Hainan sporic scorpion antibiotic and preparation method and application

A technology of antimicrobial peptide and scorpion venom, which is applied in the separation and identification of scorpion venom antibacterial peptide and scorpion antimicrobial peptide gene, and the field of preparation of the antimicrobial peptide gene, which can solve problems that do not involve the antibacterial function of scorpion toxin

Inactive Publication Date: 2007-10-31
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, there are currently no patents related to scorpion toxin genes such as Hainan spot, but there are only 7 patents related to East Asian scorpion venom adenotoxin genes: ①The application of East Asian scorpion toxin BmKAS in pharmaceuticals, the invention involves East Asian scorpion toxin BmKAS The application in the preparation of anti-peripheral injury drugs, and the application of the East Asian scorpion toxin BmKAS in the preparation of anti-hyperalgesia drugs; ② A recombinant baculovirus containing a bivalent insect-resistant gene, the invention relates to the specificity of East Asian scorpion insects Neurotoxin gene BmKIT and an insect chitinase gene (Chi) to obtain a recombinant baculovirus AcNPV-BmKIT-Chi containing a bivalent insect-resistant gene; ③ Scorpion antiarrhythmic peptide recombinant Escherichia coli and its construction method, the The invention relates to a scorpion anti-arrhythmic peptide BmKIM gene recombinant Escherichia coli, which is used for mass production of soluble recombinant peptides with anti-arrhythmic activity; ④ scorpion anti-arrhythmic peptide and its preparation method and application. The invention discloses a A scorpion anti-arrhythmic peptide BmKIM and its preparation method and application. The recombinant BmKIM polypeptide obtained in the present invention has high-efficiency anti-arrhythmic activity, good solubility, and high yield; ⑤An artificially synthesized scorpion chloride ion channel neurotoxin gene- rBmKCTa, the patent relates to the chloride ion channel neurotoxin BmKCT gene, the improved recombinant scorpion chloride ion channel neurotoxin obtained by genetic engineering has an inhibitory effect on glial cells, and can be used to prepare diseases that can be treated by inhibiting glial cells 6. the purposes of a transitional scorpion toxin BmKabT, the purposes of the transitional scorpion toxin BmKabT disclosed by the present invention, it can be used as a unique sodium channel modulator, a probe for studying the composition, structure and function of the sodium channel, Modulators and medicines that can regulate and treat sodium channel-related diseases, and can be prepared to regulate or treat hyperkalemic paralysis, congenital myotonia and other skeletal muscle diseases, long QT interval disorder type III (LQT3), primary ventricular Modulators or medicines for fibrillation and other heart diseases; ⑦A recombinant scorpion toxin and its soluble expression and purification method, the invention relates to a recombinant scorpion insect toxin rBmKIT and its soluble expression and purification method
[0006] Although the patents ①②③④⑤⑥⑦ are all about the toxin of East Asian scorpion scorpion, and the scorpions used in the present invention are scorpions such as Hainan spot, the scorpion toxin genes BmKAS, BmKIT, BmKIM, BmKCT, BmKabT involved in the patent and the ImKAMP1 gene obtained by the present invention are Completely different toxin genes, and none of the patented inventions ①②③④⑤⑥⑦ involves the antibacterial function of scorpion toxin

Method used

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  • Hainan sporic scorpion antibiotic and preparation method and application
  • Hainan sporic scorpion antibiotic and preparation method and application
  • Hainan sporic scorpion antibiotic and preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Extraction of scorpion venom total RNA (Trizol LS one-step method)

[0051] ① Grind 500mg of scorpion gland into fine powder in liquid nitrogen, add 10ml TRIZOL reagent (purchased from Invitrogen, USA) and mix well, and let it stand at room temperature (20-25℃, the same below) for 5 minutes; ②Then add 2ml chloroform and mix for 15 seconds , place at room temperature for 2-3 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; ④precipitate with 5ml 75% ethanol Wash and centrifuge at 7500g for 5 minutes; ⑤The RNA pellet is dried and dissolved in DEPC-treated water, and incubated 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 RNAIsolation) Reagent Kit. The quality of the prepared scorpion venom glan...

Embodiment 2

[0052] Embodiment 2: Separation and purification of mRNA

[0053] 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 5 μl of Oligo (dT) at a concentration of 250 pM, add distilled water to 50 μl; add 1.6 ml of preheated dilution buffer (32 μl β-mercaptoethanol has been added to the dilution buffer), mix with RNA, and incubate at 70°C for 5 minute. ③Activation of ma...

Embodiment 3

[0054] Example 3: First strand cDNA synthesis

[0055] 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, and after brief centrifugation, add the following components: 4μl 5X first strand buffer; 2μl 0.1M DTT; 1μl 10mM dNTPs; 1μl DEPC·H 2 O. Gently mix and then briefly centrifuge, put at 37°C for 2 minutes; ②Add 5μl reverse transcriptase, take 2μl after mixing, add 1μl [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 for 3 times, each time for 5 minutes, wash 1 time with 95% ethanol, after air drying, put it into 1.5ml scintillation fluid (for 1# sample); After drying, put 1.5ml scintillation flu...

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PUM

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Abstract

The invention discloses a preparing method of HaiNan speckle equal scorpion antibiotic peptide gene and appliance, which comprises the following steps: restructuring bacillus coli Escherichia coli DH5a/ImAMP1, CCTCC NO: M207034; constructing HaiNan speckle equal scorpion ioterium cell cDNA library; designing primer; choosing PCR method; sieving positive colony of scorpion antibiotic peptide gene from ioterium cDNA library; sequence-analyzing coding trait of antibiotic peptide gene; assuring amino acid sequence of the antibiotic peptide gene; adopting chemosynthesis antibiotic peptide; possessing inhibition of diverse density for Gram's bacterium. This peptide can be used to medicine to prevent and cure Gram's bacterium.

Description

technical field [0001] The invention belongs to the field of biotechnology, and the invention relates to a scorpion venom antibacterial peptide such as Hainan spot, a preparation method of the antibacterial peptide gene, application of the gene, molecular design and antibacterial function identification of the highly active antibacterial peptide. Specifically, the present invention relates to the isolation and identification of a scorpion antimicrobial peptide gene such as Hainan spot; it relates to the antibacterial spectrum identification of a scorpion antibacterial peptide: the scorpion venom antibacterial peptide produced by chemical synthesis means is effective against Gram-positive bacteria Has broad-spectrum resistance, but has no effect on Gram-negative bacteria; involves scorpion antimicrobial peptides that are effective against clinically isolated drug-resistant hemolytic staphylococci; also involves a highly active molecular design and antibacterial function identifi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/12C07K14/435A61K38/17A61P31/04C12R1/19
Inventor 曹志贱李文鑫吴英亮马一保戴潮
Owner WUHAN UNIV
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