Method for high-efficiency expression of all subtype mature proteins of IL-37 by utilizing tobaccos

A high-efficiency expression and IL-37 technology, applied in the field of genetic engineering and protein expression, can solve problems such as misfolding, protease degradation, and loss of activity

Inactive Publication Date: 2017-02-22
马生武 +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, a possible method is to express only the gene encoding mature IL-37 in plant cells, but this method has obvious disadvantages, that is, the low expression level and low activity of the target protein, because the newly synthesized protein is due to the lack of signal Peptide-mediated transport is prone to misfolding, which not only makes it inactive but also susceptible to degradation by proteases in the cytoplasm of plant cells
[0006] Another possible method is to replace the original signal peptide sequence of the IL-37 gene with a plant protein signal peptide so that the newly synthesized target protein molecule can achieve transmembrane transport and produce a mature gene by cutting off the grafted plant protein signal peptide in the process. The target protein with a natural N-terminus. But it is necessary to test the signal peptides of many different plant proteins to find a suitable signal peptide, because different signal peptides have different functions and have a significant impact on protein expression

Method used

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  • Method for high-efficiency expression of all subtype mature proteins of IL-37 by utilizing tobaccos
  • Method for high-efficiency expression of all subtype mature proteins of IL-37 by utilizing tobaccos
  • Method for high-efficiency expression of all subtype mature proteins of IL-37 by utilizing tobaccos

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Design and Synthesis of Sp-TEV-CS-IL-37b, Sp-TEV-CS-37a, Sp-TEV-CS-IL-37d, Sp-TEV-CS-IL-37c and Sp-TEV-CS -IL-37e fusion gene

[0035] First obtain the cDNA sequence of each protein subtype of IL-37 in GenBank of NCBI website, coded as Gen Bank accession NO.NM_014439.3 for IL37b; Gen Bank accession NO.NM_173205.1 for IL-37a; Gen Bank accession NO.NM_173204 .1 for IL-37c; Gen Bank accession NO.NM_173202.1 for IL-37d and Gen Bank accession NO.NM_173203.1 for IL-37e. Similarly, the nucleic acid sequence encoding TEV protease was obtained from Gen Bank, coded as Gen Bank accession NO.NP_062908.1.

[0036] Design of IL-37-TEV fusion gene.

[0037] As shown in Figure 1, the basic structural sequence of the fusion gene is: its own original signal peptide-flexible linker peptide (GGGGSx3)-TEV protease-TEV enzyme recognition site-mature IL-37 coding sequence.

[0038] According to the preference of tobacco codons, the designed TEV-IL-37 fusion gene was optimized wi...

Embodiment 2

[0039] Example 2: Recombinant expression vectors pBI-Sp-TEV-CS-IL-37b, pBI-Sp-TEV-CS-IL-37a, pBI-Sp-TEV-CS-IL-37d, pBI-Sp-TEV-CS - Construction of IL-37c and pBI-Sp-TEV-CS-IL-37e

[0040] (1) Construction of recombinant expression vector pBI-Sp-TEV-CS-IL-37b: The construction method is shown in Figure 2, including the following main steps:

[0041] The transition vector pRTL2-GUS was digested with Nco I+ Xba I to recover the 4.2 Kb vector fragment with the GUS gene removed. Plasmid pRTL2-GUS contains E35S promoter (enhanced 35S promoter) - 5' untranslated TEV leader sequence - GUS- Nos3' terminator GUS gene expression cassette.

[0042] The pUC57-Sp-TEV-CS-IL-37b plasmid was digested with Pcil and Xba I, the fusion gene fragment was recovered and inserted into pRTL2-GUS, and the vector fragment recovered after digestion with Nco I+Xba I obtained the transitional expression vector pRTL2 - Sp-TEV-CS-IL-37b.

[0043] Use Hind III to digest the transitional expression vector pR...

Embodiment 3

[0049] Example 3: Stable transformation of tobacco

[0050] The constructed recombinant expression vector plasmids were respectively transformed into Agrobacterium LBA4404 by tri-parental mating.

[0051] Tobacco culture. Tobacco seeds (N. tabacum CV.81V9) were sterilized with 70% alcohol, planted in a plant tissue culture box (magenta vessel) containing MS+ 8g / L agar medium, and cultured in an incubator. The temperature is 25±1°C, the humidity is about 70%, and the culture is carried out under the light cycle of 16 hours of light / 8 hours of darkness. After 3-4 weeks, when the seedling grows to 3-4 true leaves, the sterile plant leaves can be taken as genetic transformation materials.

[0052] Genetic transformation of tobacco with recombinant expression vector. The plasmids of the present invention were introduced with reference to the leaf transformation method described by Horsch et al. (Science 1985. 227, 1229-1231). Taking the recombinant vector PBI-Sp-TEV-CS-IL-37 b ...

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Abstract

The invention discloses a method for high-efficiency expression of all subtype mature proteins of IL-37 by utilizing tobaccos. The method comprises the following steps of: inserting coding sequences of TEV protease and enzyme cutting sites thereof between coding sequences of signal peptide and mature IL-37 proteins to form an IL-37-TEV fusion gene; according to the preference of tobacco codons, under the condition of guaranteeing no change of the amino acid sequence, optimizing the sequence of the fusion gene; connecting 5'-end upstream of the constructed IL-37-TEV fusion gene with a common promoter including other regulating areas, and connecting the 3'-end downstream with a transcription terminator; integrating a constructed recombinant expression vector onto cell chromosomes of the tobacco to culture a transgenic tobacco plant with high-efficiency stable expression of IL-37 and injecting the constructed recombinant expression vector to the tobacco leaves to instantaneously express target proteins. The method disclosed by the invention has the advantages that by construction of the plant expression vector, the mature proteins of IL-37 with high activity are expressed in the plants, and the obtained product has important application value for treatment of numerous inflammatory diseases and early detection and diagnosis of the inflammatory diseases.

Description

technical field [0001] The invention relates to the fields of genetic engineering and protein expression, in particular to a method for efficiently expressing mature proteins of IL-37 subtypes by using tobacco. Background technique [0002] Human interleukin-37 (IL-37) is a newly added member of the IL-1 family of cytokines, discovered in 2000. In contrast to other members of the IL-1 family, IL-37 is an anti-inflammatory cytokine with broad anti-inflammatory effects, playing both a role in limiting innate inflammation and suppressing acquired immune responses. plays a key role. In vitro experiments have found that inhibiting the normal expression of IL-37 in human cells will increase the expression and release of inflammatory cytokines induced by IL-1, Toll-link receptor (TRL) and uric acid crystals, including TNF-α, IL-1α, IL-1ß, IL-6 and G-CSF. Human IL-37 is active in mice, although no human equivalent IL-37 gene has been identified in mice so far. The constructed IL...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/62A01H5/00
CPCC12N15/8251C07K14/54C07K2319/02C07K2319/50C12N15/625
Inventor 马生武安托尼·迈克尔·耶尼卡邓绍平杨洪吉魏亮
Owner 马生武
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