Novel melittin variant and application thereof

A kind of melittin, a new type of technology, applied in the new variant of melittin and its application field, can solve problems such as unsatisfactory antibacterial ability

Active Publication Date: 2019-11-26
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the bacteriostatic ability of wild-type melittin to Gram-positive bacteria is not ideal. Considering wound infection, patients are often infected with more than one kind of bacteria. If wild-type melittin can res

Method used

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  • Novel melittin variant and application thereof
  • Novel melittin variant and application thereof
  • Novel melittin variant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 polypeptide design and antibacterial test

[0040] 1. Design of the polypeptide Based on the melittin polypeptide sequence and synthesis, the melittin-R sequence (R-Meli) was designed:

[0041] Melittin sequence: GIGAVLKVLTTGLPALISWIKRKRQQ-NH2

[0042] Melittin-R (R-Meli) sequence: GIGAVLRVLTTGLPALISWIRRRRQQ-NH2

[0043] 2. Antibacterial test

[0044] The minimum inhibitory concentration (MIC) of melittin-R was tested using the broth dilution method.

[0045] 2.1 MIC plate preparation

[0046] The sample solution with a concentration of 256 mg / ml was respectively injected into the first row of wells of a 96-well polystyrene plate, 100 ul per well, and each sample was repeated 3 times. Add 50ul LB liquid medium to each hole in the back row, use a row gun to draw 50ul sample solution from the first row, blow and mix it with the second row, and then draw 50ul and add it to the third plate and mix it by blowing and blowing, and then perform two-fold dilutio...

Embodiment 2

[0054] Embodiment 2 erythrocyte hemolysis experiment

[0055] 1. Experimental reagents:

[0056] TBS buffer: 605mg Tris (final concentration 10mM TRIS), 4.4gNacl (150mM NaCl), 500mlddH 2 O, pH=7.2;

[0057] Red blood cell lysate: 0.2% Triton X-100 diluted with TBS buffer.

[0058] 2. Experimental steps:

[0059] 1) The blood sample was taken from normal human blood, centrifuged at 1000g for 5min, removed the supernatant, added an equal amount of TBS buffer, mixed gently, centrifuged again at 1000g for 5min, and repeated 3-5 times until the supernatant was clear.

[0060] 2) Collect the resulting precipitate, dilute it in TBS buffer at 1:50, and gently mix it by inverting back and forth to obtain a red blood cell suspension.

[0061] 3) Adding samples to the 96-well plate: use the method of sample injection and dilution with a row gun to form a total of 8 concentration gradients of 128, 64, 32, 16, 8, 4, 2, and 1ug / ml in each sample solution of the experimental group, 100ul...

Embodiment 3

[0067] Example 3 Toxicity test of melittin-R to tumor cells

[0068] 1. Experimental reagents:

[0069] MTS cytotoxic staining reagent was purchased from Promega Company. DMEM high-glucose cell culture medium, 10% fetal bovine serum + 90% DMEM, was purchased from the Cell Resource Center of the Chinese Academy of Medical Sciences.

[0070] Tumor cells: SW620 human colon cancer cells.

[0071] 2. Experimental steps:

[0072] 1) Resuscitate SW620 cells and passage them for more than 2 generations. After the cells grow stably, take a plate of well-grown cells to digest and count the plate. Adjust the concentration of the cell suspension according to the statistical value and add it to a 96-well plate. Add cells as a blank group, and the number of cells per well is about 20,000. Treat overnight at 37°C in a 5% carbon dioxide incubator. After 12 hours, the cells complete attachment.

[0073] 2) After the cells adhere to the wall, carefully suck out the old medium in the 96-well...

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Abstract

The invention relates to a novel melittin variant and an application thereof. At current, overuse of novel antibacterials and antibiotics is clinically to be developed, melittin as a good biology antibacterial agent is one of the best substitutes of the antibiotics. The inventor finds novel melittin based on the research. The melittin has good bacteriostasis and sterilizing capacity for Gram-negative bacteria, and through sequence improvement, the variant is obtained. Based on reservation of Gram-negative bacteria resistance of wild type melittin, good Gram-negative bacteria resistance is obtained. The novel melittin and the variant thereof provided by the invention have favorable clinical application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a novel melittin variant and application thereof. Background technique [0002] Staphylococcus aureus, the most common human bacterial pathogen, is a Gram-positive coccus that can cause skin and soft tissue infections, septic arthritis, endocarditis, osteomyelitis, pneumonia, and bacteremia systemic infection such as sepsis and sepsis. Initially, the sensitivity of Staphylococcus aureus to penicillin was more than 90%, but with the extensive use of antibiotics, Penicillin-resistant Staphylococcus aureus also appeared, and it was also found to be resistant to other antibiotics, such as tetracycline, erythromycin, etc. Drug-resistant Staphylococcus aureus. For a long time, treatment of drug-resistant S. aureus infections has not been as effective as expected. In recent years, resistance to methicillin, oxacillin, cephradine and other antibiotics has emerged, as well as d...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12A61K38/17A61P31/04A61P35/00
CPCA61K38/00A61P31/04A61P35/00C07K14/43572Y02A50/30
Inventor 王晨轩邓准于兰兰张文博祖瑞涓郑明睿
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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