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Recombinant coxsackie B3 virus with fluorescent protein tag and construction method

A fluorescent protein and construction method technology, applied in the field of recombinant Coxsackie B3 virus and construction, can solve the problem of insufficient and in-depth analysis of the dynamic process of the virus, and achieve the effect of a stable and reliable research and application platform

Pending Publication Date: 2022-06-28
YICHANG NO 1 PEOPLES HOSPITAL PEOPLES HOSPITAL OF CHINA THREE GORGES UNIV
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

The current studies on the pathogenic mechanism of Coxsackie virus and the tropism of virus tissues mostly rely on immunological methods to detect virus antigens in fixed cells or tissues or use electron microscopy to detect virus particles in fixed cells or tissues. Obviously, these studies However, it is a more convenient and intuitive way to observe and study viruses directly in living cells or in vivo

Method used

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  • Recombinant coxsackie B3 virus with fluorescent protein tag and construction method
  • Recombinant coxsackie B3 virus with fluorescent protein tag and construction method
  • Recombinant coxsackie B3 virus with fluorescent protein tag and construction method

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Embodiment 1

[0036] In order to facilitate the introduction of foreign genes into the CVB3 genome, on the basis of the constructed full-length infectious clone (pCV) of CVB3, this experiment introduced the Multiple Cloning Site (MCS) into the P1 region of the CVB3 genome and P2 region, and the restriction recognition sequence of 2Apro (TTMTNTG / AFGQQSGA) was added before the multiple cloning site. According to the sequencing sequence of pCV, DNASTAR Lasergene 9.0 software was used to design 2 pairs of primers for cloning insertion of MCS and 2Apro restriction recognition sequence with a length of 42bp (Table 1-1). In order to facilitate the integration of the full-length gene into the plasmid, the XhoI restriction site was introduced into the upstream sequence of the first pair of primers, and the SpeI restriction site was designed in the downstream primer sequence of the second pair of primers. The primers used were synthesized by Shenzhen Huada Gene Company.

[0037] Table 2-1 Constructi...

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Abstract

The invention provides a recombinant coxsackie B3 virus with a fluorescent protein tag, the virus is an infectious recombinant virus rCV-CS and a recombinant virus rCV-GFP for expressing GFP, and the sequence is as shown in SEQ ID NO: 1. The construction method comprises the following steps: on the basis of a CVB3 full-length infectious cDNA cloning plasmid pCV, constructing a CVB3 full-length infectious cloning plasmid pCV-CS containing multiple cloning sites MCS, and adding a 2Apro enzyme digestion recognition sequence in front of the MCS; amplifying a GFP fragment by taking the pEGFP as a template to obtain a GFP coding gene; the method comprises the following steps: introducing a GFP coding gene into multiple cloning sites of pCV-CS, and constructing a CVB3 whole-genome infectious cloning plasmid pCV-GFP for expressing the GFP; and transfecting the pCV-GFP through a liposome, and amplifying the pCV-GFP to obtain the recombinant coxsackie B3 virus rCV-GFP with a fluorescent protein tag. The technical scheme of the invention is used for preparing tracer viruses in living animal cells, and a remarkable effect is generated.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a recombinant Coxsackie B3 virus with a fluorescent protein label and a construction method and application. Background technique [0002] Coxsackie virus B3 is a single-stranded positive-stranded small RNA virus, spherical, icosahedral, three-dimensionally symmetrical, without envelope, without protrusions, and the naked protein nucleocapsid wraps nucleic acid to form virions with a diameter of 20-30nm. Coxsackie virus mainly infects humans or animals through the fecal-oral route of transmission. The virus invades the host through the digestive tract and spreads to other tissues and organs. There are many studies on the way of poliovirus infection. Poliovirus may invade the central nervous system by infecting peripheral nerves, or spread the virus to the whole body through blood transport, or through the infection of vascular endothelial cells as a further step. The route o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/86C12N15/12A61K49/00
CPCC12N7/00C12N15/86C07K14/43595A61K49/0045A61K49/0097C12N2770/32621C12N2770/32643
Inventor 曾俊贺茜邓辉雄
Owner YICHANG NO 1 PEOPLES HOSPITAL PEOPLES HOSPITAL OF CHINA THREE GORGES UNIV
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