East-Asia scorpion antibiotic peptide gene and preparation method and application

A technology of bacteria and amino acids, applied in the biological field, can solve the problems that do not involve the antibacterial function of scorpion toxins

Active Publication Date: 2009-09-09
唐克煌
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the patents ①②③④⑤⑥⑦ are all about the East Asian scorpion toxin, the scorpion toxin genes BmKAS, BmKIT, BmKIM, BmKCT, BmKabT involved in the patent and the BmKAMP obtained by the present invention 1 The gene is a completely different toxin gene, and none of the patented inventions ①②③④⑤⑥⑦ involve the antibacterial function of scorpion toxin

Method used

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  • East-Asia scorpion antibiotic peptide gene and preparation method and application
  • East-Asia scorpion antibiotic peptide gene and preparation method and application
  • East-Asia scorpion antibiotic peptide gene and preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Extraction of total RNA from scorpion venom glands (Trizol LS one-step method: Trizol LS is purchased from Invitrogen, USA)

[0050] ① Grind 500mg of scorpion gland into fine powder in liquid nitrogen, add 10ml TRIZOL reagent and mix well, and let stand at room temperature (20-25℃, the same below) for 5 minutes; Minutes, centrifuge at 12,000g for 15 minutes at 4°C; ③Add 1 volume of isopropanol to the water phase, place at room temperature for 10 minutes, and centrifuge at 12,000g for 10 minutes at 4°C to obtain RNA precipitation; ④Wash the precipitate with 5m175% ethanol, and centrifuge at 7,500g for 5 minutes; ⑤ After the RNA precipitate was dried, it was dissolved in DEPC-treated water, and kept at 55-60°C for 10 minutes to completely dissolve the RNA. The whole process was carried out according to the method recommended by TR1ZOL (Total RNA Isolation) ReagentKit. The quality of the prepared scorpion venom gland total RNA was detected by formaldehyde denat...

Embodiment 2

[0051] Embodiment 2: Separation and purification of mRNA

[0052] The PolyA Tract mRNA isolation system (Promega, USA) was used to isolate and purify mRNA. Its working principle is based on the complementary pairing characteristics of Oligo(dT) and the poly(A) tail at the 3' end of mRNA. Oligo(dT) was labeled with biotin and passed It anneals with the mRNA 3' end poly(A) to form a hybrid, then captures and washes the biotin Oligo(dT) / mRNA hybrid with avidin-labeled magnetic beads and a magnetic separation rack, and finally uses RNase-free wxya 2 O elutes it to achieve the purpose of isolating mRNA from total RNA. ① Sample preparation: RNA was added to 800 μl GTC containing 32 μl β-mercaptoethanol. ②Annealing of the probe: Take 5μl of Oligo(dT) at a concentration of 250pM, add distilled water to 50μl; add 1.6ml of preheated dilution buffer (32μl of β-mercaptoethanol has been added to the dilution buffer), mix with RNA, and incubate at 70°C for 5 minutes . ③Activation of mag...

Embodiment 3

[0053] Example 3: First strand cDNA synthesis

[0054] ①Add 2μl Not I Primer-adapter and 6μl mRNA (including 3μg mRNA) to a 1.5ml Ep tube, incubate at 70°C for 10min, put it on ice quickly, after centrifugation, add the following components: 4μl 5X firststrand buffer; 2μl 0.1M DTT ; 1 μl 10mM dNTPs; 1 μl DEPC·H 2 O. Gently mix and centrifuge, put at 37°C for 2 min; ② Add 5 μl reverse transcriptase, take 2 μl after mixing, add 1 μl [α- 32 P] dCTP (4 μCi) (tracer tube). Incubate with the above reaction components (sample tubes) at 37°C for 1 hour, and then put them on ice to terminate the reaction; ③ For the tracer tubes, add 43 μl 20 mM EDTA and 5 μl yeast tRNA in sequence, mix well, and take two 10 μl portions respectively. On two filter membranes, wash 1 part with 10% TCA 3 times, each time for 5 minutes, wash 1 time with 95% ethanol, air dry, put into 1.5ml scintillation fluid (1 # sample); the other 1 part was air-dried and put into 1.5ml scintillation fluid (for 2 # s...

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PUM

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Abstract

The invention discloses an East Asian scorpion antimicrobial peptide gene and its preparation method and application, which is characterized in that the scorpion venom antibacterial peptide is recombinant Escherichia coli DH5a / BmKAMP1, CCTCC NO: M207036, and the steps are as follows: firstly, the East Asian scorpion venom is constructed Gland cell cDNA library; secondly, the positive clones of scorpion antimicrobial peptide gene were screened from poison gland cDNA library by PCR method; the coding characteristics of the antimicrobial peptide gene were sequence analyzed to determine the amino acid sequence of the antimicrobial peptide; Lambert bacteria have inhibitory effects at different concentrations. In particular, the East Asian scorpion antimicrobial peptide has a strong inhibitory effect on drug-resistant hemolytic staphylococcus, and has a good antibacterial effect. The antimicrobial peptide of the scorpion scorpion of the invention is specific to Gram bacteria, has high activity and is simple and convenient. Can be used as antibacterial drug application.

Description

technical field [0001] The invention belongs to the field of biotechnology, and the invention relates to a scorpion antimicrobial peptide and the scorpion antimicrobial peptide gene, and also relates to a preparation method of the East Asian scorpion toxin gene, and also relates to the application of the gene, specifically, the present invention relates to Isolation of a scorpion antimicrobial peptide gene; involving a scorpion antimicrobial peptide identification of antimicrobial spectrum: it has broad-spectrum resistance to Gram-positive bacteria, but has no effect on Gram-negative bacteria; involving point mutations, its antimicrobial peptide improves the antibacterial Resistance of Gram-negative bacteria; also involved scorpion antimicrobial peptides are effective against clinically isolated drug-resistant hemolytic staphylococci, and have potential value for the development of antibacterial drugs. Background technique [0002] East Asian pincer scorpion (Buthus martensi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/12C07K14/435A61K38/17A61P31/04C12R1/19
Inventor 曹志贱李文鑫戴潮吴英亮蒋达和毛歆刘辉
Owner 唐克煌
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