Anti-aging secretory Klotho protein and coding gene thereof, recombinant expression vector and application

An expression vector, anti-aging technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problem of lack of protein processing mechanism

Inactive Publication Date: 2021-01-08
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Represented by the expression system of Escherichia coli, the advantages are clear genetic background, low cost, high expression level and relatively simple separation and purification of expression products, etc. The disadvantag

Method used

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  • Anti-aging secretory Klotho protein and coding gene thereof, recombinant expression vector and application
  • Anti-aging secretory Klotho protein and coding gene thereof, recombinant expression vector and application
  • Anti-aging secretory Klotho protein and coding gene thereof, recombinant expression vector and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Construction and expression of embodiment 1 recombinant prokaryotic expression vector

[0048] (1) Construction of recombinant expression vector

[0049] Design primers according to the known Klotho sequence number (Gen Bank.NC_000013) and optimization and BL21(DE3) vector cloning site:

[0050] Using the pcDNA3.1-Klotho constructed above as a template and using the following sequences (upstream and downstream primers) as primers, PCR amplification was performed to obtain PCR amplification products; then the PCR amplification products were double-digested with EcoRI and XhoI enzymes, Simultaneously, the pET-28a(+) plasmid was also double-digested synchronously, and the target fragment was recovered by electrophoresis (the size of the Klotho gene was 1764bp, figure 1 ) was purified by the PCR purification kit, and then ligated with the double-digested and purified pET-28a(+) vector (purchased from EMDBiosciences (Novagen)) by T4 DNA ligase to obtain pET-28a-Klotho. Whe...

Embodiment 2

[0058] Example 2 Expression, purification and identification of recombinant prokaryotic carrier protein

[0059] (1) SDS-PAGE analysis of expressed protein

[0060] After the bacterium collected in Example 1 was broken, the precipitate was collected by centrifugation, and the precipitate was washed with PBS buffer solution containing 2M urea, and the precipitate was collected by centrifugation at 17000g at 4°C for 30 min; Wash with PBS buffer, 4°C, 17000g, 30min centrifugation to collect the precipitate; then wash with PBS buffer 2 to 3 times, 4°C, 17000g, 30min centrifugation to collect the precipitate; finally dissolve the precipitate in PBS buffer, add the final concentration 1 mM DTT (dithiothreitol), stirred overnight at 4°C. Collect the supernatant by centrifugation at 4°C, 9000rpm, for 20min. Pack the column with NI-NTA filler, wash the column with pure water for 2-3 column volumes, and equilibrate the column with PBS buffer at pH 8.0. Stir the balanced filler and th...

Embodiment 3

[0063] Anti-aging activity detection of embodiment 3 recombinant Klotho protein

[0064] (1) Establishment of cell senescence model

[0065] Human immortalized skin keratinocytes (HaCaT) and human skin fibroblasts (HSF) were irradiated with medium-wave ultraviolet light, and the aging model of cell damage (the cells were purchased from Shanghai Aiyan and Shanghai Zhongqiao Xinzhu Biotechnology Co., Ltd., aging The model was established by UVB irradiation method): cell culture was carried out at 37°C, 5% (v / v) CO 2 Routine culture in the cell incubator, placed in different energy 700 ~ 1000mJ / cm 2 Irradiate under a UVB lamp, the distance between the lamp source and the culture bottle is 15cm, after the experimental group is irradiated, add PBS buffer solution to wash, then discard the PBS buffer solution, add 100μL culture solution (DMEM medium+10% (v / v) Newborn bovine serum), normal culture; after 24h, MTT method detects the growth inhibition rate of two groups of cells%=(1-...

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Abstract

The invention discloses an anti-aging secretory Klotho protein and a coding gene thereof, a recombinant expression vector and an application. The recombinant anti-aging protein expressed by a prokaryotic expression vector is screened by adopting a genetic engineering technology and strategy, adopting high-fidelity Taq polymerase, optimizing a codon sequence of a target gene and adding a histidinetag, and the amino acid sequence of the recombinant anti-aging protein is shown as SEQ ID NO.1. The invention also provides a gene for coding the anti-aging secretory Klotho protein, and the sequenceof the gene is shown as SEQ ID NO.2. The obtained Klotho protein has a better anti-aging function, and can be used for resisting aging, regulating the generation of nitric oxide, resisting oxidative stress, regulating calcium and phosphorus metabolism, resisting inflammation, inhibiting cancer and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-aging secreted Klotho protein and its coding gene, recombinant expression vector and application. Background technique [0002] Klotho is the name of the goddess who spins the thread of life in ancient Greek mythology. It is used in the name of the gene to refer to the length of the spinning silk thread as a metaphor for its lifespan. This gene was discovered and identified on a mouse model of human aging in 1997, and it is the earliest anti-aging gene found in mammals. It was later confirmed that the product encoded by this gene is an important anti-aging protein. The gene-deficient mice show signs of aging at 4 weeks after birth, including shortened lifespan, growth arrest, vascular calcification, arteriosclerosis, skin atrophy, bone loss, emphysema, hearing impairment, motor neuron degeneration, and infertility Symptoms and tumors, etc., died of aging at the ...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/66C12N15/70C12N1/21A61K38/47A61P29/00A61P35/00A61P39/00A61P39/06C12R1/19
CPCC12N9/2402C12Y302/01031C12N15/66C12N15/70A61P29/00A61P35/00A61P39/00A61P39/06A61K38/00
Inventor 陈宏远芮雯
Owner GUANGDONG PHARMA UNIV
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