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Antineoplastic biological medicament PTD4-GFP-Apoptin fusion protein and preparation method thereof

A technology of ptd4-gfp-apoptin and pet28a-ptd4-gfp-apoptin, which is applied in the field of biotechnology drugs for the treatment of tumors, can solve problems such as unreported, achieve large killing ability, promote tumor apoptosis, and inhibit tumor growth. Effect

Inactive Publication Date: 2007-12-05
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ho et al. [17] TAT was modified by chemical synthesis, and a series of TAT-PTD polypeptides were obtained through amino acid substitutions. Among them, PTD4 has a more stable secondary structure and higher transduction efficiency, which can almost achieve a 100% success rate of target protein transduction. Moreover, the amount of protein imported into a single cell is 33 times that of TAT, but whether PTD4 can achieve transmembrane transport of fusion proteins through biosynthesis has not yet been reported.

Method used

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  • Antineoplastic biological medicament PTD4-GFP-Apoptin fusion protein and preparation method thereof
  • Antineoplastic biological medicament PTD4-GFP-Apoptin fusion protein and preparation method thereof
  • Antineoplastic biological medicament PTD4-GFP-Apoptin fusion protein and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Construction of prokaryotic expression vector pET28a-PTD4 containing PTD4 sequence

[0057] 1. Preparation of PTD4 sequence by annealing artificially synthesized oligonucleotide fragments

[0058] (1) According to Ho's report [17] , PTD4 polypeptide consists of 11 amino acids, and its base sequence is independently designed according to the characteristics of prokaryotic codons as follows:

[0059] S1: 5′-CTAGTTATGCCCGCGCGGCAGCACGACAAGCTCGAGCCC-3′

[0060] S2: 5′-CTAGGGGCTCGAGCTTGTCGTGCTGCCGCGCGGGCATAA-3′

[0061] Two oligonucleotide fragments were synthesized by Shanghai Bioengineering Company.

[0062] The two oligonucleotide fragments were mixed equimolarly, 95°C for 10 min, and then placed at room temperature for 1 h to anneal to form a double-stranded DNA encoding PTD4.

[0063] 2. Construction of recombinant pET28a-PTD4

[0064] (1) The prokaryotic expression vector pET-28a(+) was purchased from Novagen.

[0065] (2) The construction method adopts the conven...

Embodiment 2

[0073] Construction of prokaryotic expression vector pET28a-PTD4-GFP containing GFP gene

[0074] 1. Using the PCR method to amplify the GFP gene of green fluorescent protein

[0075] (1) pEGFP-C1 was purchased from CLONETECH.

[0076] (2) PCR primer sequences are as follows:

[0077] P3: 5′-ACGGATCCATGGTGAGCAAGGGCG-3′:

[0078] P4: 5′-GCGAATTCCTTGTACAGCTCGTCCATGC-3′

[0079] The 5' ends of the two primers contain recognition sites for restriction endonucleases BamH I and EcoR I, respectively.

[0080] (3) PCR amplification reaction conditions

[0081] The cycle parameters are 94°C for 5min, 94°C for 55sec, 62°C for 55sec, and 72°C for 1min, and the cycle amplification is 30 times, and finally, keep at 72°C for 10min. The PCR product was identified by 1.5% agarose gel electrophoresis, and the band size was correct.

[0082] 2. Construction of prokaryotic expression vector pET28a-PTD4-GFP

[0083] (1) For the construction of the prokaryotic expression vector pET28a-PTD4,...

Embodiment 3

[0088] Construction of prokaryotic expression vector pET28a-PTD4-GFP-VP3 containing vp3 gene

[0089] 1. Utilize the PCR method to amplify the vp3 gene of chicken anemia virus

[0090] (1) Construct the eukaryotic expression vector pcDNA-vp3 containing the vp3 gene. For specific methods, see: Wang Yuzhe, Tian Jun, Qu Shen, etc., Construction of chicken anemia virus vp3 gene and research on its apoptosis-inducing effect in vitro, Journal of Tongji Medical University, 2001 , 30(4):300-4. [18]

[0091] (2) PCR primer sequences are as follows:

[0092] P5: 5′-AGGAATTCATGAACGCTCTCCAAG-3′

[0093] P6: 5′-GCGTCGACTTACAGTCTTATACGCC-3′

[0094] The 5' ends of the two primers contain recognition sites for restriction endonucleases EcoR I and Sal I, respectively.

[0095] (3) PCR amplification reaction conditions

[0096] The cycle parameters are 94°C for 5min, 94°C for 55sec, 60°C for 50sec, and 72°C for 55sec, cycle amplification 30 times, and keep warm at 72°C for 10min. The PC...

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Abstract

The biomedicine PTD4-GFP-Apoptin fusion protein for treating tumor has its synthesized PTD4 utilized to introduce Apoptin protein into cell, and its Apoptin further utilized to induce tumor apoptosis, so as to inhibit tumor growth. At the same time, green fluorescent protein (GFP) is utilized for observing the intracellular distribution of the fusion protein and showing the aggregation of the medicine in the target position, realizing the effective monitoring and timely regulation of the treating process. Experiment shows that the fusion protein can penetrate biomembrane to kill tumor cell effectively while generating no damage to health cell. The present invention is applied in treating tumor.

Description

technical field [0001] The invention relates to biotechnological medicines, especially biotechnological medicines for treating tumors. Background technique [0002] Cancer is a great threat to human health and life. About 7 million people in the world are newly diagnosed with cancer every year, and more than 5 million people die of cancer every year. At present, about 1.5 million people are newly diagnosed with cancer every year in our country, and about 800,000 people die of cancer every year. Cancer is one of the major diseases that seriously affect human health and threaten human life. Together with cardiovascular and cerebrovascular diseases and accidents, cancer constitutes the top three causes of death in all countries in the world today. Therefore, the World Health Organization and the health departments of governments of various countries have listed conquering cancer as a top priority. Finding new safe and effective drugs and methods for treating tumors is an imp...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K38/17A61P35/00C12N15/63C12N15/62
Inventor 屈伸孙军宗义强
Owner HUAZHONG UNIV OF SCI & TECH
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