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Porphyra yezoensis manganese superoxide dismutase and its preparation method

A technology of laver and superoxide, which is applied in the directions of botanical equipment and methods, biochemical equipment and methods, enzymes, etc., can solve the problems of reduced assimilation, reduced plant quality, reduced yield, etc., and can solve the problems of promoting Effect

Inactive Publication Date: 2006-05-03
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows us create an organism called Phophora vulgaris which contains specific proteins found naturally within its cells or tissues. These special enzymes are involved in various metabolic processes such as cellular signaling pathways and energy generation. They help protect these organs against damage caused by environmental factors like UV rays or chemical agents during their lifecycle stages. Overall, they provide technical benefits over current methods of producing synthetic molecules containing harmful substances.

Problems solved by technology

This patents discusses how certain compounds called sulfite ion hydrolones (Sulfonium Hydroxy Isozopyrrolide), known collectively abbreviations SOSHYLFOXM, LCQB, CNS Drug Excipients Group IIIa, Benthiaceae Ferrucosa Yellow Leaf Extract, Phenyl Sulfonates). These substances act like molecular chelators but their effectiveness depends upon factors like pigments, sugars, amino acids, nitrilotriaccharides, phosphorylcholine, fatty bases, oxysporum polyunsaturated linolecules, iron cofactors, and unsymmetrical conjugative imbalance between these components.

Method used

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  • Porphyra yezoensis manganese superoxide dismutase and its preparation method
  • Porphyra yezoensis manganese superoxide dismutase and its preparation method
  • Porphyra yezoensis manganese superoxide dismutase and its preparation method

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Experimental program
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Effect test

Embodiment 1

[0060] Construction of Porphyra zebra cDNA library (using oligonucleotide capping method (Olige-capping), refer to Maruyama et al., 1994):

[0061] (1), extraction of total RNA:

[0062] Total RNA was extracted using UNIQ-10 Column Trizol Total RNA Extraction Kit (purchased from Shanghai Bioengineering Co., Ltd.). The material was obtained from the Porphyra zebra filaments of the algae bank in our laboratory, and the total RNA was extracted by an improved one-step method. Specific steps are as follows:

[0063] Wash with sterile water and blot dry with filter paper, weigh 70 mg of algae, and freeze in liquid nitrogen for 20 minutes. After fully grinding with liquid nitrogen, transfer the algae powder to a pre-cooled 1.5ml EP tube, add 450μl Trizol, vortex vigorously for 30s, and mix thoroughly. with 26-G # The needle was cut twice to form a homogenate. Transfer the homogenate to a new 1.5ml RNase-free EP tube, add 100μl chloroform / isoamyl alcohol, vortex vigorously for 30...

Embodiment 2

[0118] Database construction:

[0119] Porphyra zebra cDNA library constructed by oligonucleotide capping method (Olige-capping) was used as the library source for sequencing EST. The DNA sequencing work was completed by Hangzhou Huada Genome Research Center. The universal primer used is M13F (5'-GTAAAACGACGGCCAGT-3'), and the automatic sequencer is MegaBace TM 1000.

[0120] After removing the carrier sequence in the sequence and correcting the ambiguous bases detected at the 3' end, the sequence with a fragment length of less than 100 is regarded as an invalid sequence, and the rest of the sequence is processed using EST Analysis Pipeline System TM Assist with bioinformatics analysis to determine function and reduce redundancy. The program combines common tools for bioinformatics analysis, including BLAST, Phred, Pfam, Crossmatch, Phrap, Cap3, etc. A total of 469 EST sequences representing different genes were obtained, and the non-redundant genes were searched for homol...

Embodiment 3

[0122] Select the required strain, save, sequence and similarity analysis:

[0123] According to the results of EST analysis, the Mn-SOD genes related to the stress resistance of Porphyra were selected. The host cell of the strain is Escherichia coli competent cell DH5α, and the plasmid vector is pMD18-T. The strain preservation number is CGMCC No.1414.

[0124]The sequence of the inserted fragment of the strain was determined by Shanghai Yingjun Biotechnology Co., Ltd. Most of the sequence was determined by bidirectional sequencing, and a 958bp nucleotide sequence was obtained, which is the cDNA of the Mn-SOD gene of Porphyra zebra sequence. The sequence has been submitted to the International Gene Sequence Bank (GenBank) with the sequence number DQ146477. The open reading frame of the sequence is 675bp (ATG-TAA), can translate 224 amino acid residues and stop codon UAA, and the predicted molecular weight is 24469.09Da (application analysis software DNAsis). See SEQ NO.1 ...

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Abstract

The invention relates to a Porphyra yezoensis superoxide dismutase with preparation method, which comprises 224 amino residues with predictive molecular weight as 24469.09Da. Wherein, the most of 24 residues on N-end are hydrophobic amino acid, this peptide segment plays an role in transfering the enzyme to mitochondria; the histidine (H) on 27th, 84th, 178th sites of the sequence, and aspartic acid (D) on 174th site, are all the combination sites with Mn2+; there is '' DVWEHAYYL '' as conservative metal combination structure domain; compared with the sequence of Mn-SOD amino acid, this enzyme has 50-60% isogeny. This invention uses enzyme cDNAsequence and expression carrier pSE380 to construct recombinant plasmid pSE380/MnSOD and express int escherichia coli DH5alpha; the method is precise and reasonable and exploits the material resource for superoxide dismutase.

Description

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Claims

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Application Information

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Owner OCEAN UNIV OF CHINA
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