CN116410992A - mRNA and vaccine for preventing and/or treating novel coronavirus, and preparation method and application thereof - Google Patents

Abstract

The invention discloses an mRNA vaccine for preventing and/or treating novel coronaviruses, which comprises mRNA with a sequence shown in any one of SEQ ID NO.3 to SEQ ID NO. 7. The mRNA transfected cells can express high levels, have correct conformation and can be combined with the Spike protein of hACE2, mRNA vaccines prepared from the mRNA provide effective immunoprotection for novel coronavirus Omicron mutant strains, subtype mutant strains and delta strains, so that the mRNA vaccines can generate broad-spectrum effective immunoprotection for Omicron strains and delta strains of different subtypes, can provide immunoprotection for epidemic strains of novel coronaviruses with continuous mutation prevention and control, and have important clinical application value.

Description

mRNA and vaccine for preventing and/or treating novel coronavirus, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicine, and in particular relates to mRNA (messenger ribonucleic acid) and vaccine for preventing and/or treating novel coronavirus as well as a preparation method and application thereof.

Background

SARS-CoV-2 is a novel coronavirus, causing global pandemic of coronavirus disease in 2019 (COVID-19). SARS-CoV-2 is an enveloped positive-strand RNA virus. Spike (S) protein (Spike protein) is the major envelope protein of SARS-CoV-2, entering cells by recognizing ACE2 protein of the human host. Spike proteins consist of S1 and S2 subunits. Wherein the cell receptor binding domain (Receptor binding domain, RBD) of the Spike protein domain is located at the receptor ACE2 protein that S1 is responsible for recognizing cells. The Spike protein plays a role in binding and fusing the virus and host cell membrane receptors, and is an important action site of a host neutralizing antibody and a key target point of vaccine design. SARS-CoV-2 recognizes the ACE2 protein of a human host into cells through the virus surface Spike protein, and amino acid variation of Spike protein leads to changes in the species tropism and infection characteristics of the virus.

Armikovia (omacron) and its subtypes are currently the major mutants widely spread worldwide. Compared with other novel coronavirus mutants, the amikan contains more mutation sites, and mutation of the sites causes the novel mutants to have the characteristics of stronger binding force with ACE2, stronger transmission capability and immunity escape enhancement.

The SARS-CoV-2 vaccine can effectively reduce the severity of disease and slow down the transmission. The evolution rate of SARS-CoV-2 on immune response also presents challenges to drive the continuous renewal of vaccines and broad-spectrum immune protection.

mRNA vaccines are directed to the introduction of mRNA encoding a disease-specific antigen into the body, which utilizes the protein synthesis machinery of the host cell to produce the antigen, thereby triggering an immune response. The mRNA sequence of specific antigen can be constructed according to different diseases, the specific antigen is wrapped and transported into cells by the novel lipid nano carrier particles, then the human ribosome is utilized to translate the mRNA sequence to generate antigen protein of the diseases, and the immune system is activated to recognize and generate immune response, so that the effect of preventing the diseases is achieved. mRNA vaccines have advantages in hair-center response, tfh activation, neutralizing antibody production, specific memory B cells, long-life plasma cells (long-lived plasma cells), etc. At present, aiming at the characteristics of rapid mutation of SARS-CoV-2 and rapid evolution speed of immune response, there is a great need to develop a safe and effective novel anti-SARS-CoV-2 vaccine, in particular an mRNA vaccine with broad-spectrum protection effect.

Disclosure of Invention

The present invention aims to provide an mRNA vaccine which is capable of preventing and/or treating novel coronaviruses.

The technical scheme for achieving the aim comprises the following steps.

In a first aspect of the invention, there is provided an mRNA for translating a Spike protein, the coding region of said mRNA comprising the nucleotide sequence shown in any one of SEQ ID NO.3 to SEQ ID NO. 7.

In some of these embodiments, the coding region of the mRNA comprises the nucleotide sequence set forth in SEQ ID NO. 5.

In some of these embodiments, the mRNA further comprises a cap structure.

In a second aspect of the present invention, there is provided a coding sequence encoding a Spike protein, the coding sequence being any one of SEQ ID NO.8 to SEQ ID NO. 12.

In some of these embodiments, the coding sequence is the nucleotide sequence set forth in SEQ ID NO. 10.

In a third aspect of the present invention, there is provided a coding gene for expressing Spike protein, wherein the coding sequence of the coding gene is any one of the coding sequences described above.

In a fourth aspect of the invention, there is provided the use of an mRNA as described above for the preparation of a novel coronavirus vaccine for prophylaxis and/or treatment.

In some of these embodiments, the novel coronavirus is one or more of omacron and its subtype strains, delta strains.

In a fifth aspect of the invention there is provided an mRNA vaccine for the prophylaxis and/or treatment of novel coronaviruses, said mRNA vaccine being prepared from an mRNA as described above and a vector.

In some of these embodiments, the novel coronavirus is one or more of omacron and its subtype strains, delta strains.

In some of these embodiments, the carrier is a liposome nanoparticle.

In some embodiments, the mRNA vaccine is administered by intramuscular injection, intradermal injection, intravenous injection, or arterial injection.

In a sixth aspect of the invention, there is provided a Spike protein, wherein the amino acid sequence of the Spike protein is shown in SEQ ID NO. 2.

In a seventh aspect, the present invention provides the use of an mRNA vaccine as described above for the preparation of a medicament for the prophylaxis and/or treatment of novel coronaviruses.

In some of these embodiments, the novel coronavirus is one or more of omacron and its subtype strains, delta strains.

According to an eighth aspect of the present invention, there is provided a method for preparing an mRNA vaccine as described above, comprising the steps of:

dissolving the mRNA in a buffer solution to prepare a water phase, wherein the concentration of the mRNA is 90 ng/ul-110 ng/ul;

preparing the carrier into an ethanol phase by taking absolute ethanol as a solvent, wherein the concentration of the carrier is 9 mg/ml-11 mg/ml;

the aqueous phase and the ethanol phase are compared with the aqueous phase according to the flow rate: the alcohol phase is 2.5-5.5:1, the total flow rate is 10-14mL/min, coating is carried out, nano carrier particles loaded with mRNA are collected, and the mRNA vaccine particles are obtained after filtration and purification.

In some of these embodiments, the carrier is a Lipid Nanoparticle (LNP) comprising an ionizable cationic liposome, a neutral phospholipid, a sterol lipid, a pegylated phospholipid in a mass ratio of 49-51:9-11:38-39: 1.4-1.6.

In some of these embodiments, the ionizable cationic liposome is SM102, the neutral phospholipid is DSPC, and the sterol lipid is cholesterol; the PEGylated phospholipid is DMG-PEG2000.

In some of these embodiments, the flow rate is greater than that of the aqueous phase: the alcohol phase is 2.5-3.5:1, and the total flow rate is 11-13mL/min.

In the present invention, by studying and selecting an appropriately mutated mRNA, the transfected mRNA cells are capable of expressing high levels, have the correct conformation, and are capable of binding to the Spike protein of hACE2, wherein the S4-1889mRNA expresses the Spike protein at the highest level. The mRNA vaccine prepared from the S4-1889mRNA has high immune efficacy on novel coronavirus Omicron mutant strains, subtype mutant strains and Delta strains, and immune-induced serum neutralizing antibodies not only have high-level neutralizing activity on Omicron BA.1 strains, but also have high-level cross neutralizing activity on strains of various subtypes of Omicron and Delta strains, so that the mRNA vaccine can generate broad-spectrum effective immune protection effects on Omicron strains and Delta strains of different subtypes, and has important clinical application value on preventing and controlling epidemic strains of continuously mutated novel coronaviruses.

Drawings

FIG. 1 is a plasmid map of recombinant plasmid S4-1889-pNeoCura-Bvac of example 1.

FIG. 2 is a graph showing the result of agarose gel electrophoresis of the recombinant plasmid of example 1 before digestion; wherein lane 1 is the molecular weight standard; lane 2: plasmid S4-0432-pNeoCura-Bvac before cleavage; lane 3: after the plasmid S4-0432-pNeoCura-Bvac is digested; lane 4: plasmid S4-0445-pNeoCura-Bvac before cleavage; lane 5: after the plasmid S4-0445-pNeoCura-Bvac is digested; lane 6: plasmid S4-1889-pNeoCura-Bvac before cleavage; lane 7: after the plasmid S4-1889-pNeoCura-Bvac is digested; lane 8: plasmid S4-0932-pNeoCura-Bvac before cleavage; lane 9: after the plasmid S4-0932-pNeoCura-Bvac is digested; lane 10: plasmid S4-0792-pNeoCura-Bvac before cleavage; lane 11: plasmid S4-0792-pNeoCura-Bvac was digested.

FIG. 3 shows the result of agarose gel electrophoresis of mRNA of example 1; wherein lane 1 is the molecular weight standard; lane 2: s4-0432 mRNA; lane 3: s4-0445 mRNA; lane 4: s4-1889 mRNA; lane 5: s4-0932 mRNA; lane 6: s4-0792 mRNA.

FIG. 4 shows the Mean Fluorescence Intensity (MFI) of binding of Spike proteins expressed on the cell surface to R001 after transfection of HEK293T cells with mRNA of each of the sequences in example 1.

FIG. 5 shows the binding of Spike proteins expressed on the cell surface to neutralizing antibodies AM122 and hACE2 after transfection of HEK293T cells with S4-1889mRNA as described in example 2.

FIG. 6 shows the Western blot results of Spike proteins expressed after transfection of S4-1889mRNA into HEK293T cells in example 2.

FIG. 7 is a graph showing the LNP Zeta potential result of the S4-1889mRNA in example 3.

FIG. 8 is a graph showing the results of the LNP Zeta size of the S4-1889mRNA in example 3.

FIG. 9 LNP cryoelectron micrograph of S4-1889mRNA from example 3.

FIG. 10 is a drawing of the specific binding of Spike protein in serum induced by immunization of Babl/c mice with the S4-1889mRNA LNP vaccine of example 4.

FIG. 11 is S4-1889mRNA LNP vaccine of example 4 immunized with Babl/c mice induced serum neutralizing antibodies (pseudovirus neutralizing antibody titer assay).

FIG. 12 spleen IFN-. Gamma. + cell response of the S4-1889mRNA LNP vaccine of example 4 immunized Babl/c mice.

FIG. 13S 4-1889mRNA LNP vaccine of example 4 immune Babl/c mice spleen IL-4T+ cell response.

FIG. 14 is a diagram of the immunization of human-ACE2 transgenic mice with S4-1889mRNA LNP vaccine of example 5 to produce live virus neutralizing antibodies to Omacron BA.1 and Delta strains.

FIG. 15 is a graph showing that immunization with the S4-1889mRNA LNP vaccine of example 5 is effective in reducing pulmonary viral load.

Figure 16 is that the S4-1889mRNA LNP vaccine immunization of example 5 was effective in reducing viral load in the upper respiratory tract (nasal swab).

FIG. 17 is a diagram of S4-1889mRNA LNP vaccine of example 5 inducing cross-reactive binding antibodies to Omacron different subtype strains (BA.1, BA.2, BA.2.12.1, BA.4& 5) in human-ACE2 transgenic mouse serum.

FIG. 18 is a pseudo-virus neutralizing antibody of S4-1889mRNA LNP vaccine of example 5 inducing cross-reactivity of human-ACE2 transgenic mice against Omacron different subtype strains (BA.1, BA.2, BA.4& 5).

Detailed Description

The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.

Unless otherwise defined, all technical and scientific terms used herein and belonging to the same

The meaning is generally understood by those skilled in the art. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

In order to facilitate an understanding of the aspects of the present invention, some terms and phrases are defined below.

mRNA (Messenger RNA), messenger RNA, is a single-stranded RNA that is transcribed from one strand of DNA as a template and carries genetic information to direct protein synthesis.

Signal peptide: short peptide chains that direct the transfer of the newly synthesized protein to the secretory pathway.

LNP: lipid nanoparticles, a lipid vesicle with a homogeneous lipid core, are widely used for delivery of small molecules and nucleic acid drugs, can encapsulate messenger ribonucleic acid (mRNA) encoding viral proteins, and facilitate transport of viral proteins into cells and protect them from damaging enzymes.

SM102:(6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate。

DMG-PEG2000:1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (1, 2-dimyristoyl-rac-glyco-3-methoxypolyethylene glycol-2000, DMG-PEG 2000).

DSPC: distearoyl phosphatidylcholine.

The present invention will be described in further detail with reference to specific examples.

Example 1

1. Construction of recombinant plasmids

According to the novel coronavirus Omicron BA.1 mutant Spike protein sequence of the sequence 1 (SEQ ID NO. 1), according to the research and accumulated experience of the inventor, 680 th amino acid arginine (R) is mutated into alanine (A), 682 th amino acid arginine (R) is mutated into alanine (A), 814 th amino acid phenylalanine (F) is mutated into proline (P), 889 th amino acid alanine (A) is mutated into proline (P), 896 th amino acid alanine (A) is mutated into proline (P), 939 th amino acid alanine (A) is mutated into proline (P), 983 rd amino acid lysine (K) is mutated into proline (P), 984 th amino acid valine (V) is mutated into proline (P), and as shown in the sequence 2 (SEQ ID NO. 2), 1 st to 13 th amino acids are signal peptides. The mRNA sequences encoding the sequence 2 protein were codon optimized to give 5 mRNA sequences, respectively mS4-0432 (SEQ ID NO. 3), mS4-0445 (SEQ ID NO. 4), mS4-1889 (SEQ ID NO. 5), mS4-0932 (SEQ ID NO. 6), mS4-0792 (SEQ ID NO. 7). The DNA sequences corresponding to the 5 mRNA sequences are DNA S4-0432 (SEQ ID NO. 8), DNA S4-0445 (SEQ ID NO. 9), DNA S4-1889 (SEQ ID NO. 10), DNA S4-0932 (SEQ ID NO. 11) and DNA S4-0792 (SEQ ID NO. 12) respectively. The DNA molecule shown in the sequence is inserted between BamHI and SacI sequences of a pNeoCura-Bvac plasmid vector through a gene synthesis scheme to obtain recombinant plasmids, and the recombinant plasmids are used as plasmid templates for preparing mRNA, wherein the corresponding recombinant plasmids are S4-0432-pNeoCura-Bvac, S4-0445-pNeoCura-Bvac, S4-1889-pNeoCura-Bvac, S4-0932-pNeoCura-Bvac and S4-0792-pNeoCura-Bvac.

Wherein the sequences of SEQ ID NO.1 to SEQ ID NO.12 are shown as follows:

SEQ ID NO.1：

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHVISGTNGTKRFDNPVLPFNDGVYFASIEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLDHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPIIVREPEDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLKGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFKGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDIFSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT。

SEQ ID NO.2：

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHVISGTNGTKRFDNPVLPFNDGVYFASIEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLDHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPIIVREPEDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLKGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHRAAASVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSPIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFKGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGPALQIPFPMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDIFSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT。

SEQ ID NO.3：

AUGUUCGUGUUCCUGGUGCUGCUGCCCCUGGUGAGCAGCCAGUGCGUGAAUCUGACAACCAGAACACAGCUGCCACCCGCCUACACCAAUAGCUUCACCAGGGGCGUGUACUACCCCGAUAAGGUGUUCAGAAGCAGCGUGCUGCACAGCACCCAGGACCUGUUCCUGCCCUUCUUUAGCAACGUGACCUGGUUCCACGUGAUCAGCGGCACCAACGGCACCAAGAGAUUCGACAAUCCCGUGCUGCCCUUCAACGACGGCGUGUACUUCGCCAGCAUCGAGAAGUCCAACAUCAUCCGGGGCUGGAUCUUCGGCACAACCCUGGACAGCAAGACCCAGAGCCUGCUGAUCGUGAACAACGCCACCAACGUGGUGAUCAAGGUGUGCGAGUUCCAGUUCUGCAACGACCCCUUCCUGGACCACAAGAACAACAAGAGCUGGAUGGAGAGCGAGUUCCGGGUGUACAGCAGCGCCAACAACUGCACCUUCGAGUACGUGAGCCAGCCAUUCCUGAUGGACCUGGAGGGCAAGCAGGGCAACUUCAAGAACCUGCGGGAGUUCGUGUUCAAGAACAUCGACGGCUACUUCAAGAUCUACAGCAAGCACACCCCAAUCAUUGUGAGAGAGCCCGAGGACCUGCCACAGGGCUUUUCUGCCCUGGAGCCACUGGUGGAUCUGCCAAUCGGCAUCAACAUCACCAGGUUCCAGACCCUGCUGGCCCUGCACAGAUCUUACCUGACACCUGGCGAUUCUAGCAGCGGCUGGACAGCUGGCGCCGCUGCCUAUUAUGUGGGCUAUCUGCAGCCCCGGACCUUCCUGCUGAAGUACAACGAGAACGGCACCAUCACAGACGCCGUGGAUUGUGCCCUGGACCCACUGAGCGAGACCAAGUGCACCCUGAAAAGCUUCACCGUGGAGAAGGGCAUCUACCAGACCAGCAACUUCAGAGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAAUAUCACCAACCUGUGCCCCUUCGACGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUAUGCCUGGAACAGGAAGAGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACCUGGCCCCAUUCUUCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUUAGAGGCGACGAGGUGAGACAGAUCGCCCCUGGCCAGACCGGCAACAUUGCCGACUACAACUACAAGCUGCCAGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAAGCUGGACAGCAAGGUGAGCGGCAACUACAACUACCUGUACCGGCUGUUCCGCAAGAGCAAUCUGAAGCCCUUCGAGAGGGACAUCAGCACCGAGAUCUACCAGGCCGGCAACAAGCCAUGCAAUGGCGUGGCCGGCUUCAAUUGCUACUUUCCCCUGCGGAGCUACAGCUUCAGACCAACCUACGGCGUGGGCCACCAGCCCUAUAGAGUUGUGGUGCUGUCUUUCGAACUGCUGCACGCCCCUGCCACAGUGUGCGGCCCAAAGAAGUCCACCAAUCUGGUGAAGAACAAGUGCGUGAACUUCAACUUCAACGGCCUGAAGGGCACCGGCGUGCUGACAGAGAGCAACAAGAAGUUUCUGCCCUUCCAGCAGUUCGGCCGGGACAUCGCUGAUACAACAGAUGCCGUGAGAGACCCACAGACCCUGGAGAUCCUGGACAUCACCCCAUGCAGCUUUGGAGGAGUGAGCGUGAUCACCCCAGGCACCAAUACCAGCAACCAGGUGGCCGUGCUGUAUCAGGGCGUGAAUUGCACCGAAGUGCCAGUGGCCAUUCAUGCCGACCAGCUGACCCCAACCUGGAGAGUGUACAGCACAGGCUCUAAUGUGUUCCAGACCAGGGCCGGCUGUCUGAUCGGCGCCGAGUACGUGAAUAAUAGCUACGAGUGCGACAUCCCUAUCGGCGCCGGCAUCUGUGCCAGCUACCAGACCCAGACAAAAUCCCACAGAGCUGCCGCCAGCGUGGCCUCUCAGAGCAUUAUCGCCUACACCAUGAGCCUGGGAGCCGAGAAUAGCGUGGCCUACAGCAACAACAGCAUCGCCAUCCCAACCAACUUCACCAUCAGCGUGACCACCGAGAUCCUGCCCGUGAGCAUGACCAAGACCAGCGUGGAUUGCACCAUGUACAUCUGCGGCGACAGCACCGAGUGCAGCAACCUCCUGCUGCAGUAUGGCAGCUUCUGCACACAGCUGAAGAGAGCCCUGACAGGCAUCGCUGUGGAGCAGGACAAGAAUACCCAGGAGGUGUUCGCCCAGGUGAAGCAGAUCUACAAGACCCCACCCAUCAAGUACUUCGGCGGCUUCAACUUCAGCCAGAUUCUGCCCGACCCUAGCAAGCCCUCUAAGAGAAGCCCAAUCGAGGACCUGCUGUUCAACAAGGUGACCCUGGCCGACGCCGGCUUCAUCAAACAGUACGGCGACUGCCUGGGAGACAUUGCCGCCAGAGACCUGAUCUGCGCCCAGAAGUUUAAGGGCCUGACCGUGCUGCCACCACUGCUGACAGACGAAAUGAUUGCCCAGUACACAAGCGCCCUGCUGGCCGGCACCAUCACAAGCGGCUGGACAUUUGGAGCUGGACCAGCCCUGCAGAUCCCAUUCCCAAUGCAGAUGGCCUACAGGUUCAACGGCAUCGGCGUGACCCAGAACGUGCUGUACGAGAACCAGAAGCUGAUCGCCAACCAGUUCAACAGCGCCAUCGGCAAGAUCCAGGACAGCCUGAGCAGCACACCUUCUGCCCUGGGCAAGCUGCAGGAUGUGGUGAACCACAACGCCCAGGCCCUGAAUACCCUGGUGAAGCAGCUGAGCAGCAAGUUCGGCGCCAUCAGCAGCGUGCUGAAUGACAUCUUCAGCAGACUGGACCCACCAGAAGCCGAGGUGCAGAUCGACAGACUGAUCACAGGCAGACUGCAGAGCCUGCAGACCUAUGUGACCCAGCAGCUGAUCAGAGCCGCCGAAAUCAGAGCCUCUGCCAACCUGGCUGCCACAAAGAUGAGCGAGUGUGUGCUGGGACAGAGCAAGCGGGUGGACUUCUGUGGCAAGGGCUACCACCUGAUGAGCUUCCCACAGUCUGCCCCACAUGGAGUGGUGUUCCUGCAUGUGACCUACGUGCCAGCCCAGGAAAAGAAUUUCACCACAGCCCCUGCCAUCUGCCACGACGGCAAAGCCCACUUUCCAAGAGAGGGCGUGUUCGUGAGCAAUGGCACCCACUGGUUCGUGACCCAGCGGAAUUUCUACGAGCCCCAGAUCAUCACCACCGACAACACCUUCGUGAGCGGCAACUGCGACGUGGUGAUCGGCAUCGUGAACAACACCGUGUACGAUCCACUGCAGCCCGAGCUGGACAGCUUCAAGGAGGAGCUGGACAAGUACUUCAAGAACCACACCAGCCCUGAUGUGGACCUGGGCGACAUCAGCGGCAUCAACGCCUCUGUGGUGAACAUCCAGAAGGAGAUCGACCGGCUGAACGAGGUGGCCAAGAACCUGAAUGAGAGCCUGAUCGACCUGCAGGAGCUGGGCAAGUACGAGCAGUACAUCAAGUGGCCCUGGUACAUCUGGCUGGGCUUCAUCGCCGGCCUGAUCGCCAUCGUGAUGGUGACCAUCAUGCUGUGCUGCAUGACCAGCUGCUGUAGCUGCCUGAAGGGCUGCUGUAGCUGCGGCAGCUGCUGCAAGUUUGACGAGGAUGACAGCGAGCCAGUGCUGAAGGGCGUGAAGCUGCACUACACCUGAUGA。

SEQ ID NO.4：

AUGUUCGUGUUCCUGGUGCUGCUGCCACUGGUGAGCAGCCAGUGUGUGAAUCUGACAACCAGAACCCAACUGCCUCCCGCCUACACCAACAGCUUCACCAGAGGCGUGUACUACCCCGACAAGGUGUUCAGAAGCAGCGUGCUGCACAGCACCCAGGACCUGUUCCUGCCCUUCUUCAGCAACGUGACCUGGUUCCACGUGAUCAGCGGCACCAACGGCACAAAGCGGUUCGACAACCCCGUGCUGCCAUUCAAUGACGGCGUGUACUUCGCCAGCAUCGAGAAGUCCAACAUCAUCCGGGGCUGGAUCUUCGGCACCACCCUGGAUAGCAAGACCCAGAGCCUGCUGAUCGUGAACAACGCCACCAACGUGGUGAUCAAGGUGUGCGAGUUCCAGUUCUGCAACGACCCCUUCCUGGACCACAAGAACAACAAGAGCUGGAUGGAGAGCGAGUUCCGGGUGUACAGCAGCGCCAAUAACUGCACCUUCGAGUACGUGAGCCAGCCAUUCCUGAUGGACCUGGAGGGCAAGCAGGGCAACUUCAAGAACCUGCGGGAGUUCGUGUUCAAGAACAUCGACGGCUACUUCAAGAUCUACAGCAAGCACACCCCAAUCAUCGUGCGGGAGCCAGAGGACCUGCCACAGGGAUUUUCUGCCCUGGAGCCCCUGGUGGACCUGCCAAUCGGAAUCAAUAUCACCAGGUUCCAGACCCUGCUGGCCCUGCACAGAAGCUACCUGACACCUGGCGAUUCAUCUAGCGGCUGGACAGCCGGCGCCGCCGCUUACUAUGUGGGCUACCUGCAGCCCAGAACCUUCCUGCUGAAGUACAACGAGAACGGCACCAUCACAGAUGCCGUGGACUGUGCCCUGGACCCACUGAGCGAGACCAAGUGCACACUGAAGUCCUUCACCGUGGAGAAGGGCAUCUACCAGACCAGCAACUUCCGGGUGCAGCCAACCGAGAGCAUCGUGAGGUUCCCCAAUAUCACCAACCUGUGCCCCUUCGACGAGGUGUUCAACGCCACCAGGUUUGCCAGCGUGUACGCCUGGAAUAGGAAGCGGAUCAGCAAUUGCGUGGCCGACUACAGCGUGCUGUACAACCUGGCCCCAUUCUUCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAAUGUGUACGCCGACAGCUUCGUGAUCAGGGGCGAUGAGGUGAGACAGAUUGCCCCAGGCCAGACCGGCAAUAUCGCCGACUACAACUACAAGCUGCCAGACGACUUCACCGGCUGUGUGAUCGCCUGGAACAGCAACAAGCUGGACAGCAAGGUGAGCGGCAACUACAACUACCUGUACCGGCUGUUCCGCAAGAGCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACCGAGAUUUACCAGGCCGGCAAUAAGCCAUGCAACGGCGUGGCCGGCUUCAACUGCUACUUCCCACUGAGGAGCUACAGCUUCAGACCAACCUACGGCGUGGGCCACCAGCCAUAUAGAGUCGUGGUGCUGAGCUUUGAACUGCUGCACGCUCCAGCCACAGUGUGCGGCCCUAAGAAGUCCACCAACCUGGUGAAGAACAAGUGCGUGAACUUCAACUUCAACGGCCUGAAGGGCACCGGCGUGCUGACCGAAAGCAACAAGAAGUUCCUGCCCUUCCAGCAGUUCGGCAGAGAUAUCGCCGACACCACAGAUGCCGUGAGAGAUCCCCAGACCCUGGAGAUCCUGGAUAUCACCCCAUGCAGCUUUGGCGGCGUGAGCGUGAUCACCCCAGGCACCAAUACCAGCAACCAGGUGGCUGUGCUGUAUCAGGGCGUGAAUUGCACAGAGGUGCCCGUGGCCAUUCACGCCGAUCAGCUGACCCCAACCUGGAGAGUGUACAGCACCGGCUCUAAUGUGUUCCAGACAAGAGCCGGCUGCCUGAUCGGCGCCGAGUACGUGAACAAUAGCUACGAGUGCGAUAUCCCAAUUGGCGCCGGCAUCUGUGCCAGCUACCAGACCCAGACAAAGUCUCACAGAGCUGCCGCCUCUGUGGCCAGCCAGAGCAUUAUCGCCUACACCAUGAGCCUGGGCGCCGAAAAUAGCGUGGCCUACAGCAACAACAGCAUCGCCAUCCCCACCAACUUCACCAUCAGCGUGACCACCGAGAUCCUGCCCGUGAGCAUGACCAAGACCAGCGUGGAUUGCACCAUGUACAUCUGCGGCGACAGCACCGAGUGCAGCAACCUCCUGCUGCAGUAUGGCAGCUUUUGCACCCAGCUGAAAAGAGCCCUGACAGGCAUCGCCGUGGAGCAAGACAAGAACACCCAGGAGGUGUUCGCCCAGGUGAAGCAGAUCUACAAGACCCCACCCAUCAAGUACUUCGGCGGCUUCAACUUCAGCCAGAUCCUGCCUGAUCCCAGCAAGCCCAGCAAGAGAAGCCCCAUCGAGGACCUGCUGUUCAAUAAGGUGACCCUGGCCGACGCUGGCUUCAUCAAACAGUACGGAGAUUGUCUGGGCGACAUCGCCGCCAGAGACCUGAUCUGCGCCCAGAAGUUCAAAGGCCUGACCGUGCUGCCACCACUGCUGACAGACGAGAUGAUUGCCCAGUACACCAGCGCCCUGCUGGCCGGCACCAUCACAAGCGGAUGGACCUUUGGAGCCGGACCCGCCCUGCAGAUCCCAUUUCCAAUGCAGAUGGCCUACAGGUUCAACGGCAUCGGCGUGACCCAGAACGUGCUGUACGAGAACCAGAAGCUGAUCGCCAACCAGUUCAACAGCGCCAUCGGCAAAAUCCAGGACAGCCUGAGCAGCACACCUUCUGCCCUGGGCAAGCUGCAGGAUGUGGUGAAUCACAACGCCCAGGCCCUGAACACCCUGGUGAAACAGCUGAGCAGCAAGUUCGGCGCCAUCAGCAGCGUGCUGAAUGAUAUCUUCAGCAGGCUGGACCCACCUGAGGCCGAGGUGCAGAUCGAUAGACUGAUCACCGGCAGACUGCAGUCUCUGCAGACCUACGUGACCCAGCAACUGAUCAGAGCCGCCGAGAUCAGAGCUUCUGCCAACCUGGCCGCCACCAAGAUGUCUGAGUGCGUGCUGGGACAGAGCAAGAGAGUGGACUUCUGUGGCAAGGGCUAUCACCUGAUGAGCUUCCCACAGUCUGCCCCACACGGAGUGGUGUUCCUGCACGUGACAUACGUGCCCGCCCAGGAGAAGAAUUUUACCACCGCCCCAGCCAUCUGUCACGACGGCAAGGCCCACUUUCCAAGAGAGGGCGUGUUCGUGAGCAACGGCACCCACUGGUUCGUGACCCAGCGGAACUUCUACGAGCCCCAGAUCAUCACCACCGACAACACCUUCGUGAGCGGCAACUGCGACGUGGUGAUCGGCAUCGUGAACAACACCGUGUACGAUCCACUGCAGCCCGAGCUGGACAGCUUCAAGGAGGAGCUGGACAAGUACUUCAAGAACCACACCAGCCCAGAUGUGGACCUGGGCGACAUCAGCGGCAUCAAUGCCAGCGUGGUGAACAUCCAGAAGGAGAUCGACCGGCUGAAUGAGGUGGCCAAGAACCUGAACGAGAGCCUGAUCGACCUGCAGGAGCUGGGCAAGUACGAGCAGUACAUCAAGUGGCCAUGGUACAUCUGGCUGGGCUUCAUCGCCGGCCUGAUCGCCAUCGUGAUGGUGACCAUCAUGCUGUGCUGCAUGACCAGCUGCUGCAGCUGUCUGAAGGGCUGCUGUAGCUGCGGCAGCUGCUGUAAGUUCGAUGAGGACGAUAGCGAGCCCGUGCUGAAGGGCGUGAAGCUGCACUACACCUGAUGA。

SEQ ID NO.5：

AUGUUCGUGUUCCUGGUGCUGCUGCCUCUGGUGAGCAGCCAGUGCGUGAACCUGACCACCAGAACCCAGCUGCCUCCCGCCUACACCAACAGCUUCACCAGAGGCGUGUACUACCCCGACAAGGUGUUCAGGAGCAGCGUGCUGCACAGCACCCAGGACCUGUUCCUGCCCUUCUUCUCCAACGUGACCUGGUUCCACGUGAUCAGCGGCACCAACGGCACAAAGCGGUUCGACAACCCCGUGCUGCCCUUCAACGACGGCGUGUACUUCGCCAGCAUCGAGAAGUCCAACAUCAUCCGGGGCUGGAUCUUCGGCACCACCCUGGACAGCAAGACCCAGAGCCUGCUGAUCGUGAACAACGCCACCAACGUGGUGAUCAAGGUGUGCGAGUUCCAGUUCUGCAACGACCCCUUCCUGGACCACAAGAACAACAAGAGCUGGAUGGAGAGCGAGUUCCGGGUGUACAGCAGCGCCAACAACUGCACCUUCGAGUACGUGAGCCAGCCCUUCCUGAUGGACCUGGAGGGCAAGCAGGGCAACUUCAAGAACCUGCGGGAGUUCGUGUUCAAGAACAUCGACGGCUACUUCAAGAUCUACAGCAAGCACACCCCUAUCAUCGUGCGGGAGCCUGAGGACCUGCCUCAGGGAUUCAGCGCUCUGGAGCCUCUGGUGGACCUGCCUAUCGGCAUCAACAUCACCCGGUUCCAGACCCUGCUGGCCCUGCACAGAAGCUACCUGACACCUGGCGACAGCAGCAGCGGAUGGACAGCUGGAGCCGCCGCCUACUACGUGGGCUACCUGCAGCCUCGGACCUUCCUGCUGAAGUACAACGAGAACGGCACAAUCACCGACGCCGUGGACUGCGCCCUGGACCCUCUGAGCGAGACAAAGUGCACCCUGAAGUCCUUCACCGUGGAGAAGGGCAUCUACCAGACCAGCAACUUCCGGGUGCAGCCCACCGAGAGCAUCGUGCGGUUCCCCAACAUCACCAACCUGUGCCCCUUCGACGAGGUGUUCAACGCCACCAGAUUCGCCAGCGUGUACGCCUGGAACCGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACCUGGCCCCUUUCUUCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAGGUGAGACAGAUCGCCCCUGGCCAGACCGGCAACAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAACAAGCUGGACAGCAAGGUGAGCGGCAACUACAACUACCUGUACCGGCUGUUCCGCAAGAGCAACCUGAAGCCCUUCGAGCGGGACAUCAGCACAGAGAUCUACCAGGCCGGCAACAAGCCUUGCAACGGCGUGGCCGGCUUCAACUGCUACUUCCCUCUGCGGAGCUACAGCUUCAGACCCACCUACGGCGUGGGCCACCAGCCUUACAGAGUGGUGGUGCUGAGCUUCGAGCUGCUGCACGCCCCUGCCACAGUGUGCGGCCCCAAGAAGUCCACAAACCUGGUGAAGAACAAGUGCGUGAACUUCAACUUCAACGGCCUGAAGGGCACCGGCGUGCUGACCGAGAGCAACAAGAAGUUCCUGCCCUUCCAGCAGUUCGGCAGAGACAUCGCCGACACAACCGACGCCGUGAGAGACCCUCAGACACUGGAGAUCCUGGACAUCACACCCUGCAGCUUCGGCGGCGUGAGCGUGAUCACCCCUGGCACAAACACAAGCAACCAGGUGGCCGUGCUGUACCAGGGCGUGAACUGCACAGAGGUGCCUGUGGCCAUCCACGCCGACCAGCUGACCCCUACCUGGAGAGUGUACAGCACCGGCAGCAACGUGUUCCAGACCAGAGCCGGCUGCCUGAUCGGCGCCGAGUACGUGAACAACAGCUACGAGUGCGACAUCCCUAUCGGCGCCGGCAUCUGCGCCAGCUACCAGACACAGACAAAGAGCCACAGAGCCGCCGCCAGCGUGGCCAGCCAGAGCAUCAUCGCCUACACAAUGAGCCUGGGCGCCGAGAACAGCGUGGCCUACAGCAACAACAGCAUCGCCAUCCCCACCAACUUCACCAUCAGCGUGACCACCGAGAUCCUGCCCGUGAGCAUGACCAAGACCAGCGUGGACUGCACCAUGUACAUCUGCGGCGACAGCACCGAGUGCAGCAACCUGCUGCUGCAGUACGGCAGCUUCUGCACCCAGCUGAAGAGAGCCCUGACAGGCAUCGCCGUGGAGCAGGACAAGAACACCCAGGAGGUGUUCGCCCAGGUGAAGCAGAUCUACAAGACCCCUCCCAUCAAGUACUUCGGCGGCUUCAACUUCAGCCAGAUCCUGCCCGACCCCAGCAAGCCCAGCAAGAGAAGCCCCAUCGAGGACCUGCUGUUCAACAAGGUGACACUGGCCGACGCCGGCUUCAUCAAGCAGUACGGCGACUGCCUGGGCGAUAUCGCCGCCAGAGACCUGAUCUGCGCCCAGAAGUUCAAGGGCCUGACCGUGCUGCCUCCUCUGCUGACAGACGAGAUGAUCGCCCAGUACACAAGCGCCCUGCUGGCCGGCACAAUCACAAGCGGCUGGACAUUCGGCGCCGGACCUGCCCUGCAGAUCCCUUUCCCUAUGCAGAUGGCCUACCGGUUCAACGGCAUCGGCGUGACCCAGAACGUGCUGUACGAGAACCAGAAGCUGAUCGCCAACCAGUUCAACAGCGCCAUCGGCAAGAUCCAGGACAGCCUGAGCAGCACACCUAGCGCUCUGGGCAAGCUGCAGGACGUGGUGAACCACAACGCCCAGGCCCUGAACACCCUGGUGAAGCAGCUGAGCAGCAAGUUCGGCGCCAUCAGCAGCGUGCUGAACGACAUCUUCAGCAGACUGGACCCUCCUGAGGCCGAGGUGCAGAUCGACAGACUGAUCACAGGCAGACUGCAGAGCCUGCAGACCUACGUGACCCAGCAGCUGAUCAGAGCCGCCGAGAUCAGAGCCAGCGCCAACCUGGCCGCCACAAAGAUGAGCGAGUGCGUGCUGGGCCAGAGCAAGAGAGUGGACUUCUGCGGCAAGGGCUACCACCUGAUGAGCUUCCCUCAGAGCGCCCCUCACGGCGUGGUGUUCCUGCACGUGACAUACGUGCCUGCCCAGGAGAAGAACUUCACCACCGCCCCUGCCAUCUGCCACGACGGCAAGGCCCACUUCCCUAGAGAGGGCGUGUUCGUGAGCAACGGCACACACUGGUUCGUGACCCAGCGGAACUUCUACGAGCCCCAGAUCAUCACCACCGACAACACCUUCGUGAGCGGCAACUGCGACGUGGUGAUCGGCAUCGUGAACAACACCGUGUACGACCCUCUGCAGCCCGAGCUGGACAGCUUCAAGGAGGAGCUGGACAAGUACUUCAAGAACCACACCAGCCCUGACGUGGACCUGGGCGACAUCAGCGGCAUCAACGCCAGCGUGGUGAACAUCCAGAAGGAGAUCGACCGGCUGAACGAGGUGGCCAAGAACCUGAACGAGAGCCUGAUCGACCUGCAGGAGCUGGGCAAGUACGAGCAGUACAUCAAGUGGCCCUGGUACAUCUGGCUGGGCUUCAUCGCCGGCCUGAUCGCCAUCGUGAUGGUGACCAUCAUGCUGUGCUGCAUGACCAGCUGCUGCAGCUGCCUGAAGGGCUGCUGCAGCUGCGGCAGCUGCUGCAAGUUCGACGAGGACGACAGCGAGCCUGUGCUGAAGGGCGUGAAGCUGCACUACACCUGAUGA。

SEQ ID NO.6：

AUGUUCGUGUUCCUGGUGCUGCUGCCACUGGUGUCUUCUCAGUGUGUGAACCUGACCACCAGAACACAGCUGCCUCCAGCCUACACCAACAGCUUCACCCGGGGAGUGUACUACCCAGACAAGGUGUUCAGGAGCAGCGUGCUGCACAGCACCCAGGACCUGUUCCUGCCAUUCUUCAGCAACGUGACCUGGUUCCACGUGAUCAGCGGCACCAACGGCACCAAGAGAUUCGACAACCCUGUGCUGCCCUUCAACGACGGCGUGUACUUCGCCAGCAUCGAGAAGUCCAACAUCAUCCGGGGCUGGAUCUUCGGCACAACACUGGACAGCAAGACCCAGAGCCUGCUGAUCGUGAACAACGCCACCAACGUGGUGAUCAAGGUGUGCGAGUUCCAGUUCUGCAACGACCCCUUCCUGGACCACAAGAACAACAAGAGCUGGAUGGAGAGCGAGUUCCGGGUGUACAGCAGCGCCAACAACUGCACCUUCGAGUACGUGAGCCAGCCCUUUCUGAUGGACCUGGAGGGCAAGCAGGGCAACUUCAAGAACCUGCGGGAGUUCGUGUUCAAGAACAUCGACGGCUACUUCAAGAUCUACAGCAAGCACACCCCAAUCAUCGUGCGGGAGCCAGAGGACCUGCCACAGGGAUUUUCUGCCCUGGAGCCCCUGGUGGAUCUGCCAAUCGGCAUCAACAUCACCAGGUUCCAGACCCUGCUGGCCCUGCACAGGAGCUAUCUGACACCAGGCGAUUCCAGCAGCGGCUGGACAGCCGGCGCAGCCGCCUAUUACGUGGGCUACCUGCAGCCCAGAACAUUCCUGCUGAAGUACAACGAGAACGGCACAAUCACAGACGCCGUGGAUUGCGCCCUCGACCCUCUGAGCGAGACCAAGUGCACCCUGAAGUCCUUCACCGUGGAGAAGGGCAUCUACCAGACCAGCAACUUCAGGGUGCAGCCUACAGAGAGCAUCGUGCGGUUCCCCAACAUCACCAAUCUGUGCCCCUUUGACGAGGUGUUCAACGCCACCCGGUUUGCUAGCGUGUACGCCUGGAACAGGAAGCGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACCUGGCCCCAUUCUUCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACUCUUUCGUGAUCAGAGGCGACGAGGUGAGACAGAUCGCCCCUGGCCAGACAGGCAAUAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUUGCCUGGAACAGCAACAAGCUGGACAGCAAGGUGAGCGGCAACUACAACUACCUGUACCGGCUGUUCCGCAAGAGCAACCUGAAGCCCUUCGAGAGAGACAUCAGCACCGAGAUCUACCAGGCCGGCAACAAGCCAUGUAACGGCGUGGCCGGCUUCAACUGCUACUUUCCCCUGCGGAGCUACAGCUUCAGACCAACCUAUGGCGUGGGCCACCAGCCCUAUAGAGUCGUGGUGCUGAGCUUUGAGCUGCUGCACGCCCCUGCUACAGUGUGUGGCCCAAAGAAGUCCACCAACCUGGUGAAGAACAAGUGCGUGAACUUCAACUUCAACGGCCUGAAGGGCACCGGCGUGCUGACCGAGAGCAAUAAGAAGUUCCUGCCCUUCCAGCAGUUCGGCAGAGACAUCGCCGACACAACCGAUGCCGUGAGAGACCCACAGACCCUGGAGAUCCUGGACAUCACCCCAUGCAGCUUUGGAGGCGUGAGCGUGAUCACACCAGGCACCAAUACCAGCAAUCAGGUGGCCGUGCUGUACCAGGGCGUGAACUGCACAGAAGUGCCAGUGGCUAUUCACGCCGACCAGCUGACCCCAACCUGGAGAGUGUACAGCACCGGAAGCAAUGUGUUCCAGACCCGGGCUGGCUGCCUGAUCGGCGCCGAGUACGUGAAUAAUAGCUACGAGUGCGACAUCCCUAUCGGAGCCGGCAUCUGCGCCAGCUAUCAGACCCAGACAAAGAGCCAUAGAGCUGCCGCCUCUGUGGCCUCUCAGAGCAUCAUCGCCUACACCAUGAGCCUGGGAGCCGAGAACAGCGUGGCCUACAGCAAUAACAGCAUCGCCAUCCCCACCAACUUCACCAUCAGCGUGACCACAGAGAUCCUGCCCGUGAGCAUGACCAAGACCAGCGUGGAUUGCACCAUGUACAUCUGCGGCGACAGCACCGAGUGUAGCAACCUCCUGCUGCAGUAUGGCAGCUUCUGCACCCAGCUGAAGAGAGCCCUGACAGGCAUCGCCGUGGAGCAGGAUAAGAAUACCCAGGAGGUGUUCGCCCAGGUGAAGCAGAUCUACAAGACCCCACCCAUCAAGUACUUCGGCGGCUUCAAUUUCAGCCAGAUCCUGCCCGACCCCAGCAAGCCCUCUAAGAGAAGCCCAAUCGAGGACCUGCUGUUCAACAAGGUGACCCUGGCCGACGCCGGCUUUAUUAAGCAGUACGGCGACUGUCUGGGAGACAUUGCCGCCAGAGAUCUGAUCUGUGCCCAGAAGUUCAAGGGCCUGACAGUGCUGCCACCCCUGCUGACCGAUGAGAUGAUCGCCCAAUACACCAGCGCCCUGCUGGCCGGCACAAUCACCAGCGGCUGGACAUUCGGCGCCGGACCAGCCCUGCAGAUCCCAUUCCCAAUGCAGAUGGCCUACAGAUUCAACGGCAUCGGCGUGACCCAGAACGUGCUGUACGAGAACCAGAAGCUGAUCGCCAACCAGUUCAACAGCGCCAUCGGCAAGAUCCAGGACAGCCUGAGCAGCACACCUUCUGCCCUGGGAAAGCUGCAGGACGUGGUGAAUCACAAUGCCCAGGCCCUGAAUACCCUGGUGAAGCAGCUGAGCAGCAAGUUCGGCGCCAUCAGCAGCGUGCUGAAUGACAUCUUCAGCAGGCUGGACCCACCUGAAGCCGAGGUGCAGAUCGACAGACUGAUCACCGGAAGACUGCAGAGCCUGCAGACCUACGUGACCCAGCAGCUGAUCAGAGCCGCCGAGAUUAGAGCCAGCGCCAACCUGGCUGCCACCAAAAUGUCUGAGUGUGUGCUGGGCCAGAGCAAGCGGGUGGAUUUCUGCGGCAAAGGCUACCACCUGAUGAGCUUCCCACAGUCUGCCCCACACGGAGUGGUGUUCCUGCACGUGACAUACGUGCCAGCCCAGGAGAAGAAUUUCACCACCGCCCCAGCCAUCUGUCAUGAUGGAAAGGCCCACUUCCCAAGGGAGGGCGUGUUUGUGAGCAACGGCACCCACUGGUUCGUGACCCAGAGGAACUUCUACGAGCCCCAGAUCAUCACCACCGACAACACCUUUGUGAGCGGCAACUGCGACGUGGUGAUCGGCAUCGUGAACAAUACCGUGUACGACCCACUGCAGCCCGAGCUGGACAGCUUUAAGGAGGAGCUGGACAAGUACUUCAAGAACCACACCAGCCCUGACGUGGAUCUGGGCGACAUCAGCGGCAUCAACGCCAGCGUGGUGAAUAUCCAGAAGGAGAUCGACCGGCUGAACGAGGUGGCCAAGAAUCUGAACGAGAGCCUGAUCGACCUGCAGGAGCUGGGCAAGUACGAGCAGUACAUCAAGUGGCCCUGGUAUAUCUGGCUGGGCUUCAUCGCCGGCCUGAUCGCCAUCGUGAUGGUGACCAUCAUGCUGUGCUGCAUGACCAGCUGCUGCAGCUGUCUGAAGGGCUGCUGUAGCUGUGGCAGCUGCUGCAAGUUUGAUGAGGACGACAGCGAGCCAGUGCUGAAGGGCGUGAAGCUGCACUACACCUGAUGA。

SEQ ID NO.7：

AUGUUCGUGUUCCUGGUGCUGCUGCCACUGGUGAGCAGCCAGUGCGUGAAUCUGACAACCAGGACACAGCUGCCACCCGCCUACACCAAUAGCUUCACCAGAGGCGUGUACUACCCAGACAAGGUGUUCAGGAGCAGCGUGCUGCACAGCACCCAGGACCUGUUCCUGCCCUUCUUCAGCAAUGUGACCUGGUUCCACGUGAUCAGCGGCACCAACGGCACCAAGAGAUUCGACAACCCCGUGCUGCCCUUCAACGACGGCGUGUACUUCGCCAGCAUCGAGAAGUCCAACAUCAUCCGGGGCUGGAUCUUCGGCACCACACUGGACAGCAAGACCCAGAGCCUGCUGAUCGUGAACAACGCCACCAACGUGGUGAUCAAGGUGUGCGAGUUCCAGUUCUGCAACGACCCCUUCCUGGACCACAAGAACAACAAGAGCUGGAUGGAGAGCGAGUUCCGGGUGUACAGCAGCGCCAACAACUGCACAUUCGAGUACGUGAGCCAGCCAUUCCUGAUGGACCUGGAGGGCAAGCAGGGCAACUUCAAGAACCUGCGGGAGUUCGUGUUCAAGAACAUCGACGGCUACUUCAAGAUCUACAGCAAGCACACCCCAAUCAUCGUGAGAGAGCCUGAGGAUCUGCCUCAGGGCUUUUCUGCUCUGGAGCCACUGGUGGACCUGCCCAUCGGCAUCAAUAUCACCAGAUUUCAGACCCUGCUGGCCCUGCACAGAAGCUACCUGACACCUGGCGAUUCUAGCAGCGGCUGGACAGCUGGAGCUGCCGCCUACUACGUGGGCUACCUGCAGCCAAGGACCUUCCUGCUGAAGUACAACGAGAACGGCACAAUCACCGACGCUGUGGAUUGUGCCCUGGACCCACUGAGCGAGACCAAGUGCACCCUGAAGUCCUUCACCGUGGAGAAGGGCAUCUACCAGACCAGCAACUUCCGGGUGCAGCCCACCGAGAGCAUCGUGAGGUUCCCAAACAUCACCAACCUGUGCCCCUUCGACGAGGUGUUUAACGCCACCCGGUUCGCCAGCGUGUACGCCUGGAACAGAAAGAGGAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACCUGGCCCCAUUCUUCACCUUCAAGUGCUACGGCGUGAGCCCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGAUGAGGUGAGACAGAUUGCCCCAGGCCAGACCGGCAAUAUCGCCGACUACAAUUACAAGCUGCCCGACGACUUCACCGGCUGCGUGAUCGCCUGGAACAGCAAUAAGCUGGACAGCAAGGUGAGCGGCAACUACAACUACCUGUACCGGCUGUUCCGCAAGAGCAACCUGAAGCCCUUCGAGAGAGACAUCAGCACCGAGAUCUACCAGGCCGGCAACAAGCCAUGCAAUGGAGUGGCCGGCUUCAACUGCUACUUCCCACUGAGGAGCUACAGCUUCAGACCCACAUACGGCGUGGGCCACCAGCCCUACAGAGUCGUGGUGCUGUCUUUUGAACUGCUGCAUGCCCCAGCCACCGUGUGCGGCCCAAAGAAGUCCACCAACCUGGUGAAGAACAAGUGCGUGAACUUCAACUUCAACGGCCUGAAGGGCACCGGCGUGCUGACCGAGAGCAACAAGAAAUUCCUGCCCUUCCAGCAGUUCGGCAGAGACAUCGCCGAUACCACCGACGCCGUGAGAGAUCCACAGACACUGGAGAUCCUGGACAUCACCCCAUGCAGCUUCGGCGGAGUGAGCGUGAUCACACCCGGCACAAACACCAGCAAUCAGGUGGCCGUGCUGUACCAGGGCGUGAAUUGUACAGAGGUGCCCGUGGCCAUUCACGCCGAUCAGCUGACCCCUACCUGGAGAGUGUACAGCACAGGAAGCAAUGUGUUCCAGACCAGGGCCGGCUGCCUGAUCGGAGCCGAGUACGUGAACAACAGCUACGAGUGCGACAUCCCAAUCGGCGCCGGCAUCUGUGCCAGCUACCAGACCCAGACAAAAUCCCACAGAGCUGCCGCUUCUGUGGCCAGCCAGAGCAUCAUCGCCUACACAAUGAGCCUGGGCGCCGAGAAUAGCGUGGCCUACAGCAAUAACAGCAUCGCCAUCCCAACCAACUUCACCAUCAGCGUGACCACCGAGAUCCUGCCCGUGAGCAUGACAAAGACCAGCGUGGACUGCACCAUGUACAUCUGCGGCGAUAGCACCGAGUGCAGCAAUCUCCUGCUGCAGUACGGCAGCUUUUGCACCCAGCUGAAGAGAGCCCUGACAGGCAUUGCCGUGGAGCAGGACAAGAACACCCAGGAGGUGUUCGCCCAGGUGAAGCAGAUCUACAAGACCCCACCCAUCAAGUACUUCGGCGGCUUCAACUUCAGCCAGAUUCUGCCUGACCCUAGCAAGCCCAGCAAGAGAAGCCCCAUCGAGGACCUGCUGUUCAAUAAGGUGACCCUGGCCGACGCCGGCUUCAUCAAGCAGUACGGCGACUGUCUGGGCGAUAUUGCCGCCAGAGACCUGAUCUGCGCCCAGAAGUUCAAGGGCCUGACAGUGCUGCCACCCCUGCUGACCGAUGAGAUGAUCGCUCAGUACACAUCUGCCCUGCUGGCCGGCACAAUCACAAGCGGCUGGACAUUUGGCGCCGGACCUGCUCUGCAGAUCCCAUUCCCAAUGCAGAUGGCCUACAGGUUCAACGGCAUCGGCGUGACCCAGAACGUGCUGUACGAGAACCAGAAGCUGAUCGCCAACCAGUUCAACAGCGCCAUCGGCAAGAUCCAGGAUAGCCUGUCUAGCACACCAAGCGCCCUGGGCAAGCUGCAGGACGUGGUGAAUCACAACGCCCAAGCCCUGAACACCCUGGUGAAGCAGCUGAGCAGCAAGUUCGGCGCCAUCAGCAGCGUUCUGAACGACAUCUUCAGCAGACUGGACCCUCCUGAGGCCGAGGUGCAGAUCGAUAGACUGAUCACCGGAAGACUGCAGAGCCUGCAGACCUACGUGACACAGCAGCUGAUCAGAGCCGCCGAGAUCAGAGCCUCUGCCAAUCUGGCCGCCACAAAGAUGAGCGAGUGUGUGCUGGGCCAGAGCAAGAGAGUGGACUUCUGCGGCAAGGGCUACCACCUGAUGAGCUUCCCACAGUCUGCCCCACACGGCGUGGUGUUCCUGCAUGUGACAUACGUGCCAGCCCAGGAGAAGAACUUUACCACCGCCCCUGCCAUCUGCCACGAUGGCAAAGCCCACUUCCCCAGAGAGGGCGUGUUUGUGAGCAAUGGCACACACUGGUUCGUGACCCAGAGGAACUUCUACGAGCCCCAGAUCAUCACCACCGACAACACCUUCGUGAGCGGCAACUGCGACGUGGUGAUCGGCAUCGUGAACAACACCGUGUACGACCCUCUGCAGCCUGAGCUGGACAGCUUCAAGGAGGAGCUGGACAAGUACUUCAAGAACCACACCAGCCCAGAUGUGGACCUGGGCGACAUCAGCGGCAUCAAUGCCAGCGUGGUGAACAUCCAGAAGGAGAUCGACCGGCUGAACGAAGUGGCCAAGAACCUGAACGAGAGCCUGAUCGACCUGCAGGAGCUGGGCAAGUACGAGCAGUACAUCAAGUGGCCAUGGUACAUCUGGCUGGGCUUCAUCGCCGGCCUGAUCGCCAUCGUGAUGGUGACCAUCAUGCUGUGCUGCAUGACCAGCUGCUGCUCUUGCCUGAAGGGCUGCUGUAGCUGCGGCAGCUGCUGCAAGUUCGAUGAGGAUGACAGCGAGCCCGUGCUGAAGGGCGUGAAGCUGCACUACACCUGAUGA。

SEQ ID NO.8：

ATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCCAGTGCGTGAATCTGACAACCAGAACACAGCTGCCACCCGCCTACACCAATAGCTTCACCAGGGGCGTGTACTACCCCGATAAGGTGTTCAGAAGCAGCGTGCTGCACAGCACCCAGGACCTGTTCCTGCCCTTCTTTAGCAACGTGACCTGGTTCCACGTGATCAGCGGCACCAACGGCACCAAGAGATTCGACAATCCCGTGCTGCCCTTCAACGACGGCGTGTACTTCGCCAGCATCGAGAAGTCCAACATCATCCGGGGCTGGATCTTCGGCACAACCCTGGACAGCAAGACCCAGAGCCTGCTGATCGTGAACAACGCCACCAACGTGGTGATCAAGGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGACCACAAGAACAACAAGAGCTGGATGGAGAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACCTTCGAGTACGTGAGCCAGCCATTCCTGATGGACCTGGAGGGCAAGCAGGGCAACTTCAAGAACCTGCGGGAGTTCGTGTTCAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACACCCCAATCATTGTGAGAGAGCCCGAGGACCTGCCACAGGGCTTTTCTGCCCTGGAGCCACTGGTGGATCTGCCAATCGGCATCAACATCACCAGGTTCCAGACCCTGCTGGCCCTGCACAGATCTTACCTGACACCTGGCGATTCTAGCAGCGGCTGGACAGCTGGCGCCGCTGCCTATTATGTGGGCTATCTGCAGCCCCGGACCTTCCTGCTGAAGTACAACGAGAACGGCACCATCACAGACGCCGTGGATTGTGCCCTGGACCCACTGAGCGAGACCAAGTGCACCCTGAAAAGCTTCACCGTGGAGAAGGGCATCTACCAGACCAGCAACTTCAGAGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAATATCACCAACCTGTGCCCCTTCGACGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTATGCCTGGAACAGGAAGAGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACCTGGCCCCATTCTTCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATTAGAGGCGACGAGGTGAGACAGATCGCCCCTGGCCAGACCGGCAACATTGCCGACTACAACTACAAGCTGCCAGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAAGCTGGACAGCAAGGTGAGCGGCAACTACAACTACCTGTACCGGCTGTTCCGCAAGAGCAATCTGAAGCCCTTCGAGAGGGACATCAGCACCGAGATCTACCAGGCCGGCAACAAGCCATGCAATGGCGTGGCCGGCTTCAATTGCTACTTTCCCCTGCGGAGCTACAGCTTCAGACCAACCTACGGCGTGGGCCACCAGCCCTATAGAGTTGTGGTGCTGTCTTTCGAACTGCTGCACGCCCCTGCCACAGTGTGCGGCCCAAAGAAGTCCACCAATCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGAAGGGCACCGGCGTGCTGACAGAGAGCAACAAGAAGTTTCTGCCCTTCCAGCAGTTCGGCCGGGACATCGCTGATACAACAGATGCCGTGAGAGACCCACAGACCCTGGAGATCCTGGACATCACCCCATGCAGCTTTGGAGGAGTGAGCGTGATCACCCCAGGCACCAATACCAGCAACCAGGTGGCCGTGCTGTATCAGGGCGTGAATTGCACCGAAGTGCCAGTGGCCATTCATGCCGACCAGCTGACCCCAACCTGGAGAGTGTACAGCACAGGCTCTAATGTGTTCCAGACCAGGGCCGGCTGTCTGATCGGCGCCGAGTACGTGAATAATAGCTACGAGTGCGACATCCCTATCGGCGCCGGCATCTGTGCCAGCTACCAGACCCAGACAAAATCCCACAGAGCTGCCGCCAGCGTGGCCTCTCAGAGCATTATCGCCTACACCATGAGCCTGGGAGCCGAGAATAGCGTGGCCTACAGCAACAACAGCATCGCCATCCCAACCAACTTCACCATCAGCGTGACCACCGAGATCCTGCCCGTGAGCATGACCAAGACCAGCGTGGATTGCACCATGTACATCTGCGGCGACAGCACCGAGTGCAGCAACCTCCTGCTGCAGTATGGCAGCTTCTGCACACAGCTGAAGAGAGCCCTGACAGGCATCGCTGTGGAGCAGGACAAGAATACCCAGGAGGTGTTCGCCCAGGTGAAGCAGATCTACAAGACCCCACCCATCAAGTACTTCGGCGGCTTCAACTTCAGCCAGATTCTGCCCGACCCTAGCAAGCCCTCTAAGAGAAGCCCAATCGAGGACCTGCTGTTCAACAAGGTGACCCTGGCCGACGCCGGCTTCATCAAACAGTACGGCGACTGCCTGGGAGACATTGCCGCCAGAGACCTGATCTGCGCCCAGAAGTTTAAGGGCCTGACCGTGCTGCCACCACTGCTGACAGACGAAATGATTGCCCAGTACACAAGCGCCCTGCTGGCCGGCACCATCACAAGCGGCTGGACATTTGGAGCTGGACCAGCCCTGCAGATCCCATTCCCAATGCAGATGGCCTACAGGTTCAACGGCATCGGCGTGACCCAGAACGTGCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGATCCAGGACAGCCTGAGCAGCACACCTTCTGCCCTGGGCAAGCTGCAGGATGTGGTGAACCACAACGCCCAGGCCCTGAATACCCTGGTGAAGCAGCTGAGCAGCAAGTTCGGCGCCATCAGCAGCGTGCTGAATGACATCTTCAGCAGACTGGACCCACCAGAAGCCGAGGTGCAGATCGACAGACTGATCACAGGCAGACTGCAGAGCCTGCAGACCTATGTGACCCAGCAGCTGATCAGAGCCGCCGAAATCAGAGCCTCTGCCAACCTGGCTGCCACAAAGATGAGCGAGTGTGTGCTGGGACAGAGCAAGCGGGTGGACTTCTGTGGCAAGGGCTACCACCTGATGAGCTTCCCACAGTCTGCCCCACATGGAGTGGTGTTCCTGCATGTGACCTACGTGCCAGCCCAGGAAAAGAATTTCACCACAGCCCCTGCCATCTGCCACGACGGCAAAGCCCACTTTCCAAGAGAGGGCGTGTTCGTGAGCAATGGCACCCACTGGTTCGTGACCCAGCGGAATTTCTACGAGCCCCAGATCATCACCACCGACAACACCTTCGTGAGCGGCAACTGCGACGTGGTGATCGGCATCGTGAACAACACCGTGTACGATCCACTGCAGCCCGAGCTGGACAGCTTCAAGGAGGAGCTGGACAAGTACTTCAAGAACCACACCAGCCCTGATGTGGACCTGGGCGACATCAGCGGCATCAACGCCTCTGTGGTGAACATCCAGAAGGAGATCGACCGGCTGAACGAGGTGGCCAAGAACCTGAATGAGAGCCTGATCGACCTGCAGGAGCTGGGCAAGTACGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCTGTGCTGCATGACCAGCTGCTGTAGCTGCCTGAAGGGCTGCTGTAGCTGCGGCAGCTGCTGCAAGTTTGACGAGGATGACAGCGAGCCAGTGCTGAAGGGCGTGAAGCTGCACTACACCTGATGA。

SEQ ID NO.9：

ATGTTCGTGTTCCTGGTGCTGCTGCCACTGGTGAGCAGCCAGTGTGTGAATCTGACAACCAGAACCCAACTGCCTCCCGCCTACACCAACAGCTTCACCAGAGGCGTGTACTACCCCGACAAGGTGTTCAGAAGCAGCGTGCTGCACAGCACCCAGGACCTGTTCCTGCCCTTCTTCAGCAACGTGACCTGGTTCCACGTGATCAGCGGCACCAACGGCACAAAGCGGTTCGACAACCCCGTGCTGCCATTCAATGACGGCGTGTACTTCGCCAGCATCGAGAAGTCCAACATCATCCGGGGCTGGATCTTCGGCACCACCCTGGATAGCAAGACCCAGAGCCTGCTGATCGTGAACAACGCCACCAACGTGGTGATCAAGGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGACCACAAGAACAACAAGAGCTGGATGGAGAGCGAGTTCCGGGTGTACAGCAGCGCCAATAACTGCACCTTCGAGTACGTGAGCCAGCCATTCCTGATGGACCTGGAGGGCAAGCAGGGCAACTTCAAGAACCTGCGGGAGTTCGTGTTCAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACACCCCAATCATCGTGCGGGAGCCAGAGGACCTGCCACAGGGATTTTCTGCCCTGGAGCCCCTGGTGGACCTGCCAATCGGAATCAATATCACCAGGTTCCAGACCCTGCTGGCCCTGCACAGAAGCTACCTGACACCTGGCGATTCATCTAGCGGCTGGACAGCCGGCGCCGCCGCTTACTATGTGGGCTACCTGCAGCCCAGAACCTTCCTGCTGAAGTACAACGAGAACGGCACCATCACAGATGCCGTGGACTGTGCCCTGGACCCACTGAGCGAGACCAAGTGCACACTGAAGTCCTTCACCGTGGAGAAGGGCATCTACCAGACCAGCAACTTCCGGGTGCAGCCAACCGAGAGCATCGTGAGGTTCCCCAATATCACCAACCTGTGCCCCTTCGACGAGGTGTTCAACGCCACCAGGTTTGCCAGCGTGTACGCCTGGAATAGGAAGCGGATCAGCAATTGCGTGGCCGACTACAGCGTGCTGTACAACCTGGCCCCATTCTTCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAATGTGTACGCCGACAGCTTCGTGATCAGGGGCGATGAGGTGAGACAGATTGCCCCAGGCCAGACCGGCAATATCGCCGACTACAACTACAAGCTGCCAGACGACTTCACCGGCTGTGTGATCGCCTGGAACAGCAACAAGCTGGACAGCAAGGTGAGCGGCAACTACAACTACCTGTACCGGCTGTTCCGCAAGAGCAACCTGAAGCCCTTCGAGCGGGACATCAGCACCGAGATTTACCAGGCCGGCAATAAGCCATGCAACGGCGTGGCCGGCTTCAACTGCTACTTCCCACTGAGGAGCTACAGCTTCAGACCAACCTACGGCGTGGGCCACCAGCCATATAGAGTCGTGGTGCTGAGCTTTGAACTGCTGCACGCTCCAGCCACAGTGTGCGGCCCTAAGAAGTCCACCAACCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGAAGGGCACCGGCGTGCTGACCGAAAGCAACAAGAAGTTCCTGCCCTTCCAGCAGTTCGGCAGAGATATCGCCGACACCACAGATGCCGTGAGAGATCCCCAGACCCTGGAGATCCTGGATATCACCCCATGCAGCTTTGGCGGCGTGAGCGTGATCACCCCAGGCACCAATACCAGCAACCAGGTGGCTGTGCTGTATCAGGGCGTGAATTGCACAGAGGTGCCCGTGGCCATTCACGCCGATCAGCTGACCCCAACCTGGAGAGTGTACAGCACCGGCTCTAATGTGTTCCAGACAAGAGCCGGCTGCCTGATCGGCGCCGAGTACGTGAACAATAGCTACGAGTGCGATATCCCAATTGGCGCCGGCATCTGTGCCAGCTACCAGACCCAGACAAAGTCTCACAGAGCTGCCGCCTCTGTGGCCAGCCAGAGCATTATCGCCTACACCATGAGCCTGGGCGCCGAAAATAGCGTGGCCTACAGCAACAACAGCATCGCCATCCCCACCAACTTCACCATCAGCGTGACCACCGAGATCCTGCCCGTGAGCATGACCAAGACCAGCGTGGATTGCACCATGTACATCTGCGGCGACAGCACCGAGTGCAGCAACCTCCTGCTGCAGTATGGCAGCTTTTGCACCCAGCTGAAAAGAGCCCTGACAGGCATCGCCGTGGAGCAAGACAAGAACACCCAGGAGGTGTTCGCCCAGGTGAAGCAGATCTACAAGACCCCACCCATCAAGTACTTCGGCGGCTTCAACTTCAGCCAGATCCTGCCTGATCCCAGCAAGCCCAGCAAGAGAAGCCCCATCGAGGACCTGCTGTTCAATAAGGTGACCCTGGCCGACGCTGGCTTCATCAAACAGTACGGAGATTGTCTGGGCGACATCGCCGCCAGAGACCTGATCTGCGCCCAGAAGTTCAAAGGCCTGACCGTGCTGCCACCACTGCTGACAGACGAGATGATTGCCCAGTACACCAGCGCCCTGCTGGCCGGCACCATCACAAGCGGATGGACCTTTGGAGCCGGACCCGCCCTGCAGATCCCATTTCCAATGCAGATGGCCTACAGGTTCAACGGCATCGGCGTGACCCAGAACGTGCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAAATCCAGGACAGCCTGAGCAGCACACCTTCTGCCCTGGGCAAGCTGCAGGATGTGGTGAATCACAACGCCCAGGCCCTGAACACCCTGGTGAAACAGCTGAGCAGCAAGTTCGGCGCCATCAGCAGCGTGCTGAATGATATCTTCAGCAGGCTGGACCCACCTGAGGCCGAGGTGCAGATCGATAGACTGATCACCGGCAGACTGCAGTCTCTGCAGACCTACGTGACCCAGCAACTGATCAGAGCCGCCGAGATCAGAGCTTCTGCCAACCTGGCCGCCACCAAGATGTCTGAGTGCGTGCTGGGACAGAGCAAGAGAGTGGACTTCTGTGGCAAGGGCTATCACCTGATGAGCTTCCCACAGTCTGCCCCACACGGAGTGGTGTTCCTGCACGTGACATACGTGCCCGCCCAGGAGAAGAATTTTACCACCGCCCCAGCCATCTGTCACGACGGCAAGGCCCACTTTCCAAGAGAGGGCGTGTTCGTGAGCAACGGCACCCACTGGTTCGTGACCCAGCGGAACTTCTACGAGCCCCAGATCATCACCACCGACAACACCTTCGTGAGCGGCAACTGCGACGTGGTGATCGGCATCGTGAACAACACCGTGTACGATCCACTGCAGCCCGAGCTGGACAGCTTCAAGGAGGAGCTGGACAAGTACTTCAAGAACCACACCAGCCCAGATGTGGACCTGGGCGACATCAGCGGCATCAATGCCAGCGTGGTGAACATCCAGAAGGAGATCGACCGGCTGAATGAGGTGGCCAAGAACCTGAACGAGAGCCTGATCGACCTGCAGGAGCTGGGCAAGTACGAGCAGTACATCAAGTGGCCATGGTACATCTGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCTGTGCTGCATGACCAGCTGCTGCAGCTGTCTGAAGGGCTGCTGTAGCTGCGGCAGCTGCTGTAAGTTCGATGAGGACGATAGCGAGCCCGTGCTGAAGGGCGTGAAGCTGCACTACACCTGATGA。

SEQ ID NO.10：

ATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGAGCAGCCAGTGCGTGAACCTGACCACCAGAACCCAGCTGCCTCCCGCCTACACCAACAGCTTCACCAGAGGCGTGTACTACCCCGACAAGGTGTTCAGGAGCAGCGTGCTGCACAGCACCCAGGACCTGTTCCTGCCCTTCTTCTCCAACGTGACCTGGTTCCACGTGATCAGCGGCACCAACGGCACAAAGCGGTTCGACAACCCCGTGCTGCCCTTCAACGACGGCGTGTACTTCGCCAGCATCGAGAAGTCCAACATCATCCGGGGCTGGATCTTCGGCACCACCCTGGACAGCAAGACCCAGAGCCTGCTGATCGTGAACAACGCCACCAACGTGGTGATCAAGGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGACCACAAGAACAACAAGAGCTGGATGGAGAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACCTTCGAGTACGTGAGCCAGCCCTTCCTGATGGACCTGGAGGGCAAGCAGGGCAACTTCAAGAACCTGCGGGAGTTCGTGTTCAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACACCCCTATCATCGTGCGGGAGCCTGAGGACCTGCCTCAGGGATTCAGCGCTCTGGAGCCTCTGGTGGACCTGCCTATCGGCATCAACATCACCCGGTTCCAGACCCTGCTGGCCCTGCACAGAAGCTACCTGACACCTGGCGACAGCAGCAGCGGATGGACAGCTGGAGCCGCCGCCTACTACGTGGGCTACCTGCAGCCTCGGACCTTCCTGCTGAAGTACAACGAGAACGGCACAATCACCGACGCCGTGGACTGCGCCCTGGACCCTCTGAGCGAGACAAAGTGCACCCTGAAGTCCTTCACCGTGGAGAAGGGCATCTACCAGACCAGCAACTTCCGGGTGCAGCCCACCGAGAGCATCGTGCGGTTCCCCAACATCACCAACCTGTGCCCCTTCGACGAGGTGTTCAACGCCACCAGATTCGCCAGCGTGTACGCCTGGAACCGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACCTGGCCCCTTTCTTCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAGGTGAGACAGATCGCCCCTGGCCAGACCGGCAACATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAAGCTGGACAGCAAGGTGAGCGGCAACTACAACTACCTGTACCGGCTGTTCCGCAAGAGCAACCTGAAGCCCTTCGAGCGGGACATCAGCACAGAGATCTACCAGGCCGGCAACAAGCCTTGCAACGGCGTGGCCGGCTTCAACTGCTACTTCCCTCTGCGGAGCTACAGCTTCAGACCCACCTACGGCGTGGGCCACCAGCCTTACAGAGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCTGCCACAGTGTGCGGCCCCAAGAAGTCCACAAACCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGAAGGGCACCGGCGTGCTGACCGAGAGCAACAAGAAGTTCCTGCCCTTCCAGCAGTTCGGCAGAGACATCGCCGACACAACCGACGCCGTGAGAGACCCTCAGACACTGGAGATCCTGGACATCACACCCTGCAGCTTCGGCGGCGTGAGCGTGATCACCCCTGGCACAAACACAAGCAACCAGGTGGCCGTGCTGTACCAGGGCGTGAACTGCACAGAGGTGCCTGTGGCCATCCACGCCGACCAGCTGACCCCTACCTGGAGAGTGTACAGCACCGGCAGCAACGTGTTCCAGACCAGAGCCGGCTGCCTGATCGGCGCCGAGTACGTGAACAACAGCTACGAGTGCGACATCCCTATCGGCGCCGGCATCTGCGCCAGCTACCAGACACAGACAAAGAGCCACAGAGCCGCCGCCAGCGTGGCCAGCCAGAGCATCATCGCCTACACAATGAGCCTGGGCGCCGAGAACAGCGTGGCCTACAGCAACAACAGCATCGCCATCCCCACCAACTTCACCATCAGCGTGACCACCGAGATCCTGCCCGTGAGCATGACCAAGACCAGCGTGGACTGCACCATGTACATCTGCGGCGACAGCACCGAGTGCAGCAACCTGCTGCTGCAGTACGGCAGCTTCTGCACCCAGCTGAAGAGAGCCCTGACAGGCATCGCCGTGGAGCAGGACAAGAACACCCAGGAGGTGTTCGCCCAGGTGAAGCAGATCTACAAGACCCCTCCCATCAAGTACTTCGGCGGCTTCAACTTCAGCCAGATCCTGCCCGACCCCAGCAAGCCCAGCAAGAGAAGCCCCATCGAGGACCTGCTGTTCAACAAGGTGACACTGGCCGACGCCGGCTTCATCAAGCAGTACGGCGACTGCCTGGGCGATATCGCCGCCAGAGACCTGATCTGCGCCCAGAAGTTCAAGGGCCTGACCGTGCTGCCTCCTCTGCTGACAGACGAGATGATCGCCCAGTACACAAGCGCCCTGCTGGCCGGCACAATCACAAGCGGCTGGACATTCGGCGCCGGACCTGCCCTGCAGATCCCTTTCCCTATGCAGATGGCCTACCGGTTCAACGGCATCGGCGTGACCCAGAACGTGCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGATCCAGGACAGCCTGAGCAGCACACCTAGCGCTCTGGGCAAGCTGCAGGACGTGGTGAACCACAACGCCCAGGCCCTGAACACCCTGGTGAAGCAGCTGAGCAGCAAGTTCGGCGCCATCAGCAGCGTGCTGAACGACATCTTCAGCAGACTGGACCCTCCTGAGGCCGAGGTGCAGATCGACAGACTGATCACAGGCAGACTGCAGAGCCTGCAGACCTACGTGACCCAGCAGCTGATCAGAGCCGCCGAGATCAGAGCCAGCGCCAACCTGGCCGCCACAAAGATGAGCGAGTGCGTGCTGGGCCAGAGCAAGAGAGTGGACTTCTGCGGCAAGGGCTACCACCTGATGAGCTTCCCTCAGAGCGCCCCTCACGGCGTGGTGTTCCTGCACGTGACATACGTGCCTGCCCAGGAGAAGAACTTCACCACCGCCCCTGCCATCTGCCACGACGGCAAGGCCCACTTCCCTAGAGAGGGCGTGTTCGTGAGCAACGGCACACACTGGTTCGTGACCCAGCGGAACTTCTACGAGCCCCAGATCATCACCACCGACAACACCTTCGTGAGCGGCAACTGCGACGTGGTGATCGGCATCGTGAACAACACCGTGTACGACCCTCTGCAGCCCGAGCTGGACAGCTTCAAGGAGGAGCTGGACAAGTACTTCAAGAACCACACCAGCCCTGACGTGGACCTGGGCGACATCAGCGGCATCAACGCCAGCGTGGTGAACATCCAGAAGGAGATCGACCGGCTGAACGAGGTGGCCAAGAACCTGAACGAGAGCCTGATCGACCTGCAGGAGCTGGGCAAGTACGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCTGTGCTGCATGACCAGCTGCTGCAGCTGCCTGAAGGGCTGCTGCAGCTGCGGCAGCTGCTGCAAGTTCGACGAGGACGACAGCGAGCCTGTGCTGAAGGGCGTGAAGCTGCACTACACCTGATGA。

SEQ ID NO.11：

ATGTTCGTGTTCCTGGTGCTGCTGCCACTGGTGTCTTCTCAGTGTGTGAACCTGACCACCAGAACACAGCTGCCTCCAGCCTACACCAACAGCTTCACCCGGGGAGTGTACTACCCAGACAAGGTGTTCAGGAGCAGCGTGCTGCACAGCACCCAGGACCTGTTCCTGCCATTCTTCAGCAACGTGACCTGGTTCCACGTGATCAGCGGCACCAACGGCACCAAGAGATTCGACAACCCTGTGCTGCCCTTCAACGACGGCGTGTACTTCGCCAGCATCGAGAAGTCCAACATCATCCGGGGCTGGATCTTCGGCACAACACTGGACAGCAAGACCCAGAGCCTGCTGATCGTGAACAACGCCACCAACGTGGTGATCAAGGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGACCACAAGAACAACAAGAGCTGGATGGAGAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACCTTCGAGTACGTGAGCCAGCCCTTTCTGATGGACCTGGAGGGCAAGCAGGGCAACTTCAAGAACCTGCGGGAGTTCGTGTTCAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACACCCCAATCATCGTGCGGGAGCCAGAGGACCTGCCACAGGGATTTTCTGCCCTGGAGCCCCTGGTGGATCTGCCAATCGGCATCAACATCACCAGGTTCCAGACCCTGCTGGCCCTGCACAGGAGCTATCTGACACCAGGCGATTCCAGCAGCGGCTGGACAGCCGGCGCAGCCGCCTATTACGTGGGCTACCTGCAGCCCAGAACATTCCTGCTGAAGTACAACGAGAACGGCACAATCACAGACGCCGTGGATTGCGCCCTCGACCCTCTGAGCGAGACCAAGTGCACCCTGAAGTCCTTCACCGTGGAGAAGGGCATCTACCAGACCAGCAACTTCAGGGTGCAGCCTACAGAGAGCATCGTGCGGTTCCCCAACATCACCAATCTGTGCCCCTTTGACGAGGTGTTCAACGCCACCCGGTTTGCTAGCGTGTACGCCTGGAACAGGAAGCGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACCTGGCCCCATTCTTCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACTCTTTCGTGATCAGAGGCGACGAGGTGAGACAGATCGCCCCTGGCCAGACAGGCAATATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATTGCCTGGAACAGCAACAAGCTGGACAGCAAGGTGAGCGGCAACTACAACTACCTGTACCGGCTGTTCCGCAAGAGCAACCTGAAGCCCTTCGAGAGAGACATCAGCACCGAGATCTACCAGGCCGGCAACAAGCCATGTAACGGCGTGGCCGGCTTCAACTGCTACTTTCCCCTGCGGAGCTACAGCTTCAGACCAACCTATGGCGTGGGCCACCAGCCCTATAGAGTCGTGGTGCTGAGCTTTGAGCTGCTGCACGCCCCTGCTACAGTGTGTGGCCCAAAGAAGTCCACCAACCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGAAGGGCACCGGCGTGCTGACCGAGAGCAATAAGAAGTTCCTGCCCTTCCAGCAGTTCGGCAGAGACATCGCCGACACAACCGATGCCGTGAGAGACCCACAGACCCTGGAGATCCTGGACATCACCCCATGCAGCTTTGGAGGCGTGAGCGTGATCACACCAGGCACCAATACCAGCAATCAGGTGGCCGTGCTGTACCAGGGCGTGAACTGCACAGAAGTGCCAGTGGCTATTCACGCCGACCAGCTGACCCCAACCTGGAGAGTGTACAGCACCGGAAGCAATGTGTTCCAGACCCGGGCTGGCTGCCTGATCGGCGCCGAGTACGTGAATAATAGCTACGAGTGCGACATCCCTATCGGAGCCGGCATCTGCGCCAGCTATCAGACCCAGACAAAGAGCCATAGAGCTGCCGCCTCTGTGGCCTCTCAGAGCATCATCGCCTACACCATGAGCCTGGGAGCCGAGAACAGCGTGGCCTACAGCAATAACAGCATCGCCATCCCCACCAACTTCACCATCAGCGTGACCACAGAGATCCTGCCCGTGAGCATGACCAAGACCAGCGTGGATTGCACCATGTACATCTGCGGCGACAGCACCGAGTGTAGCAACCTCCTGCTGCAGTATGGCAGCTTCTGCACCCAGCTGAAGAGAGCCCTGACAGGCATCGCCGTGGAGCAGGATAAGAATACCCAGGAGGTGTTCGCCCAGGTGAAGCAGATCTACAAGACCCCACCCATCAAGTACTTCGGCGGCTTCAATTTCAGCCAGATCCTGCCCGACCCCAGCAAGCCCTCTAAGAGAAGCCCAATCGAGGACCTGCTGTTCAACAAGGTGACCCTGGCCGACGCCGGCTTTATTAAGCAGTACGGCGACTGTCTGGGAGACATTGCCGCCAGAGATCTGATCTGTGCCCAGAAGTTCAAGGGCCTGACAGTGCTGCCACCCCTGCTGACCGATGAGATGATCGCCCAATACACCAGCGCCCTGCTGGCCGGCACAATCACCAGCGGCTGGACATTCGGCGCCGGACCAGCCCTGCAGATCCCATTCCCAATGCAGATGGCCTACAGATTCAACGGCATCGGCGTGACCCAGAACGTGCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGATCCAGGACAGCCTGAGCAGCACACCTTCTGCCCTGGGAAAGCTGCAGGACGTGGTGAATCACAATGCCCAGGCCCTGAATACCCTGGTGAAGCAGCTGAGCAGCAAGTTCGGCGCCATCAGCAGCGTGCTGAATGACATCTTCAGCAGGCTGGACCCACCTGAAGCCGAGGTGCAGATCGACAGACTGATCACCGGAAGACTGCAGAGCCTGCAGACCTACGTGACCCAGCAGCTGATCAGAGCCGCCGAGATTAGAGCCAGCGCCAACCTGGCTGCCACCAAAATGTCTGAGTGTGTGCTGGGCCAGAGCAAGCGGGTGGATTTCTGCGGCAAAGGCTACCACCTGATGAGCTTCCCACAGTCTGCCCCACACGGAGTGGTGTTCCTGCACGTGACATACGTGCCAGCCCAGGAGAAGAATTTCACCACCGCCCCAGCCATCTGTCATGATGGAAAGGCCCACTTCCCAAGGGAGGGCGTGTTTGTGAGCAACGGCACCCACTGGTTCGTGACCCAGAGGAACTTCTACGAGCCCCAGATCATCACCACCGACAACACCTTTGTGAGCGGCAACTGCGACGTGGTGATCGGCATCGTGAACAATACCGTGTACGACCCACTGCAGCCCGAGCTGGACAGCTTTAAGGAGGAGCTGGACAAGTACTTCAAGAACCACACCAGCCCTGACGTGGATCTGGGCGACATCAGCGGCATCAACGCCAGCGTGGTGAATATCCAGAAGGAGATCGACCGGCTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCGACCTGCAGGAGCTGGGCAAGTACGAGCAGTACATCAAGTGGCCCTGGTATATCTGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCTGTGCTGCATGACCAGCTGCTGCAGCTGTCTGAAGGGCTGCTGTAGCTGTGGCAGCTGCTGCAAGTTTGATGAGGACGACAGCGAGCCAGTGCTGAAGGGCGTGAAGCTGCACTACACCTGATGA。

SEQ ID NO.12：

ATGTTCGTGTTCCTGGTGCTGCTGCCACTGGTGAGCAGCCAGTGCGTGAATCTGACAACCAGGACACAGCTGCCACCCGCCTACACCAATAGCTTCACCAGAGGCGTGTACTACCCAGACAAGGTGTTCAGGAGCAGCGTGCTGCACAGCACCCAGGACCTGTTCCTGCCCTTCTTCAGCAATGTGACCTGGTTCCACGTGATCAGCGGCACCAACGGCACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGCGTGTACTTCGCCAGCATCGAGAAGTCCAACATCATCCGGGGCTGGATCTTCGGCACCACACTGGACAGCAAGACCCAGAGCCTGCTGATCGTGAACAACGCCACCAACGTGGTGATCAAGGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGACCACAAGAACAACAAGAGCTGGATGGAGAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACATTCGAGTACGTGAGCCAGCCATTCCTGATGGACCTGGAGGGCAAGCAGGGCAACTTCAAGAACCTGCGGGAGTTCGTGTTCAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACACCCCAATCATCGTGAGAGAGCCTGAGGATCTGCCTCAGGGCTTTTCTGCTCTGGAGCCACTGGTGGACCTGCCCATCGGCATCAATATCACCAGATTTCAGACCCTGCTGGCCCTGCACAGAAGCTACCTGACACCTGGCGATTCTAGCAGCGGCTGGACAGCTGGAGCTGCCGCCTACTACGTGGGCTACCTGCAGCCAAGGACCTTCCTGCTGAAGTACAACGAGAACGGCACAATCACCGACGCTGTGGATTGTGCCCTGGACCCACTGAGCGAGACCAAGTGCACCCTGAAGTCCTTCACCGTGGAGAAGGGCATCTACCAGACCAGCAACTTCCGGGTGCAGCCCACCGAGAGCATCGTGAGGTTCCCAAACATCACCAACCTGTGCCCCTTCGACGAGGTGTTTAACGCCACCCGGTTCGCCAGCGTGTACGCCTGGAACAGAAAGAGGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACCTGGCCCCATTCTTCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGATGAGGTGAGACAGATTGCCCCAGGCCAGACCGGCAATATCGCCGACTACAATTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAATAAGCTGGACAGCAAGGTGAGCGGCAACTACAACTACCTGTACCGGCTGTTCCGCAAGAGCAACCTGAAGCCCTTCGAGAGAGACATCAGCACCGAGATCTACCAGGCCGGCAACAAGCCATGCAATGGAGTGGCCGGCTTCAACTGCTACTTCCCACTGAGGAGCTACAGCTTCAGACCCACATACGGCGTGGGCCACCAGCCCTACAGAGTCGTGGTGCTGTCTTTTGAACTGCTGCATGCCCCAGCCACCGTGTGCGGCCCAAAGAAGTCCACCAACCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGAAGGGCACCGGCGTGCTGACCGAGAGCAACAAGAAATTCCTGCCCTTCCAGCAGTTCGGCAGAGACATCGCCGATACCACCGACGCCGTGAGAGATCCACAGACACTGGAGATCCTGGACATCACCCCATGCAGCTTCGGCGGAGTGAGCGTGATCACACCCGGCACAAACACCAGCAATCAGGTGGCCGTGCTGTACCAGGGCGTGAATTGTACAGAGGTGCCCGTGGCCATTCACGCCGATCAGCTGACCCCTACCTGGAGAGTGTACAGCACAGGAAGCAATGTGTTCCAGACCAGGGCCGGCTGCCTGATCGGAGCCGAGTACGTGAACAACAGCTACGAGTGCGACATCCCAATCGGCGCCGGCATCTGTGCCAGCTACCAGACCCAGACAAAATCCCACAGAGCTGCCGCTTCTGTGGCCAGCCAGAGCATCATCGCCTACACAATGAGCCTGGGCGCCGAGAATAGCGTGGCCTACAGCAATAACAGCATCGCCATCCCAACCAACTTCACCATCAGCGTGACCACCGAGATCCTGCCCGTGAGCATGACAAAGACCAGCGTGGACTGCACCATGTACATCTGCGGCGATAGCACCGAGTGCAGCAATCTCCTGCTGCAGTACGGCAGCTTTTGCACCCAGCTGAAGAGAGCCCTGACAGGCATTGCCGTGGAGCAGGACAAGAACACCCAGGAGGTGTTCGCCCAGGTGAAGCAGATCTACAAGACCCCACCCATCAAGTACTTCGGCGGCTTCAACTTCAGCCAGATTCTGCCTGACCCTAGCAAGCCCAGCAAGAGAAGCCCCATCGAGGACCTGCTGTTCAATAAGGTGACCCTGGCCGACGCCGGCTTCATCAAGCAGTACGGCGACTGTCTGGGCGATATTGCCGCCAGAGACCTGATCTGCGCCCAGAAGTTCAAGGGCCTGACAGTGCTGCCACCCCTGCTGACCGATGAGATGATCGCTCAGTACACATCTGCCCTGCTGGCCGGCACAATCACAAGCGGCTGGACATTTGGCGCCGGACCTGCTCTGCAGATCCCATTCCCAATGCAGATGGCCTACAGGTTCAACGGCATCGGCGTGACCCAGAACGTGCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGATCCAGGATAGCCTGTCTAGCACACCAAGCGCCCTGGGCAAGCTGCAGGACGTGGTGAATCACAACGCCCAAGCCCTGAACACCCTGGTGAAGCAGCTGAGCAGCAAGTTCGGCGCCATCAGCAGCGTTCTGAACGACATCTTCAGCAGACTGGACCCTCCTGAGGCCGAGGTGCAGATCGATAGACTGATCACCGGAAGACTGCAGAGCCTGCAGACCTACGTGACACAGCAGCTGATCAGAGCCGCCGAGATCAGAGCCTCTGCCAATCTGGCCGCCACAAAGATGAGCGAGTGTGTGCTGGGCCAGAGCAAGAGAGTGGACTTCTGCGGCAAGGGCTACCACCTGATGAGCTTCCCACAGTCTGCCCCACACGGCGTGGTGTTCCTGCATGTGACATACGTGCCAGCCCAGGAGAAGAACTTTACCACCGCCCCTGCCATCTGCCACGATGGCAAAGCCCACTTCCCCAGAGAGGGCGTGTTTGTGAGCAATGGCACACACTGGTTCGTGACCCAGAGGAACTTCTACGAGCCCCAGATCATCACCACCGACAACACCTTCGTGAGCGGCAACTGCGACGTGGTGATCGGCATCGTGAACAACACCGTGTACGACCCTCTGCAGCCTGAGCTGGACAGCTTCAAGGAGGAGCTGGACAAGTACTTCAAGAACCACACCAGCCCAGATGTGGACCTGGGCGACATCAGCGGCATCAATGCCAGCGTGGTGAACATCCAGAAGGAGATCGACCGGCTGAACGAAGTGGCCAAGAACCTGAACGAGAGCCTGATCGACCTGCAGGAGCTGGGCAAGTACGAGCAGTACATCAAGTGGCCATGGTACATCTGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCATCATGCTGTGCTGCATGACCAGCTGCTGCTCTTGCCTGAAGGGCTGCTGTAGCTGCGGCAGCTGCTGCAAGTTCGATGAGGATGACAGCGAGCCCGTGCTGAAGGGCGTGAAGCTGCACTACACCTGATGA。

2. preparation of mRNA

1. Linearization template preparation

Recombinant plasmids S4-0432-pNeoCura-Bvac, S4-0445-pNeoCura-Bvac, S4-1889-pNeoCura-Bvac, S4-0932-pNeoCura-Bvac and S4-0792-pNeoCura-Bvac were taken, and restriction enzymes BspQ I (NEB, cat# R0712L) were used to perform cleavage, and VAHTS DNA Clean Beads (Nuo' S praise, N411-01) was used to purify the linearized plasmid after cleavage. The plasmid map of the recombinant plasmid S4-1889-pNeoCura-Bvac before enzyme digestion is shown in figure 1; the agarose gel electrophoresis results of the linear plasmids before cleavage and after cleavage of the respective recombinant plasmids were purified are shown in FIG. 2.

2. In vitro transcription to RNA

RNA was obtained by in vitro transcription using the in vitro transcription kit T7 High Yield RNA Transcription Kit (N1-Me-pseudoUTP) (Norpran, DD 4202). RNA was purified using VAHTS RNA Clean Beads (Vazyme, N412-01).

3. Capping and purification

The purified RNA was subjected to capping using the capping kit mRNA Cap 2' -O-methyl transfer ferase (Northenzan, DD 4110). The capped RNA was purified using VAHTS RNA Clean Beads (Norflu, cat# N412-01) to give mRNA of the corresponding sequence, and the agarose gel electrophoresis results are shown in FIG. 3.

3. In vitro mRNA expression

HEK293T cell plating

Cell inoculation 24 pore plate (2.5-3×10) ⁵ Well)) using DMEM medium (Thermo Fisher Scientific) with 10% serum and 1% diabody at 37 ℃,5% CO ₂ Culturing for 16-18 hr to reach cell density of 80% fusion before transfection.

2. Transfection

Replace with serum-free Opti-MEM Medium (Thermo Fisher Scientific), mRNA (0.3. Mu.g, 1. Mu.g RNA/well) and Lipofectamine ^TM Messenger MAX ^TM After mixing the transfection reagent (Thermo Fisher Scientific) HEK293T cells were transfected and cultured for 6 hours, the medium was replaced with DMEM medium (Thermo Fisher Scientific) containing 10% serum and 1% penicillin and the culture was continued for 24 hours.

3. Flow cytometry to detect expression of cell surface Spike proteins

24 hours after transfection, 1000g was centrifuged for 10 minutes, and cells were collected and incubated with anti-SARS-CoV-2 Spike RBD monoclonal neutralizing antibody AM122 (Beijing Baips Biotech Co., ltd., S1N-M122). Flow cytometry (BD, FACS Celesta) detects the Mean Fluorescence Intensity (MFI) of cell surface expressed Spike protein binding to neutralizing antibody AM 122. In vitro mRNA expression showed that the expression level of Spike protein on the surface of HEK293T cells transfected with the sequence S4-1889mRNA was significantly higher than that of other optimized sequences (see FIG. 4 for results).

Example 2 in vitro expression of S4-1889mRNA and verification of protein Properties

HEK293T cell plating

Cell inoculation 24 pore plate (2.5-3×10) ⁵ Well)) using DMEM medium (Thermo Fisher Scientific) with 10% serum and 1% diabody at 37 ℃,5% CO ₂ Culturing for 16-18 hr to reach cell density of 80% fusion before transfection.

2. Transfection

Replaced with serum-free Opti-MEM medium (Thermo Fisher Scientific), GFP mRNA (encoding green fluorescent protein GFP mRNA, control mRNA) and S4-1889mRNA (0.3 ug or 1 ug RNA/well) with Lipofectamine after 1 hour ^TM Messenger MAX ^TM After the transfection reagent (Thermo Fisher Scientific) was mixed and the cells were transfected, the medium was replaced with DMEM medium (Thermo Fisher Scientific) containing 10% serum and 1% penicillin and cultured for another 24 hours.

Conformational verification of F protein expressed by S4-1889mRNA transfected cells and experiments on binding to hACE2

Cells after 24 hours of S4-1889mRNA transfection were centrifuged at 1000g for 10 minutes, and the cells were collected and incubated with anti-SARS-CoV-2 Spike RBD monoclonal neutralizing antibody AM122 (S1N-M122, beijing Baips Biotechnology Co., ltd.) and hACE2 (Sino Biological, 10108-H02H), respectively. Cell surface expressed Spike protein was tested for its ability to bind to neutralizing antibodies AM122 and hACE2 by flow (BD, FACS Celesta) (see fig. 5 for results). The results show that: s4-1889mRNA transfected cells expressed Spike protein that bound to neutralizing antibodies AM122 and hACE 2.

4. Protein molecular weight validation

After 24 hours of transfection HEK293T cells were lysed, western blot was used to detect S protein in the lysate supernatant. Western blot uses monoclonal neutralizing antibody R001 (Sino Biological, 40592-R001) of anti-SARS-CoV-2 Spike protein, positive control protein is commercial SARS-CoV-2 Omicron BA.1Spike Trimer Protein (Beijing Bai Sai Biotech Co., ltd., product number: SPN-C522 a), and Western blot results are shown in FIG. 6. The results showed that bands of expected molecular weight (150-200 Kda) were detected between 130Kda and 250Kda, indicating that S4-1889mRNA transfected cells expressed Spike full-length protein.

EXAMPLE 3S 4-1889mRNA LNP (lipid nanoparticle) preparation

1. Dissolving the prepared S4-1889mRNA on ice, and diluting to 100ng/ul with a prepared 50mM sodium citrate buffer solution to obtain an aqueous phase; ethanol phase was prepared using absolute ethanol as solvent according to SM102: DSPC: cholesterol: DMG-pe2000=50: 10:38.5:1.5 was formulated so that the SM102 concentration in the ethanol phase was 6.22mg/ml and the liposome concentration was 10.87mg/ml. (SM 102: xiaomenobang Biotechnology Co., ltd., CAS number: 06040008800; DSPC: ai Weita (Shanghai) medical technology Co., ltd., CAS number: S01005; CHO-HP (cholesterol) Ai Weita (Shanghai) medical technology Co., ltd., CAS number: O01001; DMG-PEG2000: ai Weita (Shanghai) medical technology Co., ltd., CAS number: O02005).

2. The aqueous phase and the ethanol phase are compared with the aqueous phase according to the flow rate: alcohol phase = 3:1, total flow rate of 12mL/min, coating using a nano-drug preparation system (INano L, micana (Shanghai) instrument technologies limited) and collecting LNP. Diluting the collected LNP 3 times with RNase-free water, placing into a 100kd dialysis bag, and dialyzing the dialysis bag in 200 times volume of LNP 1 XPBS with a magnetic stirrer at 300rpm at room temperature for 18h; collecting LNP in the dialysis bag, and sterilizing and filtering by using a 0.22um filter membrane; the LNP after filtration was centrifuged at 3000rpm and 4℃for 90min using a 100kd ultrafiltration tube, the LNP in the ultrafiltration tube was collected, the residual LNP in the tube was rinsed with 1 XPBS, and the LNP was mixed with the previous LNP to obtain S4-1889mRNA-LNP, which was stored at 2-8℃in the dark.

3. Detecting PDI, particle size and Zeta potential of S4-1889mRNA LNP by using a nano particle size meter (Litesizer 500, anton Paar); particle size: 93.76nm; PDI:19.1%; zeta potential: 28mV. The S4-1889mRNA LNP was shown to have a stable structure. The potential measurement results are shown in FIG. 7, and the particle size results are shown in FIG. 8.

4.S4-1889 mRNA LNP was taken and the encapsulation efficiency was calculated using Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific) and was 92.73%.

5. The S4-1889mRNA LNP is taken, and the form and structure of the LNP are observed by using a cryoelectron microscope, which shows that the S4-1889mRNA LNP has uniform spherical form and structure and the grain diameter is 100-120 nm. See fig. 9.

Example 4S 4-1889mRNA LNP vaccine immunization of Babl/c mice induced humoral and T cell immune responses.

1. Immunization protocol

Female Babl/c mice of 6-8 weeks of age were randomly divided into 4 groups of 10 mice each. Negative control (empty LNP), S4-1889mRNA LNP low dose, medium dose, high dose. Each group of mice was immunized by intramuscular injection on day 1 (Prime) of the experiment, day 21 (Boost) and the immunization volume was 50 μl. The immunization groups and dosages are shown in the following table:

negative control group	Injection of empty LNP
		Low dose group	Injection of S4-1889mRNA LNP prepared in example 3, 1. Mu.g
Medium dose group	Injection of S4-1889mRNA LNP prepared in example 3, 3. Mu.g
		High dose group	Injection of S4-1889mRNA LNP prepared in example 3, 10. Mu.g

Note that: the dose of each dose group is the dose of RNA.

S4-1889mRNA LNP vaccine immunization induced the serum of Babl/c mice to produce Spike protein-specific binding antibodies.

On experiment day 14 (14 days post-Prime), experiment day 35 (14 days post-Boost) were bled through the mouse retroorbital venous plexus, serum samples were isolated, heated at 56℃for 30 minutes for inactivation, and the inactivated serum was subjected to gradient dilution with PBS buffer to obtain test serum samples. Antigen-specific antibodies in mouse serum were detected using ELISA kit (Vazyme, cat# DD 3208), 3 multiplex wells were set per well of test serum sample, and the procedure was referred to the instructions provided by the manufacturer. The results show that: each dose group S4-1889mRNA LNP induced high titers of Omacron BA.1spike specific IgG in mice after primary immunization (Prime), the antibody titers increased 10-100 fold 14days after Boost, and the serum antibody titers induced by the high dose group immunization were significantly higher than those of the low dose group. No antibodies were generated by the negative control group (see fig. 10 for results).

S4-1889mRNA LNP vaccine immunization of Babl/c mice induced serum neutralizing antibodies (pseudovirus neutralizing antibody titre detection)

Mouse inactivated serum isolated on day 35 of the experiment (14 days post-Boost) was taken and the neutralizing antibody titer to Omicron (ba.1) strain in mouse immune serum was detected using a pseudo virus system based on HIV expressing Omicron (ba.1) strain Spike protein, respectively. Half-median neutralization titers (pNT 50) results are shown in fig. 11, which shows that: s4-1889mRNA LNP immunization of mice induced the production of high titer neutralizing antibodies against the Omacron BA.1 strain pseudovirus.

S4-1889mRNA LNP vaccine immunization of Babl/c mice induced spleen T cell responses.

On experiment day 49, mice were sacrificed to obtain spleens, elispot detected IFN-gamma and IL-4T cell responses in the spleens of the mice, the pool of peptides stimulating spleen cells was a pool of scanning peptides against Omacron BA.1spike, and the Elispot detection procedure was as described in the instructions provided by the commercial company (Mouse IFN-gamma Precoated ELISPOT Kit (strips), dayou, accession number 2210006; mouse IL-4Precoated ELISPOT Kit, dayou, accession number 2210403). The Elispot results are shown in FIGS. 12 and 13, which show that: s4-1889mRNA LNP immunization of mice induced T cell responses producing IFN-. Gamma. + in the spleen against the Omacron BA.1spike response, did not produce T cell responses producing IL-4+ against the Omacron BA.1spike response, indicating that S4-1889mRNA LNP immunization induced Th1 and CD8+ T cell responses in the spleen of mice, and did not induce Th2 responses.

Example 5S 4-1889mRNA LNP vaccine induces immunoprotection in human-ACE2 transgenic mice.

1. Immunization protocol

Female human-ACE2 transgenic mice (Jiangsu Jiugang biotechnology Co., ltd., product number: T037657) of 6-8 weeks old were randomly divided into 3 groups of 6-7 mice each. Negative control (empty LNP), S4-1889mRNA LNP low dose, high dose. Each group of mice was immunized by intramuscular injection on day 1 (Prime) of the experiment, day 21 (Boost) and the immunization volume was 50 μl.

Negative control group	Injection of empty LNP
		Low dose group	Injection of S4-1889mRNA LNP,3ug prepared in example 3
High dose group	Injection of S4-1889mRNA LNP,30ug prepared in example 3

Note that: the doses of each of the S4-1889mRNA LNP dose groups were RNA doses.

S4-1889mRNA LNP vaccine induced human-ACE2 transgenic mice to produce neutralizing antibodies to Omicron BA.1 and Delta strains.

The live virus neutralizing antibody titers against omacron ba.1 and Delta strains in the mouse immune serum were measured separately from the mouse inactivated serum isolated on day 35 of the experiment (14 days post-Boost). Half-median neutralization titers (NT 50) results are shown in fig. 14, which shows that: s4-1889mRNA LNP immunized mice induced high titers of neutralizing antibodies against the Omacron BA.1 strain, while also having high levels of cross-neutralizing antibody titers against the Delta strain.

3. Toxicity attack protection experiment

The human-ACE2 transgenic mice were challenged (2X 10) by nasal drip with Omacron BA.1 virus or Delta virus (supplied by the national Kunming high-grade biosafety laboratory center P3 laboratory) at day 30 (day 51) after the second immunization ³ TCID ₅₀ ) Viral load in mouse nasal swabs was measured 5 consecutive days after challenge (fig. 15), mice were sacrificed on day 5 post challenge, and pulmonary (fig. 16) viral load was measured. The results show that the S4-1889mRNA LNP vaccine immunization significantly reduces the pulmonary viral load of human-ACE2 transgenic mice and significantly reduces the viral load (Omicron BA.1 viral load reduction) in the upper respiratory tract (nasal swab) of the mice>5log copies/g, delta viral load reduction>2log copies/g). The S4-1889mRNA LNP vaccine immunization effectively reduces the pulmonary viral load and has immune protection effect on mice; the virus passing through the upper respiratory tract is cleared and inhibited, so that the transmission risk is reduced.

S4-1889mRNA LNP vaccine induces cross-reactive binding antibodies in serum of human-ACE2 transgenic mice to the Spike protein of the different subtypes of Omicron strain (BA.1, BA.2, BA.2.12.1, BA.4& 5).

ELISA kit (product number: RAS-T060, RAS-T089, RAS-T114, RAS-T121) was used to detect binding antibodies cross-reactive against different subtypes of Omicron mutant strain (BA.1, BA.2, BA.2.12.1, BA.4& 5) in mouse serum at day 35, respectively, and 3 multiplex wells were set per test serum sample, and the procedure was referred to the instructions provided by the manufacturer. The results show that: the mice were induced to produce high levels of antibodies recognizing Spike proteins from different omacron subtypes (BA.1, BA.2, BA.2.12.1, BA.4& 5) following LNP immunization with S4-1889mRNA in each dose group, and no antibodies were produced in the negative control group. (results see FIG. 17)

S4-1889mRNA LNP vaccine induces pseudo-virus neutralizing antibodies of human-ACE2 transgenic mice cross-reactive against Omicron different subtype strains (BA.1, BA.2, BA.4& 5).

Mouse inactivated serum isolated on day 35 of the experiment (14 days post-Boost) was taken and the neutralizing antibody titers to the Omicron different subtype strains in the mouse immune serum were detected using a pseudovirus system based on HIV expressing Omicron BA.1, BA.2, BA.4&5 strain Spike, respectively. The results of pseudovirus half-neutralization titers (pNT 50) are shown in fig. 18, which shows that: s4-1889mRNA LNP vaccine immunization induced high levels of pseudovirus neutralizing antibodies against different Omacron subtypes (BA.2 and BA.4/5), suggesting that S4-1889mRNA LNP vaccine could produce a broad spectrum of potent immunoprotection against different subtypes of Omacron strains.

In conclusion, mRNA of any one of the sequences shown in SEQ ID No.3 to SEQ ID No.7 can express Spike protein and can induce organism to form immune response. Further, the S4-1889mRNA LNP vaccine was able to provide effective immunoprotection against omacron ba.1, reducing the risk of transmission by reducing the upper respiratory viral load. S4-1889mRNA LNP vaccine immunization induces neutralizing antibodies to high levels of different subtypes and Delta strains of Omicron, which suggests that S4-1889mRNA LNP vaccine can produce broad-spectrum effective immunoprotection to different subtypes of Omicron strains and Delta strains.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (10)

1. An mRNA for translating a Spike protein, wherein the coding region of said mRNA comprises a nucleotide sequence set forth in any one of SEQ ID No.3 to SEQ ID No. 7.

2. The mRNA of claim 1, wherein the coding region of the mRNA comprises the nucleotide sequence set forth in SEQ ID No. 5.

3. A coding sequence for coding Spike protein, characterized in that the coding sequence is any one of SEQ ID No.8 to SEQ ID No. 12.

4. A coding sequence according to claim 3, wherein the coding sequence is the nucleotide sequence set forth in SEQ ID No. 10.

5. A coding gene for expressing Spike protein, wherein the coding sequence of the coding gene is any one of the coding sequences of claim 3 or 4.

6. The Spike protein is characterized in that the amino acid sequence of the Spike protein is shown as SEQ ID NO. 2.

7. Use of an mRNA according to claim 1 or 2 for the preparation of a vaccine for the prevention and/or treatment of novel coronaviruses.

8. An mRNA vaccine for preventing and/or treating a novel coronavirus, wherein the mRNA vaccine is prepared from the mRNA of claim 1 or 2 and a vector.

9. Use of the mRNA vaccine of claim 8 for the preparation of a medicament for the prevention and/or treatment of novel coronaviruses.

10. A method of preparing an mRNA vaccine according to claim 8, comprising the steps of:

dissolving the mRNA in a buffer solution to prepare a water phase, wherein the concentration of the mRNA is 90 ng/ul-110 ng/ul;

preparing the carrier into an ethanol phase by taking absolute ethanol as a solvent, wherein the concentration of the carrier is 9 mg/ml-11 mg/ml;

the aqueous phase and the ethanol phase are compared with the aqueous phase according to the flow rate: the alcohol phase is 2.5-5.5:1, the total flow rate is 10-14mL/min, coating is carried out, nano carrier particles loaded with mRNA are collected, and the mRNA vaccine particles are obtained after filtration and purification.

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