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Label-free human galectin 13 as well as preparation method and application thereof

A galectin, label-free technology, applied in the field of label-free human galectin 13 and its preparation, to achieve the effect of lowering blood pressure

Pending Publication Date: 2021-02-09
NORTHEAST NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Up to now, there is no report on the overexpression of untagged human galectin-13 and the purification of the protein in Escherichia coli

Method used

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  • Label-free human galectin 13 as well as preparation method and application thereof
  • Label-free human galectin 13 as well as preparation method and application thereof

Examples

Experimental program
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preparation example Construction

[0040] The preparation method of the unlabeled human galectin-13 of the present invention specifically comprises the following steps:

[0041]Step 1. Insert the human galectin-13 gene into the plasmid pET15b, the restriction endonuclease sites are NcoI and XhoI or BamHI, and the end of the human galectin-13 cDNA is added with a stop codon TAA, The obtained plasmid was named pET15b_Galectin-13;

[0042] Among them, the gene of human galectin 13 is derived from human (Homo sapiens) or derived from gene synthesis.

[0043] Among them, the gene of human galectin 13 is cDNA or a synthetic gene.

[0044] Step 2, transforming the plasmid pET15b_Galectin-13 into Escherichia coli BL21, Escherichia coli BL21(DE3) or Escherichia coli BL21(DE3)pLysS;

[0045] Step 3: Pick a number of Escherichia coli colonies and place them in LB medium, and culture them in a shaker, the temperature in the shaker is 37°C, and the rotation speed is 150-220rpm; wait for OD 600 To 0.2-4, add 0.1-10mM IPTG...

Embodiment 1

[0073] Example 1 Preparation of unlabeled human galectin 13

[0074] (1) Insert the cDNA of human galectin 13 into the plasmid pET15b, the restriction endonuclease sites are NcoI and XhoI or BamHI, and the end of the cDNA of human galectin 13 is added with a stop codon TAA, The obtained plasmid was named pET15b_Galectin-13;

[0075] (2) Transform the plasmid pET15b_Galectin-13 into Escherichia coli BL21;

[0076] (3) Pick a number of Escherichia coli colonies and place them in LB medium, and place them in a shaker for cultivation. The temperature in the shaker is 37° C., and the rotation speed is 150 rpm; 600 To 0.2-4, add 10mM IPTG to induce protein expression, and the induction time is 24h; the bacteria are collected by centrifugation at 6000g;

[0077] (4) Add bacterial lysate (10mM Hepes, pH7.0) and ultrasonically lyse for 30min;

[0078] (5) Centrifuge at 12000g to remove the precipitate, collect the supernatant, which contains unlabeled human galectin-13, and the oper...

Embodiment 2

[0090] Example 2 Preparation of unlabeled human galectin 13

[0091] (1) Insert the cDNA of human galectin 13 into the plasmid pET15b, the restriction endonuclease sites are NcoI and XhoI or BamHI, and the end of the cDNA of human galectin 13 is added with a stop codon TAA, The obtained plasmid was named pET15b_Galectin-13;

[0092] (2) Transform the plasmid pET15b_Galectin-13 into Escherichia coli BL21 (DE3);

[0093] (3) Pick a number of Escherichia coli colonies and place them in LB medium, place them in a shaker for cultivation, the temperature in the shaker is 37°C, and the rotation speed is 220rpm; 600 To 0.2-4, add 0.1mM IPTG to induce protein expression, the induction time is 2h; 5000g centrifuge to collect bacteria;

[0094] (4) Add bacterial lysate (100mM Hepes, pH7.0) and ultrasonically lyse for 2min;

[0095] (5) Centrifuge at 8000g to remove the precipitate, collect the supernatant, which contains unlabeled human galectin-13, and the operating temperature is 4°...

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Abstract

The invention discloses label-free human galectin 13 as well as a preparation method and application thereof, and relates to the field of protein preparation. The method comprises the following stepsof firstly, obtaining a gene of the human galectin 13, constructing an overexpression plasmid, inducing the human galectin 13 to be expressed in escherichia coli, and subjecting protein to ammonium sulfate precipitation method purification, ion exchange column purification and gel filtration and purification, so as to obtain the label-free human galectin 13. The human galectin 13 prepared by the method is high in purity and free of labels, naturally exists in a human body, and has certain safety to the human body. In addition, animal experiments prove that the prepared label-free human galectin 13 has the function of reducing blood pressure of female rats and female rhesus monkeys, so that the label-free human galectin 13 can be used as an effective component for treating pregnancy hypertension, preeclampsia and HELLP syndromes.

Description

technical field [0001] The invention relates to the technical field of protein preparation, in particular to an unlabeled human galectin-13, its preparation method and application. Background technique [0002] Human galectin 13 (uniport number Q9UHV8) is mainly expressed in and secreted from placental syncytiotrophoblast cells. Upregulation of human galectin-13 expression in early pregnancy is essential for normal pregnancy. If the expression of human galectin-13 is inhibited, or the gene of human galectin-13 is mutated, preeclampsia or HELLP syndrome will be caused. [0003] Currently, there are two main sources of galectin-13 used in reported studies: 1. Purified from human placenta tissue; 2. Purified from Escherichia coli (with a tag or with a few extra amino acids left over). The first method is not economical enough and has the problem of high cost. The second method is relatively simple, saves material and financial resources, and is low in cost. However, the rep...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K1/36C07K1/34C07K1/30C07K1/18C07K1/16C12N15/12C12N15/70A61K38/17A61P9/12A61P15/00
CPCC07K14/4726C12N15/70A61P9/12A61P15/00A61K38/00
Inventor 苏纪勇姚圆
Owner NORTHEAST NORMAL UNIVERSITY
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