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Angiotensin II immunogen and preparation method thereof

An angiotensin and immunogen technology, applied in the field of immunity, can solve the problems of reducing the reactogenicity of angiotensin II molecules, unfavorable activities of angiotensin II molecules, and the steric hindrance effect of carrier proteins, so as to reduce the steric hindrance Effect, Improving Immunogenicity, Effect of Enhancing Immunogenicity

Active Publication Date: 2019-01-25
NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the existing angiotensin II immunogen preparation technology, the N-terminal is directly introduced into the Cys-coupled carrier protein, and the antigen-binding site of the angiotensin II antibody obtained after immunization of animals can easily contain Cys amino acid residues, thereby reducing the effect on vascular tension. reactogenicity of the angiotensin II molecule; and the previously reported conventional 2-6 carbon chain linking arm is basically a rigid peptide chain, which is not conducive to the activity of the angiotensin II molecule, easily causes the steric hindrance effect of the carrier protein, and reduces the antigen immunity Originality

Method used

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  • Angiotensin II immunogen and preparation method thereof
  • Angiotensin II immunogen and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Preparation of angiotensin II immunogen: first design angiotensin II derivative sequence, sequence: Cys-(Gly)2-(Ser)3-(Gly)2-Asp-Arg-Val-Tyr-Ile-His- Pro-Phe, ExPASy-ProtParam predicted molecular weight is 1.57×103Da, isoelectric point PI is 6.74. The designed sequence was handed over to Shanghai Jier Biochemical Co., Ltd. to synthesize the above peptide chain with an automatic peptide synthesizer. The purity of the synthetic peptide is identified by high-performance liquid chromatography and hydrolysis amino acid analysis, and the purity is ≥98%. After concentration and freeze-drying, the freeze-dried powder of the synthetic peptide is obtained and delivered in 2mg / tube.

[0025] The lyophilized powder of angiotensin II derivatives was first dissolved in 100 μl of acetonitrile, then mixed with 400 μl of deionized water to prepare a 4 mg / ml solution of angiotensin II derivatives. Dissolve 0.5 mg cross-linker Sulfo-SMCC in 100 μl deionized water. 5mgKLH (4.0×106Da) was...

Embodiment 2

[0031] Immunization of mice with angiotensin II immunogen: Dilute the angiotensin II immunogen prepared in Example 1 to 1 mg / ml with 0.01M pH7.4 phosphate buffer, mix with an equal volume of Freund's complete adjuvant, Fully emulsify with MXR-C350 emulsifier (Shanghai Muxuan), and subcutaneously immunize 5 mice at multiple points on the abdomen and back, each with 50 μg. After 3 weeks, take 1 mg / ml of angiotensin II immunogen and Freund’s incomplete adjuvant The mixture was fully emulsified after mixing, and the mice were boosted for the first time, followed by three booster immunizations every 2 weeks, and blood was taken from the eyes of the mice to measure the titer. In this experiment, the immunogen KLH-ANGII without tether and the immunogen KLH-C6-ANGII with 6 carbon rigid tethers were also immunized with 3 mice for comparison, and the serum of one unimmunized mouse was used as a negative control.

Embodiment 3

[0033] Competitive inhibition ELISA method was used to detect the reactivity of antiserum and angiotensin II molecules. Prepare coating buffer (weigh 1.59g Na2CO3, 2.93g NaHCO3 and dilute to 1L with pure water), wash buffer (weigh 8.0g NaCl, KCl 0.2g, Na2HPO4·12H2O 2.9g, KH2PO4 0.2g, Tween-20 0.5mL, dilute to 1L with pure water), blocking solution (weigh NaCl 8.0g, KCl 0.2g, Na2HPO4·12H2O 2.9g, KH2PO4 0.2g , 50g of skimmed milk powder and dilute to 1L with pure water).

[0034]Dilute OVA-G2S3G2-ANGII to 1 mg / L with coating buffer, spread 100 μL per well into a 96-well ELISA plate, and incubate at 37 °C for 1 hour; discard the sample, wash 3 times with 200 μL per well of washing buffer, and shoot Dry the liquid in the well, add 200 μL of blocking solution to each well, and incubate at 37°C for 1 hour; discard the blocking solution, and wash 3 times with 200 μL of washing buffer per well. 50 μL of the serum of each mouse diluted with blocking solution at a ratio of 1:2000 was m...

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Abstract

The invention provides angiotensin II immunogen and a preparation method thereof. According to the angiotensin II immunogen and the preparation method thereof, when the N-end of angiotensin II is connected to carrier protein, a derivative of the angiotensin II is firstly synthesized, in the derivative of the angiotensin II, a segment of polypeptide is added to the N-end of the angiotensin II to serve as a connecting arm, the connecting arm comprises a flexible peptide, the flexible peptide is composed of Gly and Ser amino acid residues, the position of the Gly are distributed on the two sidesof the Ser, and therefore the torsion of the connecting arm is facilitated; and the derivative of the angiotensin II can effectively reduce the steric-hindrance effect of the carrier protein on smallmolecule hapten of the angiotensin II, and -OH on side chains of the Ser can improve the water solubility of molecules of the angiotensin II to a certain extent. The prepared angiotensin II immunogenhas the advantages of being simple in preparation method, low in cost, strong in immunogenicity and suitable for preparation of an angiotensin II antibody.

Description

technical field [0001] The invention relates to the technical field of immunization, in particular to an angiotensin II immunogen and a preparation method thereof. Background technique [0002] Angiotensin is a peptide hormone that causes vasoconstriction and blood pressure increase. It is a component of the renin-angiotensin system (RAS). Angiotensin is a group of degradation products derived from angiotensinogen, mainly including angiotensin Angiotensin I (AngI), Angiotensin II (Ang II), Angiotensin III (Ang III), Angiotensin IV (Ang IV), Ang(1-9), Ang(1-7), Ang(1 -5) etc. AngII is the main biologically active peptide in RAS. By binding to specific AngII receptors, it has a significant impact on systemic circulation hemodynamics and cardiovascular structure and function. Angiotensin II (Ang II) can directly contract vascular smooth muscle, increase the release of norepinephrine from sympathetic nerve endings, reduce the reuptake of norepinephrine, and promote the release...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K16/26C07K16/06
CPCC07K7/14C07K16/06C07K16/26C07K2319/40
Inventor 邹炳德邹继华赵金华李燕鸿武强何进军贾江花
Owner NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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