Bacterial packaging strains usefuel for generation and production of recombinant double-stranded RNA nucleocapsids and uses thereof

A technology of bacteria and strains, applied in the direction of bacteria, sugar derivatives, and vector-borne diseases, which can solve the problem of not providing recombinant fragment integration, not providing rdsRP composition, rdsRP replication and stable preparation, etc. question

Active Publication Date: 2011-08-10
AERAS GLOBAL TB VACCINE FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, 20040132678 does not provide a specific method to stably integrate the recombinant fragment into dsRP, and has little reference to the specific methods of subsequent replication and stable preparation of rdsRP
Also, 20040132678 does not provide an rdsRP composition lacking both wild-type Fragment-M and Fragment-S
Finally, 20040132678 also did not provide packaging strains expressing Fragment-L and producing procapsids, and the resulting strains were able to initiate rdsRP by a de novo synthesis method and stably produce resRP

Method used

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  • Bacterial packaging strains usefuel for generation and production of recombinant double-stranded RNA nucleocapsids and uses thereof
  • Bacterial packaging strains usefuel for generation and production of recombinant double-stranded RNA nucleocapsids and uses thereof
  • Bacterial packaging strains usefuel for generation and production of recombinant double-stranded RNA nucleocapsids and uses thereof

Examples

Experimental program
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Embodiment 1

[0230] Example 1: Recombinant DNA method

[0231] Restriction enzymes ("RE" herein); (New England Biolabs, Beverly, MA), T4 DNA ligase (New England Biolabs, Beverly, MA) and Taq polymerase (Life Technologies, Gaithersburg) were used according to the manufacturer's instructions. , MD); plasmid DNA was prepared using a small-scale (Qiagen MiniprepR kit, Santa Clarita, CA) or large-scale (Qiagen MidiprepR kit, Santa Clarita, CA) plasmid DNA purification kit (Qiagen, Santa Clarita, CA) according to the manufacturer's instructions. ); nuclease-free, molecularly pure deionized water, Tris-HCl (pH 7.5), EDTA pH 8.0, 1M MgCl 2 , 100% (v / v) ethanol, ultrapure agarose, and agarose gel running buffer were purchased from Life Technologies, Gaithersburg, MD. Restriction enzyme digestion, PCR, DNA ligation reaction and agarose gel electrophoresis were performed according to known methods (Sambrook, et al., supra, 1989); (Ausubel, et al., supra, 1990) . In the following examples, the veri...

Embodiment 2

[0237] Example 2: Compensation for the asd mutation and the construction of rdsRNA fragments expressing fluorescent reporters and Mycobacterium tuberculosis antigens and LCMV antigens

[0238] The purpose of this study was to develop a recombinant fragment capable of integrating into a prototype rdsRN based on the phi-8 dsRNA genome (Mindich et al., J.Bacterid, 181:4505; 1999); (Mindich, Microbiol.MoI.Biol.Rev, 63: 149; 1999); (Hoogstraten et al., Virology, 272:218; 2000); (Sun et al., Virology, 308:354; 2003). As mentioned above, the genome of phi-8 consists of three segments: S, M and L. A prototype rdsRN can be constructed so that the RNA-dependent RNA polymerase-expressed passenger gene encoded by wild-type fragment-L (referred to herein as "wtL") is cloned into recombinant fragment-M and -S (in referred to herein as "rM" and "rS", respectively). Both rM and rS encode the wild-type aspartate semialdehyde dehydrogenase gene (referred to herein as "asd", GenBank # V00262) ...

Embodiment 3

[0257] Example 3: Construction of prototype packaging and delivery strains

[0258] The purpose of this study was to construct a prototype bacterial packaging strain. Shigella flexneri possesses an irreversible chromosomal asd marker indel mutation, resulting in a defect in the inability to produce aspartate-semialdehyde dehydrogenase (referred to herein as "ASD"), and is produced by This lacks the ability to synthesize the cell wall component diaminopimelic acid (referred to herein as "DAP") (Sizemore et al., Vaccine. 1997 Jun;15(8):804-7). Growth in the absence of genetic complementation required supplementation of the medium with 50 μg / ml DAP (Sigma-Aldrich, St. Louis, MO, Cat. No. D1377).

[0259]Although Shigella was chosen as an example only, its invasive characteristics and natural tropism for mucosal immune cells also make it an ideal delivery vehicle. Just as the asd mutation needs to be compensated by the rdsRN encoding the asd allele, it is also necessary to const...

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Abstract

Bacterial packaging strains useful for generating recombinant double-stranded RNA nucleocapsids (rdsRNs) are provided. The packaging strains are useful for the production of RNA encoding vaccine antigens, bioactive proteins, immunoregulatory proteins, antisense RNAs, and catalytic RNAs in eukaryotic cells or tissues. Recombinant ssRNA is introduced into the strains and packaged to form rdsRNs de novo.

Description

field of invention [0001] The present invention provides bacterial packaging strains suitable for producing recombinant double-stranded RNA nucleocapsids (rdsRNs), which can be used to produce RNAs encoding vaccine antigens, bioactive proteins, immunomodulatory proteins, antisense RNAs and catalytic RNA. In particular, the present invention also provides a bacterial packaging strain into which recombinant ssRNA can be introduced and packaged to form rdsRN in a de novo synthesis manner, and the rdsRN can be replicated in the packaging strain and thereby produce the target RNA. Background of the invention [0002] Viral nucleocapsids, the nucleoprotein core of viruses, possess a number of features that make them valuable for expressing heterologous gene sequences in biological systems. Lacking the outer membrane and adhesins of intact viruses, nucleocapsids are noninfectious particles composed of the proteins and genetic material of the viral core, which retain the ability to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C07H21/02
CPCY02A50/30
Inventor 大卫·迈克尔·霍恩约翰·富尔克松杰拉尔德·C·萨多夫大卫·奥尼亚贝米谢勒·斯通
Owner AERAS GLOBAL TB VACCINE FOUND
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