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Solution for preserving magnetosome and application thereof

A magnetosome, solution technology, applied in ferrous oxides, nanotechnology for materials and surface science, nanotechnology, etc., can solve the problems of separation and cultivation of magnetotactic bacteria, etc.

Active Publication Date: 2014-04-30
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, it is difficult to isolate and cultivate magnetotactic bacteria that synthesize and secrete BMPs, and only a few domestic laboratories can successfully do so.

Method used

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  • Solution for preserving magnetosome and application thereof
  • Solution for preserving magnetosome and application thereof
  • Solution for preserving magnetosome and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Preparation and preservation methods of two solutions for preserving magnetosomes

[0030] A 150 mM NaCl solution at pH 7.00 and a 0.01 M phosphate buffer saline (PBS) solution at pH 7.26 were prepared.

[0031] The preparation method of 150mM NaCl solution with a pH value of 7.00: Weigh 0.8775g of NaCl, dissolve it in 80ml of double distilled water, adjust the pH value to 7.00, and finally add double distilled water to make up to 100ml.

[0032] The preparation method of 0.01M phosphate buffer with a pH value of 7.26: Weigh 7.9g NaCl, 0.2g KCl, 0.24g KH 2 PO4 (or 1.44g Na 2 HPO 4 ) and 1.8g K 2 HPO 4 , dissolved in 800ml of distilled water, adjusted the pH value of the solution to 7.26 with HCl, and finally added distilled water to distill the volume to 1L.

[0033] The solution prepared above was autoclaved, 1ml was aliquoted, and stored at 4°C until use.

[0034] Weigh 500 μg of purified magnetosomes (BMPs) (BMPs are prepared with reference to the con...

Embodiment 2

[0035] Example 2 Connection and incubation of BMPs-PEI / DNA ternary gene transfer complex

[0036] (1) Adjust the concentrations of BMPs (magnetosomes), PEI (polyethyleneimine), and pDNA (pEGFP-N1, purchased from Addgene, USA) to PEI: 2 μg / μl, BMPs: 1 μg / μl, and pDNA: 1μg / μl, OD260 / OD230≥1.80.

[0037] (2) Dilute 0.8 μg of pDNA and 1.6 μg of PEI into 48.7 μl of 0.01M PBS (pH 7.26) prepared in Example 1, and gently vortex to mix. Then slowly add the PEI diluent to the DNA diluent drop by drop (the order of addition cannot be changed), mix gently and then incubate at room temperature for 10 minutes to allow PEI and DNA to self-assemble to form a PEI / DNA binary nanocomposite.

[0038] (3) Take 1 μl of the BMPs solution with a concentration of 1 μg / μl stored in the 150mM NaCl and 0.01M PBS solutions in Example 1, and add to the 99 μl PEI / DNA complex that has been connected and assembled in step (2) after ultrasonic mixing Mix again in an ultrasonic water bath, and incubate at roo...

Embodiment 3

[0039] Example 3 Comparison of application effects of magnetosomes preserved in two solutions in transfected cells

[0040] Inoculate Hela, Hep-G2, Cos-7 and C2C12 cells (the above cells were purchased from Peking Union Medical College Cell Resource Center) 12 hours in advance in 24-well plates at 37°C, 5% CO 2 , cultured in a 100% humidity cell incubator. Cell seeding density for Hela: 3×10 5 / hole, Hep-G2: 4×10 5 / hole; Cos-7: 4×10 5 / hole and C2C12: 2×10 5 / hole.

[0041] After the BMPs-PEI / DNA ternary gene transfer complexes of the two groups of A and B in Example 2 are connected, the old culture medium of the cells whose fusion degree has reached nearly 80% after being cultivated for 12 hours is completely aspirated, and then used 0.01 M's PBS (pH 7.26) washed the cells three times, and added 300 μl of serum-free DMEM medium (purchased from GIBCO, USA, Cat. No. C11995) to each well. Add 100 μl of the BMPs-PEI / DNA ternary gene transfer complex connected in Example 2...

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Abstract

The invention provides a solution for preserving magnetosome and an application thereof and belongs to the application field of biomaterials. The solution for preserving magnetosome is a phosphatic buffer solution (PBS). During the process of preserving magnetosome, the structure and functions of magnetosome are the least influenced and interfered by the solution. A BMPs-PEI / DNA composite obtained by connection of preserved magnetosome can reach higher PEI, nucleic acid fragments immobilization and ligation efficiency. By the adoption of the solution, cell transfection ratios of Hela, Hep-G2, Cos-7 and C2C12 are respectively higher than cell transfection ratio of magnetosome preserved by a routine method. The BMPs preserving solution provided by the invention is of great practical significance for more efficiently preserving BMPs for longer and more widely performing its application value.

Description

technical field [0001] The invention relates to the application field of biological materials, in particular to a solution for preserving magnetosomes and its application. Background technique [0002] Magnetosomes (BMPs, Bacterial Magnetic Particles) are novel superparamagnetic nanoparticles synthesized and secreted by magnetotactic bacteria, mainly composed of Fe 3 o 4 Constituted and surrounded by a lipid membrane that resembles a cell membrane. As a real biomineralization synthetic material, since its discovery, BMPs are being favored by more and more researchers due to their great potential in gene therapy. BMPs are pure in composition, unique in shape and uniform in size. The gene transfer complex formed by linking nucleic acids with polymers can not only perform cell transfection in vitro to improve transfection efficiency and reduce transfection toxicity, but also deliver drugs in vivo. Or nucleic acid for cancer treatment or gene therapy. At present, studies have...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C01G49/08B82Y30/00
Inventor 方美英康晓龙杨万杰
Owner CHINA AGRI UNIV
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