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Plant oil-body expression vector and process of expressing human epidermal growth factor with plant oil-body

A technology of epidermal growth factor and expression vector, which is applied in the fields of botany equipment and methods, biochemical equipment and methods, plant gene improvement, etc. It can solve protein denaturation, chemical synthesis product purity and yield that cannot meet industrial production, and production cost advanced questions

Active Publication Date: 2007-06-27
浙江美仁谷生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, EGF is mainly expressed in Escherichia coli, which is basically an injection drug, and the production cost is high
[0004] In summary, the advantages of the present invention are as follows: due to the existence of disulfide bonds and other various functional groups in the hEGF molecule, the purity and yield of the chemically synthesized product cannot meet the requirements of industrial production.
At present, the production of hEGF is mainly obtained in Escherichia coli and yeast through genetic engineering technology, but the expression product often forms inclusion bodies, and the separation and purification involves protein denaturation and renaturation, as well as multi-step reproduction for post-treatment and purification processes operate

Method used

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  • Plant oil-body expression vector and process of expressing human epidermal growth factor with plant oil-body

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: Preparation and sequence determination of EGF gene

[0025] Taking the hEGF gene as a blueprint, first remodel the base sequence of hEGF according to the codons preferred by plants, and eliminate the enzyme cutting sites used in this experiment according to the degeneracy of the codons, use the online software Primer Finder and DNA STAR Primers were designed, and the EGF gene was synthesized by bridge PCR.

[0026] EGF full-length gene synthesis

[0027] PCR system one:

[0028] 10×pfu buffer 10μl

[0029] dNTP (2.5mM) 8μl

[0030] Primer F1 (10μmol / L) 2μl

[0031] Primer R1 (10μmol / L) 2μl

[0032] pfu DNA polymerase 0.5μl

[0033] wxya 2 O 77.5 μl

[0034]

[0035] Total 100μl

[0036] PCR system two:

[0037] 10×pfu buffer 10μl

[0038] dNTP (2.5mM) 8μl

[0039] Primer F2 (10μmol / L) 2μl

[0040] Primer R2 (10μmol / L) 2μl

[0041] pfu DNA polymerase 0.5μl

[0042] wxya 2 O 77.5 μl

[0043] ...

Embodiment 2

[0061] Example 2: Construction of recombinant oil body expression vector

[0062] Using the total DNA of Brassica napus as a template, primers were designed based on the sequence of the promoter of the oleosin gene. Primer 1 contains a HindIII restriction site, and primer 2 introduces XbaI and EcoRI restriction sites. After PCR amplification, about 900bp The promoter of 20KD oleosin gene (PCR reaction conditions: 94°C, 5 minutes; 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute; after 25 cycles, 72°C extension for 5 minutes), the PCR product was treated with HindIII and After EcoRI double digestion, it was connected to pUC19 to obtain pUCON, and the sequencing results showed that the cloned product was the promoter of rapeseed oleosin gene.

[0063] 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...

Embodiment 3

[0065] Example 3: Construction of recombinant oil body expression vector containing hEGF gene

[0066] After the pUCEGF plasmid was double-digested with KpnI and SpeI, the obtained hEGF fragment was connected with the recombinant oil body expression vector p1390ONE to obtain the recombinant plant oil body expression vector p1390ONE-EGF.

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Abstract

The present invention is gene recombination process, in which human epidermal growth factor, (hEGF) and oil-body protein gene are fused to constitute one plant oil-body expressing vector. Root nodule agrobacterium is used to transform Arabidopsis thalianuum, safflower, soybean, black bean and rape to establish genetic expressing system of fusion expressed oil-body protein gene and the target gene and to obtain transgenic plant. The molecular biology detection on transgenic safflower and rape proves the integration and expression of exogenous gene, and the activity detection proves the bioactivity corresponding to the standard hEGF activity of obtained recombinant protein. The present invention also provides the method of expressing hEGF in plant seed, and the method may be used in producing hEGF at low cost and developing new medicine.

Description

technical field [0001] The invention relates to the biological field, in particular to a method for expressing human epidermal growth factor by using plant oil body expression vector. Background technique [0002] The use of transgenic plants to produce medicinal proteins has attracted more and more attention in recent years. To express medicinal proteins in plant oil bodies, the target gene is inserted into the N-terminus or C-terminus of the oil body protein gene to construct a fusion protein gene. Since the oil body protein is embedded on the surface of the oil body, the constructed fusion protein gene can be specifically expressed in the oil body of the recipient plant seed, which can greatly increase the expression level of the foreign protein in the plant tissue, and can utilize the oil body affinity Lipid-hydrophobic characteristics, after the seeds are crushed, the oil phase can be recovered through steps such as liquid extraction and centrifugation, and the fusion p...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/62C12N15/09C12N15/29C12N15/12
Inventor 李校堃肖业臣庞实锋柯实张驰
Owner 浙江美仁谷生物技术有限公司
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