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CN-120365442-B - Method for expressing human tumor necrosis factor-alpha by alfalfa

CN120365442BCN 120365442 BCN120365442 BCN 120365442BCN-120365442-B

Abstract

The invention provides a method for recombining transmembrane human tumor necrosis factor-alpha expressed by alfalfa, belonging to the technical field of biology. The invention constructs a plant binary expression vector containing a transmembrane human tumor necrosis factor-alpha gene, and further utilizes agrobacterium containing the plant binary expression vector to transform a receptor alfalfa, thereby preparing the recombinant transmembrane human tumor necrosis factor. The invention uses alfalfa as recombinant human tumor necrosis factor chassis plant, provides an excellent cell system for recombinant transmembrane human tumor necrosis factor-alpha expression, and provides a new solution for the market demand of recombinant transmembrane trimer tumor necrosis factor-alpha.

Inventors

  • LI JIKAI
  • Tan Zirui
  • ZHANG JUNMING
  • NIE MINGXUAN

Assignees

  • 河北绶肽生物科技有限公司

Dates

Publication Date
20260508
Application Date
20250523

Claims (7)

  1. 1. The preparation method of the recombinant human tumor necrosis factor-alpha is characterized in that after a receptor plant alfalfa is transformed by utilizing a gene containing the human tumor necrosis factor-alpha, the obtained genetic transformation positive plant is cultured to prepare the human tumor necrosis factor-alpha, wherein the human tumor necrosis factor-alpha exists in a trimer form; the amino acid sequence of the human tumor necrosis factor-alpha is shown as SEQ ID No. 1, and a PR1b signal peptide coding sequence is added at the N end and a KDEL coding sequence is integrated at the C end; The amino acid sequence of the PR1b signal peptide is shown as SEQ ID NO. 2; the nucleotide sequence of the optimized human tumor necrosis factor-alpha gene is shown as SEQ ID NO. 3.
  2. 2. The method of claim 1, wherein the amino acid sequence of human tumor necrosis factor- α is further fused to a tag sequence.
  3. 3. The method of claim 2, wherein the fused amino acid sequence is shown in SEQ ID NO. 4.
  4. 4. A method according to any one of claims 1 to 3, further comprising: identifying the insertion of human tumor necrosis factor-alpha gene in the genetically transformed positive plant; And/or protein identification of recombinant human tumor necrosis factor-alpha in genetically transformed positive plants.
  5. 5. The method of claim 4, wherein the recipient plant is transformed by Agrobacterium-mediated transformation with a plant binary expression vector comprising the human tumor necrosis factor-alpha gene.
  6. 6. The method of claim 5, wherein the plant binary expression vector comprising the human tumor necrosis factor-alpha gene is a pCambia1300-35S vector.
  7. 7. A method according to any one of claims 1 to 3, further comprising the step of preparing said human tumor necrosis factor- α from the isolated cultured product.

Description

Method for expressing human tumor necrosis factor-alpha by alfalfa Technical Field The invention belongs to the field of biotechnology, and relates to a method for expressing recombinant transmembrane human tumor necrosis factor-alpha by alfalfa. Background Tumor necrosis factor alpha (TNF- α) is a pleiotropic immune cytokine which is produced mainly by cells of the innate immune system, such as macrophages and NK cells, and cells of the adaptive immune system, in particular activated T cells. Its main function is to regulate immune and inflammatory reactions, and has the effect of killing or inhibiting tumor cells. In infection, tissue injury or immune response, the tumor necrosis factor content increases, triggering a downstream series of immune response responses by binding to its receptor. Tumor necrosis factor alpha exists in vivo in two forms, one is a transmembrane precursor protein form (membrane-bound precursor protein, tmTNF-alpha) with a molecular weight of 26Kda and the other is a soluble form (sTNF-alpha) after cleavage, with a molecular weight of 18Kda. Both forms of TNF- α can form homotrimers to bind to its receptor (TNFR 1 or TNFR 2) to activate signaling pathways such as inflammation, apoptosis and cell proliferation. The recombinant human tumor necrosis factor-alpha sold in the market at present is mostly produced by the expression of a prokaryotic system, but the prokaryotic system expresses the human tumor necrosis factor-alpha and has the following disadvantages that (1) the prokaryotic system does not carry out post-translational modification on the human tumor necrosis factor-alpha protein produced by the expression of the prokaryotic system due to the lack of a precise protein post-translational modification system, so that the human tumor necrosis factor-alpha protein is incomplete in function and is in a monomer form, (2) the endotoxin is released by escherichia coli in the process of purifying the human tumor necrosis factor-alpha by cell rupture, the existence of the endotoxin not only influences the quality of the product and brings safety risks, but also the cost of the product can be greatly increased by removing the endotoxin. Meanwhile, most of recombinant human tumor necrosis factor-alpha (TNF-alpha) on the market is a soluble form after cleavage, but the current molecular biology proves that a transmembrane precursor protein form of TNF-alpha (hereinafter abbreviated as tmTNF-alpha) plays an important role in signal paths such as immunoregulation and the like. Plants have been studied for nearly thirty years as expression and production systems for pharmaceutical proteins. Compared with single-cell microorganisms, the multicellular plant system is rich in an endomembrane system and various organelles, glandular hairs on the surface of the plant are important sites for synthesizing and storing metabolites, the complex space-time characteristic provides the most suitable environment for synthesizing different types of enzymes and metabolites, is favorable for maintaining the activity and yield of protein, and the complexity of the multicellular plant system also provides an excellent model system for carrying out synthesis biological research. Plants are rich in a large number of metabolites and can directly provide precursors for synthesizing plant active molecules. Therefore, the synthesis of important humanized proteins and naturally active small molecules by plants has become a necessary path for technological development. Alfalfa is a perennial leguminous forage grass which is the earliest in cultivation, the largest in planting area and the largest in planting country in the world, and is also the forage grass mainly cultivated in China, and the alfalfa is characterized by high yield, good quality and strong adaptability, is the cultivated forage grass with the highest feeding value and economic significance, and enjoys the reputation of 'forage grass king'. Compared with other plants, the alfalfa has the advantages that (1) the yield of the alfalfa is high, the yield per mu can reach 10 tons and is greatly higher than the yield of the chassis of common plants such as tobacco, rice and the like, and (2) the alfalfa is long in utilization period and can be recovered to be alive quickly after mowing, and the alfalfa can grow for many years after one-time sowing, so that the labor capacity is greatly reduced. Currently, plant expression systems have been considered as a promising alternative to animal cell and microorganism culture for large-scale production of recombinant proteins. The production method of recombinant proteins using plants as a chassis can be seen, for example, in chinese patents CN 107827975 and CN 106554971, U.S. patent No. 5750871 and No. 5565347, and the like. However, no report is reported on the expression of recombinant human tumor necrosis factor by using plants. Disclosure of Invention In view of the above problems, the present invention provid