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Unlabeled human galectin 10 crystal and its preparation method and application

A galectin, label-free technology, applied in the field of label-free human galectin 10 crystals and their preparation, to achieve the effect of improving safety

Active Publication Date: 2021-10-22
NORTHEAST NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] So far, there is no report on overexpressing untagged human galectin-10 and purifying the protein using E. coli

Method used

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  • Unlabeled human galectin 10 crystal and its preparation method and application
  • Unlabeled human galectin 10 crystal and its preparation method and application
  • Unlabeled human galectin 10 crystal and its preparation method and application

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preparation example Construction

[0044] Such as figure 1 As shown, the preparation method of the unlabeled human galectin-10 crystal of the present invention specifically includes the following steps:

[0045] Step 1. Insert the gene of human galectin 10 or human galectin 10 mutant A28V into the plasmid pET22b, the restriction endonuclease sites are NdeI and BamHI, the cDNA of human galectin 10 The stop codon TAA was added at the end, and the obtained plasmid was named pET22b_Galectin-10.

[0046] The gene of human galectin 10 is derived from human (Homo sapiens) or derived from gene synthesis.

[0047] The gene of human galectin 10 mutant A28V is derived from human (Homo sapiens) or derived from gene synthesis.

[0048] The gene of human galectin 10 is cDNA or a synthetic gene.

[0049] The gene of human galectin 10 mutant A28V is cDNA or synthetic gene.

[0050] Step 2, transforming the plasmid pET22b_Galectin-10 into Escherichia coli BL21(DE3).

[0051] Step 3. Pick a number of E. coli colonies and pl...

Embodiment 1

[0074] Example 1 Preparation of unlabeled human galectin-10 crystals

[0075] (1) Insert the cDNA of human galectin 10 into the plasmid pET22b, the restriction endonuclease sites are NdeI and BamHI, and the end of the cDNA of human galectin 10 is added with a stop codon TAA, the obtained The plasmid was named pET22b_Galectin-10.

[0076] (2) Transform the plasmid pET22b_Galectin-10 into Escherichia coli BL21(DE3).

[0077] (3) Pick a number of Escherichia coli colonies and place them in 1L2×YT or LB medium, and place them in a shaker for cultivation, the temperature in the shaker is 37°C, and the rotation speed is 185rpm; 600 To 0.2-4, add 5mM IPTG to induce protein expression, and the induction time is 12h; the next day, the bacteria are collected by centrifugation at 5000g.

[0078] (4) Add 50ml of bacterial lysate PBS (8gNaCl, 0.2g KCl, 3.63g NaCl, pH 7.2 2 HPO 4 12H 2 O, 0.24g KH 2 PO 4 , pH 6.8-7.6), sonicated for 16 minutes.

[0079] (5) Centrifuge at 6000g to re...

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Abstract

The unlabeled human galectin 10 crystal and its preparation method and application relate to the field of protein crystal preparation. First obtain the gene of human galectin 10 or human galectin 10 mutant A28V, construct an overexpression plasmid, induce the expression of human galectin 10 in E. coli, and purify human galactose by protein crystallization and ion exchange column Lectin 10, human galectin 10 was obtained and finally crystallized. The crystal has the characteristics of high purity and no label, naturally exists in the human body, and has certain safety for the human body. Therefore, the human galectin-10 crystal of the present invention can be used as a new type of vaccine adjuvant. The crystals can be stored in deionized water, distilled water, 1‑30% (v / v) ethanol, PBS, 1‑20% (v / v) DMSO, 1‑10% (m / v) glucose or 0.1‑5% (m / v) in sodium chloride.

Description

technical field [0001] The invention relates to the technical field of protein crystal preparation, in particular to an unlabeled human galectin-10 crystal and its preparation method and application. Background technique [0002] Human galectin-10 crystals are a type 2 vaccine adjuvant that elicits a strong immune response. In the human body, human galectin-10 can automatically form crystals. Its other Chinese name is Charcot Leyden Crystal Protein (CLC), and the uniport number is Q05315. [0003] Human galectin-10 is a protein containing 142 amino acids, and its mRNA is abundantly transcribed in bone marrow, which suggests that human galectin-10 can participate in regulating the function of immune cells. Human galectin-10 is also overexpressed in eosinophils and can be present in basophils and macrophages. Intracellularly, human galectin-10 can form crystals. [0004] At present, there are three main sources of galectin-10 used in reported studies: 1. Purified from human...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/70C07K14/47C07K1/34A61K39/39
CPCA61K39/39A61K2039/55516C07K14/4726C12N15/70
Inventor 苏纪勇姚圆
Owner NORTHEAST NORMAL UNIVERSITY
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