CN116804210A - Enterovirus vector and application thereof - Google Patents

Abstract

The invention relates to an enterovirus vector and its application, belonging to the technical field of biomedicine. The present invention provides an enterovirus vector. The enterovirus vector uses EV71 replicon EV71replicon as a backbone, and its nucleotide sequence is shown in SEQ ID No. 1. The present invention provides an enterovirus vector that can express other membrane proteins or secreted proteins, and a construction method. The present invention also provides a CA16 infectious clone (CA16-memHiBiT) carrying a membrane protein reporter gene, and rescues the recombinant virus. The present invention overcomes the limitations of the enterovirus vector itself and enables it to express various forms of foreign proteins including intracellular, extracellular and membrane, thereby expanding the application scope of the enterovirus vector.

Description

An enterovirus vector and its application

Technical field

The invention relates to an enterovirus vector and its application, belonging to the technical field of biomedicine.

Background technique

The genus Enterovirus belongs to the single-stranded positive-strand RNA virus family of the family Picornaviridae. The genus Enterovirus contains 15 species, namely EnterovirusA-I and RhinovirusA-C. Among them, the common enterovirus is EnterovirusA71 (EVA71 ), CoxsackievirusA10 (CVA10), Human rhinovirusA1 (HRVA1), Poliovirus (PV), etc. The viral genome is about 7.5kb in length and consists of a 5’ non-coding region (5’UTR), a coding region and a 3’ non-coding region (3’UTR). After the virus infects cells, genomic RNA is released into the cytoplasm where it is replicated and translated. The viral genome contains a reading frame, which is first translated in the cytoplasm to form a polyprotein including structural proteins and non-structural proteins. Then the virus-encoded proteases 2A and 3C cleave the polyprotein to form four mature structural proteins - VP1 and VP2. , VP3 and VP4 and 7 non-structural proteins - 2A, 2B, 2C, 3A, 3B, 3C and 3D. The replication process of the enterovirus genome occurs in the replication complex (Replicationorganelle, RO) located in the cytoplasm. Mature replication-related proteins enter the RO to assist in the replication of RNA. On the other hand, structural proteins wrap the viral RNA to form a mature icosahedron. Viral particles are released outside the cell.

Enterovirus infection can induce systemic mucosal immune responses, making it a highly potential viral vector. Vector research based on enterovirus genomes is divided into two categories according to different vector structures: one is using the full length of the viral genome as a vector, directly inserting foreign genes into the reading frame of the virus. This type of vector can self-replicate and express Foreign proteins will also produce mature virus particles at the same time; the other type uses a replicon (Replicon (a sequence that deletes part or all of the structural protein genome)) as a vector to place the sequence of the foreign protein in the viral structural protein coding region. This type of vector can self-replicate, but will not produce mature viral particles due to the lack of viral structural proteins when expressing foreign proteins. Since the life cycle of enteroviruses is completely completed in the cytoplasm, although enterovirus genome-based vectors can express exogenous cytoplasmic proteins, they are difficult to express exogenous proteins carrying signal peptides, such as membrane proteins and secreted proteins. . For example, according to literature reports, influenza virus hemagglutinin (HA) cannot be directly expressed using PVreplicon as a vector. Only when the vector is designed as a bicistronic vector can the HA protein be successfully expressed. However, studies have also shown that the genome of this type of bicistronic enterovirus has poor stability and will be completely lost after several passages. Therefore, unimproved enterovirus vectors are difficult to express various types of immunogens that are mainly glycoproteins (membrane proteins or secreted proteins with signal peptides), which limits the development and application of enterovirus vectors.

Gluc luciferase is a new type of luciferase isolated from large marine copepods. Gluc catalyzes the oxidation reaction of the substrate coelenterazine with a stronger luminous intensity than the firefly luciferase (Fluc) reaction. Unlike traditional Fluc, Gluc contains a secreted signal peptide and is a natural secreted protein. As a foreign protein reporter gene carrying a signal peptide, it is particularly suitable for vector research.

The present invention uses Gluc as a reporter gene and optimizes the transmembrane region sequence of the foreign protein, so that the enterovirus vector can successfully express the foreign membrane protein. Furthermore, based on the design of the transmembrane region, exogenous secreted proteins were successfully expressed by adding Furin recognition and T2A cleavage sites. In addition, the present invention also successfully prepared a CA16 infectious clone with a membrane protein reporter gene for the first time and rescued the CA16-memHiBiT reporter virus. The above designs enable enterovirus vectors to overcome their own limitations and express any subcellularly localized foreign protein, thereby broadening the application scope of enterovirus vectors.

Contents of the invention

The purpose of the present invention is to solve the technical problem of how to stably express foreign proteins carrying signal peptides in enterovirus vectors.

The present invention provides an enterovirus vector, which uses EV71 replicon EV71replicon as a backbone, and its nucleotide sequence is shown in SEQ ID No. 1.

The invention provides an application of enterovirus vector in preparing vaccines and antiviral drugs.

The present invention provides an enterovirus vector that can express other membrane proteins. The enterovirus vector at least includes the EV71 replicon and the transmembrane region fragment of the nucleotide sequence shown in SEQ ID No. 1; the transmembrane region The region fragment at least includes PDGFRβTM with the nucleotide sequence shown in SEQ ID NO.3, SpikeTM with the nucleotide sequence shown in SEQ ID NO.4, CD28TM with the nucleotide sequence shown in SEQ ID NO.5, such as PDGFRβ+SpikeTM with the nucleotide sequence shown in SEQ ID NO.6 or Linker+TM+Linker with the nucleotide sequence shown in SEQ ID NO.7.

The present invention provides an enterovirus vector that can express other membrane proteins and is used in the preparation of vaccines and antiviral drugs.

The present invention provides an enterovirus vector that can express secreted proteins. The enterovirus vector includes the above-mentioned enterovirus vector that can express other membrane proteins and a cleavage site fragment; the cleavage site fragment at least includes The Furin recognition region of the nucleotide sequence shown in SEQ ID NO.8, the mFurin recognition region of the nucleotide sequence shown in SEQ ID NO.9, or the T2A of the nucleotide sequence shown in SEQ ID NO.10.

The invention provides an application of an enterovirus vector capable of expressing secreted proteins in the preparation of vaccines and antiviral drugs.

The present invention provides a method for constructing a CA16 infectious clone carrying a membrane display reporter gene. Using pSVA-CA16 as a template, the memHiBiT membrane display reporter gene is introduced, and through seamless recombination and cloning, a correctly sequenced pSVA-CA16-memHiBiT is obtained. Infectious clone; the nucleotide sequence of CA16-memHiBiT is shown in SEQ ID NO. 11; the memHiBi membrane display reporter gene fragment is inserted between the 5'UTR and the P1 fragment.

The present invention provides the application of a CA16 infectious clone carrying a membrane display reporter gene in screening antiviral drugs.

The present invention provides a method for constructing an enterovirus vector that can express other membrane proteins. The vector constructed using the genome of a serotype virus in the genus Enterovirus as a skeleton is used as a template, different transmembrane region fragments are amplified through PCR, and then the vector is amplified through PCR without Seam cloning, construct clones with different transmembrane region fragments, screen the correct clones verified by sequencing and extract plasmids for storage; the transmembrane region fragment at least includes PDGFRβTM with the nucleotide sequence shown in SEQ ID NO.3, such as SpikeTM with the nucleotide sequence shown in SEQ ID NO.4, CD28TM with the nucleotide sequence shown in SEQ ID NO.5, PDGFRβ+SpikeTM with the nucleotide sequence shown in SEQ ID NO.6 or SEQ ID NO. .Linker+TM+Linker of the nucleotide sequence shown in .7.

The invention provides a method for constructing an enterovirus vector capable of expressing secreted proteins, which includes the following steps:

Step 1: Using the vector one constructed with the genome of enterovirus serotypes as the backbone as a template, amplify different transmembrane region fragments through PCR, and then construct a vector two with different transmembrane region fragments through seamless cloning. Screen the correct clones verified by sequencing and extract the plasmid for storage; the transmembrane region fragment at least includes PDGFRβTM with the nucleotide sequence shown in SEQ ID NO.3, SpikeTM with the nucleotide sequence shown in SEQ ID NO.4, CD28TM with the nucleotide sequence shown in SEQ ID NO.5, PDGFRβ+SpikeTM with the nucleotide sequence shown in SEQ ID NO.6 or Linker+TM+Linker with the nucleotide sequence shown in SEQ ID NO.7 ;

Step 2: Using the vector two obtained in step 1 as a template, use seamless cloning technology to introduce cutting site fragments respectively; thereby constructing an enterovirus vector three that can express secreted proteins; the cutting site fragments at least include SEQ. The Furin recognition region of the nucleotide sequence shown in ID NO.8, the mFurin recognition region of the nucleotide sequence shown in SEQ ID NO.9, or the T2A of the nucleotide sequence shown in SEQ ID NO.10.

Compared with the existing technology, the present invention has the following beneficial effects:

1. The EV71replicon vector in the present invention is constructed based on PL451 as the backbone, and the luciferase reporter gene is inserted between the 5’UTR and P2 gene of the replican, which is beneficial to the efficient expression of foreign genes;

2. Based on the life history characteristics of enterovirus, the present invention designs a transmembrane region suitable for all enterovirus vectors. The foreign protein carrying this transmembrane region can be translated normally and does not affect the replication and translation of the enterovirus itself. ;

3. Based on the design of the transmembrane region, the enterovirus vector can express secreted proteins by introducing the Furin recognition region or T2A sequence; in addition, the combination of T2A and Furin recognition regions can further promote the efficiency of secretion;

4. The present invention overcomes the limitations of traditional enterovirus vectors and uses the optimized transmembrane region design and the introduction of T2A and Furin sites to realize the expression of exogenous membrane-displayed proteins and secreted proteins by enteroviruses. Most of the common proteins with immunogenicity are glycoproteins. The design of the present invention enables the enterovirus vector to be applied in many aspects such as vaccine development and tumor immunotherapy;

5. The present invention utilizes the design of the above-mentioned novel transmembrane region to construct a CA16 infectious clone carrying a membrane display reporter gene: pSVA-CA16-memHiBiT. After the RNA formed by in vitro transcription of this clone is transfected into cells, CA16-memHiBiT virus particles carrying the memHiBiT luciferase reporter gene can be produced. The activity of the reporter gene carried by the recombinant virus has been verified through cell infection experiments, and it has been confirmed that the virus can also induce cytopathic effects in cultured cell lines. This shows that the recombinant virus has similar growth characteristics and other virological characteristics to the parent virus. At the same time, the new CA16 infectious clone can also be used as a powerful tool for basic and applied research related to CA16 viruses;

6. The present invention verified through luciferase activity detection that the CA16-memHiBiT virus rescued by the CA16 infectious clone carrying the memHiBiT luciferase reporter gene constructed in the present invention can be stably passaged more than 20 times in Vero cells in vitro, and There was no decrease in luciferase activity. According to existing reports, viruses rescued by enterovirus infectious clones carrying exogenous genes can only be stably passaged five times in cells at most. The CA16-memHiBiT virus rescued by the present invention has a high degree of genetic stability and is stable. The CA16-memHiBiT virus will provide a more efficient and easy-to-use tool for subsequent viral mechanism research and antiviral drug screening.

Description of the drawings

Figure 1 is a schematic diagram of the optimization scheme of the transmembrane region in the EV71replicon vector;

Among them, Figure A is a schematic diagram of the optimization plan for the transmembrane region; Figure B is a graph of luciferase activity after transfection of cells with 71rep-Gluc-TM carrying different transmembrane regions;

Figure 2 is a schematic diagram of EV71replicon vector expressing secreted proteins;

Among them, Figure A is a schematic diagram of the design of secreted proteins; Figure B shows the expression of 71rep-Gluc-TM, 71rep-Gluc-Furin-TM, and 71rep-Gluc-mFurin-TM in the slurry, membrane surface and supernatant after transfection of cells. The proportion of expressed luciferase; Picture C shows the luciferase expressed in the slurry, membrane surface and supernatant after transfection of cells with 71rep-Gluc-T2A-TM and 71rep-Gluc-T2A-Furin-TM. proportion chart;

Figure 3 is a schematic diagram of the preparation of the pSVA-CA16-memHiBiT infectious clone and the passage stability of the rescued CA16-memHiBiT virus;

in,

Picture A shows the design of the pSVA-CA16-memHiBiT infectious clone;

Picture B shows the cytopathic changes caused by CA16 and CA16-memHiBiT transfection of cells;

Picture C shows the viral genome copy number and luciferase results at different time points after transfection of cells with CA16 and CA16-memHiBiT;

Picture D shows the luciferase activity of different generations of CA16-memHiBiT virus after serial passage.

Detailed ways

In order to make the present invention more obvious and understandable, the preferred embodiments are described in detail as follows together with the accompanying drawings:

The purpose of the present invention is to provide an enterovirus vector. The enterovirus vector uses the EV71 replicon EV71replicon as the backbone, and its nucleotide sequence is shown in SEQ ID No. 1.

The invention provides an application of enterovirus vector in preparing vaccines and antiviral drugs.

The present invention provides an enterovirus vector that can express other membrane proteins. The enterovirus vector at least includes the EV71 replicon and the transmembrane region fragment of the nucleotide sequence shown in SEQ ID No. 1; the transmembrane region The region fragment at least includes PDGFRβTM with the nucleotide sequence shown in SEQ ID NO.3, SpikeTM with the nucleotide sequence shown in SEQ ID NO.4, CD28TM with the nucleotide sequence shown in SEQ ID NO.5, such as PDGFRβ+SpikeTM with the nucleotide sequence shown in SEQ ID NO.6 or Linker+TM+Linker with the nucleotide sequence shown in SEQ ID NO.7.

The present invention provides an enterovirus vector that can express other membrane proteins and is used in the preparation of vaccines and antiviral drugs.

The present invention provides an enterovirus vector that can express secreted proteins. The enterovirus vector includes the above-mentioned enterovirus vector that can express other membrane proteins and a cleavage site fragment; the cleavage site fragment at least includes The Furin recognition region of the nucleotide sequence shown in SEQ ID NO.8, the mFurin recognition region of the nucleotide sequence shown in SEQ ID NO.9, or the T2A of the nucleotide sequence shown in SEQ ID NO.10.

The invention provides an application of an enterovirus vector capable of expressing secreted proteins in the preparation of vaccines and antiviral drugs.

The present invention provides a method for constructing a CA16 infectious clone carrying a membrane display reporter gene. Using pSVA-CA16 as a template, the memHiBiT membrane display reporter gene is introduced, and through seamless recombination and cloning, a correctly sequenced pSVA-CA16-memHiBiT is obtained. Infectious clone; the nucleotide sequence of CA16-memHiBiT is shown in SEQ ID NO. 11; the memHiBi membrane display reporter gene fragment is inserted between the 5'UTR and the P1 fragment.

The present invention provides the application of a CA16 infectious clone carrying a membrane display reporter gene in screening antiviral drugs.

The present invention provides a method for constructing an enterovirus vector that can express other membrane proteins. The vector constructed using the genome of a serotype virus in the genus Enterovirus as a skeleton is used as a template, different transmembrane region fragments are amplified through PCR, and then the vector is amplified through PCR without Seam cloning, construct clones with different transmembrane region fragments, screen the correct clones verified by sequencing and extract plasmids for storage; the transmembrane region fragment at least includes PDGFRβTM with the nucleotide sequence shown in SEQ ID NO.3, such as SpikeTM with the nucleotide sequence shown in SEQ ID NO.4, CD28TM with the nucleotide sequence shown in SEQ ID NO.5, PDGFRβ+SpikeTM with the nucleotide sequence shown in SEQ ID NO.6 or SEQ ID NO. .Linker+TM+Linker of the nucleotide sequence shown in .7.

The invention provides a method for constructing an enterovirus vector capable of expressing secreted proteins, which includes the following steps:

Step 1: Using the vector one constructed with the genome of enterovirus serotypes as the backbone as a template, amplify different transmembrane region fragments through PCR, and then construct a vector two with different transmembrane region fragments through seamless cloning. Screen the correct clones verified by sequencing and extract the plasmid for storage; the transmembrane region fragment at least includes PDGFRβTM with the nucleotide sequence shown in SEQ ID NO.3, SpikeTM with the nucleotide sequence shown in SEQ ID NO.4, CD28TM with the nucleotide sequence shown in SEQ ID NO.5, PDGFRβ+SpikeTM with the nucleotide sequence shown in SEQ ID NO.6 or Linker+TM+Linker with the nucleotide sequence shown in SEQ ID NO.7 ;

Step 2: Using the vector two obtained in step 1 as a template, use seamless cloning technology to introduce cutting site fragments respectively; thereby constructing an enterovirus vector three that can express secreted proteins; the cutting site fragments at least include SEQ. The Furin recognition region of the nucleotide sequence shown in ID NO.8, the mFurin recognition region of the nucleotide sequence shown in SEQ ID NO.9, or the T2A of the nucleotide sequence shown in SEQ ID NO.10.

The purpose of the present invention is to overcome the shortcomings of enterovirus vectors and propose a series of novel enterovirus vector design ideas and potential applications. These new enteroviral vector designs allow enteroviral vectors to express foreign membrane proteins and secreted proteins. In addition, the present invention also constructed a CA16-memHiBiT infectious clone and successfully rescued the reporter virus. The present invention can provide important tools and reference ideas for basic and applied research on enterovirus-based vectors and other similar enteroviruses.

The object of the present invention can be achieved through the following technical solutions:

An optimization scheme for the transmembrane region of the EV71replicon vector. The nucleotide sequence of the EV71replicon vector is shown in SEQ ID NO.1, the nucleotide sequence of PDGFRβTM is shown in SEQ ID NO.3, and the nucleotide sequence of Spike TM is shown in SEQ ID NO.1. SEQ ID NO.4 is shown, the nucleotide sequence of CD28TM is shown in SEQ ID NO.5, the nucleotide sequence of PDGFRβ+SpikeTM is shown in SEQ ID NO.6, the nucleotide sequence of Linker+TM+Linker As shown in SEQ ID NO.7.

EV71replicon vector can express secreted proteins.

CA16 infectious clones can carry membrane-displayed reporter genes.

An optimization scheme for the transmembrane region in the EV71replicon vector, the steps are as follows:

S1: PL451-71rep-Gluc clone construction;

S2: Construction of PL451-71rep-Gluc-TM clones with different transmembrane regions;

S3: in vitro transcription;

S4: Gluc luciferase expression detection.

In step S1, use a kit to extract the respectively amplified Gluc gene fragment and PL451-71rep vector fragment. The obtained cDNA is verified by agarose gel electrophoresis. The correct size fragment is recovered from the gel and the concentration is determined. The two fragments of Gluc and PL451-71rep are Recombinant construction of PL451-71rep-Gluc clone.

In step S2, use the PL451-71rep-Gluc clone prepared in step S1 as a template to amplify the PDGFRβTM, SpikeTM, CD28TM, PDGFRβ+SpikeTM and Linker+TM+Linker transmembrane regions respectively, and use seamless cloning technology to construct the correct sequence PL451-71rep-Gluc-TM clones with different transmembrane regions.

PCR amplify the 71rep-Gluc-TM region from the PL451-71rep-Gluc-TM clone with different transmembrane regions in step S2. The obtained cDNA is verified by agarose gel electrophoresis. The correct size fragments are recovered from the gel and the concentration is determined. Preparation of in vitro transcription templates. Use the T7 in vitro transcription kit to prepare 71rep-Gluc-TMmRNA. The LiCl-purified mRNA is verified by agarose gel electrophoresis. The correct mRNA is verified to be aliquoted and stored at -80°C.

In step S4, RD cells are seeded into a 96-well plate and cultured overnight. After transfection with 71rep-Gluc-TMmRNA with different transmembrane regions, luciferase activity is detected using a luciferase detection kit.

The EV71replicon vector can be designed to express secreted proteins. The steps are as follows:

S1: PL451-71rep-Gluc-Furin-TM clone construction;

S2: PL451-71rep-Gluc-mFurin-TM clone construction;

S3: PL451-71rep-Gluc-T2A-TM clone construction;

S4: PL451-71rep-Gluc-T2A-Furin-TM clone construction;

S5: in vitro transcription;

S6: Gluc luciferase expression detection.

In step S1, PL451-71rep-Gluc-TM is used as the template, and the PCR primer introduces the Furin recognition site in the template. The fragments of the correct size are recovered from the gel and the concentration is measured. The correct clone for sequencing is PL451-71rep-Gluc-Furin-TM. clone.

In step S2, the PL451-71rep-Gluc-Furin-TM clone prepared in step S1 is used as a template. The point mutation primer mutates the Furin site in the template to mFurin. The fragments with the correct size are recovered from the gel and the concentration is determined. The correct fragments are sequenced. The clone was the PL451-71rep-Gluc-mFurin-TM clone.

Use the PL451-71rep-Gluc-Furin-TM clone prepared in step S2 as a template. The PCR primer introduces T2A into the template and deletes the Furin recognition site. The fragments of the correct size are recovered from the gel and the concentration is determined. The correct clone for sequencing is PL451. -71rep-Gluc-T2A-TM clone.

In step S4, use the PL451-71rep-Gluc-Furin-TM clone prepared in step S2 as a template. The PCR primer introduces the T2A sequence into the template. The fragments of the correct size are recovered from the gel and the concentration is determined. The correct clone for sequencing is PL451-71rep. -Gluc-T2A-Furin-TM clone.

In step S5, the clones prepared in steps S1, S2, S3 and S4 are PCR amplified. The obtained cDNA is verified by agarose gel electrophoresis. Fragments of the correct size are recovered from the gel and the concentration is determined to prepare an in vitro transcription template. The T7 in vitro transcription kit was used to prepare mRNA. The LiCl-purified mRNA was verified by agarose gel electrophoresis. The correct mRNA was verified to be aliquoted and stored at -80°C.

In step S6, RD cells are inoculated into a 96-well plate and cultured overnight. After transfecting with mRNA, luciferase activity is detected using a luciferase detection kit.

A method for constructing a CA16 infectious clone carrying a membrane display reporter gene, the steps are as follows:

S1: pSVA-CA16-memHiBiT clone construction;

S2: Rescue of CA16-memHiBiT virus and virus growth characteristics;

S3: CA16-memHiBiT virus passage and genetic stability verification;

In step S1, the pSVA-CA16 infectious clone stored in the laboratory is used as a template, and the PCR primers amplify the pSVA-CA16 vector fragment. The pCDNA-memHiBiT clone stored in the laboratory is used as a template, and the PCR primers amplify the memHiBiT insert fragment. The cDNA was verified by agarose gel electrophoresis. The correct size fragments were recovered from the gel and the concentration was determined. The two fragments of memHiBiT and pSVA-CA16 were recombined to construct the pSVA-CA16-memHiBiT infectious clone.

In step S2, PCR amplify the pSVA-CA16-memHiBiT infectious clone prepared in step S1 and the pSVA-CA16 infectious clone stored in the laboratory respectively. The obtained cDNA is verified by agarose gel electrophoresis, and the fragments of the correct size are recovered from the gel. And determine the concentration to prepare in vitro transcription template. The T7 in vitro transcription kit was used to prepare mRNA. The LiCl-purified mRNA was verified by agarose gel electrophoresis. The correct mRNA was verified to be aliquoted and stored at -80°C.

In step S3, CA16 and CA16-memHiBiTmRNA are transfected into Vero cells, and the cells are harvested at different time points for RNA extraction and q-PCR quantification.

In step S4, after CA16-memHiBiTmRNA is transfected into Vero cells, it is passaged every 48 hours, and the viral supernatant of each passage is collected. Then the viral supernatant of different passages is infected into Vero cells in a 96-well plate, and fluorescence is measured using a luciferase detection kit. Detection of enzyme activity.

Example

In the following examples, the nucleotide sequence is as follows:

The nucleotide sequence of EV71replicon is shown in SEQ ID NO.1,

The nucleotide sequence of Gluc is shown in SEQ ID NO.2,

The nucleic acid sequence of PDGFRβTM is shown in SEQ ID NO.3,

The nucleic acid sequence of SpikeTM is shown in SEQ ID NO.4,

The nucleic acid sequence of CD28TM is shown in SEQ ID NO.5,

The nucleic acid sequence of PDGFRβ+SpikeTM is shown in SEQ ID NO.6,

The nucleic acid sequence of Linker+TM+Linker is shown in SEQ ID NO.7.

The nucleic acid sequence of the Furin recognition region is shown in SEQ ID NO.8,

The nucleic acid sequence of the mFurin recognition region is shown in SEQ ID NO.9,

The nucleic acid sequence of T2A is shown in SEQ ID NO.10.

The nucleic acid sequence of CA16-memHiBiT is shown in SEQ ID NO. 11.

in:

The nucleotide sequence of EV71replicon is shown in SEQ ID NO.1:

TTAAAACAGCCTGTGGGTTGCACCCACTCACAGGGCCCACGTGGCGCTAGCACTCTGATTCTACGGAATCTTTGTGCGCCTGTTTTACAACCCCCCCAATTTGCAACTTAGAAGCAATACACAACACTGATCAACAGCAGGCATGGCGCACCAGCCATGTCTTGATCAAGCACTTCTGTTTCCCCGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTCCGTTATCCGACTAACTTCGAGAA ACCTAGTAGCACCATTGAAGCTGCGGGTTTTCGCTCGGCACTTCCCCCGTGTAGATCAGGCCGATGAGTCACTGCAATCCCCACGGGCCGACCGTGGCAGTGGCTGCGCTGCGGCCTGCCTATGGGGCAACCCATAGGACGCTCTAATGTGGACATGGTGCGAAGAGTCTATTGAGCTAGTTAGTACCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACATGCCCTCCAACCCAGAGGGTAGTGT GTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATTCTTACATTGGCTGCTTATGGTGACAATCACAGAATTGTTACCATATAGCTATTGGATTGGCCATCCGGTGTGCAATAGAGCTATTATATACCTATTTGTTGGCTTTGTACCACTAACCTTAAAATCTATAACCACCCTCAACTTTATATTAACTCTCAATACAGTCAAACATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGG TGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTT CAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGC GACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGCCATTACTACCCTTGGAAAGTTCGGCCAGCAATCTGGGGCCATCTACGTGG GCAACTTCAGAGTGGTTAATCGTCACCTCGCTACTCATAATGACTGGGCGAATTCGTCTGGGAAG ACAGCTCCCGCGACCTACTAGTGTCGTCTACCACTGCCCAGGGCTGTGATACAATTGCACGTTGTGACTGTCAAACAGGAGTGTACTATTGTAACTCTAAGAGAAAGCACTATCCAGTCAGCTTCTCTAAACCTAGCCTCATTTATGTGGAAGCTAGCGAGTATTACCCTGCTAGATACCAGTCTCATCTCATGCTTGCGGCGGGCCATTCTGAACCAGGAGATTGTGGTGGCATCCTTAGGTGCCAGCGGTGTTGTCG GCATTGTGTCCACTGGTGGCAATGGGCTTGTTGGCTTTGCAGATGTCAGGGATCTCCTATGGTTGGATGAAGAAGCAATGGAGCAGGGTGTGTCCGACTACATTAAGGGACTTGGTGATGCGTTTGGAACTGGCTTCACCGATGCAGTTTCCAGAAGTTGAGGCTCTTAAGAACCACCTTATTGGGTCTGAGGGAGCTGTCGAGAAGATCCTAAAGAATTTGATCAAATTAATCCTGCTTTAGTCATCGTGATT AGGAGTGATTATGACATGGTCACTCTCACAGCAACTTTAGCTCTGATAGGGTGCCCACGGTAGTCCCTGGGCATGGATTAAAGCAAAAACAGCTTCCATTCTAGGCATCCCTATTGCCCAGAAGCAAAGTGCGTCCTGGCTTAAAAAGTTTAACGATATGGCCAACGCCGCCAAGGGGTTAGAGTGGATCTCCAGTAAGATTAGTAAATTCATCGATTGGCTCAAAGAGAAAATCATACCAGCGGCTAGGGAGAAGGTGGAGTTT CTGAACAACTTAAAACAGTTGCCGTTGTTGGAGAATCAGATCTCAAATCTAGAGCAGTCTGCTGCTTCACAAGAAGATCTTGAAGCCATGTTCGGAAATGTGTCGTACCTAGCTCACTTCTGCCGCAAATTCCAACCACTGTACGCTGCAGAAGCCAAAAGGGTCTATGCTTTGGAGAAAAGAATGAACAACTACATGCAGTTCAAGAGCAAACACCGAATTGAACCTGTATGTCTTATTATTAGGGGTTCTCCGGGCACCGGG AAATCACTAGCAACTGGCATCATTGCTCGGGCAATAGCAGACAAGTATCACTCAAGTGTGTACTCACTCCCACCAGACCCGGATCACTTTGATGGATACAAACAGCAGGTGGTCACGGTCATGGATGACTTATGTCAGAACCCTGATGGCAAAGATATGTCATTGTTCTGCCAGATGGTGTCCACAGTGGATTTCATCCCACCAATGGCTTCCCTAGAAGAGAAAGGAGTTTCTTTCACATCTAAATTTGTCATTGCATCTACCAATTCCAGCAA CATCATAGTACCAACAGTGTCTGATTCTGATGCAATTCGCCGTAGGTTCTATATGGATTGTGATATCGAAGTCACGGATTCATATAAAACGGACTTGGGTAGGTTAGATGCTGGGCGAGCCGCCAAATTGTGCTCTGAGAATAACACAGCAAATTTCAAGCGTTGTAGCCCACTAGTGTGTGGGAAGGCCATTCAGCTGAGAGATAGAAAGTCCAAGGTCAGGTACAGTGTGGACACGGTGGTTTCTGAGCTCATAAGGGAATA CAACAACAGGTCTGCTATTGGTAACACAATTGAAGCACTATTCCAAGGTCCGCCTAAGTTTAGACCCATAAGGATCAGTCTTGAAGAAAAGCCAGCCCCAGACGCCATTAGTGACCTTTCTTGCTAGTGTGGATAGCGAGGAGGTACGCCAGTACTGTAGAGATCAAGGTTGGATCATACCAGAGACTCCTACAAATGTTGAACGGCACCTCAACAGAGCTGTGCTAGTCATGCAATCCATCGCTACAGTTGTGGCAGTCGTTTCACT GGTGTATGTCATCTACAAGCTCTTCGCTGGATTCCAAGGCGCGTACTCTGGTGCTCCCAAGCAGATACTCAAGAAACCCGTTTCCGCAGGCAACAGTACAGGGTCCAAGTCTCGATTTCGCTCTGTCTCTACTGAGGAGGAATATCAGGC AAGTGCAAACAGATCAAGGACATTTCACCATGCTGGGTGTCAGGGACCGTTTGGCTGTTCTTCCACGCCACTCCCAGCCCGGCAAAACAATCTGGGTAGAGCACAAACTCG TAAACATTCTGGACGCTGTTGAACTGGTGGATGAACAAGGGGTTAATTTGGAGCTAACCCTAATCACCCTTGACACTAATGAGAAATTCAGAGACATTACTAAGTTCATCCCAGAGAGCATCAGTGCTGCAAGCGATGCTACCCTAGTGATCAACACAGAGCACATGCCCTCAATGTTTGTGCCGGTGGGAGACGTTGTGCAATATGGTTTTCTCAACCTCAGTGGAAAGCCAACCCACCGTACCATGATGTACAACTTTCCCACCAAA GGCAGGGCAGTGTGGAGGGGTGGTGACATCAGTTGGAAAGGTCATTGGTATACACATAGGTGGTAACGGCAGACAAGGTTTCTGTGCGGGACTTAAGAGGAGCTACTTCGCCAGCGAGCAAGGAGAGATCCAGTGGGTCAAGCCCAATAAAGAAACTGGGAGACTCAACATCAATGGGCCAACTCGCACTAAGCTCGAACCCAGTGTATTTCATGATGTTTTTGAGGGAAACAAGGAGCCAGCAGTTCTACACAGTAAA GATCCCCGCCTCGAGGTGGATTTTGAGCAGGCTTTGTTCTCCAAGTACGTAGGGAACACACTATATGAGCCTGATGAGTACATTAAGGAGGCAGCTCTTCACTATGCAAATCAGTTGAAACAGCTAGACATTGACACCTCCCAGATGAGCATGGAGGAAGCCTGTTACGGCACTGAGAACCTTGAGGCCATTGATCTTCACACTAGTGCAGGCTACCCATACAGTGCTCTAGGAATAAAGAAAAGAGACATCCTAGATTCT ACTACTAGGGATGTGAGCAAGATGAAATTTTATATGGACAAGTATGGCTTGGACCTTCCCTACTCCACCTATGTCAAGGATGAGCTACGCTCGATAGATAAGATCAAGAAGGGGAAGTCTCGTTTGATTGAAGCTAGCAGCTTAAATGATTCTGTGTACCTTAGGATGACTTTTGGGCACCTTTATGAAACCTTCCATGCAAACCCTGGAACTGTGACCGGCTCAGCCGTGGGATGCAATCCGGATACATTCTGGAGTAAAACTACC CATCTTACTCCCTGGCTCACTCTTTGCTTTCGACTATTCAGGATATGATGCTAGTCTTAGCCCTGTCTGGTTCAGAGCATTGGAATTAGTTCTTAGGGAAATAGGCTACAGTGAGGAAGCAGTTTCACTTGTTGAGGGGATCAATCACACACACCATGTATACCGCAACAAAACTTACTGTGTGCTTGGTGGCATGCCCTCTGGTTGCTCAGGAACATCCATATTCAATTCAATGATCAATAACATTATTATTAGAGCACTGCTCAT CAAAACGTTCAAGGGCATTGATTTAGACGAGCTCAACATGGTTGCCTACGGGGATGATGTGCTTGCCAGTTACCCTTTTCCAATTGACTGTTTGGAGTTGGCAAGAACAGGCAAGGAGTACGGTTTAACCATGACTCCTGCAGACAAATCCCCATGCTTCAATGAAGTCAATTGGGATAACGCAACCTTTCTCAAAAGAGGCTTCTTGCCCGATGAACAATTTCCATTCCTTATCCACCCTACCATGCCAATGAAAGAAATCCACGA GTCCATACGGTGGACCAAGGACCGCGAAATACCCAAGATCACGTGCGGTCCTTGTGCCTTCTGGCATGGCACAATGGTAAGCAGGAATATGAAAAGTTTGTGAGCGCAATTAGATCAGTACCAGTAGGAAAAGCATTGGCTATTCCAAATTATGAGAATCTGAGACGCAATTGGCTCGAACTATTCTAGAGGTTAAGTATATACCTCAACCCCACCAGGAATCTGGTCGTGAACATGACTGGTGGGGGTAAATTTGTTATAACC AGAATAGCAGAAAAAAAAAAAAAAAAAAAAAA

The nucleotide sequence of Gluc is shown in SEQ ID NO.2:

ATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCGAAGCTGCCGCTGGAGGTGCTCAAAGAGCTTGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACCGCCCAA GATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGATGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCATTGAGCAGTTCATCGCACAGGTCGATCTGTGGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATC CAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGATGAC

The nucleic acid sequence of PDGFRβTM is shown in SEQ ID NO.3:

GCTGTGGGCCAGGACACGCAGGAGGTCATCGTGGTGCCACACTCCTTGCCCTTTAAGGTGGTGGTGATCTCAGCCATCCTGGCCCTGGTGGTGCTCACCATCATCTCCCTTATCATCCTCATCATGCTTTGGCAGAAGAAGCCACGT

The nucleic acid sequence of SpikeTM is shown in SEQ ID NO.4:

GACGTGGATCTGGGCGACATCAGCGGCATCAATGCCTCCGTGGTGAACATCCAGAAGGAGATCGACAGGCTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCGATCTGCAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCCTGGTATATCTGGCTGGGCTTCATCGCCGGCCTGATCGCTATCGTGATGGTGACCATCATGCTGTGCTGTATGACATCCTGCTGTTCTTGCCTGAAGGGCTGC TGTAGCTGTGGCTCCTGCTGTAAGTTTGATGAGGACGATTCCGAGCCAGTGCTGAAGGGCGTGAAGCTGCACTACACC

The nucleic acid sequence of CD28TM is shown in SEQ ID NO.5:

ATCGAGGTGATGTACCCTCCCCCTTACCTGGACAACGAGAAGAGCAACGGCACCATCATCCACGTGAAGGGCAAGCACCTGTGCCCTAGCCCCCTGTTCCCCGGACCTAGCAAGCCCTTTTGGGTGCTGGTGGTGGTGGGCGGCGTGCTGGCCTGTTACTCCCTGCTGGTGACCGTGGCCTTCATTATCTTCTGGGTGAGGAGCAAGAGGAGCAGGCTGCTGCACAGCGACTACATGAACATGACACCCAGGAGACCTGGCCCC ACCAGAAAGCACTACCAGCCCTATGCCCCCCCCAGAGACTTTGCCGCCTACAGAAGC

The nucleic acid sequence of PDGFRβ+SpikeTM is shown in SEQ ID NO.6:

GACGTGGATCTGGGCGACATCAGCGGCATCAATGCCTCCGTGGTGAACATCCAGAAGGAGATCGACAGGCTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCGATCTGCAGGAGCTGGGCAAGTATGAGCAGTACATCGCTGTGGGCCAGGACACGCAGGAGGTCATCGTGGTGCCACACTCCTTGCCCTTTAAGGTGGTGGTGATCTCAGCCATCCTGGCCCTGGTGGTGCTCACCATCATCTCCC TTATCATCCTCATCATGCT TTGGCAGAAGAAGCCACGTCTGTGCTGTATGACATCCTGCTGTTCTTGCCTGAAGGGCTGCTGTAGCTGTGGCTCCTGCTGTAAGTTTGATGAGGACGATTCCGAGCCAGTGCTGAAGGGCGTGAAGCTGCACTACACC

The nucleic acid sequence of Linker+TM+Linker is shown in SEQ ID NO.7:

GAATTCGGAGGCAGTGGAGGAGGTAGTAATGGAGGCGGTAGTGGAGGCGGAGGTAGTAGAGGAGGAGGAGGCAGTGGAGGAGGTAGTAATGGAGGCGGAAGTGGAGGCGGAGGTAGTAGAGGAGGAGGAGGCAGTGGAGGAGGTAGTAATGCTGTGGGCCAGGACACGCAGGAGGTCATCGGTGGTGCCACACTCCTTGCCCTTTAAGGTGGTGGTGATCAGCCATCCCTGGCCCTGGTGGTGC TCACCATCATCTCCCTTATCATCCTCATCATGCTTTGGCAGAAGAAGCCACGTGGAGGCGGTAGTGGAGGCGGAGGTAGTAGAGGAGGAGGAGGCAGTGGAGGAGGTAGTAATGGAGGCGGAAGTGGAGGCGGAGGTAGTAGAGGAGGAGGAGGCAGTGGAGGAGGTAGTAATGGTACC

The nucleic acid sequence of Furin is shown in SEQ ID NO.8:

CGCCGAAAGAGA

The nucleic acid sequence of mFurin is shown in SEQ ID NO.9:

GCCCGAAAGGCT

The nucleic acid sequence of T2A is shown in SEQ ID NO.10:

GAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCA

The nucleic acid sequence of CA16-memHiBiT is shown in SEQ ID NO.11:

TTAAAACAGCCTGTGGGTTGTTCCCACCCACAGGGCCCACTGGGCGCTAGCACACTGGTTCTGCGGGACCTTTGTGCGCCTGTTTTATAACCCTTCCCTAAGCAGTAACTTAGAAGCTTTGTACAATCATGGCCAATAGTGGGCGTGGCGCGCCAGTCACGTCTTGGTCAAGCACTTCTGTATCCCCGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAACGTTCGTTACCCGGCTAGCTACTTCGAGAAACCTAGTAGCACCATGAAAGTTGCGGAGTGTTTCGCTCAGCACTTCCCCCGTGTAGATCAGGTCGATGAGTCACTGTAAACCCCACGGGCGACCGTGACAGTGGCTGCGTTGGCGGCCTGCCCATGGGGTAACCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTAGCAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCAGAGCGCGCACCCTCAACCCAGGGGGCGGCGCGTCGTAATGGGTAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATTCTTTATTGGCTGCTTATGGTGACAATTGAAAAGTTGTTACCATATAGCTATTGGATTGGCCATCCGGTGTCTAACAGAGCTATTGTTTACCTATTCATTGGCTTCGTCCCTCTTAATCTCAAGGCCATTCAAACTCTTGATTACATATTGCTCCTCAACTGTAAGAAATGGGATCCCAGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGCCTGCTCCTGGTGTTGCCTGC TGCCTTCCCTGCCCCAGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGTGGCGGTAGTAATGACGTGGATCTGGGCGACATCAGCGGCATCAATGCCTCCGTGGTGAACATCCAGAAGGAGATCGACAGGCTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCGATCTGCAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCCTGGTATATCTGGCTGGGCTTCATCGCCGGCCTGATCGCTATCGTGATGGTGACCATCATGCTGTGCTGTATGACATCCTGCTGTTCTTGCCTGAAGGGCTGCTGTAGCTGTGGCTCCTGCTGTAAGTTTGATGAGGACGATTCCGAGCCAGTGCTGAAGGGCGTGAAGCTGCACTACACCGCCATTACTACCCTTGGGTCACAAGTCTCCACCCAGCGATCCGGGTCACATGAGAACTCAAACTCTGCATCGGAGGGATCGACCATAAATTACACAACTATAAACTACTACAAGGATGCATACGCTGCAAGTGCGGGGCGCCAGGATATGTCCCAAGACCCGAAGAAATTTACTGACCCTGTCATGGATGTTATACATGAGATGGCCCCACCGCTAAAGTCTCCAAGCGCTGAGGCGTGTGGTTACAGTGATCGTGTGGCTCAGCTTACCATTGGAAACTCCACTATCACGACACAGGAAGCAGCTAACATAGTCATAGCCTATGGAGAGTGGCCTGAGTACTGCCCAGACACAGATGCAACGGCAGTCGACAAACCCACTCGACCTGACGTATCAGTGAATAGATTTTTCACGTTAGACACTAAATCTTGGGCCAAGGATTCAAAGGGTTGGTATTGGAAATTCCCTGATGTTTTGACAGAGGTAGGTGTTTTCGGTCAAAATGCTCAGTTCCACTACCTGTATCGATCTGGGTTTTGCGTGCACGTTCAGTGCAACGCTAGTAAATTTCACCAAGGTGCCTTACTAGTGGCTGTGCTGCCTGAGTATGTACTCGGTACTATCGCAGGAGGGACCGGGAACGAGAATTCTCATCCTCCCTACGCCACCACACAGCCTGGTCAGGTTGGCGCAGTTCTGACGCACCCTTACGTGCTAGACGCAGGGATCCCTTTAAGCCAATTAACTGTGTGCCCACACCAGTGGATTAATTTAAGAACTAATAATTGTGCAACCATTATAGTTCCATATATGAACACAGTTCCTTTTGACTCAGCTCTCAATCACTGCAACTTTGGTCTACTGGTTATCCCAGTGGTGCCATTAGATTTCAACACAGGTGCCACATCTGAAATTCCCATCACAGTCACCATAGCTCCCATGTGTGCGGAGTTTGCGGGTCTGCGCCAGGCGGTAAAGCAAGGTATACCAACAGAGCTTAAACCTGGTACCAATCAGTTTCTTACTACTGATGATGGTGTCTCCGCACCAATTCTGCCAGGTTTCCATCCAACTCCGCCTATACATATACCAGGGGAAGTACACAACCTATTGGAAATATGTAGAGTGGAGACCATCCTGGAAGTCAATAACCTGAAGACCAATGAGACCACCCCCATGCAGCGCTTGTGTTTCCCAGTGTCGGTGCAGAGTAAAACGGGCGAATTATGTGCTGCTTTCAGAGCAGACCCTGGAAGAGATGGCCCGTGGCAGTCTACAATACTAGGTCAACTCTGCAGATACTACACCCAGTGGTCAGGTTCACTGGAGGTGACATTCATGTTTGCAGGTTCATTCATGGCCACAGGAAAAATGCTCATCGCCTACACCCCACCTGGGGGAAATGTGCCTGCGGACAGAATCACAGCAATGCTAGGAACACATGTGATCTGGGACTTCGGATTGCAGTCTTCTGTGACGTTGGTCGTGCCATGGATCAGTAATACACACTACAGGGCACACGCCCGCGCTGGGTACTTTGACTATTACACCACTGGCATCATAACCATATGGTATCAAACTAACTATGTCGTACCCATTGGTGCTCCCACTACAGCATATATCGTGGCTTTGGCAGCAGCTCAGGATAACTTCACTATGAAACTGTGCAAGGA TACCGAGGACATTGAGCAAACGGCTAACATACAAGGGGATCCCATTGCAGATATGATTGACCAAACTGTAAACAATCAAGTGAACCGCTCCTTGACTGCGATGCAAGTACTACCTACAGCTGCCAACACTGAGGCAAGTAGTCACAGATTAGGCACTGGTGTTGTACCAGCACTACAAGCTGCGGAGACAGGGGCGTCGTCTAATGCTAGTGACAAGAACCTCATTGAGACTAGATGTGTGTTGAACCATCATTCCACGCAAGAGACAGCCATTGGGAATTTCTTTAGCCGTGCTGGTCTTGTCAGCATCATCACAATGCCCACCACGGGTACACAGAATACAGATGGTTATGTTAATTGGGACATTGATTTAATGGGATATGCTCAGCTGCGGCGGAAATGCGAGTTATTCACCTACATGCGCTTTGATGCTGAATTCACATTTGTCGTAGCCAAACCCAATGGTGAGCTAGTCCCCCAATTACTCCAGTACATGTATGTCCCGCCAGGGGCTCCGAAACCTACTTCCAGAGATTCGTTTGCTTGGCAGACAGCCACCAACCCATCTGTGTTTGTGAAAATGACGGACCCGCCAGCTCAAGTGTCAGTCCCCTTCATGTCACCAGCTAGTGCATATCAATGGTTTTATGATGGTTATCCCACCTTTGGTGAGCACCTCCAAGCAAATGACCTGGATTATGGTCAATGCCCGAATAATATGATGGGCACTTTTAGCATTAGGACAGTAGGGACTGAGAAGTCACCACACTCCATTACCCTGAGGGTGTATATGAGAATCAAACACGTCAGGGCATGGATCCCAAGACCTCTGAGAAATCAACCCTATTTGTTTAAGACTAACCCAAATTATAAAGGAAATGATATTAAGTGTACCAGCACTAGTAGAGACAAGATAACAACGCTGGGAGAGTTTGGACAGCAATCGGGCGCCATATATGTGGGCAACTATAGGGTGGTGAATCGACACCTTGCCACACACAACGACTGGTCAAACCTTGTGTGGGAGGACAGCTCTAGGGACTTGTTAGTCTCCTCCACCACCGCCCAGGGATGCGACACCATCGCTAGGTGCGATTGTCAAACCGGTGTATACTATTGCAGCTCCAAAAGGAAACACTACCCGGTTAGTTTCACCAAGCCCAGTCTGATATTTGTGGAAGCTAGTGAGTATTACCCAGCTAGATATCAGTCCCATCTCATGCTTGCTGTGGGTCACTCGGAACCTGGTGACTGTGGTGGCATCCTCAGATGCCAGCATGGTGTGGTAGGAATTGTCTCCACTGGTGGAAATGGTCTTGTGGGGTTTGCCGATGTCAGAGACCTTCTATGGCTGGATGAAGAAGCAATGGAGCAGGGAGTGTCTGACTATATCAAAGGCCTCGGTGACGCTTTTGGTATGGGCTTCACTGATGCAGTGTCTAGGGAAGTAGAGGCATTGAAGAACCATCTAATTGGCTCAGAAGGAGCTGTTGAGAAGATCTTGAAGAACTTGGTGAAACTAATCTCGGCTTTGGTCATAGTTGTTAGGAGTGACTATGACATGGTCACCCTCACGGCTACGCTAGCCCTGATTGGGTGTCATGGAAGCCCTTGGGCATGGATTAAAGCGAAAACAGCCTCTATTCTTGGCATTCCCATAGTGCAGAAACAGAGCGCTTCATGGCTAAAGAAGTTTAATGATATGGCCAACGCCGCGAAAGGGCTCGAGTGGATTTCTAGTAAAATTAGCAAGTTCATTGACTGGCTTAAAGAAAAGATTATTCCGGCCGCTAAAGAGAAAGTTGAATTCTTAAACAACCTGAAACAGCTTCCCTTGTTGGAGAACCAGATCTCAAACCTTGAACAGTCTGCTGCCTCGCAAGAAGATCTAGAAGCTATGTTTGGCAATGTGTCATATTTAGCCCACTTCTGCCGCAAGTTCCAGCCACTCTATGCGGTTGAAGCCAAAAGGGTTTATGCCCTAGAGAAAAGGATGAATAACTACATGCAGTTCAAGAGCAAACACCGTATTGAACCTGTATGCTTAGTCATTAGAGGCTCCCCAGGAAC AGGTAAATCACTTGCTACGGGCATCATTGCTAGAGCTATTGCTGACAAATACCACTCTAGCGTTTACTCACTCCCTCCAGATCCAGACCATTTTGATGGATACAAGCAACAAGTAGTCACTGTTATGGATGATCTCTGTCAGAACCCAGATGGAAAGGACATGTCCCTATTTTGCCAAATGGTTTCTACAGTGGACTTTATACCACCCATGGCATCATTGGAAGAGAAAGGAGTGTCTTTCACCTCCAAGTTTGTCATCGCATCGACCAATGCTAGCAATATTGTAGTTCCCACAGTTTCAGATTCTGATGCGATTCGCAGGCGGTTTTATATGGACTGCGATATAGAGGTGACAGACTCTTACAAGACGGACCTTGGCCGACTCGACGCAGGCAGGGCCGCTAAGCTTTGCACAGAAAACAACACTGCTAATTTTAAGAGATGCAGCCCATTAGTGTGTGGCAAGGCCATTCAATTAAGAGACAGGAAGTCTAAAGTGAGATATAGTATTGACACTGTGGTGTCAGAGTTAATCAGAGAGTATAATAATAGGTCTGCCATTGGGAACACCATAGAAGCCCTCTTCCAAGGACCCCCTAAGTTCAGGCCTATAAGAATTAGCCTTGAAGAAAAGCCGGCCCCAGATGCCATCAGTGACTTACTTGCTAGTGTGGATAGCGAGGAGGTTCGACAATACTGCAGGGAACAAGGGTGGATAATTCCAGAAACATCGACCAATGTGGAACGTCACCTCAACAGAGCAGTGTTGGTGATGCAGTCCATTGCCACCGTGGTTGCGGTTGTGTCTCTCGTCTATGTCATTTATAAATTGTTTGCTGGATTTCAAGGTGCCTATTCTGGTGCACCTAAGCAAGCTCTCAAAAAGCCTGTGCTAAGGACAGCCACAGTCCAAGGACCGAGCTTGGACTTTGCCTTATCCCTTCTAAGGCGCAACATCAGACAGGTGCAAACTGACCAAGGACATTTCACTATGTTAGGAGTGCGAGATCGCCTAGCCATTTTGCCGCGCCACTCGCAACCAGGAAAAACTATCTGGGTGGAGCACAAATTAATCAATGTGTTAGATGCTGTTGAATTGGTGGATGAGCAAGGTGTAAATTTGGAACTTACACTAGTAACCTTGGACACCAACGAAAAATTTAGAGATGTCACCAAGTTTATTCCAGAGACGATCACCGGGGCAAGCGACGCAACCTTGGTCATCAACACTGAGCACATGCCCTCTATGTTCGTCCCAGTGGGTGATGTTGTACAGTATGGGTTTCTAAATCTCAGCGGTAAGCCCACACACCGAACCATGATGTACAATTTCCCCACAAAGGCAGGGCAGTGTGGAGGTGTGGTCACTTCAGTCGGTAAGATTATTGGAATTCATATCGGTGGGAATGGACGCCAAGGCTTCTGTGCTGGACTGAAGAGAGGTTACTTTGCCAGTGAACAAGGAGAGATCCAATGGATGAAGTCCAATAAAGAAACTGGGAGACTGAATATTAATGGTCCAACACGTACCAAATTGGAGCCCAGTGCATTCTACGATGTGTTTGAGGGCAGCAAAGAACCAGCAGTCTTAACCAGTAAGGATCCCAGACTTGAGGTTGATTTTGAGCAAGCTTTGTTTTCCAAATATGTAGGAAATACCCTGCATGAGCCTGACGAGTATGTGACACAGGCTGCTCTCCACTATGCAAACCAGCTAAAGCAATTAGATATAAACACTAATAAGATGAGTATGGAAGAAGCATGCTACGGCACTGAATATCTAGAGGCTATAGACTTGCACACCAGTGCCGGGTACCCCTATAGTGCCCTGGGTGTCAAGAAAAGAGACATACTTGACCCAACCACTAGAGATACTACCAAAATGAAATTCTACATGGATAAATATGGGTTAGACTTGCCCTATTCCACCTATGTGAAAGATGAGCTTAGATCCTTAGATAAGATTAAGAAAGGGAAATCCCGCTTGATTGAAGCCAGTAGTCTAAATGACTCAGTCTACCTTAGGATGACTTTCGGGCATCTCTATGAAACTTTTCATGCCAACCCGGG GACTGTGACTGGGTCTGCAGTAGGGTGTAATCCTGATGTGTTCTGGAGTAAATTACCAATCCTGTTGCCAGGATCGCTCTTTGCATTTGACTATTCAGGATATGATGCAAGTCTCAGCCCAGTATGGTTTAGAGCTTTGGAAGTGGTTCTCCGAGAGATCGGATACTCAGAGGAAGCTGTATCACTAATAGAAGGGATCAACCACACCCATCATGTGTATCGGAACAGGACGTATTGTGTCCTTGGTGGAATGCCTTCAGGTTGTTCCGGCACTTCCATCTTCAATTCCATGATCAATAACATAATAATCAGAACCCTTTTGATTAAAACTTTTAAGGGGATCGATTTAGATGAGCTGAATATGGTAGCTTATGGAGATGATGTGTTAGCTAGCTATCCATTCCCTATTGACTGCTCGGAGCTAGCCAGAACTGGTAAAGAGTATGGATTAACAATGACACCTGCTGACAAGTCACCTTGCTTTAATGAAGTTACCTGGGAAAATGCTACATTCTTAAAGAGAGGCTTCCTGCCAGATCATCAGTTCCCATTTCTTATCCATCCTACCATGCCCATGAGGGAGATCCACGAGTCCATTCGCTGGACTAAAGACGCACGCAACACTCAGGACCACGTGCGCTCTCTGTGCCTCTTAGCGTGGCATAATGGGAAGGAGGAATATGAAAAATTTGTGAGCACAATTAGATCAGTTCCTATTGGAAAAGCCTTGGCTATACCAAATTTTGAGAACTTGAGAAGAAATTGGCTCGAGTTATTTTAATATACAGTTTAAAGCTGAACCCCACCAGAAGTCTGGTCGTGTTAATGACTGGTGGGGGTAAATTTGTTATAACCAGAATAGCAGAAAAAAAAAAAAAAAAAAAAAAAAA

Example 1

The steps for optimizing the transmembrane region in an EV71replicon vector are:

1. Construct PL451-71rep-Gluc clone:

Use the PL451-71rep vector as a template and primers to amplify the PL451-71rep fragment; use pcDNA-Gluc as a template and primers to amplify the Gluc fragment. The amplified fragment is electrophoresed using agarose gel with a concentration of 0.7% (w/v). , according to the DNAMarker bands, cut the PL451-71rep and Gluc amplification products of the correct size and use the Tiangen enhanced gel recovery kit to recover and purify the DNA fragments.

The purified PL451-71rep and Gluc fragments were recombined using a two-fragment recombination kit (purchased from Shanghai Yisheng Biotechnology Co., Ltd.). The recombinant products were transferred into Trans2-Blue E. coli competent cells, and single clone colonies were picked and processed. The correct clone was initially identified by PCR, the culture was expanded, and the plasmid was extracted and sequenced for verification. The correct plasmid was identified as the PL451-71rep-Gluc clone.

The upstream amplification primer of PL451-71rep, the sequence is shown in SEQ ID NO.12:

5’-CTCAATACAGTCAAACGCCACCATGGGAGTCAAAG-3’;

The downstream amplification primer of PL451-71rep has the sequence shown in SEQ ID NO.13:

5’-GTGGGTGATCGCGTCATCACCGGCCCCCCTT-3’;

The upstream amplification primer of Gluc has the sequence shown in SEQ ID NO.14:

5’-GATGACGCGATCACCACTCTTGGAAAGTTCG-3’;

The downstream amplification primer of Gluc has the sequence shown in SEQ ID NO.15:

5’-GTTTGACTGTATTGAGAGTTAATATAAAGTTGAGGGTG-3’;

The recombination reaction system is: PL451-71rep: 1μl, Gluc: 1μl, vector: 2μl, water: 6μl; 2×Hieff MultiSEnzymePremix: 10μl, 50℃, 30min.

2. Optimization of the transmembrane region in PL451-71rep-Gluc cloning:

Use PL451-71rep-Gluc in step 1 as a template, use primers to amplify PL451-71rep-Gluc, and use primers to amplify PDGFRβTM, SpikeTM, CD28TM, PDGFRβ+SpikeTM and Linker+TM+Linker transmembrane regions. After amplification, The fragments were electrophoresed using agarose gel with a concentration of 0.7% (w/v). According to the DNAMarker bands, the correct sizes of PL451-71rep-Gluc, PDGFRβTM, SpikeTM, CD28TM, PDGFRβ+SpikeTM and Linker+TM+Linker were cut. In the membrane area, amplify the product and use Tiangen enhanced gel recovery kit to recover and purify DNA fragments.

The purified PL451-71rep-Gluc was recombined with PDGFRβTM, SpikeTM, CD28TM, PDGFRβ+SpikeTM and Linker+TM+Linker transmembrane region fragments using a two-fragment recombination kit (purchased from Shanghai Yisheng Biotechnology Co., Ltd.). After recombination The products were transferred into Trans2-Blue Escherichia coli competent cells, and the correct clones were preliminarily identified by PCR after picking single clones. The bacteria were expanded and the plasmids were extracted and sequenced for verification. The correct plasmid was PL451-71rep-Gluc-PDGFRβTM. , PL451-71rep-Gluc-SpikeTM, PL451-71rep-Gluc-CD28TM, PL451-71rep-Gluc-PDGFRβ+SpikeTM, PL451-71rep-Linker+TM+Linker clone (Figure A in Figure 1).

The upstream amplification primer of PL451-71rep-Gluc, the sequence is shown in SEQ ID NO.16:

5’-GCCACGTGGATCACCACTCTTGGAAAGTTCG-3’;

The downstream amplification primer of PL451-71rep-Gluc has the sequence shown in SEQ ID NO.17:

5’-CCCACAGCGTCATCACCGGCCCCCTT-3’;

The upstream amplification primer of PDGFRβTM has the sequence shown in SEQ ID NO.18:

5’-GTGATGACCGCTGTGGGCCAGGACAC-3’;

The downstream amplification primer of PDGFRβTM has the sequence shown in SEQ ID NO.19:

5’-GTGATCGCACGTGGCTTCTTCTGCCAAAG-3’;

The upstream amplification primer of SpikeTM has the sequence shown in SEQ ID NO.20:

5’-TGTGATGACGGAGGCGGATCAGGTGGC-3’;

The downstream amplification primer of SpikeTM has the sequence shown in SEQ ID NO.21:

5’-CAAGAGTGGTGATCGCGGTGTAGTGCAGCTTCACGC-3’;

The upstream amplification primer of CD28TM has the sequence shown in SEQ ID NO.22:

5’-GCCGGTGATGACATCGAGGTGATGTACCCTCCC-3’;

The downstream amplification primer of CD28TM has the sequence shown in SEQ ID NO.23:

5’-GTGATCGCGCTTCTGTAGGCGGCAAAGTC-3’;

The upstream amplification primer of PDGFRβ+SpikeTM has the sequence shown in SEQ ID NO.24:

5’-GTGATGACGACGTGGATCTGGGCGACA-3’;

The downstream amplification primer of PDGFRβ+SpikeTM has the sequence shown in SEQ ID NO.25:

5’-CAAGAGTGGTGATCGCGGTGTAGTGCAGCTTCACGC-3’;

The upstream amplification primer of Linker+TM+Linker, the sequence is shown in SEQ ID NO.26:

5’-GGTGATGACGAATTCGGAGGCAGTGGAGG-3’;

The downstream amplification primer of Linker+TM+Linker has the sequence shown in SEQ ID NO.27:

5’-GTGGTGATCGCGGTACCATTACTACCTCCTCCACTGC-3’;

3. In vitro transcription:

Using PL451-71rep-Gluc-PDGFRβTM, PL451-71rep-Gluc-SpikeTM, PL451-71rep-Gluc-CD28TM, PL451-71rep-Gluc-PDGFRβ+SpikeTM, PL451-71rep-Linker+TM+Linker plasmids as templates, PCR amplification The full-length DNA template is added, and the PCR product is subjected to gel recovery, proteinase K treatment, and DNA product purification, and then T7 in vitro transcription is performed to obtain mRNA. After the mRNA is purified by LiCl precipitation, agarose gel electrophoresis with a concentration of 0.7% (w/v) is used. Check the quality of the mRNA. After the bands are correct, aliquot the mRNA and store it at -80°C.

The upstream primer for full-length amplification has the sequence shown in SEQ ID NO.28:

5’-GTGCCACCTGACCGTCTAAGAAAC-3’;

The downstream primer for full-length amplification has the sequence shown in SEQ ID NO.29:

5’-TTTTTTTTTTTTTTTTTTTTTTTCTGCTATTCTGGTTATAACAAATTTACCCCC-3’;

T7 in vitro transcription reaction system: Template: 8μl, A: 1.5μl, G: 1.5μl, C: 1.5μl, U: 1.5μl, Buffer: 2μl, T7Enzyme: 1μl; RNase-freeddH2O: 3μl, 37℃, 2h.

4. Gluc luciferase expression detection:

2.5*10 ⁴ RD cells were seeded into a 96-well plate, cultured at 37°C overnight, and transfected with 71rep-Gluc-TMmRNA carrying different transmembrane regions into the RD cells. After continuing to culture for 24 hours, add 20 μl of lysis buffer to each well and incubate at room temperature for 5 minutes. , then add 20 μl of substrate and detect the fluorescence value immediately. By comparing the fluorescence values produced after transfecting cells with PL451-71rep-Gluc-TM carrying different transmembrane regions, we can reflect the expression of foreign proteins in the vector (Figure B in Figure 1).

Example 2:

EV71replicon vector is designed to express secreted proteins.

The steps are:

1.PL451-71rep-Gluc-Furin-TM clone construction:

Using PL451-71rep-Gluc-Linker+TM+Linker as the template, use PCR primers to directly introduce Furin sites into the template. The amplified fragments are electrophoresed using agarose gel with a concentration of 0.7% (w/v). According to DNAMarker bands, cut amplification products of correct size and use Tiangen enhanced gel recovery kit to recover and purify DNA fragments.

The purified DNA fragments were treated with DMT enzyme (purchased from Shanghai Yisheng Biotechnology Co., Ltd.). The products treated with DMT enzyme were transferred to Trans2-Blue E. coli competent cells. After picking single clone colonies, the correct clones were preliminarily identified by PCR. The culture was expanded, and the plasmid was extracted and sequenced for verification. The correctly sequenced plasmid was the PL451-71rep-Furin-TM clone (Panel A in Figure 2).

PL451-71rep-Gluc-Linker+TM+Linker upstream amplification primer, the sequence is shown in SEQ ID NO.30:

5’-CGCCGAAAGAGAGGCTCCGGCGAGGGCAGGGGAAGTCTTCTAAC-3’;

PL451-71rep-Gluc-Linker+TM+Linker downstream amplification primer, the sequence is shown in SEQ ID NO.31:

5’-CTTCTCTTTCGGCGATTACTACCGCCACCACTACC-3’;

2.PL451-71rep-Gluc-mFurin-TM clone construction:

Using PL451-71rep-Gluc-mFurin-TM as a template, use point mutation PCR primers to directly introduce mFurin sites into the template. The amplified fragments are electrophoresed using agarose gel with a concentration of 0.7% (w/v). According to DNAMarker bands, cut amplification products of correct size and use Tiangen enhanced gel recovery kit to recover and purify DNA fragments.

The purified DNA fragments were treated with DMT enzyme (purchased from Shanghai Yisheng Biotechnology Co., Ltd.). The products treated with DMT enzyme were transferred to Trans2-Blue E. coli competent cells. After picking single clone colonies, the correct clones were preliminarily identified by PCR. The culture was expanded and the plasmid was extracted and sequenced for verification. The correctly sequenced plasmid was the PL451-71rep-mFurin-TM clone.

PL451-71rep-Gluc-mFurin-TM upstream amplification primer, the sequence is shown in SEQ ID NO.32:

5’-CGAAAGGCTGGTTCTGGCGACGTGGATCTGGGCGACAT-3’;

PL451-71rep-Gluc-mFurin-TM downstream amplification primer, the sequence is shown in SEQ ID NO.33:

5’-CAGAACCAGCCTTTCGGGCATTACTACCGCCACCACTACCG-3’;

3.PL451-71rep-Gluc-T2A-TM clone construction:

Using PL451-71rep-Gluc-Furin-TM as the template, use PCR primers to directly delete the Furin site in the template and introduce the T2A site. The amplified fragment is used in an agarose gel with a concentration of 0.7% (w/v). Electrophoresis, according to the DNAMarker band, cut the amplification product of the correct size and use the Tiangen enhanced gel recovery kit to recover and purify the DNA fragments.

The purified DNA fragments were treated with DMT enzyme (purchased from Shanghai Yisheng Biotechnology Co., Ltd.). The products treated with DMT enzyme were transferred to Trans2-Blue E. coli competent cells. After picking single clone colonies, the correct clones were preliminarily identified by PCR. The culture was expanded, and the plasmid was extracted and sequenced for verification. The correctly sequenced plasmid was the PL451-71rep-T2A-TM clone.

The amplification primer upstream of the template has the sequence shown in SEQ ID NO.34:

5’-CTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCAGACGTGGATCTGGGCGA-3’;

The amplification primer downstream of the template has the sequence shown in SEQ ID NO.35:

5’-CGTCCCCGCATGTTAGAAGACTTCCCCTGCCCTCGCCGGAGCCGTCATCAC-3’;

4.PL451-71rep-Gluc-T2A-Furin-TM clone construction

Using PL451-71rep-Gluc-Furin-TM as the template, use PCR primers to directly introduce the T2A site into the template. The amplified fragments are electrophoresed using agarose gel with a concentration of 0.7% (w/v). According to the DNAMarker strips Band, cut the amplification product of correct size and use Tiangen enhanced gel recovery kit to recover and purify the DNA fragments.

The purified DNA fragments were treated with DMT enzyme (purchased from Shanghai Yisheng Biotechnology Co., Ltd.). The products treated with DMT enzyme were transferred to Trans2-Blue E. coli competent cells. After picking single clone colonies, the correct clones were preliminarily identified by PCR. The culture was expanded, and the plasmid was extracted and sequenced for verification. The correctly sequenced plasmid was the PL451-71rep-T2A-Furin-TM clone.

The template upstream amplification primer has the sequence shown in SEQ ID NO.36:

5’-GTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCAGACGTGGATCTGGGCGAC-3’;

The amplification primer downstream of the template has the sequence shown in SEQ ID NO.37:

5’-CGCATGTTAGAAGACTTCCCCTGCCCTCGCCGGAGCCTCTCTTTCGG-3’;

5. In vitro transcription

Using PL451-71rep-Gluc-Furin-TM, PL451-71rep-Gluc-mFurin-TM, PL451-71rep-Gluc-T2A-TM and PL451-71rep-Gluc-T2A-Furin-TM plasmids as templates, PCR amplified the whole After the long DNA template and PCR product were gel recovered, proteinase K treated and DNA product purified, T7 in vitro transcription was performed to obtain mRNA. After the mRNA was purified by LiCl precipitation, 0.7% (w/v) agarose gel electrophoresis was used to detect the mRNA. After the quality and bands are correct, the mRNA is aliquoted and stored at -80°C.

The upstream primer for full-length amplification has the sequence shown in SEQ ID NO.28.

The sequence of the downstream primer for full-length amplification is shown in SEQ ID NO. 29.

6. Gluc luciferase expression detection

2.5*10 ⁴ RD cells were seeded into a 96-well plate, cultured at 37°C overnight, and transfected with 71rep-Gluc-Furin-TM, 71rep-Gluc-mFurin-TM, 71rep-Gluc-T2A-TM and 71rep-Gluc- respectively. T2A-Furin-TMmRNA was transfected into RD cells. Each sample was transfected into 6 replicate wells (A, B, C, D, E, F). After continuing to culture for 24 hours, 20 μl of supernatant was taken from wells A, B, and C and then Add 20 μl of substrate and detect the fluorescence value immediately; then completely remove the supernatant from wells A, B, and C, wash once with PBS, and immediately add 20 μl of substrate to detect the fluorescence value; finally, remove the supernatant from wells D, E, and F. , wash once with PBS, add 20 μl of lysis buffer, incubate at room temperature for 5 min, add 20 μl of substrate to detect the fluorescence value. By comparing the fluorescence values produced after transfecting cells with PL451-71rep-Gluc-TM carrying different transmembrane regions, we can reflect the expression of foreign proteins in the vector (Panels B and C in Figure 2).

Example 3:

A method for constructing a CA16 infectious clone carrying a membrane display reporter gene. The steps are:

1.Construction of CA16-memHiBiT infectious clones:

Using the pSVA-CA16 infectious clone stored in the laboratory as a template, PCR primers amplify the pSVA-CA16 vector fragment. Using the pCDNA-memHiBiT clone stored in the laboratory as a template, PCR primers amplify the memHiBiT insert fragment. The amplified fragment is used Agarose gel electrophoresis with a concentration of 0.7% (w/v) was performed. According to the DNAMarker bands, the amplification products of the correct size were cut and the DNA fragments were recovered and purified using Tiangen enhanced gel recovery kit.

After the purified DNA fragment pSVA-CA16 and memHiBiT were recombined, the product was transferred to the Trans2-Blue E. coli competent state. After picking the single clone colony, the correct clone expanded strain was preliminarily identified by PCR. The plasmid was extracted and sequenced for verification. The sequencing was correct. The plasmid is the pSVA-CA16-memHiBiT infectious clone (Panel A in Figure 3).

pSVA-CA16 upstream amplification primer, the sequence is shown in SEQ ID NO.38:

5’-CTACACCGCCATTACTACCCTTGGGTCAC-3’;

pSVA-CA16 downstream amplification primer, the sequence is shown in SEQ ID NO.39:

5’-GGGATCCATTTCTTACAGTTGAGGAGCAATATGTAATCAAG-3’;

memHiBiT upstream amplification primer, the sequence is shown in SEQ ID NO.40:

5’-GTAAGAAATGGGATCCCAGAACTCCTTTCTCCACAAGCGC-3’;

memHiBiT downstream amplification primer, the sequence is shown in SEQ ID NO.41:

5’-GTAGTAATGGCGGTGTAGTGCAGCTTCACGC-3’;

2. Rescue of CA16-memHiBiT virus and virus growth characteristics:

Using the pSVA-CA16-memHiBiT plasmid as a template, PCR amplifies the full-length DNA template. After the PCR product is gel recovered, proteinase K treated and DNA product purified, T7 in vitro transcription is performed to obtain mRNA. After the mRNA is purified by LiCl precipitation, the concentration used is: Detect the quality of the mRNA by 0.7% (w/v) agarose gel electrophoresis. After the band is correct, the mRNA is aliquoted and stored at -80°C.

Inoculate 2.5*10 ⁵ Vero cells in each well of a 12-well plate, culture at 37°C overnight, transfect 1 μg of CA16-memHiBiTmRNA into Vero cells, and transfect CA16mRNA as a control group, with 5 duplicate wells for each sample; observe after transfection Cytopathological changes (Panel B in Figure 3); cells were collected at 12h, 24h, 36h and 48h respectively. After extracting sample RNA, one-step RT-PCR was used to quantitatively detect the viral gene copy number and analyze the virus growth curve; transfection was carried out for 12h at the same time. , 24h, 36h and 48h CA16-memHiBiT sample supernatant was infected into Vero cells, and luciferase activity was measured after 24h. The growth characteristics of the virus were analyzed based on the quantitative results and luciferase results (Panel C in Figure 3).

CA16-memHiBiT full-length upstream amplification primer, the sequence is shown in SEQ ID NO.42:

5’-GCGTATCACGAGGCCCTTTC-3’;

CA16-memHiBiT full-length downstream amplification primer, the sequence is shown in SEQ ID NO.43:

5’-TTTTTTTTTTTTTTTTTTTTTTTCTGCTATTCTGGTTATAACAAATTTACCCCC-3’;

The reaction system of TR-PCR is: Oligo(dT)23VN (10μM): 1μl, RNA: 3μl, RNase-free ddH2O: 4μl, 65℃, 5min;

The above mixed solution: 8 μl, 2×RTbuffer: 10 μl, Enzyme: 2 μl; 50°C, 45min; 85°C, 5min.

3.CA16-memHiBiT virus passage and genetic stability verification:

Use Lipo3000 transfection reagent to transfect CA16-memHiBiTmRNA into Vero cells. The Vero cells transfected with the mRNA will continue to be cultured and the cytopathic effects will be observed. After more than 80% of the cells develop cytopathic effects, freeze and thaw twice at -80°C twice. The virus supernatant was collected by centrifugation at 4000g as the P0 generation. The P0 generation virus liquid continued to infect Vero cells. When the cell lesions exceeded 80%, the virus liquid was collected as the P1 generation virus. The same steps were continued to the P20 generation. The harvested virus liquid was stored at -80°C.

2.5*10 ⁴ Vero cells were seeded into a 96-well plate, cultured at 37°C overnight, and infected with different generations of CA16-memHiBiT virus. After continuing to culture for 24 hours, add 20 μl of lysis buffer to each well, incubate at room temperature for 5 minutes, and then add 20 μl of bottom. substance and detect the fluorescence value immediately. The stability of the virus is reflected by comparing the fluorescence values produced by different generations of CA16-memHiBiT viruses after infecting cells (Figure 3-D).

The above are only preferred embodiments of the present invention, and do not limit the present invention in any form or substance. It should be pointed out that those of ordinary skill in the art can also make other modifications without departing from the present invention. Several improvements and additions are made, and these improvements and additions should also be regarded as the protection scope of the present invention. Those skilled in the art who are familiar with the art can make slight changes, modifications and equivalent changes based on the technical content disclosed above without departing from the spirit and scope of the invention. Equivalent embodiments; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims (10)

1. The enterovirus vector is characterized in that the enterovirus vector takes EV71replicon as a framework, and the nucleotide sequence of the enterovirus vector is shown as SEQ ID No. 1.

2. The use of an enterovirus vector according to claim 1 for the preparation of vaccines and antiviral drugs.

3. An enterovirus vector capable of expressing other membrane proteins, which is characterized by at least comprising an EV71replicon with a nucleotide sequence shown as SEQ ID No.1 and a transmembrane region fragment; the transmembrane region segment at least comprises PDGFRbeta TM with a nucleotide sequence shown as SEQ ID NO.3, spike TM with a nucleotide sequence shown as SEQ ID NO.4, CD28TM with a nucleotide sequence shown as SEQ ID NO.5, PDGFRbeta+Spike TM with a nucleotide sequence shown as SEQ ID NO.6 or linker+TM+linker with a nucleotide sequence shown as SEQ ID NO. 7.

4. The use of an enterovirus vector capable of expressing other membrane proteins according to claim 3 for the preparation of vaccines and antiviral drugs.

5. An enterovirus vector capable of expressing a secreted protein, wherein the enterovirus vector comprises the enterovirus vector of claim 3 and a cleavage site fragment; the cleavage site fragment at least comprises a Furin recognition region of a nucleotide sequence shown as SEQ ID NO.8, a mforin recognition region of a nucleotide sequence shown as SEQ ID NO.9 or T2A of a nucleotide sequence shown as SEQ ID NO. 10.

6. The use of an enterovirus vector capable of expressing a secreted protein according to claim 5 in the preparation of vaccines and antiviral drugs.

7. A construction method of a CA16 infectious clone carrying a membrane display reporter gene is characterized in that pSVA-CA16 is taken as a template, a memHiBiT membrane display reporter gene is introduced, and pSVA-CA16-memHiBiT infectious clone with correct sequencing is obtained through seamless recombination cloning; the nucleotide sequence of the CA16-memHiBiT is shown in SEQ ID NO. 11; the memHiBi membrane display reporter fragment is inserted between the 5' UTR and the P1 fragment.

8. Use of a CA16 infectious clone carrying a membrane-displayed reporter gene in screening antiviral drugs.

9. The construction method of enterovirus vector capable of expressing other membrane proteins is characterized by taking a carrier constructed by taking a serotype virus genome in enterovirus as a framework as a template, amplifying different transmembrane region fragments by PCR, constructing clones with different transmembrane region fragments by seamless cloning, screening and verifying correct clones by sequencing, extracting plasmids and storing; the transmembrane region segment at least comprises PDGFRbeta TM with a nucleotide sequence shown as SEQ ID NO.3, spike TM with a nucleotide sequence shown as SEQ ID NO.4, CD28TM with a nucleotide sequence shown as SEQ ID NO.5, PDGFRbeta+Spike TM with a nucleotide sequence shown as SEQ ID NO.6 or linker+TM+linker with a nucleotide sequence shown as SEQ ID NO. 7.

10. The construction method of the enterovirus vector capable of expressing the secretion protein is characterized by comprising the following steps:

step 1: the method comprises the steps of taking a vector I constructed by taking a serotype virus genome in enterovirus as a framework as a template, amplifying different transmembrane region fragments through PCR, constructing a vector II with different transmembrane region fragments through seamless cloning, screening clones which are verified to be correct through sequencing, extracting plasmids and storing; the transmembrane region segment at least comprises PDGFRbeta TM with a nucleotide sequence shown as SEQ ID NO.3, spike TM with a nucleotide sequence shown as SEQ ID NO.4, CD28TM with a nucleotide sequence shown as SEQ ID NO.5, PDGFRbeta+Spike TM with a nucleotide sequence shown as SEQ ID NO.6 or linker+TM+linker with a nucleotide sequence shown as SEQ ID NO. 7;

step 2: taking the second vector obtained in the step 1 as a template, and respectively introducing cutting site fragments by using a seamless cloning technology; thus constructing enterovirus vector III capable of expressing secretion protein; the cleavage site fragment at least comprises a Furin recognition region of a nucleotide sequence shown as SEQ ID NO.8, a mforin recognition region of a nucleotide sequence shown as SEQ ID NO.9 or T2A of a nucleotide sequence shown as SEQ ID NO. 10.