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High expression of tenebrio molitor antibacterial peptide TmAMP3m in escherichia coli and application of TmAMP3m

A kind of high-efficiency expression, antibacterial peptide technology

Inactive Publication Date: 2013-04-17
XINJIANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although Tenebrio molitor is widely distributed and rich in resources, due to the low content of natural antibacterial peptides, the extraction rate is low (0.005%), and the cost is high, industrial production has not been realized.

Method used

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  • High expression of tenebrio molitor antibacterial peptide TmAMP3m in escherichia coli and application of TmAMP3m
  • High expression of tenebrio molitor antibacterial peptide TmAMP3m in escherichia coli and application of TmAMP3m
  • High expression of tenebrio molitor antibacterial peptide TmAMP3m in escherichia coli and application of TmAMP3m

Examples

Experimental program
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Effect test

Embodiment 1

[0074] Example 1: High expression of recombinant mutant Tenebrio molitor antimicrobial peptide TmAMP3m in Escherichia coli

[0075] See attached Figure 1-4 :

[0076] (1) Design specific primers using the antimicrobial peptide sequence of Tenebrio molitor (Accession No. TMU21482) published by GenBank:

[0077] Upstream primer TF1: 5′-ATGAAAACATTCGTGATTTGCTTGATTCTGG-3′,

[0078] Downstream primer TR1: 5′-TTAATGA CCATGTGTCTTGTACCCTCCTTGG-3′;

[0079] (2) The antimicrobial peptide gene tenecin3 was cloned from Tenebrio molitor by RT-PCR

[0080] Before the operation, the laboratory bench was irradiated with ultraviolet light for 30 minutes, and a 5-year-old Tenebrio molitor larva was taken and ground in an RNase-free mortar with liquid nitrogen. The powder was transferred into an Eppendorf tube containing 1 mL of Trizol reagent, and the cap was tightly shaken. 15s, place at room temperature at 15-30°C for 3-5min, centrifuge at 12000r / min at 4°C for 15min, carefully transfer ...

Embodiment 2

[0107] Example 2: Prokaryotic expression of recombinant plasmid pET30a-TmAMP3m

[0108] Mix the extracted plasmid DNA DH5α / pET30a-TmAMP3m with 35 μL of competent cells E.coliBL21, place in an ice bath for 30 minutes, then heat shock in a water bath at 42°C for 45 seconds, and quickly remove from the ice bath for 3 minutes (do not shake the EP tube). Add 800 μL of fresh LB culture medium without antibiotics to each tube, mix well, and incubate at 37°C for 1 h with gentle shaking. 4°C, 3000rpm, centrifuge for 1min, discard 600μL supernatant, gently pipette and mix the remaining bacterial solution, spread evenly on the prepared solid LB medium (containing kanamycin), and culture overnight at 37°C. The next day, pick a single clone and culture it in 6mL LB medium (containing kanamycin) for 12-16h, then transfer it to fresh medium (containing Kana) at a volume ratio of 1% to 2%, at 37°C, 220r Shake the bacterial solution at 0.4-0.6 per minute until the OD600 is 0.4-0.6, and pipett...

Embodiment 3

[0109] Example 3: Antibacterial Activity Detection Method of Recombinant Mutant Tenebrio Molitor Antimicrobial Peptide TmAMP3m

[0110] First, 3 μl of the antimicrobial peptide obtained by expression was taken out. Take Staphylococcus aureus as the experimental strain, culture in LB liquid medium until OD600=0.5, refer to the agar hole diffusion method, take 10μl of bacterial suspension and 3ml of solid medium (containing 0.8% agar) and mix at 45°C , and spread it in a sterile petri dish with a diameter of 6cm, and store it at 4°C after solidification; when in use, make a number of small holes with a diameter of 2mm in the dish, and drop 3μl of the product to be tested into the holes, and store at 37°C Cultivate under conditions for 12-16h. Measure the size of the zone of inhibition. According to the size of the inhibition zone to determine the activity of antimicrobial peptides, see the attached Figure 7 .

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Abstract

The invention discloses tenebrio molitor antibacterial peptide TmAMP3m and a method for efficiently expressing the TmAMP3m in escherichia coli. A tenebrio molitor antibacterial peptide sequence published by a GenBank is utilized to design a specific primer, an antibacterial peptide gene is cloned from a tenebrio molitor body by adopting an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method and is mutated into TmAMP3m, the gene TmAMP3m is subcloned into a pET-30a expression carrier and is transformed into E.coli BL21 to express fusion protein, and the fusion protein is purified bycarrying out affinity chromatography with a Ni<2+> column to obtain the TmAMP3m, the TmAMP3m has broad spectrum bacteriostatic activity, can kill drug fastbacteria, has strong thermostability and is resistant to strong acid and strong alkali. The invention provides guarantee for realizing high antibacterial activity and strong stability, difficultly inactivating antibacterial peptide in an industrialized production process and producing and fermenting in a large scale and has a wide application value.

Description

field of invention [0001] The invention relates to the technical field of microbial secondary substance metabolism. Specifically, the present invention relates to the high-level expression of Tenebrio molitor antimicrobial peptide in Escherichia coli and the application of high-level expression thereof. Background technique [0002] Antimicrobial peptides widely exist in various organisms, such as bacteria, fungi, insects, plants and vertebrates, etc. They have broad-spectrum antibacterial activity, have inhibitory and killing activities against bacteria, fungi, viruses, and protozoa, and can protect the body against external The invasion of source microorganisms is an important component of the innate immune system in the host. Therefore, antimicrobial peptides are also called host defense peptides. In addition, antimicrobial peptides also play multiple functions in immune response (inflammatory response, wound healing, regulation of acquired immune system) and maintenance...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/70A61K38/17A61P31/04C12R1/19
Inventor 刘忠渊张富春唐馨
Owner XINJIANG UNIVERSITY
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