Preparation method of cold shock protein

A technology for cold shock protein and protein solution, applied in the field of preparation, can solve problems such as no biological activity, and achieve the effects of simplified preparation conditions and large yield

Inactive Publication Date: 2014-11-05
FIRST HOSPITAL AFFILIATED TO GENERAL HOSPITAL OF PLA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The protein expressed and purified by the existing technology is an inclusion body, which only has the primary structure of the protein and can be used for antibody production, and has no biological activity

Method used

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  • Preparation method of cold shock protein
  • Preparation method of cold shock protein
  • Preparation method of cold shock protein

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Embodiment Construction

[0018] The preferred embodiments of the present invention are given below in conjunction with the accompanying drawings to describe the technical solution of the present invention in detail.

[0019] The preparation method of the cold shock protein of the present invention comprises the following steps:

[0020] Step 1: Extract total RNA by TRIzol method; grind mouse tissue samples with a mortar, add 1ml of lysate for every 50-100mg of tissue, rinse cells with PBS, add 1ml of TRIzol reagent, blow and mix, transfer to 1.5ml EP tube, Then add chloroform for extraction, fully wash the precipitate with 75% ethanol, remove the supernatant after centrifugation, dissolve with DEPC water after drying, and store at -70°C for future use.

[0021] Step 2, eDNA first-strand synthesis Add 5 μl RNA solution, 1 μl Oligo(dT), 1 μl dNTP to 20 μl reaction system, react at 65°C for 5 minutes, and quickly cool on ice. Then add 4 μl buffer (buffer), 2 μl DTT (dithiothreitol), 1 μl Rnase inhibitor...

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Abstract

The invention discloses a preparation method of a cold shock protein. The preparation method is characterized by comprising the following steps of 1, TRIzol method-based total RNAs extraction: grinding a mouse tissue sample by a mortar and adding 1ml of a pyrolysis liquid into each 50-100mg of the tissue, 2, cDNA first-chain synthesis: adding 5 microliters of a RNA solution, 1 microliter of Oligo and 1 microliter of dNTP into 20 microliters of a reaction system and carrying out a reaction process at a temperature of 65 DEG C for 5min, 3, a reaction process: adding 1 microliter of cDNA, 2 microliters of dNTP, 1 microliter of TapDNA polymerase, 1 microliter of an upstream primer, 1 microliter of an downstream primer and 2 microliters of 10*buffer into 20 microliters of the reaction system, 5, inducible expression, 6, preliminary purification and 7, high-degree purification. The cold shock protein which is coldinducible RNA-binding protein (CIRP) has effects of protecting nervous tissue cells and resisting nervous tissue cell apoptosis.

Description

technical field [0001] The invention relates to a preparation method, in particular to a preparation method of cold shock protein. Background technique [0002] The protein expressed and purified by the prior art is an inclusion body, which only has the primary structure of the protein and can be used for antibody production, and has no biological activity. Contents of the invention [0003] The technical problem to be solved by the present invention is to provide a method for preparing cold shock protein, the prepared protein is soluble expression, has biological activity, and has protective effect on nerve cells. [0004] The present invention solves the above-mentioned technical problems through the following technical scheme: a method for preparing cold shock protein, which is characterized in that it comprises the following steps: [0005] Step 1: Extract total RNA by TRIzol method; grind mouse tissue samples with a mortar, add 1ml of lysate for every 50-100mg of tis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/66C12N15/70C07K14/47C07K1/36C07K1/34C07K1/18
Inventor 刘嘉霖张志文
Owner FIRST HOSPITAL AFFILIATED TO GENERAL HOSPITAL OF PLA
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