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Mutated e. coli heat-labile enterotoxin

A heat-resistant enterotoxin, Escherichia coli technology, applied in the direction of viruses, antibacterial drugs, antiviral agents, etc., can solve problems such as high toxicity restricting clinical applications

Active Publication Date: 2010-03-31
DEV CENT FOR BIOTECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the high toxicity of wild-type LT limits its clinical application

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Construction of encoding wild-type LT A and LT mutant LT A gene

[0020] A 1.8-kb DNA fragment of the LT gene, including subunit A and subunit B, was isolated from human enterotoxigenic E. coli H10407 and cloned into pBluescript IIKS(-) vector (pBluescript-LThWT). The nucleotide sequence (SEQ ID NO: 6) of the LT gene (encoding subunits A and B) and the amino acid sequence (SEQ ID NO: 5) of the subunit A of the LT are as follows:

[0021] Nucleotide sequence of LT (SEQ ID NO: 6) (subunit A: 1-777; subunit B: 774-1148):

[0022] atgaaaaata taactttcat tttttttatt ttattagcat cgccattata tgcaaatggc 60

[0023] gacaaattat accgtgctga ctctagaccc ccagatgaaa taaaacgttc cggaggtctt

[0024] 120

[0025] atgcccagag ggcataatga gtacttcgat agaggaactc aaatgaatat taatctttat

[0026] 180

[0027] gatcacgcga gaggaacaca aaccggcttt gtcagatatg atgacggata tgtttccact

[0028] 240

[0029] tctcttagtt tgagaagtgc tcacttagca ggacagtcta tattatcagg atattccact

[0030] 300

[0031]...

Embodiment 2

[0206] Example 2: Preparation of wild type LT and mutant LT A LT

[0207]E. coli HB101 was transformed with the pBluescript II KS(-) vector containing native or mutant LT genes (including subunit A and subunit B genes). Native and mutant LTs were purified from cultures grown overnight in 3-liter shake flasks containing L-broth supplemented with 100 μg / ml ampicillin. Cells were collected by centrifugation, resuspended in TEAN buffer (0.2M NaCl, 50 mM Tris, 1 mM EDTA and 3 mM NaN3, pH 7.4), and lysed with a microfluidizer (Microfluidics Corporation, USA). After clarification of the lysate by centrifugation, LT was fractionated by addition of solid ammonium sulfate to 65% saturation. The preparation was then suspended in TEAN buffer, extensively dialyzed against the same buffer, and used as crude LT. Crude LT was chromatographed at 4°C on an immobilized D-galactose (Pierce, Rockford, IL) column equilibrated with TEAN buffer (Uesaka et al., 1994, Microbial Pathogenesis 16:71-76...

Embodiment 3

[0213] Example 3: Determination of wild-type LT and mutant-containing LT A Effect of LT on intracellular cAMP levels

[0214] Caco-2 cells (ATCC HTB-37) at 5×10 per well 4 Concentrations of cells were maintained in 24-well plates in MEM-α medium supplemented with 20% FBS, grown to near confluence, and incubated in 5% CO 2 Incubate for 30 min in MEM-α containing 1% FBS and 1 mM 3-isobutyl-1-methylxanthine (IBMX), then add toxin (Grant et al., 1994, Infection and Immunity 62:4270-4278) . Native or mutant LT was added to each well and incubated for 4 hours. Cells were then washed twice with cold PBS. Intracellular cAMP was extracted by adding 200 [mu]l 0.1 N HCl to each well and incubating for 15 minutes at room temperature. Supernatants of cell lysates were collected after addition of 0.1 N NaOH to each well (Cheng et al., 2000, Vaccine 18:38-49; Park et al., 1999, Experimental and Molecular Medicine 31:101-107). cAMP was measured with a cAMP enzyme immunoassay kit (Assay ...

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PUM

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Abstract

This invention relates to a mutant E. coli heat-labile enterotoxin (LT) subunit A that can be used as an adjuvant. This subunit A mutant contains an amino acid substitution at a position correspondingto position 61 of a wild-type LT. An LT containing this mutated subunit A exhibits reduced toxicity compared to its wild type counterpart.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Patent Application Serial No. 11 / 779,419, filed July 18, 2007, the contents of which are hereby incorporated by reference. Background technique [0003] Enterotoxigenic E. coli strains cause diarrhea in humans and domesticated animals by producing two types of enterotoxins, heat-labile toxin (LT) and heat-stable toxin (ST) (Hofstra et al., 1984, J. Bio. Chem. 259:15182-15187). LT is functionally, structurally and immunologically related to cholera toxin (CT) (Clements et al., 1978, Infect. Immun. 21: 1036-1039). LT and CT are synthesized as holotoxin molecules consisting of five identical subunits B and one enzymatically active subunit A (AB 5 ) (Spangler, 1992, Microbio. Rev. 56(4): 622-647). The B pentamer binds to the ganglioside GM1 in the membrane of intestinal epithelial cells or any other GM1-containing cell (van Heyningen, 1974, Science 183:656-657). After subunit B bin...

Claims

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Application Information

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IPC IPC(8): A61K39/02
CPCA61K2039/543A61K2039/5252C12N2760/16134A61K39/145A61K39/0258C07K14/425A61K39/39A61K2039/55544A61K39/12A61P31/04A61P31/12A61P31/14A61P31/16A61P31/18A61P31/20Y02A50/30C07K14/245A61K39/02C12N15/11C12N15/63
Inventor 徐悠深林阳生阮大同
Owner DEV CENT FOR BIOTECHNOLOGY
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