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Expression method and application of recombinant human CYTL1 protein

An expression method and protein technology, applied in the field of recombinant human CTYL1 protein expression, can solve the problems of ossification of new cartilage tissue, complex functions, high price, etc., and achieve the effect of promoting proliferation

Inactive Publication Date: 2014-10-08
广州市红十字会医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, this type of cytokines has complex functions and multiple regulatory functions. While promoting the repair of cartilage tissue, it is also easy to cause the proliferation of other tissues (such as fibrous tissue), leading to ossification or fibrosis of new cartilage tissue and affecting the regeneration of tissue. biological function
In addition, commercialized exogenous cytokines mostly rely on foreign imports, which are very expensive

Method used

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  • Expression method and application of recombinant human CYTL1 protein
  • Expression method and application of recombinant human CYTL1 protein
  • Expression method and application of recombinant human CYTL1 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] A method for expressing recombinant human CYTL1 protein, the specific steps are as follows:

[0034] 1. Preparation of human chondrocyte CYTL1 protein cDNA

[0035] 1.1 RNA extraction: Shake and mix the human chondrocyte line C28112 cells until the cells are completely lysed. After standing at room temperature for about 10 minutes, add 200 μl of chloroform to each 1ml of TRIzol, cover the lid, shake vigorously for about 1 minute, and let stand at room temperature. 2 to 5 minutes, then refrigerated and centrifuged at 12,000g for 15 minutes (6°C), and the sample was carefully taken out. Through observation, it can be found that the sample is divided into three layers, and the uppermost layer contains the RNA sample.

[0036] 1.2 RNA Precipitation: Carefully pipette about 450 μl of the supernatant (the amount absorbed per 1 ml of TRIzol) into a new RNase Free centrifuge tube containing 600 μl of frozen isopropanol, mix well, place at -20°C for about 10 minutes, and centrif...

Embodiment 2

[0101] MTS method to determine the activity of recombinant human CYTL1 in promoting the proliferation of human chondrocytes

[0102] (1) The human chondrocyte cell line C28112 was seeded in a 96-well plate, and the number of cells was 5×10 3cells / mL, 200 μl per well.

[0103] (2) Recombinant human CYTL1 protein was added to the experimental group, and the addition amount was 4000, 2000, 1000, 500, 250 and 125 (ng / ml); Recombinant human CYTL1 protein was used as a control.

[0104] (3) Add DMEM-F12 culture solution containing 2% fetal bovine serum, and incubate at 37°C and 5% CO 2 cultured in an incubator for one day.

[0105] Use the MTS method (operate according to the instructions), read the absorbance value (OD value) at 490nm in the multi-mode detection system, and use the OD value to represent the growth activity of the cells, such as Figure 8 , the recombinant human CYTL1 protein with a concentration of 1000ng / ml had the strongest activity.

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Abstract

The invention discloses an expression method of a recombinant human CYTL1 protein. The method comprises a step of performing prokaryotic expression on SEQ ID:NO.1. The method adopts a gene recombination technique to prepare the CYTL1 protein in a scale for the first time, the expressed recombinant human source interleukin 1-CYTL1 protein is soluble protein, and moreover the recombinant protein can promote amplification of cartilage cells.

Description

technical field [0001] The invention relates to a protein expression method, in particular to a recombinant human CTYL1 protein expression method and application. Background technique [0002] Articular cartilage is a unique connective tissue composed of chondrocytes and cartilage matrix. The repair of cartilage damage depends on the secretion of chondrocytes. Because there is no blood vessel in cartilage tissue, chondrocytes are embedded in a dense solid matrix composed of type II collagen and proteoglycan, and cannot move to the defect site. Therefore, even a small defect is difficult to repair by itself. Articular cartilage injury is more common clinically. At present, its treatment is divided into two types: prevention and symptomatic treatment. Symptomatic patients can be intervened with drugs and surgical treatment, but these methods cannot fundamentally prevent the development of the disease, so people have been Actively looking for more effective treatment and preve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N5/077C07K14/52
Inventor 杨小红李斯明崔树良梁伟国
Owner 广州市红十字会医院