Construction method and application of efficient expression system based on Seali forest virus replicon

Abstract

The invention provides a construction method and application of a high-efficiency expression system based on a Seali forest virus replicon. The method comprises the following steps: constructing a saRNA system for expressing a reporter gene; constructing to obtain replicase providing plasmids in the taRNA system; constructing to obtain an antigen providing plasmid in the taRNA system; the method comprises the following steps: respectively linearizing pSFV-CMV-T7-X, pSFV-CMV-T7-TR-X and pSFV-CMV-T7-replicase, and then carrying out in vitro transcription to prepare mRNA (Messenger Ribonucleic Acid); transfecting the prepared mRNA into a host eukaryotic cell for expression according to a certain proportion to obtain an antigen protein; and detecting the antigen protein to evaluate the expression efficiency of the saRNA and taRNA system based on the SFV replicon. According to the self-amplification DNA and RNA vaccine based on the virus replicon, the development period can be shortened, and the vaccine antigen can be expressed at a high level.

Description

Technical Field

[0001] The present invention relates to the field of virus vaccine vector design, and in particular to a construction method and application of a high-efficiency expression system based on Semliki Forest Virus replicon. Background Art

[0002] Currently, two main types of RNA are used as vaccines to prevent diseases: one is self-amplifying RNA (saRNA), and the other is non-replicating mRNA (nrRNA). saRNA vaccine is a new type of vaccine based on RNA replicons. The RNA replicons it contains are RNA derived from the RNA viral genome and can replicate autonomously, thereby achieving rapid and massive self-replication of mRNA in the cytoplasm. The viruses most commonly used to develop replicons are Alphaviruses in the Togaviridae family, such as Sindbis virus (SIN) and Semliki forest virus (SFV).

[0003] SFV replicon vaccines are primarily derived from the bicistronic genome of the Semliki Forest virus. For vaccine engineering, genes encoding nonstructural proteins in the SFV genomic RNA are retained, while structural protein genes under the control of subgenomic promoters are replaced with vaccine antigens of interest. Nonstructural proteins autocatalytically mature and assemble into a multienzyme replicase complex that is able to replicate its own template RNA. The replicase acts as an RNA-dependent RNA polymerase and amplifies the RNA in cis, resulting in high vector copy numbers in the inoculated host cells, resulting in high levels of vaccine antigens. Therefore, lower dose levels of saRNA than nrRNA may be sufficient to induce similar immune responses.

[0004] The latest research shows that replicase can also amplify trans RNA, replicase and antigen can be split into two RNAs, where the gene of interest is expressed by RNA lacking replicase, and is expressed in trans through non-replicating mRNA. The specificity of the interaction between replicase and RNA carrying exogenous genes is guaranteed by the conserved sequence elements in the 5' and 3' end regions inherited by the RNA lacking replicase from the parent saRNA. This type of vaccine is called a trans-amplifying RNA (trans-amplifying taRNA) vaccine based on a binary fission SFV system. In this system, the vaccine antigen and replicase components are encoded on independent vectors for splitting, which can increase the level of antigen expression by adjusting the ratio of the two RNAs, and can also develop bivalent or even multivalent vaccines by increasing the types of antigen protein RNA.

[0005] saRNA vaccines encode not only antigenic genes but also genes required for RNA replication, including RNA-dependent RNA polymerase. Typically, saRNA is a viral genome that lacks some or all of the viral structural genes. Due to the lack of viral structural genes, the assembly of infectious viral particles does not occur. At the same time, the genes necessary for replicating viral RNA are retained, and these partial viral RNAs can replicate in cells. Due to this self-replication ability, repRNA itself can act as an adjuvant and can elicit a strong immune response. Self-amplifying RNA has been effective as a candidate vaccine in several animal species, including mice, non-human primates, and humans. In addition, it has been reported that even with 1 / 64 thousand of the amount of RNA used, saRNA vaccines can achieve the same degree of protection as synthetic mRNA vaccines. The ability of repRNA vaccines to produce strong immunity at a small dose makes them a candidate vaccine that can reduce manufacturing costs and quickly end the COVID-19 pandemic.

[0006] Traditional inactivated or attenuated vaccines require a long development cycle, and traditional nucleic acid vaccines have low antigen expression efficiency, which is difficult to meet the demand. Due to the presence of viral replicase, self-amplifying RNA (saRNA) vaccines based on viral replicons can not only shorten the development cycle, but also express vaccine antigens at high levels. Therefore, saRNA vaccines based on viral replicons are expected to break the bottleneck of vaccine development.

[0007] At present, the research on RNA vaccine systems developed based on SFV replicons is not in-depth enough, and there is little research on taRNA vaccine systems. Many antigenic proteins have a large molecular weight, such as IBVS protein and PEDV S protein, and their expression efficiency in general vectors is very low, or even cannot be expressed. Therefore, the present invention hopes to propose a saRNA and taRNA vaccine system based on SFV replicons that can increase the number of high vector copies and mRNA in host cells through replicase, which is safe, efficient, and can induce protective immunity. This vaccine is expected to produce a large number of vaccines quickly and cost-effectively according to the needs of rapidly evolving or emerging viral pathogens. Summary of the invention

[0008] The purpose of the present invention is to propose a construction method and application of a high-efficiency expression system based on the Semliki Forest Virus replicon, which aims to insert avian infectious bronchitis virus S protein, Newcastle disease HN gene, etc. into the saRNA or taRNA system, and after in vitro transcription into mRNA, both systems can efficiently express the inserted antigen protein and have high safety, and can be used in the development of poultry-related vaccines.

[0009] In a first aspect, the present invention provides a method for constructing a high-efficiency expression system based on a Semliki Forest virus replicon, the method comprising:

[0010] (1) Using pSFVCs-CMV-EGFP as material, construct the saRNA system pSFV-CMV-T7-EGFP expressing the reporter gene;

[0011] (2) pSFV-CMV-T7-EGFP was split and optimized to construct the replicase-providing plasmid pSFV-CMV-T7-replicase in the taRNA system;

[0012] (3) pSFV-CMV-T7-EGFP was split and optimized to construct the antigen providing plasmid pSFV-CMV-T7-TR-EGFP in the taRNA system;

[0013] (4) replacing the reporter gene EGFP with the antigen protein gene sequence X to construct pSFV-CMV-T7-X or pSFV-CMV-T7-TR-X;

[0014] (5) pSFV-CMV-T7-X, pSFV-CMV-T7-TR-X, and pSFV-CMV-T7-replicase were linearized and then transcribed in vitro to prepare mRNA;

[0015] (6) transfecting the prepared mRNA into host eukaryotic cells at a certain ratio for expression to obtain antigenic protein;

[0016] (7) Collect the cells after electrotransfection, obtain the test samples, and detect the antigen protein to evaluate the expression efficiency of the saRNA and taRNA systems based on the SFV replicon.

[0017] Furthermore, the saRNA system constructed in step (1) comprises retaining the CMV promoter in pSFVCs-CMV-EGFP and adding a T7 promoter;

[0018] The saRNA system constructed in step (1) comprises deleting the CE3 portion of the SFV structural protein;

[0019] The construction method of step (1) is to construct the saRNA system pSFV-CMV-T7-EGFP expressing EGFP by segmented PCR of pSFVCs-CMV-EGFP through homologous recombination;

[0020] Specifically, the fragment A1 is amplified from the sequence as shown in SEQ ID NO. 1 by primers A1-nsp1-F and A2-nsp4-R; the vector fragment A2 is cut out by restriction endonucleases BamH I and Xba I; the fragment A1 and the fragment A2 are homologously recombined and connected by homologous recombinase to obtain the saRNA system pSFV-CMV-T7-EGFP expressing EGFP;

[0021] The sequence of the T7 promoter is shown in SEQ ID NO.1; the sequence of A1-nsp1-F is shown in SEQ ID NO.6; and the sequence of A2-nsp4-R is shown in SEQ ID NO.7.

[0022] Furthermore, the sequence of the SFV replicase in step (2) is shown as SEQ ID NO.2;

[0023] The optimization in step (2) includes optimization of 5'UTR and 3'UTR;

[0024] The optimization in step (2) included extending the ploy A tail;

[0025] The optimization in step (2) includes the change of the linearization restriction site, and the same restriction site on the plasmid is subjected to a synonymous mutation;

[0026] The construction method in step (2) constructs the SFV replicase into the recombinant vector A through homologous recombination.

[0027] Furthermore, the 5'UTR optimized sequence is selected from human α-globin 5'UTR, and its sequence is shown in SEQ ID NO.3:

[0028] The 3'UTR represents a fusion of a sequence selected from the amino-terminal enhancer of split (AES) mRNA and mitochondrial encoded 12SrRNA (mtRNR1), the sequence of which is shown in SEQ ID NO. 4;

[0029] The poly A tail is extended by extending 65 A in the template to 100 A, and a short connecting sequence is connected after the initial 30 A, and then 70 A are connected, totaling 100 A. The sequence of the poly A is shown in SEQ ID NO.5;

[0030] The linearization restriction site was changed from the original spe I to the IIS type restriction enzyme BsaΙ, so that a BsaΙ site was inserted after the poly A tail. The sequence of the linearization restriction site is shown in SEQ ID NO.8, and the initial three BsaΙ sites in the recombinant vector were synonymously mutated.

[0031] Further, the homologous recombination steps in step (2) are as follows: fragment B1 is amplified from pSFVCs-CMV-EGFP using primers 5'UTR-F and B1-BsaI-mut-R; vector fragment B2 is amplified from pSFVCs-CMV-EGFP using primers B2-BsaI-mut-F and B2-BsaI-mut-R; fragment B3 is amplified from pSFVCs-CMV-EGFP using primers B3-BsaI-mut2-F and B3-BsaI-mut-R; 3'UTR optimized sequence and optimized polyA tail sequence are removed from the sequence as shown in SEQ ID NO: Fragment B4 was cut out by the enzyme shown in NO.3; fragment B5 was amplified from pSFVCs-CMV-EGFP by primers A5-F and B5-BsaI-mut3-R; fragment B6 was amplified from pSFVCs-CMV-EGFP by primers B6-BsaI-mut3-F and A6-5'UTR-R; fragments B1, B2, B3, B5 and B6 were homologously recombined and connected using homologous recombinase with fragment B4 as a vector to obtain the plasmid pSFV-CMV-T7-replicase that provides replicase in the taRNA system;

[0032] The sequence of B1-5'UTR-F is shown in SEQ ID NO.9, the sequence of B1-BsaI-mut1-R is shown in SEQ ID NO.10, the sequence of B2-BsaI-mut1-F is shown in SEQ ID NO.11, the sequence of B2-BsaI-mut2-R is shown in SEQ ID NO.12, the sequence of B3-BsaI-mut2-F is shown in SEQ ID NO.13, the sequence of B3-R is shown in SEQ ID NO.14, the sequence of B5-F is shown in SEQ ID NO.15, the sequence of B5-BsaI-mut3-R is shown in SEQ ID NO.16, the sequence of B6-BsaI-mut3-F is shown in SEQ ID NO.17, and the sequence of B6-5'UTR-R is shown in SEQ ID NO.18.

[0033] Furthermore, the optimization in step (3) is to retain part of the SFV replicon sequence, including 221 bp at the 5' end of nsp1 and 984 bp at the 3' end of nsp4 of the SFV replicon, and delete the rest of the replicase part.

[0034] The construction method in step (3) constructs the recombinant vector pSFV-CMV-T7-TR-EGFP through homologous recombination.

[0035] The homologous recombination in step (3) is performed by amplifying fragment C1 from pSFVCs-CMV-EGFP using primers A1-nsp1-F and A1-nsp1-R; fragment C2 is amplified from pSFVCs-CMV-EGFP using primers A2-nsp4-F and A2-nsp4-R: fragments C1 and C2 are homologously recombined and connected using a homologous recombinase to obtain a recombinant vector pSFV-CMV-T7-TR-EGFP;

[0036] The sequence of A1-nsp1-F is shown in SEQ ID NO.19, the sequence of A1-nsp1-R is shown in SEQ ID NO.20, the sequence of A2-nsp4-F is shown in SEQ ID NO.21, and the sequence of A2-SFV-promoter-R is shown in SEQ ID NO.22.

[0037] Furthermore, the construction method in step (4) is to construct the DNA sequence containing the antigen protein into a vector expressing the exogenous gene through homologous recombination;

[0038] The steps of homologous recombination in step (4) are specifically as follows:

[0039] Using cDNAs of avian infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and infectious bursal disease virus (IBDV) of cell-adapted strains IBV-P65, A96, MH20, and H120 as templates, IBVP65S, A96S, MH20S, H120S, NDV-HN, and IBDV VP2 cDNA sequences were obtained by amplification with the primers listed in Table 12;

[0040] The vectors pSFV-CMV-T7 and pSFV-CMV-T7-TR were cut into linear fragments by BamHΙ and SmaΙ, and then homologously recombined with the amplified IBV P65S, A96S, MH20S, H120S, NDV-HN, and IBDV VP2 cDNA sequences, respectively.

[0041] Furthermore, in step (2), the pSFV-CMV-T7-X and pSFV-CMV-T7-TR-X plasmids are linearized by digestion with Spe I, and the pSFV-CMV-T7-replicase plasmid is linearized by digestion with Bsa I;

[0042] The in vitro transcription to prepare mRNA in step (5) also includes an mRNA capping process and an mRNA purification process;

[0043] The detection of the antigen protein in step (7) includes western blotting.

[0044] Furthermore, the host eukaryotic cell in step (6) is at least one of human kidney epithelial cells 293T and Syrian hamster kidney cells BHK;

[0045] The pSFV-CMV-T7-X RNA in step (6) is purified and quantified, and then transfected according to the required RNA amount;

[0046] The pSFV-CMV-T7-TR-X RNA and pSFV-CMV-T7-replicase RNA in step (6) were purified and quantified, and then transfected at a mass ratio of 1:4;

[0047] In step (6), the pSFV-CMV-T7-TR-X transfected is simultaneously transfected with 2-3 species, wherein X is at least one of NDV HN, IBV S, and IBDV VP2.

[0048] In the second aspect, the present invention also proposes a taRNA system obtained by the construction method of the high-efficiency expression system based on the Semliki Forest virus replicon as mentioned above, or the use of the taRNA system in the preparation of vaccines for mammals.

[0049] In summary, according to the above-mentioned method for constructing an efficient expression system based on the Semliki Forest virus replicon, two efficient expression systems were constructed by using the Semliki Forest virus replicon (SFV) as a replicon vector. In this system, the genes encoding non-structural proteins in the SFV genomic RNA are retained, and the structural protein genes under the control of the subgenomic promoter are replaced by the vaccine antigen of interest. The non-structural proteins autocatalytically mature and assemble into a multi-enzyme replicase complex that can replicate its own template RNA. As an RNA-dependent RNA polymerase, the replicase amplifies RNA in cis, resulting in a high vector copy number in the inoculated host cell, thereby resulting in a high level of vaccine antigen. At the same time, a taRNA vaccine system was constructed, which split the replicase and antigen into two RNAs, in which the antigen protein was expressed by RNA lacking the replicase, and expressed in trans by non-replicating mRNA. The specificity of the interaction between the replicase and the RNA carrying the antigen gene is guaranteed by the conserved sequence elements in the 5' and 3' end regions inherited by the RNA lacking the replicase from the parent saRNA. At present, many antigen proteins have large molecular weights, such as IBVS protein and PEDV S protein, and their expression efficiency in general vectors is very low, or even cannot be expressed. The mRNA vaccine system provided by the present invention not only encodes antigen genes, but also encodes RNA-dependent RNA polymerase required for RNA replication, thereby continuously amplifying and generating more RNA with RNA as a template, so that antigen proteins can be expressed efficiently. Among them, the reverse amplification RNA vaccine system can also express two or more antigen proteins at the same time. This type of vaccine has the ability to produce strong immunity with a small dose, and makes the vaccine safer and more efficient and simplifies the mRNA vaccine production process, which helps to deal with rapidly evolving pathogens and emerging infectious diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0051] Figure 1 This is the plasmid map of pSFVCs-CMV-EGFP;

[0052] Figure 2 Map of the saRNA system plasmid pSFV-CMV-T7-EGFP plasmid constructed by homologous recombination technology;

[0053] Figure 3 is a map of the taRNA system constructed by homologous recombination technology, a. map of pSFV-CMV-T7-replicase plasmid, b. map of pSFV-CMV-T7-TR-EGFP plasmid;

[0054] Figure 4 To construct the nucleic acid map of saRNA system fragments and overexpression fluorescence detection, a is the PCR amplification product of saRNA system; b is the double enzyme digestion identification product of pSFV-CMV-T7-EGFP Sma I and EcoR V; is the fluorescence expression of HEK-293T cells transfected with pSFV-CMV-T7-EGFP (scale: 200 μm);

[0055] Figure 5 To construct the saRNA system fragment nucleic acid map and overexpression fluorescence detection map, a is the plasmid pSFV-CMV-T7-replicase PCR amplification product for the construction of replicase; b is the PCR amplification product for the construction of pSFV-CMV-T7-TR-EGFP; c is the fluorescence expression of HEK-293T cells transfected with the taRNA system (scale: 200 μm);

[0056] Figure 6 Figure 2 is a diagram of RNA nucleic acid gel detection and mRNA transfection expression of in vitro transcribed RNA inserted into other antigen saRNA systems, where a1 is pSFV-CMV-T7-EGFP mRNA; b1-3 are pSFV-CMV-T7-H120 S, pSFV-CMV-T7-A96S, and pSFV-CMV-T7-MH20S mRNA, respectively; c is the expression of green fluorescent protein after pSFV-CMV-T7-EGFP mRNA transfected cells (scale: 200 μm); d is the WB detection result after pSFV-CMV-T7-IBV S transfected cells; e is the WB detection result after cells were transfected with saRNA expressing H120 S, MH20 S, and A96 S;

[0057] Figure 7 Schematic diagram of RNA nucleic acid gel detection and RNA transfection expression of in vitro transcribed taRNA system inserted with other antigens, where a and b are in vitro transcribed RNA nucleic acid gel detection, where a1-3 are in vitro transcribed mRNAs of pSFV-CMV-T7-EGFP, pSFV-CMV-T7-replicase, and pSFV-CMV-T7-TR-EGFP, respectively; b1-4 are in vitro transcribed mRNAs of pSFV-CMV-T7-replicase, pSFV-CMV-T7-IBVS, pSFV-CMV-T7-TR-IBVS, and pSFV-CMV-T7-TR-H120S, respectively. c is the expression of green fluorescent protein after pSFV-CMV-T7-EGFP mRNA transfected cells (scale: 200μm); d is the WB detection result after b is transfected into BHK cells with taRNA system mRNA expressing H120 S and IBV S;

[0058] Figure 8 The figure is a schematic diagram of the Western Blot detection results of the taRNA system expressing multiple antigen proteins at the same time, wherein a is the case where the taRNA system simultaneously expresses IBV S protein and NDV HN protein; b is the case where the taRNA system simultaneously expresses IBV S protein, NDV HN protein and IBDV VP2 protein;

[0059] Fig. 9 Schematic diagram of the mRNA vaccine skeleton for expressing NDVF protein in saRNA and taRNA systems, where a is the pSFV-CMV-T7-NDV F mRNA vaccine skeleton; b is the pSFV-CMV-T7-TR-NDV F and pSFV-CMV-T7-replicase vaccine skeleton;

[0060] Fig.10 Expression verification diagram of NDV mRNA vaccine prepared by saRNA and taRNA systems, where Figure a is the in vitro transcription RNA nucleic acid gel detection of two systems expressing NDV F protein, a1-3: pSFV-CMV-T7-NDV F, pSFV-CMV-T7-replicase, pSFV-CMV-T7-TR-NDV F; Figure b is the Western Blot protein detection diagram of two NDV mRNA vaccines;

[0061] Fig.11 This is a schematic diagram of the ELISA antibody level detection of the NDV mRNA vaccine group, where the y-axis is the absorbance at 450 nm, n=4.

[0062] Fig.12 Schematic diagram of the neutralizing antibody detection of the NDV mRNA vaccine group, where the y-axis is the neutralizing antibody titer, where n=4;

[0063] Fig.13 Schematic diagram of the PEDV DNA vaccine backbone based on the SFV replicon;

[0064] Fig.14 This is a schematic diagram of the Western Blot detection of PEDV DNA vaccine;

[0065] Fig.15 The results of serum-specific antibody test for PEDV DNA vaccine, where n=5, the y-axis is the absorbance at 450 nm; the significance analysis is a schematic diagram of the results compared with the PBS group;

[0066] Fig.16 The results of mouse spleen lymphocyte proliferation experiment, where n = 3, the y-axis is the stimulation index (SI) of mouse spleen lymphocytes;

[0067] Fig.17 This is a schematic diagram of the secretion levels of IFN-γ and IL4 in the cell supernatant of splenic lymphocytes of immunized mice after stimulation with specific antigens. DETAILED DESCRIPTION

[0068] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

[0069] The following detailed description is an exemplary description, which is intended to provide further detailed description of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. The terms used in the present invention are only for describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present invention.

[0070] Example 1

[0071] This embodiment provides a method for constructing an efficient expression system based on the Semliki Forest Virus replicon, specifically a saRNA system pSFV-CMV-T7-EGFP expressing the reporter gene EGFP, specifically comprising the following steps:

[0072] According to the gene sequence of pSFVCs-CMV-EGFP (whose sequence is shown in SEQ ID NO.1), primers containing homology arms for adding T7 promoter and deleting part of SFV structural protein CE3 were designed. The primer sequences were synthesized by Jinweizhi Biotechnology Co., Ltd., as shown in Table 1; primers A1-nsp1-F and A2-nsp4-R were designed using homologous recombination technology to amplify vector fragment A1 containing SFV replicon; restriction endonucleases BamH I and Xba I were used to cut out vector fragment A2. The two fragments were homologously recombined by homologous recombination enzyme. The connection reaction system is shown in Table 2, and the reaction conditions are shown in Table 3. The connection product was transformed into Escherichia coli trans10 competent cells by heat shock method, and an appropriate amount of transformation product was spread and cultured in LB solid culture medium containing 100 micrograms per milliliter of ampicillin. Several single clones were selected for plasmid extraction, and two fragments of 7871bp and 3981bp were obtained as target plasmids by EcoRV and Sma I restriction enzyme digestion. The plasmid was further verified to be successful, and finally the positive plasmid was selected and sent to the company for sequencing verification, and the positive plasmid pSFV-CMV-T7-EGFP was obtained. The constructed positive plasmid map is shown in Figure 2 The results of PCR and restriction digestion are shown in Figure 4 As shown in a and b.

[0073] Verification of overexpression of the saRNA system pSFV-CMV-T7-EGFP expressing EGFP: prepare two 1.5mL centrifuge tubes, add 100μL of serum-free and dual-antibody-free Opti-MEM reduced medium to each centrifuge tube, and then add 2μg of plasmid to one of the tubes as needed, and gently mix after adding; then add twice the volume of the total amount of plasmid to the other tube (add 4μL of transfection reagent), let it stand for 15 minutes, and then add the mixture drop by drop into the corresponding wells of a twelve-well plate with a cell density of 70%-80%. After shaking, place it in an incubator at 37°C and 5% CO2 for 4-6 hours, replace the medium and continue to culture. After 24 hours, observe the expression of EGFP under a fluorescent inverted microscope. The results are shown in Figure 4 c.

[0074] Table 1 Amplification primer sequences for constructing pSFV-CMV-T7-EGFP plasmid

[0075]

[0076] Table 2 Homologous recombination reaction system

[0077]

[0078]

[0079] Table 3 Homologous recombination reaction conditions

[0080] Reaction temperature Reaction time 50℃ 30min 4℃ 3min

[0081] Example 2

[0082] This embodiment provides a method for constructing an efficient expression system based on the Semliki Forest Virus replicon, specifically providing a plasmid pSFV-CMV-T7-replicase for the replicase in the taRNA system, specifically comprising the following steps:

[0083] According to the gene sequence of pSFV-CMV-T7-EGFP, primers containing T7 promoter and human α-globin 5'UTR were designed. The primers are shown in Table 4. The homologous recombination technology was used to design primers B1-5'UTR-F (adding human α-globin 5'UTR sequence to the homology arm to replace SFV 5'UTR) and B1-BsaI-mut1-R to amplify vector fragment B1; primers B2-BsaI-mut1-F and B2-BsaI-mut2-R for point mutation of BsaI restriction site were designed to amplify fragment B2; primers B3-BsaI-mut2-F and B3-R for point mutation of Bsa1 restriction site 2 were designed to amplify fragment B3; the AES-mtRNR1-polyA-Bsal fragment synthesized by the company was double-digested with Hind III and Xho I, and the 3'UTR optimized sequence and the optimized poly A tail sequence were removed from the sequence as shown in SEQ ID NO.3 was used to cut out fragment B4; primers B5-F and B5-BsaI-mut3-R were designed to amplify fragment B5; primers B6-BsaI-mut3-F and B6-5'UTR-R for point mutation Bsa1 restriction site 3 were designed to amplify fragment B6; the fragments were homologously recombined and connected using homologous recombinase with restriction fragment B4 as a vector. The connection reaction system is shown in Table 5, and the reaction conditions are shown in Table 6; the connection product was transformed into Escherichia coli trans10 competent cells by heat shock method, and an appropriate amount of the transformation product was spread and cultured in LB solid culture medium containing 100 μg / ml ampicillin, and several single clones were selected for plasmid extraction, and the plasmid was extracted by EcoR V and SmaΙ enzyme digestion, obtained 7110bp and 3535bp fragments as the target plasmid; further verify whether the plasmid is successful, finally select the positive plasmid to send to the company for sequencing verification, determine the positive plasmid pSFV-CMV-T7-replicase, the constructed positive plasmid map is as follows Figure 3a As shown, PCR amplification is as follows Figure 5 As shown in a.

[0084] Table 4 Amplification primer sequences for constructing pSFV-CMV-T7-replicase plasmid

[0085]

[0086]

[0087] Table 5 Homologous recombination reaction system

[0088]

[0089]

[0090] Table 6 Homologous recombination reaction conditions

[0091] Reaction temperature Reaction time 50℃ 60min 4℃ 3min

[0092] Example 3

[0093] This embodiment provides a process for constructing an antigen providing plasmid pSFV-CMV-T7-TR-EGFP in a taRNA system and overexpression verification, and the specific process is as follows:

[0094] Amplification primers were designed based on the gene sequence of pSFV-CMV-T7-EGFP. The primer sequences were synthesized by GENEWIZ Biotechnology Co., Ltd. The primer sequences are shown in Table 7. Primers A1-nsp1-F and A1-nsp1-R were designed using homologous recombination technology to amplify the fragment C1 containing the 221 bp at the 5' end of the SFV replicon nsp1. Primers A2-nsp4-F and A2-SFV-promoter-R were designed to amplify the vector fragment C2 containing the 984 bp at the 3' end of the SFV replicon nsp4. The PCR amplification results are shown in Table 7. Figure 5 As shown in b, the two fragments were homologously recombined and connected by homologous recombination enzyme. The connection reaction system is shown in Table 1, and the reaction conditions are shown in Table 2; the connection product was transformed into Escherichia coli trans10 competent cells by heat shock method, and an appropriate amount of transformation product was spread and cultured in LB solid culture medium containing 100 micrograms per milliliter of ampicillin. Finally, several single colonies were selected to verify whether the cloning was successful by PCR method; several single clones were selected from them for plasmid extraction, and two fragments of 1777bp and 3981bp were obtained as target plasmids by EcoR V and SmaΙ restriction enzyme digestion; further verify whether the plasmid was successful, and finally select positive plasmids to send to the company for sequencing verification, and determine that the positive plasmid pSFV-CMV-T7-X was obtained. The constructed positive plasmid map is shown in Figure 3b shown.

[0095] Overexpression verification of the taRNA system expressing EGFP: prepare two 1.5mL centrifuge tubes, add 100μL of serum-free and double-antibody-free Opti-MEM reduced medium to each centrifuge tube, and then add plasmids to one of the tubes as needed (including pSFV-CMV-T7-TR-EGFP 700ng, pSFV-CMV-T7-replicase 2.1μg), and mix gently after adding; then add twice the volume of the total amount of plasmids to the other tube (add 5.6μL transfection reagent), let it stand for 15min, and then add the mixture drop by drop into the corresponding wells of the twelve-well plate with a cell density of 70%-80%, shake well, and place it in an incubator at 37°C and 5% CO2 for 4-6h, then replace the medium and continue to culture, and observe the expression of EGFP under a fluorescent inverted microscope after 24h. The results are shown in the figure. Figure 5 c.

[0096] Table 7 Amplification primer sequences for constructing pSFV-CMV-T7-TR-EGFP plasmid

[0097]

[0098] Table 8 Homologous recombination reaction system

[0099]

[0100] Table 9 Homologous recombination reaction conditions

[0101] Reaction temperature Reaction time 50℃ 30min 4℃ 3min

[0102] Example 4

[0103] This example provides a process for constructing different antigen gene sequences into the saRNA system. The specific steps are as follows:

[0104] Viral RNA was extracted from cell samples infected with IBV virus with cell adaptability in this laboratory (i.e., avian infectious bronchitis virus (IBV), purchased from American Type Culture Collection, ATCC, first adapted to 10-day-old chicken embryos for 3 generations, and then transferred to African green monkey kidney cells (Vero, purchased from American Type Culture Collection, ATCC) for continuous infection for 65 generations (P65), to obtain the adapted strain IBV-P65 (hereinafter referred to as IBVS to represent IBVP65S). The experimental samples were obtained by infecting Vero cells with the adapted strain IBV-P65, and when all cells developed pathological effects, the cells were lysed with Trizol and the samples were collected) and IBV wild-type strains A96, MH20, and H120 samples isolated from chicken farms, and reverse transcribed into cDNA. The reverse transcription system is shown in Table 10, and the reaction conditions are shown in Table 11; a vector with the same pSFV-CMV-T7 vector (the vector was transcribed by BamH I and Sma I restriction enzyme digestion of pSFV-CMV-T7-EGFP, gel recovery of pSFV-CMV-T7 linear fragments) primers with the same homology arms were used to amplify IBV S, IBV wild-type strains A96, MH20, and H120 S protein gene fragments by PCR using cDNA as a template. The primers for amplifying these fragments are shown in Table 12, the reaction system is shown in Table 13, and the reaction conditions are shown in Table 14; the fragments and the linearized vector pSFV-CMV-T7-X were homologously recombined and connected by homologous recombination (the reaction system and conditions were the same as in Example 1); the connection products were transformed into Escherichia coli trans10 competent cells by heat shock method, and an appropriate amount of the transformation product was spread and cultured in LB solid culture medium containing 100 micrograms per milliliter of ampicillin, and finally several single colonies were selected to verify whether the cloning was successful by PCR; several single clones were selected from them for plasmid extraction, and the EcoR pSFV-CMV-T7-IBVS, pSFV-CMV-T7-A96S, pSFV-CMV-T7-MH20S, and pSFV-CMV-T7-H120S were digested with V and SmaΙ to obtain two fragments of 10643bp+3981bp, 10652bp+3981bp, 10652bp+3981bp, and 10616bp+3981bp respectively as the target plasmids to verify whether the plasmid construction was successful. Finally, the positive plasmids were selected and sent to the company for sequencing verification to determine whether the positive plasmids pSFV-CMV-T7-IBVS, pSFV-CMV-T7-MH20S, pSFV-CMV-T7-A96S, and pSFV-CMV-T7-H120S were obtained.

[0105] Table 10 Reverse transcription reaction system

[0106]

[0107] Table 11 Reverse transcription reaction conditions

[0108]

[0109]

[0110] Table 12 Primer sequences

[0111]

[0112]

[0113] Table 13 Homologous recombination reaction system

[0114]

[0115]

[0116] Table 14 Homologous recombination reaction conditions

[0117] Reaction temperature Reaction time 50℃ 45min 4℃ 3min

[0118] Example 5

[0119] This embodiment provides a process for constructing different antigen gene sequences into the taRNA system, and the specific steps are as follows:

[0120] The cDNA fragments of IBV-P65 and H120 and Newcastle disease virus extracted in Example 4 were used as templates for amplifying the S and Newcastle disease virus HN protein gene sequences, and the pXJ40-IBDVVP2 plasmid containing infectious bursal disease virus antigens preserved in this laboratory was used as a template for amplifying the IBDV VP2 protein gene. Primers with the same homology arms as the pSFV-CMV-T7-TR vector (the vector was obtained by digesting pSFV-CMV-T7-TR-EGFP with BamH1 and Sma1, and gel recovery of pSFV-CMV-T7-TR linear fragments) were designed, and the above gene fragments were amplified by PCR. The primer sequences for amplifying these fragments are shown in Table 12, and the primer sequences for amplifying NDV HN and IBDVVP2 protein genes are shown in Table 15. The fragment and the linearized vector pSFV-CMV-T7-TR were homologously recombined and connected by homologous recombination, and pSFV-CMV-T7-TR-IBVS, pSFV-CMV-T7-TR-H120S, pSFV-CMV-T7-TR-NDV HN, and pSFV-CMV-T7-TR-IBDV VP2 were digested and verified by EcoR V and BamH Ι; after sequencing, positive plasmids pSFV-CMV-T7-IBVS, pSFV-CMV-T7-TR-H120S, pSFV-CMV-T7-TR-NDV HN and pSFV-CMV-T7-TR-IBDV VP2 were obtained, and the method for constructing the plasmid was the same as that in Example 4.

[0121] Table 15 Primer sequences

[0122]

[0123]

[0124] Example 6

[0125] This example provides the process of in vitro transcription and RNA transfection of the saRNA system, and the specific steps are as follows:

[0126] pSFV-CMV-T7-EGFP, pSFV-CMV-T7-IBVS, pSFV-CMV-T7-A96S, pSFV-CMV-T7-MH20S, and pSFV-CMV-T7-H120S plasmids were linearized using speΙ digestion, and the reaction system is shown in Table 16:

[0127] Table 16 System of speΙ enzyme digestion of saRNA system plasmid

[0128]

[0129] The linearized plasmid was transcribed in vitro to obtain the corresponding mRNA; co-transcriptional capping was performed using the Novagen in vitro transcription kit (TR101-01, purchased from Novagen) and NEB m7G(5)ppp(5)G RNA cap analog (S1411S, purchased from NEB), and in vitro transcription was performed according to the in vitro transcription system in Table 17.

[0130] Table 17 In vitro transcription system of saRNA system.

[0131]

[0132]

[0133] After the above system is prepared, transcribe at 37℃ for 1h, then add 2μL GTP, 0.5μL 10X buffer and 2.5μL Nuclease-free water and continue transcription at 37℃ for 3.5h. After the transcription is completed, take 0.5μL of the transcription product, add 4μL Nuclease-free water and 1μL 6x loading buffer for dilution, and operate aseptically to prevent RNA degradation. Then perform electrophoresis in 1% agarose gel to verify whether the band is correct.

[0134] Test results such as Figure 6 As shown, Figure 6 Lane 1 in a shows the gel run of RNA transcribed in vitro from pSFV-CMV-T7-EGFP plasmid. Figure 6 Lanes 2-4 in b are the gel run of RNA transcribed in vitro from pSFV-CMV-T7-H120S, pSFV-CMV-T7-A96S, and pSFV-CMV-T7-MH20S, respectively. The above results show that the transcription results are consistent with the theoretical values.

[0135] (3) RNA purification

[0136] 1. Perform RNA purification according to the instructions of Macherey-Nagel RNA clean up kit (Cat. No.: 740948.50). First, fill the in vitro transcribed RNA sample to 100 μL with depc water;

[0137] 2. Prepare a 1:1 buffer RA1 ethanol premix. For every 100 μL RNA sample, mix 300 μL buffer RA1 and 300 μL ethanol (96% to 100%).

[0138] 3. Take an RNA purification column placed in a collection tube and add 700 μL of the above lysis solution;

[0139] 4. Centrifuge at 8000xg for 30 seconds, discard the collection tube, and place the purification column into a new collection tube.

[0140] 5. Add 700 μL of buffer RA3 to the column, centrifuge at 8000 x g for 30 seconds, discard the collection tube, and return the purification column to a new collection tube;

[0141] 6. Add 350 μL of buffer RA3 to the column and centrifuge at 8000 x g for 2 min;

[0142] 7. Transfer the column to a nuclease-free collection tube, open the centrifuge column cover, and air dry for 3 minutes to remove the ethanol;

[0143] 8. According to experimental needs, add 60-120 μL of depc water to the middle of the membrane of the centrifugal column and centrifuge at 8000xg for 1 min.

[0144] 9. Dilute the eluted RNA 10 times with depc water and measure the RNA concentration using a NanoDrop One ultra-micro-volume UV spectrophotometer.

[0145] (4) Electroporation of the in vitro transcribed mRNA of pSFV-CMV-T7-EGFP into BHK cells

[0146] 1. Transfer BHK cells to 100 mm cell culture dishes in advance and conduct experiments when the cell confluence reaches 80%;

[0147] 2. Pre-cool 1.5 mL EP tubes and PBS on ice, pre-cool 2 mm electrode cups in a 4°C refrigerator, add 4 mL of culture medium (containing 3% FBSDMEM) to a 60 mm culture dish and pre-warm it in a 37°C incubator.

[0148] 3. Trypsinize the cells in 100 mm culture medium and resuspend in 5 mL complete medium;

[0149] Centrifuge at 4.500 g and 4°C for 5 min, discard the supernatant, and wash the cells with 10 mL of pre-cooled PBS.

[0150] Centrifuge at 500 g and 4°C for 5 min, and wash again with 10 mL of pre-cooled PBS;

[0151] Discard the supernatant and completely remove the remaining PBS, and resuspend the cells with 200 μL pre-cooled PBS. After mixing, pipette the cell suspension into a pre-cooled 1.5 mL EP tube;

[0152] Add 30 μg of purified RNA to the cell suspension and let it stand on ice for two minutes.

[0153] Transfer the above system to a pre-cooled electrode cup, and shock once at 110V, 25ms. After shocking, place on ice for two minutes.

[0154] The cell suspension after electroporation in the electrode cup was aspirated into a preheated 60 mm culture dish, gently shaken, and placed in a 37°C, 5% CO2 incubator for 24 hours. The expression of the reporter gene EGFP was observed under a fluorescence microscope and cell protein samples were collected. The fluorescence expression was as follows: Figure 6 As shown in c.

[0155] Example 7

[0156] This embodiment provides the process of in vitro transcription and RNA transfection of the taRNA system, and the specific steps are as follows:

[0157] The pSFV-CMV-T7-replicase plasmid was linearized by BsaΙ digestion, and the pSFV-CMV-T7-TR-EGFP, pSFV-CMV-T7-TR-IBVS, pSFV-CMV-T7-TR-H120S, pSFV-CMV-T7-TR-HN, and pSFV-CMV-T7-TR-IBDV VP2 plasmid were linearized by speΙ digestion. The reaction system is shown in Tables 17-18.

[0158] Table 17Bsa I digestion of pSFV-CMV-T7-replicase plasmid

[0159] Element Volume (μL) BsaΙ 1.5 pSFV-CMV-T7-replicase 30 (30 μg plasmid) Quick cut buffer 6 Nuclease-free water 22.5

[0160] Table 18speΙ enzyme digestion taRNA system antigen provides plasmid

[0161]

[0162] The linearized replicase providing plasmid pSFV-CMV-T7-replicase and the above antigen providing plasmid were transcribed in vitro to obtain the corresponding mRNA; the co-transcription capping was performed using the Novagen in vitro transcription kit (TR101-01, purchased from Novagen) and NEB m7G(5)ppp(5)G RNA cap analog (S1411S, purchased from NEB). The in vitro transcription system and method were the same as those in Example 6. The test results are shown in FIG. Figure 7 a and Figure 7 As shown in b, Figure 7 Lanes 1-3 in a are the results of in vitro transcribed RNA gel run of pSFV-CMV-T7-EGFP, pSFV-CMV-T7-replicase and pSFV-CMV-T7-TR-EGFP plasmids. Figure 7Lanes 1-4 in b are the results of in vitro transcribed RNA of pSFV-CMV-T7-replicase, pSFV-CMV-T7-IBVS, pSFV-CMV-T7-TR-IBVS, and pSFV-CMV-T7-TR-H120S, respectively. The above results show that the transcription results are consistent with the theoretical values.

[0163] The RNA after in vitro transcription was purified using the same purification method as in Example 6.

[0164] The mRNA provided by the replicase plasmid pSFV-CMV-T7-replicase in the trans-amplification mRNA system and the in vitro transcribed mRNA from the antigen-providing plasmids pSFV-CMV-T7-TR-EGFP, pSFV-CMV-T7-TR-IBVS, and pSFV-CMV-T7-TR-H120S were co-electroporated into BHK cells, respectively. In each system, the ratio of the antigen-provided mRNA to the replicase-provided plasmid pSFV-CMV-T7-replicase mRNA was 1:3, for example, 10 μg of the antigen-provided mRNA and 30 μg of the replicase-provided plasmid pSFV-CMV-T7-replicase mRNA were mixed and then electroporated into BHK cells.

[0165] The electroporation ratio of the taRNA system for simultaneously amplifying multiple antigen TR-RNAs is as follows: the total TR-RNA amount of multiple antigens and the pSFV-CMV-T7-replicase RNA amount are electroporated at a ratio of 1:2.

[0166] Simultaneously electroporate pSFV-CMV-T7-TR-HN and pSFV-CMV-T7-TR-IBV: according to their protein size ratio, for example, IBVS is 150KDa, HN protein is 62.8KDa, and VP2 is 37KDa, then use a 1:2 ratio (if the total antigen RNA amount is 30μg, then IBVS protein RNA is 20μg, NDV HN protein RNA is 10μg) and pSFV-CMV-T7-replicase RNA is 60μg.

[0167] At the same time, pSFV-CMV-T7-TR-HN, pSFV-CMV-T7-TR-IBVS, and pSFV-CMV-T7-TR-IBDVVP2 were electrotransferred: according to the protein size ratio, for example, IBVS is 150KDa, HN protein is 62.8KDa, and VP2 is 37KDa, then pSFV-CMV-T7-replicase RNA is 60μg at a ratio of 1:2 (if the total antigen RNA amount is 30μg, then IBVS protein RNA is 18μg, NDV HN protein RNA is 7.5μg, and IBDV VP2 is 4.5μg), and it is electrotransferred to a large dish of BHK cells, and 1 / 15 of the sample is taken for loading after the protein sample is collected later. The remaining electrotransfer steps are the same as the method in Example 6.

[0168] Example 8

[0169] This embodiment provides a process for detecting protein expression of saRNA vaccine system and taRNA vaccine system protein immunoassay, and the specific steps are as follows:

[0170] The protein expression was tested by Western Blot, specifically, the protein samples collected in Examples 6 and 7 were taken out, boiled in a metal bath at 95°C for 10 minutes, an equal amount of each protein was taken for protein electrophoresis in SDS-PAGE gel, and finally incubated overnight with the corresponding primary and secondary antibodies, and then imaged on a gel imaging instrument.

[0171] Western blot test results are as follows Figure 6 de, 7d and Figure 8 As shown. Figure 6 d Lane 2 is the IBVS protein band of electroporated pSFV-CMV-T7-IBVS mRNA, about 150KD. Figure 6 Lanes 2-4 are the H120 S, A96S, and MH20S protein bands of electroporated pSFV-CMV-T7-H120S, pSFV-CMV-T7-A96S, and pSFV-CMV-T7-MH20S mRNA, respectively, with two bands of approximately 150KD and 75KD. Figure 7d Lanes 3-6 are the IBVS protein bands of electroporated pSFV-CMV-T7-IBVS mRNA, about 150KD; the H120S protein bands of electroporated pSFV-CMV-T7-TR-H120 SmRNA; the IBVS protein bands of electroporated pSFV-CMV-T7-TR-IBVS mRNA (the ratio of antigen-provided mRNA to replicase-provided mRNA is 1:3); the IBVS protein bands of electroporated pSFV-CMV-T7-TR-IBVS mRNA (the ratio of antigen-provided mRNA to replicase-provided mRNA is 1:4). (Since the antibody only targets the S2 part of the IBV P65S protein, the affinity of the antibody to the S protein of different genotypes is limited, and the actual protein expression should be greater than the bands developed by the antibody. After sequence analysis, the antibody affinity is ranked as IBV P65S>H120S>MH20S>A96S) Figure 8 As can be seen in a, the expression of IBVS (about 150 KD) and NDV HN (about 62.5 KD) can be detected simultaneously in the sample in which pSFV-CMV-T7-TR-IBVS and pSFV-CMV-T7-TR-NDV HN RNA were simultaneously electroporated. Figure 8 As can be seen in b, the expression of IBVS, NDV HN and IBDV VP2 (about 37KD) can be simultaneously detected in the samples that were simultaneously electroporated with pSFV-CMV-T7-TR-IBVS, pSFV-CMV-T7-TR-NDV HN and pSFV-CMV-T7-TR-IBDV VP2RNA, indicating that the taRNA system can express at least 2-3 proteins simultaneously and has the potential to prepare multivalent vaccines.

[0172] The above results show that the two saRNA and taRNA systems based on SFV replicons are feasible and effective.

[0173] Example 9

[0174] This embodiment provides a process for the preparation of saRNA vaccines and taRNA vaccines against Newcastle disease virus and animal immunization, and the specific steps are as follows:

[0175] Referring to Examples 5 and 6, the RNA of the Newcastle disease virus preserved in this laboratory was extracted for reverse transcription, and the antigenic protein NDV F protein was amplified using the primers in Table 19 and inserted into the saRNA and taRNA systems to prepare saRNA vaccines and taRNA vaccines for Newcastle disease virus. The vaccine design is shown in Fig.11, prepare an uncapped in vitro transcription system with reference to Example 7, wherein 2 μL of GTP is used but no m7G(5)ppp(5)G RNA cap analog component is added, and DEPC water is used to make up to 20 μL, and the system is placed in a PCR instrument and transcribed at 37°C for 3.5 hours to obtain uncapped mRNA molecules, which are subsequently capped using the Cap 1 Capping System. Referring to the instructions for use of the kit, all prepared RNAs are first purified using the Nucleospin RNA Clean-up (column purification) of MACHEREY-NAGEL. The RNA prepared after the uncapped in vitro transcription and purification is placed at 65°C and heated for 5 minutes, and then quickly placed on ice to remove the higher-order structure of the RNA. Then, the RNA is capped according to the Cap 1 Capping System instructions of Suzhou Nearshore Protein Technology Co., Ltd. to prepare RNA molecules containing Cap 1. The capped RNA is purified to obtain mRNA with Cap 1. The concentration of mRNA is measured using a Nanodrop UV spectrophotometer. The two vaccines were electroporated into BHK cells according to the methods of Examples 7 and 8, and protein detection was performed 24 hours later. The protein detection results are shown in Fig.10 The other part is used for animal immunization.

[0176] Animal grouping: 5-6 week old female Balb / c mice were randomly divided into 4 groups, 4 mice in each group. They were taRNA vaccine group: pSFV-CMV-T7-TR-NDVF+pSFV-CMV-T7-replicase, pSFV-CMV-T7-TR-NDVF+

[0177] pSFV-CMV-T7-replicase was delivered using B-PEI. saRNA vaccine group: pSFV-CMV-T7-NDVF was delivered using B-PEI. And PBS negative control group. The immunization cycle was 0 and 4 weeks for a total of two immunizations. All immunizations were injected into the thigh muscle. 100 μL of vaccine was injected into the unilateral leg muscle. The B-PEI delivery group was used and the N / P ratio was 16 for packaging. The immunization dose is shown in Table 22 below (see the immunization procedure for details). Figure 4 Serum samples were obtained from the taRNA and saRNA vaccine groups at 0, 42, and 49 days after the initial immunization (serum samples were taken before each immunization).

[0178] Table 19 Amplification of NDV F Primers

[0179]

[0180]

[0181] Table 20 Grouping of mice immunized with NDV mRNA vaccines based on saRNA and taRNA systems

[0182]

[0183] Sampling: 500 μL of whole blood was obtained by collecting blood from the orbital venous plexus of mice. The blood sample was placed in a 37°C incubator for 1 hour and then placed in a 4°C refrigerator overnight. The next day, the serum was separated by centrifugation at 3000 r / min and 4°C for 10 minutes.

[0184] Detection: NDV F IgG in mouse serum was detected using the NDV whole virus coated plate in the Biochek kit, and the serum sample to be tested was diluted 1:30 using sample diluent.

[0185] Test results: The protein expression of the protein samples of RNA prepared by electroporation of pSFV-CMV-T7-NDV F and pSFV-CMV-T7-TR-NDVF+ppSFV-CMV-T7-replicase in vitro transcription was as follows Fig.10 As shown in Figure 2, the expression of NDV F protein (about 59KD) can be detected in both groups of samples. The specific antibody results of mouse serum are shown in Figure 2. Fig.11 As shown in the figure, compared with the PBS group, both groups of taRNA vaccines produced specific IgG on the 42nd and 49th days after immunization, and the specific IgG level on the 49th day was slightly higher than that on the 42nd day. The specific IgG of the taRNA vaccine group delivered by B-PEI was lower than that of the naked taRNA vaccine group injected directly. Only the naked taRNA vaccine group was significantly different from the PBS group (P<0.01). Only a few mice in the saRNA vaccine group were detected with specific IgG. The results of the virus neutralization experiment of mouse serum are shown in the figure. Fig.12 As shown in the figure, compared with the PBS group, lower levels of neutralizing antibodies were detected in the saRNA vaccine group and the two taRNA vaccine groups after immunization injection. The neutralization titer of the two groups of taRNA vaccines was approximately 1:12, which was significantly different from the PBS group (P<0.001), but there was no significant difference between the taRNA vaccine groups using PEI delivery and direct injection (P>0.05). The neutralization titer of saRNA vaccines was lower. This may be related to the larger molecules of saRNA vaccines and easier degradation.

[0186] Example 10

[0187] This example provides a process for the preparation of porcine epidemic diarrhea virus DNA vaccine and animal immunization based on the saRNA system, as follows:

[0188] Referring to Examples 5 and 6, the RNA of porcine epidemic diarrhea virus GDS01 preserved in this laboratory was extracted for reverse transcription, and its antigen protein PEDV S protein was inserted into the saRNA system using the primers to prepare a DNA vaccine against porcine epidemic diarrhea virus. The vaccine plasmid was extracted in large quantities using an endotoxin-free plasmid extraction kit. 2 μg of vaccine plasmid was transfected into HEK-293T cells, and protein samples were collected after 48 hours for western blot detection. The vaccine design was as follows: Fig.13 The protein expression is shown in Fig.14 The rest were subjected to animal immunization experiments.

[0189] Animal grouping: 5-6 week old female Balb / c mice were randomly divided into 4 groups, 5 mice in each group. They were pSFV-CMV-T7-PEDV S group, pSFV-CMV-T7-PEDV S+poly(I:C) adjuvant group, poly(I:C) group, and PBS negative control group. The immunization cycle was 0, 2, and 4 weeks, with a total of three immunizations. All immunizations were injected into the femoral muscle. 140 μL of vaccine (prepared with PBS) was injected into the unilateral leg muscle, containing 100 μg of DNA plasmid. The adjuvant group also contained 50 μg of immune adjuvant poly(I:C). Serum samples were obtained at the 2nd, 4th, and 6th week after the primary immunization (serum samples were taken before each immunization), and spleen lymphocyte samples were obtained at the 7th week. See Table 23 for details, and the immunization time and detection procedures are shown in Figure 5 .

[0190] Table 21 Amplification of PEDV S Primers

[0191]

[0192]

[0193] Table 22 Grouping of mice immunized with PEDV DNA vaccine based on saRNA system

[0194]

[0195] Sampling: 500 μL of whole blood was obtained by collecting blood from the mouse orbital venous plexus. The blood sample was placed in a 37°C incubator for 1 hour and then placed in a 4°C refrigerator overnight. The next day, the serum was separated by centrifugation at 3000 r / min and 4°C for 10 minutes. Three weeks after the last immunization, the mice were euthanized, the spleen was separated aseptically, and single spleen cells were separated by grinding with a 70 μm cell sieve. Lymphocytes were separated according to the instructions of the mouse spleen lymphocyte separation kit. After separation, lymphocytes were counted using a hemocytometer.

[0196] Detection: The PEDV S IgG in mouse serum was detected using the PEDV antibody detection kit of Zhaoqing Dahuanong Biopharmaceutical Co., Ltd., and the serum sample to be tested was diluted at 1:40 using sample diluent; the isolated mouse spleen lymphocytes were stimulated with non-specific stimulant concanavalin A (ConA) and specific stimulant formaldehyde-inactivated PEDV to perform spleen lymphocyte stimulation experiments; mouse interferon gamma (IFN-γ) and mouse interleukin 4 (IL-4) ELISA detection kits were used to determine the contents of IFN-γ and IL-4 in the culture supernatant of mouse spleen lymphocytes after inactivated PEDV stimulation.

[0197] Test results: The protein expression results of the cells transfected with the vaccine plasmid are as follows Fig.14 As shown in the figure, it can be seen that the expression of PEDV S protein (about 250KD and 100KD) can be detected in this sample, indicating that pSFV-CMV-T7-PEDV S was successfully constructed and can be expressed normally. Fig.15 As shown, after three immunizations, the absorbance at 450nm of the immune groups increased. Compared with the PBS group, the pSFV-CMV-T7-PEDV S+poly(I:C) group increased the fastest (P<0.0001), followed by the pSFV-CMV-T7-PEDV S group (P<0.001). In the three tests, the OD450 of the PBS group and the poly(I:C) group was close to 0.1, and no antibody production was detected. The above results show that the PEDV DNA vaccine based on the SFV replicon can induce Balb / c mice to produce antibodies against PEDV.

[0198] Mouse spleen lymphocyte proliferation assay Fig.16 As shown in the results, under the stimulation of ConA, the SI value of spleen lymphocytes in the PBS group was significantly lower than that in the pSFV-CMV-T7-PEDV S+poly(I:C) group (P<0.001). Under the stimulation of PEDV, the SI value of spleen lymphocytes in the PBS group was significantly lower than that in the pSFV-CMV-T7-PEDV S+poly(I:C) group (P<0.05). The above data indicate that the pSFV-CMV-T7-PEDV SDNA vaccine constructed in this experiment can effectively induce specific T cell immune response in Balb / C mice. Poly(I:C) adjuvant can enhance the cellular immune effect to a certain extent.

[0199] Test results such as Fig.17As shown, compared with the PBS group, the IFN-γ levels of both experimental groups increased after three immunizations, among which the IFN-γ level detected in the cell supernatant of the pSFV-CMV-T7-PEDV S group was the highest (P<0.0001). In the IL-4 assay, except for the significant difference in IL-4 levels between the pSFV-CMV-T7-PEDV S+poly(I:C) group and the PBS group (P<0.05), there was no significant difference between the other groups (P>0.05). These data indicate that the PEDV DNA vaccine elicited an antigen-specific cellular immune response in mice, mainly inducing a Th 1 type cellular immune response.

[0200] In summary, the present invention provides a method for constructing an efficient expression system based on the Semliki Forest virus replicon, which is a self-amplifying RNA (saRNA) system for expressing replicase and heterologous antigen in one molecule and a trans-amplifying RNA (taRNA) system comprising a replicase vector and an antigen providing vector, respectively. Since both systems have CMV and T7 promoters, they can be used as DNA or RNA vaccine vectors. In addition, the present invention uses pSFVCs-CMV-EGFP as material to construct a saRNA system pSFV-CMV-T7-EGFP expressing a reporter gene; then it is split and optimized to construct a dual-carrier taRNA system. That is, the vector pSFV-CMV-T7-replicase expressing replicase and the vector pSFV-CMV-T7-TR-EGFP expressing antigen protein; then the antigen protein gene is constructed into the target protein expression vector pSFV-CMV-T7 or pSFV-CMV-T7-TR (i.e., replacing EGFP). The two system plasmids are linearized and then transcribed in vitro to prepare RNA vaccines, and then the prepared two system RNAs are co-electrotransfected into host eukaryotic cells at a certain ratio to express antigenic proteins. In addition, the present invention discloses preliminary applications of developing NDV RNA candidate vaccines with a taRNA system and developing PEDV DNA candidate vaccines with a saRNA system. Due to the presence of viral replicase, the self-amplifying DNA and RNA vaccines based on viral replicons provided by the present invention can not only shorten the development cycle, but also express vaccine antigens at high levels. Among them, the taRNA system can also express two or more antigenic proteins at the same time. This type of vaccine has the ability to produce strong immunity in small doses, and makes vaccines safer, more efficient and simplifies the mRNA vaccine production process, which helps to deal with rapidly evolving pathogens and emerging infectious diseases.

[0201] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

[0202] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for constructing an efficient expression system based on Semliki Forest virus replicon, characterized in that: The method comprises: (1) Using pSFVCs-CMV-EGFP as material, construct the saRNA system pSFV-CMV-T7-EGFP expressing the reporter gene; (2) pSFV-CMV-T7-EGFP was split and optimized to construct the replicase-providing plasmid pSFV-CMV-T7-replicase in the taRNA system; (3) pSFV-CMV-T7-EGFP was split and optimized to construct the antigen providing plasmid pSFV-CMV-T7-TR-EGFP in the taRNA system; (4) replacing the reporter gene EGFP with the antigen protein gene sequence X to construct pSFV-CMV-T7-X or pSFV-CMV-T7-TR-X; (5) pSFV-CMV-T7-X, pSFV-CMV-T7-TR-X, and pSFV-CMV-T7-replicase were linearized and then transcribed in vitro to prepare mRNA; (6) transfecting the prepared mRNA into host eukaryotic cells at a certain ratio for expression to obtain antigenic protein; (7) Collect the cells after electroporation to obtain test samples, and detect the antigen protein to evaluate the expression efficiency of the saRNA and taRNA systems based on the SFV replicon.

2. The method for constructing a high-efficiency expression system based on Semliki Forest virus replicon according to claim 1, characterized in that: The saRNA system constructed in step (1) includes retaining the CMV promoter in pSFVCs-CMV-EGFP and adding a T7 promoter; The sequence of the T7 promoter is shown in SEQ ID NO.1; the sequence of A1-nsp1-F is shown in SEQ ID NO.6; the sequence of A2-nsp4-R is shown in SEQ ID NO.7; The saRNA system constructed in step (1) comprises deleting the CE3 portion of the SFV structural protein; The construction method of step (1) is to construct the saRNA system pSFV-CMV-T7-EGFP expressing EGFP by segmented PCR of pSFVCs-CMV-EGFP through homologous recombination; Specifically, fragment A1 was amplified from the sequence as shown in SEQ ID NO.1 using primers A1-nsp1-F and A2-nsp4-R; restriction endonucleases BamH I and Xba I were used to cut out vector fragment A2; fragment A1 and fragment A2 were homologously recombined and connected using a homologous recombinase to obtain the saRNA system pSFV-CMV-T7-EGFP expressing EGFP.

3. The method for constructing a high-efficiency expression system based on Semliki Forest virus replicon according to claim 1, characterized in that: The sequence of the SFV replicase in step (2) is shown in SEQ ID NO.2; The optimization in step (2) includes optimization of 5'UTR and 3'UTR; The optimization in step (2) included extending the ploy A tail; The optimization in step (2) includes the change of the linearization restriction site, and the same restriction site on the plasmid is subjected to a synonymous mutation; The construction method in step (2) constructs the SFV replicase into the recombinant vector A through homologous recombination.

4. The method for constructing a high-efficiency expression system based on Semliki Forest virus replicon according to claim 3, characterized in that: The 5'UTR optimized sequence is selected from human α-globin 5'UTR, and its sequence is shown in SEQ ID NO.3: The 3'UTR represents a fusion of a sequence selected from the amino-terminal enhancer of split (AES) mRNA and mitochondrial encoded 12SrRNA (mtRNR1), the sequence of which is shown in SEQ ID NO. 4; The poly A tail is extended by extending 65 A in the template to 100 A, and a short connecting sequence is connected after the initial 30 A, and then 70 A are connected, totaling 100 A. The sequence of the poly A is shown in SEQ ID NO.5; The linearization restriction site was changed from the original spe I to the IIS type restriction enzyme BsaΙ, so that a BsaΙ site was inserted after the poly A tail. The sequence of the linearization restriction site is shown in SEQ ID NO.8, and the initial three BsaΙ sites in the recombinant vector were synonymously mutated.

5. The method for constructing a high-efficiency expression system based on Semliki Forest virus replicon according to claim 4, characterized in that: The steps of homologous recombination in step (2) are as follows: fragment B1 is amplified from pSFVCs-CMV-EGFP using primers 5'UTR-F and B1-BsaI-mut-R; vector fragment B2 is amplified from pSFVCs-CMV-EGFP using primers B2-BsaI-mut-F and B2-BsaI-mut-R; fragment B3 is amplified from pSFVCs-CMV-EGFP using primers B3-BsaI-mut2-F and B3-BsaI-mut-R; 3'UTR optimized sequence and optimized poly A tail sequence are removed from the sequence as shown in SEQ ID NO: 1 by double digestion with Hind III and Xho I. Fragment B4 was cut out by the enzyme shown in NO.3; fragment B5 was amplified from pSFVCs-CMV-EGFP by primers A5-F and B5-BsaI-mut3-R; fragment B6 was amplified from pSFVCs-CMV-EGFP by primers B6-BsaI-mut3-F and A6-5'UTR-R; fragments B1, B2, B3, B5 and B6 were homologously recombined and connected using homologous recombinase with fragment B4 as a vector to obtain the plasmid pSFV-CMV-T7-replicase that provides replicase in the taRNA system; The sequence of B1-5'UTR-F is shown in SEQ ID NO.9, and the sequence of B1-BsaI-mut1-R is shown in SEQ ID NO.

10. The sequence of B2-BsaI-mut1-F is shown in SEQ ID NO.11, the sequence of B2-BsaI-mut2-R is shown in SEQ ID NO.12, the sequence of B3-BsaI-mut2-F is shown in SEQ ID NO.13, the sequence of B3-R is shown in SEQ ID NO.14, the sequence of B5-F is shown in SEQ ID NO.15, the sequence of B5-BsaI-mut3-R is shown in SEQ ID NO.16, the sequence of B6-BsaI-mut3-F is shown in SEQ ID NO.17, and the sequence of B6-5'UTR-R is shown in SEQ ID NO.

18.

6. The method for constructing a high-efficiency expression system based on Semliki Forest virus replicon according to claim 1, characterized in that: The optimization in step (3) is to retain part of the SFV replicon sequence, including 221 bp at the 5' end of nsp1 and 984 bp at the 3' end of nsp4 of the SFV replicon, and delete the rest of the replicase. The construction method in step (3) constructs the recombinant vector pSFV-CMV-T7-TR-EGFP through homologous recombination. The homologous recombination in step (3) is performed by amplifying fragment C1 from pSFVCs-CMV-EGFP using primers A1-nsp1-F and A1-nsp1-R; fragment C2 is amplified from pSFVCs-CMV-EGFP using primers A2-nsp4-F and A2-nsp4-R: fragments C1 and C2 are homologously recombined and connected using a homologous recombinase to obtain a recombinant vector pSFV-CMV-T7-TR-EGFP; The sequence of A1-nsp1-F is shown in SEQ ID NO.19, the sequence of A1-nsp1-R is shown in SEQ ID NO.20, the sequence of A2-nsp4-F is shown in SEQ ID NO.21, and the sequence of A2-SFV-promoter-R is shown in SEQ ID NO.

22.

7. The method for constructing a high-efficiency expression system based on Semliki Forest virus replicon according to claim 1, characterized in that: The construction method in step (4) is to construct the DNA sequence containing the antigen protein into the vector expressing the foreign gene through homologous recombination; The steps of homologous recombination in step (4) are specifically as follows: Using cDNAs of avian infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and infectious bursal disease virus (IBDV) of cell-adapted strains IBV-P65, A96, MH20, and H120 as templates, IBVP65S, A96S, MH20S, H120S, NDV-HN, and IBDV VP2 cDNA sequences were obtained by amplification with the primers listed in Table 12; The vectors pSFV-CMV-T7 and pSFV-CMV-T7-TR were cut into linear fragments by BamHΙ and SmaΙ, and then homologously recombined with the amplified IBV P65S, A96S, MH20S, H120S, NDV-HN, and IBDV VP2 cDNA sequences, respectively.

8. The method for constructing a high-efficiency expression system based on Semliki Forest virus replicon according to claim 1, characterized in that: In step (2), the pSFV-CMV-T7-X and pSFV-CMV-T7-TR-X plasmids are linearized by digestion with Spe I, and the pSFV-CMV-T7-replicase plasmid is linearized by digestion with Bsa I; The in vitro transcription to prepare mRNA in step (5) also includes an mRNA capping process and an mRNA purification process; The detection of the antigen protein in step (7) includes western blotting.

9. The method for constructing a high-efficiency expression system based on Semliki Forest virus replicon according to claim 1, characterized in that: The host eukaryotic cell in step (6) is at least one of human kidney epithelial cells 293T and Syrian hamster kidney cells BHK; The pSFV-CMV-T7-X RNA in step (6) is purified and quantified, and then transfected according to the required RNA amount; The pSFV-CMV-T7-TR-X RNA and pSFV-CMV-T7-replicase RNA in step (6) were purified and quantified, and then transfected at a mass ratio of 1:4; In step (6), the pSFV-CMV-T7-TR-X transfected is simultaneously transfected with 2-3 species, wherein X is at least one of NDV HN, IBV S, and IBDV VP2.

10. A taRNA system obtained by the construction method of an efficient expression system based on the Semliki Forest virus replicon as described in any one of claims 1 to 9 or an application of the taRNA system in the preparation of a vaccine for a mammal.

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