Anti tumor translocation peptide of scorpion, preparation method and application

An anti-tumor metastasis, scorpion venom gland technology, applied in anti-tumor drugs, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problem of low yield and achieve the effect of inhibiting chloride ion channels

Inactive Publication Date: 2007-07-25
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Recently, Zhang Shoutao et al. (Zhang Shoutao, Guo Aiguang, Xiao Leyi, Dong Jianjun, Zhao Tianzeng, Xue Qigeng, Kong Tianhan, Guo Wei "Journal of Northwest A&F University" 2002 Volume 30 No. 6: 30-33) through reverse transcription-polymerase chain reaction ( RT-PCR) method to amplify the cDNA of the neurotoxin BmKCT of the East Asian pincer scorpion and clone it into the pTrcHisA expression vector and transfer it to Escherichia coli BL21 (DE3) to express and produce the scorpion BmKCT peptide. However, the yield is not high, and most of them form inclusion bodies. insoluble

Method used

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  • Anti tumor translocation peptide of scorpion, preparation method and application
  • Anti tumor translocation peptide of scorpion, preparation method and application
  • Anti tumor translocation peptide of scorpion, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Design of primers and PCR amplification of scorpion anti-tumor metastasis peptide gene

[0073] Design PCR primers according to the sequence of the scorpion anti-tumor metastasis peptide gene (BmKCTL gene) provided by SEQID NO: 1, for example: A1, 5'-GCCGGATCCCCGATGACGATGACAAGTGTGGGCCTTGCTTTAC-3', A2: 5'-GCCCTCGAGTCATTCACGGTTACACAGACATTG-3', to obtain from scorpion venom gland cells The clone of the BmKCTL gene (SEQID NO: 1) obtained by screening in the cDNA library was used as a template to carry out PCR reaction to amplify the scorpion anti-tumor metastasis peptide gene in large quantities. PCR reaction conditions: 1 μl Taq polymerase (1U), 0.5 μl four (adenine, guanine, cytosine, thymine) deoxymononucleotide mixture (10mmol / L), 16.5 μl sterile double distilled Water, 2.5 μl 10-fold PCR buffer, 1.5 μl magnesium chloride (25 mmol / L), A1 (10 μmol / L) and A2 (10 μmol / L) primers, and 1 μl each of BmKCTL gene template in a total volume of 25 μl. PCR reaction pro...

Embodiment 2

[0074] Embodiment 2: Construction of recombinant expression vector (rBmKCTL / pGEX)

[0075] The PCR product obtained in Example 1 was extracted with restriction endonucleases through phenol: chloroform: isopentyl glycol (25: 24: 1), precipitated with absolute ethanol (2.5 times volume) and washed with 50 μl TE buffer (recorded After this example) to dissolve the precipitate. The recovered PCR product and expression vector pGEX-5x-1 plasmid were digested with restriction endonucleases Bam HI and Xho I (products of Takara Company). Enzyme digestion reaction: 1 μl each of BamHI (14U / μl) and XhoI (20U / μl), 2.5 μl of 10-fold buffer, 50-100 ng of PCR product or pGEX-5x-1 plasmid, add sterile water to a total volume of 25 μl, 37 ℃ water bath for 5 hours, the digested product was extracted with phenol-chloroform, precipitated with absolute ethanol (2.5 times the volume) and washed with T 4 DNA ligase connects the PCR product with the expression vector pGEX-5x-1 to form a recombinant ...

Embodiment 3

[0077] Example 3: Obtaining recombinant Escherichia coli BL21 (rBmKCTL / pGEX)

[0078] The ligation product in Example 2 was transformed into Escherichia coli BL21. Inoculate Escherichia coli BL21 in 3ml of LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L), and culture on a shaker at 37°C until OD 600 When it reaches 0.4, centrifuge at 12000 rpm for 5 minutes, remove the supernatant, add ice-precooled 0.1MCaCl 2 120 μl of resuspended bacteria. Add 4 μl (<20 ng) of the ligation product, place in an ice bath at 10°C for 30 minutes, heat shock at 42°C for 90 seconds, and quickly transfer to an ice bath at 10°C for 1-2 minutes. Add 1ml of SOC (tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, potassium chloride 2.5mmol / L, magnesium chloride 10mmol / L, glucose 20mmol / L) solution, shake at 37°C Incubate on the bed for 1 hour, centrifuge at 1200 rpm for 5 minutes, remove the supernatant, add 100 μl of SOC solution to resuspend the bacteria, and...

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PUM

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Abstract

This invention discloses a method for preparing anti-tumor peptide of Buthus martensii, and its application. The method comprises: (1) screening cDNA sequence of anti-tumor peptide gene from cDNA library of Buthus martensii venom gland, designing primers, and performing PCR with the screened cDNA as a template to obtain anti-tumor peptide gene or gene fragment containing a digestion site of restriction endonuclease; (2) digesting the PCR product and expression plasmid; (3) ligating with T4 DNA ligase to obtain recombinant vector rBmKCTL / pGEX; (4) transferring into Eschericia coli BL21 to obtain engineering Eschericia coli BL21 / rBmKCTL / pGEX (CCTCC No. M206094). The method has such advantages as simple process, high safety, low cost, easy purification, and high product purity. The anti-tumor peptide can be used to manufacture drugs for preventing or treating tumor metastasis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and the invention relates to a scorpion anti-tumor metastasis peptide and a method for producing the scorpion anti-tumor metastasis peptide, specifically, the present invention relates to a recombinant E. The invention relates to a preparation method of highly efficient biologically active scorpion anti-tumor metastasis peptide, a method for producing scorpion anti-tumor metastasis peptide by means of genetic engineering, and the application of the generated scorpion anti-tumor metastasis peptide in the preparation of medicines for treating or preventing anti-tumor metastasis. Background technique [0002] The East Asian scorpion is the most widely distributed scorpion species in China. Because of the complex components and properties of the scorpion venom, it produces a variety of physiological and pharmacological activities. Cardiovascular disease has an important therapeutic effect. However, due ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/12C07K14/435A61K38/17A61P35/00C12R1/19
Inventor 蒋达和李文鑫曹志贱毛歆范少忠刘辉
Owner WUHAN UNIV
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