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Method for expressing metallothionein of human by using body of vegetable oil

A metallothionein and oil body protein technology, applied in the field of metallothionein and its expression in vegetable oil bodies, can solve the problems of increased production cost and cycle, nucleic acid, protein and lipid damage, and reduced target protein yield, etc. The effect of preventing UV damage, preventing aging, and reducing content

Inactive Publication Date: 2009-12-30
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] 3. Eliminate free radicals in the body and prevent the aging of the body. Oxygen free radicals can be produced spontaneously in the body through enzymatic or non-enzymatic reactions. Some normal physiological processes or biochemical reactions require a certain amount of oxygen free radicals to participate, but excess free radicals The base has a strong oxidation ability in the body and can cause damage to nucleic acids, proteins and lipids.
However, since the exogenous protein expressed by plant oil body protein is a fusion protein coupled with oil body protein, whether this fusion protein will affect the biological activity of MT, if it is necessary to separate MT and oil body protein, it will be difficult to a certain extent It will increase its production cost and cycle, and will cause a decrease in the yield of the target protein
However, there is no report on the expression of MT using the plant oil body expression system

Method used

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  • Method for expressing metallothionein of human by using body of vegetable oil
  • Method for expressing metallothionein of human by using body of vegetable oil
  • Method for expressing metallothionein of human by using body of vegetable oil

Examples

Experimental program
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Effect test

Embodiment 1

[0066] Embodiment 1: Preparation and sequence determination of MT gene

[0067] First, modify the nucleic acid sequence encoding the human MT protein found in GENEBANK according to the plant-preferred codons, and replace the rare amino acid codons in plants with plant-preferred codons without changing MT Amino acid sequence in primary structure. Specific modification method reference www.kazusa.or.jp / codon / related list. According to the degeneracy of codons, the enzyme cutting sites used in this experiment were eliminated, and primers were designed using the online software Primerfinder and DNASTAR. In order to synthesize the full-length MT gene with plant preference, six primers were designed and synthesized, each Primer length is about 57~59bp: F 1 , F 2 , F 3 For the forward primer, R 1 , R 2 , R 3 is the reverse primer, where F 1 and R 1 ;F 2 and R 2 is a complementary chimeric primer (with 20 complementary bases); R 3 Including the nucleic acid sequence enc...

Embodiment 2

[0123] Example 2: Construction of recombinant oil body expression vector

[0124]Using the total DNA of Brassica napus as a template, primers were designed with the promoter sequence of oleosin gene, primer 1 contains Hind III restriction site, primer 2 introduces Xba I and EcoR I restriction site, obtained by PCR amplification About 900 bp of the promoter of the 20KD oleosin gene (PCR reaction conditions: 94°C, 5 minutes; 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute; 72°C extension for 5 minutes after 25 cycles), PCR product After double digestion with Hind III and EcoRI, it was connected to pUC19 to obtain pUCON, and the sequencing results showed that the cloned product was the promoter of rapeseed oil body protein gene.

[0125] Primers were designed based on the soybean 24kD oil body protein gene sequence: the sequence of primer 1 was: 5′-GAA TCT AGA GAT GAT GAT GAT AAG ATG ACC ACA CAAGTA CC, and the sequence of primer 2 was: 5′-GCC GGT ACC ATC ATC ATC ATC A...

Embodiment 3

[0127] Example 3: Construction of recombinant oil body expression vector containing MT gene

[0128] After the pUC-MT plasmid constructed in Example 1 was double-digested with KpnI and SpeI, the obtained MT fragment was ligated with the recombinant oil body expression vector p1390ONE to obtain the recombinant plant oil body expression vector p1390ONE-MT.

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Abstract

This invention provides a method for expressing human metallothionein (MT) by using plant oil. The method comprises: designing and synthesizing human MT gene according to the preference of plant codon, fusing human MT gene with plant oil protein gene, constructing an expression vector with the fused genes, converting recipient plant by using the expression vector to obtain converted plant, and efficiently expressing human MT by using the seed of the converted plant. Activity experiments show that purified recombinant MT has comparable activity with human origin MT. The method utilizes plant bioreactor to produce MT, thus can avoid possible attack by animal pathogen and toxin in E. coli. The method can largely simplify the separation and purification process of human MT, and lower the production cost, this is advantageous for MT industrialization.

Description

technical field [0001] The present invention relates to a kind of metallothionein (Metallothionein, MT) and its expression method in plant oil body, particularly relates to the method for producing metallothionein by utilizing plant oil body protein-purpose protein composition fusion protein gene expression system. Background technique [0002] Metallothionein (Metallothionein, MT) is a kind of metal-binding protein with low molecular weight (2-7kD), rich in cysteine ​​(20%-30%), capable of binding heavy metals and induced by metals. The name of this protein comes from the extremely high amount of metal and sulfhydryl groups it contains. Since Margoshes and Valee first isolated metallothionein in horse kidney cortex in 1957, subsequent studies have found that MT widely exists in the tissues of animals, plants, and microorganisms, such as in other mammals, plants, fungi, cyanobacteria, fish, etc. species, protozoa, etc. A typical MT has the following characteristics: [00...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/82C12P21/02C07K1/14
Inventor 李校堃张驰肖业臣柯实庞实锋
Owner JILIN AGRICULTURAL UNIV
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