Novel use and preparation method of human beta-defensin 3

A technology for defensins and new uses, which is applied in the field of new uses and preparations of active small molecular proteins, and can solve the problems of difficulty in ensuring the biological activity of polypeptides, high cost, and low yield.

Inactive Publication Date: 2018-11-20
GUIZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing synthetic method of human β-defensin 3 has the problems of high cost, low yield and difficulty in ensuring the biological activity of the polypeptide

Method used

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  • Novel use and preparation method of human beta-defensin 3
  • Novel use and preparation method of human beta-defensin 3
  • Novel use and preparation method of human beta-defensin 3

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0027] Experimental example 1: Construction experiment of expressing engineered bacteria Rosseta-gami(2)-pET32a(+) / HBD3

[0028] 1. Construction of pET32a(+) / HBD3 recombinant plasmid

[0029] (1) Extraction of plasmid pET32a(+): Culture Escherichia coli JM109 (containing pET32a(+) plasmid) by shaking with LB medium, collect bacterial cells, and operate with a plasmid extraction kit to obtain pET32a(+) plasmid (pET32a (+) The schematic diagram of the plasmid structure is shown in figure 1 ).

[0030] (2) Restriction endonuclease reaction: The synthesized HBD3 mature peptide gene and pET32a(+) plasmid were digested with BamHI and XholⅠ under appropriate conditions, and the reaction products were gel-recovered after agarose electrophoresis to obtain enzyme digestion product.

[0031] (3) Ligation reaction: The above-mentioned digested products were recovered and purified by gel, and then ligation reaction was carried out. Under the action of T4 DNA ligase reaction, the molecu...

experiment example 2

[0040] Experimental example 2: Induced expression, identification and optimization of induction conditions of HBD3 fusion protein

[0041] 1. Induced expression and identification of HBD3 fusion protein

[0042] (1) Induced expression of HBD3 fusion protein: the glycerol-preserved expression strains Rossetta-gami(2)-pET32a(+) / HBD3 and Rossetta-gami(2)-pET32a(+) were inoculated in 5 mL LB / AMP liquid medium respectively, Shake culture at 37°C overnight, take 50 μL of bacterial solution and re-inoculate 5 mL of LB / AMP liquid medium at an inoculum size of 1:100, shake at 37°C and 200 rpm for 4 hours, until the bacteria reach the logarithmic growth phase (A600≈0.6) , as a positive control, no IPTG was added, and the rest were added with IPTG to a final concentration of 0.5mM, then continued to culture at 37°C for 4h, centrifuged to remove the supernatant, washed the bacterial cells twice with 0.01mol / LPBS buffer 1mL, and then added 0.5mL of PBS Resuspended cells.

[0043] (2) SDS...

experiment example 3

[0055] Experimental example 3: Purification of recombinant HBD3 polypeptide

[0056] 1. Affinity chromatography purification of HBD3 fusion protein

[0057] (1) Inoculate the glycerin-preserved expression strain Rossetta-gami(2)-pET32a(+) / HBD3 in 5 mL LB / AMP liquid medium, culture with shaking at 37°C overnight, take 50 μL of bacterial liquid and re-inoculate with an inoculum volume of 1:100 In 1L LB / AMP liquid medium, shake culture at 37°C and 200rpm for 4h, until the bacteria reach the logarithmic growth phase (A600≈0.6), add IPTG to a final concentration of 0.5mM, then continue to culture at 34°C for 6h, centrifuge to remove After the supernatant, wash the bacterial cells twice with 1 mL of 0.01 mol / LPBS buffer, add the bacterial cells to the ratio of 10 mL of culture to 1 mL of Binding Buffer, and resuspend the bacterial cells.

[0058] (2) Put the supernatant on ice and sonicate (probe No. 6, 100 power, sonicate for 3 s, interval 3 s, act for 30 min), centrifuge at 4°C a...

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Abstract

The invention discloses a novel use and a preparation method of human beta-defensin 3, and provides application of the human beta-defensin 3 in preparation of medicines for treating fungal, especiallyfilamentous fungal infectious diseases. The invention provides a preparation method of a recombinant human beta-defensin 3 polypeptide, wherein the preparation method comprises the following steps: inserting an HBD3 mature peptide gene into a pET32a(+) plasmid; transferring the correctly constructed recombinant plasmid into Escherichia coli to obtain expression engineering bacteria; inducing andculturing the expression engineering bacteria to express an HBD3 fusion protein; and finally obtaining the recombinant human beta-defensin 3 polypeptide by nickel affinity chromatography, thrombin digestion, purification and concentration. The invention proposes the novel use of the human beta-defensin 3. The preparation method provided by the invention has characteristics of low production cost,high yield and easy assurance of biological activity of the polypeptide.

Description

technical field [0001] The present invention relates to a new application of active small molecule protein and its preparation method, especially the new application of human β-defensin 3 and its preparation method. Background technique [0002] In recent years, with the widespread application of broad-spectrum antibiotics, hormones, and immunosuppressants, as well as the spread of AIDS, the promotion and application of organ transplantation, and interventional techniques, the incidence of fungal infections has shown an upward trend. In 2014, the fungal infection rate was 20. Several times in the 1990s, Candida and Aspergillus are currently the most common fungal infection pathogens, and the fatality rate of systemic infections caused by some fungi is as high as 90%. For AIDS patients (AIDS), fungal infection is the most common opportunistic infection, and its morbidity and mortality are high. Although antifungal drugs with different mechanisms of action have played an impo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61P31/10C12N15/70C07K14/47
CPCA61K38/1729A61P31/10C07K14/4723C12N15/70
Inventor 江滟沈祥春杨红宇易旭罗红兰露莎
Owner GUIZHOU MEDICAL UNIV
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