Islet-targeted protein and eukaryotic expression method thereof

An expression method and protein technology, which are applied in the field of pancreatic islet-targeted protein and eukaryotic expression of pancreatic islet-targeted protein, can solve the problem of preparing soluble IGRP206-214MHC complex, prokaryotic expression system without glycosylation function, and unable to truly simulate protein features, etc.

Inactive Publication Date: 2017-10-27
李凯
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, the prokaryotic expression system cannot truly simulate the characteristics of the protein because it does not have the function of glycosylation
Currently, there is no method for preparing soluble IGRP206-214MHC complexes using eukaryotic expression systems

Method used

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  • Islet-targeted protein and eukaryotic expression method thereof
  • Islet-targeted protein and eukaryotic expression method thereof
  • Islet-targeted protein and eukaryotic expression method thereof

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Embodiment Construction

[0029] Preferred embodiments of the present invention will be described in more detail below. Wherein, various reagents used can be obtained commercially, and the specific operation method is carried out with reference to the operation manual attached to the commercially available reagents.

[0030] The term abbreviations, English full names and Chinese full names involved in the present invention are shown in Table 1.

[0031] Table 1

[0032]

[0033] 1. IGRP 206-214 - Construction of dtSCT eukaryotic expression system and protein preparation

[0034] amplified IGRP 206-214 The full-length coding gene, adding a biotinylated site and 6×his tag at the end, and adding a restriction site at the beginning and end, inserted into the eukaryotic expression plasmid, and introduced into Pasteur yeast to construct a eukaryotic expression system for the protein, And use methanol culture to induce the expression of the target protein.

[0035] Use Oligo 7 to design the upstream a...

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Abstract

The invention provides an islet-targeted protein and a eukaryotic expression method thereof. The method comprises the following steps of (1) constructing plasmids containing a protein coding gene IGRP206-214-dtSCT; (2) recombining the plasmids into a chromosome of saccharomyces pastorianus; and (3) obtaining a target protein through methanol fermentation induction. A soluble IGRP206-214MHC compound is prepared through an eukaryotic expression system. Compared with the previously prepared protein for prokaryotic expression, the islet-targeted protein has galactosylated modification, and is closer to an in-vivo protein crystal structure, and the recognition rate of specific CTL on the protein and the bonding stability of TCR and an MHC molecule are improved, the endocytosis amount of viruses coupled to the protein is increased, the efficacy of the recombinant protein is improved and the recombinant protein is more stable in an external environment after galactosylated modification, and the storage period is prolonged.

Description

technical field [0001] The invention belongs to the field of biological protein preparation, and more specifically relates to a method for eukaryotic expression of a pancreatic islet-targeted protein and the pancreatic islet-targeted protein obtained by the method. Background technique [0002] NOD (no-obese diabetes) mice are animal models for studying type 1 diabetes. At the age of 13 weeks, IGRP206-214 antigenic peptides appear on the surface of pancreatic β cells, which are presented to cytotoxic T cells (CTL) through MHC packaging. After recognition, it kills pancreatic beta cells by secreting granzyme, perforin, etc., resulting in decreased insulin secretion and increased blood sugar. The research group has previously prepared the islet-targeting protein soluble IGRP through the prokaryotic expression system 206-214 -dtSCT complex, prepared into a tetramer, coupled with toxin-saporin, recognized by specific CTL, endocytized and necrotic after endocytosis, so as to del...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/81
CPCC12N9/16C12N15/815C12Y301/03009
Inventor 王宇航李凯
Owner 李凯
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