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Preparation method and application for invertebrate recombinant ferritin based anti-tumor nano arsenic sphere

A technology of recombining ferritin and invertebrates, which is applied in the field of preparation of anti-tumor nano-arsenic spheres, can solve problems such as unsatisfactory therapeutic effects, side effects, and death

Active Publication Date: 2019-09-06
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the therapeutic effect of low-dose arsenic is not satisfactory, while higher doses have greater toxic side effects on normal cells and cause death.

Method used

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  • Preparation method and application for invertebrate recombinant ferritin based anti-tumor nano arsenic sphere
  • Preparation method and application for invertebrate recombinant ferritin based anti-tumor nano arsenic sphere
  • Preparation method and application for invertebrate recombinant ferritin based anti-tumor nano arsenic sphere

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Experimental program
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Effect test

specific Embodiment 1

[0033] Preparation of recombinant ferritin based on invertebrates

[0034] 1. Cloning of ferritin gene sequence

[0035] 1.1 Gene amplification

[0036] Using the Fer147 sequence (SUMO+ferritin gene) as a template, use Primer Primer 5.0 software to design the upstream expression primer FER-F: 5′- CCGCTCGAGAATTAGGAGGAAGTCCAAGA-3′; and the downstream expression primer FER-R: 5′-CGCCATATGTCGGACTCAGAAGTCAATCA-3′, PCR The target gene of Fer147 was amplified, and the PCR product was recovered and purified by gel recovery kit, and after confirmation by sequencing comparison, the target gene of Fer147 was obtained. (Fer147 is a new protein—Fer147, which can interact with ferritin of D. pachyrhiza, which was screened out by the research group using the yeast two-hybrid system. Through sequence analysis, it was found that Fer147 is a member of the ferritin family, and its full-length DNA sequence contains 916 bases, composed of 174 amino acids. The gene sequence of Fer147 has not been...

specific Embodiment 2

[0055] enrichment of heavy metals

[0056] 1. Based on the pre-experimental results, after determining the concentration range of the metal solution for ferritin enrichment of heavy metal ions, 5 mM metal solution was selected as the enrichment concentration. Put the purified ferritin without the SUMO enzyme tag into the dialysis bag, in 5 mM sodium arsenite (NaAsO 2 ) solution, and a magnetic stirrer was used to simulate flowing liquid during the whole enrichment process. After 12 h of dialysis, the dialysis bag was dialyzed in protein buffer (25 mM Tris, 150 mM NaCl, pH 8.0) for 12 h, during which The protein buffer was changed every 4 h to remove heavy metal ions not adsorbed by the recombinant protein. The protein solution in the dialysis bag was taken out and placed in an ultrafiltration tube. According to the requirements of subsequent experiments, the protein was concentrated and stored at -80 °C for future use.

[0057] 2. Determination of Fer147 enriched arsenic con...

specific Embodiment 3

[0067] Crystal preparation and resolution

[0068] 1. Preparation of protein samples

[0069] (1) Thaw frozen ferritin samples on ice from a -80°C refrigerator (centrifuge directly for fresh samples), centrifuge the thawed protein samples in a high-speed refrigerated centrifuge at 4°C, 12,000 rpm for 15 min, and remove Denatured protein precipitates and impurities, and ensure the uniformity of the sample. Transfer the supernatant solution after centrifugation to a new pre-cooled centrifuge tube. If there are many foams in the solution during the transfer process, centrifuge for 5 minutes to remove the foam. ;

[0070] (2) Determination of protein concentration: Use the BCA protein concentration determination kit to measure the protein concentration, determine the concentration of the remaining protein after centrifugation, and provide a reference for the subsequent determination of the optimal protein crystallization concentration.

[0071] 2. Primary screening of crystalliz...

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Abstract

The invention discloses a preparation method and application for an invertebrate recombinant ferritin based anti-tumor nano arsenic sphere. The preparation method is characterized in that the method includes the following steps: (1) obtaining invertebrate recombinant ferritin successively through gene amplification, the construction and prokaryotic expression of prokaryotic expression vectors andprotein purification; and (2) placing the invertebrate recombinant ferritin into a dialysis bag, performing dialysis in a 5 mM sodium arsenite solution, using a magnetic stirrer to simulate flowing liquid during a whole enrichment process, placing the dialysis bag into a protein buffer solution to perform dialysis for 12 h after 12 h of the dialysis, replacing the protein buffer solution every 4 h, taking the protein solution in the dialysis bag, and obtaining invertebrate recombinant ferritin based anti-tumor nano arsenic spheres after concentration. The preparation method has significant inhibition effects on the propagation of chronic granulocyte white blood cell K562 cells.

Description

technical field [0001] The invention relates to a method for preparing heavy metal nanospheres by using recombinant ferritin, in particular to a preparation method and application of anti-tumor nanoarsenic balls based on invertebrate recombinant ferritin. Background technique [0002] Star worms, nematodes, mud clams, razor clams, sea cucumbers and sea cucumbers are representatives of economic invertebrates. They are typical benthic organisms and can tolerate high concentrations of heavy metals in the environment. This characteristic is inseparable from the enrichment function of ferritin in their bodies for heavy metals. Therefore, studying the biological functions of heavy metal enrichment in these invertebrates opens up possibilities for applications in fields such as biomedicine and materials chemistry, as well as bionanotechnology. In recent years, ferritin has been used for targeted drug delivery. [0003] Chronic myeloid leukemia is a malignant tumor originating fro...

Claims

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Application Information

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IPC IPC(8): A61K33/36A61K9/16A61K47/42A61P35/02C07K14/435C12N15/70C12R1/19
CPCA61K9/1658A61K33/36A61P35/02C07K14/43504C12N15/70
Inventor 明庭红苏秀榕吴燕蒋琴琴郇恒尚周君芦晨阳
Owner NINGBO UNIV
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