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Use of ligands derived from receptor-binding domain of porcine endogenous retrovirus type b for diagnosing SMVT-related diseases

A technology for infectious diseases and ligands used in the field of measuring SMVT levels in biological samples, which can solve problems such as restricting development

Pending Publication Date: 2021-10-15
METAFORA BIOSYST +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, to date, limited knowledge of potential targets on the human cell surface recognized by various RBDs has limited the development of probes based on such interactions.

Method used

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  • Use of ligands derived from receptor-binding domain of porcine endogenous retrovirus type b for diagnosing SMVT-related diseases
  • Use of ligands derived from receptor-binding domain of porcine endogenous retrovirus type b for diagnosing SMVT-related diseases
  • Use of ligands derived from receptor-binding domain of porcine endogenous retrovirus type b for diagnosing SMVT-related diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0350] Example 1: Automated screening for PERVB-RBD identifies SLC5A6 as a PERVB-RBD cognate receptor

[0351] Materials and Methods

[0352] Three independent screens were performed on PERV-B-RBD (SEQ ID NO: 13) binding of a total of 172 solute carrier protein (SLC) family members using the Robot, images and fluorescence intensity parameters were acquired by Cellomics (ArrayScan XTI HCS Thermo Scientific).

[0353] 20,000 quail QT6 cells per well were seeded in 96-well plates coated with poly-D-lysine. After 24 hours, cells were transfected with 100 ng of SLC expression vector using JetPrime transfection reagent (Polypus Transfection 114-15) according to the manufacturer's instructions. QT6 cells were chosen because the spontaneous binding of PERV-B.RBD ligand was the lowest of the cell lines considered and tested. The next day cells were washed with PBS and incubated in fresh DMEM / FBS for 48 hours prior to binding assay. Binding assays were performed on transfected ad...

Embodiment 2

[0356] Example 2: Binding specificity of PERV-B.RBD ligands to SMVT / SLC5A6

[0357] Materials and Methods

[0358] Will 5x10 5 HEK293T cells were seeded on 6-well plates and transfected with siRNA against firefly luciferase gene (siLUC, 5'-CUUACGCUGAGUACUUCGA-3'-SEQ ID NO: 15), or siRNA against SLC5A6 gene (siSMVT, 5'-GGAUGAGUCUUGGUGUGUUTT-3'-SEQ ID NO: 16). Forty-eight hours after transfection, cells were deplated with PBS containing 1 mM EDTA. For binding assays, 1x10 5 3 cells were resuspended in 100 μl of PBA (PBS with 2% FBS) containing saturating concentrations of PERV-B.RBD (SEQ ID NO: 13) or human T-cell leukemia type 2 known to bind SLC2A1 / GLUT1 Virus (HTLV2)-RBD and incubated at 37°C for 30 minutes. Cells were then washed twice with 100 μl of PBA, incubated with Alexa488-conjugated anti-mouse IgG1 for 20 min at 4°C, washed twice and resuspended in 200 μl of PBA. Results were acquired by flow cytometry on a FACSCalibur (Becton Dickinson) and analyzed by FlowJo...

Embodiment 3

[0361] Example 3: Binding specificity of PERV-B.RBD ligands to SMVT / SLC5A6, assessed using secondary anti-SMVT siRNA

[0362] Materials and Methods

[0363] Will 5x10 5 HEK293T cells were seeded on 6-well plates and transfected with: siRNA against the firefly luciferase gene (siLUC, 5'-CUUACGCUGAGUACUUCGA-3'-SEQ ID NO: 15), or against the SLC5A6 gene and different from the example siRNA of the siRNA used in 2 (siSMVT A, 5'GCAGGAUCAUGCCAGAAAUTT-3'-SEQ ID NO: 23). Forty-eight hours after transfection, cells were deplated with PBS containing 1 mM EDTA. For binding assays, 1x10 5 Each cell was resuspended in 100 μl of PBA (PBS containing 2% FBS) containing a predetermined saturating concentration of PERV-B.RBD (SEQ ID NO: 13) or RBD from a heterophile murine retrovirus (XRBD, which receptor known as XPR1 / SLC53A1) or HTLV2-RBD (H2, whose receptor is known as GLUT1 / SLC2A1) or RBD derived from bovine leukemia virus (BLV) (whose receptor is known as CAT1 / SLC7A1) or not Control ...

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Abstract

The present invention relates to methods for detecting and / or measuring the level of sodium-dependent multivitamin transporter (SMVT) in a biological sample, wherein said method comprises the steps of: a. contacting said biological sample with a PERV-B.RBD ligand, a variant and / or a fragment thereof; and, b. detecting and / or measuring the binding of said PERV-B.RBD ligand, variant and / or fragment thereof to SMVT.

Description

technical field [0001] The present invention relates to the diagnosis of diseases associated with sodium-dependent multivitamin transporter / solute carrier family 5 member 6 (SMVT / SLC5A6). In particular, the present invention relates to methods of measuring SMVT levels in biological samples using receptor binding domain (RBD) ligands associated with the envelope protein of porcine endogenous retrovirus type B (PERV-B). Background technique [0002] The sodium-dependent multivitamin transporter (SMVT), the product of the solute carrier family 5 member 6 (SLC5A6) gene, is a 12-transmembrane protein responsible for the cellular uptake of biotin, pantothenic acid, and lipoic acid (Wang et al., J Biol Chem. 1999 May 21; 274(21): 14875-83; Prasad et al., J Biol Chem. 1998 Mar 27; 273(13): 7501-6). Therefore, it plays a vital role in the synthesis and metabolism of proteins, carbohydrates and lipids. Biotin deficiency results in a variety of clinical abnormalities including, for e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68A61K49/00C07K14/005
CPCG01N33/6845G01N33/6872G01N2333/15C07K14/005A61K49/00
Inventor M·西特邦V·佩蒂特S·伊万诺瓦J-L·巴蒂尼V·库诺德D·焦万尼尼J·莱扎
Owner METAFORA BIOSYST
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