Immobilized nuclease and preparing method thereof

A nuclease and solid-phase carrier technology, which is applied in the field of immobilized nuclease and its preparation, can solve problems such as unexpectedness, reduction of catalytic efficiency, and disturbance of microenvironment.

Active Publication Date: 2014-12-24
HANGZHOU JUNFENG BIOENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0022] (2) Steric hindrance effect
When there is a steric hindrance effect, it is difficult for the substrate to contact the active center of the enzyme, resulting in a decrease in the catalytic efficiency of the enzyme
[0023] (3) Microenvironment disturbance
[0025] (5) Diffusion limitation effect
However, th

Method used

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  • Immobilized nuclease and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] The combination of embodiment one nuclease and agarose Sepharose-4B

[0059] Take an appropriate amount of Sepharose-4B gel, pump out the protective solution in a G3 sand core funnel, filter with a vacuum pump, weigh 50g of Sepharose-4B gel in wet weight, rinse with 500ml, 0.5mol / L NaCl solution, and remove the gel Clean the protective agent inside with distilled water.

[0060] In the fume hood, put on rubber gloves, carefully weigh 15g of BrCN, add 15ml of acetonitrile to dissolve it.

[0061] Transfer 50g of gel to a 500ml beaker, add 75ml of 0.2mol / L, pH10.0 Na 2 CO 3 -NaHCO 3 buffer, then place the beaker in an ice bath and stir slowly with a stirrer. Take a dropper and add BrCN solution dropwise to the beaker to make it react with Sepharose-4B gel, and take another dropper to add 6mol / L NaOH dropwise to the beaker to keep the pH in the reaction solution at about 10.0. Measured with a pH meter, continue to react for 5 minutes after adding BrCN. At this moment...

Embodiment 2

[0067] The combination of embodiment two nuclease and Sepharose CL-6B

[0068] Take 50mL of Sepharose CL-6B medium stored in 20% ethanol, wash repeatedly with excess distilled water to remove ethanol, then place it in a G3 sintered glass funnel and vacuum-dry it for 5min; weigh 10g of the medium after suction filtration, and use 20% 50% and 70% dimethyl sulfoxide (Dimethyl Sulfoxide, DMSO) aqueous solution cleaning, after each cleaning, vacuum in the cleaning solution; add 6.5ml2mol / L NaOH, 1,5ml epichlorohydrin to the treated medium (ECH), 15ml 56% 1,4-dioxane. The suspension of the medium was shaken and reacted at 170r / min for 2 hours on a constant temperature air bath shaker, and the reaction temperature was 45°C. After the reaction, it was washed with a large amount of deionized water until no epoxy group was detected in the cleaning solution. The epoxy group in the cleaning solution is measured by thiosulfate, using phenolphthalein as the indicator. If the cleaning solut...

Embodiment 3

[0072] The quantitative analysis of embodiment three epoxy group modification density

[0073] The epoxy-modified density of the agarose gel was determined by sodium thiosulfate titration. The activated medium after washing with deionized water was placed in a G3 funnel and vacuum-dried for 5 minutes, and then weighed 0.5 g and placed in a ground-mouth Erlenmeyer flask. Add about 3 mL of 1.3 mol / L sodium thiosulfate and 1-2 drops of phenolphthalein indicator, seal the Erlenmeyer flask and shake it at room temperature for 30 minutes. The reacted solution is titrated with 0.1 mol / L hydrochloric acid standard solution until the solution From red to colorless. According to the volume of the consumed hydrochloric acid standard solution, calculate the epoxy modification density: S=M*(V 0 -V 1 ) / W / ρ=91.2

[0074] Wherein: S is epoxy group modification density (mol / mL);

[0075] M is the concentration of HCl (mol / L);

[0076] V0, V1 is the volume (mL) of HCl before and after titra...

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Abstract

The invention belongs to the field of biological medicines and relates to an immobilized nuclease and a preparing method thereof. The immobilized nuclease comprises a nuclease and a solid-phase vector. The nuclease is connected to groups on the solid-phase vector through covalent bonds. The activity of the immobilized nuclease is higher than 500 U/g. A functional pH range of the immobilized nuclease is 6-8. The proper function temperature of the immobilized nuclease is 37 DEG C. The invention also discloses the preparing method of the immobilized nuclease. The immobilized nuclease is applied in processes of medicine products, avoids nuclease residue in a final product during product separation and purification steps, and improves the product quality.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a nuclease and a preparation method thereof, in particular to an immobilized nuclease and a preparation method thereof. Background technique [0002] When biotechnology products are highly expressed, the host cells or bacteria also proliferate in large quantities. This process is often accompanied by the amplification of a large number of host or bacteria nucleic acids. In the subsequent isolation process, a large number of nucleic acids are released. Purification increases the difficulty, and at the same time, it is difficult to remove some residual host DNA combined with the product. The DNA of the host cell entering the human body together with the drug will produce unpredictable side effects. The residual DNA may cause the insertion mutation of the DNA of the body, resulting in the inactivation of the tumor suppressor gene, the activation of the oncogene, etc., which may cause the dru...

Claims

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Application Information

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IPC IPC(8): C12N11/12C12N11/10C12N11/08C12N11/02
Inventor 刘国安徐辉方芳
Owner HANGZHOU JUNFENG BIOENG
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