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ShRNA (short hairpin Ribonucleic Acid) expression vector for interfering BVDV (Bovine Viral Diarrhea Virus) copying and constructed recombinant cell

A technology of expressing vectors and expression cassettes, which is applied in the field of genetic engineering, can solve the problems of vaccination failure and antigen diversity, and achieve the effect of reducing viral RNA expression and virus production

Inactive Publication Date: 2012-08-08
NORTHWEST A & F UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although a commercial BVDV vaccine is available, the antigenic diversity of BVDV leads to vaccination failure

Method used

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  • ShRNA (short hairpin Ribonucleic Acid) expression vector for interfering BVDV (Bovine Viral Diarrhea Virus) copying and constructed recombinant cell
  • ShRNA (short hairpin Ribonucleic Acid) expression vector for interfering BVDV (Bovine Viral Diarrhea Virus) copying and constructed recombinant cell
  • ShRNA (short hairpin Ribonucleic Acid) expression vector for interfering BVDV (Bovine Viral Diarrhea Virus) copying and constructed recombinant cell

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Embodiment Construction

[0030] The present invention first constructs the expression vector pARNG-2shRNA-BVDV containing two shRNA fragments that inhibit BVDV replication and the red fluorescent protein (RFP) gene, and then introduces the exogenous expression vector pARNG-2shRNA-BVDV into the bovine fetus by electroporation Fibroblasts, observe the expression of the marker gene RFP with a fluorescent microscope, and obtain positive cells after G418 screening. After PCR identification, it is confirmed that two pieces of BVDV interference sequence shRNA are integrated into the genome of bovine fetal fibroblasts; finally, the interference BVDV replication will be included The bovine fetal fibroblasts of the shRNA interference fragment were transferred into bovine enucleated oocytes as nuclear donors to obtain transgenic cloned embryos. The present invention will be further described in detail in conjunction with the construction and detection of specific vectors below, which is an explanation of the pres...

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Abstract

The invention discloses a shRNA (short hairpin Ribonucleic Acid) expression vector for interfering BVDV (Bovine Viral Diarrhea Virus) copying and a constructed recombinant cell. Different shRNA fragments are designed specific to different regions of a BVDV. SiRNA (small interfering Ribonucleic Acid) is formed by transcribing shRNA in a host cell and shearing, and acts on different regions of a virus RNA to interfere the copying and expression of viruses. BVDV cell surface receptors exists in bovine kidney cells, testicle cells and the like, so that BVDV is most sensitive to fetal bovine kidney cells and testicle cells, a complete transgenic cloned cattle individual is formed by performing induced rearrangement on a fetal bovine fibroblast serving as a somatic cell nuclear transplanting donor cell, and BVDV sensitive organs such as kidney and the like have the capabilities of suppressing virus expression.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a site-specifically integrated expression vector, in particular to an shRNA expression vector that interferes with BVDV replication and a constructed recombinant cell. Background technique [0002] Bovine viral diarrhea virus (BVDV) is a folded, positive-sense, single-stranded RNA virus belonging to the Pestivirus genus of the Flaviviridae family. BVDV can cause lifelong persistent infection in cattle herds and is an important factor causing significant economic losses in the cattle industry. The associated symptoms of BVDV range from mild to severe in clinical development, involving the respiratory system, intestinal tract, reproductive system, immune system and endocrine system. Although there are commercialized BVDV vaccines, the antigenic diversity of BVDV leads to the failure of vaccination. Furthermore, despite widespread culling of positive cattle after selective testing...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63C12N5/10
Inventor 佟琪张涌余源王勇胜权富生苏峰刘军
Owner NORTHWEST A & F UNIV
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