Human leukocyte protease inhibitory factor as well as recombinant preparation and application thereof

A protease inhibition and white blood cell technology, applied in the field of biomedicine, can solve the problems of lack of effective drugs, etc., and achieve the effect of inhibiting the weight gain of adipose tissue, inhibiting lipid accumulation, and inhibiting hepatic steatosis

Active Publication Date: 2020-04-07
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There are currently no effective drugs for the treatment of various metabolic diseases such as obesity, type 2 diabetes and non-alcoholic fatty liver disease

Method used

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  • Human leukocyte protease inhibitory factor as well as recombinant preparation and application thereof
  • Human leukocyte protease inhibitory factor as well as recombinant preparation and application thereof
  • Human leukocyte protease inhibitory factor as well as recombinant preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] A method for constructing, expressing and purifying recombinant human leukocyte protease inhibitor WAP, SWAP1, and SWAP2 proteins, the method comprising the following steps:

[0051] (1) Construction of recombinant plasmids pET-WAP, pET-SWAP1, pET-SWAP2 A1) According to the sequence published on the biological information website, design and synthesize primers to construct pet-WAP plasmid: pET-6*his-enterokinase site -WAP, the primer sequences are respectively SEQ ID NO:4 and SEQ ID NO:5; pET-SWAP1 plasmid: pet-6*his-enterokinase site-SWAP1, the primer sequences are respectively SEQ ID NO:6 and SEQ ID NO :7; pET-SWAP2 plasmid: pet-6*his-enterokinase site-SWAP2, the primer sequences are respectively SEQ ID NO:8 and SEQ ID NO:9; the role of the enterokinase site is in the process of protein refolding Promote the correct folding of proteins and increase the renaturation yield of proteins;

[0052] A2) Extract the DNA of human lung tissue and use it as a template, and use ...

Embodiment 2

[0068] WAP protein, SWAP1 protein and SWAP2 protein increased hWAP content in serum of mice.

[0069] The C57BL / 6 mice used in the examples of the present invention were purchased from the Experimental Animal Center of Hubei Province. Feeding mice with a high-fat diet is a classic mouse model that mimics type 2 diabetes. Before WAP injection, 4-week-old C57BL / 6 mice were fed a high-fat diet for 14 weeks. The mice in the HFD group were randomly divided into 4 groups: the HFD group was injected with normal saline (Saline group), the HFD group was injected with WAP full-length protein (WAP group); the HFD group was injected with WAP short peptide 1 (SWAP1 group); the HFD group was injected with WAP short peptide 2 (SWAP2 group), the injected WAP protein, SWAP1 protein and SWAP2 protein sequences are as follows figure 2 shown. Formal injection of WAP and its two short peptides, subcutaneous injection, each injection at a dose of 0.2 μg / g body weight, once a day, for a total of...

Embodiment 3

[0072] WAP protein, SWAP1 protein and SWAP2 protein reduced the body weight of mice, but did not affect the food intake of mice.

[0073] After 5 days of injection of WAP protein, SWAP1 protein and SWAP2 protein, statistical calculation was performed on the body weight change (△BW) of the mice compared with that before injection: compared with the HFD+Saline group, the WAP protein, SWAP1 protein and SWAP2 protein group The body weight of the mice decreased significantly ( Figure 6); Simultaneously, the food intake (Food intake) of each group of mice was counted, and there was no significant difference between the food intake of each group of mice ( Figure 7 ), indicating that the short-term injection of WAP protein, SWAP1 protein and SWAP2 protein can significantly reduce the body weight of C57BL / 6 mice fed with high-fat diet. At the same time, the injection of WAP protein, SWAP1 protein and SWAP2 protein does not affect the body weight of mice.

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Abstract

The invention relates to a human leukocyte protease inhibitory factor as well as recombinant preparation and an application thereof, which belong to the field of biological medicines. The amino acid sequence contained in the human leukocyte protease inhibitory inhibitor is as follows: (1) an amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; (2) an amino acid sequence obtained by connecting a label to the N end or/and C end of the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; and (3) an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 3 and has the activity of the human leukocyte protease inhibitory factor. The preparation method comprises the following steps: constructing plasmids, carrying out inducible expression to obtain fusion protein, purifying, and carrying out renaturation concentration. The protein provided by the invention has important application in preparation of drugs for treating metabolic diseases, and can reduce weight, inhibit increase of adipose tissue weight and inhibit liver fatty degeneration.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to human leukocyte protease inhibitory factor and its recombinant preparation and application, in particular to the application of recombinant human WAP protein, SWAP1 protein and SWAP2 protein for preparing medicines for preventing and treating obesity and metabolic diseases. Background technique [0002] Metabolic diseases, represented by obesity and diabetes, are major health problems facing our country and even the world, bringing serious social impact and economic burden to human beings. The number of diabetic and obese patients in China ranks first in the world, with approximately 114 million diabetics and 89 million obese patients. At the same time, according to conservative estimates, there are about 140 million people in China who are pre-diabetic, that is, people with abnormal glucose tolerance, and these people will gradually develop into diabetic patients. Therefore, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/70A61K38/57A61P1/16A61P3/00A61P3/04A61P3/06A61P3/10A61P9/10
CPCA61K38/00A61P1/16A61P3/00A61P3/04A61P3/06A61P3/10A61P9/10C07K14/47C12N15/70
Inventor 黄昆郑凌陈红刘成宇
Owner HUAZHONG UNIV OF SCI & TECH
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