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A kind of high-efficiency preparation method of antibacterial peptide cath-bf30 of golden snake

An antibacterial peptide, -cath-bf30 technology, which is applied in the field of efficient preparation of the antibacterial peptide Cath-BF30, can solve the problems of difficult synthesis, low yield of antibacterial peptide Cath-BF30, and difficulty in popularization and application. To achieve the effect of ensuring correct secretion and expression, good industrial performance, and clear genetic background

Active Publication Date: 2017-10-31
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In actual work, due to the limited source of snake venom, the yield of the antimicrobial peptide Cath-BF30 is low and the cost is high, while the chemical synthesis of antimicrobial peptides also has the problem of high cost, difficult synthesis, expensive price, and difficult to popularize and apply

Method used

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  • A kind of high-efficiency preparation method of antibacterial peptide cath-bf30 of golden snake
  • A kind of high-efficiency preparation method of antibacterial peptide cath-bf30 of golden snake
  • A kind of high-efficiency preparation method of antibacterial peptide cath-bf30 of golden snake

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0074]Example 2 Construction of the cloning vector of Cath-BF30 antimicrobial peptide

[0075] 1. The genetic engineering operation was carried out according to the molecular cloning experiment guide method, and the Cath-BF30 target gene synthesized by PCR and the vector plasmid PGAPZa were respectively used xho I and Xba I double digestion, and then ligation transformation. The specific procedure is as follows:

[0076] (1) Obtaining the vector plasmid PGAPZa: Cultivate Escherichia coli containing the PGAPZa plasmid overnight at 37°C and 170 rpm, extract the plasmid with a plasmid mini-extraction kit, perform gel electrophoresis, and store at -20°C.

[0077] (2) Double digestion: The vector plasmid PGAPZa and the Cath-BF30 gene were respectively digested with xho I and Xba I double enzyme digestion, 37 ℃ water bath for 2 hours, gel electrophoresis, observe whether the double enzyme digestion is complete, if the target fragment and the carrier have been double enzyme...

Embodiment 3

[0082] Example 3 Construction of engineering bacteria SMD-pGAPZα-30

[0083] 1. Linearization of recombinant expression plasmid pGAPZα-Cath-BF30

[0084] A large number of pGAPZα-Cath-BF30 plasmids were extracted with the plasmid extraction kit, and the Bln I enzyme linearizes it. 200μL linearization system: 10μL BlnI , 40 μL pGAPZα-Cath-BF30 (about 20 μg), 20 μL 10× buffer, 130 μL ddH 2 O. After mixing well, digest overnight at 37°C, take 2 μL of the digested product and do 1% agarose gel electrophoresis to observe whether it is completely linearized.

[0085] 2. Extract the fully linearized plasmid with phenol-chloroform to reach the concentration required for electroporation. The specific steps are as follows:

[0086] (1) Add 300 μL ddH to 200 μL enzyme digestion system 2 O to make a total volume of 500 μL.

[0087] (2) Add 500 μL of phenol chloroform to extract once, 12000 rpm, 10 min, carefully take the upper layer in the stratification.

[0088] (3) Add 1 / 10 ...

Embodiment 4

[0138] Example 4 Fermentation of engineering bacteria SMD-pGAPZα-30

[0139] 1. Observation of the effect of pH value on the secretion and expression of Cath-BF

[0140] Performed at the shake flask level, adjust the pH to 4.0, 5.0, 6.0 with phosphate buffer, and adjust the pH 6.8 YPD medium without phosphate buffer, inoculate the engineered bacteria SMD-pGAPZα- at 1% (v / v) 30 seed liquids, 28°C, 170rpm shaking culture for 96h, take 0h, 24h, 48h, 72h, 96h samples, measure the corresponding pH, cell density OD 600 and the size of the inhibition zone to determine the optimum pH of the expression product.

[0141] The results are shown in Table 2. The results showed that the pH of the engineered bacteria SMD-pGAPZα-30 culture decreased significantly after 24 hours of culture, and then increased significantly in the middle and late stages of culture. When the pH value was between 4.0 and 5.0, the culture supernatant The antibacterial activity of the sample was high, and the anti...

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Abstract

The invention discloses a high-efficiency preparation method of Cath-BF30 antibacterial peptide Cath-BF30, the steps are as follows: design and synthesize the Cath-BF30 gene, whose sequence is SEQ ID NO.1; use XhoI and XbaI double enzyme digestion to synthesize The gene fragment was cloned into the pGAPZα vector to obtain the recombinant expression plasmid pGAPZα‑Cath‑BF30; the recombinant expression plasmid was transformed into Pichia pastoris cells to obtain the engineering bacteria SMD‑pGAPZα‑30; the engineering bacteria were fermented at a pH lower than 5; After the fermentation supernatant was filtered through a 0.45 μm filter membrane to remove impurities, it was separated and purified by SP Sepharose FF column. This method can achieve high-efficiency secreted expression, and ensure that the secreted product has a natural N-terminal sequence, and the secreted expressed product has the activity of inhibiting Bacillus acnes, and the fermentation supernatant only needs one-step ion exchange to obtain high-purity Cath‑BF30.

Description

technical field [0001] The invention belongs to the technical field of polypeptide medicine. More specifically, it relates to a high-efficiency preparation method of the antibacterial peptide Cath-BF30. Background technique [0002] Acne is a common pilosebaceous gland inflammatory skin disease, which is mainly caused by anaerobic Propionibacterium acnes. At present, antibiotics are mainly used for treatment, but the curative effect is not satisfactory, and it can lead to the production of resistant bacteria and hypersensitivity reactions. Recent studies have found that the antimicrobial peptide Cath-BF isolated from the venom of golden krait can effectively inhibit the growth of Propionibacterium acnes and other acne-infecting bacteria in mouse models, and inhibit the related inflammatory hyperplasia, so it plays an important role in the prevention and treatment of acne. application value. The antimicrobial peptide Cath-BF isolated from the venom of krait snake belongs to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/81C12N15/12C07K14/46C07K1/18C12R1/84
Inventor 李黄金赵林郝思怡
Owner GUANGDONG PHARMA UNIV
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