Replicase and application thereof

Abstract

The invention provides replicase and application thereof. Based on the sequence of a wild replicase, the replicase is mutated, so that a salt bridge, a hydrophobic bond or electrostatic interaction is formed in nsP1-4 protein. The main mutation sites of the gene comprise T372K of the protein nsP1, S54R of the protein nsP2, C157P, A161L, L168E, E404F of the protein nsP4 and the like, and the structural stability of the nsP1-4 protein can be remarkably improved, so that the novel replicase is prepared. According to the invention, the structural stability of replicase is improved, and more stable and massive amplification and translation of target protein can be realized. When the compound is used for preparing vaccines, the expression of a target protein can be remarkably increased in vitro under the same dosage, and higher specific antibody titer aiming at a target antigen and stronger cellular immune response are induced in vivo.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a replicase and an application thereof. Background Art

[0002] Coronavirus disease 19 (COVID-19), caused by SARS-CoV-2, RSV is the leading cause of acute lower respiratory tract infection (LRTI), and worldwide, an estimated 118,200-149,400 children under 5 years old die from RSV each year. The development of vaccines for respiratory infectious diseases is necessary and urgent. Several recent studies have shown that the proportion of coinfection with RSV in people who have been infected with SARS-CoV-2 is about 3%; the coinfection rate in children under 24 months of age in Brazil is as high as 18.7%; and 9.7% of children who died from SARS-CoV-2 in Iran were coinfected with RSV. More importantly, coinfection may be closely associated with increased severity compared with single infection. Therefore, in the current world health environment, a vaccine that can simultaneously prevent multiple viruses is urgently needed.

[0003] In recent years, the production technology of mRNA vaccines has gradually matured, and lipid nanoparticles (LNP) have also become one of the effective carriers for the delivery of mRNA in vivo. Vaccines based on mRNA have developed rapidly, among which two mRNA vaccines for the prevention of SARS-CoV-2, mRNA-1273 and BNT162b, produced by Moderna and BioNTech, respectively, have been very successful. However, vaccines or drugs based on mRNA also have certain limitations. The mode of action of mRNA is to stimulate the immune system or express proteins for treatment. Therefore, the level of protein expression is closely related to the therapeutic effect. One of the disadvantages of traditional mRNA is that its structure is unstable and it will be degraded in cells within a few days, resulting in unsustainable protein expression levels. If used for the treatment of long-term diseases, patients may need to inject large amounts of mRNA, which will increase the toxic side effects of mRNA therapy.

[0004] Self-amplifying RNA (saRNA) can effectively solve this problem. Compared with non-replicating mRNA, saRNA can use its own RNA sequence as a template for self-amplification. Specifically, in addition to expressing the target protein, saRNA also carries a sequence that can express RNA polymerase (RNA-dependent RNA polymerase, RdRP). After this RNA polymerase is produced, it can continue to produce more copies of saRNA using saRNA as a template. Studies have shown that in preclinical vaccine experiments, saRNA induces protective immunity at a much lower dose than non-replicating mRNA, and saRNA has been shown to be a promising vaccine platform against a variety of pathogens. However, saRNA also faces unique challenges in research and development. Because saRNA connects the sequence encoding RNA polymerase to the sequence of the target protein, the molecular weight of the entire RNA molecule is much larger than that of traditional mRNA, and excessive molecular weight may lead to reduced stability and translation efficiency.

[0005] Trans-replicating RNA (taRNA) is a new technology based on saRNA. taRNA combines non-replicating mRNA encoding alphavirus replicase and trans-replicon (TR, trans-replicon) RNA encoding antigen. After translation, the replicase amplifies the TR encoding the antigen, so that a minimum amount of TR is required for immunization. The TR amplification of the replicase follows a complex mechanism that is finely regulated by the genomic and subgenomic promoters (SGPs) and generates genomic and subgenomic amplicons, only the latter of which are translated into the target protein. The taRNA strategy can effectively improve the efficiency of RNA translation and avoid the limitation of saRNA's excessive molecular weight.

[0006] Self-replicating saRNA or trans-self-replicating taRNA systems can be used to express target proteins or to prepare replicating or trans-replicating mRNA vaccines, respectively. Only a small amount of TR is needed to achieve a good immune effect. However, when the existing self-replicating or trans-replicating systems are used to prepare mRNA vaccines, there is usually an unsatisfactory effect of expressing the target protein and failing to achieve the expected expression amount. Therefore, it is urgent to find a replicase that can express the target protein more efficiently, so as to achieve a better immune effect when expressing the target protein or preparing the vaccine. Summary of the invention

[0007] In order to solve the technical problem, the present invention provides a replicase and its application. Based on the sequence of the wild-type replicase, it is mutated to form salt bridges, hydrophobic bonds or electrostatic effects in its nsP1-4 proteins. The main mutation sites include T372K of protein nsP1, S54R of protein nsP2, C157P, A161L, L168E, E404F, etc. of protein nsP4, which can significantly improve the structural stability of nsP1-4 proteins, thereby preparing a new replicase or self-replicase, improving the structural stability of the replicase, and achieving more stable and large-scale amplification and translation of target proteins. When used for preparing vaccines, at the same dose, it can significantly increase the expression of the target protein in vitro, and can induce higher specific antibody titers against the target antigen and stronger cellular immune responses in vivo.

[0008] On the one hand, the present invention provides a novel replicase, the sequence of which is based on the sequence of a wild-type replicase and has a mutation site, wherein the mutation site forms a salt bridge, a hydrophobic bond or an electrostatic interaction within the nsP1-4 protein of the replicase.

[0009] Since the structure of the CHIKV nsP1-4 complex has been resolved and the interaction mechanism at the molecular level is relatively clear, the present invention conducts research and design on the CHIKV nsP1-4 complex, hoping to form salt bridges, hydrophobic bonds or electrostatic effects in the nsP1-4 protein of the trans-replicase through mutation sites, thereby increasing the stability of the replicase protein, thereby allowing the target antigen protein to be continuously expressed and have high stability. When used for preparing vaccines, the body's ability to recognize antigens is enhanced, long-term immunity of the body is achieved, and the immune effect of the vaccine is improved.

[0010] The present invention analyzes the electron microscopic structure of the CHIKV nsP1-4 complex and finds a series of mutation sites that play a decisive role in stabilizing the structure of the CHIKV nsP1-4 complex. The nsP1-4 complex is stabilized by mutation-induced salt bridges, hydrophobic bonds or electrostatic interactions, thereby constructing a replicase with a more stable structure. When used to express a target protein, the expression amount of the target protein can be significantly increased.

[0011] Furthermore, the mutation sites include any one or more of T372K of protein nsP1, S54R of protein nsP2, C157P, A161L, L168E, and E404F of protein nsP4.

[0012] The mutation site T327K refers to the mutation of "T" at position 327 of the amino acid sequence to "K". Other mutation sites are also named in this way.

[0013] Based on the electron microscopy structure of the CHIKV nsP1-4 complex in SCIENCE ADVANCES, 2022 (DOI: 10.1126 / sciadv.add2536), the present invention analyzes the interaction sites at the binding interface of nsP1 and nsP4 and the binding interface of nsP2 and nsP4, and selects amino acids that play a decisive role in stabilizing the structure of the complex but are far away from the central site of nsP4 enzyme activity, such as E226 and E304 of nsP4, etc., in the hope of stabilizing the nsP1-4 complex by inducing salt bridges through mutations.

[0014] By comparing the sequences of replicase homologous family proteins VEEV nsP1-4, it was found that the designed S54R (nsP2) mutation can form a new electrostatic interaction with E225 (nsP4), and the T372K (nsP1) mutation can form a new electrostatic interaction with E303 (nsP4). It can be seen that the S54R mutation or the T372K mutation can form a new electrostatic interaction and lead to the formation of new salt bridge pairs, thereby improving the stability of the CHIKV nsP1-4 complex.

[0015] In addition, since CHIKV hydrogen bond interactions occur in multiple nsP1:nsP4 interfaces, such as V367:C158; V368:A162; R374:L169, etc., and Y60:E405 in the nsP2:nsP4 interface; therefore, the present invention designs a strong hydrophobic bond to enhance the VEEV interaction, ultimately forming: V367 (nsP1):C157P (nsP4); V368 (nsP1):A161L (nsP4); Y60 (nsP2):E404F (nsP4) and enhancing the electrostatic interaction pair: R374 (nsP1):L168E (nsP4). It can be seen that the C157P mutation, the A161L mutation, the L168E mutation, or the E404F mutation all have the effect of enhancing the VEEV interaction or enhancing the electrostatic interaction, and can improve the stability of the CHIKV nsP1-4 complex.

[0016] In some embodiments, the wild-type replicase is from an alphavirus, including any one of Semliki Forest virus (SFV), Venezuelan equine encephalitis virus (VEEV), Sindbis virus (SINV), and Chikungunya virus (CHIKV).

[0017] In some embodiments, the alphavirus is Venezuelan equine encephalitis virus (VEEV).

[0018] Compared with the wild type, the mutation sites provided by the present invention add new salt bridge pairs, or form strong hydrophobic bonds, enhance electrostatic interactions, and improve the stability of the CHIKV nsP1-4 complex, thereby improving the stability of the replicase structure. It is also understandable that mutations in any of the six mutation sites can improve the stability of the CHIKV nsP1-4 complex to a certain extent, so that when used to express the target protein, they can all play a role in increasing the expression amount of the target protein to a certain extent.

[0019] The wild-type replicase may come from different alphaviruses. Different wild-type replicase sequences are different, but the structures of their nsP1-4 complexes are similar. The stability of the nsP1-4 complexes can be improved by mutating the relevant sites of the nsP1-4 complexes, thereby improving the replication and expression of the target protein.

[0020] Further, the replicase includes a target RNA sequence that can be replicated in cis or trans. The replicase can replicate the target RNA sequence in cis or trans respectively through a self-replicating saRNA or a trans-self-replicating taRNA system.

[0021] In some embodiments, the sequence of the wild-type replicase has an amino acid sequence as shown in SEQ ID No. 1 in the sequence listing.

[0022] In some embodiments, the nucleotide sequence of the wild-type replicase is shown in SEQ ID NO.2.

[0023] In some embodiments, the T372K mutation of the protein nsP1 is performed on the basis of the wild-type replicase, and the amino acid sequence of the obtained replicase mutant is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4.

[0024] In some embodiments, the S54R mutation of the protein nsP2 is performed on the basis of the wild-type replicase, and the amino acid sequence of the obtained trans-replicase mutant is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6.

[0025] In some embodiments, the C157P mutation of the protein nsP4 is performed on the basis of the wild-type replicase, and the amino acid sequence of the obtained replicase mutant is shown in SEQ ID NO.7, and the nucleotide sequence is shown in SEQ ID NO.8.

[0026] In some embodiments, the A161L mutation of the protein nsP4 is performed on the basis of the wild-type replicase, and the amino acid sequence of the obtained replicase mutant is shown in SEQ ID NO.9, and the nucleotide sequence is shown in SEQ ID NO.10.

[0027] In some embodiments, the L168E mutation of the protein nsP4 is performed on the basis of the wild-type replicase, and the amino acid sequence of the obtained replicase mutant is shown in SEQ ID NO.11, and the nucleotide sequence is shown in SEQ ID NO.12.

[0028] In some embodiments, the E404F mutation of the protein nsP4 is performed on the basis of the wild-type replicase, and the amino acid sequence of the obtained replicase mutant is shown in SEQ ID NO.13, and the nucleotide sequence is shown in SEQ ID NO.14.

[0029] The present invention has been shown through research that by selecting several mutation sites among the six mutation sites for combination, it is expected to further improve the stability of the CHIKV nsP1-4 complex, thereby further improving the effect of the replicase.

[0030] In some embodiments, the combination comprises T372K of protein nsP1 and S54R of protein nsP2.

[0031] In some embodiments, the combination comprises T372K of protein nsP1 and C157P of protein nsP4.

[0032] In some embodiments, the combination comprises T372K of protein nsP1 and A161L of protein nsP4.

[0033] In some embodiments, the combination comprises T372K of protein nsP1 and L168E of protein nsP4.

[0034] In some embodiments, the combination comprises T372K of protein nsP1 and E404F of protein nsP4.

[0035] In some embodiments, the combination comprises T372K of protein nsP1, S54R of protein nsP2, and C157P of protein nsP4.

[0036] In some embodiments, the combination comprises T372K of protein nsP1, C157P of protein nsP4, and A161L of protein nsP4.

[0037] In some embodiments, the combination comprises T372K of protein nsP1, C157P of protein nsP4, and L168E of protein nsP4.

[0038] In some embodiments, the combination comprises T372K of protein nsP1, C157P of protein nsP4, and C157P and E404F of protein nsP4.

[0039] In addition, there are many other combinations. Selecting any 1 to 6 of the six mutation sites for combination, or combining with other mutation sites that are expected to improve the stability of the replicase or the replication expression effect, all have certain beneficial effects.

[0040] Furthermore, the mutation sites include a combination of two mutation sites, T372K of protein nsP1 and S54R of protein nsP2; or a combination of four mutation sites, C157P, A161L, L168E and E404F of protein nsP4; or a combination of six mutation sites, T372K of protein nsP1, S54R of protein nsP2 and C157P, A161L, L168E and E404F of protein nsP4.

[0041] The present invention has been verified by cells, and three preferred combinations have been screened out to construct replicase mutants for use in preparing vaccines, which can significantly improve the immune effect of the vaccine.

[0042] Furthermore, the replicase comprising two mutation site combinations has an amino acid sequence as shown in SEQ ID No.15 in the sequence listing; the replicase comprising three mutation site combinations has an amino acid sequence as shown in SEQ ID No.17 in the sequence listing; the replicase comprising six mutation site combinations has an amino acid sequence as shown in SEQ ID No.19 in the sequence listing.

[0043] In some embodiments, the amino acid sequence of the replicase comprising the combination of two mutation sites is shown in the sequence listing as SEQ ID No. 15, and the nucleotide sequence is shown in SEQ ID NO. 16.

[0044] In some embodiments, the amino acid sequence of the replicase comprising the combination of three mutation sites is shown as SEQ ID NO.17, and the nucleotide sequence is shown as SEQ ID NO.18.

[0045] In some embodiments, the amino acid sequence of the replicase comprising the combination of six mutation sites is shown as SEQ ID NO.19, and the nucleotide sequence is shown as SEQ ID NO.20.

[0046] Furthermore, it includes a combination of six mutation sites, having an amino acid sequence as shown in SEQ ID No. 19 of the sequence listing, and a nucleotide sequence as shown in SEQ ID No. 20 of the sequence listing.

[0047] In vitro cell validation and animal experiment validation have shown that the most preferred replicase mutant has six mutation sites: T372K of nsP1, S54R of protein nsP2, and C157P, A161L, L168E and E404F of protein nsP4.

[0048] Furthermore, the replicase also includes 5'UTR (untranslated region) and 3'UTR (untranslated region) sequences, which can replicate the target RNA sequence in cis or in trans.

[0049] It is understood that the specific 5'UTR and 3'UTR sequences are connected to the two ends of the replicase sequence, respectively, and can be used to prepare a cis or trans replication system, respectively, and can perform cis or trans replication on the target RNA sequence, respectively. That is to say, the difference between the cis replicase and the trans replicase is that they have different 5'UTR and 3'UTR sequences, and the replicase sequences between the 5'UTR and 3'UTR sequences can be the same.

[0050] The 5'UTR and 3'UTR sequences used to prepare the cis-replicase and the 5'UTR and 3'UTR sequences used to prepare the trans-replicase are all known conventional sequences. The replicase mutant sequence provided by the present invention can be used by simply connecting the specific 5'UTR and 3'UTR sequences, respectively.

[0051] Therefore, the replicase mutant provided by the present invention can be used to prepare cis replicase, and then perform cis replication on the target RNA sequence through the cis replication system, or can be used to prepare trans replicase, and then perform trans replication on the target RNA sequence through the trans replication system.

[0052] In some embodiments, the 5'UTR and 3'UTR sequences of the trans-replicase are shown as SEQ ID No. 27 and SEQ ID No. 28, respectively.

[0053] In another aspect, the present invention provides a composition comprising an RNA construct encoding the replicase as described above, and a replicon RNA for expressing a target protein.

[0054] Furthermore, the replicon RNA includes a cis replicon or a trans replicon, and the cis or trans replicon contains a recognition sequence of a replicase and can perform cis or trans replication through the replicase.

[0055] In some embodiments, the replicon RNA includes a target RNA sequence and a 5'CSE, a 3'CSE, and a subgenomic promoter sequence.

[0056] The 5'CSE, 3'CSE and subgenomic promoter sequences in the cis-replicon and the trans-replicon are also known sequences and can be used normally only by combining with the replicase mutant provided by the present invention.

[0057] Furthermore, the RNA construct is based on the nucleotide sequence of the wild-type replicase and is mutated, having a nucleotide sequence as shown in the sequence listing SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10, SEQ ID No.12, SEQ ID No.14, SEQ ID No.16, SEQ ID No.18, and SEQ ID No.20.

[0058] Furthermore, the target protein includes any one or more of antigens, antibodies, fusion proteins, and polypeptides.

[0059] Furthermore, the target proteins include novel coronavirus spike protein and respiratory syncytial virus.

[0060] In some embodiments, the spike protein has an amino acid sequence as shown in SEQ ID No.21 in the sequence listing, and the respiratory syncytial virus has an amino acid sequence as shown in SEQ ID No.23 in the sequence listing.

[0061] In some embodiments, the spike protein has a nucleotide sequence as shown in SEQ ID No.22 in the sequence listing, and the respiratory syncytial virus has a nucleotide sequence as shown in SEQ ID No.24 in the sequence listing.

[0062] In some embodiments, the trans-replicon RNA expressing the novel coronavirus spike protein has a nucleotide sequence as shown in SEQ ID No.25 in the sequence listing, and the trans-replicon RNA expressing the respiratory syncytial virus has a nucleotide sequence as shown in SEQ ID No.26 in the sequence listing.

[0063] Furthermore, the outer layer of the drug is coated with lipid nanoparticles.

[0064] In another aspect, the present invention provides an expression vector, comprising the composition as described above, and the expression vector is capable of expressing a target protein in a prokaryotic or eukaryotic host cell.

[0065] In still another aspect, the present invention provides a vaccine comprising the composition as described above.

[0066] It is understandable that the vaccine provided by the present invention can be used to prepare vaccines of various viral antigen proteins.

[0067] Furthermore, the vaccine is a monovalent, bivalent or multivalent vaccine.

[0068] In some embodiments, the vaccine is a bivalent vaccine, comprising a trans-replicon RNA encoding a novel coronavirus spike protein (S protein) and a trans-replicon RNA encoding a respiratory syncytial virus (F protein) antigen.

[0069] In some ways, the vaccine is designed based on a trans-replicon that can be recognized and replicated by a replicase, the nucleic acid sequence encoding the replicase is on one chain, and the nucleic acid sequences encoding the S protein and F protein antigens are on the other two chains respectively. After the replicase mRNA is translated in the cell, the trans-replicon RNA of the S protein and the trans-replicon RNA of the F protein can be amplified, thereby achieving continuous expression of the two proteins in the cell, inducing higher specific antibody titers for the two target antigens and stronger cellular immune responses in vivo. The bivalent vaccine prepared using the optimally designed trans-replicon system provided by the present invention can achieve a better immune effect with the same immune dose, and has important clinical transformation potential.

[0070] The number of trans-replicon RNAs can be 1 to n, where n is an integer greater than 1. n can be selected according to the actual needs of the product. Generally, n<8 helps to improve the replication effect.

[0071] In some embodiments, the vaccine further comprises LNP, and the bivalent taRNA is encapsulated in the LNP.

[0072] In some embodiments, the lipids include cationic lipids, neutral phospholipids, steroidal lipids and polyethylene glycol lipids. The molar percentage of cationic lipids in the lipid component is 20-60%, the molar percentage of neutral phospholipids in the lipid component is 5%-25%, the molar percentage of steroidal lipids in the lipid component is 25%-55%; the molar percentage of polyethylene glycol lipids in the lipid component is 0.1%-15%.

[0073] Furthermore, the vaccine also includes a pharmaceutically acceptable adjuvant. The designed sequence is synthesized, reverse transcribed, purified, and encapsulated to obtain the corresponding taRNA-LNP complex.

[0074] On the other hand, the present invention provides a use of a replicase for preparing a reagent for increasing the expression amount of mRNA, wherein the replicase uses the sequence of a wild-type replicase as an initial sequence and has a mutation site, wherein the mutation site includes any one or more of T372K of protein nsP1, S54R of protein nsP2, C157P, A161L, L168E, and E404F of protein nsP4.

[0075] In some embodiments, the replicase acts in trans on the replicon sequence.

[0076] On the other hand, the present invention provides a use of a replicase for preparing a reagent for increasing the expression amount of a vaccine, wherein the replicase has a sequence of a wild-type replicase as an initial sequence and has a mutation site, wherein the mutation site includes any one or more of T372K of protein nsP1, S54R of protein nsP2, C157P, A161L, L168E, and E404F of protein nsP4.

[0077] In some embodiments, the replicase acts in trans on the replicon sequence.

[0078] Compared with the prior art, the present invention has the following technical effects or advantages:

[0079] 1. The present invention obtains a series of novel replicases through mutation, which have a more stable protein structure than the wild-type replicase, and the stability of the replicase is significantly improved, so that the target protein can be expressed more efficiently at the same dose;

[0080] 2. Based on the sequence of the wild-type replicase, the present invention screened out a series of mutation sites that can improve the stability of the protein structure, constructed a series of mutants, and verified the effects of the self-replication, trans-replication and expression of the target protein of the novel replicase through in vivo and in vitro experiments, and screened out better mutants;

[0081] 3. The newly constructed replicase is used to prepare vaccines and other products, which can achieve better immune effects with the same or less immune doses and has important clinical transformation potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0083] Figure 1 A schematic diagram of the mRNA of the replicase provided by the present invention;

[0084] Figure 2 Schematic diagram of the preparation of taRNA bivalent vaccine in the present invention;

[0085] Figure 3 The levels of S protein and F protein expressed by the taRNA bivalent vaccine in HEK293 cells;

[0086] Figure 4 The specific humoral immune response levels of S protein and F protein after mice were immunized with taRNA bivalent vaccine;

[0087] Figure 5 The levels of S protein and F protein-specific cellular immune responses after mice were immunized with the taRNA bivalent vaccine;

[0088] Figure 6 The levels of S protein and F protein-specific cellular immune responses after mice were immunized with the taRNA bivalent vaccine; Figure 7 To maintain the long-term humoral immune response levels of S protein and F protein specificity after mice are immunized with the taRNA bivalent vaccine. DETAILED DESCRIPTION

[0089] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0090] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0091] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0092] Example 1. Screening of mutation sites

[0093] This example is based on the electron microscopic structure of the CHIKV nsP1-4 complex in SCIENCE ADVANCES, 2022 (DOI: 10.1126 / sciadv.add2536), and analyzes the interaction sites of the nsP1 and nsP4 binding interface and the nsP2 and nsP4 binding interface, and selects amino acids that play a decisive role in stabilizing the structure of the complex but are far away from the center of nsP4 enzyme activity, such as E226 and E304 of nsP4. It is hoped that the nsP1-4 complex can be stabilized by inducing a salt bridge form through mutation.

[0094] By comparing the sequences of replicase homologous family proteins VEEV nsP1-4, it was found that the designed S54R (nsP2) mutation can form a new electrostatic interaction with E225 (nsP4), and the T372K (nsP1) mutation can form a new electrostatic interaction with E303 (nsP4). It can be seen that the S54R mutation or the T372K mutation can form a new electrostatic interaction and cause the formation of new salt bridge pairs, thereby improving the stability of the CHIKV nsP1-4 complex.

[0095] In addition, since CHIKV hydrogen bond interactions occur in multiple nsP1:nsP4 interfaces, such as V367:C158; V368:A162; R374:L169, etc., and Y60:E405 in the nsP2:nsP4 interface; therefore, this embodiment designs a strong hydrophobic bond to enhance the VEEV interaction, ultimately forming: V367 (nsP1):C157P (nsP4); V368 (nsP1):A161L (nsP4); Y60 (nsP2):E404F (nsP4) and enhancing the electrostatic interaction pair: R374 (nsP1):L168E (nsP4). It can be seen that the C157P mutation, the A161L mutation, the L168E mutation, or the E404F mutation all have the effect of enhancing the VEEV interaction or enhancing the electrostatic interaction, and can improve the stability of the CHIKV nsP1-4 complex.

[0096] Finally, a total of six mutation sites that may improve the stability of the CHIKV nsP1-4 complex were screened out, namely: T372K of protein nsP1, S54R of protein nsP2, C157P, A161L, L168E, and E404F of protein nsP4.

[0097] Example 2: Effect of mutation on replicase stability

[0098] In this example, according to the six mutation sites provided in Example 1, a trans-replicase mutant and a self-replicase mutant were constructed respectively.

[0099] Among them, based on the wild replicase (SEQ ID No. 1), 9 replicase mutants were constructed: mutant 1: mutation T372K (nsP1); mutant 2: mutation S54R (nsP2); mutant 3: mutation C157P (nsP4); mutant 4: mutation A161L (nsP4); mutant 5: mutation L168E (nsP4); mutant 6: mutation E404F (nsP4); mutant 7: mutation (nsP1) and S54R (nsP2); mutant 8: mutation C157P, A161L, L168E and E404F (nsP4); mutant 9: mutation T372K (nsP1), S54R (nsP2) and C157P, A161L, L168E and E404F (nsP4), and 9 replicases were obtained by in vitro expression.

[0100] At the same time, this example also adaptively adjusted the mutation sites of another wild replicase (SEQ ID No. 29) in the same mutation manner for different wild replicases, and obtained 9 mutants. Mutant 1': mutation T372K (nsP1); mutant 2': mutation S54R (nsP2); mutant 3': mutation C158P (nsP4); mutant 4': mutation A162L (nsP4); mutant 5': mutation L169E (nsP4); mutant 6': mutation E405F (nsP4); mutant 7': mutation (nsP1) and S54R (nsP2); mutant 8': mutation C158P, A162L, L169E and E405F (nsP4); mutant 9: mutation T372K (nsP1), S54R (nsP2) and C158P, A162L, L169E and E405F (nsP4), 9 replicases were obtained by in vitro expression.

[0101] The stability of the above replicase was tested by the mutant thermal stability-Tm value determination method and compared with the wild-type replicase. The test results are shown in Table 1. Tm represents the midpoint temperature of thermal denaturation of the protein, that is, the temperature at which the protein unfolds 50%, reflecting the trend of protein conformation change during temperature change.

[0102] Table 1. Stability of replicase mutants

[0103] Replicase Tm(℃) Mutant 1 63 Mutant 2 65 Mutant 3 62.5 Mutant 4 67 Mutant 5 61 Mutant 6 62 Mutant 7 68 Mutant 8 70 Mutant 9 72.5 Wild-type replicase 1 52 Mutant 1' 59 Mutant 2' 59.5 Mutant 3' 56 Mutant 4' 60.5 Mutant 5' 57 Mutant 6' 53 Mutant 7' 61.5 Mutant 8' 62 Mutant 9' 66 Wild-type replicase 2 50

[0104] As can be seen from Table 1, compared with the wild-type replicase, the thermal stability of the replicase mutants is significantly improved, among which the thermal stability of the trans-replicase after combined mutation is higher than the stability of a single mutation site, and the replicase mutant 9 has the highest thermal stability.

[0105] At the same time, it was found that after mutation, the thermal stability of the replicase mutants constructed for the same mutation site and the two different wild replicases was significantly improved, among which the thermal stability of the mutant constructed for wild replicase 1 was more significantly improved. Moreover, the thermal stability of the two wild replicases was not much different, but the effects after mutation were different.

[0106] The higher the thermal stability, the more stable the spatial conformation of the replicase, and the stronger the replication activity of the prepared replicase mutant. At the same time, subsequent embodiments of the present invention also found that the replicase with higher thermal stability can replicate the target RNA more effectively, thereby increasing the yield of expressed protein, and when used to prepare vaccines, it can express antigens more stably and persistently, thereby improving the immune effect.

[0107] Example 3: Preparation of the replicase and replicon RNA provided by the present invention

[0108] like Figure 2 As shown, the steps for preparing the replicase and replicon RNA provided in this embodiment are mainly divided into the following steps:

[0109] (I) RNA synthesis

[0110] The DNA sequence encoding the replicase was commissioned to be synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. Among them, the RNA of the replicase, based on the wild-type replicase (SEQ ID No.2), also has 9 mutants: taRNA 1: mutation T372K (nsP1) (SEQ ID No.4); taRNA 2: mutation S54R (nsP2) (SEQ ID No.6); taRNA 3: mutation C157P (nsP4) (SEQ ID No.8); taRNA 4: mutation A161L (nsP4) (SEQ ID No.10); taRNA 5: mutation L168E (nsP4) (SEQ ID No.12); taRNA 6: mutation E404F (nsP4) (SEQ ID No.14); taRNA 7: mutation (nsP1) and S54R (nsP2) (SEQ ID No.16); taRNA 8: mutation C157P, A161L, L168E and E404F (nsP4) (SEQ IDNo.18); taRNA 9: Mutations T372K (nsP1), S54R (nsP2) and C157P, A161L, L168E and E404F (nsP4) (SEQ ID No. 19); Schematic diagram of the mRNA structure of the replicase is shown in Figure 1 As shown, the 5'UTR and 3'UTR sequences are SEQ ID No.27 and SEQ ID No.28 respectively.

[0111] This embodiment also synthesizes the DNA sequences (SEQ ID No.25, SEQ ID No.26) corresponding to the trans-replicon RNA expressing the novel coronavirus S protein and the trans-replicon RNA expressing the F protein (including all sequences such as 5'CSE, 3'CSE, subgenomic promoter, etc. in the trans-replication system). Among them, the S protein sequence is based on the spike of the wild type of SARS-CoV-2, and the mutation corresponding to the Omicron mutant strain (B.1.1.529) is added; the F protein sequence is based on the Ds-CaV1 form of the F protein of the RSV A2 strain (P03420). Among them, the amino acid sequence of the S protein is shown in SEQ ID No.21, and the DNA sequence is shown in SEQ ID No.22; the amino acid sequence of the F protein is shown in SEQ ID No.23, and the DNA sequence is shown in SEQID No.24.

[0112] The above sequence is directly synthesized into the pUC57-T7 plasmid vector, located between the T7 promoter binding sequence and the polyA site. The synthesized plasmid is transferred into E. coli (E.Coli) and the positive clones with stable polyA are selected. The bacteria are collected after the shake flask expansion culture. After alkaline lysis, neutralization, clarification, concentration and liquid replacement, purification, linearization and enzyme digestion and template purification, a high-quality template is obtained for in vitro transcription.

[0113] The template is transcribed in vitro using optimized T7 RNA polymerase and transcription conditions to obtain the respective mRNAs.

[0114] (II) RNA purification

[0115] The synthesized RNA was purified by oligo-dT affinity, buffer exchanged to sodium acetate by lateral flow filtration, and sterile filtered. RNA integrity was determined by microfluidic parallel capillary electrophoresis (Fragment Analyzer system 5200, Agilent), and the solution concentration, pH, residual DNA, protein, and double-stranded RNA impurities were also determined. The 5'-end capping efficiency and 3'-polyadenosine (poly A) tail of mRNA were analyzed by liquid chromatography-mass spectrometry (LC-MS).

[0116] Example 4: Levels of taRNA expressing S protein in HEK293 cells

[0117] The different taRNAs synthesized in Example 3 were transfected in HEK293T cells, and the groups were set as: PBS control group, taRNA-WT, taRNA1, taRNA2, taRNA3, taRNA4, taRNA5, taRNA6, taRNA7, taRNA8 and taRNA9. The total amount of RNA transfected in each well was 0.1 μg, including 0.08 μg replicase RNA + 0.02 μg S protein trans-replicon RNA. Cells were collected 72 hours after transfection, and the expression of target antigens in cells was detected by flow cytometry. The specific experimental method is as follows:

[0118] 1. HEK293T cells were seeded in 24-well plates and cultured overnight;

[0119] 2. When the cell growth density reaches 80%, the target protein RNA is transfected into the cells. The transfection reagent is purchased from Changchun Jinchuan Technology Co., Ltd. Every 1 μg RNA is mixed with 2 μL of transfection reagent, and the mixture is added to the cells after standing at room temperature for 5 minutes;

[0120] 3. Collect cells 72 hours after transfection;

[0121] 4. Fix the cells with 4% paraformaldehyde (100 μL) for 15 min at room temperature;

[0122] 5. PBS (500 μL) was used to wash away 4% paraformaldehyde, and the cells were permeabilized with 0.2% Saponin for 10 minutes;

[0123] 6. Wash away 0.2% Saponin with PBS (500 μL), add spike (S) protein-specific antibody (purchased from SinoBiological, 1:200), and incubate at room temperature for 60 min;

[0124] 7. Wash off the antibody in step 6 with PBS, add FITC-conjugated goat anti-rabbit IgG antibody (purchased from Abcam, 1:500), and incubate at room temperature for 60 minutes;

[0125] 8. Wash off the antibody in step 7 with PBS and resuspend the cells with PBS;

[0126] 9. Use flow cytometer to detect the fluorescence intensity and positive cell ratio.

[0127] The results are as follows Figure 3 As shown, compared with the wild-type replicase, the expression of S protein was significantly increased in both single mutation taRNA1-6 and combined mutation taRNA7-9, among which the expression of S protein in combined mutation taRNA7-9 was even higher than that in single mutation taRNA1-6.

[0128] This embodiment also attempts to use replicase mutant taRNA1-9 to express other proteins, and finds that compared with wild-type replicase, replicase mutants can increase protein expression. The space is limited, and the experimental data are omitted. It can be understood that the replicase mutant taRNA1-9 provided in this embodiment can be used to express any protein, and it also has the effect of significantly increasing protein expression.

[0129] At the same time, this embodiment also uses the above mutants to design a traditional cis-replicase system, and different saRNAs are transfected in HEK293T cells. The groups are set as: PBS control group, saRNA-WT, saRNA1, saRNA2, saRNA3, saRNA4, saRNA5, saRNA6, saRNA7, saRNA8 and saRNA9. When the two replicase systems, namely the traditional cis-replicase system and the wild-type cis-replicase system, are used to express the S protein, it is found that whether it is a single mutation or a combined mutation, the cis-replicase mutant saRNA1-9 can express the target protein, and compared with the wild-type cis-replicase, it also has the effect of improving the expression level, wherein the S protein expression amount of the single mutant cis-replicase mutant can be relatively increased by about 10%, and the S protein expression amount of the combined cis-replicase mutant can be relatively increased by about 30%. However, the trans-replicase system prepared by the mutant provided by the present invention is more obviously improved for expressing the target protein, and is more suitable for preparing trans-replicase and trans-replicase system.

[0130] Example 5. Preparation of taRNA bivalent vaccine particles

[0131] 1. Dissolve SM102, DSPC, cholesterol and DMG-PEG2000 in ethanol solution at a concentration of 10 mg / ml. According to the ratio of SM102: DSPC: cholesterol: DMG-PEG2000 of 50:10:38.5:1.5 (molar ratio), mix the four lipids and add anhydrous ethanol to control the total lipid concentration to 10 mM.

[0132] 2. Dilute different antigen RNAs in a citrate buffer (pH 4.0) at a mass ratio of 1:1, where the input amount of RNA and lipid must satisfy the N / P of 6. The prepared organic phase (lipid) and aqueous phase (RNA) are used to prepare RNA-encapsulated LNP vaccine particles through a microfluidic instrument, where the flow rate of the aqueous phase is 12 mL / min and the flow rate of the organic phase is 4 mL / min.

[0133] 3. Immediately after obtaining the LNP vaccine particles, they were diluted with sterile ultrapure water, and the dilution volume was 10 times. After dilution, the vaccine particles were ultrafiltered using a 100kDa ultrafiltration tube, and the centrifugation conditions were 3000rpm and 4°C. When the liquid was centrifuged to 1mL, sterile ultrapure water was added to 15mL, and centrifugation was continued for 3 times to reduce the ethanol content in the vaccine particles.

[0134] 4. Centrifuge for the last time and concentrate the vaccine particles to 1 mL to ensure that the required immune dose requirements for the experiment are met. Before immunization, the concentrated vaccine particles were adjusted to isotonicity using 10×PBS. Among them, the bivalent vaccine synthesized by wild-type replicase RNA was named taRNA-WT-LNP, the bivalent vaccine synthesized by replicase RNA7 (SEQ ID No.16) was named taRNA-1-LNP, the bivalent vaccine synthesized by replicase RNA 8 (SEQ ID No.18) was named taRNA-2-LNP, and the bivalent vaccine synthesized by replicase RNA 9 (SEQ IDNo.20) was named taRNA-3-LNP.

[0135] Example 6: Levels of S and F proteins expressed by taRNA bivalent vaccine in HEK293 cells

[0136] 1. Different vaccine particles were transfected in HEK293T cells, and the groups were set as: PBS control group, taRNA-WT-LNP, taRNA-1-LNP, taRNA-2-LNP and taRNA-3-LNP (prepared in Example 5). The total RNA amount transfected in each well was 0.1 μg, including 0.08 μg replicase RNA + 0.01 μg S protein replicon RNA + 0.01 μg F protein replicon RNA. Cells were collected 24, 48, 72, and 96 hours after transfection, and the expression of target antigens in cells was detected by flow cytometry. The specific experimental method is as follows:

[0137] 2. HEK293T cells were seeded in 24-well plates and cultured overnight;

[0138] 3. When the cell growth density reaches 80%, transfect the taRNA bivalent vaccine particles into the cells;

[0139] 4. Collect cells 24, 48, 72, and 96 hours after transfection;

[0140] 5. Fix the cells with 4% paraformaldehyde (100 μL) for 15 min at room temperature;

[0141] 6. PBS (500 μL) was used to wash away 4% paraformaldehyde, and the cells were permeabilized with 0.2% Saponin for 10 minutes;

[0142] 7. Wash away 0.2% Saponin with PBS (500 μL), add spike (S) protein-specific antibody (purchased from SinoBiological, 1:200) or fusion (F) protein-specific antibody (purchased from Cambridge Bio, 1:100), and incubate at room temperature for 60 min;

[0143] 8. Wash off the antibody in step 7 with PBS, add FITC-conjugated goat anti-rabbit IgG antibody (purchased from Abcam, 1:500), and incubate at room temperature for 60 min;

[0144] 9. Wash off the antibody in step 8 with PBS and resuspend the cells with PBS;

[0145] 10. Use flow cytometer to detect the fluorescence intensity and positive cell ratio.

[0146] The results are as follows Figure 4 As shown, compared with taRNA-WT-LNP, taRNA-1-LNP and taRNA-2-LNP, the expression of the two target proteins of taRNA-3-LNP was significantly improved, indicating that the optimized replicase is more stable than the wild-type replicase, among which taRNA-3-LNP is the most preferred, which can remain basically stable within 98 hours.

[0147] Example 7, detection of humoral immunity level of taRNA bivalent vaccine

[0148] 6-week-old BALB / c mice were randomly divided into groups: blank control group, taRNA-WT-LNP and taRNA-3-LNP (prepared in Example 5). The vaccine was injected into the mice by intramuscular injection on the outside of the right hind leg. The injection RNA dose was 1 μg / mouse, including 0.8 μg replicase RNA + 0.1 μg S protein replicon RNA + 0.1 μg F protein replicon RNA, and the injection volume was 50 μL / mouse. The blank control group was injected with only the same volume of normal saline. On the 21st day, booster immunization was performed in the same manner with the same dose. On the 28th day, 100 μL of whole blood from each group of mice was taken, the serum was separated, and the binding antibody titer in the serum was detected. The specific experimental operation steps are as follows:

[0149] 1. Coat the whole proteins of S protein and F protein in ELISA plate, dilute to 1μg / mL and 2μg / mL with PBS, 100μL per well, and incubate at 4℃ overnight;

[0150] 2. The next day, wash three times with PBS, discard the washing solution, add PBS blocking solution containing 5% fetal bovine serum, 200 μL per well, and place at room temperature at 37°C for 2 hours;

[0151] 3. Discard the blocking solution, add 1:200 diluted mouse serum, 3-fold gradient dilution, set up 7 gradients in total, 100 μL per well, incubate at room temperature for 1 hour;

[0152] 4. Discard the supernatant, wash three times with PBST solution, 200 μL per well, add HRP-labeled goat anti-mouse antibody (dilution ratio of 1:10000), 100 μL per well, and incubate at room temperature for 1 hour;

[0153] 5. Add 100 μL TMB colorimetric solution and color at room temperature in the dark for 8 min. Add 50 μL TMB stop solution to stop colorimetric development and measure the absorbance at 450 nm using an ELISA reader.

[0154] 6. The criteria for positive results are: the sample color value is greater than the corresponding dilution multiple control color value by more than 0.2, and the sample color value is more than 2 times the corresponding dilution multiple control color value, so that the color result of the sample at this dilution multiple is considered positive. Calculate the GMT value of the antibody produced by the mouse.

[0155] The results are as follows Figure 5 It was shown that compared with taRNA-WT-LNP, taRNA-3-LNP induced significantly more binding antibodies in mouse serum, significantly increased the antibody titer, and showed a stronger humoral immune response.

[0156] Example 8, Detection of cellular immunity level of taRNA bivalent vaccine

[0157] 6-week-old BALB / c mice were randomly divided into groups: blank control group, taRNA-WT-LNP and taRNA-3-LNP (prepared in Example 5). The mRNA vaccine was injected into mice by intramuscular injection on the outside of the right hind leg, and the injection RNA dose was 1 μg / mouse, including 0.8 μg replicase RNA + 0.1 μg S protein replicon RNA + 0.1 μg F protein replicon RNA, and the injection volume was 50 μL / mouse. The blank control group was injected with only the same volume of normal saline. Booster immunization was performed in the same way on the 21st day, with the same dose.

[0158] On the 28th day, spleens of mice in each group were taken to detect cell-specific immune responses. The specific experimental methods are as follows:

[0159] 1. Place the spleen in a small dish, add 500 μL PBS, cover with a 200-mesh sieve, and use a 1 mL syringe needle to grind the spleen through the sieve.

[0160] 2. After grinding, resuspend in 4 mL PBS and transfer the resuspension into a 15 mL EP tube.

[0161] 3. Centrifuge at 1800 rpm and 4°C for 5 min.

[0162] 4. Resuspend the pellet with 1 mL of red blood cell lysis buffer, lyse at room temperature for 7 minutes, and then add 10 mL of PBS to terminate the lysis.

[0163] 5. Centrifuge at 1800 rpm and 4°C for 5 min, resuspend the precipitate with 1 mL PBS, transfer the resuspension into a 1.5 mL EP tube for later use, and pass it through a 200-mesh sieve once during the process to remove visible flocs.

[0164] 6. Count the cells of the spleen single cell suspension prepared above.

[0165] 7. Prepare RPMI1640 complete medium containing peptide pool: dilute S protein peptide pool or F protein peptide pool with 1640 complete medium respectively, and the final concentration of peptide pool is 0.2 μg / mL.

[0166] 8. Remove the ELISPOT plate from the kit (purchased from Dakoway Biotechnology Co., Ltd.), add 200 μL 1640 culture medium to the plate for activation, let it stand at room temperature for 10 minutes, and then remove it.

[0167] 9. Add 1640 complete medium containing peptide pool to the activated ELISPOT plate, 150 μL / well. For the positive control group, add 140 μL 1640 complete medium and 10 μL positive stimulator from the kit (prepared with 500 μL PBS as described in the instructions) to the plate. For the positive control group, only 150 μL 1640 complete medium was added to the plate.

[0168] 10. Take out 5×10 5 The cells were added to the ELISPOT well plate and the added cells were evenly dispersed in the culture.

[0169] 11. Place the ELISPOT plate in a cell culture incubator and incubate for 18-24 hours.

[0170] 12. After incubation, drain the liquid in the well plate, add 200 μL of ice-cold deionized water, and incubate at 4°C for 10 minutes to fully lyse the cells.

[0171] 13. Remove the liquid from the plate, add 200 μL 1×washing buffer (the 50×washing buffer in the kit is diluted 50 times with deionized water) and wash three times, leaving the plate in 1×washing buffer for 1 min each time.

[0172] 14. Add 100 μL of diluted primary antibody solution (the biotin-labeled antibody in the kit is diluted 100 times with Dilutionbuffer R) and incubate at 37°C for 1 hour.

[0173] 15. Remove the liquid from the plate and add 200 μL 1×washing buffer to wash 5 times, leaving the plate in 1×washing buffer for 1 min each time.

[0174] 16. Add 100 μL of diluted enzyme-labeled avidin (diluted 100 times with Dilution buffer R in the kit) and incubate at 37°C for 1 hour.

[0175] 17. Remove the liquid from the plate and add 200 μL 1×washing buffer to wash 5 times, leaving the plate for 1 min each time.

[0176] 18. Add 100 μL of prepared ACE colorimetric solution (mix 20×AEC solution I, 20×AEC solution II, 200×AEC solution III, and AEC dilution in the kit in a ratio of 10:10:1:180) and incubate at 37°C for 15-30 minutes.

[0177] 19. Observe with the naked eye until the color develops to an appropriate level (observe every 5 minutes), remove the liquid in the hole, clean the inside of the hole plate and the base with deionized water, and place it in a dark place to dry naturally.

[0178] 20. Count the spots in the wells using an automated ELISPOT plate reader (Mabtech IRIS FluoroSpot / ELISpot reader).

[0179] The results are as follows Figure 6 It was shown that compared with taRNA-WT-LNP, the number of cells secreting IFN-γ in the spleen of mice immunized with taRNA-3-LNP was significantly increased, indicating a stronger antigen-specific cellular immune response.

[0180] Example 9, detection of the ability to maintain the level of S protein and F protein-specific long-term humoral immune response after immunization of mice with the taRNA bivalent vaccine.

[0181] 6-week-old BALB / c mice were randomly divided into groups: blank control group, taRNA-WT-LNP and taRNA-3-LNP (prepared in Example 5). The vaccine was injected into mice by intramuscular injection on the outside of the right hind leg. The injection RNA dose was 1 μg / mouse, including 0.8 μg replicase RNA + 0.1 μg S protein replicon RNA + 0.1 μg F protein replicon RNA, and the injection volume was 50 μL / mouse. The blank control group was injected with only the same volume of normal saline. On the 21st day, booster immunization was performed in the same manner with the same dose. 100 μL of whole blood from each group of mice was taken on days 0, 21, 35, 49, 120 and 250, the serum was separated, and the binding antibody titer in the serum was detected. The specific experimental method is as follows:

[0182] 1. Coat the whole proteins of S protein and F protein in ELISA plate, dilute to 1μg / mL and 2μg / mL with PBS, 100μL per well, and incubate at 4℃ overnight;

[0183] 2. The next day, wash with PBS three times, discard the washing solution, add PBS blocking solution containing 5% fetal bovine serum, and place at room temperature 37℃ for 2h;

[0184] 3. Discard the blocking solution, add 1:200 diluted mouse serum, 3-fold gradient dilution, set up 7 gradients in total, 100 μL per well, incubate at room temperature for 1 hour;

[0185] 4. Discard the supernatant, wash with PBST buffer 3 times, add HRP-labeled goat anti-mouse antibody (dilution ratio 1:10000), 100 μL per well, and incubate at room temperature for 1 hour;

[0186] 5. Add 100 μL TMB colorimetric solution and color at room temperature in the dark for 8 minutes. Add 50 μL TMB stop solution to stop colorimetric development, and measure the absorbance at 450 nm with an ELISA reader;

[0187] 6. The criteria for positive results are: the sample color value is greater than the corresponding dilution multiple control color value by more than 0.2, and the sample color value is more than 2 times the corresponding dilution multiple control color value, so that the color result of the sample at this dilution multiple is considered positive. Calculate the GMT value of the antibody produced by the mouse.

[0188] The results are as follows Figure 7 The results showed that after immunization with taRNA-3-LNP, the binding antibodies induced in mice were significantly higher than those produced by taRNA-WT-LNP at all detection time points until 250 days after immunization.

[0189] In summary, the taRNA bivalent vaccine containing mutant replicase provided by the present invention can achieve higher target antigen expression and better immune effect at the same immune dose compared to the wild-type replicase, and has important clinical transformation potential.

[0190] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0191] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0192] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

[0193] Sequence Listing

[0194] SEQ ID No.1:

[0195] Amino acid sequence of wild-type replicase:

[0196]

[0197] SEQ ID No.2:

[0198] DNA sequence of wild-type replicase:

[0199]

[0200] SEQ ID No.3:

[0201] The amino acid sequence of the first replicase mutant:

[0202]

[0203] SEQ ID No.4:

[0204] The first replicase mutant DNA sequence:

[0205]

[0206] SEQ ID No.5:

[0207] The second replicase mutant amino acid sequence:

[0208]

[0209] SEQ ID No.6:

[0210] The second replicase mutant DNA sequence:

[0211] ATGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGC

[0212] TTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTT

[0213] CGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGGACCCATCCGACACGATCCTTGACATTGGAA

[0214] GTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCATTGTATCTGTCCGATGAGATGTGCGGA

[0215] AGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAAAACTGTAAGGAAATAACTGATAA

[0216] GGAATTGGACAAGAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACCCTGACCTGGAAACTGAGA

[0217] CTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGTTTACCAGGATGTATA

[0218] CGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTCGCCTACTGGAT

[0219] AGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACTCTACCAACT

[0220] GGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGAGCGGT

[0221] CACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTGAAACCATCCAACAATGTTCTATTCTCTGTT

[0222] GGCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCAC

[0223] TTACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTA

[0224] AAAGAATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGA

[0225] GGGATTCTTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCAC

[0226] GTATGTGCCAGCTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGA

[0227] CGCGCAAAACTGCTGGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACA

[0228] CCAATACCATGAAAAAATTACCTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATA

[0229] TAAGGAAGATCAAGAAGATGAAAGGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTG

[0230] TTGGGCTTTTAGAAGGCACAAGATAACATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAA

[0231] GTGAACAGCGATTTCCACTCATTCGTGCTGCCCAGGATAGGCAGTAACACATTGGAGATCGGGCTG

[0232] AGAACAAGAATCAGGAAAATGTTAGAGGAGCACAAGGAGCCGTCACCTCTCATTACCGCCGAGGA

[0233] CGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGAGGTGCGTGAAGCCGAGGAGTTGCGCG

[0234] CAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTGGAAGCCGATGTCGACTTGATGTT

[0235] ACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAAAGGTTACCAGCTACGATGG

[0236] CGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAAGAGTGAAAAATTATCT

[0237] TGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACCGGGGCCGAAAAGGGCGTTATGCC

[0238] GTGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGGACTTTCAA

[0239] GCTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCACCAT

[0240] ATTGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAG

[0241] CACGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGG

[0242] GCTAGGGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAAC

[0243] ACGACCAGCCGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTC

[0244] TGGCATCATTAAAAGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTG

[0245] CAGAAATTATAAGGGACGTCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCA

[0246] GTGCTCTTGAATGGATGCAAACACCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATG

[0247] CAGGTACTCTCAGAGCGCTCATAGCCATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCA

[0248] AACAGTGCGGTTTTTTTAACATGATGTGCCTGAAAGTGCATTTTAACCACGAGATTTGCACACAAGT

[0249] CTTCCACAAAAGCATCTCTCGCCGTTGCACTAAATCTGTGACTTCGGTCGTCTCAACCTTGTTTTAC

[0250] GACAAAAAAATGAGAACGACGAATCCGAAAGAGACTAAGATTGTGATTGACACTACCGGCAGTAC

[0251] CAAACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAGGGTGGGTGAAGCAGTTGCAAATAGA

[0252] TTACAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCTGACCCGTAAAGGTGTGTATGC

[0253] CGTTCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAACATGTGAACGTCCTACT

[0254] GACCCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGATAAAAACACTGA

[0255] CTGCCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGATGCCATCA

[0256] TGAGGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGTTGGG

[0257] CCAAGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTG

[0258] TGGATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTT

[0259] CTTTGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATC

[0260] ACTGGGATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCG

[0261] CAGGTACCCACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACT

[0262] GCGCAATTATGATCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCC

[0263] ACCATAATGAACACCCACAGGTGACTTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCT

[0264] GGTGGTCGGGGAAAAGTTGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGG

[0265] CTACCTTCAGAGCTCGGCTGGATTTAGGCATCCCAGGTGATGTCCAAATATGACATAATATTTGTT

[0266] AATGTGAGGACCCCATATAAATACCATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCAT

[0267] GTTGACCAAGAAAGCTTGTCTGCATCTGAATCCCGCGGAACCTGTGTCAGCATAGGTTATGGTTAC

[0268] GCTGACAGGGCCAGCGAAAGCATCATTGGTGCTATAGCGCGGCAGTTTCAAGTTTTCCCGGGTATGC

[0269] AAACCGAAATCCTCACTTGAAGAGACGGAAGTTCTGTTTGTATTCATTGGGTACGATCGCAAGGCCC

[0270] GTACGCACAATCCTTACAAGCTTTCATCAACCTTGACCAACATTTATACAGGTTCCAGACTCCACGA

[0271] AGCCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGATATTGCCACGGCCACCGAAGGAGTGATT

[0272] ATAAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTGTGCGGAGCGCTGTATAAGAAATTC

[0273] CCGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGACTGGTCAAAGGTGCAGCTAA

[0274] ACATATCATTCATGCCGTAGGACCAACTTCAACAAAGTTTCGGAGTTGAAGGTGACAAAACAGTT

[0275] GGCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCAGTAGCGATTCCA

[0276] CTGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACCATTTGCTGA

[0277] CAGCTTTAGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAATGACTCT

[0278] CAAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATGCATATCCGACGACTCTTCAGTGA

[0279] CAGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTAC

[0280] AGCACAAGCGATGGCAAAACTTTCTCTATTTGGAAGGGACCAAGTTTCACCAGGCGGCCAAGGAT

[0281] ATAGCAGAAATTAATGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCC

[0282] TCGGAGAAAGCATGAGCAGTATTAGGTCGAAATGCCCCGTCGAGAGTCGGAAGCCTCCACACCAC

[0283] CTAGCACGCTGCCTTGCTTGTGCATCCATGCCATGACTCCAGAAAGAGTACAGCGCCTAAAAGCCTC

[0284] ACGTCCAGAACAAATTACTGTGTGCTCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAG

[0285] AAGATCCAATGCTCCCAGCCTATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTA

[0286] TCTCGTGGAAACACCACCGGTAGACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGG

[0287] GGACACCTGAACAACCACCACTTATAACCGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCA

[0288] TCATCGAAGAGGAAGAAGAGGATAGCATAAGTTTGCTGTCAGATGGCCCGACCCACCAGGTGCTGC

[0289] AAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTATCTAGCTCATCCTGGTCCATTCCTCATGCATCC

[0290] GACTTTGATGTGGACAGTTTATCCATACTTGACACCCTGGAGGGAGCTAGCGTGACCAGCGGGGCA

[0291] ACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAGTTTCTGGCGCGACCGGTGCCTGCG

[0292] CCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAAGAACACCGTCACTTGCACCC

[0293] AGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAATAGGGTGATCACTAGA

[0294] GAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGAACCAGCCTGGTC

[0295] TCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAGCACAACAA

[0296] CAATGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACAACAAA

[0297] AATCAGTAAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTATG

[0298] CCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTG

[0299] CTAACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTC

[0300] TGCAAGGCCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTG

[0301] TTCCTTTGTATTCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAA

[0302] CGCCATGTTGAAAGAGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATT

[0303] TGGACATGGTTGACGGAGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGCTGCGCA

[0304] GCTTTCCAAAGAAACACTCCTATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGA

[0305] ACACGCTCCAGAACGTCCTGGCAGCTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAA

[0306] TTGCCCGTATTGGATTCGGCGGCCTTTAATGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATA

[0307] TTGGGAAACGTTTAAAGAAAACCCCATCAGGCTACTGAAAAAACGTGGTAAAATTACATTACCAA

[0308] ATTAAAAGGACCAAAGCTGCTGCTCTTTTTGCGAAGACACATAATTTGAATATGTTGCAGGACATA

[0309] CCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGTGAAAGTGACTCCAGGAAAAAAACATACT

[0310] GAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCGCTAGCAACAGCGTATCTGTGCGGA

[0311] ATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGAACATTCATACACTGTTTGATAT

[0312] GTCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGGGGATTGTGTTCTGGAAACT

[0313] GACATCGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCGTTAATGATTCTGGAA

[0314] GACTTAGGTGTGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAATTTCATCAATAC

[0315] ATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGAGAAATCTGGAATGTTCCTCACACTGTTT

[0316] GTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGATCACCA

[0317] TGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAGAC

[0318] AGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCT

[0319] TATTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCC

[0320] TAAAAAGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAA

[0321] GGGCATTGCATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAG

[0322] TAGAATCAAGGTATGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGT

[0323] GTTAAATCATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAA

[0324] SEQ ID No.7:

[0325] Amino acid sequence of the 3rd replicase mutant:

[0326]

[0327] SEQ ID No.8:

[0328] The third replicase mutant DNA sequence:

[0329]

[0330] SEQ ID No.9:

[0331] Amino acid sequence of the fourth replicase mutant:

[0332]

[0333] SEQ ID No.10:

[0334] The fourth replicase mutant DNA sequence:

[0335]

[0336] SEQ ID No.11:

[0337] The amino acid sequence of the fifth replicase mutant:

[0338]

[0339] SEQ ID No.12:

[0340] The 5th replicase mutant DNA sequence:

[0341] ATGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGC

[0342] TTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTT

[0343] CGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGGACCCATCCGACACGATCCTTGACATTGGAA

[0344] GTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCATTGTATCTGTCCGATGAGATGTGCGGA

[0345] AGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAAAACTGTAAGGAAATAACTGATAA

[0346] GGAATTGGACAAGAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACCCTGACCTGGAAACTGAGA

[0347] CTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGTTTACCAGGATGTATA

[0348] CGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTCGCCTACTGGAT

[0349] AGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACTCTACCAACT

[0350] GGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGAGCGGT

[0351] CACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTGAAACCATCCAACAATGTTCTATTCTCTGTT

[0352] GGCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCAC

[0353] TTACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTA

[0354] AAAGAATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGA

[0355] GGGATTCTTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCAC

[0356] GTATGTGCCAGCTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGA

[0357] CGCGCAAAACTGCTGGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACA

[0358] CCAATACCATGAAAAAATTACCTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATA

[0359] TAAGGAAGATCAAGAAGATGAAAGGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTG

[0360] TTGGGCTTTTAGAAGGCACAAGATAACATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAA

[0361] GTGAACAGCGATTTCCACTCATTCGTGCTGCCCAGGATAGGCAGTAACACATTGGAGATCGGGCTG

[0362] AGAACAAGAATCAGGAAAATGTTAGAGGAGCACAAGGAGCCGTCACCTCTCATTACCGCCGAGGA

[0363] CGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGAGGTGCGTGAAGCCGAGGAGTTGCGCG

[0364] CAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTGGAAGCCGATGTCGACTTGATGTT

[0365] ACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAAAGGTTACCAGCTACGATGG

[0366] CGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAAGAGTGAAAAATTATCT

[0367] TGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAAGGGCGTTATGCCG

[0368] TGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGGACTTTCAAG

[0369] CTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCACCATAT

[0370] TGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAGCA

[0371] CGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGC

[0372] TAGGGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACAC

[0373] GACCAGCCGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTG

[0374] GCATCATTAAAAGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCA

[0375] GAAATTATAAGGGACGTCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGT

[0376] GCTCTTGAATGGATGCAAACACCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCA

[0377] GGTACTCTCAGAGCGCTCATAGCCATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAA

[0378] CAGTGCGGTTTTTTTAACATGATGTGCCTGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCT

[0379] TCCACAAAAGCATCTCTCGCCGTTGCACTAAATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGA

[0380] CAAAAAAATGAGAACGACGAATCCGAAAGAGACTAAGATTGTGATTGACACTACCGGCAGTACCA

[0381] AACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAGGGTGGGTGAAGCAGTTGCAAATAGATTA

[0382] CAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCTGACCCGTAAAGGTGTGTATGCCGT

[0383] TCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAACATGTGAACGTCCTACTGAC

[0384] CCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGATAAAAACACTGACTG

[0385] CCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGATGCCATCATGA

[0386] GGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGTTGGGCCA

[0387] AGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTGTGG

[0388] ATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTT

[0389] TGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACT

[0390] GGGATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCA

[0391] GGTACCCACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGC

[0392] GCAATTATGATCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCAC

[0393] CATAATGAACACCCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTG

[0394] GTGGTCGGGGAAAAGTTGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCT

[0395] ACCTTCAGAGCTCGGCTGGATTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAA

[0396] TGTGAGGACCCCATATAAATACCATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGT

[0397] TGACCAAGAAAGCTTGTCTGCATCTGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGC

[0398] TGACAGGGCCAGCGAAAGCATCATTGGTGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAA

[0399] ACCGAAATCCTCACTTGAAGAGACGGAAGTTCTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGT

[0400] ACGCACAATCCTTACAAGCTTTCATCAACCTTGACCAACATTTATACAGGTTCCAGACTCCACGAAG

[0401] CCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGATATTGCCACGGCCACCGAAGGAGTGATTAT

[0402] AAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTGTGCGGAGCGCTGTATAAGAAATTCC

[0403] CGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGACTGGTCAAAGGTGCAGCTAAA

[0404] CATATCATTCATGCCGTAGGACCAAACTTCCAACAAAGTTTCGGAGGTTGAAGGTGACAAACAGTTG

[0405] GCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCAGTAGCGATTCCAC

[0406] TGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACCATTTGCTGAC

[0407] AGCTTTGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAATGACTCTC

[0408] AAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATACCCGACGACTCTTCAGTGAC

[0409] AGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACA

[0410] GCACAAGCGATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCCAAGGATA

[0411] TAGCAGAAAATTATGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCT

[0412] CGGAGAAAGCATGAGCAGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCAC

[0413] CTAGCACGCTGCCTTGCTTGTGCATCCATGCCATGACTCCAGAAAGGTACAGCGCCTAAAAGCCTC

[0414] ACGTCCAGAACAAATTACTGTGTGCTCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAG

[0415] AAGATCCAATGCTCCCAGCCTATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTA

[0416] TCTCGTGGAAACACCACCGGTAGACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGG

[0417] GGACACCTGAACAACCACCACTTATAACCGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCA

[0418] TCATCGAAGAGGAAGAAGAGGATAGCATAAGTTTGCTGTCAGATGGCCCGACCCACCAGGTGCTGC

[0419] AAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTATCTAGCTCATCCTGGTCCATTCCTCATGCATCC

[0420] GACTTTGATGTGGACAGTTTATCCATACTTGACACCCTGGAGGGAGCTAGCGTGACCAGCGGGGCA

[0421] ACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAGTTTCTGGCGCGACCGGTGCCTGCG

[0422] CCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAAGAACACCGTCACTTGCACCC

[0423] AGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAATAGGGTGATCACTAGA

[0424] GAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGAACCAGCCTGGTC

[0425] TCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAGCACAACAA

[0426] CAATGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACAACAAA

[0427] AATCAGTAAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTATG

[0428] CCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTG

[0429] CTAACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTC

[0430] TGCAAGGCCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTG

[0431] TTCCTTTGTATTCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAA

[0432] CGCCATGTTGAAAGAGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATT

[0433] TGGACATGGTTGACGGAGCTTCATGCTGCTTAGACACTGCCAGTTTTTGCCCTGCAAAGGAGCGCA

[0434] GCTTTCCAAAGAAACACTCCTATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGA

[0435] ACACGCTCCAGAACGTCCTGGCAGCTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAA

[0436] TTGCCCGTATTGGATTCGGCGGCCTTTAATGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATA

[0437] TTGGGAAACGTTTAAAGAAAACCCCATCAGGCTACTGAAAAAACGTGGTAAAATTACATTACCAA

[0438] ATTAAAAGGACCAAAGCTGCTGCTCTTTTTGCGAAGACACATAATTTGAATATGTTGCAGGACATA

[0439] CCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGTGAAAGTGACTCCAGGAAAAAAACATACT

[0440] GAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCGCTAGCAACAGCGTATCTGTGCGGA

[0441] ATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGAACATTCATACACTGTTTGATAT

[0442] GTCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGGGGATTGTGTTCTGGAAACT

[0443] GACATCGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCGTTAATGATTCTGGAA

[0444] GACTTAGGTGTGGACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAATTTCATCAATAC

[0445] ATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGAGAAATCTGGAATGTTCCTCACACTGTTT

[0446] GTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGATCACCA

[0447] TGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAGAC

[0448] AGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCT

[0449] TATTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCC

[0450] TAAAAAGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAA

[0451] GGGCATTGCATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAG

[0452] TAGAATCAAGGTATGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGT

[0453] GTTAAATCATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAA

[0454] SEQ ID No.13:

[0455] Amino acid sequence of the 6th replicase mutant:

[0456]

[0457] SEQ ID No.14:

[0458] The sixth replicase mutant DNA sequence:

[0459]

[0460] SEQ ID No.15:

[0461] Amino acid sequence of the seventh replicase mutant:

[0462]

[0463] SEQ ID No.16:

[0464] The seventh replicase mutant DNA sequence:

[0465]

[0466] SEQ ID No.17:

[0467] The amino acid sequence of the eighth replicase mutant:

[0468]

[0469] SEQ ID No.18:

[0470] The DNA sequence of the 8th replicase mutant:

[0471] ATGGAGAAAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCAGAGCTTTGCAGCGGAGC

[0472] TTCCCGCAGTTTGAGGTAGAAGCCAAGCAGGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTT

[0473] CGCATCTGGCTTCAAAACTGATCGAAACGGAGGTGGACCCATCCGACACGATCCTTGACATTGGAA

[0474] GTGCGCCCGCCCGCAGAATGTATTCTAAGCACAAGTATCATTGTATCTGTCCGATGAGATGTGCGGA

[0475] AGATCCGGACAGATTGTATAAGTATGCAACTAAGCTGAAGAAAAACTGTAAGGAAATAACTGATAA

[0476] GGAATTGGACAAGAAAATGAAGGAGCTCGCCGCCGTCATGAGCGACCCTGACCTGGAAACTGAGA

[0477] CTATGTGCCTCCACGACGACGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGTTTACCAGGATGTATA

[0478] CGCGGTTGACGGACCGACAAGTCTCTATCACCAAGCCAATAAGGGAGTTAGAGTCGCCTACTGGAT

[0479] AGGCTTTGACACCACCCCTTTTATGTTTAAGAACTTGGCTGGAGCATATCCATCATACTCTACCAACT

[0480] GGGCCGACGAAACCGTGTTAACGGCTCGTAACATAGGCCTATGCAGCTCTGACGTTATGGAGCGGT

[0481] CACGTAGAGGGATGTCCATTCTTAGAAAGAAGTATTGAAACCATCCAACAATGTTCTATTCTCTGTT

[0482] GGCTCGACCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCCGTCTGTATTTCAC

[0483] TTACGTGGCAAGCAAAATTACACATGTCGGTGTGAGACTATAGTTAGTTGCGACGGGTACGTCGTTA

[0484] AAAGAATAGCTATCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGATGCACCGCGA

[0485] GGGATTCTTGTGCTGCAAAGTGACAGACACATTGAACGGGGAGAGGGTCTCTTTTCCCGTGTGCAC

[0486] GTATGTGCCAGCTACATTGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGACGA

[0487] CGCGCAAAACTGCTGGTTGGGCTCAACCAGCGTATAGTCGTCAACGGTCGCACCCAGAGAAACA

[0488] CCAATACCATGAAAAAATTACCTTTGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATA

[0489] TAAGGAAGATCAAGAAGATGAAAGGCCACTAGGACTACGAGATAGACAGTTAGTCATGGGGTGTTG

[0490] TTGGGCTTTTAGAAGGCACAAGATAACATCTATTTATAAGCGCCCGGATACCCAAACCATCATCAAA

[0491] GTGAACAGCGATTTCCACTCATTCGTGCTGCCCAGGATAGGCAGTAACACATTGGAGATCGGGCTG

[0492] AGAACAAGAATCAGGAAAATGTTAGAGGAGCACAAGGAGCCGTCACCTCTCATTACCGCCGAGGA

[0493] CGTACAAGAAGCTAAGTGCGCAGCCGATGAGGCTAAGGAGGTGCGTGAAGCCGAGGAGTTGCGCG

[0494] CAGCTCTACCACCTTTGGCAGCTGATGTTGAGGAGCCCACTCTGGAAGCCGATGTCGACTTGATGTT

[0495] ACAAGAGGCTGGGGCCGGCTCAGTGGAGACACCTCGTGGCTTGATAAAGGTTACCAGCTACGATGG

[0496] CGAGGACAAGATCGGCTCTTACGCTGTGCTTTCTCCGCAGGCTGTACTCAAGAGTGAAAAATTATCT

[0497] TGCATCCACCCTCTCGCTGAACAAGTCATAGTGATAACACACTCTGGCCGAAAAGGGCGTTATGCCG

[0498] TGGAACCATACCATGGTAAAGTAGTGGTGCCAGAGGGACATGCAATACCCGTCCAGGACTTTCAAG

[0499] CTCTGAGTGAAAGTGCCACCATTGTGTACAACGAACGTGAGTTCGTAAACAGGTACCTGCACCATAT

[0500] TGCCACACATGGAGGAGCGCTGAACACTGATGAAGAATATTACAAAACTGTCAAGCCCAGCGAGCA

[0501] CGACGGCGAATACCTGTACGACATCGACAGGAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGC

[0502] TAGGGCTCACAGGCGAGCTGGTGGATCCTCCCTTCCATGAATTCGCCTACGAGAGTCTGAGAACAC

[0503] GACCAGCCGCTCCTTACCAAGTACCAACCATAGGGGTGTATGGCGTGCCAGGATCAGGCAAGTCTG

[0504] GCATCATTAAAAGCGCAGTCACCAAAAAAGATCTAGTGGTGAGCGCCAAGAAAGAAAACTGTGCA

[0505] GAAATTATAAGGGACGTCAAGAAAATGAAAGGGCTGGACGTCAATGCCAGAACTGTGGACTCAGT

[0506] GCTCTTGAATGGATGCAAACACCCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCA

[0507] GGTACTCTCAGAGCGCTCATAGCCATTATAAGACCTAAAAAGGCAGTGCTCTGCGGGGATCCCAAA

[0508] CAGTGCGGTTTTTTTAACATGATGTGCCTGAAAGTGCATTTTAACCACGAGATTTGCACACAAGTCT

[0509] TCCACAAAAGCATCTCTCGCCGTTGCACTAAATCTGTGACTTCGGTCGTCTCAACCTTGTTTTACGA

[0510] CAAAAAAATGAGAACGACGAATCCGAAAGAGACTAAGATTGTGATTGACACTACCGGCAGTACCA

[0511] AACCTAAGCAGGACGATCTCATTCTCACTTGTTTCAGAGGGTGGGTGAAGCAGTTGCAAATAGATTA

[0512] CAAAGGCAACGAAATAATGACGGCAGCTGCCTCTCAAGGGCTGACCCGTAAAGGTGTGTATGCCGT

[0513] TCGGTACAAGGTGAATGAAAATCCTCTGTACGCACCCACCTCAGAACATGTGAACGTCCTACTGAC

[0514] CCGCACGGAGGACCGCATCGTGTGGAAAACACTAGCCGGCGACCCATGGATAAAAACACTGACTG

[0515] CCAAGTACCCTGGGAATTTCACTGCCACGATAGAGGAGTGGCAAGCAGAGCATGATGCCATCATGA

[0516] GGCACATCTTGGAGAGACCGGACCCTACCGACGTCTTCCAGAATAAGGCAAACGTGTGTTGGGCCA

[0517] AGGCTTTAGTGCCGGTGCTGAAGACCGCTGGCATAGACATGACCACTGAACAATGGAACACTGTGG

[0518] ATTATTTTGAAACGGACAAAGCTCACTCAGCAGAGATAGTATTGAACCAACTATGCGTGAGGTTCTT

[0519] TGGACTCGATCTGGACTCCGGTCTATTTTCTGCACCCACTGTTCCGTTATCCATTAGGAATAATCACT

[0520] GGGATAACTCCCCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGCTCTCTCGCA

[0521] GGTACCCACAACTGCCTCGGGCAGTTGCCACTGGAAGAGTCTATGACATGAACACTGGTACACTGC

[0522] GCAATTATGATCCGCGCATAAACCTAGTACCTGTAAACAGAAGACTGCCTCATGCTTTAGTCCTCCAC

[0523] CATAATGAACACCCACAGAGTGACTTTTCTTCATTCGTCAGCAAATTGAAGGGCAGAACTGTCCTG

[0524] GTGGTCGGGGAAAAGTTGTCCGTCCCAGGCAAAATGGTTGACTGGTTGTCAGACCGGCCTGAGGCT

[0525] ACCTTCAGAGCTCGGCTGGATTTAGGCATCCCAGGTGATGTGCCCAAATATGACATAATATTTGTTAA

[0526] TGTGAGGACCCCATATAAATACCATCACTATCAGCAGTGTGAAGACCATGCCATTAAGCTTAGCATGT

[0527] TGACCAAGAAAGCTTGTCTGCATCTGAATCCCGGCGGAACCTGTGTCAGCATAGGTTATGGTTACGC

[0528] TGACAGGGCCAGCGAAAGCATCATTGGTGCTATAGCGCGGCAGTTCAAGTTTTCCCGGGTATGCAA

[0529] ACCGAAATCCTCACTTGAAGAGACGGAAGTTCTGTTTGTATTCATTGGGTACGATCGCAAGGCCCGT

[0530] ACGCACAATCCTTACAAGCTTTCATCAACCTTGACCAACATTTATACAGGTTCCAGACTCCACGAAG

[0531] CCGGATGTGCACCCTCATATCATGTGGTGCGAGGGGATATTGCCACGGCCACCGAAGGAGTGATTAT

[0532] AAATGCTGCTAACAGCAAAGGACAACCTGGCGGAGGGGTGTGCGGAGCGCTGTATAAGAAATTCC

[0533] CGGAAAGCTTCGATTTACAGCCGATCGAAGTAGGAAAAGCGCGACTGGTCAAAGGTGCAGCTAAA

[0534] CATATCATTCATGCCGTAGGACCAAACTTCCAACAAAGTTTCGGAGGTTGAAGGTGACAAACAGTTG

[0535] GCAGAGGCTTATGAGTCCATCGCTAAGATTGTCAACGATAACAATTACAAGTCAGTAGCGATTCCAC

[0536] TGTTGTCCACCGGCATCTTTTCCGGGAACAAAGATCGACTAACCCAATCATTGAACCATTTGCTGAC

[0537] AGCTTTGACACCACTGATGCAGATGTAGCCATATACTGCAGGGACAAGAAATGGGAAATGACTCTC

[0538] AAGGAAGCAGTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATACCCGACGACTCTTCAGTGAC

[0539] AGAACCTGATGCAGAGCTGGTGAGGGTGCATCCGAAGAGTTCTTTGGCTGGAAGGAAGGGCTACA

[0540] GCACAAGCGATGGCAAAACTTTCTCATATTTGGAAGGGACCAAGTTTCACCAGGCGGCCCAAGGATA

[0541] TAGCAGAAAATTATGCCATGTGGCCCGTTGCAACGGAGGCCAATGAGCAGGTATGCATGTATATCCT

[0542] CGGAGAAAGCATGAGCAGTATTAGGTCGAAATGCCCCGTCGAAGAGTCGGAAGCCTCCACACCAC

[0543] CTAGCACGCTGCCTTGCTTGTGCATCCATGCCATGACTCCAGAAAGGTACAGCGCCTAAAAGCCTC

[0544] ACGTCCAGAACAAATTACTGTGTGCTCATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAG

[0545] AAGATCCAATGCTCCCAGCCTATATTGTTCTCACCGAAAGTGCCTGCGTATATTCATCCAAGGAAGTA

[0546] TCTCGTGGAAACACCACCGGTAGACGAGACTCCGGAGCCATCGGCAGAGAACCAATCCACAGAGG

[0547] GGACACCTGAACAACCACCACTTATAACCGAGGATGAGACCAGGACTAGAACGCCTGAGCCGATCA

[0548] TCATCGAAGAGGAAGAAGAGGATAGCATAAGTTTGCTGTCAGATGGCCCGACCCACCAGGTGCTGC

[0549] AAGTCGAGGCAGACATTCACGGGCCGCCCTCTGTATCTAGCTCATCCTGGTCCATTCCTCATGCATCC

[0550] GACTTTGATGTGGACAGTTTATCCATACTTGACACCCTGGAGGGAGCTAGCGTGACCAGCGGGGCA

[0551] ACGTCAGCCGAGACTAACTCTTACTTCGCAAAGAGTATGGAGTTTCTGGCGCGACCGGTGCCTGCG

[0552] CCTCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCGCGCACAAGAACACCGTCACTTGCACCC

[0553] AGCAGGGCCTGCTCGAGAACCAGCCTAGTTTCCACCCCGCCAGGCGTGAATAGGGTGATCACTAGA

[0554] GAGGAGCTCGAGGCGCTTACCCCGTCACGCACTCCTAGCAGGTCGGTCTCGAGAACCAGCCTGGTC

[0555] TCCAACCCGCCAGGCGTAAATAGGGTGATTACAAGAGAGGAGTTTGAGGCGTTCGTAGCACAACAA

[0556] CAATGACGGTTTGATGCGGGTGCATACATCTTTTCCTCCGACACCGGTCAAGGGCATTTACAACAAA

[0557] AATCAGTAAGGCAAACGGTGCTATCCGAAGTGGTGTTGGAGAGGACCGAATTGGAGATTTCGTATG

[0558] CCCCGCGCCTCGACCAAGAAAAAGAAGAATTACTACGCAAGAAATTACAGTTAAATCCCACACCTG

[0559] CTAACAGAAGCAGATACCAGTCCAGGAAGGTGGAGAACATGAAAGCCATAACAGCTAGACGTATTC

[0560] TGCAAGGCCTAGGGCATTATTTGAAGGCAGAAGGAAAAGTGGAGTGCTACCGAACCCTGCATCCTG

[0561] TTCCTTTGTATTCATCTAGTGTGAACCGTGCCTTTTCAAGCCCCAAGGTCGCAGTGGAAGCCTGTAA

[0562] CGCCATGTTGAAAGAGAACTTTCCGACTGTGGCTTCTTACTGTATTATTCCAGAGTACGATGCCTATT

[0563] TGGACATGGTTGACGGAGCTTCATGCCCCTTAGACACTCTCAGTTTTTGCCCTGCAAAGGAGCGCA

[0564] GCTTTCCAAAGAAACACTCCTATTTGGAACCCACAATACGATCGGCAGTGCCTTCAGCGATCCAGA

[0565] ACACGCTCCAGAACGTCCTGGCAGCTGCCACAAAAAGAAATTGCAATGTCACGCAAATGAGAGAA

[0566] TTGCCCGTATTGGATTCGGCGGCCTTTAATGTGGAATGCTTCAAGAAATATGCGTGTAATAATGAATA

[0567] TTGGGAAACGTTTAAAGAAAACCCCATCAGGCTACTGAAAAAACGTGGTAAAATTACATTACCAA

[0568] ATTAAAAGGACCAAAGCTGCTGCTCTTTTTGCGAAGACACATAATTTGAATATGTTGCAGGACATA

[0569] CCAATGGACAGGTTTGTAATGGACTTAAAGAGAGACGTGAAAGTGACTCCAGGAAAAAAACATACT

[0570] GAAGAACGGCCCAAGGTACAGGTGATCCAGGCTGCCGATCCGCTAGCAACAGCGTATCTGTGCGGA

[0571] ATCCACCGAGAGCTGGTTAGGAGATTAAATGCGGTCCTGCTTCCGAACATTCATACACTGTTTGATAT

[0572] GTCGGCTGAAGACTTTGACGCTATTATAGCCGAGCACTTCCAGCCTGGGGATTGTGTTCTGGAAACT

[0573] GACATCGCGTCGTTTGATAAAAGTGAGGACGACGCCATGGCTCTGACCGCGTTAATGATTCTGGAA

[0574] GACTTAGGTGTGGACGCAGAGCTGTTGACGCTGATTTTCGCGGCTTTCGGCGAAATTTCATCAATAC

[0575] ATTTGCCCACTAAAACTAAATTTAAATTCGGAGCCATGAGAAATCTGGAATGTTCCTCACACTGTTT

[0576] GTGAACACAGTCATTAACATTGTAATCGCAAGCAGAGTGTTGAGAGAACGGCTAACCGGATCACCA

[0577] TGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGTCAAATCGGACAAATTAATGGCAGAC

[0578] AGGTGCGCCACCTGGTTGAATATGGAAGTCAAGATTATAGATGCTGTGGTGGGCGAGAAAGCGCCT

[0579] TATTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGCGTGCCGTGTGGCAGACCCCC

[0580] TAAAAAGGCTGTTTAAGCTTGGCAAACCTCTGGCAGCAGACGATGAACATGATGATGACAGGAGAA

[0581] GGGCATTGCATGAAGAGTCAACACGCTGGAACCGAGTGGGTATTCTTTCAGAGCTGTGCAAGGCAG

[0582] TAGAATCAAGGTATGAAACCGTAGGAACTTCCATCATAGTTATGGCCATGACTACTCTAGCTAGCAGT

[0583] GTTAAATCATTCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAA

[0584] SEQ ID No.19:

[0585] Amino acid sequence of the 9th replicase mutant:

[0586]

[0587] SEQ ID No.20:

[0588] The ninth replicase mutant DNA sequence:

[0589]

[0590] SEQ ID No.21:

[0591] S protein amino acid sequence:

[0592]

[0593] SEQ ID No.22:

[0594] S protein DNA sequence:

[0595]

[0596] SEQ ID No.23:

[0597] Amino acid sequence of F protein:

[0598] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0599] SEQ ID No.24:

[0600] DNA sequence of F protein:

[0601]

[0602] SEQ ID No.25:

[0603] DNA sequence corresponding to the S protein trans-replicon:

[0604]

[0605] SEQ ID No.26:

[0606] DNA sequence corresponding to the F protein trans-replicon:

[0607]

[0608] SEQ ID No.27

[0609] 5’ UTR sequence

[0610] AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC

[0611] SEQ ID No.28

[0612] 3’ UTR sequence

[0613] CTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGC

[0614] SEQ ID No.29

[0615] Another wild-type replicase amino acid sequence

[0616]

Claims

1. A replicase, characterized in that Based on the sequence of the wild-type replicase and having mutation sites, the mutation sites form salt bridges, hydrophobic bonds or electrostatic interactions within the nsP1-4 proteins of the replicase.

2. The replicase according to claim 1, characterized in that The mutation sites include any one or more of T372K of protein nsP1, S54R of protein nsP2, C157P, A161L, L168E, and E404F of protein nsP4.

3. The replicase according to claim 2, characterized in that The mutation sites include any one or more of the following combinations: (i) T372K of protein nsP1; (ii) S54R of protein nsP2; (iii) C157P, A161L, L168E and E404F of protein nsP4.

4. The replicase according to claim 3, characterized in that The wild-type replicase is derived from an alphavirus, which includes any one or more of Semliki Forest virus, Venezuelan equine encephalitis virus, Sindbis virus, and Chikungunya virus.

5. The replicase according to claim 4, characterized in that The alphavirus is Venezuelan equine encephalitis virus.

6. The replicase according to claim 5, characterized in that The sequence of the wild-type replicase is as shown in SEQ ID No. 1 in the sequence listing.

7. The replicase according to claim 6, characterized in that The replicase comprising two mutation site combinations has an amino acid sequence as shown in SEQ ID No.15 in the sequence listing; the replicase comprising three mutation site combinations has an amino acid sequence as shown in SEQ ID No.17 in the sequence listing; the replicase comprising six mutation site combinations has an amino acid sequence as shown in SEQ ID No.19 in the sequence listing.

8. The replicase according to claim 7, characterized in that The replicase also includes a 5' untranslated region and a 3' untranslated region sequence.

9. A composition, characterized in that It comprises an RNA construct for encoding the replicase according to any one of claims 1 to 8, and a replicon RNA for expressing a target protein.

10. The composition according to claim 9, characterized in that The replicon RNA comprises a cis replicon or a trans replicon, and the cis or trans replicon comprises a recognition sequence of the replicase according to any one of claims 1 to 8.

11. The composition according to claim 9, characterized in that The RNA construct is mutated based on the nucleotide sequence of the wild-type replicase and has a nucleotide sequence as shown in SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10, SEQ ID No.12, SEQ ID No.14, SEQ ID No.16, SEQ ID No.18 or SEQ ID No.20 in the sequence listing.

12. The composition according to claim 10, characterized in that The target protein includes any one or more of antigens, antibodies, fusion proteins, and polypeptides.

13. The composition according to claim 12, characterized in that The target protein includes the novel coronavirus spike protein and / or respiratory syncytial virus.

14. The composition according to claim 13, characterized in that The trans-replicon RNA expressing the novel coronavirus spike protein has a nucleotide sequence as shown in SEQ ID No. 25 in the sequence listing, and the trans-replicon RNA expressing the respiratory syncytial virus has a nucleotide sequence as shown in SEQ ID No. 26 in the sequence listing.

15. The composition according to claim 10, characterized in that The outer layer of the composition is coated with lipid nanoparticles.

16. An expression vector, characterized in that: Comprising the composition according to any one of claims 9 to 15, the expression vector is capable of expressing the target protein in a prokaryotic or eukaryotic host cell.

17. A vaccine, characterized in that Comprising the composition according to any one of claims 9 to 15.

18. The vaccine according to claim 17, characterized in that The vaccine is a monovalent, bivalent or multivalent vaccine.

19. A use of a replicase for preparing a reagent for increasing the expression amount of a target protein, characterized in that: The replicase uses the sequence of the wild-type replicase as the initial sequence and has mutation sites, wherein the mutation sites include any one or more of T372K of protein nsP1, S54R of protein nsP2, C157P, A161L, L168E, and E404F of protein nsP4.

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