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Genetic engineering tumor targeting KCT-W1 polypeptides and preparation method and application

A KCT-W1, genetic engineering technology, applied in the direction of peptide preparation methods, antineoplastic drugs, chemical instruments and methods, etc., can solve the problems of inability to early screen and diagnose tumors, achieve high stability, simple operation, and value huge effect

Inactive Publication Date: 2009-03-11
WUHAN MORE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Even the most advanced magnetic resonance imaging (MRI) can only detect tumor lesions with more than 1 million cancer cells, and cannot perform early screening and diagnosis of tumors

Method used

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  • Genetic engineering tumor targeting KCT-W1 polypeptides and preparation method and application
  • Genetic engineering tumor targeting KCT-W1 polypeptides and preparation method and application
  • Genetic engineering tumor targeting KCT-W1 polypeptides and preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Molecular design of KCT-W1 polypeptide sequence

[0032] Through protein structure prediction (http: / / swissmodel.expasy.org / / SWISS-MODEL.html) SWISS-MODEL program and protein structure analysis (http: / / swissmodel.expasy.org / spdbv / ) Swiss-PdbViewer program, based on With a deep understanding of the diversity and mechanism of interactions between scorpion venom peptides and channels, a protein peptide sequence specifically acting on chloride channels was designed: MCMPCFTTRHQMARKCRKCCGGKGRGKCYGPRCLCR (KCT-W1). Each KCT-W1 polypeptide molecule has 4 pairs of disulfide bonds. The existence of 4 pairs of disulfide bonds makes the KCT-W1 polypeptide have strong structure and property stability.

Embodiment 2

[0033] Embodiment 2: design primer and PCR amplification KCT-W1 gene

[0034] According to the KCT-W1 polypeptide sequence, the nucleotide sequence encoding the protein was deduced, and on this basis, primers were designed for PCR amplification to obtain the KCT-W1 gene. 合成KCT-W1多肽基因的4条引物如下:正向引物P1(5'GCCGGATCCCCGATGACGATGACAAAATGTGTATGCCGTGCTTCACTACC3'),正向引物P2(5'GGCATCGTAAATGGTGCTGGAAACGGTGGTGTCGTAAATGCTGTAAATGC3'),反向引物P3(5'TACGGCACGAAGTGATGGGCAGTGGTCTACCGTGCATTTACAGCATTTAC3')和反向引物P4( 5'GCCCTCGAGTCAACGGCACAGACAACGCGGACCGTAGCATTTTACCACGAC3'). Forward primer P2 and reverse primer P3 are used in the first round of PCR amplification, and forward primer P1 and reverse primer P4 are used in the second round of PCR amplification. The reagents and conditions of the first round of PCR reaction are as follows: 1 μl Taq polymerase (1 unit), 0.5 μl four kinds (adenine, guanine, cytosine, thymine) deoxymononucleotide equal ratio mixture (10mmol / L), 17.5 μl sterile double distilled water, ...

Embodiment 3

[0035] Example 3: Double digestion and connection of KCT-W1 gene and pGEX-6p-1

[0036] The PCR product obtained in Example 2 was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) (volume ratio), precipitated with absolute ethanol (2.5 times the volume), and then dissolved with 50 μl of sterile water. The recovered PCR product and expression vector pGEX-6p-1 plasmid were digested with restriction endonucleases BamHI and XhoI (products of Takara Company). Enzyme digestion reaction: 1 μl each of BamHI (14U / μl) and XhoI (20U / μl), 2.5μl of 10-fold buffer, 50-100ng of PCR product or pGEX-6p-1 plasmid, add sterile water to a total volume of 25μl . 37 ℃ water bath for 5 hours, the digested product was extracted with phenol: chloroform: isoamyl alcohol, precipitated with absolute ethanol (2.5 times volume) and washed with T 4 DNA ligase ligated the PCR product to the expression vector pGEX-6p-1. Ligation reaction: T 4 DNA ligase (1U / μl) 1μl, the molar ratio of PCR product...

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Abstract

The invention discloses gene engineering tumor targeted polypeptide and a preparation method and application thereof. The method separates a polypeptide, a sequence of the a polypeptide is an amino acid sequence shown in SEQ ID NO: 1. A primer that the gene engineering relates to inserts a nucleotide sequence of KCT-W1 into an expression vector pGEX-6p-1 to form recombinant expression plasmids; the recombinant expression plasmids transform colon bacillus Rossetta(DE3) and then obtain recombinant KCT-W1 polypeptide through cell lysis and GST affinity chromatography, the cutting of small intestine kinase and chromatogram purification. Cy5.5NHS easter is used to mark the KCT-W1 polypeptide to obtain a steady KCT-W1 polypeptide fluorescence complex. The KCT-W1 polypeptide fluorescence complex has special target function for neuroglioma transplanted under the oxter of a big mouse, and can perform accurate tracing and positioning to tumor. The invention also discloses a use of the KCT-W1 polypeptide fluorescence complex in the preparation of medicine used to treat or prevent incipient tumor. The method is simple and easy, convenient in operation, highly efficient and sensitive.

Description

technical field [0001] The invention belongs to the field of biotechnology, and the invention relates to a sequence of a tumor-targeting scorpion venom polypeptide KCT-W1, a genetic engineering preparation method of a tumor-targeting scorpion venom polypeptide, and an efficient and sensitive tumor detection KCT-W1 polypeptide fluorescent compound The preparation of the drug also relates to the tumor targeting visualization of the scorpion venom polypeptide KCT-W1 and its use in the preparation of drugs for treating or preventing early tumors. Background technique [0002] Tumor is a disease that seriously threatens human life and health. In 2002, a research report published by the World Health Organization showed that the global cancer situation will become increasingly serious, the number of new patients will increase from the current 10 million per year to 15 million in the next 20 years, and the number of deaths from cancer will also increase from 6 million per year. to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/11C12N15/70C07K1/13A61K38/16A61P35/00
Inventor 李文鑫曹志贱孙正博吴英亮刘辉
Owner WUHAN MORE BIOTECH
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