Pichia pastoris genetic engineering hybrid antibacterial peptide CC29 and preparing method thereof

A technology of hybrid antimicrobial peptides and Pichia pastoris, which is applied in genetic engineering and biological fields, can solve the problems of large molecular weight and low content of antimicrobial peptides, which are difficult to meet large-scale production, and achieve high antibacterial activity.

Inactive Publication Date: 2015-07-22
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Abstract
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AI Technical Summary

Problems solved by technology

So far, thousands of natural antimicrobial peptides have been discovered, but there are relatively few species with good medical selection index. Therefore, how to obtain antibacterial peptides with good antibacterial effect and high yield has been widely concerned by researchers.
At present, there are three main ways to obtain antimicrobial peptides: (1) directly extracting natural peptides from organisms, but the content is very small, and the extraction process is complicated, the cost is high, and it is difficult to meet the requirements of large-scale production; (2) chemical methods synthesis
This method is only suitable for very short antimicrobial peptides, but most antimicrobial peptides have relatively large molecular weights and are not suitable for chemical synthesis; (3) Most antimicrobial peptides with relatively large molecular weights use genetic engineering methods, which have been thoroughly studied , and is an effective way to produce antimicrobial peptides

Method used

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  • Pichia pastoris genetic engineering hybrid antibacterial peptide CC29 and preparing method thereof
  • Pichia pastoris genetic engineering hybrid antibacterial peptide CC29 and preparing method thereof

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preparation example Construction

[0051] ② Preparation of Competent Cells: Use TakaRa’s Competent Cell Preparation Kit to prepare Escherichia coli DH5α competent cells. The specific method is as follows:

[0052] a Strain purification: use LB plate medium. Pick Escherichia coli DH5α (preserved in glycerol at -80°C) with a sterile inoculation needle, grade and streak on the plate medium, place it upside down in a constant temperature incubator at 37°C, and cultivate overnight, so as to be able to grow a single colony.

[0053] b bacteria culture:

[0054] a Take 20mL of φb×broth and transfer it into a 100mL Erlenmeyer flask, plug it with a cotton plug, sterilize it under high temperature and high pressure, and then cool it to room temperature; pick a single colony on the streaked plate, and inoculate it into the sterilized and cooled medium above; Shaking (120rmp) culture at 37°C;

[0055] b Measure the OD600 value, when the OD600 value reaches 0.35-0.5 (about 5 hours of cultivation), place it on ice to stop ...

Embodiment 1

[0110] Bacteriostatic Activity of Hybrid Antimicrobial Peptides by Zone of Inhibition Method

[0111] The antibacterial activity of the target protein was preliminarily detected by the agar hole diffusion method. The strains used in the experiments included Escherichia coli, Staphylococcus aureus, Salmonella gallinarum, Streptococcus dysgalactiae, Lactobacillus brevis, Lactobacillus delbrueckii and Lactobacillus crispatus. Inoculate the above-mentioned various bacteria on appropriate solid medium respectively, and after a single colony grows, carry out shaking culture in the corresponding liquid medium. Using the plate dilution method, dilute the above-mentioned suspensions of various strains in the logarithmic growth phase to 108 / μL, inoculate them into a suitable solid medium at 1%, mix well and invert the plate. After the plate was condensed, holes were punched with an autoclaved punch (3 mm), and 20 μL of liquid was added dropwise to each hole. After incubating at 37°C f...

Embodiment 2

[0114] Determination of minimum inhibitory concentration (MIC)

[0115] Pick a single colony of Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, Salmonella, Lactobacillus brevis, Lactobacillus delbrueckii and Lactobacillus crispatus in the plate and culture them overnight at 37°C and 200rpm air shaking in a suitable liquid medium. The 6 hybrid antimicrobial peptides were diluted stepwise into 8 gradients: 200, 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 mg / mL. Dilute the overnight cultured bacteria to 2×105-7×105 CFU / mL, add the diluted test bacteria solution to the 96-well culture plate respectively, 50 μL per well for 1-12 wells in each row, and then add dropwise to each well 50 μL of hybrid antimicrobial peptides. Three replicates were set for each sample, and no test bacteria was used as a negative control, and Amp was used as a positive control; cultured at a constant temperature at 37°C with a rotation speed of 100rpm for 24h; the absorbance was measur...

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Abstract

A pichia pastoris genetic engineering hybrid antibacterial peptide CC29 and a preparing method thereof are disclosed. Musca domestica cecropin obtained by cloning in a laboratory and rana chensinensis Chensirin are adopted as parent peptides and are subjected to hybrid designing to obtain the novel hybrid peptide with low hemolysis and high antibacterial activity and a fermentation strain capable of efficiently expressing the novel hybrid peptide. Compared with natural peptides, the peptide has huge advantages. A foundation of large-scale production of the antibacterial peptide is laid.

Description

technical field [0001] The invention relates to the fields of genetic engineering and biotechnology, in particular to a Pichia pastoris genetically engineered hybrid antibacterial peptide CC29 and a method for obtaining the same. Background technique [0002] Antimicrobial peptides are an important part of the biological innate immune system. They are small molecular polypeptides that are induced by immunity and have biological activity against external pathogen infection. Antimicrobial peptides have a broad-spectrum antibacterial effect, especially for killing some pathogenic bacteria that produce antibiotic resistance, which has aroused people's interest in it. Antimicrobial peptides have great application potential in agriculture, medical treatment, food and other fields. So far, thousands of natural antimicrobial peptides have been discovered, but there are relatively few species with good medical selection index. Therefore, how to obtain antibacterial peptides with goo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/81
Inventor 张爱忠姜宁张晨雪蔡鹏朱双刘晓明祁丽
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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