CN114908122B

CN114908122B - Novel influenza virus vector for expressing exogenous protein and construction method thereof

Info

Publication number: CN114908122B
Application number: CN202110168832.4A
Authority: CN
Inventors: 陈凌; 杨臣臣; 韩璐杰
Original assignee: Guangzhou N Biomed Ltd
Current assignee: Guangzhou N Biomed Ltd
Priority date: 2021-02-07
Filing date: 2021-02-07
Publication date: 2024-05-07
Anticipated expiration: 2041-02-07
Also published as: WO2022166176A1; CN114908122A

Abstract

The invention constructs a vector taking influenza virus PB2, PB1, PA, NP, HA, NA, M or NS genes as a framework, truncates PB2, PB1, PA, NP, HA, NA, M or NS gene sequences, and connects NA gene truncated fragments to PB2, PB1, PA, NP, HA, NA, M or NS truncated gene sequences, so that a novel vector capable of accommodating exogenous genes larger than the traditional influenza vector is prepared. Based on the characteristics of the vector for accommodating large fragment genes, the vector has wider applicability in the selection of exogenous genes, including fluorescent reporter genes, antigen genes, therapeutic genes and the like. The vector construction method provided by the experiment is mature, simple and convenient, convenient to operate and high in success rate. The constructed novel vector can efficiently express a plurality of exogenous proteins.

Description

Novel influenza virus vector for expressing exogenous protein and construction method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a novel influenza virus vector for expressing exogenous proteins and a preparation method thereof.

Background

Influenza viruses are single negative strand RNA viruses, and thus direct injection of viral genomic vRNA into cells cannot produce infectious viral particles. The progress of reverse genetics rescue system makes it possible to obtain infectious virus particles directly from cDNA, so that it has fast development of influenza virus vaccine, high research speed of influenza virus biological property and powerful technological support for modifying influenza virus into useful gene carrier. The development of influenza virus reverse genetics rescue systems provides a convenient and powerful technical support for the development of influenza virus vectors. Throughout the development history of influenza virus vectors, reverse genetics systems are one of the key technologies to whether vectors can be developed successfully. The influenza virus vector has very broad application prospect, but the development of the influenza virus vector is still immature at present.

Disclosure of Invention

It is a first object of the present invention to provide an influenza virus vector.

The second object of the present invention is to provide a method for constructing an influenza virus vector.

A third object of the present invention is to provide an application of the above influenza virus in expression of foreign proteins.

A fourth object of the present invention is to provide an influenza virus vector carrying a foreign gene.

A fifth object of the present invention is to provide a method for constructing an influenza virus vector carrying a foreign gene.

A sixth object of the present invention is to provide a method for expressing a foreign protein.

The technical scheme adopted by the invention is as follows:

in a first aspect, the present invention provides an influenza virus vector comprising:

A truncated gene sequence of the influenza virus gene PB2, PB1, PA, NP, HA, NA, M or NS, the truncated gene sequence comprising a coding region packaging signal sequence of the 3 'and 5' ends of the influenza virus gene PB2, PB1, PA, NP, HA, NA, M or NS; and, a step of, in the first embodiment,

And an NA gene truncated segment inserted into the PB2, PB1, PA, NP, HA, NA, M or NS truncated gene sequence, wherein the NA gene truncated segment comprises a 3' -end coding region packaging signal of the NA gene.

Preferably, the NA gene truncated fragment is inserted in the open reading frame of the PB2, PB1, PA, NP, HA, NA, M or NS truncated gene sequence.

NA is the second glycoprotein on the surface of influenza virus particles, a typical type II glycoprotein, with the amino terminus facing the interior of the virus particle. The NA protein is structurally formed as a tetramer, each protein monomer consisting of a short non-conserved amino-terminal cytoplasmic region, a hydrophobic transmembrane region, a stem region, and a globular head. The invention selects 1 st-201 st nucleotide of N end, and the segment contains packaging signal (183 nucleotides) of NA 3' end coding region. This fragment is inserted into a truncated PB2, PB1, PA, NP, HA, NA, M or NS gene sequence. At this time, the NA gene truncated segment is used as a base and is connected with the inserted exogenous gene, so that the aim of bringing the exogenous gene to the surface of influenza virus is fulfilled.

The nucleotide sequence of the NA gene truncated segment is shown as SEQ ID NO.1, and the encoded amino acid sequence is shown as SEQ ID NO. 2.

Preferably, as follows: the influenza virus vector is derived from an influenza a or b strain, preferably an H1N1 subtype influenza strain, e.g., a PR8 influenza strain.

As one embodiment of the present invention, the influenza virus vector comprises a truncated PB2 gene sequence, wherein the truncated PB2 gene sequence retains a packaging signal of 36 nucleotides at the 3 'and 5' ends, respectively, and the other portions are all truncated; a truncated NA gene fragment (SEQ ID NO. 1) was inserted into the truncated PB2 gene sequence.

In a second aspect, the present invention provides a method for constructing an influenza virus vector, comprising the steps of:

(1) Truncating at least one of the influenza virus PB2, PB1, PA, NP, HA, NA, M and NS genes, and preserving the coding region packaging signal sequences of the 3 'and 5' ends of said genes;

(2) Inserting the NA gene truncated segment into the truncated PB2, PB1, PA, NP, HA, NA, M or NS gene sequence to obtain an influenza virus vector; the NA gene truncated segment comprises a 3' -end coding region packaging signal;

Preferably, the nucleotide sequence of the truncated NA gene fragment is:

atgaatccaaatcagaaaataacaaccattggatcaatctgtctggtagtcggactaattagcctaatattgcaaatagggaatataatctcaatatggattagccattcaattcaaactggaagtcaaaaccatactggaatatgcaaccaaaacatcattacctataaaaatagcacctgggtaaaggacacaacttca(SEQ ID NO.1)

The amino acid sequence encoded by the NA gene truncated fragment is as follows:

MNPNQKITTIGSICLVVGLISLILQIGNIISIWISHSIQTGSQNHTGICNQNIITYKNSTWVKDTTS(SEQ ID NO.2)

In a third aspect, the present invention provides the use of an influenza virus vector according to the first aspect for expressing a foreign protein.

Preferably, the protein expression may be performed in chicken embryos or eukaryotic cells.

Preferably, the expressed protein may be in the human body:

Inducing an immune response; or (b)

Generating a biological reporter; or (b)

A tracking molecule for detection; or (b)

Regulating gene function; or (b)

As therapeutic molecules.

In a fourth aspect, the invention provides an influenza virus vector carrying a foreign gene comprising a truncated PB2, PB1, PA, NP, HA, NA, M or NS gene sequence which retains the coding region packaging signal sequences at the 3 'and 5' ends; and the truncated gene sequence is inserted with a fusion gene of an exogenous gene and an NA gene truncated segment, wherein the NA gene truncated segment comprises a 3' -end coding region packaging signal.

Preferably, the amino acid sequence encoded by the NA gene truncated fragment is shown as SEQ ID NO. 2;

preferably, the nucleotide sequence of the truncated NA gene fragment is shown in SEQ ID NO. 1.

Preferably, the exogenous gene is linked to the truncated NA gene segment by a linker sequence (linker).

Commonly used linker are GGGSG, GGGGS or GSG. Of course, other commonly used fusion protein linkers may also be used.

Preferably, the influenza virus vector is derived from an influenza a or b strain, preferably an H1N1 subtype influenza strain.

In a fifth aspect, the present invention provides a method for constructing an influenza virus vector carrying a foreign gene, comprising the steps of:

(1) Truncating at least one of the influenza virus genome PB2, PB1, PA, NP, HA, NA, M and NS genes, and reserving coding region packaging signal sequences of 3 'and 5' ends;

(2) Connecting exogenous genes with NA gene truncated fragments, and then inserting the exogenous genes into PB2, PB1, PA, NP, HA, NA, M or NS gene sequences truncated by influenza viruses to obtain an influenza virus vector carrying exogenous genes; the NA gene truncated segment comprises a 3' -end coding region packaging signal;

In a sixth aspect, the present invention provides a method for expressing a foreign protein, comprising the steps of:

1) Truncating at least one of the influenza virus genome PB2, PB1, PA, NP, HA, NA, M and NS genes, and reserving coding region packaging signal sequences of 3 'and 5' ends;

2) Connecting a foreign protein coding gene with the NA gene truncated segment, and then inserting the foreign protein coding gene into PB2, PB1, PA, NP, HA, NA, M or NS gene sequences truncated by influenza viruses;

3) Performing amplification culture on the recombinant influenza virus obtained in the step 2;

Preferably, the truncated NA gene segment comprises a 3' coding region packaging signal.

Further preferably, the amino acid sequence encoded by the truncated NA gene fragment is shown as SEQ ID NO. 2;

Further preferably, the nucleotide sequence of the truncated NA gene fragment is shown in SEQ ID NO. 1;

further preferably, the exogenous gene is linked to the truncated NA gene segment by a linker sequence (linker).

Preferably, the expressed protein may be in the human body:

Inducing an immune response; or (b)

Generating a biological reporter; or (b)

A tracking molecule for detection; or (b)

Regulating gene function; or (b)

As therapeutic molecules.

The beneficial effects of the invention are as follows: a vector taking the PB2, PB1, PA, NP, HA, NA, M or NS gene sequence of the influenza virus as a framework is constructed, the vector truncates the PB2, PB1, PA, NP, HA, NA, M or NS gene sequence, and then NA gene truncated fragments are connected to the PB2, PB1, PA, NP, HA, NA, M or NS truncated gene sequence, so that a novel vector capable of accommodating exogenous genes larger than the traditional influenza vector is prepared. Based on the characteristics of the vector for accommodating large fragment genes, the vector has wider applicability in the selection of exogenous genes, including fluorescent reporter genes, antigen genes, therapeutic genes and the like. The vector construction method provided by the experiment is mature, simple and convenient, convenient to operate and high in success rate. The constructed novel vector can efficiently express a plurality of exogenous proteins.

Drawings

FIG. 1pM-PR8-NA construction flow chart.

FIG. 2pM-PR8-PB2 construction flow chart.

FIG. 3pM-PR8-PB2 skeleton structure.

FIG. 4 is a block diagram of influenza virus vector pMPR-PB 2-NA.

FIG. 5 is a schematic diagram of a vector construction design.

FIG. 6pMPR A flowchart of the construction of PB 2-NA-EGFP.

FIG. 7pMPR PCR amplification of the 8-PB2-NA-EGFP backbone and fragment.

FIG. 8 PCR amplification characterization of plasmid pMPR-PB 2-NA-EGFP.

FIG. 9 shows green fluorescence from cells when transfected with plasmid pMPR-PB 2-NA-EGFP in 293T cells under a fluorescence microscope.

Detailed Description

In order that the technical contents of the present invention may be more clearly understood, the following embodiments are specifically described with reference to the accompanying drawings. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

Example 1 novel influenza Virus vector

A novel influenza virus vector comprising a truncated PB2 gene sequence that retains only 36 nucleotides of packaging signals at the 3 'and 5' ends respectively; and an NA gene truncated segment is inserted into the open reading frame of the truncated gene sequence, and the NA gene truncated segment comprises a 3' -end coding region packaging signal of the NA gene. The specific construction method is as follows:

construction of influenza 1 vector pMPR-NA

Searching PR8-NA sequence (gene sequence number is AF 389120.1) in NCBI to obtain and synthesize NA gene; synthetic pM vectors (pM is a known universal and highly expressed vector, which can be synthesized by biological companies); performing PCR amplification by taking the synthesized NA gene as a template and na_F and na_R as primers to obtain NA gene segments, and performing gel recovery and purification; PCR (polymerase chain reaction) amplification is carried out by taking a synthetic pM carrier as a template and pM1_F and pM1_R as primers to obtain a pM skeleton fragment, and glue recovery and purification are carried out;

homologous recombination is carried out on the NA gene and the pM vector skeleton by Exnase enzyme, and the transformed Top10 is inoculated with an Amp-resistant LB solid medium to obtain a clone plasmid pMPR-NA (figure 1).

na_F：gttggggccagcgaaagcaggggtttaaa(SEQ ID NO.3)，

na_R：ggttattagtagaaacaaggagttttttgaacaga(SEQ ID NO.4)，

PCR conditions: 98 ℃ for 3min;98 ℃ for 10s;62 ℃,10s;72 ℃,30s; cycles 30;72 ℃ for 5min; 10min at 4 ℃.

pM1_F：tttctactaataacccggcggcccaaaat(SEQ ID NO.5)，

pM1_R：ttcgctggccccaacttcggaggtcgaccag(SEQ ID NO.6)，

PCR conditions: 98 ℃ for 3min;98 ℃ for 10s;62 ℃,20s;72 ℃,30s; cycles 30;72 ℃ for 5min; 10min at 4 ℃.

PCR identification of 2-positive plasmid pMPR-NA

na_F：gttggggccagcgaaagcaggggtttaaa(SEQ ID NO.7)，

na_R：ggttattagtagaaacaaggagttttttgaacaga(SEQ ID NO.8)，

Construction of influenza 3 vector pMPR-PB 2

Searching PR8-PB2 sequence (the gene sequence number is AF 389115.1) in NCBI to obtain and synthesize PB2 gene; synthetic pM vectors (pM is a known universal and highly expressed vector, which can be synthesized by biological companies); using synthetic PB2 gene as a template, using pb2_F and pb2_R as primers for PCR amplification to obtain PB2 gene fragments, and performing gel recovery and purification; PCR (polymerase chain reaction) amplification is carried out by taking a synthetic pM carrier as a template and pM2_F and pM2_R as primers to obtain a pM skeleton fragment, and glue recovery and purification are carried out;

The PB2 gene and the pM vector backbone were subjected to homologous recombination with Exnase enzyme, and the transformed Top10 was inoculated with Amp-resistant LB solid medium to obtain clone plasmid pMPR-PB 2 (FIG. 2).

pb2_F：tggggccagcgaaagcaggtcaattatatt(SEQ ID NO.9)，

pb2_R：gggttattagtagaaacaaggtcgttttta(SEQ ID NO.10)，

PCR conditions: 98 ℃ for 3min;98 ℃ for 10s;62 ℃,15s;72 ℃,30s; cycles 30;72 ℃ for 5min; 10min at 4 ℃.

pM2_F：ttctactaataacccggcggcccaaaatgc(SEQ ID NO.11)，

pM2_R：ctttcgctggccccaacttcggaggtcg(SEQ ID NO.12)，

PCR identification of 4-positive plasmid pMPR-PB 2

pb2_F：tggggccagcgaaagcaggtcaattatatt(SEQ ID NO.13)，

pb2_R：gggttattagtagaaacaaggtcgttttta(SEQ ID NO.14)，

Construction of influenza 5 vector pMPR-PB 2-NA

PCR amplification is carried out by taking pM-PR8-NA plasmid as a template and NA1_F and NA1_R as primers to obtain NA gene truncated fragment (SEQ ID NO. 1), and gel recovery and purification are carried out;

The pM-PR8-PB2 plasmid is used as a template, dM-PB2_F and dM-PB2_R are used as primers, a pM-PR8-PB2 skeleton is obtained through PCR amplification, and the mixture is recovered and purified by glue. The PB2 gene in the pM-PR8-PB2 backbone was truncated, leaving only a packaging signal of 36 nucleotides at the 3 'and 5' ends each (FIG. 3).

Homologous recombination is carried out on the NA gene truncated fragment (SEQ ID NO. 1) and the pM-PR8-PB2 skeleton fragment by Exnase enzyme, and the transformed Top10 is inoculated with an Amp-resistant LB solid medium to obtain a clone plasmid pMPR-PB 2-NA (FIG. 4).

NA1_F：taatatcgatgaatccaaatcagaaaataac(SEQ ID NO.15)，

NA1_R：tgtgccgctgcctgaagttgtgtcctttacccag(SEQ ID NO.16)，

PCR conditions: 98 ℃ for 3min;98 ℃ for 10s;62 ℃,5s;72 ℃,30s; cycles 30;72 ℃ for 5min; 10min at 4 ℃.

dM-PB2_F：tcaggcagcggcacagcgaccaaaagaattcgg(SEQ ID NO.17)，

dM-PB2_R：ggattcatcgatattagatttcttagttctt(SEQ ID NO.18)，

PCR identification of 6-Positive plasmid pMPR-PB 2-NA

PB2-NCR_F：CAGCGAAAGCAGGTCAATTATATTCA(SEQ ID NO.19)，

PB2-NCR_R：AAACAAGGTCGTTTTTAAACTATTCGACA(SEQ ID NO.20)，

PCR conditions: 98 ℃ for 3min;98 ℃ for 10s;56 ℃ for 5s;72 ℃,30s; cycles 30;72 ℃ for 5min; 10min at 4 ℃.

Example 2 construction of novel influenza vector carrying EGFP Gene

1 Construction pMPR-PB 2-NA-EGFP plasmid

The EGFP fragment is obtained by PCR amplification using pCDNA3.1-EGFP (pCDNA3.1 is a known general and high-expression vector, pCDNA3.1-EGFP can be synthesized by biological company) as a template and EGFP1_F and EGFP1_R as primers, and is recovered and purified by gel.

The pMPR-PB 2-NA plasmid is used as a template, dM-PB2-NA_F and dM-PB2-NA_R are used as primers, a pM-PR8-PB2-NA framework is obtained through PCR amplification, and gel recovery and purification are carried out.

Homologous recombination is carried out on the EGFP fragment and the pM-PR8-PB2-NA framework fragment by Exnase enzyme, and the converted Top10 is inoculated into an Amp-resistant LB solid medium to obtain a cloning plasmid pMPR-PB 2-NA-EGFP.

EGFP-1_F：tcaggcagcggcggcttggtgagcaagggcgagg(SEQ ID NO.21)，

EGFP-1_R：gtcgctgttttacttgtacagctcgtcca(SEQ ID NO.22)，

PCR conditions: 98 ℃ for 3min;98 ℃ for 10s;56 ℃ for 5s;72 ℃,20s; cycles 30;72 ℃ for 5min; 10min at 4 ℃.

dM-PB2-NA_F：caagtaaaacagcgaccaaaagaattcgg(SEQ ID NO.23)，

dM-PB2-NA_R：gccgccgctgcctgaagttgtgtcctttaccca(SEQ ID NO.24)，

PCR conditions: 98 ℃ for 3min;98 ℃ for 10s;56 ℃ for 20s;72 ℃,20s; cycles 30;72 ℃ for 5min; 10min at 4 ℃.

PCR identification of 4-positive plasmid pMPR-PB 2-NA-EGFP

PB2-NCR_F：CAGCGAAAGCAGGTCAATTATATTCA(SEQ ID NO.25)，

PB2-NCR_R：AAACAAGGTCGTTTTTAAACTATTCGACA(SEQ ID NO.26)，

EGFP protein expression verification after transfection of 293T cells with 5 plasmid pMPR-PB 2-NA-EGFP

Plasmid pMPR-PB 2-NA-EGFP was transfected into 293T cells in an amount of 2.5ug, and after 48h, the cells were observed to carry green fluorescence under a fluorescence microscope. As shown in FIG. 9, the results indicate that EGFP fluorescent protein expression can be detected after transfection of 293T cells with plasmid pMPR-PB 2-NA-EGFP.

SEQUENCE LISTING

<110> Guangzhou Enbao biomedical technology Co., ltd

<120> A novel influenza virus vector for expressing foreign proteins and method for constructing the same

<130>

<160> 26

<170> PatentIn version 3.5

<210> 1

<211> 201

<212> DNA

<213> influenza virus

<400> 1

atgaatccaa atcagaaaat aacaaccatt ggatcaatct gtctggtagt cggactaatt 60

agcctaatat tgcaaatagg gaatataatc tcaatatgga ttagccattc aattcaaact 120

ggaagtcaaa accatactgg aatatgcaac caaaacatca ttacctataa aaatagcacc 180

tgggtaaagg acacaacttc a 201

<210> 2

<211> 67

<212> PRT

<213> influenza virus

<400> 2

Met Asn Pro Asn Gln Lys Ile Thr Thr Ile Gly Ser Ile Cys Leu Val

1 5 10 15

Val Gly Leu Ile Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile

20 25 30

Trp Ile Ser His Ser Ile Gln Thr Gly Ser Gln Asn His Thr Gly Ile

35 40 45

Cys Asn Gln Asn Ile Ile Thr Tyr Lys Asn Ser Thr Trp Val Lys Asp

50 55 60

Thr Thr Ser

65

<210> 3

<211> 29

<212> DNA

<213> Artificial sequence

<400> 3

gttggggcca gcgaaagcag gggtttaaa 29

<210> 4

<211> 35

<212> DNA

<213> Artificial sequence

<400> 4

ggttattagt agaaacaagg agttttttga acaga 35

<210> 5

<211> 29

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<213> Artificial sequence

<400> 5

tttctactaa taacccggcg gcccaaaat 29

<210> 6

<211> 31

<212> DNA

<213> Artificial sequence

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ttcgctggcc ccaacttcgg aggtcgacca g 31

<210> 7

<211> 29

<212> DNA

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gttggggcca gcgaaagcag gggtttaaa 29

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<213> Artificial sequence

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ggttattagt agaaacaagg agttttttga acaga 35

<210> 9

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<212> DNA

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tggggccagc gaaagcaggt caattatatt 30

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<212> DNA

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gggttattag tagaaacaag gtcgttttta 30

<210> 11

<211> 30

<212> DNA

<213> Artificial sequence

<400> 11

ttctactaat aacccggcgg cccaaaatgc 30

<210> 12

<211> 28

<212> DNA

<213> Artificial sequence

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ctttcgctgg ccccaacttc ggaggtcg 28

<210> 13

<211> 30

<212> DNA

<213> Artificial sequence

<400> 13

tggggccagc gaaagcaggt caattatatt 30

<210> 14

<211> 30

<212> DNA

<213> Artificial sequence

<400> 14

gggttattag tagaaacaag gtcgttttta 30

<210> 15

<211> 31

<212> DNA

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taatatcgat gaatccaaat cagaaaataa c 31

<210> 16

<211> 34

<212> DNA

<213> Artificial sequence

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tgtgccgctg cctgaagttg tgtcctttac ccag 34

<210> 17

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<212> DNA

<213> Artificial sequence

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tcaggcagcg gcacagcgac caaaagaatt cgg 33

<210> 18

<211> 31

<212> DNA

<213> Artificial sequence

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ggattcatcg atattagatt tcttagttct t 31

<210> 19

<211> 26

<212> DNA

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cagcgaaagc aggtcaatta tattca 26

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<211> 29

<212> DNA

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aaacaaggtc gtttttaaac tattcgaca 29

<210> 21

<211> 34

<212> DNA

<213> Artificial sequence

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tcaggcagcg gcggcttggt gagcaagggc gagg 34

<210> 22

<211> 29

<212> DNA

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gtcgctgttt tacttgtaca gctcgtcca 29

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<211> 29

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<213> Artificial sequence

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caagtaaaac agcgaccaaa agaattcgg 29

<210> 24

<211> 33

<212> DNA

<213> Artificial sequence

<400> 24

gccgccgctg cctgaagttg tgtcctttac cca 33

<210> 25

<211> 26

<212> DNA

<213> Artificial sequence

<400> 25

cagcgaaagc aggtcaatta tattca 26

<210> 26

<211> 29

<212> DNA

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<400> 26

aaacaaggtc gtttttaaac tattcgaca 29

Claims

1. An influenza virus vector, characterized in that: the carrier comprises:

a truncated influenza virus PB2 gene sequence, wherein the truncated influenza virus PB2 gene sequence retains packaging signals of 36 nucleotides at the 3 'end and the 5' end respectively, and the other parts are completely truncated; and, a step of, in the first embodiment,

The NA gene truncated segment is inserted into the truncated influenza virus PB2 gene sequence, and the nucleotide sequence of the NA gene truncated segment is shown as SEQ ID NO. 1;

the GenBank accession number of the PB2 gene of the influenza virus is AF389115.1.

2. The influenza virus vector according to claim 1, wherein: the influenza virus vector is derived from a PR8 influenza virus strain.

3. A construction method of an influenza virus vector comprises the following steps:

(1) Truncating the PB2 gene of the influenza virus, reserving the packaging signals of 36 nucleotides at the 3 'end and the 5' end of the gene, and truncating all other parts to obtain a truncated PB2 gene sequence of the influenza virus;

(2) Inserting the NA gene truncated segment into the truncated influenza virus PB2 gene sequence to obtain an influenza virus vector; the nucleotide sequence of the NA gene truncated segment is shown as SEQ ID NO. 1;

4. Use of the influenza virus vector of any one of claims 1-2 for in vitro expression of a foreign protein.

5. The use according to claim 4, characterized in that: the protein expression is carried out in chicken embryo.

6. The use according to claim 4, characterized in that: the protein expression is performed in eukaryotic cells.

7. The use according to claim 4, characterized in that: the expressed protein can be in human:

Inducing an immune response; or (b)

Generating a biological reporter; or (b)

A tracking molecule for detection; or (b)

Regulating gene function; or (b)

As therapeutic molecules.

8. An influenza virus vector carrying a foreign gene comprising a truncated influenza virus PB2 gene sequence that retains packaging signals of 36 nucleotides each at the 3 'and 5' ends, the remainder being truncated entirely; the fusion gene of the exogenous gene and the truncated NA gene segment is inserted into the truncated influenza virus PB2 gene sequence, and the nucleotide sequence of the truncated NA gene segment is shown as SEQ ID NO. 1;

9. The influenza virus vector of claim 8 wherein the exogenous gene is linked to the truncated NA gene segment by a linker sequence.

10. The influenza virus vector according to any one of claims 8 to 9, wherein: the influenza virus vector is derived from a PR8 influenza virus strain.

11. A construction method of an influenza virus vector carrying exogenous genes comprises the following steps:

(2) Connecting an exogenous gene with the NA gene truncated segment, and then inserting the exogenous gene into the truncated influenza virus PB2 gene sequence to obtain an influenza virus vector carrying the exogenous gene; the nucleotide sequence of the NA gene truncated segment is shown as SEQ ID NO. 1;

12. An in vitro expression method of an exogenous protein, comprising the following steps:

(2) Connecting a foreign protein coding gene with the NA gene truncated segment, and then inserting the foreign protein coding gene into the truncated influenza virus PB2 gene sequence;

(3) Performing amplification culture on the recombinant influenza virus obtained in the step (2);

The nucleotide sequence of the NA gene truncated segment is shown as SEQ ID NO. 1;

13. The in vitro expression method according to claim 12, wherein: the protein expression is carried out in chicken embryo.

14. The in vitro expression method according to claim 12, wherein: the protein expression is performed in eukaryotic cells.

15. The in vitro expression method according to claim 12, wherein: the expressed protein can be in human:

Inducing an immune response; or (b)

Generating a biological reporter; or (b)

A tracking molecule for detection; or (b)

Regulating gene function; or (b)

As therapeutic molecules.