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Construction and application of infectious clone of bovine foamy virus

A technology for infectious cloning, bovine foamy virus, applied in the direction of microorganism-based method, determination/inspection of microorganisms, introduction of foreign genetic material using vectors, etc., to achieve high safety effects

Inactive Publication Date: 2013-06-19
TIANJIN INT JOINT ACADEMY OF BIOTECH & MEDICINE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the cellular and humoral immune responses induced by FV are still insufficiently understood, and perhaps this is the key to answering the non-pathogenicity of FVs

Method used

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  • Construction and application of infectious clone of bovine foamy virus
  • Construction and application of infectious clone of bovine foamy virus
  • Construction and application of infectious clone of bovine foamy virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Obtaining the genomic cDNA of Chinese bovine foamy virus BFV3026.

[0057] Use the method of extracting Hirt DNA to extract the DNA of the virus that has not integrated into the cell genome and use it as a template for PCR. The specific method is as follows:

[0058] After the retrovirus infects the host cell, the RNA is first reverse-transcribed into double-stranded cDNA, and then integrated into the host genome. During rapid viral replication, large numbers of non-integrated linear or circular cDNA molecules are often present in the nucleus, especially for foamy viruses. When conventional methods extract host genome cDNA, the proportion of viral cDNA is low (<15%), while the method established by Hirt can significantly increase the abundance of viral cDNA in the host cell DNA sample (commonly referred to as Hirt DNA), the highest Up to 80%. In this paper, referring to Hirt's method with some changes (Hirt, 1967; Bieniasz et al, 1995a; Nandi, 1999), high-copy BFV3026...

Embodiment 2

[0077] Construction of infectious full-length clones of BFV3026.

[0078] The purpose is to construct the full-length clone of Chinese bovine foamy virus BFV3026 isolated in our laboratory by molecular cloning. The pBlueScript SK(-) prokaryotic cloning vector was selected, and the BFV3026 genomic cDNA was ligated into it through restriction sites to obtain several strains of full-length BFV3026 clone pBFV. Specific operations include: purification of PCR products, cloning of PCR products, screening and identification of BFV3026 genome cDNA fragment clones, construction and identification of full-length BFV3026 clone pBFV, and mass preparation of full-length plasmid pBFV.

[0079] First, the purification scheme of PCR products:

[0080] After the PCR product was electrophoresed on a 1% agarose gel by mass percentage, it was purified and recovered using a QIAGEN gel recovery kit. Purified PCR samples were stored at -20°C.

[0081]

[0082] PCR(S1) PCR(S2)

[0083]

[0...

Embodiment 3

[0135] The purpose of screening and phenotypic analysis of bovine foamy virus BFV3026 with infectivity in BFVL cells is to detect the virus activity of all constructed full-length BFV3026 clone pBFV. The BFVL cell line was transfected with the pBFV plasmid, the subsequent luciferase activity was analyzed, and clones with trans-transcription activation activity were initially screened.

[0136] First, the transfection of the plasmid.

[0137] 1. Inoculate BFVL cells in a six-well plate at a density of 2×105 / well, add 2ml of DMEM containing 10% fetal bovine serum and 1% penicillin, and culture in a CO2 incubator at 37°C for 18-24h. Start the transfection operation when the cells cover 50% to 80% of the bottom surface of the well;

[0138] 2. Preparation of DNA / PEI Mixture

[0139] 1) Solution A: Dilute 1 μg of the plasmid to be transfected to 70 μl with DMEM without serum and antibiotics (negative control: pBS plasmid;

[0140] 2) Take 4 μL of PEI (1 mg / Ml, Polysciences, Inc....

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Abstract

The invention relates to a construction and an application of infectious clone of bovine foamy virus. An original viral genome cDNA of the bovine foamy virus 3026 (BFV3026) which is obtained and isolated in China is constructed into infectious clone pBFV. The bovine foamy virus 3026 (BFV3026) is named as Escherichia coli in a classified manner and is preserved at China General Microbiological Center Culture Collection Center at NO.3, NO.1 Courtyard, West Beichen Road, Chaoyang District, Beijing, China on June, 29th, 2011, and the preservation number of the bacterial strain is CGMCC No. 4994. The characteristics of the infection life cycle of the bovine foamy virus 3026 (BFV3026) can be simulated by the infectious clone; active bovine foamy virus can be produced by infection, and a foundation for further studying interactions between the bovine foamy virus and host cells is lain. Besides, the infectious clone can play a role in a process for constructing gene vectors of the bovine foamy virus.

Description

technical field [0001] The technical scheme of the invention relates to the construction and application of bovine foamy virus infectious clones. Background technique [0002] Foamy virus (Foamy virus, FVs) is a special retrovirus, first discovered in 1954, because it does not show any disease, and it is even difficult to link it with any disease, so its research lags far behind Other retroviruses, until recent years, with the development of gene therapy, the non-pathogenicity and unique gene expression regulation mechanism of FVs endowed them with incomparable advantages for the construction of transfer vectors, making their research develop rapidly. [0003] Foamy virus (FV) is named for its ability to induce endoplasmic reticulum swelling and vacuole formation in sensitive cells during tissue culture in vitro, resulting in foam-like lesions. However, the relative foamy virus can cause severe lesions at the cellular level and even lead to cell disintegration, and it has n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C12Q1/70C12Q1/68C12N15/86C12R1/93
Inventor 谈娟乔文涛耿运琪邴铁军
Owner TIANJIN INT JOINT ACADEMY OF BIOTECH & MEDICINE
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