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Cloning and expression of arnox protein transmembrane 9 superfamily (tm9sf), methods and utility

A protein and DNA molecule technology used in molecular biology and biochemistry to solve problems such as linking changes in cells and tissues

Inactive Publication Date: 2012-07-11
NOX TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has been difficult to link changes in mtDNA itself to other forms of cellular and tissue changes associated with aging

Method used

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  • Cloning and expression of arnox protein transmembrane 9 superfamily (tm9sf), methods and utility
  • Cloning and expression of arnox protein transmembrane 9 superfamily (tm9sf), methods and utility
  • Cloning and expression of arnox protein transmembrane 9 superfamily (tm9sf), methods and utility

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0411] Example 1. Cloning and expression of soluble arNOX protein transmembrane 9 superfamily (TM9SF) isoform 4

[0412] The pET11b vector and BL21(DE3) competent cells were purchased from Novagen (Madison, WI). A plasmid carrying the TM9SF4 sequence was prepared by inserting the soluble TM9SF4 coding sequence into the pET11b vector (between the NheI and BamHI sites). The TM9SF4 sequence was amplified from full-length cDNA by PCR. The primers used were 5'-GATATACATATGGCTAGCATGGCGACGGCGATGGAT-3' (forward) (SEQ ID NO: 11) and 5'-TTGTTAGCAGCCGGATCCTCAGTCTATCTAGC-3' (reverse) (SEQ ID NO: 12). The PCR product was then double digested with NheI and BamHI and ligated into pET11B vector.

[0413] The DNA sequence of the ligation product (pET11b-TM9SF4) was confirmed by DNA sequencing. Then pET11b-TM9SF4 was transformed into BL21(DE3) competent cells. Single clones were picked and inoculated into 5ml LB+ampicillin (LB / AM) medium. The overnight culture (1 ml) was diluted into 100 m...

Embodiment 2

[0420] Example 2. Characterization of recombinant arNOX

[0421]The reduction of ferricytochrome c by superoxide has been adopted as a standard measure of superoxide formation (Mayo, L.A. and Curnutte, J., 1990, Meth. Enzymol. 186:567-575; Butler, J. et al. al., 1982, J. Biol. Chem. 257:10747-10750). This method is generally accepted for measuring superoxide production when used in conjunction with superoxide dismutase inhibition. The assay reagent is dissolved in PBSG buffer solution (8.06g NaCl, 0.2g KCl, 0.18g NaCl) by 150μl 2 HPO 4 , 0.13g CaCl 2 , 0.1g MgCl 2 , 1.35g of glucose dissolved in 1000ml of deionized water, adjusted to pH 7.4, filtered and stored at 4 ° C) in the buffer coating material composition. The reduction of ferricytochrome c by superoxide was monitored by the increase in absorbance at 550 nm compared to the absorbance at 540 nm (Butler et al., 1982). As another control for the specificity of arNOX activity, 60 units of superoxide dismutase (SOD) w...

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Abstract

Described are cell surface and circulating markers for aging related disorders (specific isoforms of NADH oxidase (arNOX)). Recombinant age-related NADH oxidase isoforms and their coding sequences and methods for detecting arNOX isoform presence and quantitation in tissues and in blood, sera, urine, saliva, perspiration and in other body fluids, are provided. Recombinant arNOX proteins are useful in preparing antigens for use in the generation of monoclonal and polyclonal antibodies as well as immunogenic compositions for diagnosis and treatment of aging disorders. DNA probes based on the DNA sequence information provide may be used to identify individuals at risk for aging disorders and for development of therapeutic interventions or anti-aging cosmetic or other formulations of benefit in slowing the aging process in mammals.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application 61 / 234,368, filed August 17, 2009, which is incorporated herein by reference to the extent not inconsistent with the present disclosure. Background technique [0003] The present disclosure relates to the fields of molecular biology and biochemistry, in particular, the present invention relates to the prevention or treatment of conditions caused by oxidative damage using aging-specific isoforms of NADH oxidase (arNOX) , and relates to its use as a circulating marker for aging-related disorders, recombinant expression, and screening assays for its expression or inhibitors. [0004] The present inventors have elucidated a cell surface protein (named NOX) with hydroquinone (NADH) oxidase activity, which functions as a terminal oxidase for plasma membrane electron transport to complete a process involving cytosolic hydroquinone reductase, located at the plasma me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/00C12P21/00
CPCG01N2333/90209C07K16/28C12N9/0036A61K39/00G01N33/573A61P37/04A61P43/00
Inventor D·J·摩尔唐晓宇S·迪克C·梅德思D·M·摩尔
Owner NOX TECH