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Recombinant virus, escherichia coli retaining the same and a process for production thereof

A technology for recombining viruses and viruses, applied in the directions of viruses/phages, biochemical equipment and methods, viruses, etc., can solve the problem of not getting Escherichia coli and other problems, and achieve the effect of simple virus modification

Inactive Publication Date: 2013-03-27
THE UNIV OF TOKYO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, with regard to HSV-2, Escherichia coli containing a recombinant virus substantially maintaining the full length of the genome has not been obtained

Method used

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  • Recombinant virus, escherichia coli retaining the same and a process for production thereof
  • Recombinant virus, escherichia coli retaining the same and a process for production thereof
  • Recombinant virus, escherichia coli retaining the same and a process for production thereof

Examples

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Embodiment 1

[0058] (Method for preparing recombinant virus in which fluorescent protein expression cassette is inserted in intergenic region)

[0059] (1) Construction of transfer plasmid

[0060] The 270bp fragment containing the UL26.5 promoter region (nt 51388 to nt 51673) of p26.5-Venus, HSV-1 (F), Venus fluorescent protein gene, and the bidirectional polyA signal of HSV-1 UL21 and UL22 was cloned into pBluescript II KS+ (Stratagene). The SacI-KpnI fragment of p26.5-Venus was cloned into the BamHI site of pBR442 (J. Virol. 65:938-944, 1991) to prepare the transfer plasmid p26.5-Venus in UL3-4. Preparation of a PacI site-introduced PacI site in the region between the polyA signals of UL50 and UL51 by cloning the Bg1II-EcoRI fragment (nt 106750 to nt 110095) of HSV-1 (F) containing UL50, UL51 into pBluescript II KS+ pUL50-51pac. The SacI-KpnI fragment of p26.5-Venus was cloned into the PacI site of pUL50-51pac to prepare the transfer plasmid p26.5-Venus in UL50-51. Preparation of a ...

Embodiment 2

[0078] (1) Construction of pBS246GFPBAC

[0079] pEGFP-C1 (Clontech) was digested with BglII, BamHI. Afterwards, the ends are blunted and ligated. The AseI-Af1III fragment was blunt-ended and cloned into the SmaI site of pBS246 (Invitrogen). Next, the SalI fragment of pBe11oBAC11 (Research Genetics) was blunt-ended and cloned into the EcoRV site. ( Image 6 )

[0080] (2) Construction of pBS-2NC-EGFP / BAC

[0081] Genomic DNA Not I-ClaI fragment of HSV-2 strain 186 (n.n.106473 to n.n.110443) was cloned into the Not I-ClaI site (pBS-2NC) of pBliuescriptII KS+ (Stratagene). The NdeI site of the obtained plasmid was blunted, and the fragment obtained by blunting the end of the NotI fragment of pBS246GFPBAC was cloned to construct pBS-2NC-EGFP / BAC.

[0082] (3) Construction of YK351

[0083] Rabbit skin cells (RSC) were transfected with pBS-2NC-EGFP / BAC and viral DNA of HSV-2 strain 186. Afterwards, under a fluorescent microscope, the recombinant virus YK351, which was gene...

Embodiment 3

[0084] Embodiment 3 (the construction of YEbac356)

[0085] Vero cells were infected with YK351, and circular virus DNA was extracted from the infected cells by the Hirt method. The extracted circovirus DNA was introduced into Escherichia coli DH10B (Invitrogen) by electroporation, and screened on a medium supplemented with chloramphenicol to obtain Escherichia coli YEbac356 which maintained the YK351 genome. ( Figure 7 )

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Abstract

To provide a method for producing a recombinant virus retaining the characteristics of the genome of a target virus. The method for producing a recombinant virus including inserting a foreign gene into a region between polyA signals of two genes encoded in directions opposing one another in the genome of a target virus.

Description

technical field [0001] The present invention relates to a method for preparing a recombinant virus useful for vaccine development or gene therapy vector development, a recombinant virus that maintains the full-length genome of herpes simplex virus type II, and Escherichia coli that maintain the recombinant virus. Background technique [0002] In order to prepare recombinant viruses known as gene therapy vectors or multivalent vaccines, foreign genes must be inserted in any region of the viral genome. However, there is a problem that the obtained recombinant virus lacks or changes the gene of the virus genome of the original gene therapy vector or vaccine. [0003] As conventional methods for producing recombinant viruses, homologous recombination in cultured cells using marker (TK, LacZ) genes (Non-Patent Document 1) and homologous recombination in cultured cells using cosmids are known. Recombination method (Non-Patent Document 2), etc., but these existing methods are all ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N1/21C12N7/02
CPCC12N2710/16643C12N15/85C12N15/86C12N2800/101C12N7/00
Inventor 川口宁森本智美
Owner THE UNIV OF TOKYO