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Preparation of fibrin magnetic micrometer material for separating mRNA and application thereof

A cellulose and magnetic technology, applied in magnetic materials, DNA preparation, magnetic objects, etc., can solve the problems of easy leakage of antibodies, strong non-specificity, and reduced separation effect of target objects, so as to ensure biological activity, simplify activation steps, The effect of improving work efficiency

Inactive Publication Date: 2010-02-03
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The physical method has low binding efficiency, strong non-specificity, and easy leakage of antibodies
At present, there have been reports on magnetic microbeads used for mRNA separation. Surface activation groups include streptavidin (SA) and carboxyl groups, etc., but their activation methods all use chemical coupling. The introduction of this organic chemical reagent, The risk of the process is increased, and the chemical reagents will reduce or inactivate the activity of the coupled protein; at the same time, chemical direct coupling will inevitably lead to the length and density of the active group arm, so that even if the protein activity is not affected , due to the existence of its steric hindrance, it also reduces the separation effect of the target

Method used

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  • Preparation of fibrin magnetic micrometer material for separating mRNA and application thereof
  • Preparation of fibrin magnetic micrometer material for separating mRNA and application thereof
  • Preparation of fibrin magnetic micrometer material for separating mRNA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1, Preparation of CBD-SA bifunctional fusion protein

[0032] 1. Extraction of Genomic DNA from Streptomyces lilacinus ZG0429

[0033] Treat Streptomyces lilacinus ZG0429 suspended in 100 μL lysate (50mmol / LTris, pH8, 25mmol / LEDTA, 3% SDS (m / v), 1.2% PVP (m / v)) in a 600W microwave oven by microwave method , time 2min, add 400μL 65℃ preheated extract solution (10mmol / L Tris, pH8, 1mmol / LEDTA, 0.3mol / LNaAc, 1.2%PVP(m / v)), incubate at 65℃ for 20min, then add etc. Volume of phenol: chloroform: isoamyl alcohol (25:24:1) and chloroform: isoamyl alcohol (24:1) for extraction to remove protein, and finally add 2 times the volume of -20 ° C pre-cooled absolute ethanol, in After standing at -20°C for 1 hour, the DNA was collected by centrifugation at 12,000 rpm, dissolved in 50 μL of sterilized water and stored at -20°C for later use.

[0034] 2. PCR amplification of streptavidin (SA) gene

[0035] According to the SA gene sequence (sequence number X03591) of Streptomy...

Embodiment 2

[0044] Embodiment 2, the preparation of cellulose magnetic micron material

[0045] Take 2g of magnetic fluid and add it to 20mL of cellulose yellow gum solution. After fully stirring, use a homogenizer to homogenize for 2min under the conditions of 24000, 19000, 16000, 12000, 6000 and 3000rpm, and then add 100mL of organic phase (chlorobenzene and Carbon tetrachloride was mixed according to the volume ratio of 9:2, containing 2% Span-80 (v / v)), and fully homogenized at the same rotation speed for 2 minutes. Then transferred to 400mL organic phase, mechanically stirred at 400rpm. Heat in a 50°C hot water bath, and raise the temperature to 80°C, and keep it warm for 2.5 hours. Discard the organic phase, rinse with absolute ethanol 8 times, then alternately rinse with absolute ethanol and water, and finally fully rinse with water to obtain average particle sizes of MS1: 3.46, MS2: 5.82, MS3: 9.93, MS4 : 14.03, MS5: 38.04 and MS6: 61.86μm cellulose magnetic micro-materials, whe...

Embodiment 3

[0046] Example 3, Protein coupling efficiency of cellulose magnetic micron materials with different particle sizes

[0047] Take 100 μL each of the cellulose magnetic micro-materials with six particle sizes prepared in Example 2, add them to the fermentation lysate containing CBD-EGFP, and shake at room temperature. Measure the fluorescence emission intensity of the original fermentation lysate with a fluorescence detector at 30min, measure the fluorescence emission intensity of the fermented lysate after the adsorption of the above-mentioned cellulose magnetic micron material at the same time for 30min, calculate the fluorescence emission intensity consumed by the cellulose magnetic material, i.e. even connection efficiency. like Figure 4 As shown, the particle size of the cellulose magnetic material with the highest coupling efficiency is 5.82 μm, and it is determined that the optimum homogenate preparation speed is 19000 rpm.

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Abstract

The invention discloses preparation of fibrin magnetic micrometer material for separating mRNA and application thereof. The invention adopts a gene engineering technology to directionally clone the streptavidin and the gene of streptomyces lavendulae to pET series carrier which contains fibrin binding domain label, builds difunctional fusion protein CBD-SA carrier with biotin combination activityand fibrin combination activity, transfers E.coli BL21 competent bacterial strain, is expressed by means of IPTG low-temperature induction, and obtains high-content CBD-SA fused expression protein. CBD-SA is taken as biological bridging agent, the traditional physical adsorption method and a chemical bonding method are replaced by a biological specificity coupling method, so that the fibrin magnetic micrometer material is actived, agent with high-effeceicy combination biotinylation can be obtained after the activation, and magnetic micrometer material for separating protein or nucleic acid canbe obtained. The fibrin magnetic micrometer material can be used for separating and distilling the mRNA in biological samples, etc.

Description

【Technical field】: [0001] The invention relates to an activation method and application of a novel cellulose magnetic micron material. Specifically, the present invention relates to the preparation of bifunctional fusion protein CBD-SA, the bioactivation of cellulose magnetic micron materials, and the separation of mRNA in cell or tissue samples of cellulose magnetic materials after bioactivation, which belongs to the field of biotechnology. 【Background technique】: [0002] Magnetic microspheres are polymer microspheres with magnetic responsiveness. The cellulose magnetic micron material is a micron-level bioactive material with superparamagnetic properties prepared by using cellulose as the main material. Due to its superparamagnetism, low cost, excellent material and high biocompatibility, it has been widely used in the field of biomedicine. [0003] The activation of magnetic micro-materials is an important means to realize the biological characteristics of magnetic mic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): H01F1/00C12N15/70C12N15/10C12P21/02C12R1/19C07H21/02
Inventor 张奇白钢高智慧汤宇王龙白芳曹宇
Owner NANKAI UNIV
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