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Method for expressing human humanin protein in peanut seed

A peanut and protein technology, applied in the field of expressing human humanin protein, to achieve the effects of wide cultivation area, easy recovery and purification, and convenient transportation

Inactive Publication Date: 2012-01-18
山东省农业科学院高新技术研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Human humanin protein has broad clinical application prospects. After searching, there is no report on the method of expressing human humanin protein in peanut seeds using the oil body protein expression system

Method used

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  • Method for expressing human humanin protein in peanut seed
  • Method for expressing human humanin protein in peanut seed
  • Method for expressing human humanin protein in peanut seed

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Cloning of a promoter-containing peanut oil body protein gene

[0036] According to the promoter and coding gene sequence of peanut oil body protein in Genbank (gene accession number: EF695400), the forward primer primer1 containing EcoRI restriction site (GAATTC) was designed: AGAAT TCAGG TCAAC TACCA TTCGT; Open reading frame sequence, remove stop codon, and design reverse primer primer2 with KpnI restriction site (GGTACC): GGTACCAGTTCTCTTTGAATCCTG.

[0037] The genomic DNA of peanut (Luhua 14) was extracted as follows:

[0038] (1) get 1g of peanut young leaves, grind into powder with liquid nitrogen, and place in the Eppendof tube of liquid nitrogen precooling;

[0039] (2) Preheat CTAB extract (2% CTAB, 1.4mol / L NaCl, 20mmol / L EDTA (pH8.0), 100mmol / L Tris-Cl (pH8.0), 2%pvp- 40);

[0040] (3) To estimate the mass of the sample tissue, add 700 μL of pre-warmed CTAB extract to every 200 mg of sample, mix quickly, and incubate at 65°C for 10-30 min, mixing ...

Embodiment 2

[0056] Example 2 Synthesis of Human Humanin Gene

[0057] According to the sequence of humanin gene (NP_001177635) in Genbank, two partially complementary single-stranded DNA primers were designed and synthesized, wherein the forward primer contained KpnI (GGTACC) site and enteropeptidase recognition sequence (GATGATGATGATAAG) primer3: GAAGGTACCG ATGATGATGA TAAGATGGCT CCACGAGGGT TCAGCTGTCT CTTACTTTTA ACCAGTGAAA TTGACCTGCC; the reverse primer contains His tag sequence (CACCAC CACCAC CACCAC) and SalI restriction site (GTCGAC) primer4: ACGCGTCGAC TTAGTGGTGG TGGTGGTGGT GTGCCCGCCT CTTCACGGGC AGGTCAATTT CACTGGTTAA AAGTAAGAGA.

[0058] Use TAQ enzyme for annealing and extension, primer3 (100 μM) 1 μl, primer4 (100 μM) 1 μl, the program is: 94 ℃ for 3 min; 40 ℃ for 1 min; 72 ℃ for 2 min; Observing the results under the ultraviolet lamp, the gel recovered the target band, and the recovered product was connected to the T carrier, transformed into Escherichia coli DH5α cells according to...

Embodiment 3

[0059] Example 3 Construction of plant expression vector

[0060] The construction process of plant expression vector is as follows: figure 1 shown:

[0061] (1) The peanut oil body protein promoter and ORF were connected to the pCAMBIA2300-35S-OCS plant expression vector. The skeleton of this plant expression vector came from Cambia Company and was transformed by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (Luo et al. Journal of Integrative plant biology, 2005, 47(6), 745-752): The pCAMBIA2300-35S-OCS plasmid and the T-AhOleosin plasmid were digested with EcoRI and KpnI, respectively, the vector sequence and the AhOleosin sequence were recovered, and the two were ligated with T4 DNA ligase. ligase: 10×T4D NA ligase buffer 1 μl, AhOleosin 6 μl, pCAMBIA2300 2 μl, T4 DNA ligase 1 μl, ligated overnight at 16°C, the ligation product was transformed into E. coli DH5α according to the method in Example 1, and verified by double digestion with Ec...

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Abstract

The invention discloses a method for expressing human humanin protein in peanut seeds, which comprises the following steps: connecting humanin protein coding gene to a 3' end of an oleosin gene by a genetic engineering means, establishing an 'oleosin-erepsin-human humanin protein' plant expression vector driven by an oleosin promoter, transforming peanuts to realize high-level expression of recombinant protein in peanut seeds with the accumulation of oil body, then performing centrifugation of fusion protein, digesting the fusion protein by exo-protease, performing centrifugation, removing the oil phase and recovering water phase, performing purification to obtain human humanin protein. The recombinant protein obtained by the method of the invention is safe, has high activity, is very easy to be recovered and purified, can be stored in peanut seeds for a long term, is convenient for transportation, and greatly increases the crop added value.

Description

technical field [0001] The invention relates to a method for expressing humanin protein, in particular to a method for expressing humanin protein in peanut seeds by utilizing oil body protein system, and belongs to the field of biotechnology. Background technique [0002] Alzheimer's disease (AD) is a common disease of the elderly with progressive memory loss, cognitive impairment and dementia as clinical manifestations. Another serious threat to human health after disease, cancer and stroke. In the early 21st century, Japanese scholars first isolated a short peptide called Humanin (HN), which consists of 24 amino acids. It can effectively inhibit AD-related genes: amyloid precursor (APP) gene, preseniln1 (PS1) gene, preseniln2 (preseniln2, PS2) gene mutation, and amyloid β peptide (amyloid) peptide, Aβ)-induced neuronal cell death [1-3] . The primary structure of HN has the characteristics of signal peptide structure, which can be secreted to the outside of the cell. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84
Inventor 夏晗王兴军毕玉平肖寒赵传志李长生李爱芹
Owner 山东省农业科学院高新技术研究中心
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