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shRNA transgenic recombinant plasmid for inhibiting swine influenza viruses and use thereof

A technology of swine influenza virus and recombinant plasmid, applied in the field of genetic engineering

Inactive Publication Date: 2015-10-28
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

Current antiviral drugs and vaccine immunity have limitations, which make the virus resistant to the drug, and the virus will mutate rapidly under selective pressure to escape drug or vaccine immunity; researchers choose RNAi methods to inhibit the replication of influenza virus, usually targeting influenza virus The conservative sequence of the gene is used as the target sequence. Due to the high transferability of the interfering sequence, RNAi technology also encounters technical barriers in the application of influenza virus prevention and control.

Method used

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  • shRNA transgenic recombinant plasmid for inhibiting swine influenza viruses and use thereof
  • shRNA transgenic recombinant plasmid for inhibiting swine influenza viruses and use thereof
  • shRNA transgenic recombinant plasmid for inhibiting swine influenza viruses and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Construction of shRNA transgene recombinant plasmid capable of inhibiting swine influenza virus replication and infection

[0041] 1. Design of shRNA target sites and primers that can inhibit the replication and infection of swine influenza virus

[0042] In this example, the shRNA that can inhibit the replication and infection of swine influenza virus is based on the genome sequence of influenza virus, and selects PA, PB1, PB2 and NP, which are highly conserved in influenza virus, as target sequences to design and synthesize siRNAs, as shown in Table 1. . The specific positions of the selected conserved sequences (indicated by numbers in the primers) are the corresponding siRNAs. The selected target gene fragments are named after the original gene.

[0043] Table 1 shRNA target sites and primer design

[0044]

[0045] 2. Construction of NP, PB2, PB1 and PA gene shRNA expression cassettes

[0046] Dissolve single-stranded DNA primers (2OD) in 60 μL ann...

Embodiment 2

[0050] Example 2 Construction of recombinant lentiviral vector and establishment of cell line stably expressing shRNA

[0051] 1. Construction of recombinant lentiviral vector

[0052] The pLV-EGFP lentiviral vector was double-digested with ClaI and XbaI, and the large fragment recovered from the gel was connected to the synthetic multiple cloning site 5'-ClaI-Xmal-XhoI-MluI-XbaI-3', and the constructed lentiviral expression vector was named pLV-MCS, the expression cassette vector and lentiviral expression vector were cut with XhoI and XbaI, named pLV-EGFP-shNPAPB1PB2, the three-gene expression cassette and lentiviral expression vector were named pLV-EGFP-shPAPB1PB2, pLV-EGFP-shNPAPB1 , pLV-EGFP-shNPAPB2, pLV-EGFP-shNPB1PB2, the construction principle is as follows figure 1 shown.

[0053] The tandem shRNA gene and reporter gene arrangement constructed in the present invention are as follows figure 2 shown. Three-gene shRNA and four-gene shRNA are arranged in tandem on th...

Embodiment 3

[0059] Example 3 Comparison of three-gene and four-gene cell lines inhibiting the replication of H1N1 swine influenza virus

[0060] 6 passages of shPAPB1PB2-MDCK, shNPAPB1-MDCK, shNPAPB2-MDCK, shNPB1PB2-MDCK, shNPAPB1PB2-MDCK, Mock-MDCK and MDCK that were subcultured synchronously, with 1×10 5 The number of cells was planted in a 12-well cell culture plate. When the cell confluence reached 70%-80%, it was diluted with infection medium (penicillin 100 U / mL, streptomycin 100 μg / mL, TPCK-Trypsin 4 μg / mL) H1N1 swine influenza virus (LM / 2004 strain, 1000TCID 50 ) to inoculate cells, incubate at room temperature for 1 hour, remove virus dilution, add 1 mL infection medium to each well, and absorb cell supernatant at 12, 24, 36, 48, and 60 hours after infection to make 10 -1 -10 -8 Gradual dilution, each dilution was inoculated with three 9-10-day-old common chicken embryos, and after 72 hours (LM / 2004), the allantoic fluid was drawn to detect the virus titer.

[0061] H1N1 swine...

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Abstract

The invention provides a shRNA transgenic recombinant plasmid for inhibiting swine influenza viruses. The plasmid contains four shRNA expression genes for simultaneously coding four specific shRNAs capable of respectively targeting influenza virus Pa, PB1, PB2 and NP gene conserved regions. The plasmid is suitable for a plurality of transgenic technologies. After the plasmid is transferred into a MDCK cell cultivated in vitro by a lentivirus carrier, a challenge assay proves that the screened transgenic cell line has a function of resisting infection caused by different subtypes of influenza viruses. The plasmid is injected into a pigling with 4 week old by intravenous injection and intramuscular injection and then the pigling is infected by H3N2 subtype of pig influenza viruses, and the test result shows that an influenza virus replication inhibition function of the blood injection group is better. RNAi is used for pig influenza virus research and pig influenza prevention and treatment so that necessary experiment data is accumulated and thus a base is established for researching influenza resistance of a transgenic pig.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an shRNA transgene recombinant plasmid capable of inhibiting swine influenza virus and its application. Background technique [0002] Swine Influenza (SI) is an acute, highly contagious respiratory disease caused by Orthomyxoviridae, Influenzavirus, and Influenza A virus (IAV). The clinical symptoms are fever, Cough, runny nose, conjunctivitis, anorexia and lack of energy. Swine Influenza Virus (SIV) can replicate in epithelial cells of the upper respiratory tract of pigs, causing pig infection, resulting in changes in the homeostasis of the pig's immune system, resulting in secondary or mixed infections of bacteria, mycoplasma and other viruses, causing epidemics Aggravating, the death rate of sick pigs increases, bringing great economic losses to the pig industry. More and more data show that pigs, as the "mixer" of influenza virus, play an important r...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/79C12N5/10A61K48/00A61P31/16
Inventor 孟庆文王伟陈洪岩
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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