Immunogenic peptide of humvoltage-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.
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Embodiment 1
[0030] Synthesis of Example 1 Antigen
[0031] According to the hydrophilicity, exposure and flexibility of the amino acids of the extracellular loop peptide between the transmembrane fragments S5 and S6 of the hKv1.5 channel α subunit, the Jameson-Wolf algorithm was used to select the inclusions with high antigenic index by computer. A peptide segment of 13 consecutive amino acids-hKv1.5 extracellular loop 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, PSSM8 automatic peptide synthesizer was used to synthesize hKv1.5 channel extracellular loop peptide E313 by solid-phase method. The peptide-BSA complete antigen was synthesized by using glutaraldehyde coupling method with BSA as the carrier protein. After the antigen synthesis was completed, it was stored in a -70°C refrigerator for later use.
Embodiment 2
[0032] Example 2 Preparation of Anti-hKv1.5 Extracellular Loop Peptide E313 Antibody
[0033] (1) Animal immunity
[0034] The prepared antigen was immunized together with complete or incomplete Freund's adjuvant to New Zealand white rabbits (clean grade, body weight 2-3 kg) to prepare anti-hKv1.5 extracellular loop peptide E313 antibody. Set up the immunization group and the pseudo-immunization group, and the immunization group: take 800 μg of antigen per rabbit for the initial immunization, and 400 μg of antigen per rabbit for subsequent immunization, add an equal volume of Freund’s adjuvant to both, complete Freund’s adjuvant for the first immunization, and use Incomplete Freund's adjuvant, fully emulsified, injected subcutaneously on the back and hind legs of rabbits. Booster immunization every two weeks, a total of 5 times. Sham immunization group: Sham immunization with normal saline plus Freund's adjuvant, the dose and method were the same as the immunization group. ...
Embodiment 3
[0039] Example 3 Detection of anti-hKv1.5 extracellular loop peptide E313 antibody titer by ELISA
[0040] The serum of healthy New Zealand white rabbits with pseudo-immunization was used as negative control, and the serum of rabbits at different time periods was tested. The synthesized peptide was coated on the enzyme-linked plate at 1 μg per well, and the prepared primary antibody and HRP-labeled goat anti-rabbit IgG, TMB / H were added successively by square matrix titration. 2 o 2 color, dilute H 2 SO 4 Stop the reaction. Measure the absorbance OD value at a wavelength of 450mm in a microplate reader, and take (OD value of the well to be tested-OD value of the blank well) / (OD value of the negative control well-OD value of the blank well)≥2.1 as positive, and <2.1 as negative. Three negative controls were set up for each experiment. Take 1 positive sample and repeat the test within the same plate or between different plates.
[0041] The ELISA test found that the antibo...
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