Dipeptide derivative for detection of angiotensin Ⅱ and preparation method and application thereof
A derivative, phenylalanine dipeptide technology, applied in biological testing, biomaterial analysis, peptides, etc., can solve the problems of low detection sensitivity, poor specificity, and undetectability, and achieve high immunogenicity and high accuracy , The effect of improving the detection efficiency
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Embodiment 1
[0056] Example 1. Synthesis of proline-phenylalanine dipeptide derivatives
[0057] The chemical structure of proline-phenylalanine dipeptide derivative is shown in formula (I):
[0058]
[0059] The synthetic route and preparation steps of the above-mentioned proline-phenylalanine dipeptide derivatives are as follows:
[0060]
[0061] Concrete synthetic steps are as follows:
[0062] (1) Synthesis of Compound 2
[0063] Weigh 40g (93.6mmol) of compound 1 and dissolve it in 400mL of DCM, then add 24g (112mmol) of compound A, 25.2g (187.2mmol) of 1-hydroxybenzotriazole (HOBT), 36g (187.2mmol) Carbodiimide (EDCI) and 47.2g (480mmol) triethanolamine (TEA) were prepared as reaction mixture solution I. The reaction mixture I was stirred overnight at 30°C. After the reaction, an appropriate amount of purified water was added to the reaction mixture solution I, and then 150 mL of DCM was used for extraction, and the extraction step was repeated 4 times. The extracted orga...
Embodiment 2
[0075] Example 2. Synthesis of Proline-Phenylalanine Dipeptide Immunogen
[0076] The proline-phenylalanine dipeptide immunogen is composed of bovine serum albumin (Bovine Serum Albumin, BSA) and -CO-(CH 2 ) 3 It is formed by connecting -CO- groups, and its structural formula is shown in the following formula (II):
[0077]
[0078] The concrete steps of the synthetic method of this immunogen are as follows:
[0079] 1. Dissolve 200mg bovine serum albumin in 50ml 0.2M, pH 8.5 phosphate buffer;
[0080] 2. Add the following chemicals into a small beaker and stir to dissolve: 200mg of the proline-phenylalanine dipeptide derivative synthesized in Example 1, 3.5ml of dimethylformamide, 3.5ml of ethanol, 7.0ml of 10mM, pH 5.0 potassium phosphate buffer, 200mg 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide, 50mg N-hydroxysulfosuccinimide, these chemicals were stirred and dissolved at room temperature React for 30 minutes;
[0081] 3. Add the dissolved solution dropwise to th...
Embodiment 3
[0082] Example 3. Preparation of anti-angiotensin II specific antibody
[0083] The proline-phenylalanine dipeptide immunogen prepared in Example 2 was inoculated into experimental animal rabbits by conventional methods, and the antiserum was taken after booster immunization. The specific steps were as follows:
[0084] 1. Dilute the proline-phenylalanine dipeptide immunogen synthesized above to 1.0 mg / ml with PBS to obtain an antigen solution, then mix 1.0 ml of the antigen solution with Freund's complete adjuvant, and conduct the experiment on rabbits injection.
[0085] 2. After 2 to 3 weeks, inject 1.0ml of the same antigen solution and incomplete Freund's adjuvant to the above-mentioned experimental rabbits once, and then inject once every four weeks, a total of 4 injections.
[0086] 3. Take blood from the experimental animal rabbit in step 2, separate and purify to obtain anti-angiotensin II specific antibody with a titer of 1:30000-1:50000.
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