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Method for expressing human osteogenic protein-1 in peanut seeds

A technology of osteogenic protein and peanut, applied in the direction of animal/human protein, osteogenic protein, osteogenic factor, etc., to achieve the effect of simple method, convenient transportation and stable recombinant protein

Inactive Publication Date: 2010-12-08
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

AI Technical Summary

Problems solved by technology

Peanut has a large planting area in China, India, Nigeria, the United States, etc., so it has a wide-spread basis as a plant reactor. The method of osteogenic protein-1 has not been reported yet

Method used

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  • Method for expressing human osteogenic protein-1 in peanut seeds
  • Method for expressing human osteogenic protein-1 in peanut seeds
  • Method for expressing human osteogenic protein-1 in peanut seeds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Cloning of the soybean oleosin gene comprising a promoter

[0025] According to the sequence of the promoter of soybean oil body protein in NCBI and the gene sequence of oil body protein (gene accession number: GMU09118), the forward primer sequence containing the NcoI restriction site (CCATGG) was designed: primer1: 5'CATGCCATGGTGTTTATCTTTCTTGCTTTTCTG 3'; According to the open reading frame sequence of soybean oil body protein gene, the stop codon was removed, and the reverse primer primer2 with EcoRI restriction site (GAATTC) and enteropeptidase recognition site (TTATCATCATCATC) was designed: 5'CCGGAATTCCCTTATCATCATCATCTGCGGTTGCGGTTGTTGCTGTCA T 3 '.

[0026] Extract the genomic DNA of soybean (Zhonghuang 13), the method is as follows:

[0027] (1) Take 100 mg of young soybean leaves, grind them into powder with liquid nitrogen, or use them directly or store them at -80°C;

[0028](2) Preheat CTAB extract (0.2M Tris-HCl, pH 9.0; 0.4M LiCl; 25mM EDTA; 1% SDS...

Embodiment 2

[0042] Example 2 Acquisition of Human Osteogenic Protein Mature Peptide cDNA Gene bmp7

[0043] According to the mature peptide sequence of human osteogenic protein-1 gene in NCBI (gene accession number: NM_001719), primers containing EcoRI site primer3: 5'CCGAATTCATATGACCGGCAGCAAACAG 3' and primer4 with HindIII site: 5'GAAAGCTTGATCC TTACATGTCGCAGCCACAGGC 3' were designed .

[0044] collect 1x10 6 According to the standard procedure of One-Step RT-PCR Kit (Clonetech), the following PCR reactions were carried out: incubation at 45°C for 1h, denaturation at 45°C for 5min, annealing at 51°C for 1min, 72°C extension for 1 min) for 30 cycles, 72°C extension for 7 minutes.

[0045] 5 μL of the PCR product was subjected to 1% agarose gel electrophoresis, the results were observed under ultraviolet light, and positive clones were selected for sequencing (see Sequence Table 4).

Embodiment 3

[0046] The construction of embodiment 3 plant expression vectors

[0047] The construction process of plant expression vector is as follows: figure 1 Shown:

[0048] (1) Linkage of oleosin gene and human osteogenic protein gene (oleo-bmp7)

[0049] The PCR product was digested with the following system:

[0050] PCR product 10.0 μL, 10x Buffer H 3.0 μL, EcoRI 1.0 μL, ddH 2 O 16.0 μL. Incubate at 37°C for 4 hours, perform agarose electrophoresis, and recover oil body protein and osteogenic protein gene fragments respectively.

[0051] Ligate the oleosin gene and the human osteogenic protein gene with T4 DNA ligase: 1.0 μL of 10x T4DNA ligasebuffer, 4.0 μL of Oleosin, 4.0 μL of Bmp7, 1.0 μL of T4DNA ligase. Ligate overnight at 16°C, take 1.0 μL of the ligation product for PCR amplification, the system is as follows: 2x pfu mix 10.0 μl, primer1 (10 μm) 0.5 μl, primer4 (10 μm) 0.5 μl, ddH 2 O 8.0 μl. The PCR instrument (TaKaRa TP650) was set at 94°C for 5min, followed by 94...

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Abstract

The invention discloses a method for expressing human osteogenic protein-1 in peanut seeds, which comprises the following steps of: utilizing a gene engineering way to connect an osteogenic protein encoding gene to an 3' terminal of an oleosin gene, constructing an 'oleosin-erepsin-human osteogenic protein' plant expression vector driven by an oleosin promoter, transforming peanuts, making recombinant protein perform high-level expression in the peanut seeds along with the accumulation of an oil body, separating and purifying the recombinant protein through floating centrifugation, and then purifying to obtain the protein after the digestion of exoproteinase. The recombinant protein obtained by the method has the advantages of safety, high activity, and easy recovery and purification, canbe stored in the peanut seeds for a long time, has convenient transportation, and can increase the added value of farm products.

Description

technical field [0001] The invention relates to a method for expressing human osteogenic protein-1, in particular to a method for expressing human osteogenic protein-1 in peanut seeds by using an oil body protein expression system, and belongs to the field of biotechnology. Background technique [0002] Human Osteogenic Protein 1 (hOP-1), also known as Bone Morphogenetic Protein 7 (BMP-7), plays an important role in eye development, kidney function regulation, and hindbrain development. It has a wide range of clinical applications. However, due to the limited source of human bone and the complex procedure of extracting hOP-1 from bone tissue, it is difficult to obtain pure products. The osteoinductive activity of hOP-1 isolated and purified through cumbersome procedures is often not high, which seriously limits clinical application [1, 2, 3 ]. With the development of genetic engineering technology, heterologous expression of osteogenic proteins becomes possible. At presen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C07K14/51C07K1/14
Inventor 王兴军赵传志毕玉平夏晗李爱芹李长生
Owner 山东省农业科学院高新技术研究中心
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