Novel shuttle vector

一种穿梭载体、表达载体的技术，应用在抗肿瘤剂领域

Active Publication Date: 2007-10-31
AZUSAPHARMA SCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since there is a possibility that the plasmid with unmodified DNA will be cut by restriction enzymes in the microorganism during transfection, a higher transformation rate is required in the cloning of foreign genes

Method used

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  • Novel shuttle vector
  • Novel shuttle vector
  • Novel shuttle vector

Examples

Experimental program
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preparation example Construction

[0061] In the preparation of liquid preparations suitable for oral administration, sugars such as water, sucrose, sorbitol, and fructose; alcohols such as polyethylene glycol and propylene glycol; oils such as sesame oil, olive oil, and soybean oil can be used; Additives for preparations such as p-hydroxybenzoate and other preservatives. In addition, in the manufacture of solid preparations such as capsules, tablets, powders, or granules, for example, excipients such as lactose, glucose, sucrose, and mannose; disintegrants such as starch and sodium alginate; stearic acid Lubricants such as magnesium and talc; Binders such as polyvinyl alcohol, hydroxypropyl cellulose and gelatin; Surfactants such as fatty acid esters; Plasticizers such as glycerin.

[0062] Among preparations suitable for parenteral administration, preparations for intravascular administration such as injections and spot drops can be preferably prepared using an aqueous solvent that is isotonic with human bloo...

Embodiment 1

[0069] (Structure Analysis of Plasmid pTB6)

[0070] [Materials and methods]

[0071] 1. Strains, plasmids and media

[0072] The bacterial strains and plasmids used in the present invention are listed in Table 1. In LB broth medium (10 g of Bacto-tryptone), 5 g of yeast extract, 5 g of NaCl and 0.1% glucose / liter), Escherichia coli was cultured aerobically at 37° C. Colonies formed in broth. Supplemented 50mM sucrose, 0.34% cysteine, 0.02% sodium ascorbate MRS broth medium (manufactured by Difco Laboratories, U.S.), cultivated Bifidobacterium longum 105-A anaerobically at 37°C (referring to non-patent Literature 12). Antibiotics (50 μg / ml ampicillin (Ap) and / or 75 μg / ml spectinomycin (sp)) were added as needed. According to the method described in Non-Patent Document 12, colonies were formed on a medium containing 1.5% agar using a Gas-Pak anaerobic system (manufactured by BBL, USA).

[0073] [Table 1]

[0074] strain or plasmid

relevant features

Liter...

Embodiment 2

[0110] (Construction of Shuttle Vector pAV001)

[0111] [Construction of plasmid]

[0112] From pBLES100, the sequence containing spectinomycin adenyltransferase (AAD box) derived from Enterococcus faecalis (Enterococcus faecalis) was amplified by PCR, subcloned into pCR-BluntII-TOPO vector (manufactured by Invitrogen), and pCRTOPO- ScaI-AAD-Eaml105I. In addition, ScaI was added to the forward primer, and an Eaml105I restriction enzyme site was added to the reverse primer.

[0113] As shown in Figure 5, the cloning vector pGFPuv purchased from Invitrogen (DEFINITION: Cloning vector pGFPuv. Accession number: U62636 version: U62636.1 GI: 1490528) consists of the GFPuv gene and the multiple cloning sites (Multi-Cloning Site , MCS), ampicillin resistance gene, OriC (pUC Ori) composition of E. coli plasmid replication origin.

[0114] The ampicillin resistance gene locus of this pGFPuv was cleaved with restriction enzymes Eaml105I and ScaI to prepare a removed long fragment. Si...

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Abstract

The present invention provides a shuttle vector for a microorganism of the genus Bifidobacterium (BM) and Escherichia coli having a wide host range and a large copy number in BM and capable of highly expressing a desired protein when used as an expression vector; an expression vector capable of expressing a desired gene in BM by use of the shuttle vector; BM transformed with the expression vector; and an antitumor agent comprising the BM as an active ingredient. The vector comprises a pTB6-derived region portion comprising a replication origin (oriV)-repB region of pTB6 but not comprising MembB, MobA, OrfI, and oriT regions of pTB6 and an Escherichia coli-derived plasmid portion comprising a replication origin (oriC) region of Escherichia coli but having deleted DNA encoding an N-terminal region of an ampicillin resistance gene (ampR) expression product -lactamase, and constitute a shuttle vector for Escherichia coli with a BM having an average copy number of 6 to 30.

Description

technical field [0001] The present invention relates to a shuttle vector between microorganisms of the genus Bifidobacterium and Escherichia coli, an expression vector capable of expressing a desired gene in microorganisms of the genus Bifidobacterium using the shuttle vector, and microorganisms of the genus Bifidobacterium transformed with the shuttle vector And an antitumor agent using the microorganism of the genus Bifidobacterium as an active ingredient. Background technique [0002] Bifidobacterium longum (Bifidobacterium longum) is a Gram-positive anaerobic bacterium whose genome has a high GC content (see, for example, Non-Patent Document 1). Such Bifidobacterium longum is non-pathogenic, and constitutes most of the normal microbiota in the large intestine of humans and other animals (see, for example, Non-Patent Document 2). It is generally believed that it can: improve immune response (for example, refer to Non-Patent Document 3), have an inhibitory effect on the o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/00C12N15/09A61K35/74A61K38/51A61K48/00A61P35/00C12N1/21C12R1/01A61K35/745C12N15/70C12N15/74
CPCC12N15/70A61K38/00C12N15/746A61P35/00A61P43/00C12N15/64
Inventor 加纳康生滨地芳典藤森实佐佐木贵之河野享子天野纯谷口俊一郎
Owner AZUSAPHARMA SCI INC
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