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Heterometrus spinifer poison antibacterial polypeptide gene and preparation method and application thereof

A technology of scorpion venom and gene, which is applied in the isolation and identification of antibacterial polypeptide gene of Asian rainforest scorpion, the application of the antibacterial polypeptide gene of scorpion venom, and the preparation field of antibacterial polypeptide gene of scorpion venom can solve the problem of low resistance, systemic or local fungus Infection and other problems, to achieve low production cost, fast sterilization speed, good effect

Inactive Publication Date: 2012-07-04
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Fungi are conditional pathogens for healthy humans, but when the body's resistance is low and the external conditions are poor, it may cause systemic or local fungal infections

Method used

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  • Heterometrus spinifer poison antibacterial polypeptide gene and preparation method and application thereof
  • Heterometrus spinifer poison antibacterial polypeptide gene and preparation method and application thereof
  • Heterometrus spinifer poison antibacterial polypeptide gene and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0051] ① Take 500mg of scorpion gland and grind it 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°C, the same below) for 5 minutes; ② Then add 2ml of chloroform and mix for 15 seconds, place at room temperature for 2-3 minutes, and centrifuge at 12000g for 15 minutes at 4°C; Take the water phase and add 1 times the volume of isopropanol, let it stand at room temperature for 10 minutes, and centrifuge at 12000g for 10 minutes at 4°C to obtain RNA precipitation; The precipitate was washed with 5m175% (volume ratio, the same below) ethanol, and centrifuged at 7500g for 5 minutes; After the RNA pellet was dried, it was 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 m...

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 properties 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 of adsorption buffer containing 32 μl of β-mercaptoethanol. ② Annealing 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 minutes...

Embodiment 3

[0054] Example 3: First strand cDNA synthesis

[0055] ① Add 2 μl to a 1.5ml Ep tube not Primer-adapter and 6μl mRNA (including 3μg mRNA), incubated at 70°C for 10min, quickly placed on ice, after brief centrifugation, added the following components: 4μl 5X first strand buffer; 2μl 0.1M DTT; 1μl 10mM dNTPs; 1μl DEPC· h 2 O. Gently mix and centrifuge briefly, and place at 37°C for 2 minutes; ② Add 5 μl reverse transcriptase, take 2 μl after mixing, add 1 μl [α- 32 P]dCTP (4 μCi) (tracer tube). Incubate at 37°C for 1 hour with the above reaction components (sample tubes), then place on ice to terminate the reaction; ③ For the tracer tube, add 43 μl 20 mM EDTA and 5 μl yeast tRNA in sequence, after mixing, take two parts of 10 μl and spot on the two filter membranes, one part with 10% (volume ratio, the same below) TCA (trichloroacetic acid) ) for 3 times, 5 min each time, 1 time for 95% ethanol, air-dried, put into 1.5ml scintillation fluid (1 # sample); the other ...

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Abstract

The invention discloses a heterometrus spinifer poison antibacterial polypeptide gene and a preparation method and application thereof. The preparation method comprises the following steps of: A, constructing a heterometrus spinifer poison gland cDNA library which comprises separation of total RNA by poison gland thereof, grinding caudal gland into fine powder in liquid nitrogen, and preparing spinifer poison gland total RNA through a Trizol method; B, separating and purifying mRNA by adopting a PolyATractmRNA separation system through the prepared spinifer poison gland total RNA, and measuring the prepared spinifer poison gland mRNA by using an ultraviolet spectrophotometer; C, using RNA as an initial amount, and synthesizing a first chain and a second chain of cDNA; D, continuously connecting double-chain cDNA with a SalI adapter, digesting the double-chain cDNA through NotI, and removing excessive SalI adapters and restriction enzyme cutting fragments from double-chain cDNA molecules; and E, connecting the double-chain cDNA with a pSPORT1 vector, identifying the poison gland cell cDNA library, selecting clones from the heterometrus spinifer poison gland cell cDNA library to prepare the spinifer poison antibacterial polypeptide gene. The method has the advantages of operability, low cost and high water solubility, and the heterometrus spinifer antibacterial polypeptide has the advantages of high sterilization speed and good effect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and the invention relates to an Asian rainforest scorpion venom antibacterial polypeptide gene, a preparation method of the scorpion venom antibacterial polypeptide gene, and the application of the scorpion venom antibacterial polypeptide gene, and functional identification of highly active antibacterial polypeptide . Specifically, the present invention relates to the isolation and identification of an Asian rainforest scorpion antibacterial polypeptide gene; it relates to the identification of an antibacterial spectrum of a scorpion antibacterial polypeptide: the Asian rainforest scorpion venom antibacterial polypeptide produced by chemical synthesis has broad-spectrum resistance to fungi; It also relates to the antifungal function identification of a scorpion antibacterial polypeptide, which has the value of developing potential antifungal drugs. Background technique [0002] Fungi are conditional...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/10C07K7/08A61K38/10A61P31/04A61P31/10
Inventor 李文鑫曹志贱吴英亮曹路扬樊拯
Owner WUHAN UNIV
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