Killer cell antibiosis polypeptide activated by lymphokine and its application

An antibacterial polypeptide and α-helix technology, applied in the field of genetic engineering, can solve the problem that the characteristics of small molecule polypeptides are not clearly defined and can not be applied.

Inactive Publication Date: 2005-03-30
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The inventors of the present invention found in the research work before carrying out the research work of the present invention: there are multiple small molecular polypeptides with strong antibacterial activity in the cytoplasmic granules of human LAK cells, that is, the killer cells activated by human lymphokines, but due to lack of Separating and purifying them, so the characteristics and biological effects of these small molecule peptides have not been clarified, let alone their application

Method used

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  • Killer cell antibiosis polypeptide activated by lymphokine and its application
  • Killer cell antibiosis polypeptide activated by lymphokine and its application
  • Killer cell antibiosis polypeptide activated by lymphokine and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, the isolation culture of human LAK cell

[0034] Human peripheral blood mononuclear lymphocytes were isolated according to the common experimental methods of immunology. Dilute the white blood cell suspension with an equal volume of PBS, slowly add it to the lymphocyte separation medium along the wall of the centrifuge tube, and centrifuge at 2000r / min for 30min with a horizontal centrifuge. After centrifugation, milky white turbidity can be seen at the interface of the upper and middle layers layer of mononuclear cells. Aspirate the mononuclear cells in the interface layer with a capillary tube, add 5 times the volume of PBS to wash the cells by centrifugation, and repeat twice. Finally, the cell pellet was filled with complete RPMI1640 medium (containing r-IL 2100U / ml, PHA100μg / ml) was diluted to a concentration of 2×10 6 / ml for culture, the medium was changed once after three days, and the cells were collected by centrifugation after seven days. If...

Embodiment 2

[0035] Embodiment 2, the preparation of human LAK cell acid-soluble extract

[0036] Add an appropriate amount of 5% acetic acid to the cell pellet and homogenize it electrically in an ice bath. The homogenate is centrifuged at 13000r / min×6min at 4°C to collect the supernatant. The pellet was homogenized repeatedly until no complete cells could be seen under the microscope. The collected supernatant was transferred into a dialysis bag with a cut-off flow rate of 3.5KD, dialyzed at 4°C for 48 hours to remove acetic acid, and freeze-dried to obtain an acid-soluble crude extract of LAK cells, which was stored at -20°C for future use.

Embodiment 3

[0037] Example 3, preliminary screening of antibacterial active ingredients of human LAK cell acid-soluble extract

[0038] 1. Acid-Urea polyacrylamide Gel Electrophoresis (AU-PAGE)

[0039] Prepare the gel according to the Panyim method, the thickness of the gel is 0.75 mm, the gel concentration is 12.5%, and the pre-electrophoresis is performed at 150 V for 1.5 hours to remove TEMED and ammonium persulfate in the gel. Take 2 mg LAK acid crude extract, dissolve in 50 μl 0.01% acetic acid solution, and then add an equal amount of 2× loading buffer to dilute. Prepare 1 mg / ml lysozyme in the loading buffer, add 10 μl lysozyme solution and LAK crude extract solution to the gel comb wells on both sides. 150V constant voltage electrophoresis for 45min, the gel was taken out, one side was stained with Coomassie brilliant blue, and the other side was washed with 10mMPBS for 5min×3 times to remove impurities such as acetic acid and urea, and then used for sterilization experiments. ...

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Abstract

An antibacterial polypeptide HLP-3P21, which is separated from the kytoplasm of the killer cell activated by human lymphokine, is disclosed. Its cDNA sequence and amino acid sequence are measured. It has high activity to pathogenic colibacillus and Pseudomonas aeruginosa.

Description

1. Technical field [0001] The invention belongs to the technical field of genetic engineering, more specifically, the invention relates to the technology of separating and purifying polypeptides with activity against pathogenic microorganisms from the cytoplasmic granules of human LAK cells (lymphokine-activated killer cells) and their gene recombinant microbial expression Preparation technology of preparations. 2. Background technology [0002] The resistance of pathogenic microorganisms to traditional antibiotics is one of the global problems in clinical medicine today, and people have always doubted the efficacy of traditional antibiotics in the treatment of "opportunistic" pathogenic bacteria infections. For this reason, people need to start research and development of new anti-infective drugs and measures. Endogenous antimicrobial peptides have become the focus of scholars' research and development of anti-infective drugs and measures. [0003] Antimicrobial peptides ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/17A61P31/04C07K14/435C07K14/47
CPCY02A50/30
Inventor 王伯瑶吴琦杨华蓉
Owner SICHUAN UNIV
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