Construction and application of plant transient expression vector

A transient expression and plant technology, applied in vectors, nucleic acid vectors, and the use of vectors to introduce foreign genetic material, etc.

Pending Publication Date: 2020-10-27
浙江华缔药业集团医药开发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the current stage, domestic plant exogenous protein expression vectors are mainly used in the field of basic scientific research, and there is still little exploration of application vectors that can be used for efficient commercial production

Method used

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  • Construction and application of plant transient expression vector
  • Construction and application of plant transient expression vector
  • Construction and application of plant transient expression vector

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The construction of embodiment 1 pEAQE carrier

[0030] (1), using the plant expression vector pEAQ as the basic skeleton, performing 2-hour digestion with two restriction sites of PacI and XhoI, and then recovering a 9200bp fragment by DNA gel electrophoresis;

[0031] (2), synthesis contains artificial sequence as shown in SEQ ID No.1, including 5'UTR region, MCS region, SEKDEL sequence;

[0032] (3), design amplification primers for amplifying the artificial sequence gene fragment, the upstream primer is as shown in SEQ ID No.2, and the downstream primer is as shown in SEQ ID No.3;

[0033] (4) KOD One of TOYOBO TM PCRMasterMix for amplification reaction, the amplification system is: 22ul ddH 2 O. 25ul KOD One TM PCRMasterMix, 1ul 10uM upstream primer, 1ul 10uM downstream primer, 1ul 100ng / ul plasmid template containing the artificially synthesized sequence SEQ ID No.1. PCR amplification program: 98 degrees for 30 seconds; 28 cycles: 98 degrees for 10 seconds; ...

Embodiment 2

[0042] Embodiment 2 inserts GFP gene on the pEAQE vector

[0043] (1), using the plant expression vector pEAQE as the basic skeleton, using the SmaI restriction site for 2 hours of digestion, and then recovering a 10469bp fragment by DNA gel electrophoresis;

[0044] (2), design amplification primers for amplifying artificial sequence gene fragments, upstream primer SEQ ID No.4, downstream primer SEQ ID No.5;

[0045] (3) KOD One of TOYOBO TM PCRMasterMix for amplification reaction, the amplification system is: 22ul ddH 2 O. 25ul KOD One TM PCRMasterMix, 1ul 10uM upstream primer, 1ul 10uM downstream primer, 1ul 100ng / ul plasmid template containing GFP. PCR amplification program: 98 degrees for 30 seconds; 28 cycles: 98 degrees for 10 seconds; 55 degrees for 30 seconds; 68 degrees for 10 seconds; the last 68 degrees for 5 minutes; 4 degrees for 10 minutes;

[0046] (4) The final amplified gene fragments are recovered by gel electrophoresis through gel electrophoresis; sin...

Embodiment 3

[0054] Example 3 Infiltration

[0055] (1) The pEAQE-GFP plasmid was introduced into the competent Agrobacterium by electric shock method, and spread on the Kana and rifampicin double-antibody medium.

[0056] (2), pick the single clone and shake the bacteria with the culture medium, wait for the OD 600 When =0.8, with 8000rpm centrifugation 2 minutes, the bacterium liquid of precipitation is washed with infiltration buffer (10mM MES, 10mM Mgcl 2 , 1uM acetosyringone) resuspended and adjusted OD 600 = 0.6.

[0057] (3) Infiltrate the bacterial solution into the tobacco leaves with a negative pressure vacuum pump, and observe the fluorescence after 2 days.

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Abstract

The invention relates to the biotechnology field, in particular to construction and application of a plant transient expression vector. An artificially synthesized sequence is adopted to improve a transcriptional quantity of the vector, meanwhile, an amino acid sequence is selectively added to improve the translation level and stability of target protein, and finally, a function of improving a cell accumulation quantity of the target protein in plant leaves can be integrally achieved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the construction and application of plant transient expression vectors. Background technique [0002] Like mammalian cell expression system and microbial fermentation system, plant expression system is a biological protein expression reactor with broad application prospects. Mammalian cell expression system has the advantages of high yield, mature system, and similarity to human cells, so it is widely promoted, but the operation price is relatively expensive; microbial fermentation system technology is also quite complete, while high yield and low price, but due to differences in cell types However, the microbial fermentation system cannot well express the target protein that requires specific protein modification. The plant expression system has inherent advantages, such as the limited biomass of plants, fast growth, low cost and mammalian expression system. At the same time, plant...

Claims

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Application Information

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IPC IPC(8): C12N15/82C07K14/435
CPCC12N15/8202C07K14/43595C12N2800/80
Inventor 周昕郑蔚翟璐赵启超濮云飞
Owner 浙江华缔药业集团医药开发有限公司
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