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 nucleic acid, protein and lipid damage, reduced yield of target protein, increased production cost and cycle, etc. The effect of preventing aging, reducing content, preventing UV damage
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Embodiment 1
[0065] Embodiment 1: Preparation and sequence determination of MT gene
[0066] 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 enco...
Embodiment 2
[0117] Example 2: Construction of recombinant oil body expression vector
[0118] 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 20 KD 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 After the product was double-digested with Hind III and EcoR I, 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.
[0119] 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...
Embodiment 3
[0121] Example 3: Construction of recombinant oil body expression vector containing MT gene
[0122] After the pUC-MT plasmid constructed in Example 1 was double-digested with Kpn I and Spe I, 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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