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Aptamer functionalized UCST type temperature-sensitive block polymer and application thereof in synchronous separation and purification and immobilization of cytochrome C

A temperature-sensitive block and aptamer technology, applied in the application field of immobilized cytochrome C, can solve the problems of high cost and complicated operation, and achieve the effect of good stability

Pending Publication Date: 2022-04-15
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the actual operation process, there are still shortcomings such as complicated operation and high cost

Method used

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  • Aptamer functionalized UCST type temperature-sensitive block polymer and application thereof in synchronous separation and purification and immobilization of cytochrome C
  • Aptamer functionalized UCST type temperature-sensitive block polymer and application thereof in synchronous separation and purification and immobilization of cytochrome C
  • Aptamer functionalized UCST type temperature-sensitive block polymer and application thereof in synchronous separation and purification and immobilization of cytochrome C

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] (1) Preparation of RAFT chain transfer agent CTA, the process is as follows figure 1 As shown in the middle route (1);

[0053] 3-Mercaptopropionic acid (2.5 mL, 29.0 mmol) was added dropwise into an aqueous potassium hydroxide solution (1.8 mol / L, 32 mL), and then carbon disulfide (4 mL) was added dropwise under vigorous stirring. After the obtained orange-yellow liquid was stirred at room temperature for 5 h, benzyl bromide (5 g, 58.0 mmol) was added to the reaction solution, and then the mixture was placed in an oil bath at 85° C. and stirred under reflux for 12 h. After the reaction was completed and cooled to room temperature, the mixture solution was poured into 70 mL of chloroform, and hydrochloric acid was added dropwise to acidify until the organic phase turned yellow. Subsequently, the organic phase was washed with excess deionized water. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, added an appropriate amou...

Embodiment 2

[0068] PEG 45 -b-PAAm 176 -b-P(GMA 21 -co-AAc 140 )-Apt's Response Performance and Self-Assembly Behavior

[0069] (1) Determination of PEG by ultraviolet-visible spectrometer (UV-Vis) 45 -b-PAAm 176 -b-P(GMA 21 -co-AAc 140 )-Apt aqueous solution light transmittance at different temperatures, according to the light transmittance of 50% to determine PEG 45 -b-PAAm 176 -b-P(GMA 21 -co-AAc 140 )-UCST and pH response of Apt aqueous solution. Such as Figure 7 As shown in (a), after modifying the aptamer Apt, PEG 45 -b-PAAm 176 -b-P(GMA 21 -co-AAc 140 )-Apt had a pH response of 3.1, with no significant change. Such as Figure 7 As shown in (b), PEG 45 -b-PAAm 176 -b-P(GMA 21 -co-AAc 140 ) The UCST of the polymer solution is 22.3°C. After modifying the aptamer Apt, PEG 45 -b-PAAm 176 -b-P(GMA 21 -co-AAc 140 )-Apt solution UCST decreased to 17.6 °C

[0070] (2)PEG 45 -b-PAAm 176 -b-P(GMA 21 -co-AAc 140 )-Apt polymer has temperature response properties, ...

Embodiment 3

[0072] PEG 45 -b-PAAm 176 -b-P(GMA 21 -co-AAc 140 )-Apt synchronous separation and purification, Cyt c in immobilized porcine cardiomyocytes

[0073] (1) Preparation of Cyt c crude extract in pig cardiomyocytes

[0074] Freshly purchased pig heart, after removing impurities such as fat and blood vessels, cut it into small pieces and wash it for later use. Weigh a small piece of myocardial minced meat (40g), add H 2 SO 4 The acidified water (40mL, pH 3.6) after acidification of the solution (1mol / L) was crushed with a high-speed tissue grinder until the blocky myocardium was crushed to a homogenate state. Then, pour the myocardial homogenate into a beaker and let it stand in a water bath at a temperature of 30°C for 3 hours, filter out the precipitate with gauze, add acidified water (20 mL) to the precipitate and mash it again, and let it stand for 3 hours Filter through gauze, and combine the two filtrates. use NH 3 ·H 2 The pH of the above filtrate was adjusted to 6...

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Abstract

The invention aims to provide a preparation method of an aptamer functionalized UCST type temperature-sensitive block polymer and application of the aptamer functionalized UCST type temperature-sensitive block polymer in synchronous separation and purification and immobilization of cytochrome C (Cyt c). According to the invention, the high specific recognition and high affinity of the aptamer to enzyme and the temperature stimulation response performance of the UCST type temperature-sensitive block polymer are combined, the limitation of the existing separation and purification technology is broken through, and efficient and selective synchronous separation, purification and immobilization of Cyt c in the porcine myocardial cell crude extract are realized. Under the optimal condition, the separation efficiency of the Cyt c is 90.2%, and the obtained immobilized Cyt c shows good stability in a high-temperature alkaline environment and shows the advantages of homogeneous catalysis, heterogeneous high efficiency and rapid recovery. The prepared aptamer functionalized UCST type temperature-sensitive block polymer can be widely applied to efficient and selective synchronous separation, purification and enzyme immobilization, and is an effective method for reducing the cost of industrial enzymes.

Description

technical field [0001] The invention belongs to the technical field of polymers and preparation methods thereof, and relates to the preparation of aptamer functionalized UCST type thermosensitive polymers and the application of simultaneous separation and purification and immobilized cytochrome C (Cyt c). Background technique [0002] Enzyme preparations are mainly protein products with certain purity and catalytic activity extracted, separated, purified and processed from animals, plants and microorganisms. Enzyme crude extracts contain a large amount of miscellaneous proteins, small molecular sugars, amino acids and other components. It is extremely challenging to separate and purify target enzymes with higher purity from crude extracts with complex components. The commonly used separation method is to first use coarse separation methods such as salting out precipitation method and organic solvent precipitation method to remove most of the impurities; secondly, use membran...

Claims

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Application Information

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IPC IPC(8): C08F293/00C08F8/32C07K14/80C07K1/14C07K17/08
Inventor 刘园园李利利兰会玲王蕾韩娟王赟刘怡涵
Owner JIANGSU UNIV
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