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 effect of increasing added value, low cost and convenient transportation

Inactive Publication Date: 2013-01-30
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 the peanut oleosin gene comprising a promoter

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

[0037] Extract the genomic DNA of peanut (Luhua 14), the method is as follows:

[0038] (1) Take 1 g of young peanut leaves, grind them into powder with liquid nitrogen, and place them in an Eppendof tube pre-cooled by liquid nitrogen;

[0039] (2) Preheat CTAB extract (2% CTAB, 1.4mol / L NaCl, 20mmo / 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 preheated CTAB extract to each 200 mg sam...

Embodiment 2

[0056] Synthesis of embodiment 2 people's Humanin gene

[0057] According to the humanin gene (NP_001177635) sequence in Genbank, two partially complementary single-stranded DNA primers were designed and synthesized, in which 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 ° C for 3 min; 40 ° C for 1 min; 72 ° C for 2 min; store the product at 16 ° C; the product is electrophoresed on 1% agarose gel, Observe the results under ultraviolet light, recover the target band from the gel, connect the recovered produc...

Embodiment 3

[0059] The construction of embodiment 3 plant expression vectors

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

[0061] (1) Connect the peanut oleosin promoter and ORF to the pCAMBIA2300-35S-OCS plant expression vector. The plant expression vector backbone comes from Cambia Company and is 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): pCAMBIA2300-35S-OCS plasmid and T-AhOleosin plasmid were digested with EcoRI and KpnI respectively, the vector sequence and AhOleosin sequence were recovered, and the two were combined with T4DNA ligase. Ligation: 1 μl of 10×T4D NA ligase buffer, 6 μl of AhOleosin, 2 μl of pCAMBIA2300, 1 μl of T4 DNA ligase, ligation overnight at 16°C, the ligation product was transformed into E. coli DH5α according to the method in Example 1, and verified by double enzyme digestion with EcoRI and ...

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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 human humanin protein, in particular to a method for expressing human humanin protein in peanut seeds by using an oil body protein system, and belongs to the field of biotechnology. Background technique [0002] Alzheimer's disease (Alzheimer disease, AD) is a common disease of the elderly with progressive memory loss, cognitive impairment and dementia as the clinical manifestations. Another disease that seriously endangers human health after disease, cancer and stroke. At the beginning of the 21st century, Japanese scholars first isolated a short peptide called Humanin (HN), consisting of 24 amino acids. It can effectively inhibit AD-related genes: amyloid precursor (amyloid precursor, APP) gene, preseniln1 (preseniln1, PS1) gene, preseniln2 (preseniln2, PS2) gene mutation, and beta amyloid peptide (amyloid Nerve cell death induced by peptide, Aβ [1-3] . The primary structure of HN has the character...

Claims

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Application Information

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Patent Type & Authority Patents(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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