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Method for instantaneously expressing acidic fibroblast growth factor in plant

A fibroblast and growth factor technology, applied in the field of plant genetic engineering, can solve the problems of high price, low yield, and affecting the biological activity of aFGF, and achieve the effects of low production cost, good protein activity, and short cycle

Inactive Publication Date: 2008-11-19
东北师范大学遗传与细胞研究所
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, the traditional method to obtain aFGF is to extract from animal tissue and prokaryotic expression in Escherichia coli. However, the first method has low yield and high price. The second method is to produce recombinant protein by prokaryotic expression system. Phosphorylation and other post-translational modifications, and often form inclusion bodies to affect the biological activity of the resulting aFGF

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  • Method for instantaneously expressing acidic fibroblast growth factor in plant

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Effect test

Embodiment 1

[0014] 1. Construction of HaFGF plant potato virus X expression vector

[0015] Primers were designed according to the hafgf sequence, and the hafgf fragment was amplified by PCR. PCR parameters: 94°C for 3min; 20 cycles of 94°C for 35sec, 54°C for 30sec, and 72°C for 1min; 72°C for 1min; 4°C for 10min.

[0016] The hafgf sequence was connected into the plant potato X virus expression vector pGR107 digested with Sma I by blunt end ligation. Constructed into pGR107-haFGF. The plasmid was transformed into competent Agrobacterium GV3101 by freeze-thaw method to obtain GV3101.pGR107-haFGF.

[0017] 2. Transient expression of HaFGF in tobacco

[0018] Tobacco seedlings with a growth period of 40 days (five-leaf stage or six-leaf stage) were used as materials for infection.

[0019] Pick a single colony of transformed and identified Agrobacterium GV3101.pGR107-haFGF, inoculate it in 5ml LB liquid medium containing 50mg / L kanamycin, 12.5mg / L tetracycline and 50mg / L rifampicin, ov...

Embodiment 2

[0023] 1. Construction of expression vector of HaFGF plant Peanut early brown virus

[0024] Primers were designed according to the hafgf sequence, and the hafgf fragment was amplified by PCR. PCR parameters: 94°C for 3min; 20 cycles of 94°C for 35sec, 54°C for 30sec, and 72°C for 1min; 72°C for 1min; 4°C for 10min.

[0025] The hafgf sequence was linked into the expression vector pCAPE2 of plant pea early brown virus which was cut with Nco I and EcoR I. Constructed into pCAPE2-haFGF. The plasmid was transformed into competent Agrobacterium GV3101 by freeze-thaw method to obtain GV3101.pCAPE2-haFGF.

[0026] 2. Transient expression of HaFGF in pea

[0027] The infestation was carried out on pea seedlings with a growth period of 2 weeks.

[0028] Pick transformed and identified Agrobacterium GV3101.pCAPE2-haFGF single colonies and GV3101.pCAPE1 respectively, inoculate them in 5ml LB liquid medium containing 50mg / L kanamycin and 50mg / L rifampicin, and culture them at 28°C fo...

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Abstract

The invention discloses a method for realizing instant expression of acidic fibroblast growth factors in plant, relating to an improvement in the process for making acidic fibroblast growth factor and further belonging to the plant genetic engineering technical field. The method is as follows: firstly, the construction of the plant virus expression vector of anthropogenic acidic fibroblast growth factors is carried out; a coded anthropogenic acidic fibroblast growth factor DNA sequence 468bp is connected with a reconstructive plant virus expression vector, and then is transformed to agrobacterium GV3101 so as to be used in the instant expression in plant; a plant material of proper seedling age is selected to carry out agrobacterium infection, that is, slight scars are respectively scraped at both sides of a main vein on the back side of a leaf by a syringe needle; then, bacterial liquid is injected into the leaf from the scars by a syringe with the needle removed so as to ensure that plant virus carrying target gene hafgf can be quickly diffused, expressed and aggregated in a plant body; when the aggregation reaches to a peak value, plants are collected to carry out the extraction of target protein.

Description

technical field [0001] The invention discloses a method for transiently expressing acidic fibroblast growth factor in plants, relates to the improvement of the preparation process of acidic fibroblast growth factor, and further belongs to the technical field of plant genetic engineering. Background technique [0002] The fibroblast growth factor superfamily has so far discovered 23 members. Acidic fibroblast growth factor (acidic fibroblast growth factors, referred to as: aFGF) is one of them, it has a strong ability to promote mitosis, it can stimulate DNA synthesis and proliferation of a variety of cells, including fibroblasts, epithelial cells, Endothelial cells, smooth muscle cells, myoblasts, chondrocytes and glial cells. In addition, it is also believed that aFGF can directly stimulate the migration of endothelial cells and fibroblasts. aFGF plays an important role in ontogeny, many stages of morphogenesis, angiogenesis and wound healing. Acidic fibroblast growth fa...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/12
Inventor 王兴智朱筱娟刘晶莹麻鹏达
Owner 东北师范大学遗传与细胞研究所
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