CN118978590B

CN118978590B - A method and application of preparing hybridoma monoclonal antibody by immunizing mice with recombinant influenza virus

Info

Publication number: CN118978590B
Application number: CN202411422289.6A
Authority: CN
Inventors: 宋文俊; 卢帅; 李月
Original assignee: Beijing Subenyuanhe Biotechnology Co ltd
Current assignee: Beijing Subenyuanhe Biotechnology Co ltd
Priority date: 2024-10-12
Filing date: 2024-10-12
Publication date: 2025-01-21
Anticipated expiration: 2044-10-12
Also published as: CN118978590A

Abstract

The application provides a method for preparing hybridoma monoclonal antibodies by utilizing a recombinant influenza virus immunized mouse and application thereof, wherein the recombinant influenza virus PA-antigen fusion gene used in the method sequentially comprises a PA gene, a codon conversion signal peptide sequence, a 2A sequence, an IL-2 signal peptide sequence, an antigen gene sequence and a CD 80C-terminal sequence from a 5' segment. The application uses recombinant influenza virus to immunize mice by means of nasal drip infection, the influenza virus efficiently presents target antigens in the upper respiratory tract of the mice in the infection immunization process, and simultaneously stimulates the immune system of the mice, so that plasma cells of the secreted antibody are mature rapidly, the antibody titer can be detected within about 10 days, the time for preparing monoclonal antibodies can be shortened greatly, and meanwhile, the monoclonal antibodies obtained by the immunization method disclosed by the application have wider binding regions with antigens and higher affinity.

Description

Method for preparing hybridoma monoclonal antibodies by immunizing mice with recombinant influenza virus and application

Technical Field

The application belongs to the technical field of biology, and particularly provides a method for preparing hybridoma monoclonal antibodies by immunizing mice with recombinant influenza viruses and application thereof.

Background

Influenza viruses are widely prevalent in birds, mammals and humans. The host is widely distributed and quick in variation, and a large disease burden is often brought to human and animal breeding such as poultry and pigs. From the taxonomic perspective, influenza viruses belong to enveloped RNA viruses whose RNA is the negative strand. The inner core of the influenza virus particle is ribosomal nucleoprotein coated with single-stranded RNA, and the inner outer core is coated with matrix protein M1. The influenza virus genome consists of eight segmented linear gene segments (influenza C virus HAs only seven gene segments), and the eight gene segments encode ten proteins in total, respectively, RNA-dependent RNA polymerase (PB 2, PB1, PA), nucleocapsid forming nucleoprotein NP, matrix membrane protein (M1, M2), two surface membrane proteins (hemagglutinin HA and neuraminidase NA), non-structural protein NS1 and nuclear transport protein NEP. Transcription and replication of viral genes occurs in the nucleus of the host cell, and viral assembly and budding occurs in the host cell membrane. Gene recombination occurs when two different influenza viruses infect one cell at a time. Eight gene segments of influenza virus encode a total of nine structural proteins (PB 2, PB1, PA, HA, NP, NA, M1, M2, NEP), a non-structural protein NS1.

Influenza viruses produce many natural gene deletions or insertions during genetic evolution, but are most commonly used primarily for deletion. These deletions or insertions have an important role in viral pathogenicity, expansion of host range, and adaptation to new hosts. Gene deletions are mainly concentrated in the NA gene and the NS1 gene, especially in the case of animal-derived influenza viruses, which frequently occur during infection and adaptation to new hosts, and the specific location and number of deletions may vary from strain to strain. Insertion occurs mainly when a low pathogenic influenza virus changes to a high pathogenic influenza virus, inserting multiple basic amino acids, such as KRRK, etc., before the cleavage site/GLF motif of the HA gene. The insertion of multiple basic amino acids in such HA proteins occurs only in H5 and H7 subtype influenza viruses. These deletions and insertions of influenza viruses indicate that the genome of the virus can tolerate variations at these positions, and attempts have therefore been made to engineer influenza viruses, such as NS1 gene deleted viruses, by the location of the deletion.

Meanwhile, research shows that the NS1 deletion region can be added with exogenous genes, such as human interleukin 15 (IL 15) genes, and the recombinant viruses can express the target protein IL15, so that the influenza viruses are used as an expression system of the exogenous genes and play a certain role in-vivo immunoregulation. The deletion of NA is utilized to add GFP protein gene on NA gene, so that the recombinant influenza virus can express GFP in the process of replication, and the recombinant virus has biological characteristics similar to those of wild virus, thereby achieving the effect of visualization when the virus is infected in vivo. The exogenous gene carried by the modified influenza virus is used as a more sensitive report virus, so that antiviral drugs or pathogenesis researches are screened.

The hybridoma monoclonal antibody technology is to immunize a mouse by using a proper antigen, and then take the immunized mouse (PEG) and spleen cells (antibody forming cells) of the mouse or take the spleen cells immunized in vitro, and fuse the immunized mouse and the spleen cells by means of a polyethylene glycol myeloma cell line to form the hybridoma secreting the monoclonal antibody. The method is currently the mainstream technology of monoclonal antibody preparation screening, wherein the quality and success rate of monoclonal antibody preparation are important factors when antigen is selected and applied.

Disclosure of Invention

The invention utilizes the 5' and 3' end virus particle packaging signal sequences of the A-type influenza virus polymerase gene PA, clones the gene fragment (influenza virus H7N9 subtype hemagglutinin HA, herpes zoster virus surface glycoprotein gE, norovirus nucleocapsid protein VP 1) of the encoding target protein to the 3' end of the influenza virus PA gene by a genetic engineering method, and uses reverse genetic technology to rescue and package recombinant influenza virus with infection and replication activity as an immunogen, wherein the recombinant virus is named FluVec-H7, fluVec-gE, fluVec-NoV/VP1 respectively. The recombinant virus is used for immunizing a mouse through a nasal drip infection way, spleen cells of the mouse are collected 2 weeks after the mouse is immunized, fused with SP20, and monoclonal hybridoma cell strains secreting target proteins are screened. Compared with the traditional method for screening hybridomas by immunizing mice with protein antigens, the method for immunoscreening the mouse monoclonal antibodies has the advantages of large quantity of screened monoclonal antibodies, high quality and greatly shortened time required by the whole process. Has very high application prospect.

In one aspect, the present application provides a method for preparing hybridoma monoclonal antibodies by immunizing mice with recombinant influenza virus, wherein the recombinant influenza virus PA-antigen fusion gene used in the method comprises PA gene, codon conversion signal peptide sequence, 2A sequence, IL-2 signal peptide sequence, antigen gene sequence and CD 80C-terminal sequence in sequence from 5' segment.

Further, the antigen is influenza virus H7N9 subtype hemagglutinin HA, herpes zoster virus surface glycoprotein gE or norovirus nucleocapsid protein VP1.

Further, the sequence of the PA gene is shown as SEQ ID NO. 13.

Further, the sequence of the codon conversion signal peptide sequence is shown in SEQ ID NO. 14.

Further, the sequence of the 2A sequence is shown as SEQ ID NO. 15.

Further, the sequence of the IL-2 signal peptide sequence is shown as SEQ ID NO. 16.

Further, the sequence of the C-terminal sequence of CD80 is shown in SEQ ID NO. 17.

Further, the antigen gene sequence is selected from any one of SEQ ID NO. 9-12.

Further, the recombinant influenza virus is obtained by rescue preparation through a reverse genetics system.

Further, the method includes the step of immunizing a mouse with a recombinant influenza virus.

Further, mice were immunized by nasal drip at a dose of 1X 10 ⁴-10⁶ pfu.

The methods of the application are useful for antibody production.

In the traditional mouse hybridoma monoclonal antibody technology, protein antigen and adjuvant are matched in the immune process, and then the immune process is carried out through intramuscular injection or subcutaneous injection, so that the immune process usually needs to be enhanced for 6 weeks in order to achieve higher immune titer against the antigen, and the reaction titer of the antibody and the target antigen is measured by collecting mouse serum before spleen fusion, and the fusion can be carried out to screen more specific monoclonal antibodies usually up to 1:100000 times. The invention uses recombinant influenza virus to immunize mice by means of nasal drip infection, and the influenza virus efficiently presents target antigens in the upper respiratory tract of the mice in the infection immune process and stimulates the immune system of the mice at the same time, so that plasma cells of the secreted antibody are rapidly mature, the antibody titer can be detected in about 10 days, and the time for preparing monoclonal antibodies can be greatly shortened. In addition, through in-depth analysis of monoclonal antibodies screened by different immunization methods of the same antigen, the monoclonal antibodies obtained by using the immunization method disclosed by the invention have wider binding regions and higher affinity with the antigen, and have wide space in scientific research, clinical and industrial application.

Drawings

FIG. 1 is a diagram showing the pattern of genetic modification of the PA gene (exemplified by the H7 subtype influenza virus HA).

Detailed Description

EXAMPLE 1 preparation of the vector

The pHW2000 empty vector is described in ,A DNA transfection system for generation of influenza A virus from eight plasmids. PNAS (97) 11;6108-6113; publicly available from Zoonogen innovation development center. The pHW2000 vector was modified to change the sequence carrying the BsmBI recognition site into the influenza A general sequence uni12 (5- 'AGCGAAAGCAGG-3') and uni13 (5'-AGTAGAAACAAGG-3'). The Uni12 and Uni13 sequences are joined to form a StuI recognition sequence (AGG/CCT) therebetween, and the vector is designated pHKU _FluA vector, which is described in the following literature ,Multiple basic amino acids in the cleavage site of H7N9 hemagglutinin contribute to high virulence in mice J Thorac Dis 2021;13(8):4650-4660;. and is publicly available from Zoonogen Innovative research and development center.

The pHKU _FluA vector, rCutSmart cleavage buffer, stuI 10U, total volume 50. Mu.L, 37℃were digested overnight. And (3) recovering fragments with the size of about 3000bp from the electrophoresis gel to obtain the linearized pHW2000 vector. The linearized and the PCR products derived from the A/Puerto Rico/8/1934 (H1N 1, PR 8) HA gene are used for in-fusion ligation to obtain a recombinant vector pHW2000-PR8_HA (through sequencing, the vector is a recombinant vector obtained by inserting the H1 gene shown in the sequence 1 into the pHW2000 vector);

sequence 1:

agcaaaagcaggggaaaataaaaacaaccaaaatgaaggcaaacctactggtcctgttatgtgcacttgcagctgcagatgcagacacaatatgtataggctaccatgcgaacaattcaaccgacactgttgacacagtactcgagaagaatgtgacagtgacacactctgttaacctgctcgaagacagccacaacggaaaactatgtagattaaaaggaatagccccactacaattggggaaatgtaacatcgccggatggctcttgggaaacccagaatgcgacccactgcttccagtgagatcatggtcctacattgtagaaacaccaaactctgagaatggaatatgttatccaggagatttcatcgactatgaggagctgagggagcaattgagctcagtgtcatcattcgaaagattcgaaatatttcccaaagaaagctcatggcccaaccacaacacaaacggagtaacggcagcatgctcccatgaggggaaaagcagtttttacagaaatttgctatggctgacggagaaggagggctcatacccaaagctgaaaaattcttatgtgaacaaaaaagggaaagaagtccttgtactgtggggtattcatcacccgcctaacagtaaggaacaacagaatctctatcagaatgaaaatgcttatgtctctgtagtgacttcaaattataacaggagatttaccccggaaatagcagaaagacccaaagtaagagatcaagctgggaggatgaactattactggaccttgctaaaacccggagacacaataatatttgaggcaaatggaaatctaatagcaccaatgtatgctttcgcactgagtagaggctttgggtccggcatcatcacctcaaacgcatcaatgcatgagtgtaacacgaagtgtcaaacacccctgggagctataaacagcagtctcccttaccagaatatacacccagtcacaataggagagtgcccaaaatacgtcaggagtgccaaattgaggatggttacaggactaaggaacattccgtccattcaatccagaggtctatttggagccattgccggttttattgaagggggatggactggaatgatagatggatggtatggttatcatcatcagaatgaacagggatcaggctatgcagcggatcaaaaaagcacacaaaatgccattaacgggattacaaacaaggtgaacactgttatcgagaaaatgaacattcaattcacagctgtgggtaaagaattcaacaaattagaaaaaaggatggaaaatttaaataaaaaagttgatgatggatttctggacatttggacatataatgcagaattgttagttctactggaaaatgaaaggactctggatttccatgactcaaatgtgaagaatctgtatgagaaagtaaaaagccaattaaagaataatgccaaagaaatcggaaatggatgttttgagttctaccacaagtgtgacaatgaatgcatggaaagtgtaagaaatgggacttatgattatcccaaatattcagaagagtcaaagttgaacagggaaaaggtagatggagtgaaattggaatcaatggggatctatcagattctggcgatctactcaactgtcgccagttcactggtgcttttggtctccctgggggcaatcagtttctggatgtgttctaatggatctttgcagtgcagaatatgcatctgagattagaatttcagagatatgaggaaaaacacccttgtttctact

After linearizing pHW2000 vector with StuI, carrying out in-fusion ligation with NA gene PCR product derived from A/PR/8/1934 (H1N 1) to obtain recombinant vector pHW2000-PR8_NA (the vector is obtained by inserting N1 gene shown in sequence 2 into PHW2000 vector after sequencing);

Sequence 2:

agcaaaagcaggagtttaaaatgaatccaaatcagaaaataataaccattggatcaatctgtctggtagtcggactaattagcctaatattgcaaatagggaatataatctcaatatggattagccattcaattcaaactggaagtcaaaaccatactggaatatgcaaccaaaacatcattacctataaaaatagcacctgggtaaaggacacaacttcagtgatattaaccggcaattcatctctttgtcccatccgtgggtgggctatatacagcaaagacaatagcataagaattggttccaaaggagacgtttttgtcataagagagccctttatttcatgttctcacttggaatgcaggaccttttttctgacccaaggtgccttactgaatgacaagcattcaagtgggactgttaaggacagaagcccttatagggccttaatgagctgccctgtcggtgaagctccgtccccgtacaattcaagatttgaatcggttgcttggtcagcaagtgcatgtcatgatggcatgggctggctaacaatcggaatttcaggtccagataatggagcagtggctgtattaaaatacaacggcataataactgaaaccataaaaagttggaggaagaaaatattgaggacacaagagtctgaatgtgcctgtgtaaatggttcatgttttactataatgactgatggcccgagtgatgggctggcctcgtacaaaattttcaagatcgaaaaggggaaggttactaaatcaatagagttgaatgcacctaattctcactatgaggaatgttcctgttaccctgataccggcaaagtgatgtgtgtgtgcagagacaattggcatggttcgaaccggccatgggtgtctttcgatcaaaacctggattatcaaataggatacatctgcagtggggttttcggtgacaacccgcgtcccgaagatggaacaggcagctgtggtccagtgtatgttgatggagcaaacggagtaaagggattttcatataggtatggtaatggtgtttggataggaaggaccaaaggtcacagttccagacatgggtttgagatgatttgggatcctaatggatggacagagactgatagtaagttctctgtgaggcaagatgttgtggcaatgactgattggtcagggtatagcggaagtttcgttcaacatcctgagctgacagggctagactgtatgaggccgtgcttctgggttgaattaatcaggggacgacctaaagaaaaaacaatctggactagtgcgagcagcatttctttttgtggcgtgaatagtgatactgtagattggtcttggccagacggtgctgagttgccattcagcattgacaagtagtctgttcaaaaaactccttgtttctact

after linearizing pHW2000 vector with StuI, carrying out in-fusion ligation with PB2 gene PCR product derived from A/PR/8/1934 (H1N 1) to obtain recombinant vector pHW2000-PR 8-PB 2 (the vector is a recombinant vector obtained by inserting gene PB2 shown in sequence 3 into PHW2000 vector after sequencing);

Sequence 3:

agcgaaagcaggtcaattatattcaatatggaaagaataaaagaactacgaaatctaatgtcgcagtctcgcacccgcgagatactcacaaaaaccaccgtggaccatatggccataatcaagaagtacacatcaggaagacaggagaagaacccagcacttaggatgaaatggatgatggcaatgaaatatccaattacagcagacaagaggataacggaaatgattcctgagagaaatgagcaaggacaaactttatggagtaaaatgaatgatgccggatcagaccgagtgatggtatcacctctggctgtgacatggtggaataggaatggaccaataacaaatacagttcattatccaaaaatctacaaaacttattttgaaagagtcgaaaggctaaagcatggaacctttggccctgtccattttagaaaccaagtcaaaatacgtcggagagttgacataaatcctggtcatgcagatctcagtgccaaggaggcacaggatgtaatcatggaagttgttttccctaacgaagtgggagccaggatactaacatcggaatcgcaactaacgataaccaaagagaagaaagaagaactccaggattgcaaaatttctcctttgatggttgcatacatgttggagagagaactggtccgcaaaacgagattcctcccagtggctggtggaacaagcagtgtgtacattgaagtgttgcatttgactcaaggaacatgctgggaacagatgtatactccaggaggggaagtgaggaatgatgatgttgatcaaagcttgattattgctgctaggaacatagtgagaagagctgcagtatcagcagatccactagcatctttattggagatgtgccacagcacacagattggtggaattaggatggtagacatccttaggcagaacccaacagaagagcaagccgtggatatatgcaaggctgcaatgggactgagaattagctcatccttcagttttggtggattcacatttaagagaacaagcggatcatcagtcaagagagaggaagaggtgcttacgggcaatcttcaaacattgaagataagagtgcatgagggatatgaagagttcacaatggttgggagaagagcaacagccatactcagaaaagcaaccaggagattgattcagctgatagtgagtgggagagacgaacagtcgattgccgaagcaataattgtggccatggtattttcacaagaggattgtatgataaaagcagtcagaggtgatctgaatttcgtcaatagggcgaatcaacgattgaatcctatgcatcaacttttaagacattttcagaaggatgcgaaagtgctttttcaaaattggggagttgaacctatcgacaatgtgatgggaatgattgggatattgcccgacatgactccaagcatcgagatgtcaatgagaggagtgagaatcagcaaaatgggtgtagatgagtactccagcacggagagggtagtggtgagcattgaccgttttttgagaatccgggaccaacgaggaaatgtactactgtctcccgaggaggtcagtgaaacacagggaacagagaaactgacaataacttactcatcgtcaatgatgtgggagattaatggtcctgaatcagtgttggtcaatacctatcaatggatcatcagaaactgggaaactgttaaaattcagtggtcccagaaccctacaatgctatacaataaaatggaatttgaaccatttcagtctttagtacctaaggccattagaggccaatacagtgggtttgtaagaactctgttccaacaaatgagggatgtgcttgggacatttgataccgcacagataataaaacttcttcccttcgcagccgctccaccaaagcaaagtagaatgcagttctcctcatttactgtgaatgtgaggggatcaggaatgagaatacttgtaaggggcaattctcctgtattcaactataacaaggccacgaagagactcacagttctcggaaaggatgctggcactttaactgaagacccagatgaaggcacagctggagtggagtccgctgttctgaggggattcctcattctgggcaaagaagacaagagatatgggccagcactaagcatcaatgaactgagcaaccttgcgaaaggagagaaggctaatgtgctaattgggcaaggagacgtggtgttggtaatgaaacggaaacgggactctagcatacttactgacagccagacagcgaccaaaagaattcggatggccatcaattagtgtcgaatagtttaaaaacgaccttgtttctact

after linearizing pHW2000 vector with StuI, and carrying out in-fusion ligation with PB1 gene PCR product derived from A/PR/8/1934 (H1N 1), recombinant vector pHW2000-PR 8-PB 1 was obtained (the vector was a recombinant vector obtained by inserting gene PB1 shown in sequence 4 into pHW2000 vector via sequencing);

Sequence 4:

agcgaaagcaggcaaaccatttgaatggatgtcaatccgaccttacttttcttaaaagtgccagcacaaaatgctataagcacaactttcccttatactggagaccctccttacagccatgggacaggaacaggatacaccatggatactgtcaacaggacacatcagtactcagaaaagggaagatggacaacaaacaccgaaactggagcaccgcaactcaacccgattgatgggccactgccagaagacaatgaaccaagtggttatgcccaaacagattgtgtattggaggcgatggctttccttgaggaatcccatcctggtatttttgaaaactcgtgtattgaaacgatggaggttgttcagcaaacacgagtagacaagctgacacaaggccgacagacctatgactggactctaaatagaaaccaacctgctgcaacagcattggccaacacaatagaagtgttcagatcaaatggcctcacggccaatgagtctggaaggctcatagacttccttaaggatgtaatggagtcaatgaacaaagaagaaatggggatcacaactcattttcagagaaagagacgggtgagagacaatatgactaagaaaatgataacacagagaacaatgggtaaaaagaagcagagattgaacaaaaggagttatctaattagagcattgaccctgaacacaatgaccaaagatgctgagagagggaagctaaaacggagagcaattgcaaccccagggatgcaaataagggggtttgtatactttgttgagacactggcaaggagtatatgtgagaaacttgaacaatcagggttgccagttggaggcaatgagaagaaagcaaagttggcaaatgttgtaaggaagatgatgaccaattctcaggacaccgaactttctttcaccatcactggagataacaccaaatggaacgaaaatcagaatcctcggatgtttttggccatgatcacatatatgaccagaaatcagcccgaatggttcagaaatgttctaagtattgctccaataatgttctcaaacaaaatggcgagactgggaaaagggtatatgtttgagagcaagagtatgaaacttagaactcaaatacctgcagaaatgctagcaagcatcgatttgaaatatttcaatgattcaacaagaaagaagattgaaaaaatccgaccgctcttaatagaggggactgcatcattgagccctggaatgatgatgggcatgttcaatatgttaagcactgtattaggcgtctccatcctgaatcttggacaaaagagatacaccaagactacttactggtgggatggtcttcaatcctctgacgattttgctctgattgtgaatgcacccaatcatgaagggattcaagccggagtcgacaggttttatcgaacctgtaagctacttggaatcaatatgagcaagaaaaagtcttacataaacagaacaggtacatttgaattcacaagttttttctatcgttatgggtttgttgccaatttcagcatggagcttcccagttttggggtgtctgggatcaacgagtcagcggacatgagtattggagttactgtcatcaaaaacaatatgataaacaatgatcttggtccagcaacagctcaaatggcccttcagttgttcatcaaagattacaggtacacgtaccgatgccatataggtgacacacaaatacaaacccgaagatcatttgaaataaagaaactgtgggagcaaacccgttccaaagctggactgctggtctccgacggaggcccaaatttatacaacattagaaatctccacattcctgaagtctgcctaaaatgggaattgatggatgaggattaccaggggcgtttatgcaacccactgaacccatttgtcagccataaagaaattgaatcaatgaacaatgcagtgatgatgccagcacatggtccagccaaaaacatggagtatgatgctgttgcaacaacacactcctggatccccaaaagaaatcgatccatcttgaatacaagtcaaagaggagtacttgaggatgaacaaatgtaccaaaggtgctgcaatttatttgaaaaattcttccccagcagttcatacagaagaccagtcgggatatccagtatggtggaggctatggtttccagagcccgaattgatgcacggattgatttcgaatctggaaggataaagaaagaagagttcactgagatcatgaagatctgttccaccattgaagagctcagacggcaaaaatagtgaatttagcttgtccttcatgaaaaaatgccttgtttctact

After linearizing pHW2000 vector with StuI, carrying out in-fusion ligation with PA gene PCR product derived from A/PR/8/1934 (H1N 1) to obtain recombinant vector pHW2000-PR8_PA (the vector is a recombinant vector obtained by inserting gene PA shown in sequence 5 into pHW2000 vector after sequencing);

Sequence 5

agcgaaagcaggtactgatccaaaatggaagattttgtgcgacaatgcttcaatccgatgattgtcgagcttgcggaaaaaacaatgaaagagtatggggaggacctgaaaatcgaaacaaacaaatttgcagcaatatgcactcacttggaagtatgcttcatgtattcagattttcacttcatcaatgagcaaggcgagtcaataatcgtagaacttggtgatccaaatgcacttttgaagcacagatttgaaataatcgagggaagagatcgcacaatggcctggacagtagtaaacagtatttgcaacactacaggggctgagaaaccaaagtttctaccagatttgtatgattacaaggagaatagattcatcgaaattggagtaacaaggagagaagttcacatatactatctggaaaaggccaataaaattaaatctgagaaaacacacatccacattttctcgttcactggggaagaaatggccacaaaggcagactacactctcgatgaagaaagcagggctaggatcaaaaccagactattcaccataagacaagaaatggccagcagaggcctctgggattcctttcgtcagtccgagagaggagaagagacaattgaagaaaggtttgaaatcacaggaacaatgcgtaagcttgccgaccaaagtctcccgccgaacttctccagccttgaaaattttagagcctatgtggatggattcgaaccgaacggctacattgagggcaagctgtctcaaatgtccaaagaagtaaatgctagaattgaaccttttttgaaaacaacaccacgaccacttagacttccgaatgggcctccctgttctcagcggtccaaattcctgctgatggatgccttaaaattaagcattgaggacccaagtcatgaaggagagggaataccgctatatgatgcaatcaaatgcatgagaacattctttggatggaaggaacccaatgttgttaaaccacacgaaaagggaataaatccaaattatcttctgtcatggaagcaagtactggcagaactgcaggacattgagaatgaggagaaaattccaaagactaaaaatatgaagaaaacaagtcagctaaagtgggcacttggtgagaacatggcaccagaaaaggtagactttgacgactgtaaagatgtaggtgatttgaagcaatatgatagtgatgaaccagaattgaggtcgcttgcaagttggattcagaatgagtttaacaaggcatgcgaactgacagattcaagctggatagagctcgatgagattggagaagatgtggctccaattgaacacattgcaagcatgagaaggaattatttcacatcagaggtgtctcactgcagagccacagaatacataatgaagggagtgtacatcaatactgccttgcttaatgcatcttgtgcagcaatggatgatttccaattaattccaatgataagcaagtgtagaactaaggagggaaggcgaaagaccaacttgtatggtttcatcataaaaggaagatcccacttaaggaatgacaccgacgtggtaaactttgtgagcatggagttttctctcactgacccaagacttgaaccacataaatgggagaagtactgtgttcttgagataggagatatgcttataagaagtgccataggccaggtttcaaggcccatgttcttgtatgtgagaacaaatggaacctcaaaaattaaaatgaaatggggaatggagatgaggcgttgcctcctccagtcacttcaacaaattgagagtatgattgaagctgagtcctctgtcaaagagaaagacatgaccaaagagttctttgagaacaaatcagaaacatggcccattggagagtcccccaaaggagtggaggaaagttccattgggaaggtctgcaggactttattagcaaagtcggtattcaacagcttgtatgcatctccacaactagaaggattttcagctgaatcaagaaaactgcttcttatcgttcaggctcttagggacaacctggaacctgggacctttgatcttggggggctatatgaagcaattgaggagtgcctgattaatgatccctgggttttgcttaatgcttcttggttcaactccttccttacacatgcattgagttagttgtggcagtgctactatttgctatccatactgtccaaaaaagtaccttgtttctact

After linearizing pHW2000 vector with StuI, carrying out in-fusion ligation with NP gene PCR product derived from A/PR/8/1934 (H1N 1) to obtain recombinant vector pHW2000-PR 8-NP (the vector is a recombinant vector obtained by inserting gene NP shown in sequence 6 into pHW2000 vector after sequencing);

sequence 6

agcaaaagcagggtagataatcactcactgagtgacatcaaaatcatggcgtctcaaggcaccaaacgatcttacgaacagatggagactgatggagaacgccagaatgccactgaaatcagagcatccgtcggaaaaatgattggtggaattggacgattctacatccaaatgtgcaccgaactcaaactcagtgattatgagggacggttgatccaaaacagcttaacaatagagagaatggtgctctctgcttttgacgaaaggagaaataaataccttgaagaacatcccagtgcggggaaagatcctaagaaaactggaggacctatatacaggagagtaaacggaaagtggatgagagaactcatcctttatgacaaagaagaaataaggcgaatctggcgccaagctaataatggtgacgatgcaacggctggtctgactcacatgatgatctggcattccaatttgaatgatgcaacttatcagaggacaagagctcttgttcgcaccggaatggatcccaggatgtgctctctgatgcaaggttcaactctccctaggaggtctggagccgcaggtgctgcagtcaaaggagttggaacaatggtgatggaattggtcagaatgatcaaacgtgggatcaatgatcggaacttctggaggggtgagaatggacgaaaaacaagaattgcttatgaaagaatgtgcaacattctcaaagggaaatttcaaactgctgcacaaaaagcaatgatggatcaagtgagagagagccggaacccagggaatgctgagttcgaagatctcacttttctagcacggtctgcactcatattgagagggtcggttgctcacaagtcctgcctgcctgcctgtgtgtatggacctgccgtagccagtgggtacgactttgaaagggagggatactctctagtcggaatagaccctttcagactgcttcaaaacagccaagtgtacagcctaatcagaccaaatgagaatccagcacacaagagtcaactggtgtggatggcatgccattctgccgcatttgaagatctaagagtattaagcttcatcaaagggacgaaggtgctcccaagagggaagctttccactagaggagttcaaattgcttccaatgaaaatatggagactatggaatcaagtacacttgaactgagaagcaggtactgggccataaggaccagaagtggaggaaacaccaatcaacagagggcatctgcgggccaaatcagcatacaacctacgttctcagtacagagaaatctcccttttgacagaacaaccattatggcagcattcaatgggaatacagaggggagaacatctgacatgaggaccgaaatcataaggatgatggaaagtgcaagaccagaagatgtgtctttccaggggcggggagtcttcgagctctcggacgaaaaggcagcgagcccgatcgtgccttcctttgacatgagtaatgaaggatcttatttcttcggagacaatgcagaggagtacgacaattaaagaaaaatacccttgtttctact

After linearizing pHW2000 vector with StuI, and performing in-fusion ligation with M gene PCR product derived from A/PR/8/1934 (H1N 1), recombinant vector pHW2000-PR8_M (the vector is a recombinant vector obtained by inserting gene NP shown in sequence 7 into pHW2000 vector via sequencing);

Sequence 7

agcaaaagcaggtagatattgaaagatgagtcttctaaccgaggtcgaaacgtacgtactctctatcatcccgtcaggccccctcaaagccgagatcgcacagagacttgaagatgtctttgcagggaagaacaccgatcttgaggttctcatggaatggctaaagacaagaccaatcctgtcacctctgactaaggggattttaggatttgtgttcacgctcaccgtgcccagtgagcgaggactgcagcgtagacgctttgtccaaaatgcccttaatgggaacggggatccaaataacatggacaaagcagttaaactgtataggaagctcaagagggagataacattccatggggccaaagaaatctcactcagttattctgctggtgcacttgccagttgtatgggcctcatatacaacaggatgggggctgtgaccactgaagtggcatttggcctggtatgtgcaacctgtgaacagattgctgactcccagcatcggtctcataggcaaatggtgacaacaaccaatccactaatcagacatgagaacagaatggttttagccagcactacagctaaggctatggagcaaatggctggatcgagtgagcaagcagcagaggccatggaggttgctagtcaggctagacaaatggtgcaagcgatgagaaccattgggactcatcctagctccagtgctggtctgaaaaatgatcttcttgaaaatttgcaggcctatcagaaacgaatgggggtgcagatgcaacggttcaagtgatcctctcactattgccgcaaatatcattgggatcttgcacttgacattgtggattcttgatcgtctttttttcaaatgcatttaccgtcgctttaaatacggactgaaaggagggccttctacggaaggagtgccaaagtctatgagggaagaatatcgaaaggaacagcagagtgctgtggatgctgacgatggtcattttgtcagcatagagctggagtaaaaaactaccttgtttctact

After linearizing pHW2000 vector with StuI, carrying out in-fusion ligation with NS gene PCR product derived from A/PR/8/1934 (H1N 1) to obtain recombinant vector pHW2000-PR 8-NS (the vector is obtained by inserting gene NP shown in sequence 8 into pHW2000 vector after sequencing);

sequence 8

Agcaaaagcagggtgacaaaaacataatggatccaaacactgtgtcaagctttcaggtagattgctttctttggcatgtccgcaaacgagttgcagaccaagaactaggcgatgccccattccttgatcggcttcgccgagatcagaaatccctaagaggaaggggcagtactctcggtctggacatcaagacagccacacgtgctggaaagcagatagtggagcggattctgaaagaagaatccgatgaggcacttaaaatgaccatggcctctgtacctgcgtcgcgttacctaactgacatgactcttgaggaaatgtcaagggactggtccatgctcatacccaagcagaaagtggcaggccctctttgtatcagaatggaccaggcgatcatggataagaacatcatactgaaagcgaacttcagtgtgatttttgaccggctggagactctaatattgctaagggctttcaccgaagagggagcaattgttggcgaaatttcaccattgccttctcttccaggacatactgctgaggatgtcaaaaatgcagttggagtcctcatcggaggacttgaatggaatgataacacagttcgagtctctgaaactctacagagattcgcttggagaagcagtaatgagaatgggagacctccactcactccaaaacagaaacgagaaatggcgggaacaattaggtcagaagtttgaagaaataagatggttgattgaagaagtgagacacaaactgaagataacagagaatagttttgagcaaataacatttatgcaagccttacatctattgcttgaagtggagcaagagataagaactttctcgtttcagcttatttagtactaaaaaacacccttgtttctact

Specifically:

StuI linearized pHW2000 vector and each gene PCR product were subjected to 1% agarose electrophoresis buffer, respectively, and the target fragment and vector were excised and recovered into 1.5mL EP tubes weighed and purified by QIAEXII Gel Extraction Kit by adding 3 volumes of Buffer QXl (100 mg plus 300. Mu.L QX 1). 30 μ L Buffer QIAEX II was added and then the mixture was subjected to a 50℃water bath for 10 minutes, during which time it was vortexed every two minutes. Centrifugation at 13,000 rpm for 30 seconds, the supernatant was discarded, washed once with 500. Mu.L of QIAEX I, twice with 500. Mu.L of Buffer PE, and air dried for 15 minutes until the pellet became white. The mixture was vortexed with 20. Mu.L TE for 30 seconds to incubate for 5 minutes, centrifuged at 13,000 rpm for 30 seconds, and the supernatant was transferred to a 1.5mL EP tube to measure the concentration.

According to the principle of the in-fusion technology, the linearized pHW2000 vector and the HA isogenic fragment have 15 base coincidence in-fusion technology, so that the linearized pHW2000 vector and the HA isogenic fragment can be directionally spliced according to the coincidence part. The procedure of the in-fusion kit of Clonetech was as follows, 5 Xin-fusion buffer 2. Mu.L, enzyme 1. Mu.L, linearized pHW2000 vector, target gene fragment, water, and total volume 10. Mu.L. The amount of carrier to each fragment was such that the molar ratio was 2:1. The in-fusion mixture was treated at 37℃for 15 minutes, at 50℃for 15 minutes, 40. Mu.L of TE buffer was added to make up 50. Mu.L, and 2.5. Mu.L was transformed into competent cells. The transformed competent cells were placed in ice for 30 minutes and after heating at 42℃for 45 seconds, placed in ice for another 1 minute. LB medium was added and cultured at 37℃for 60 minutes with shaking. 100. Mu.L of LB agar plate medium coated with ampicillin was cultured at 37℃for 16 hours to form a single colony, the single colony was picked up to the ampicillin-containing LB medium and cultured for 8 hours, the bacterial solution cultured for 8 hours was subjected to QIAPREP SPIN MINIPREP KIT-hour extraction with a QIAPREP SPIN MINIPREP KIT-hour plasmid, the bacterial solution was poured into a labeled 1.5-mL centrifuge tube at 13,000 rpm for 3 minutes, the supernatant was discarded by centrifugation, 250. Mu.L of Buffer P1 was added, uniformly mixed, 250. Mu.L of Buffer P2 was added, the solution was rapidly and gently reversed 4-6 times, 350. Mu.L of Buffer N3 was added, 4-6 times of the mixing was reversed, white floccules were precipitated, 13,000 rpm,4℃for 10 minutes, QIAPREP SPIN column was placed in a 1.5-mL EP tube (waste liquid was collected), the supernatant was then transferred into a QIAPREP SPIN column at 200 rpm for 4℃for 1 minute, the waste liquid was discarded, 750. Mu.L of Buffer PE was added, 13,000 rpm,4℃was removed, 1 minute, 1.000℃of the liquid was removed, 1,000 column was added, and the supernatant was centrifuged at 1.5℃for 1 minute, and the supernatant was placed in a centrifuge tube for 1.5 minutes.

Sequencing and identification are correct, and recombinant vector pHW2000-PR8_HA,pHW2000-PR8_NA,pHW2000-PR8_PB2,pHW2000-PR8_PB1,pHW2000-PR8_PA,pHW2000-PR8_NP,pHW2000-PR8_M,pHW2000-PR8_NS and pHW2000-PR 8-PA-H7, pHW2000-PR 8-PA-gE, pHW2000-PR 8-PA-VP 1 are obtained.

The gene sequences adopted by the invention for preparing recombinant influenza virus and expressing corresponding antigens are respectively from Flu/A/H7 HA: genBankEPI2474083, VZV/gE PDB access: P09259, noV/VP1: genBank: JQ911594.1

Sequence 9 Flu/A/H7 HA gene sequence:

GACAAAATCTGCCTCGGACATCATGCCGTGTCAAACGGAACCAAAGTAAACACATTAACTGAAAGAGGAGTGGAAGTCGTCAATGCAACTGAAACAGTGGAACGAACAAACACCCCCAGGATCTGCTCAAAAGGGAAAAGGACAGTTGACCTCGGCCAATGTGGACTCCTGGGGACAATCACTGGACCACCTCAATGTGACCAATTCCTAGAATTTTCGGCCGATTTAATTATTGAGAGGCGAGAAGGAAGTGATGTCTGTTATCCTGGAAAATTCGTGAATGAAGAAGCTCTGAGGCAAATTCTCAGAGAATCAGGCGGAATTGACAAGGAACCCATGGGATTCACATACAATGGAATAAGAACTAATGGGGTGACCAGTGCATGTAGGAGATCAGGATCTTCATTCTATGCAGAAATGAAATGGCTCCTGTCAAACACAGATAATGCTGCATTCCCGCAGATGACTAAGTCATATAAAAATACAAGAGAAAGCCCAGCTATAATAGTATGGGGGATCCATCATTCCGTTTCAACTGCAGAGCAAACCAAGCTATATGGGAGTGGAAACAAACTGGTGACAGTTGGGAGTTCTAATTATCAACAATCTTTCGTACCGAGTCCAGGAGCAAGACCACAAGTTAATGGTCAATCTGGAAGAATTGACTTTCATTGGCTAATACTAAATCCCAATGATACAGTCACTTTCAGTTTCAATGGGGCTTTCATAGCTCCAGACCGTGCAAGCTTCCTGAGAGGAAAATCTATGGGAATCCAGAGTGGAGTACAGGTTGATGCCAATTGTGAAGGGGACTGCTATCATAGTGGAGGGACAATAATAAGTAACTTGCCATTTCAGAACATAGATAGCAGGGCAGTTGGAAAATGTCCGAGATATGTTAAGCAAAGGAGTCTTCTGCTGGCAACAGGGATGAAGAATGTTCCTGAGGTTCCAAAGAGAAAACGGACTGCGAGAGGCCTATTTGGTTAA

Sequence(s) 10 VZV/gE: tcagtgcttagatatgatgatttccatacagatgaagataaacttgatacaaattcagtgtatgaaccatattatcattcagatcatgcagaatcatcatgggtgaatagaggagaatcatcaagaaaagcatatgatcataattcaccatatatatggccaagaaatgattatgatggattccttgaaaatgcacatgaacatcatggagtgtataatcaaggaagaggaatagattcaggagaaagacttatgcaaccaacacaaatgtcagcacaagaagatcttggagatgatacaggaatacatgtgataccaacacttaatggagatgatagacataaaatagtgaatgtggatcaaagacaatatggagatgtgttcaaaggagatcttaatccaaaaccacaaggacaaagacttatagaagtgtcagtggaagaaaatcatccattcacacttagagcaccaatacaaagaatatatggagtgagatatacagaaacatggtcattccttccatcacttacatgcacaggagatgcagcaccagcaatacaacatatatgccttaaacatacaacatgcttccaagatgtggtggtggatgtggattgcgcagaaaatacaaaagaagatcaacttgcagaaatatcatatagattccaaggaaaaaaagaagcagatcaaccatggatagtggtgaatacatcaacacttttcgatgaacttgaacttgatccaccagaaatagaaccaggagtgcttaaagtgcttagaacagaaaaacaatatcttggagtgtatatatggaatatgagaggatcagatggaacatcaacatatgcaacattccttgtgacatggaaaggagatgaaaaaacaagaaatccaacaccagcagtgacaccacaaccaagaggagcagaattccatatgtggaattatcattcacatgtgttctcagtgggagatacattctcacttgcaatgcatcttcaatataaaatacatgaagcaccattcgatcttcttcttgaatggctttatgtgccaatagatccaacatgccaaccaatgagactttattcaacatgcctttatcatccaaatgcaccacaatgcctttcacatatgaattcaggatgcacattcacatcaccacatcttgcacaaagagtggcatcaacagtgtatcaaaattgcgaacatgcagataattatacagcatattgccttggaatatcacatatggaaccatcattcggacttatacttcatgatggaggaacaacacttaaattcgtggatacaccagaatcactttcaggactttatgtgttcgtggtgtatttcaatggacatgtggaagcagtggcatatacagtggtgtcaacagtggatcatttcgtgaatgcaatagaagaaagaggattcccaccaacagcaggacaaccaccagcaacaacaaaaccaaaagaaataacaccagtgaatccaggaacatcaccacttcttaga

Sequence 11 NoV/VP1GII.4 (used in subsequent experiments):

atgaagatggcgtcgagtgacgccaacccatctgatgggtccgcagccaacctcgtcccagaggtcaacaatgaggttatggctctggagcccgttgttggtgccgccattgcggcacctgtagcgggccaacaaaatgtaattgacccctggattagaaacaattttgtacaagcccctggtggagagtttacagtgtcccctagaaacgctccaggtgaaatactatggagcgcgcccttgggccctgatctaaatccctacttatctcatttggccagaatgtacaatggttatgcaggtggttttgaagtgcaggtaattctcgcggggaacgcgttcaccgccgggaaggtcatatttgcagcagtcccaccaaattttccaactgaaggcttgagccccagccaggtcactatgttcccccatatagtagtagatgttaggcaactagaacctgtgttgattcccttacccgatgttaggaataatttctatcattacaatcaatcaaatgaccccactattaagttgatagcaatgttgtatacaccacttagggctaataatgctggggatgatgtcttcacagtttcttgccgagttctcacgaggccatcccccgattttgactttatatttctagtgccacccacagttgagtcaagaactaaaccattctctgtcccagttttaactgttgaggagatgaccaattcaagattccccattcctttggaaaagttgttcacgggtcccagcagtgcctttgttgtccaaccacaaaacggtaggtgcacgactgatggcgtgctcctaggcaccacccaactgtctcctgtcaacatctgcaccttcagaggagatgtcacccatatcacaggtagtcataactacacaatgaatttggcttctcaaaattggagcaattatgacccaacagaagaaattccagcccctctagggactccagactttgtggggaagattcaaggcatgcttacccaaaccacaaggacagatggctcaacacgcggccacaaagccacagtgtacactgggagcgccgactttgctccaaaactgggtagagttcaatttgaaactgacacaaacaatgattttgaagctaaccaaaacacaaagttcaccccagttggcgtcatccaagatggtggcaccacccaccgaaatgaaccccaacagtgggtgctcccaagttactcaggcaggaacactcctaatgtgcatctggctcccgctgtagcccccacttttccgggtgagcaactcctcttcttcagatccaccatgcccggatgcagcgggtaccccaacatggatttggattgtctgctcccccaggaatgggtgcagtacttctaccaagaggcagccccagcacaatctgatgtggctctgctaagatttgtgaatccagacacaggtagggttttgtttgaatgtaagcttcataaatcaggctatgttacagtggctcacactggccaacatgatttggttatcccccccaatggttattttaggtttgattcctgggtcaaccagttttacacgcttgcccccatgggaaatggagcggggcgtagacgtgcacta

sequence 12 NoV/VP1 GII.17:

atgaagatggcgtcgaatgacgccgctccatctaatgatggtgctgctggtctcgtaccagagggcaacaacgagacccttcccctagaaccagttgcgggcgcagctatagccgcacccgtcactggccaaaataacataattgacccctggattagaacaaattttgtgcaagcaccaaatggagagttcacagtgtcacccagaaactctcctggagaaattttattaaacttagagttgggtcctgatttgaacccttatttggctcatttgtcaaggatgtacaatgggtatgctggtggagtggaagttcaggttctcctggcagggaacgcgttcactgccggaaagatcctcttcgccgccgtcccgccaaatttcccagtggaattcttaagcccagcccagatcacaatgctcccacatttaatagtagatgttaggactcttgaaccaattatgatcccactccctgatgttaggaatacattctttcattatagtaaccagcctaacagccgcatgagattagtggctatgctctataccccactcagatctaatggctcaggtgatgatgtctttactgtctcttgcagggttttgactaggcctactcctgattttgagttcacttatttagtgccaccttctgttgaatctaaaactaagcctttttccttacctattttaaccctttctgagctcacaaattcgaggttcccagtccccatcgattcgcttttcaccgcccagaataatgtgttgcaggtgcagtgtcaaaatggcaggtgtacacttgatggtgagttacaaggcacaacccagttgctcccatctggcatctgtgcattcagaggacgggtgacagcacaaattaaccaacgtgacaggtggcacatgcaactgcaaaacctcaatggtacaacatatgacccaactgatgatgtgccagccccgctgggaacacctgatttcaagggcgtcgtgtttgggatggtgagccaaagaaatgtgggtactgacgcgcctggctcaaccagagcccaacaggcgtgggtttcaacctatagcccccaatttgtccccaaattaggttctgtcaatcttagaattagtgataatgatgatttccagttccagccgacaaaattcacaccagtgggcgtcaatgatgacgatgatggccacccgttcagacaatgggaattaccaaactattcaggggagcttaccttgaatatgaatcttgcccccccagttgctccaaattttcctggtgaacaattgttattcttcagatctttcgtgccatgctcaggaggttacaaccaaggtattatagattgtcttattcctcaagaatggatccaacacttctatcaggaatcagcaccctctcagtcagacgtggccctaatcaggtatgttaaccccgatacgggacgcacactgtttgaagcaaaattgcacagatctggttacattactgtggctcactctggagactatcctcttgttgttccggctaatggacattttagatttgattcttgggtaaatcagttttactcactcgccccaatgggaactgggaatgggcgaaggagggctcag

The recombinant vector pHW2000-PR8_PA is used as a template, a mutated packaging sequence (CSPS), a PTV-1-2A sequence, a signal peptide sequence, three target antigen genes (FluVec-H7, fluVec-gE, fluVec-NoV/VP 1) and a PB8-PA gene packaging signal sequence are sequentially linked through an in-fusion method to obtain the recombinant vector pHW2000-PR8_PA-H7, pHW2000-PR8_PA-gE and pHW2000-PR8_PA-VP1, wherein the recombinant vector is obtained by inserting three genes shown in a sequence 9-11 into the pHW2000 vector through sequencing.

Recombinant PA plasmid sequence 1, sequence 13 for reverse genetics:

AGCGAAAGCAGGTACTGATCCAAAATGGAAGATTTTGTGCGACAATGCTTCAATCCGATGATTGTCGAGCTTGCGGAAAAAACAATGAAAGAGTATGGGGAGGACCTGAAAATCGAAACAAACAAATTTGCAGCAATATGCACTCACTTGGAAGTATGCTTCATGTATTCAGATTTTCACTTCATCAATGAGCAAGGCGAGTCAATAATCGTAGAACTTGGTGATCCAAATGCACTTTTGAAGCACAGATTTGAAATAATCGAGGGAAGAGATCGCACAATGGCCTGGACAGTAGTAAACAGTATTTGCAACACTACAGGGGCTGAGAAACCAAAGTTTCTACCAGATTTGTATGATTACAAGGAGAATAGATTCATCGAAATTGGAGTAACAAGGAGAGAAGTTCACATATACTATCTGGAAAAGGCCAATAAAATTAAATCTGAGAAAACACACATCCACATTTTCTCGTTCACTGGGGAAGAAATGGCCACAAAGGCAGACTACACTCTCGATGAAGAAAGCAGGGCTAGGATCAAAACCAGACTATTCACCATAAGACAAGAAATGGCCAGCAGAGGCCTCTGGGATTCCTTTCGTCAGTCCGAGAGAGGAGAAGAGACAATTGAAGAAAGGTTTGAAATCACAGGAACAATGCGTAAGCTTGCCGACCAAAGTCTCCCGCCGAACTTCTCCAGCCTTGAAAATTTTAGAGCCTATGTGGATGGATTCGAACCGAACGGCTACATTGAGGGCAAGCTGTCTCAAATGTCCAAAGAAGTAAATGCTAGAATTGAACCTTTTTTGAAAACAACACCACGACCACTTAGACTTCCGAATGGGCCTCCCTGTTCTCAGCGGTCCAAATTCCTGCTGATGGATGCCTTAAAATTAAGCATTGAGGACCCAAGTCATGAAGGAGAGGGAATACCGCTATATGATGCAATCAAATGCATGAGAACATTCTTTGGATGGAAGGAACCCAATGTTGTTAAACCACACGAAAAGGGAATAAATCCAAATTATCTTCTGTCATGGAAGCAAGTACTGGCAGAACTGCAGGACATTGAGAATGAGGAGAAAATTCCAAAGACTAAAAATATGAAGAAAACAAGTCAGCTAAAGTGGGCACTTGGTGAGAACATGGCACCAGAAAAGGTAGACTTTGACGACTGTAAAGATGTAGGTGATTTGAAGCAATATGATAGTGATGAACCAGAATTGAGGTCGCTTGCAAGTTGGATTCAGAATGAGTTTAACAAGGCATGCGAACTGACAGATTCAAGCTGGATAGAGCTCGATGAGATTGGAGAAGATGTGGCTCCAATTGAACACATTGCAAGCATGAGAAGGAATTATTTCACATCAGAGGTGTCTCACTGCAGAGCCACAGAATACATAATGAAGGGAGTGTACATCAATACTGCCTTGCTTAATGCATCTTGTGCAGCAATGGATGATTTCCAATTAATTCCAATGATAAGCAAGTGTAGAACTAAGGAGGGAAGGCGAAAGACCAACTTGTATGGTTTCATCATAAAAGGAAGATCCCACTTAAGGAATGACACCGACGTGGTAAACTTTGTGAGCATGGAGTTTTCTCTCACTGACCCAAGACTTGAACCACATAAATGGGAGAAGTACTGTGTTCTTGAGATAGGAGATATGCTTATAAGAAGTGCCATAGGCCAGGTTTCAAGGCCCATGTTCTTGTATGTGAGAACAAATGGAACCTCAAAAATTAAAATGAAATGGGGAATGGAGATGAGGCGTTGCCTCCTCCAGTCACTTCAACAAATTGAGAGTATGATTGAAGCTGAGTCCTCTGTCAAAGAGAAAGACATGACCAAAGAGTTCTTTGAGAACAAATCAGAAACATGGCCCATTGGAGAGTCCCCCAAAGGAGTGGAGGAAAGTTCCATTGGGAAGGTCTGCAGGACTTTATTAGCAAAGTCGGTATTCAACAGCTTGTATGCATCTCCACAACTAGAAGGATTTTCAGCTGAATCAAGAAAACTGCTTCTTATCGTTCAGGCTCTTAGGGACAACCTCGAGCCAGGAACGTTCGACCTAGGAGGACTGTACGAGGCGATCGAAGAATGTCTCATAAACGACCCTTGGGTATTACTAAACGCATCATGGTTTAACTCGTTCCTGACTCACGCTTTAAGCGGATCTGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCTATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCG

mutated packaging sequence, sequence 14:

CTCGAGCCAGGAACGTTCGACCTAGGAGGACTGTACGAGGCGATCGAAGAATGTCTCATAAACGACCCTTGGGTATTACTAAACGCATCATGGTTTAACTCGTTCCTGACTCACGCTTTAAGC

2A sequence, sequence 15:

GGATCTGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCT

IL-2 signal peptide sequence, SEQ ID NO 16:

ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCG

c-terminal 237-306 of CD80, SEQ ID NO 17:

CCACCAGAAGATCCACCAGATTCAAAAAATACACTTGTGCTTTTCGGAGCAGGATTCGGAGCAGTGATAACAGTGGTGGTGATAGTGGTGATAATAAAATGCTTCTGCAAACATAGATCATGCTTCAGAAGAAATGAAGCATCAAGAGAAACAAATAATTCACTTACATTCGGACCAGAAGAAGCACTTGCAGAACAAACAGTGTTCCTTTAA

PA packaging sequence, sequence 18:

CTGGAACCTGGGACCTTTGATCTTGGGGGGCTATATGAAGCAATTGAGGAGTGCCTGATTAATGATCCCTGGGTTTTGCTTAATGCTTCTTGGTTCAACTCCTTCCTTACACATGCATTGAGTTAGTTGTGGCAGTGCTACTATTTGCTATCCATACTGTCCAAAAAAGTACCTTGTTTCTACT

Recombinant PA plasmid sequence 2, sequence 19 for reverse genetics:

CCACCAGAAGATCCACCAGATTCAAAAAATACACTTGTGCTTTTCGGAGCAGGATTCGGAGCAGTGATAACAGTGGTGGTGATAGTGGTGATAATAAAATGCTTCTGCAAACATAGATCATGCTTCAGAAGAAATGAAGCATCAAGAGAAACAAATAATTCACTTACATTCGGACCAGAAGAAGCACTTGCAGAACAAACAGTGTTCCTTTAACTGGAACCTGGGACCTTTGATCTTGGGGGGCTATATGAAGCAATTGAGGAGTGCCTGATTAATGATCCCTGGGTTTTGCTTAATGCTTCTTGGTTCAACTCCTTCCTTACACATGCATTGAGTTAGTTGTGGCAGTGCTACTATTTGCTATCCATACTGTCCAAAAAAGTACCTTGTTTCTACT

an example of the pattern of genetic engineering of the PA gene is shown in figure 1.

EXAMPLE 2 transfection rescue Virus

For details of the preparation and identification method of recombinant viruses, refer to patent CN113073115B of the related invention of this company.

And packaging the recombinant vector obtained by the construction into mixed cells of the 293T cells transfected by the following combinations to obtain the recombinant virus.

The pHW2000-PR8_HA,pHW2000-PR8_NA,pHW2000-PR8_PB2,pHW2000-PR8_PB1,pHW2000-PR8_NP,pHW2000-PR8_M,pHW2000-PR8_NS and 0.5 mug of one of three target genes (pHW 2000-PR8_PA-H7, pHW2000-PR8_PA-gE and pHW2000-PR8_PA-VP 1) are mixed to prepare an 8 plasmid system, so that a mixed vector A required by packaging viruses is obtained;

1) 293T cells (ATCC-CRL-3216) with good growth were taken as one bottle, digested with 0.25% EDTA-pancreatin (EDTA-Trypsin), and centrifuged at 1,000 rpm for 10min. After centrifugation, the particles were broken up and resuspended in 10mL of Opti-MEM. The resuspended 293T was made up to Opti-MEM to 20mL, a six-well plate was used, 3mL of the resuspended 293T cell suspension (5X 10 ⁴) was added to each well and placed in the well for 37 months.

2) Mixing the recombinant vector obtained in the step 2 by 0.5 mug respectively, so that the vectors are mixed in equal mass;

3) The above mixed carrier A and added liposome were each 10. Mu.L and left at room temperature for 20min, and then Opti-MEM was added to 1 mL. The waste solution in the six-well plate was aspirated, opti-MEM mixed with plasmid was added, and placed in a 37℃5% CO ₂ incubator overnight. The following day, 1mL of Opti-MEM containing 2. Mu.g/mL TPCK-Trypsin was filled into each well, and the cells were harvested after culturing in a 5% CO ₂ incubator at 37℃in a secondary biosafety laboratory for 48 hours. The collected transfection supernatant was E0-generation virus, and its hemagglutination titer was determined using 1% turkey erythrocytes.

4) SPF chick embryos of 9 to 11 days old were selected and examined to observe the viability status of the chick embryos. And (3) avoiding blood vessels at the boundary edge of the embryo surface and the air chamber, and marking the head of the chick embryo, namely, inoculating an injection point. Sterilizing the marked place and surrounding with 75% alcohol, drilling holes with an egg drill without damaging the shell membrane.

5) Chick embryo allantoic cavity inoculation was performed at a cell transfection suspension level of 0.2 mL/chick embryo, two chick embryos were inoculated per sample.

6) Sealing the chick embryo with wax, culturing in a 37 ℃ incubator for 48-72 hours, collecting chick embryo allantoic fluid, and measuring the hemagglutination titer (World Health Organization,Manual on Animal Influenza Diagnosis and Surveillance, Serologic diagnosis of influenza virus infections by hemagglutination inhibition,edition 4,78-79,2004). by using 1% turkey red blood cells to obtain the recombinant virus PB1-RBD obtained by packaging, wherein the virus generation number is 1 generation (E1) of the chick embryo. The obtained recombinant viruses have the hemagglutination titer of more than 128 HAU/50  L (namely 2 ⁷).

And (3) splitting the chick embryo, continuously transmitting the recombinant virus for five generations, amplifying the antibody genes by nucleic acid, and sequencing to verify that the antibody genes have no mutation.

Detecting exogenous genes of recombinant influenza viruses:

Transgenic fragments in Influenza A Virus (IAV) rgH1N1 (PR 8) -PA-H7, rgH1N1 (PR 8) -PA-gE, rgH1N1 (PR 8) -PA-VP1 were detected.

Identification of recombinant influenza virus expression target antigen:

The colloid Jin Shi paper produced by our company is used for definite and qualitative determination of target antigen expressed by recombinant influenza virus in the virus amplification process, and the allantoic fluid is subjected to antigen qualitative detection (blank and irrelevant recombinant virus are used as contrast) after 48-72 hours of chick embryo culture, so that the result shows that the average three target antigen paper is positive, and the target antigen paper is successfully expressed by the recombinant virus in the chick embryo replication process.

The prepared recombinant influenza viruses are named FluVec-H7, fluVec-gE, fluVec-NoV/VP1, respectively.

EXAMPLE 3 immunization and antibody screening experiments

Insect baculovirus is used for infecting insect cells sf9 to express influenza virus H7 subtype HA trimer antigen, escherichia coli is used for prokaryotic expression of VZV virus gE antigen and norovirus VP1 antigen, and the gene sequences for expressing the three recombinant antigens are consistent with the gene sequence (4. ①.1) for packaging recombinant influenza virus at the protein level. The expressed recombinant protein is subjected to biological activity identification and is used for traditional subcutaneous muscle immunity and antigen coating.

Immunization of mice:

Nasal drip immunization of recombinant influenza virus

Mice were immunized with 25 μl of recombinant influenza virus containing 10 ⁵ pfu (i.n.) in a nasal drip under isoflurane inhalation anesthesia to 8-year old female BALB/C mice, 5 mice were immunized per recombinant influenza virus antigen nasal drip, and about 30 μl of serum was collected from the orbit of the mice on day 12 post-infection, and the antibody titer against the antigen of interest in the serum was determined as described in 4 ③.

Subcutaneous/intramuscular immunization with recombinant protein antigens

40. Mu.g of recombinant protein was mixed thoroughly with an equal volume of Freund's complete adjuvant to a total volume of 100. Mu.l (Sigma-Aldrich, CAT: F5881), and the mice were immunized by subcutaneous injection at multiple spots (i.p./i.m.) on the abdomen and footpad of 8 weeks female BALB/C mice, 5 mice were immunized with each of the three antigens (reFlu/A-H7, reVZV-gE, reNoV-VP 1), and three more immunizations were performed as described above for 0, 2, and 4 weeks. Mice were taken at week 6 post-immunization to detect antibody titers. Mice with highest titers were selected for booster immunization with 20 μg of recombinant protein via the tail vein, and after 3 days the spleens of the mice were taken for fusion and hybridoma cells were prepared.

Fusion and monoclonal antibody screening:

ELISA method for identifying recombinant protein/screening positive hybridoma

96-Well microplates were coated with purified recombinant proteins (including H7 subtype HA trimer antigen: reFlu/A-H7, VZV virus gE antigen: reVZV-gE, norovirus VP1 antigen: reNoV-VP 1) and ELISA methods were used to identify the reactivity of the three antigens with the corresponding positive mouse serum or commercial monoclonal antibodies. Firstly, coating recombinant protein (coating buffer solution carbonate buffer solution sodium carbonate 1.59g, sodium bicarbonate 2.93g is fixed to volume to 1L of pure water) in a microplate, wherein the coating concentration is 1 mug/mL, 4 ℃ is overnight, 1% gelatin is used for sealing, each hole is 150 microlitres, the plate is washed for 1 time at 37 ℃ for 2 hours, the plate is patted dry, antigen-specific positive serum is diluted by PBS according to the gradient of 1:100, 1:1000, 1:10000 and 1:10000, and 50 mul of the diluted antigen-specific positive serum is added into the microplate coated with the antigen, and the reaction is carried out for 30 minutes at 37 ℃. The wells were discarded, the plates were washed 4 times with PBST wash, 50. Mu.L/well of HRP-labeled goat anti-mouse secondary antibody (diluted 1:10000 in PBS) was added after the plates were patted dry, reacted for 30min at 37℃and washed 4 times, 100. Mu.L/well of TMB developing solution was added after the plates were patted dry and developed for 10min at room temperature, finally 0.5M sulfuric acid was added, the reaction was stopped, and the OD450nm value was measured with an ELISA reader. Negative control and blank wells were set simultaneously.

Mouse spleen cells were fused with myeloma cells SP2/0 by PEG1450 (Merk) to prepare antibody-secreting hybridoma cells. After 10 days of fusion, antibodies which specifically secrete target antigens are screened according to an ELISA method described by 4 ③, positive hybridoma cells are subjected to limited dilution to a monoclonal state, cell culture supernatant titers are determined, and three antigen-specific antibody cell strains which can secrete the antigen-specific antibody are screened by taking tag proteins, irrelevant antigens and the like as background controls.

The monoclonal antibodies screened by different immune methods are identified by aiming at the same target antigen, including affinity activity, approximate binding area with the antigen, linear/conformational epitope, neutralization activity, and the like, and the monoclonal antibodies screened by the recombinant influenza virus infection immune method are found to be superior to the traditional screening method in the aspects (see table 1). The method comprises the steps of screening a plurality of monoclonal antibodies aiming at the same target antigen, namely a, high affinity (ELISA detection OD reading value is more than 1.2), b, widely combining different positions of the antigen (combining with different fragments of the antigen, such as H7 monoclonal antibody is mainly concentrated at the HA1 part, if combining with HA2, the combining position is wide, other two antigens are similar, the N end fragment and the C end fragment of the antigen are taken as references), C, cloning with neutralization activity and the like are defined as high-quality monoclonal antibodies, and the ratio of the high-quality monoclonal antibodies (the simultaneous occurrence of any two indexes in the three indexes can be calculated as one high-quality monoclonal antibody) to all positive monoclonal antibodies is called high-quality monoclonal antibody ratio, namely 'good' monoclonal antibody duty ratio.

According to the invention, three antigens are selected, and meanwhile, infection immunity by using a viral vector is compared with that of a traditional immunization method, the fusion rate of the three antigens under different immunization strategies is kept consistent, but the positive rate and the high-quality monoclonal antibody rate are obviously superior to those of the traditional immunization method in three projects (see table 2). Positive rate refers to the ratio of wells that were detected by fusion plate supernatant to antigen-specific binding to all fusion wells (5X 96X fusion rate) after 2 weeks of spleen fusion of 5 96 well plates per mouse. The fusion rate refers to the ratio of the number of wells fused with sp20 into hybridoma cells to the number of all wells (5X 96) after 10 days after 5 wells fused with spleen of each mouse.

TABLE 1 comparison of different immunization patterns

TABLE 2 comparison of infectious immunity between different items and monoclonal antibodies obtained by conventional immunization

In summary, the method for preparing the mouse monoclonal antibody by using the hybridoma technology and using the recombinant influenza virus as an immunogen for mouse immunization has obvious advantages compared with the existing hybridoma technology.

Claims

1. A method for preparing hybridoma monoclonal antibodies by utilizing a recombinant influenza virus immunized mouse is characterized in that the recombinant influenza virus PA-antigen fusion gene used in the method sequentially comprises a PA gene, a codon conversion signal peptide sequence, a 2A sequence, an IL-2 signal peptide sequence, an antigen gene sequence and a CD 80C-terminal sequence from a 5' segment, wherein the antigen is influenza virus H7N9 subtype hemagglutinin HA, and the gene sequence of the antigen is shown as SEQ ID NO. 9;

The sequence of the PA gene is shown as SEQ ID NO. 13;

The sequence of the codon conversion signal peptide sequence is shown as SEQ ID NO.14, the sequence of the 2A sequence is shown as SEQ ID NO.15, the sequence of the IL-2 signal peptide sequence is shown as SEQ ID NO.16, and the sequence of the C-terminal sequence of the CD80 is shown as SEQ ID NO. 17;

the recombinant influenza virus is obtained by a reverse genetic system, rescue preparation.

2. The method of claim 1, comprising the step of immunizing a mouse with a recombinant influenza virus, wherein the mouse is immunized using a nasal drip method at a dose of 1 x 10 ⁴-10⁶ pfu.