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Method for preparing recombinant GPx7 (glutathione peroxidase) protein

A recombinant protein and prokaryotic expression technology, which is applied in the field of preparation of GPx7 recombinant protein, can solve the problem of protein inactivation and achieve the effect of high purification degree, good protein activity and simple steps

Inactive Publication Date: 2014-08-27
上海御先医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the IMAC purification method is simple and easy, its disadvantage is likely to cause inactivation of proteins sensitive to redox reactions [Gaberc-Porekar, V., Menart, V.P Journal of Biochemical and Biophysical Methods.2001, 49 (1-3): 335 -360]

Method used

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  • Method for preparing recombinant GPx7 (glutathione peroxidase) protein
  • Method for preparing recombinant GPx7 (glutathione peroxidase) protein
  • Method for preparing recombinant GPx7 (glutathione peroxidase) protein

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1: Construction of the prokaryotic expression plasmid of recombinant human GPx7 protein

[0032] 1. Construction of human GPx7 prokaryotic expression plasmid and strain

[0033] According to the amino acid sequence analysis results of GPx7 on the protein database website http: / / www.uniprot.org / uniprot / Q96SL4, when constructing the prokaryotic expression plasmid, the N-terminus of GPx7 was removed, which may be 19 amino acids corresponding to the signal peptide. First, the eukaryotic expression plasmid pCDNA3.1-GPx7 (Mao Wenwei, Wang Xiao, He Huijuan, et al. The protective effect of in vivo expression of glutathione peroxidase-7 gene on myelosuppression Research. Chinese Medical Biotechnology, 3(4):250-257, 2008.) Amplify the target gene and introduce the Nde1 and BamH1 restriction sites required for the target gene to be inserted into the prokaryotic expression vector pET24A. Using sense primer 5'-GCG CATATGCAGCAGGAGCAGGACTTC-3' and antisense primer 5'-GC ...

Embodiment 2

[0034] Example 2 Expression, purification and identification of recombinant human GPx7

[0035] 1. Lactose-induced expression of GPx7

[0036] The BL21(DE3) strain containing the pET24A-GPx7 expression plasmid prepared in Example 1 was added to 3 ml of LB medium containing the corresponding resistant antibiotic at a final concentration of 100 μg / ul and shaken at 37°C for 10 h for recovery. Take 1ml of the resuscitated bacteria solution and add it to 255ml LB medium, and at the same time add the ingredients required for lactose induction: 10×lactose induction buffer, 25×lactose induction solution, 100×MgSO 4 and corresponding antibiotics at a final concentration of 100 μg / ul, cultured with shaking at 37° C. and induced for 7 hours.

[0037] Take 1ml of the bacterial cells for identification of Total cell protein (TCP) in SDS-PAGE. After collection by centrifugation, buffer suspension by pipetting, ultrasonication and centrifugation, the supernatant was taken for SDS-PAGE iden...

Embodiment 3

[0054] Example 3 Detection of GPx7 activity

[0055] The activity detection method of GPx7 adopts the indirect method. H2O2 was used as the oxidized substrate in the activity detection of GPx7. The reaction was carried out in 200 μl pH7.6 enzyme activity detection buffer system, and the components in the reaction were as follows: 0.25 mM NADPH, 2.1 mM reduced GSH, 0.5 U / ml GR, 84 μM purified GPx7 and 50 uM H2O2. The reaction was started by adding 50 μM H2O2 into the reaction system while detecting the change of A340 in real time.

[0056] The results pointed out that in the detection system containing GPx7 compared with the detection system not containing GPx7, the decline value of the former A340 was lower ( Figure 8 )

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Abstract

The invention relates to the field of biomedicine and particularly relates to a method for preparing a recombinant GPx7 (glutathione peroxidase) protein. The invention discloses a method for preparing a GPx7 recombinant protein. The method comprises the steps of constructing a GPx7 recombinant protein prokaryotic expression plasmid and strain; expressing the GPx7 recombinant protein; extracting the GPx7 recombinant protein and purifying the GPx7 recombinant protein. The method provided by the invention has the advantages of simple steps, low cost and high degree of purification; the prepared protein has good activity. The method is far superior to a metal affinity chromatography used in the prior art.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for preparing GPx7 recombinant protein. Background technique [0002] Glutathione peroxidase (Glutathione peroxidase, GPx) is an important peroxide-decomposing enzyme that widely exists in the body. The GPx that has been discovered so far has included 8 different subtypes, from the GPx1 subtype to the currently discovered GPx8 subtype, which can be subdivided into two main types, one is the subtype containing Sec rare amino acids, and the human source contains selenium There are 5 types of GPx (SecGPx), including GPx1, GPx2, GPx3, GPx4, and GPx6; one is a subtype that does not contain the rare amino acids of Sec, and GPx7 / 8 completely lacks the binding site for GSH. [0003] In 2004, it was reported for the first time that GPx7 has the function of alleviating polyunsaturated fatty acid-mediated apoptosis. Since GPx7 and GPx4 (PHGPx) have a high similarity in structure and are...

Claims

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Application Information

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IPC IPC(8): C12N9/08C12N15/70
CPCC12N9/0065C12N15/70C12Y111/01009
Inventor 孙永宪陈欣麟毛文伟杜伟业孙文立
Owner 上海御先医药科技有限公司
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