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Method for constructing pacing channel gene hypotype HCN2 recombination slow virus vector

A technology of recombinant lentivirus and construction method, which is applied in the field of construction of pacing channel gene subtype HCN2 recombinant lentiviral vector, can solve problems such as restricting development, and achieve the effect of low immunogenicity

Inactive Publication Date: 2011-06-22
THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Most of the current studies use the adenovirus expression system to initially explore the feasibility of biological pacing; although such research has also achieved great achievements, due to the high immunogenicity of the adenovirus expression system, its viral genome is free from the host genome In addition, foreign genes can only be expressed transiently, and foreign in vivo studies only lasted 3-10 days, which limits its development

Method used

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  • Method for constructing pacing channel gene hypotype HCN2 recombination slow virus vector
  • Method for constructing pacing channel gene hypotype HCN2 recombination slow virus vector
  • Method for constructing pacing channel gene hypotype HCN2 recombination slow virus vector

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Embodiment

[0021] Example: A method for constructing a recombinant lentiviral vector of the pacing channel gene subtype HCN2, which includes constructing a HCN2 recombinant virus vector with a shuttle plasmid carrying the HCN2 gene and a virus system. The shuttle plasmid is pCDH1-GFP-HCN2, The virus is a lentivirus;

[0022] The hHCN2 target sequence with a total length of about 3.0kb was unloaded from the pCDNA3-hHCN2 plasmid (previously constructed by our laboratory), and finally loaded onto the plasmid pCDH1-MCS1-EF1-copGFP with immunofluorescence protein to construct the shuttle plasmid pCDH1-GFP -HCN2, the method for constructing the shuttle plasmid pCDH1-GFP-HCN2 includes the following steps:

[0023] (1) Cut the target fragment HCN2 gene from pcDNA3-HCN2 and ligate it into the Puc19 plasmid to construct the plasmid Puc19-HCN2;

[0024] (2) Cut the target fragment HCN2 gene from plasmid Puc19-HCN2 and ligate it into pcDNA3.1A plasmid to construct plasmid pCDNA3.1A-HCN2;

[0025] (3) Cut t...

specific Embodiment

[0027] 1. Construction of shuttle plasmid pCDH1-GFP-HCN2:

[0028] The target sequence of hHCN2 is inserted into the pcDNA3 vector, and the insertion sites are EcoR I and Xba I. First, use EcoR I and Xba I to cut the target fragment from pCDNA3-hHCN2 plasmid and connect it to the Puc19 vector, then use EcoR I and Hind III Cut the target fragment from the plasmid Puc19 and connect it to the pcDNA3.1(A) vector, and then double cut the plasmid pcDNA3.1(A) into pCDH1-MCS1-EF1-copGFP with Xba I single enzyme. Finally, the positive plasmid was sent to Shanghai Shenggong for gene sequencing and identification to observe whether the hHCN2 gene was inserted into the shuttle plasmid.

[0029] 1. Preparation: extraction of pCDNA3-hHCN2, Puc19, pcDNA3.1(A)pCDH1-MCS1-EF1-copGFP plasmid

[0030] A small amount of plasmid was extracted by alkaline lysis method. First configure the following solutions:

[0031] Solution I (P1) Solution II (P2) Solution III (P3)

[0032] Buffer P1 (resuspension buffe...

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Abstract

The invention discloses a construction method of a recombinant lentiviral vector in a pacemaking channel gene subtype that is HCN2. The construction method comprises the following steps: shuttle plasmids provided with HCN2 genes and a virus system are adopted to constructed to HCN2 recombinant virus vectors together; the shuttle plasmids are pCDH1-GFP-HCN2, and the virus is lentivirus. In the method, the shuttle plasmids that are the pCDH1-GFP-HCN2 and provided with the HCN2 genes and the lentivirus are utilized to construct the HCN2 recombinant virus vectors together, so that the construction method becomes the basic of the cellular therapy and the gene therapy for chronic arrhythmia.

Description

Technical field [0001] The invention relates to a method for constructing a recombinant lentiviral vector of the pacing channel gene subtype HCN2. Background technique [0002] The mammalian genome encodes four HCN genes, named HCN1~HCN4. These four isoforms have been found in the heart, but there are differences in the expression of these isoforms in different species, different heart tissues and different stages of development: adult animals of different species all express HCN4 predominantly, which accounts for approximately 80% of the total HCN of the atrial knot. Among the four isoforms, HCN1, 2, and 4 are the main part of the heart, and HCN3 is only expressed at low levels in embryonic pacemaker cells. In areas with low pacing activity (such as ventricular muscle), the expression of HCN2 is dominant; while the expression of HCN4 in areas with high pacing activity is dominant. The current research on biological pacing focuses on HCN2 and HCN4. [0003] Most of the current ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/86C12N15/12A61K48/00A61P9/06C12R1/93
Inventor 杨向军周亚峰李红霞韩莲花蒋文平
Owner THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV