Efficient bidirectional transcription/expression plasmid and application thereof in influenza virus reverse genetics

A technology of reverse genetics and viruses, applied in the direction of viruses, viruses/bacteriophages, antisense single-stranded RNA viruses, etc., can solve the problems of low virus titer, difficulty in adapting to research needs, time-consuming and labor-intensive problems, and achieve high toxin production Effect

Inactive Publication Date: 2016-08-17
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although pHW2000 and pBD both use strong CMV promoters, the titer of the rescued first-generation virus is still low, and it needs to be expanded and cultured by MDCK cells or chicken embryos, which is time-consuming and laborious, and it is difficult to adapt to many research needs

Method used

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  • Efficient bidirectional transcription/expression plasmid and application thereof in influenza virus reverse genetics
  • Efficient bidirectional transcription/expression plasmid and application thereof in influenza virus reverse genetics
  • Efficient bidirectional transcription/expression plasmid and application thereof in influenza virus reverse genetics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction and functional identification of bidirectional transcription / expression vector

[0047] 1. Construction of bidirectional transcription / expression vector

[0048] Such as figure 1 As shown, the eukaryotic expression vector pEF4 / myc-His B is used as the backbone for transformation: an essential element (Insert 1, whose sequence is shown in SEQ ID NO.11) is inserted between its KpnI and BclI restriction sites—mouse Source polI terminator (TpolI, 33bp), BsmBI restriction site and human polI promoter (PpolI, 236bp) sequence; a short Linker sequence (Insert2, Insert2, The sequence is 5'-CTGGGGATGCGGTGGGCTCTATG-3'), replacing the non-essential elements from flori to SV40poly A signal on the original vector. The transformed vector is named pEZ, and its nucleotide sequence is shown in SEQ ID NO.1.

[0049] 2. Construction of recombinant plasmid carrying reporter gene

[0050] A reporter gene was inserted into the pEZ vector to verify its bidirectional ...

Embodiment 2E

[0061] The establishment of embodiment 2EIV reverse genetic system

[0062] 1. Construction of recombinant plasmid carrying viral cDNA and introduction of molecular markers

[0063] Extract the viral RNA of the EIV JL89 strain according to the instruction manual of the viral RNA extraction kit, use uni12 as the reverse transcription primer, use the corresponding primer pair of each segment (Table 1) as the amplification primer, and use the RT-PCR method to amplify 8 genes Segments (PB2, PB1, PA, HA, NP, NA, M and NS). Each segment was cleaved with the corresponding restriction endonuclease (BsmBI or BsaI), and then directional cloned into the BsmBI of pEZ, and 8 recombinant plasmids were constructed, and the 8 gene segments contained in them—PB2, PB1, The nucleotide sequences of PA, HA, NP, NA, NS and M gene segments are respectively shown in SEQ ID NO.2-9.

[0064] Table 1 Primers for amplifying 8 segments

[0065]

[0066] In addition, wild-type (wt) HA segments were n...

Embodiment 3

[0073] Identification of embodiment 3 recombinant virus

[0074] 1. Sequence determination of recombinant virus and detection of its molecular markers

[0075] The 8 segments of rJL89 were amplified according to the primers and methods described in Method 1 of Example 2, cloned into the pMD18T vector, the sequences of each segment were determined, and compared with the sequences of each segment of JL89. At the same time, the HA segment was identified by restriction endonucleases HindIII and NdeI.

[0076] result:

[0077] Eight segments of rJL89 were cloned and sequenced. The alignment of each segment of rJL89 with JL89 showed that the HA segment contained a nonsense mutation (T 449 C and C 983 T), the identity of the HA segment of JL89 is 99.9%; while the identity of PB2, PB1, PA, NP, NA, M and NS is 100% with the corresponding segment of JL89; this shows that the genetic information of rJL89 is almost entirely derived from Since JL89.

[0078]The HA segment of rJL89 co...

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Abstract

The invention discloses an efficient bidirectional transcription/expression plasmid and application thereof in influenza virus reverse genetics. The bidirectional transcription/expression plasmid is obtained by transformation by taking a eukaryotic expression vector pEF4/myc-His B as a skeleton. In order to create a reverse genetic system of EIVs (Equine influenza viruses), the bidirectional transcription/expression plasmid obtained by structuring is used for respectively expressing EIVs PB2, PB1, PA, HA, NP, NA and NS and M genes, and a strain of the EIV and mutants thereof are successfully saved by means of cotransfection, sequencing, drawing of growth curves and enzyme digestion are obtained. Compared with existing pBD systems, the created reverse genetic system is superior to the pBD systems in toxigenic quantity. A novel technical means is provided for research on reverse genetics of the influenza viruses, and a solid foundation is laid for future in-depth study on transmission of the EIVs, pathogenesis and even screening of vaccine candidates.

Description

technical field [0001] The invention relates to a bidirectionally transcribed plasmid and its application in influenza virus reverse genetics, which belongs to the field of biotechnology. Background technique [0002] Influenza viruses belong to the Orthomyxoviridae family and can be divided into A, B and C influenza viruses according to the antigenicity of NP and M proteins. Among them, the type C influenza virus only mildly infects people, almost asymptomatic. Both influenza A and B viruses can cause seasonal flu, and influenza A viruses can cause global pandemics. In addition, the genome of influenza virus consists of 7 (type C) or 8 (types A and B) segmented single-stranded negative-sense RNA, which is prone to rearrangement and produces new strains, which makes the prevention and control of influenza There was a challenge. The study of influenza virus is of positive significance for promoting the understanding of influenza virus and the prevention and control of infl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N7/01
CPCC12N7/00C12N15/85C12N2760/16121C12N2760/16151C12N2800/107
Inventor 王晓钧张翔郭巍张振宇胡哲
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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