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Immunogenic peptide of human voltage-gated potassium channel 1.5 (hKv1.5) and purpose thereof

A voltage-gated, immunogenic technology, applied in the biological field, can solve problems such as arrhythmia, blocking Kv1.5 channel blockers, etc.

Inactive Publication Date: 2012-04-04
XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have found that the binding sites of Kv1.5 channels and cardiac repolarization HERG and KCNQ1 / KCNE1 potassium channel blockers are highly conserved, which makes some existing Kv1.5 channel blockers block Kv1.5 channels at the same time , and also block HERG and KCNQ1 / KCNE1 channels, often leading to the occurrence of drug-induced arrhythmias, which hinders the further development of some Kv1.5 channel blockers into clinical drugs
So far, there are no related reports on anti-hKv1.5 extracellular loop peptide E313 antibody

Method used

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  • Immunogenic peptide of human voltage-gated potassium channel 1.5 (hKv1.5) and purpose thereof
  • Immunogenic peptide of human voltage-gated potassium channel 1.5 (hKv1.5) and purpose thereof
  • Immunogenic peptide of human voltage-gated potassium channel 1.5 (hKv1.5) and purpose thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Synthesis of Example 1 Antigen

[0032] According to the hydrophilicity, exposure and flexibility of the amino acids of the extracellular cyclic peptide segment between S5 and S6 of the hKv1.5 channel α subunit transmembrane fragment, the Jameson-Wolf algorithm was used to select the one with high antigen index by computer. A peptide segment of 13 consecutive amino acids-hKv1.5 extracellular cyclic peptide segment E313, the specific amino acid sequence is: Glu-Ala-Asp-Asn-Gln-Gly-Thr-His-Phe-Ser-Ser-Ile-Pro. According to its amino acid sequence, a PSSM8 automatic peptide synthesizer was used to synthesize the hKv1.5 channel extracellular cyclic peptide E313 by solid-phase method. Polypeptide-BSA complete antigen was synthesized by glutaraldehyde coupling method with BSA as carrier protein. After the antigen synthesis was completed, it was placed in a -70°C refrigerator for later use.

Embodiment 2

[0033] Example 2 Preparation of anti-hKv1.5 extracellular cyclic peptide segment E313 antibody

[0034] (1) Animal immunity

[0035] New Zealand white rabbits (clean grade, body weight 2-3 kg) were immunized with the prepared antigen together with complete or incomplete Freund's adjuvant to prepare anti-hKv1.5 extracellular cyclic peptide E313 antibody. The immunization group and the sham immunization group were set up. In the immunization group, 800 μg of antigen was taken from each rabbit for the initial immunization, and 400 μg of antigen was taken from each rabbit for the subsequent immunization, and an equal volume of Freund’s adjuvant was added. Incomplete Freund's adjuvant, after being fully emulsified, was injected subcutaneously on the back and hind legs of rabbits at multiple points. Booster immunization once every two weeks, a total of 5 times. Sham immunization group: sham immunization with normal saline plus Freund's adjuvant, the dose and method are the same as...

Embodiment 3

[0040] Example 3 ELISA detection of anti-hKv1.5 extracellular cyclic peptide segment E313 antibody titer

[0041] The sera of the healthy New Zealand white rabbits of pseudo-immune were used as negative control, and the sera of rabbits at different time periods were tested. The synthesized polypeptide was coated on the enzyme-linked plate with 1 μg per well, and the prepared primary antibody and HRP-labeled goat anti-rabbit IgG were added successively by square array titration, TMB / H 2 O 2 Color development, dilute H 2 SO 4 Terminate the reaction. Measure the OD value of absorbance at the wavelength of 450 mm on the microplate reader, and take (OD value of the well to be measured - OD value of blank well) / (OD value of negative control well - OD value of blank well) ≥ 2.1 as positive, and <2.1 as negative. Three negative controls were set up in each experiment. Take 1 positive sample and repeat the test within the same plate and between different plates.

[0042] ELISA te...

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Abstract

The invention provides an immunogenic peptide of hKv1.5 and a purpose thereof. The peptide comprises continuous thirteen amino acids on an extracellular ring between an alpha-subunit transmembrane segment S5 and an alpha-subunit transmembrane segment S6 of the hKv1.5, and the amino acid sequence of the peptide is Glu-Ala-Asp-Asn-Gln-Gly-Thr-His-Phe-Ser-Ser-Ile-Pro. An antibody which aims at the peptide and is prepared with an immunological method can be used as a new specific inhibitor of the hKv1.5 and a super-rapidly activated delayed rectifier potassium channel coded by gene of the hKv1.5, and can be used to research the adjustment of functions of the hKv1.5 and the super-rapidly activated delayed rectifier potassium channel coded by the gene of the hKv1.5. The preparative monoclonal antibody and a vaccine based on the peptide are helpful for the atrial fibrillation treatment.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the synthesis of an extracellular cyclic peptide segment E313 of a human-derived voltage-gated potassium channel 1.5 and an antibody prepared against the peptide segment, and the use of the antibody as a human-derived voltage-gated potassium channel Application of channel 1.5 and specific inhibitors of ultrafast-activating delayed rectifier potassium channels encoded by its genes. Background technique [0002] Atrial fibrillation, hereinafter referred to as atrial fibrillation, is the most common arrhythmia in clinical practice, which can induce angina pectoris and heart failure, and can lead to serious complications such as vascular embolism and increase mortality. Restoring atrial fibrillation rhythm to sinus rhythm and maintaining long-term rhythm control is an important strategy in the treatment of atrial fibrillation. Studies have shown that electrical remodeling of...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K16/18A61K39/395A61P9/06
Inventor 刘坤杨晓芳连亦田戴芝银杨勇刘金平王彦富王朝晖王敏高翔廖玉华曾秋棠刘琳玲
Owner XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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