Recombinant human interleukin 15 long peptide fragment and production method thereof

Abstract

The invention discloses a recombinant human interleukin (IL) 15 long peptide fragment and a production method thereof. The production method comprises the following steps: obtaining a human IL-15 long peptide fragment gene; establishing an eukaryotic expression vector and transforming the vector into Pichia pastoris X-33; performing resistance screening on high-level secretory-expressed yeast transformants; performing induced expression, purifying the supernate through a nickel column and an ion exchange column to obtain rhIL-15L. The production method is characterized in that the amino acid residue Ala of the site Kex2P1' of the vector pPICZAlphaA is mutated into Pro and the Pichia pastoris X-33 is taken as host bacteria, and therefore, mass high-efficiency stable expression of the rhIL-15L is realized; the recombinant protein rhIL-15L produced by the method is marked with HIS and C-MYC tags and prone to protein purification and detection; besides, the recombinant protein rhIL-15L is a glycoprotein which is modified through glycosylation to a certain extent, and therefore, the recombinant protein rhIL-15L has excellent bioactivity and is capable of effectively maintaining the proliferation and the bioactivity of human NK (Natural killer) cells in vitro and of a mouse in vivo.

Description

technical field [0001] The invention relates to the technical field of using recombinant DNA technology to produce genetically engineered protein drugs, in particular to a high-efficiency recombinant human interleukin-15 long peptide segment (rhIL-15L) and a production method thereof. Background technique [0002] Interleukin 15 (also known as IL-15), as a T cell growth factor was first discovered in 1994. Similar to interleukin 2, interleukin 4, interleukin 7, interleukin 9, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor, it belongs to the 4a helical cytokine family. Interleukin-15 is constitutively expressed in various types of cells including monocytes, macrophages, dendritic cells, fibroblasts, and nerve cells. The human IL-15 gene consists of 9 exons and 8 introns. Exons 5 to 8 encode mature interleukin 15 with a length of 162 amino acids. The receptor of interleukin 15 is composed of a, β, and γc The heterotrimer of , its ...

AI Technical Summary

Problems solved by technology

The expression product of Escherichia coli usually exists in the form of inclusion bodies, and a series of complex operations such as denaturation and renaturation are required to obtain the active form of IL-15. This method greatly limits the yield of active protein; in addition, prokaryotic host expression especially It is expressed in Escherichia coli, because of the toxicity caused by the presence of LPS, it is often necessary to analyze and determine the toxicity of the expressed purified product; finally, to obtain a high-purity protein often requires multi-step purification operations, during which the protein The yield decreases, and the biological activity will also decrease

Although insect cell expression system and mammalian cell expression system can also express human recombinant interleukin-15, the production cost is high and the yield is very low

Therefore, the price of rhIL-15 currently on the market is unusually expensive

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