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New applications of PRDX2 and/or PRDX6

A use, protein technology, applied in the field of biotechnology and medicine, can solve few problems

Active Publication Date: 2013-12-04
YANTAI JUJIE BIOENGINEERING LTD CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Now there are many "antioxidant" drugs, but there are few drugs that are really effective in antioxidation and regulation of oxidation / antioxidation balance

Method used

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  • New applications of PRDX2 and/or PRDX6
  • New applications of PRDX2 and/or PRDX6
  • New applications of PRDX2 and/or PRDX6

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0146] Example 1 Construction of pET32b (+) / PRDX6 and pET32b (+) / PRDX2 expression vectors

[0147] 1.1 Construction of pET32b(+) / PRDX6 expression vector

[0148] A pair of specific primers were designed and synthesized according to the gene sequence encoding PRDX6 mature protein,

[0149] Upstream primer: 5'-tatccatatgcccggaggtctgcttc-3' (shown in SEQ ID NO:5);

[0150] Downstream primer: 5'-ttactcgagaggctggggtgtgtagcg-3' (as shown in SEQ ID NO: 6);

[0151] The enzyme cutting sites are NdeI and XhoI respectively,

[0152] Human epididymis mRNA (or human epididymis cDNA library prepared in laboratory) extracted by conventional methods was used as template for RT-PCR amplification. see results figure 1 , a 688bp amplified fragment consistent with the theoretical value was detected.

[0153] Separate the amplified fragment (recover the target fragment from the agarose gel with a gel extraction kit), and perform double enzyme digestion with NdeI and XhoI. Then, the double-d...

Embodiment 2

[0158] Expression of embodiment 2PRDX6 protein and PRDX2 protein

[0159] 2.1 Expression of PRDX6 protein

[0160] The plasmid pET32b(+) / PRDX6 was transformed into Escherichia coli Origami B(DE3) (purchased from Novagen), and positive clones were inoculated in LB medium containing 100ug / ml ampicillin, and cultured overnight at 37 degrees in a shaker.

[0161] The next day, transfer to LB medium containing 100ug / ml ampicillin at a ratio of 1:100, culture on a 37-degree shaker until the bacterial density OD600=0.6-0.8, then add IPTG to a final concentration of 0.4mM to induce the expression of the target protein PRDX6 , After 3-4h, the cells were collected by centrifugation.

[0162] 2.2 Expression of PRDX2 protein

[0163] Complete the expression of PRDX2 protein, the expression method is the same as 2.1, the difference is that PRDX2 protein is used instead of PRDX6 protein.

Embodiment 3

[0164] Purification of embodiment 3PRDX6 protein and PRDX2 protein

[0165] 3.1 Purification of PRDX6 protein

[0166] The bacteria collected by centrifugation were resuspended in buffer A (20mM phosphate buffer, 150mM sodium chloride, pH7.2), ultrasonically disrupted in an ice bath, centrifuged at 20,000g at low temperature (4°C) for 15min, and the supernatant was taken as the purified sample.

[0167] After equilibrating the nickel affinity chromatography column with buffer A, the sample was applied to the column, and then the column was washed with buffer A containing 50 mM imidazole to remove impurities (peak 1) (see figure 2 ), the target protein was eluted with buffer A containing 300mM imidazole (peak 2) (see figure 2 ).

[0168] Finally, the eluted protein was replaced with buffer A by G-25 desalting column to remove imidazole, and detected by SDS-PAGE electrophoresis.

[0169] After analysis, the PRDX6 protein with a purity of more than 95% and a molecular weigh...

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Abstract

The invention relates to new applications of PRDX2 and / or PRDX6 and concretely discloses new applications of PRDX2 protein and / or PRDX6 protein which are used for preparation of medicine compositions treating or preventing damage, aging or diseases caused by increase of reactive oxygen species (ROS). The invention also provides medicine compositions, food compositions or cosmetic compositions which employ the PRDX2 protein and / or the PRDX6 protein as active ingredients.

Description

technical field [0001] The present invention relates to the fields of biotechnology and medicine, in particular, the present invention relates to the new application of PRDX2 and / or PRDX6, and more specifically, relates to the use of PRDX2 and / or PRDX6 in the treatment or prevention of related diseases, aging or damage caused by increased ROS Applications. Background technique [0002] Free radicals, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), are produced when cells use oxygen. In our body, each cell produces 2.5×10 11 A molecule of ROS can be produced in the human body every day 40×10 21 molecules of free radicals. They not only provide us with energy for maintaining life activities, but also help us eliminate bacteria and pathogens, and remove toxins and "garbage" from the body. They are also the promoters and regulators of various metabolic and signaling pathways in our body, such as JNK / SAPK, P38MAPK, IKK / NF-KB, P13K, Akt, CD40 / CD40L, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/44A61K8/66A61P39/06A61P15/08A61P17/18A61P17/02A61P17/16A61Q19/08A61Q19/02A61Q17/04A23L1/305A23L33/17
Inventor 李建远
Owner YANTAI JUJIE BIOENGINEERING LTD CO
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