N end to the C end is tPA-GP2- (GGGGS) 3-GP4-3 xFlag, and the amino acid sequence is as follows: (SEQ ID NO. 12)
MDAMKRGLCCVLLLCGAVFVSPSPFCLASQSPVGWWSYASDWFAPRYSVRALPFTLSNYRRSYEAFLSQCQVDIPTWGVKHPLGVLWHHKVSTLIDEMVSRRMYRIMEKAGQAAWKQVVSEATLSRISGLDVVAHFQHLAAIEAETCKYLASRLPMLHNLRLTGSNVTIVYNSTLDQVFAIFPTPGSRPKLHDFQQRIPMLRSVFGFRWLGATFLLNSWGGGGSGGGGSGGGGSCKPCFSSSLSDIKTNTTAASDFVVLQDISCLRHGDSSSPTIRKSSQCRTAIGTPVYITITANVTDENYLHSSDLLMLSSCLFYASEMSEKGFKVVFGNVSGIVAVCVNFTSYVQHVKEFTQRSLVVDHVRLLHFMTPEDYKDHDGDYKDHDIDYKDDDDK；
GP2-GP4 fusion protein: the N-terminal to C-terminal connection sequence is tPA-GP2- (GGGGS) 3-GP4-3 xFlag, and the nucleotide sequence is as follows: (SEQ ID NO. 13)
ATGGACGCCATGAAGCGCGGCCTGTGCTGCGTGCTGCTGCTGTGCGGCGCCGTGTTCGTGAGCCCCAGCCCCTTCTGCCTGGCCAGCCAGAGCCCCGTGGGCTGGTGGAGCTACGCCAGCGACTGGTTCGCCCCCCGCTACAGCGTGCGCGCCCTGCCCTTCACCCTGAGCAACTACCGCCGCAGCTACGAGGCCTTCCTGAGCCAGTGCCAGGTGGACATCCCCACCTGGGGCGTGAAGCACCCCCTGGGCGTGCTGTGGCACCACAAGGTGAGCACCCTGATCGACGAGATGGTGAGCCGCCGCATGTACCGCATCATGGAGAAGGCCGGCCAGGCCGCCTGGAAGCAGGTGGTGAGCGAGGCCACCCTGAGCCGCATCAGCGGCCTGGACGTGGTGGCCCACTTCCAGCACCTGGCCGCCATCGAGGCCGAGACCTGCAAGTACCTGGCCAGCCGCCTGCCCATGCTGCACAACCTGCGCCTGACCGGCAGCAACGTGACCATCGTGTACAACAGCACCCTGGACCAGGTGTTCGCCATCTTCCCCACCCCCGGCAGCCGCCCCAAGCTGCACGACTTCCAGCAGCGCATCCCCATGCTGCGCAGCGTGTTCGGCTTCCGCTGGCTGGGCGCCACCTTCCTGCTGAACAGCTGGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCTGCAAGCCCTGCTTCAGCAGCAGCCTGAGCGACATCAAGACCAACACCACCGCCGCCAGCGACTTCGTGGTGCTGCAGGACATCAGCTGCCTGCGCCACGGCGACAGCAGCAGCCCCACCATCCGCAAGAGCAGCCAGTGCCGCACCGCCATCGGCACCCCCGTGTACATCACCATCACCGCCAACGTGACCGACGAGAACTACCTGCACAGCAGCGACCTGCTGATGCTGAGCAGCTGCCTGTTCTACGCCAGCGAGATGAGCGAGAAGGGCTTCAAGGTGGTGTTCGGCAACGTGAGCGGCATCGTGGCCGTGTGCGTGAACTTCACCAGCTACGTGCAGCACGTGAAGGAGTTCACCCAGCGCAGCCTGGTGGTGGACCACGTGCGCCTGCTGCACTTCATGACCCCCGAGGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAAG；
GP3-Nsp9 fusion protein: the connection sequence from N end to C end is tPA-GP3- (GGGGS) 3-Nsp9-3 xFlag
The amino acid sequence is (SEQ ID NO. 14):
MDAMKRGLCCVLLLCGAVFVSPSTFCFWFPLVRGNFSFELMVNYTVCPLCPTRQAAAEILEPGKSLWCRIGHDRCSENDHDELGFMVPPGLSSEGHLTSVYAWLAFLSFSYTAQFHPEIFGIGNVSQVYVDIKHQFICAVHDGDNATLPRHDNISAVFQTYYQHQVDGGNGGGGSGGGGSGGGGSFKLLAASGLTRCGRGGLVVTETAVKIVKFHNRTFTLGPVNLKVASEVELKDAVEHNQYPVARPVDGGVVLLRSAVPSLIDVLISGADASPKLLARHGPGNTGIDGTLWDFEAEATKEEVALSAQIIQACDIRRGDAPEIGLPYKLYPVRGNPERVKGVLQNTRFGDIPYKTPSDTGSPVHAAACLTPNATPVTDGRSVLATTMPSGFELYVPTIPASVLDYLDSRPDCPKQLTEHGCEDAALRDLSKYDLSTQGFVLPGVLRLVRKYLFAHVGKCPPVHRPSTYPAKNSMAGINGNRFPTKDIQSVPEIDVLCAQAVRENWQTVTPCTLKKQYCGKKKTRTILGTNNFIALAHRAALSGVTQGFMKKAFNSPIALGKNKFKELQAPVLGRCLEADLASCDRSTPAIVRWFAANLLYELACAEEHLPSYVLNCCHDLLVTQSGAVTKRGGLSSGDPITSVSNTIYSLVIYAQHMVLSYFKSGHPHGLLFLQDQLKFEDMLKVQPLIVYSDDLVLYAESPSMPNYHWWVEHLNLMLGFQTDPKKTTITDSPSFLGCRIINGRQLVPNRDRILAALAYHMKASNVSEYYASAAAILMDSCACLEYDPEWFEELVVGIAQCARKDGYSFPGPPFFLSMWEKLRSNHEDYKDHDGDYKDHDIDYKDDDDK；
GP3-Nsp9 fusion protein: the connection sequence from N end to C end is tPA-GP3- (GGGGS) 3-Nsp9-3 xFlag
The nucleotide sequence is (SEQ ID NO. 15):
ATGGACGCCATGAAGCGCGGCCTGTGCTGCGTGCTGCTGCTGTGCGGCGCCGTGTTCGTGAGCCCCAGCACCTTCTGCTTCTGGTTCCCCCTGGTGCGCGGCAACTTCAGCTTCGAGCTGATGGTGAACTACACCGTGTGCCCCCTGTGCCCCACCCGCCAGGCCGCCGCCGAGATCCTGGAGCCCGGCAAGAGCCTGTGGTGCCGCATCGGCCACGACCGCTGCAGCGAGAACGACCACGACGAGCTGGGCTTCATGGTGCCCCCCGGCCTGAGCAGCGAGGGCCACCTGACCAGCGTGTACGCCTGGCTGGCCTTCCTGAGCTTCAGCTACACCGCCCAGTTCCACCCCGAGATCTTCGGCATCGGCAACGTGAGCCAGGTGTACGTGGACATCAAGCACCAGTTCATCTGCGCCGTGCACGACGGCGACAACGCCACCCTGCCCCGCCACGACAACATCAGCGCCGTGTTCCAGACCTACTACCAGCACCAGGTGGACGGCGGCAACGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCTTCAAGCTGCTGGCCGCCAGCGGCCTGACCCGCTGCGGCCGCGGCGGCCTGGTGGTGACCGAGACCGCCGTGAAGATCGTGAAGTTCCACAACCGCACCTTCACCCTGGGCCCCGTGAACCTGAAGGTGGCCAGCGAGGTGGAGCTGAAGGACGCCGTGGAGCACAACCAGTACCCCGTGGCCCGCCCCGTGGACGGCGGCGTGGTGCTGCTGCGCAGCGCCGTGCCCAGCCTGATCGACGTGCTGATCAGCGGCGCCGACGCCAGCCCCAAGCTGCTGGCCCGCCACGGCCCCGGCAACACCGGCATCGACGGCACCCTGTGGGACTTCGAGGCCGAGGCCACCAAGGAGGAGGTGGCCCTGAGCGCCCAGATCATCCAGGCCTGCGACATCCGCCGCGGCGACGCCCCCGAGATCGGCCTGCCCTACAAGCTGTACCCCGTGCGCGGCAACCCCGAGCGCGTGAAGGGCGTGCTGCAGAACACCCGCTTCGGCGACATCCCCTACAAGACCCCCAGCGACACCGGCAGCCCCGTGCACGCCGCCGCCTGCCTGACCCCCAACGCCACCCCCGTGACCGACGGCCGCAGCGTGCTGGCCACCACCATGCCCAGCGGCTTCGAGCTGTACGTGCCCACCATCCCCGCCAGCGTGCTGGACTACCTGGACAGCCGCCCCGACTGCCCCAAGCAGCTGACCGAGCACGGCTGCGAGGACGCCGCCCTGCGCGACCTGAGCAAGTACGACCTGAGCACCCAGGGCTTCGTGCTGCCCGGCGTGCTGCGCCTGGTGCGCAAGTACCTGTTCGCCCACGTGGGCAAGTGCCCCCCCGTGCACCGCCCCAGCACCTACCCCGCCAAGAACAGCATGGCCGGCATCAACGGCAACCGCTTCCCCACCAAGGACATCCAGAGCGTGCCCGAGATCGACGTGCTGTGCGCCCAGGCCGTGCGCGAGAACTGGCAGACCGTGACCCCCTGCACCCTGAAGAAGCAGTACTGCGGCAAGAAGAAGACCCGCACCATCCTGGGCACCAACAACTTCATCGCCCTGGCCCACCGCGCCGCCCTGAGCGGCGTGACCCAGGGCTTCATGAAGAAGGCCTTCAACAGCCCCATCGCCCTGGGCAAGAACAAGTTCAAGGAGCTGCAGGCCCCCGTGCTGGGCCGCTGCCTGGAGGCCGACCTGGCCAGCTGCGACCGCAGCACCCCCGCCATCGTGCGCTGGTTCGCCGCCAACCTGCTGTACGAGCTGGCCTGCGCCGAGGAGCACCTGCCCAGCTACGTGCTGAACTGCTGCCACGACCTGCTGGTGACCCAGAGCGGCGCCGTGACCAAGCGCGGCGGCCTGAGCAGCGGCGACCCCATCACCAGCGTGAGCAACACCATCTACAGCCTGGTGATCTACGCCCAGCACATGGTGCTGAGCTACTTCAAGAGCGGCCACCCCCACGGCCTGCTGTTCCTGCAGGACCAGCTGAAGTTCGAGGACATGCTGAAGGTGCAGCCCCTGATCGTGTACAGCGACGACCTGGTGCTGTACGCCGAGAGCCCCAGCATGCCCAACTACCACTGGTGGGTGGAGCACCTGAACCTGATGCTGGGCTTCCAGACCGACCCCAAGAAGACCACCATCACCGACAGCCCCAGCTTCCTGGGCTGCCGCATCATCAACGGCCGCCAGCTGGTGCCCAACCGCGACCGCATCCTGGCCGCCCTGGCCTACCACATGAAGGCCAGCAACGTGAGCGAGTACTACGCCAGCGCCGCCGCCATCCTGATGGACAGCTGCGCCTGCCTGGAGTACGACCCCGAGTGGTTCGAGGAGCTGGTGGTGGGCATCGCCCAGTGCGCCCGCAAGGACGGCTACAGCTTCCCCGGCCCCCCCTTCTTCCTGAGCATGTGGGAGAAGCTGCGCAGCAACCACGAGGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAAG；
GP5-M fusion protein: the connection sequence from N end to C end is tPA-GP5- (GGGGS) 3-M-3 xFlag
The amino acid sequence is (SEQ ID NO. 16):
MDAMKRGLCCVLLLCGAVFVSPSSHLQLIYTLAVLVTAKYSSSSHIQLIYTLTLCELRGTDWLAQKFDWAVEAKNCMSWRYSCTRYTNFLLDTKGRLYRWRSPVIVEKGGKVEVEGHLIDLKRVVLDGSAATPLTRVSAEQWGRLGGGGSGGGGSGGGGSMGSSLDDFCNDSTAPQKVLLAFSITYTPVMIYALKVSRGRLLGLLHLLIFLNCAFTFGYMTFVHFESTNRVALTMGAVVALLWGVYSAIETWKFITSRCRLCLLGRKYILAPAHHVESAAGFHPIAANDNHAFVVRRPGSTTVNGTLVPGLKSLVLGGRKAVKQGVVNLVKYAKDYKDHDGDYKDHDIDYKDDDDK；
GP5-M fusion protein: the N-terminal to C-terminal connection sequence is tPA-GP5- (GGGGS) 3-M-3 xFlag nucleotide sequence (SEQ ID NO. 17):
ATGGACGCCATGAAGCGCGGCCTGTGCTGCGTGCTGCTGCTGTGCGGCGCCGTGTTCGTGAGCCCCAGCAGCCACCTGCAGCTGATCTACACCCTGGCCGTGCTGGTGACCGCCAAGTACAGCAGCAGCAGCCACATCCAGCTGATCTACACCCTGACCCTGTGCGAGCTGCGCGGCACCGACTGGCTGGCCCAGAAGTTCGACTGGGCCGTGGAGGCCAAGAACTGCATGAGCTGGCGCTACAGCTGCACCCGCTACACCAACTTCCTGCTGGACACCAAGGGCCGCCTGTACCGCTGGCGCAGCCCCGTGATCGTGGAGAAGGGCGGCAAGGTGGAGGTGGAGGGCCACCTGATCGACCTGAAGCGCGTGGTGCTGGACGGCAGCGCCGCCACCCCCCTGACCCGCGTGAGCGCCGAGCAGTGGGGCCGCCTGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCATGGGCAGCAGCCTGGACGACTTCTGCAACGACAGCACCGCCCCCCAGAAGGTGCTGCTGGCCTTCAGCATCACCTACACCCCCGTGATGATCTACGCCCTGAAGGTGAGCCGCGGCCGCCTGCTGGGCCTGCTGCACCTGCTGATCTTCCTGAACTGCGCCTTCACCTTCGGCTACATGACCTTCGTGCACTTCGAGAGCACCAACCGCGTGGCCCTGACCATGGGCGCCGTGGTGGCCCTGCTGTGGGGCGTGTACAGCGCCATCGAGACCTGGAAGTTCATCACCAGCCGCTGCCGCCTGTGCCTGCTGGGCCGCAAGTACATCCTGGCCCCCGCCCACCACGTGGAGAGCGCCGCCGGCTTCCACCCCATCGCCGCCAACGACAACCACGCCTTCGTGGTGCGCCGCCCCGGCAGCACCACCGTGAACGGCACCCTGGTGCCCGGCCTGAAGAGCCTGGTGCTGGGCGGCCGCAAGGCCGTGAAGCAGGGCGTGGTGAACCTGGTGAAGTACGCCAAGGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAAG。
EXAMPLE 2 mRNA sequence Synthesis and LNP encapsulation
Step 1: plasmid linearization: carrying out single enzyme digestion on the plasmid to form a linearization plasmid;
step 2: in vitro transcription and capping: linearized plasmid, nucleic acid starting material (ATP, GTP, CTP and UTP), co-transcribed capping analogue, HEPES, DTT, T RNA polymerase, RNase inhibitor, spermidine starting material were mixed and incubated for 2-6h at 37 ℃.
Step 3: and (3) liposome packaging: the ionizable cationic liposome, neutral phospholipid, sterol lipid, PEGylated phospholipid mixture and mRNA aqueous solution are mixed by a microfluidic mixing device to prepare mRNA-LNP particles (lipid nanoparticles of mRNA) with uniform size.
Codon optimization, mRNA sequence synthesis, and LNP (lipid nanoparticle of mRNA) encapsulation were done by the company.
The GP2-GP4 and GP3-NSp9 mixed immunization group is GP2-GP4 mRNA-LNP and GP3-NSp9 mRNA-LNP with equal mass ratio.
The GP3-NSp9 alone immunization group was GP3-NSp9 mRNA-LNP;
HuN4-F112 attenuated vaccine group is highly pathogenic live vaccine for porcine reproductive and respiratory syndrome (HuN-F112 strain);
the blank group was sterile DMEM.
GP2-GP4 mRNA sequence: (SEQ ID NO. 18)
AUGGACGCCAUGAAGCGCGGCCUGUGCUGCGUGCUGCUGCUGUGCGGCGCCGUGUUCGUGAGCCCCAGCCCCUUCUGCCUGGCCAGCCAGAGCCCCGUGGGCUGGUGGAGCUACGCCAGCGACUGGUUCGCCCCCCGCUACAGCGUGCGCGCCCUGCCCUUCACCCUGAGCAACUACCGCCGCAGCUACGAGGCCUUCCUGAGCCAGUGCCAGGUGGACAUCCCCACCUGGGGCGUGAAGCACCCCCUGGGCGUGCUGUGGCACCACAAGGUGAGCACCCUGAUCGACGAGAUGGUGAGCCGCCGCAUGUACCGCAUCAUGGAGAAGGCCGGCCAGGCCGCCUGGAAGCAGGUGGUGAGCGAGGCCACCCUGAGCCGCAUCAGCGGCCUGGACGUGGUGGCCCACUUCCAGCACCUGGCCGCCAUCGAGGCCGAGACCUGCAAGUACCUGGCCAGCCGCCUGCCCAUGCUGCACAACCUGCGCCUGACCGGCAGCAACGUGACCAUCGUGUACAACAGCACCCUGGACCAGGUGUUCGCCAUCUUCCCCACCCCCGGCAGCCGCCCCAAGCUGCACGACUUCCAGCAGCGCAUCCCCAUGCUGCGCAGCGUGUUCGGCUUCCGCUGGCUGGGCGCCACCUUCCUGCUGAACAGCUGGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCUGCAAGCCCUGCUUCAGCAGCAGCCUGAGCGACAUCAAGACCAACACCACCGCCGCCAGCGACUUCGUGGUGCUGCAGGACAUCAGCUGCCUGCGCCACGGCGACAGCAGCAGCCCCACCAUCCGCAAGAGCAGCCAGUGCCGCACCGCCAUCGGCACCCCCGUGUACAUCACCAUCACCGCCAACGUGACCGACGAGAACUACCUGCACAGCAGCGACCUGCUGAUGCUGAGCAGCUGCCUGUUCUACGCCAGCGAGAUGAGCGAGAAGGGCUUCAAGGUGGUGUUCGGCAACGUGAGCGGCAUCGUGGCCGUGUGCGUGAACUUCACCAGCUACGUGCAGCACGUGAAGGAGUUCACCCAGCGCAGCCUGGUGGUGGACCACGUGCGCCUGCUGCACUUCAUGACCCCCGAGGACUACAAGGACCACGACGGCGACUACAAGGACCACGACAUCGACUACAAGGACGACGACGACAAG；
GP3-NSp9 mRNA sequence: (SEQ ID NO. 19)
AUGGACGCCAUGAAGCGCGGCCUGUGCUGCGUGCUGCUGCUGUGCGGCGCCGUGUUCGUGAGCCCCAGCACCUUCUGCUUCUGGUUCCCCCUGGUGCGCGGCAACUUCAGCUUCGAGCUGAUGGUGAACUACACCGUGUGCCCCCUGUGCCCCACCCGCCAGGCCGCCGCCGAGAUCCUGGAGCCCGGCAAGAGCCUGUGGUGCCGCAUCGGCCACGACCGCUGCAGCGAGAACGACCACGACGAGCUGGGCUUCAUGGUGCCCCCCGGCCUGAGCAGCGAGGGCCACCUGACCAGCGUGUACGCCUGGCUGGCCUUCCUGAGCUUCAGCUACACCGCCCAGUUCCACCCCGAGAUCUUCGGCAUCGGCAACGUGAGCCAGGUGUACGUGGACAUCAAGCACCAGUUCAUCUGCGCCGUGCACGACGGCGACAACGCCACCCUGCCCCGCCACGACAACAUCAGCGCCGUGUUCCAGACCUACUACCAGCACCAGGUGGACGGCGGCAACGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCUUCAAGCUGCUGGCCGCCAGCGGCCUGACCCGCUGCGGCCGCGGCGGCCUGGUGGUGACCGAGACCGCCGUGAAGAUCGUGAAGUUCCACAACCGCACCUUCACCCUGGGCCCCGUGAACCUGAAGGUGGCCAGCGAGGUGGAGCUGAAGGACGCCGUGGAGCACAACCAGUACCCCGUGGCCCGCCCCGUGGACGGCGGCGUGGUGCUGCUGCGCAGCGCCGUGCCCAGCCUGAUCGACGUGCUGAUCAGCGGCGCCGACGCCAGCCCCAAGCUGCUGGCCCGCCACGGCCCCGGCAACACCGGCAUCGACGGCACCCUGUGGGACUUCGAGGCCGAGGCCACCAAGGAGGAGGUGGCCCUGAGCGCCCAGAUCAUCCAGGCCUGCGACAUCCGCCGCGGCGACGCCCCCGAGAUCGGCCUGCCCUACAAGCUGUACCCCGUGCGCGGCAACCCCGAGCGCGUGAAGGGCGUGCUGCAGAACACCCGCUUCGGCGACAUCCCCUACAAGACCCCCAGCGACACCGGCAGCCCCGUGCACGCCGCCGCCUGCCUGACCCCCAACGCCACCCCCGUGACCGACGGCCGCAGCGUGCUGGCCACCACCAUGCCCAGCGGCUUCGAGCUGUACGUGCCCACCAUCCCCGCCAGCGUGCUGGACUACCUGGACAGCCGCCCCGACUGCCCCAAGCAGCUGACCGAGCACGGCUGCGAGGACGCCGCCCUGCGCGACCUGAGCAAGUACGACCUGAGCACCCAGGGCUUCGUGCUGCCCGGCGUGCUGCGCCUGGUGCGCAAGUACCUGUUCGCCCACGUGGGCAAGUGCCCCCCCGUGCACCGCCCCAGCACCUACCCCGCCAAGAACAGCAUGGCCGGCAUCAACGGCAACCGCUUCCCCACCAAGGACAUCCAGAGCGUGCCCGAGAUCGACGUGCUGUGCGCCCAGGCCGUGCGCGAGAACUGGCAGACCGUGACCCCCUGCACCCUGAAGAAGCAGUACUGCGGCAAGAAGAAGACCCGCACCAUCCUGGGCACCAACAACUUCAUCGCCCUGGCCCACCGCGCCGCCCUGAGCGGCGUGACCCAGGGCUUCAUGAAGAAGGCCUUCAACAGCCCCAUCGCCCUGGGCAAGAACAAGUUCAAGGAGCUGCAGGCCCCCGUGCUGGGCCGCUGCCUGGAGGCCGACCUGGCCAGCUGCGACCGCAGCACCCCCGCCAUCGUGCGCUGGUUCGCCGCCAACCUGCUGUACGAGCUGGCCUGCGCCGAGGAGCACCUGCCCAGCUACGUGCUGAACUGCUGCCACGACCUGCUGGUGACCCAGAGCGGCGCCGUGACCAAGCGCGGCGGCCUGAGCAGCGGCGACCCCAUCACCAGCGUGAGCAACACCAUCUACAGCCUGGUGAUCUACGCCCAGCACAUGGUGCUGAGCUACUUCAAGAGCGGCCACCCCCACGGCCUGCUGUUCCUGCAGGACCAGCUGAAGUUCGAGGACAUGCUGAAGGUGCAGCCCCUGAUCGUGUACAGCGACGACCUGGUGCUGUACGCCGAGAGCCCCAGCAUGCCCAACUACCACUGGUGGGUGGAGCACCUGAACCUGAUGCUGGGCUUCCAGACCGACCCCAAGAAGACCACCAUCACCGACAGCCCCAGCUUCCUGGGCUGCCGCAUCAUCAACGGCCGCCAGCUGGUGCCCAACCGCGACCGCAUCCUGGCCGCCCUGGCCUACCACAUGAAGGCCAGCAACGUGAGCGAGUACUACGCCAGCGCCGCCGCCAUCCUGAUGGACAGCUGCGCCUGCCUGGAGUACGACCCCGAGUGGUUCGAGGAGCUGGUGGUGGGCAUCGCCCAGUGCGCCCGCAAGGACGGCUACAGCUUCCCCGGCCCCCCCUUCUUCCUGAGCAUGUGGGAGAAGCUGCGCAGCAACCACGAGGACUACAAGGACCACGACGGCGACUACAAGGACCACGACAUCGACUACAAGGACGACGACGACAAG；
GP5-M mRNA sequence: (SEQ ID NO. 20)
AUGGACGCCAUGAAGCGCGGCCUGUGCUGCGUGCUGCUGCUGUGCGGCGCCGUGUUCGUGAGCCCCAGCAGCCACCUGCAGCUGAUCUACACCCUGGCCGUGCUGGUGACCGCCAAGUACAGCAGCAGCAGCCACAUCCAGCUGAUCUACACCCUGACCCUGUGCGAGCUGCGCGGCACCGACUGGCUGGCCCAGAAGUUCGACUGGGCCGUGGAGGCCAAGAACUGCAUGAGCUGGCGCUACAGCUGCACCCGCUACACCAACUUCCUGCUGGACACCAAGGGCCGCCUGUACCGCUGGCGCAGCCCCGUGAUCGUGGAGAAGGGCGGCAAGGUGGAGGUGGAGGGCCACCUGAUCGACCUGAAGCGCGUGGUGCUGGACGGCAGCGCCGCCACCCCCCUGACCCGCGUGAGCGCCGAGCAGUGGGGCCGCCUGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCAUGGGCAGCAGCCUGGACGACUUCUGCAACGACAGCACCGCCCCCCAGAAGGUGCUGCUGGCCUUCAGCAUCACCUACACCCCCGUGAUGAUCUACGCCCUGAAGGUGAGCCGCGGCCGCCUGCUGGGCCUGCUGCACCUGCUGAUCUUCCUGAACUGCGCCUUCACCUUCGGCUACAUGACCUUCGUGCACUUCGAGAGCACCAACCGCGUGGCCCUGACCAUGGGCGCCGUGGUGGCCCUGCUGUGGGGCGUGUACAGCGCCAUCGAGACCUGGAAGUUCAUCACCAGCCGCUGCCGCCUGUGCCUGCUGGGCCGCAAGUACAUCCUGGCCCCCGCCCACCACGUGGAGAGCGCCGCCGGCUUCCACCCCAUCGCCGCCAACGACAACCACGCCUUCGUGGUGCGCCGCCCCGGCAGCACCACCGUGAACGGCACCCUGGUGCCCGGCCUGAAGAGCCUGGUGCUGGGCGGCCGCAAGGCCGUGAAGCAGGGCGUGGUGAACCUGGUGAAGUACGCCAAGGACUACAAGGACCACGACGGCGACUACAAGGACCACGACAUCGACUACAAGGACGACGACGACAAG；
mRNA-LNP transfection of HEK293T cells
HEK293T at 1X 10 6 6 well plates were plated per well and transfected when the cell fusion was 80%. 2. Mu.g of each of GP2-GP4 mRNA-LNP, GP3-Nsp9 mRNA-LNP and GP5-M mRNA-LNP was mixed with 200. Mu.L of DMEM and added to the cell wells, incubated at 37℃for 8 h, replaced with fresh DMEM containing 10% FBS, and incubated at 37℃for 24 h. Only LNP transfected (synthetic non-mRNA-encapsulated lipid nanoparticles) and non-transfected blank control were set.
Western blot detection of protein expression
The supernatant from each group of transfected cells obtained in step 1 was collected in 2.0 mL EP tube, the cells were washed once with PBS, 1mL of PBS was added to blow down the cells, the cells were collected in 1.5 mL EP tube, centrifuged at 5000 rpm for 5 minutes, the supernatant was discarded, and each well of cells was lysed on ice using 200. Mu.L of RIPA high efficiency lysate (containing 1% PMSF) for 20 min. The lysed cell samples were centrifuged at 12000 rpm for 10 min, and the supernatant was added to a 5 XLoading Buffer and boiled for 10 min. The transfection supernatant was also added with 5×loading Buffer and mixed well, and then boiled for 10 min to prepare SDS-PAGE samples. The above samples were subjected to SDS-PAGE, 80V for 30 min at 120V for 1 h, and after electrophoresis, PVDF membrane was transferred with albumin glue and blocked with 5% skim milk at room temperature for 1 h. Anti-Flag mouse monoclonal antibody is used as primary antibody (1:10000 dilution), and the primary antibody is incubated for 1 h at room temperature, and PBST is washed for 3 times, each time for 5 min. The Goat Anti-Mouse IgG is used as a secondary antibody (diluted 1:10000), incubated for 1 h at room temperature, PBST is washed 3 times, and scanned and imaged by an infrared fluorescence scanning imager.
Results: expression of mRNA-LNP in HEK293T cells
Three packaged mRNA-LNPs (GP 2-GP4 mRNA-LNP, GP3-Nsp9 mRNA-LNP and GP5-M mRNA-LNP) were transfected into HEK293T cells, and the cells and supernatants were collected 24h after transfection and Western blot (FIG. 2) and the results showed that the three mRNA-LNPs expressed protein only in the cells, with GP2-GP4 protein of approximately 55kDa (expected protein size 45 kDa) and GP3-Nsp9 protein of approximately 110kDa (expected protein size 94.7 kDa) and no band was detected by the GP5-M protein. One of the three optimization schemes for each protein with the highest expression level was selected for the next experiment (GP 2-GP4 mRNA-LNP and GP3-Nsp9 mRNA-LNP).
3. Immune animal experiment and efficacy evaluation
The group of 12 PRRSV antigen antibody negative pigs at 40 days of age was divided into 4 groups, 3 groups of 3 groups, respectively, GP2-GP4 and GP3-NSp9 mixed immunization group, GP3-NSp9 individual immunization group, huN F112 vaccine immunization control group and blank control group, GP2-GP4 and GP3-NSp9 mixed immunization group immunized 100. Mu.g/head pig on days 0 and 21 respectively, GP3-NSp9 individual immunization group immunized GP3-NSp9 mRNA-LNP 100. Mu.g/head pig on days 0 and 21 respectively, huN4-F112 attenuated vaccine group immunized 1 head/head pig according to the instruction, blank control group immunized DMEM 100. Mu.L/head pig on days 0 and 21 respectively. After 14 days from completion of immunization procedure, PRRSV HuN4 challenge was performed with a titer of 10 5.5 TCID 50 1mL each of the left and right nostrils of each pig was given 1mL intramuscular injection. Pig rectum temperature was measured daily after challenge for 21 consecutive days. Blood was collected on days 0, 7, 14, 21 after challenge, and PRRSV N protein antibodies in serum were detected using ELISA kits after serum separation. Meanwhile, viruses were extracted using a virus extraction Kit, RNA was reverse transcribed into cDNA using PrimeScript ™ II 1st Strand cDNA Synthesis Kit, the number of copies of viruses in serum was detected by quantitative PCR using One Step TB Green PrimeScript ™ RT-PCR Kit (Perfect Real Time), the pig lung 21 days after challenge was detected in the same manner and quantitative PCR was performed, and data was analyzed using GraphPad Prism software. Groups of surviving pigs were examined by dissection on day 21 after challenge to observe the appearance of the lungs.
Results: (1) Detecting body temperature and N protein antibody level after immune pig detoxication
After the immunization program is finished, the temperature of the rectum of the pig is detected at regular time every day, a temperature graph is drawn (figure 3A), and the result shows that the trend of the GP3-NSp9 single immunization group is consistent with that of the blank control group, the mixed immunization group of the GP2-GP4 and the GP3-NSp9 has reduced fever days compared with the GP3-NSp9 single immunization group, the body temperature of the HuN-F112 attenuated vaccine control group is the most stable, and the fever days are the shortest. The N protein antibody levels were detected on days 0, 7, 14, and 21 after challenge, and the results showed that the GP2-GP4 and GP3-Nsp9 mixed immunization group N protein antibody levels remained low at all times, demonstrating that GP2-GP4 and GP3-Nsp9 mixed immunization can effectively inhibit PRRSV replication (fig. 3B).
PRRSV N protein antibody levels
(2) Detection of viral copy number after immune pig challenge
The virus load in the serum and the lung is detected by an RT-qPCR method on days 0, 7, 14 and 21 after the immune pigs are challenged, and the result shows that the virus load in the serum of the mRNA-LNP mixed immune group is lowest on the 7 th day after the challenge, the virus load in the lung is about 1 Log value lower than that of the blank control group and is approximate to the copy number level of the HuN4-F112 attenuated vaccine control group (figure 4B) compared with the blank control group by nearly 4 Log values. The result shows that the mixed immunization of GP2-GP4 and GP3-NSp9 can effectively remove PRRSV and relieve viremia caused by PRRSV infection.
Viral load in serum
Pulmonary viral load
(3) Lung biopsy results
The lung lesions were observed after dissection of each group of pigs, and the results show that the lung congestion, the sarcoidosis and the lesions of the GP3-Nsp9 individual immune groups are lighter, and the lung of the HuN4-F112 attenuated immune group and the GP2-GP4 and GP3-Nsp9 mixed immune group are normal (figure 5).
The results prove that the immune effect of the GP2-GP4 and GP3-NSp9 mixed immune group is stronger than that of the GP3-NSp9 single immune group, and the mixed immune group has a certain resistance effect on PRRSV HuN4 infected pigs. The mixed mRNA vaccine of GP3-NSp9 and GP2-GP4 developed by the research has a certain immune protection effect on PRRSV HuN4, and lays a foundation for the development of novel safe and efficient vaccine of PRRSV.
Claims (10)
1. A sequence combination for preparing an mRNA vaccine for porcine reproductive and respiratory syndrome, wherein the sequence combination of the mRNA vaccine is obtained by mixing lipid nanoparticles of mRNA encoding recombinant protein 1 and lipid nanoparticles of mRNA encoding recombinant protein 2 according to a mass ratio of 1:1; the recombinant protein 1 is obtained by fusing GP2 protein and GP4 protein by using a Linker; the recombinant protein 2 is obtained by fusing GP3 protein and Nsp9 protein.
2. The sequence combination of mRNA vaccine according to claim 1, wherein the Linker has the amino acid sequence (GGGGS) 3 。
3. The sequence combination of an mRNA vaccine of claim 1, wherein the N-terminus of the recombinant protein is linked to a tPA secretory peptide.
4. The sequence combination of an mRNA vaccine of claim 3, wherein the amino acid sequence of the tPA secretory peptide is MDAMKRGLCCVLLLCGAVFVSAR.
5. The sequence combination of an mRNA vaccine according to claim 1, wherein the C-terminal of the recombinant protein is added with a 3 xflag tag.
6. The sequence combination of mRNA vaccine according to claim 1, wherein the amino acid sequence of the recombinant protein 1 is shown in SEQ ID No. 12.
7. The sequence combination of mRNA vaccine according to claim 1, wherein the amino acid sequence of recombinant protein 2 is shown in SEQ ID No. 14.
8. The sequence combination of mRNA vaccine according to claim 1, characterized in that the preparation method of lipid nanoparticles of mRNA is as follows:
step 1: plasmid linearization: carrying out single enzyme digestion on the plasmid to form a linearization plasmid;
step 2: in vitro transcription and capping: mixing linearized plasmid, nucleic acid material, co-transcribed capping analogue, HEPES, DTT, T RNA polymerase, rnase inhibitor and spermidine material, incubating for 2-6h at 37 ℃;
step 3: and (3) liposome packaging: the lipid nanoparticle of mRNA is prepared by mixing the mixture of the ionizable cationic liposome, neutral phospholipid, sterol lipid and PEGylated phospholipid with the aqueous solution of mRNA through a microfluidic mixing device.
9. The sequence combination of an mRNA vaccine according to claim 1, wherein the sequence of the mRNA encoding recombinant protein 1 is shown in SEQ ID No. 18; the sequence of mRNA encoding recombinant protein 2 is shown in SEQ ID NO. 19.
10. Use of a combination of sequences of an mRNA vaccine according to any one of claims 1 to 9 for the preparation of a medicament for the treatment or prevention of porcine reproductive and respiratory syndrome virus.
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| CN102321180A (en) * | 2011-08-11 | 2012-01-18 | 中国科学院微生物研究所 | Polypeptide fragment composition and application in preparation of porcine reproductive and respiratory syndrome vaccine thereof |
| US20160137699A1 (en) * | 2014-11-19 | 2016-05-19 | National Pingtung University Of Science And Technology | Recombinant fusion antigen gene, recombinant fusion antigen protein and subunit vaccine composition having the same against infection of porcine reproductive and respiratory syndrome virus |
| CN115025212A (en) * | 2022-06-20 | 2022-09-09 | 天康制药(苏州)有限公司 | mRNA vaccine for preventing porcine reproductive and respiratory syndrome and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102321180A (en) * | 2011-08-11 | 2012-01-18 | 中国科学院微生物研究所 | Polypeptide fragment composition and application in preparation of porcine reproductive and respiratory syndrome vaccine thereof |
| US20160137699A1 (en) * | 2014-11-19 | 2016-05-19 | National Pingtung University Of Science And Technology | Recombinant fusion antigen gene, recombinant fusion antigen protein and subunit vaccine composition having the same against infection of porcine reproductive and respiratory syndrome virus |
| CN115025212A (en) * | 2022-06-20 | 2022-09-09 | 天康制药(苏州)有限公司 | mRNA vaccine for preventing porcine reproductive and respiratory syndrome and preparation method thereof |
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| COMPOSITIONMARGA GARCÍA DURÁN ET AL.: "Generation of porcine reproductive and respiratory syndrome (PRRS)virus-like-particles (VLPs) with different protein composition", JOURNAL OF VIROLOGICAL METHODS, vol. 236, 18 July 2016 (2016-07-18), pages 77 - 86, XP029710168, DOI: 10.1016/j.jviromet.2016.03.021 * |
| 陈振海等: "表达PRRSV GP2a／GP2b、GP3、GP4、GP5和M 蛋白重组腺病毒的构建", 福建农业学报, vol. 22, no. 3, 31 December 2007 (2007-12-31), pages 221 - 226 * |
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