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Method for establishing transgenic cell line of target genes shRNA (short hairpin ribonucleic acid) interfering propagation of porcine reproductive and respiratory syndrome viruses

A technology of transgenic cell lines and porcine PRRS virus, applied in the direction of microorganism-based methods, cells modified by introducing foreign genetic material, antiviral agents, etc., can solve problems such as difficulty in inducing antibody levels

Inactive Publication Date: 2013-01-16
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the vaccine research on PRRS at home and abroad is very active, so far, there is still no vaccine that can completely protect pigs, and it is generally difficult to induce earlier and higher antibody levels.

Method used

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  • Method for establishing transgenic cell line of target genes shRNA (short hairpin ribonucleic acid) interfering propagation of porcine reproductive and respiratory syndrome viruses
  • Method for establishing transgenic cell line of target genes shRNA (short hairpin ribonucleic acid) interfering propagation of porcine reproductive and respiratory syndrome viruses
  • Method for establishing transgenic cell line of target genes shRNA (short hairpin ribonucleic acid) interfering propagation of porcine reproductive and respiratory syndrome viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1, the design, synthesis, vector construction of the targeting gene shRNA aimed at the proliferation and replication of porcine PRRS virus

[0070] According to the structural sequence and shRNA sequence design principles of PRRSV-ORF5 and PRRSV-ORF6 protein genes, 5 pairs of shRNA sequences were designed respectively for PRRSV-ORF5 and PRRSV-ORF6; the 10 pairs of shRNA sequences are described in Table 1 and Table 2. :

[0071] Table 1. shRNA design for PRRSV-ORF5

[0072]

[0073] Table 2. shRNA design for PRRSV-ORF6

[0074]

[0075] The 10 pairs of shRNA sequences were annealed to form double strands with cohesive ends, which were directly connected to the linearized RNAi-Ready pSIREN-RetroQ-ZsGreen vectors to construct 10 RNA interference vectors; the plasmids (i.e. the above 10 RNA interference carrier) DNA was introduced into Marc-145 cells under the mediation of transfection reagents, and fluorescence observation was carried out after 24 hours ...

Embodiment 2

[0081] Embodiment 2, the establishment of the transgenic cell line of anti-porcine PRRS virus propagation

[0082] The vector PXL-BAC2-FRT-EGFP-NEO-shRNA and helper were respectively used lipofectamine 2000, FuGENE HD and Lipofectamine LTX & PLUS three transfection reagents were used to transfect Marc-145 cells, and the transfection efficiency, clone formation rate and positive clone rate were compared to select a screening method suitable for high-efficiency shRNA transfection.

[0083] details as follows:

[0084] 2.1 Construction of recombinant vector PXL-BAC2-FRT-EGFP-NEO-shRNA

[0085] Such as figure 2 As indicated, zsGFP and NEO fragments were amplified by PCR, and the shRNA (6e) was digested (EcoRI-BamHI

[0086] ) was connected to the pMD18-T Simple vector, digested by BglII-EcoRV, recovered, and connected to the PXL-BAC2 vector to construct the recombinant vector PXL-BAC2-FRT-EGFP-NEO-shRNA, and the constructed recombinant vector was digested by And sequencing i...

Embodiment 3

[0100] Example 3. Target gene integration and expression detection in transgenic cells resistant to propagation of porcine PRRS virus

[0101] 3.1 GFP and Neomycin in the genome of transgenic cell lines r Insert detection

[0102] Using the transgenic cell line obtained in Example 2 as a material to extract the genome as a template, design upstream and downstream primers respectively, specifically: the GFP primer sequence as described in SEQ ID NO 1 (the PCR amplification product contains CMV+GFP, and the predicted molecular weight is 1400bp) and Neomycin as described in SEQ ID NO 2 r The primer sequence (the molecular weight of the product after PCR amplification with this primer is predicted to be 2000bp) was used for PCR amplification.

[0103] SEQ ID NO 1: Transgenic cell line GFP amplification primer sequence (CMV+GFP≈1400bp)

[0104] EGFP-F: 5'-GagatctgaagttcctattctctagaaagtataggaacttcTAGTTATTAATAGTAATCAATTACGG-3'

[0105] EGFP-R: 5'-GaagcttTTCACCGTCATCACCGAAA-3'

...

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Abstract

The invention discloses a method for establishing a transgenic cell line of target genes shRNA (short hairpin ribonucleic acid) interfering propagation of porcine reproductive and respiratory syndrome viruses. Target genes shRNA capable of preventing propagation and copy of the porcine reproductive and respiratory syndrome viruses effectively are designed, and a clonal cell line capable of preventing infection of the porcine reproductive and respiratory syndrome viruses effectively is obtained by the aid of genomes inserted into donorcells via transposons carriers. The invention further discloses an establishing method of a recombinant carrier PXL-BAC2-FRT-EGFP-NEO-shRNA. The establishing method includes designing a target gene shRNA-ORF6-6e, amplifying zsGFP and NEO fragments by PCR (polymerase chain reaction), the shRNA of the ORF6-6e is connected to a carrier pMD18-T Simple in an enzyme digestion manner, and is recovered and connection on a carrier PXL-BAC2 by enzyme digestion. The invention discloses application of the transgenic cell line. The transgenic cell line has a suppression function on the PRRSV (porcine reproductive and respiratory syndrome viruses).

Description

technical field [0001] The present invention designs the construction of a transgenic cell line resistant to porcine PRRS virus; specifically, the present invention designs a targeted gene shRNA that can effectively prevent the proliferation and replication of porcine PRRS virus, and inserts it into the donor through a transposon carrier The genome of the cell was obtained to obtain the Marc-145 cell clone that can effectively prevent the infection of the PRRS virus. Background technique [0002] Porcine reproductive and respiratory syndrome was first discovered in the United States in the late 1980s. Its causative agent is porcine reproductive and respiratory syndrome virus (PRRS virus, PRRSV), which mainly causes reproductive disorders in sows and the death of a large number of piglets. Piglet morbidity 100%, the mortality rate is more than 50%, and the abortion rate of sows is more than 30%, which is an important animal disease. [0003] In recent years, porcine blue-ear...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12N5/10A61P31/14C12R1/91
Inventor 缪云根周芳
Owner ZHEJIANG UNIV
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