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BASB 111 polypeptide and polynucleotide from moraxella cathraahalis

A polynucleotide and nucleotide sequence technology, applied in the field of Moraxella catarrhalis BASB111 polypeptides and polynucleotides, can solve problems such as low frequency

Inactive Publication Date: 2002-11-06
GLAXOSMITHKLINE BIOLOGICALS SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Other bacteria (Haemophilus influenzae type b, Streptococcus pyogenes, etc.) can also be isolated from the middle ear, but at a much lower frequency (2% or less)

Method used

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  • BASB 111 polypeptide and polynucleotide from moraxella cathraahalis
  • BASB 111 polypeptide and polynucleotide from moraxella cathraahalis
  • BASB 111 polypeptide and polynucleotide from moraxella cathraahalis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0207] Example 1: Discovery and confirmatory DNA sequencing of the BASB111 gene in M. catarrhalis strain ATCC 43617

[0208] The BASB111 gene sequence of M. catarrhalis strain ATCC43617 (also known as MC2931) is shown in SEQ ID NO:1. The translation of the BASB111 polynucleotide sequence is shown in SEQ ID NO:2.

Embodiment 2

[0209] Embodiment 2: Construction of the plasmid expressing recombinant BASB111

[0210] A: Cloning of BASB111

[0211] Insert the EcoRI and SalI restriction sites into forward MC-Lip2-Fn / t-RI (5′-AGGCAG AGG GAA TTC ATG AAT TTT GGT AAA ATT AAT GG-3′, SEQ ID NO: 3) and reverse MC- Lip2RCh / t-Sal (5′-AGG CAG AGG GTC GAC TTAATG GTG ATG GTG ATG GTG CCA GCC TTT GAT AAC ACC ATCTT-3′, SEQ ID NO: 4) amplification primers that allow directional cloning of PCR products into E. coli In the expression plasmid pTLZ2, the BASB111 protein can be expressed as a fusion protein containing (His)6 affinity chromatography marker at the C-terminus. The BASB111 PCR product was purified from the amplification product using a silica-based spin column (QiaGen) according to the manufacturer's instructions. To generate the EcoRI and SalI ends required for cloning, the purified PCR product was sequentially digested to completion with EcoRI and SalI restriction enzymes according to the manufacturer's (Lif...

Embodiment 4

[0218] Example 4: Production of recombinant BASB111

[0219] bacterial strain

[0220] A recombinant expression strain of E. coli JM109 containing a plasmid (pTLZ2) encoding M. catarrhalis BASB111 was used to generate a cell population for purification of the recombinant protein. Expression strains were cultured on LB agar plates containing 100 μg / ml ampicillin (Ap) to ensure the presence of the pTLZ2 plasmid. For storage at -80°C, strains were propagated in LB broth containing the same concentration of antibiotics, and then mixed with an equal volume of LB broth containing 30% (w / v) glycerol.

[0221] culture medium

[0222]The fermentation medium for recombinant protein production was 2X YT broth (Difco) containing 100 μg / ml Ap. Antifoam was added to the medium in the fermenter to 0.25ml / L (Antifoam204, Sigma). To induce the expression of BASB111 recombinant protein, IPTG (isopropyl β-D-thiogalactopyranoside) (1 mM, final concentration) was added to the fermentor.

[02...

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PUM

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Abstract

The present invention provides BASB111 polypeptides and polynucleotides encoding BASB111 polypeptides as well as methods of producing such polypeptides by recombinant technology. The invention also provides diagnostic, prophylactic and therapeutic applications.

Description

field of invention [0001] The present invention relates to polynucleotides (herein referred to as "BASB111 polynucleotides"), their encoded polypeptides (herein referred to as "BASB111" or "BASB111 polypeptides"), recombinant substances and methods for their preparation. In another aspect, the invention relates to methods of using such polypeptides and polynucleotides, including vaccines against bacterial infections. In yet another aspect, the invention relates to diagnostic assays for detecting infection by certain pathogens. Background of the invention [0002] Moraxella catarrhalis (Branhamella catarrhalis) is a Gram-negative bacterium frequently isolated from the upper respiratory tract of humans. It causes several diseases, mainly otitis media in infants and young children and pneumonia in the elderly. It also causes sinusitis and nosocomial infections, and occasionally invasive disease. [0003] Otitis media is an important childhood disea...

Claims

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Application Information

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IPC IPC(8): G01N33/53A61K39/00A61K39/02A61K48/00A61P31/04C07K14/195C07K14/21C07K16/12C12N1/15C12N1/19C12N1/21C12N5/10C12N15/09C12N15/31C12P21/02C12R1/19G01N33/569
CPCA61K39/00A61K48/00C07K14/212A61P31/00A61P31/04
Inventor J·通纳德
Owner GLAXOSMITHKLINE BIOLOGICALS SA
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