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Targeted gene disruption methods and immunogenic compositions

A technology of immunogenicity and composition, applied in the field of targeted gene destruction and immunogenic composition

Pending Publication Date: 2020-03-24
KANSAS STATE UNIV RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although random mutations have been successfully generated using transposon mutagenesis, for obligate bacteria, generation of targeted mutations in specific genes of interest followed by complementation is problematic and an obsessively pursued goal

Method used

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  • Targeted gene disruption methods and immunogenic compositions
  • Targeted gene disruption methods and immunogenic compositions
  • Targeted gene disruption methods and immunogenic compositions

Examples

Experimental program
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Effect test

Embodiment 1

[0074] Materials and methods

[0075] In Vitro Culture of Ehrlichia Chaffee . In the ISE6 tick cell line, Ixodes scapularis (I. scapularis) embryonic cell line for continuous culture of Arkansas isolates of Ehrlichia chaffae. Ehrlichia chaffeensis was also cultured using a canine macrophage cell line (DH82) following a previously reported protocol (Walker, D.H., Paddocl, C.D. and Dumler, J.S., Med Clin North Am; 92, 1345-1361 (2008)).

[0076] Construction of Homologous Recombination Plasmids and Fragments . All primers used to prepare the recombinant plasmid constructs developed for targeted mutagenesis experiments are described in Table 1. Table 2 lists the plasmids used and prepared in this study.

[0077] Table 1: List of oligonucleotides used in this study.

[0078] Capitalized sequences are gene-specific. The lowercase font sequences are overlaps assembled by Gibson.

[0079]

[0080]

[0081]

[0082]

[0083] *If a primer is listed multiple times...

Embodiment 2

[0093] Materials and methods. In Vitro Culture of Ehrlichia canis and Anaplasma phagocytophilum . In ISA6 Continuous culture of Ehrlichia canis and Anaplasma phagocytophilum in a tick cell line, an Ixodes scapularis (I. scapularis) embryonic cell line.

[0094] Construction of Homologous Recombination Plasmids and Fragments . All primers used to prepare recombinant plasmid constructs were developed for targeted mutagenesis experiments following a similar protocol to what we described for Ehrlichia chaffeensis, except that gene-targeting specific primers for this pathogen were used. Similarly, the detailed molecular steps followed to prepare constructs for allelic exchange mutagenesis experiments were the same as those for Ehrlichia chaffae by following standard molecular methods (Sambrook, J. & Russell, D.W. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. (2001)) described a similar procedure. Briefly, approximately 2...

Embodiment 3

[0100] Materials and Methods

[0101] In Vitro Culture and Recovery of Cell-Free Ehrlichia Chaffee

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Abstract

Targeted disruption of a specific gene and its subsequent restoration in obligate intracellular bacteria remains extremely challenging due to their absolute requirement for residence inside a host cell to replicate. Here, targeted allelic exchange mutations were created to inactivate two genes and then to restore one of the two genes of a rickettsial pathogen, Ehrlichia chaffeensis. These methodswere then also successfully utilized in Ehrlichia canis and Anaplasma phagocyophilum. The resultant mutated pathogens are useful in immunogenic compositions for reducing the incidence of or severity of infection with ricksettsial pathogens.

Description

[0001] This invention was made with Government support under NIH Grant No. AI070908. The government has certain rights in this invention. [0002] Background of the invention [0003] Disrupting specific gene function and subsequently restoring its activity in obligate intracellular bacteria remains extremely challenging because obligate intracellular bacteria absolutely need to reside within the host cell in order to replicate. Here, we created targeted mutations and genetically complemented one gene by allelic exchange of two genes of the rickettsial pathogen Ehrlichia chaffeensis. In principle, this method can be applied to other obligate intracellular bacteria and will enable routine structure-function analysis in intracellular bacteria. This method is also applicable to the generation of attenuated strains of obligate intracellular bacteria, which would be valuable when used as live vaccine candidates. [0004] Obligate intracellular bacteria (hereinafter referred to as ...

Claims

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Application Information

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IPC IPC(8): A61K35/74A61K39/00A61K39/118C12N1/20C12N1/21C12N15/09A61P31/04
CPCA61K39/0233A61K39/118C07K14/195C12N1/20A61K2039/522A61P31/04C07K14/29C07K14/295A61K2039/552Y02A50/30
Inventor R·R·甘塔Y·王
Owner KANSAS STATE UNIV RES FOUND
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