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Tobacco transient expression method and detection method thereof

A technology of transient expression and detection method, which is applied in the fields of plant molecular biotechnology and genetic engineering, and can solve problems such as hindering the industrialization of biopharmaceutical methods, failing to meet the requirements of pharmaceutical proteins, and restricting applications.

Pending Publication Date: 2022-04-26
TOBACCO RES INST CHIN AGRI SCI ACAD
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Transient expression technology refers to a technology in which the gene is not integrated into the plant genome and the foreign protein can be expressed in the plant in a short period of time. Functional medicinal proteins can be used in biopharmaceuticals; however, the amount of protein expressed by the current tobacco transient expression technology can only be used at the laboratory level, and cannot meet the level of mass production of medicinal proteins using this technology; severe limitations The application of this technology in the biopharmaceutical industry also hinders the industrialization of this technology as a means of biopharmaceuticals

Method used

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  • Tobacco transient expression method and detection method thereof
  • Tobacco transient expression method and detection method thereof
  • Tobacco transient expression method and detection method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1: Tobacco (containing avrPtoB-C gene SEQ:NO:1) transient expression experiment

[0032] First, spray the hydrocultured tobacco leaves containing the avrPtoB-C gene (SEQ: NO: 1, select plasmid PER8 SEQ: NO: 2 to construct the recombinant gene) with 10 μM estradiol (estradiol, EST) (such as Figure 1-3 As shown), the specific steps are: configure 10mM estradiol mother solution with DMSO, dilute 1000 times estradiol mother solution with water to make 10μM estradiol working solution 200mL for later use; take it from the hydroponic rack to induce the expression of avrPtoB-C gene (SEQ: NO: 1, select the plasmid PER8 SEQ: NO: 2 to construct the recombinant gene) material; spray 10 μ M estradiol with a watering can; cover the treated material with a plastic cover, and keep it for 24 hours;

[0033] Second, transient infection was performed using the Agrobacterium-mediated vacuum infection method (eg Figure 3-4 Shown), its concrete steps are: shake 500mL Agrobacter...

Embodiment 2

[0034] Embodiment 2: Tobacco (containing avrPtoB-C gene SEQ:NO:1) transient expression detection experiment

[0035] In this example, on the basis of Example 1, the following steps are added: replenishing the spraying of luminescent substance substrates in time, and detecting protein expression at different time points.

Embodiment 3

[0036] Example 3: Control group 1—normal tobacco (without avrPtoB-C gene SEQ:NO:1) transient expression detection experiment

[0037] The difference between this example and Example 2 is that the tobacco used is normal tobacco, ie does not contain the avrPtoB-C gene SEQ:NO:1.

[0038] This group is used as the control group experiment, and compares (such as Figure 8 ).

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Abstract

The invention belongs to the field of plant molecular biotechnology and gene engineering, relates to a tobacco transient expression method and a detection method thereof, and provides the tobacco transient expression method and the detection method thereof, which comprise the following steps: (1) spraying estradiol on tobacco leaves; (2) keeping humidity of leaves; (3) carrying out instantaneous infection by adopting an agrobacterium tumefaciens-mediated vacuum infection method; and (4) culturing plants. According to the invention, a more efficient tobacco transient expression method is established, tobacco for inducing over-expression of avrPtoB-C is taken as a transient expression material chassis, the transient expression efficiency is improved by more than 3 times, the time of protein expression at the highest point is advanced, and the method shortens the time for transient expression and purification of protein, so that the overall efficiency of the method is greatly improved, and the method is suitable for industrial production. And a technical reserve is provided for industrialization of medicinal protein production by a tobacco transient expression system.

Description

technical field [0001] The invention belongs to the fields of plant molecular biotechnology and genetic engineering, and relates to a tobacco transient expression method and a detection method thereof. Background technique [0002] Transient expression technology refers to a technology in which the gene is not integrated into the plant genome and the foreign protein can be expressed in the plant in a short period of time. Functional medicinal proteins can be used in biopharmaceuticals; however, the amount of protein expressed by the current tobacco transient expression technology can only be used at the laboratory level, and cannot meet the level of mass production of medicinal proteins using this technology; severe limitations The application of this technology in the biopharmaceutical industry has also hindered the industrialization of this technology as a means of biopharmaceuticals. Contents of the invention [0003] In order to solve the above-mentioned difficulties,...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H6/82A01H5/12G01N33/68
CPCC12N15/8205G01N33/68
Inventor 张增林郭永峰王跃驹吴荣荣
Owner TOBACCO RES INST CHIN AGRI SCI ACAD
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