Method for efficiently expressing foreign protein

An exogenous protein and protein technology, which is applied in the direction of peptide preparation methods, chemical instruments and methods, botanical equipment and methods, etc., can solve the complex interaction of genes, the uncertainty of exogenous gene insertion sites, and analysis and detection methods Difficult to accurately evaluate and other problems, to achieve the effect of high protein purity

Inactive Publication Date: 2012-12-12
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the transgenic plant reactor, the uncertainty of the insertion site of the exogenous gene and the complex interaction between genes often occur, which lead to a series of changes in the metabolism, development, and genetics of the transgenic crop,

Method used

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  • Method for efficiently expressing foreign protein
  • Method for efficiently expressing foreign protein
  • Method for efficiently expressing foreign protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1, the construction of recombinant expression vector and recombinant Agrobacterium

[0054] 1. Construction of recombinant expression vector

[0055] 1. Using Arabidopsis cDNA as a template, PCR amplification is performed with a primer pair composed of F1 and R1 to obtain a PCR amplification product.

[0056] F1: 5'-TCCCCCGGGATGGAGGATCCTGATATCAAGA-3';

[0057] R1: 5'-GGCGAGCTCTCATATTGGCTCCTTCAGGACTC-3'.

[0058] PCR amplification program: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 2 min, a total of 25 cycles; extension at 72°C for 10 min.

[0059] 2. Digest the PCR amplification product of step 1 with restriction endonucleases SmalI and SacI, and recover the digested product.

[0060] 3. Digest the pMYC2 vector in step 2 with restriction enzymes SmalI and SacI, and recover the linearized vector backbone (about 11 kbp).

[0061] 4. Ligate the digested product of step 2 and the carrie...

Embodiment 2

[0069] Example 2, Preparation and purification of COI1 protein (determination of protein expression)

[0070] 1. Tobacco cultivation before transformation

[0071] 1. Spread the seeds of Nicotiana benthamiana on MS medium, and after vernalization at 4°C for 3 days in the dark, place them in the plant room for cultivation (22°C; 16 hours of light, 8 hours of darkness) until the seedlings grow two leaves (7- 10 days).

[0072] 2. Transplant the seedlings in mixed soil (vermiculite: black soil is 1:1; mass ratio), pour enough water, and continue to cultivate (28°C; 16 hours of light, 8 hours of darkness) for 3 weeks (2-3 weeks can be ).

[0073] 2. Cultivation of recombinant Agrobacterium before transformation

[0074] 1. Put 1 ml of the recombinant Agrobacterium liquid obtained in step 2 of Example 1 into a 1.5 ml EP tube, centrifuge at 20° C. at 4000 g for 5 min, and collect the bacteria.

[0075] 2. Infect the injection with 500 μl (composed of solute and water; solute and...

Embodiment 3

[0094] Preparation and purification of embodiment 3, COI1 protein (determination of the optimal transformation time of leaves)

[0095] 1. Tobacco cultivation before transformation

[0096] Same as Step 1 of Example 2.

[0097] 2. Cultivation of recombinant Agrobacterium before transformation

[0098] Same as Step 2 of Example 2.

[0099] 3. Transformation of tobacco with recombinant Agrobacterium

[0100] On the different leaves of the same tobacco seedling obtained in step 1 (the old leaves are the leaves of 44 days of growth; the middle leaves are the leaves of 38 days of growth; the new leaves are the leaves of 32 days of growth) and inject the recombinant Agrobacterium bacterium liquid obtained in step 2 respectively , each leaf was injected with 300 μl, the timing was started after the bacterial solution was injected, and each leaf treated with injection was taken after 69 hours.

[0101] 4. Extraction of COI1 protein

[0102] Same as Step 4 of Example 2.

[0103] 5....

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Abstract

The invention discloses a method for efficiently expressing foreign protein. The method comprises the steps of cloning a foreign protein gene onto a pMYC2 carrier, enabling the foreign protein gene and an encoding genes of six MYC tags on the pMYC2 carrier to form a fused gene, and then utilizing an agrobacterium infection transformation method to express the foreign protein in tobacco leaves instantly and efficiently; utilizing affinity media carried with corresponding tag antibodies to efficiently purify the protein expressed by tobacco according to MYC affinity tags carried by recombinant protein and finally performing competitive elution of the affinity media to obtain purified target protein. The purified protein can be used for further biological function research. The method achieves protein purification in one step and is simple and quick, and the protein purity is high. The foreign recombinant protein can be used for biological safety evaluation and biological function research.

Description

technical field [0001] The invention relates to a method for highly expressing foreign protein. Background technique [0002] The use of transgenic technology to produce high value-added proteins in bioreactors has important theoretical significance and application value. Bioreactors have gone through four stages: bacterial genetic engineering, cell genetic engineering, transgenic animal bioreactors and transgenic plant bioreactors. [0003] Bacterial genetic engineering products are often not biologically active, and must undergo a series of modifications such as glycosylation and hydroxylation before they can become effective drugs. Cell genetic engineering is also expensive because of the harsh culture conditions of mammalian cells. , which limits scale production. [0004] Animal bioreactor has the characteristics of high product quality and easy purification, which makes up for the defects of other various gene expression systems, but its technology itself involves mo...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/82C07K14/00C07K1/22A01H5/00
Inventor 李海鸥闫建斌姚瑞枫谢道昕李素华白志燕彭文
Owner TSINGHUA UNIV
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