CN-121182760-B - Thioredoxin mutant and preparation method and application thereof

Abstract

The application belongs to the technical field of biology, and particularly relates to a thioredoxin mutant and a preparation method and application thereof. The mutant with high antioxidant activity is obtained by carrying out combined mutation on the wild thioredoxin, has outstanding advantages in improving the expression quantity and good solubility, has stronger antioxidant activity and anti-inflammatory and soothing effects, can be applied to skin care and washing and caring products by combining the mutant with other raw materials and auxiliary materials, and is a brand new high-efficiency active raw material.

Inventors

• TANG WEI
• YE DEWEN
• ZHU LI
• FENG SU
• SONG JIAMIN
• ZHANG XIAOWEI
• LI ZHILIN
• LIU YANG
• CAO SHENGBIAO
• TONG FEIFEI
• LU HAOPING

Assignees

• 南京诺唯赞生物科技股份有限公司
• 南京迪诺薇华生物科技有限公司

Dates

Publication Date

20260512

Application Date

20251010

Claims (10)

1. 1. A thioredoxin mutant is characterized in that the amino acid sequence is shown as SEQ ID NO.5 or SEQ ID NO. 7.
2. 2. A gene for coding the thioredoxin mutant as set forth in claim 1, characterized in that the nucleotide sequence of the gene for coding the thioredoxin mutant with the amino acid sequence shown in SEQ ID NO.5 is shown in SEQ ID NO.6, and the nucleotide sequence of the gene for coding the thioredoxin mutant with the amino acid sequence shown in SEQ ID NO.7 is shown in SEQ ID NO. 8.
3. 3. An expression vector comprising the gene of claim 2.
4. 4. The expression vector according to claim 3, wherein the expression vector is obtained by inserting the gene according to claim 2 into the expression vector through NcoI and XhoI cleavage sites.
5. 5. A recombinant genetically engineered bacterium comprising the gene of claim 2.
6. 6. The preparation method of the thioredoxin mutant is characterized by comprising the following steps of transforming the expression vector of claim 3 or 4 into competent cells, culturing to obtain genetically engineered bacteria, and performing induced culture and purification on the genetically engineered bacteria to obtain the thioredoxin mutant.
7. 7. The preparation method according to claim 6, comprising the following steps: (1) Taking a wild thioredoxin gene as a template, carrying out site-directed mutagenesis to obtain a mutant with an amino acid sequence shown as SEQ ID NO.5 or SEQ ID NO.7, amplifying the mutant gene by adopting PCR, converting the product into DH5 alpha competent cells, picking up monoclonal after coating culture, carrying out test tube culture, extracting and verifying plasmids; (2) Transforming the recombinant plasmid which is verified to be correct into E.coli BL21 (DE 3) competent cells, and culturing to obtain genetically engineered bacteria; (3) The thioredoxin mutant is obtained by the induced culture, screening and purification of the genetically engineered bacteria.
8. 8. Use of the thioredoxin mutant of claim 1 in the preparation of a skin care product or a washing product.
9. 9. The use according to claim 8, wherein the skin care or wash product is for antioxidant, anti-inflammatory and soothing.
10. 10. The use according to claim 8, wherein the skin care product is a essence, cream, lotion, emulsion or mask, and the skin care product is a facial cleanser, body wash or body lotion.

Description

Thioredoxin mutant and preparation method and application thereof Technical Field The application belongs to the technical field of biology, and particularly relates to a thioredoxin mutant and a preparation method and application thereof. Background Thioredoxin is a low molecular weight protein widely existing in prokaryotic and eukaryotic cells, and forms a thioredoxin system with thioredoxin reductase and reduced coenzyme II (NADPH), and plays roles in maintaining redox balance, signaling and the like in organisms. Almost all thioredoxins have a conserved active center WCGPC, two cysteine residues (Cys) of which are involved in disulfide bond reduction of the target protein, thereby performing important biological functions including scavenging free radicals, regulating cell antioxidant, anti-stress and anti-apoptotic, regulating protein nitrosylation, regulating transcription factor binding and immune response, etc. In recent years, the achievement of safe, efficient, natural active ingredients is a goal pursued by the cosmetic skin care industry. Thioredoxin becomes one of potential components due to multiple functions, heterologous recombinant expression is a necessary means for breaking through the limitation of large-scale application, but wild type thereof has the problems of low expression quantity, insufficient antioxidant activity and the like, and limits the application in skin care products. In the prior art, although research on genetic engineering of thioredoxin is carried out, less research on antioxidation and anti-inflammatory relief is carried out. For example, CN110257347a discloses a thioredoxin mutant, which has high-temperature solubility and thermal stability, good solubility and expression, and is beneficial to recombinant fusion protein production by means of multi-site combined mutation, but the mutant is not studied in terms of antioxidation and anti-inflammatory relief. CN117321208a discloses a modified human serum albumin-thioredoxin fusion, and also further performs site-directed combined mutation on the development of the modified fusion to obtain a serum albumin-thioredoxin fusion with improved activity and stable activity, but the application direction of the fusion is in the application of treating specific diseases, and the mutant has not been studied in terms of antioxidation and anti-inflammatory relief. Disclosure of Invention Aiming at the prior art, the application firstly combines the Asp60 and the Cys73 of the natural human thioredoxin TXN to mutate, and further firstly iterates and combines the mutation Cys62 and the mutation Cys69, and the obtained mutant has high expression, good solubility, high antioxidant activity and good application prospect. The application provides a thioredoxin mutant, the amino acid sequence of which is shown as SEQ ID NO. 5. The nucleotide sequence of the gene for encoding the thioredoxin mutant is shown as SEQ ID NO. 6. The application also provides a thioredoxin mutant, the amino acid sequence of which is shown as SEQ ID NO. 7. The nucleotide sequence of the gene for encoding the thioredoxin mutant is shown as SEQ ID NO. 8. The application also provides an expression vector containing the coding gene. The expression vector is obtained by inserting the gene into the expression vector through NcoI and XhoI restriction sites. The application also provides a recombinant genetically engineered bacterium containing the gene. The genetically engineered bacterium is selected from gram-positive bacteria, gram-negative bacteria and escherichia coli. The application also provides a preparation method of the thioredoxin mutant, which comprises the following steps of transforming the expression vector into competent cells, culturing to obtain genetically engineered bacteria, and performing induced culture and purification on the genetically engineered bacteria to obtain the thioredoxin mutant. As a technical scheme, the preparation method comprises the following steps: (1) Taking a wild thioredoxin gene as a template, amplifying the mutant gene by adopting PCR after site-directed mutagenesis, converting the product into DH5 alpha competent cells, picking up monoclonal after coating culture, and carrying out test tube culture, and extracting and verifying plasmids; (2) Transforming the recombinant plasmid which is verified to be correct into BL21 (DE 3) competent cells, and culturing to obtain genetically engineered bacteria; (3) The thioredoxin mutant is obtained by the induced culture, screening and purification of the genetically engineered bacteria. The application also provides application of the thioredoxin mutant in preparing skin care products or washing products. As a technical scheme, the cosmetic or skin care product is used for resisting oxidation, resisting inflammation and relieving, and can specifically remove ABTS free radicals and inhibit ROS generation induced by H 2O2. The skin care product can be essence, essence cream, toning