Targeted chitosan gene vector and its preparation and use

A technology of gene carrier and chitosan, applied in the direction of introducing foreign genetic material by using carrier, using micro-injection method, other methods of inserting foreign genetic material, etc., can solve the problem of low transfection efficiency and achieve high cell transfection efficiency , the result is easy, overcome the effect of low transfection efficiency

Inactive Publication Date: 2006-12-27
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The object of the present invention is to overcome the shortcoming of existing chitosan gene carrier transfection efficiency low, provide a kind of transfection

Method used

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  • Targeted chitosan gene vector and its preparation and use
  • Targeted chitosan gene vector and its preparation and use
  • Targeted chitosan gene vector and its preparation and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The synthetic method of embodiment 1 targeting molecule HA

[0035] Weigh 1.0 g of HA (Shandong Zhengda Freda Company, China) and dissolve it in newly prepared 100 ml of pH 6.5 phosphate buffer, stir it electromagnetically for 6-8 hours, and after forming a uniform and transparent hydrogel, add 31.25 mg Hyaluronidase (EC3.2.1.35type I-S, sigma company), stirred and reacted at 37° C. for 30 hours. The reacted solution is placed in boiling water and boiled for 5-10 minutes to terminate the reaction. After the solution was cooled to room temperature, it was filtered through a 0.45 micron filter membrane to remove hyaluronidase. The resulting filtrate was purified by dialysis in pure water with a dialysis membrane (cutoff molecular weight around 6000). The product was lyophilized to obtain the desired length of HA solid powder. The change of molecular weight of HA with enzyme digestion time is shown in the table below, and the results were determined by GPC.

[0036] ...

Embodiment 2

[0038] The synthetic method of embodiment 2 targeting chitosan gene carrier

[0039] The four chitosans are numbered 1, 2, 3, and 4 in order of molecular weight from small to large, and the results of their molecular weight and deacetylation degree are as follows:

[0040] Chitosan No.

[0041] The specific synthesis method is as follows: Accurately weigh 1.0 g of chitosan sample, add acetic acid and stir until completely dissolved, adjust the pH value of the solution to 4.5-5.5, filter the solution with a disposable syringe filter (0.22 micron in aperture) and set aside. Weigh 0.24 grams of HA, dissolve in 50 milliliters of 1.0 mol / liter NaCl solution, adjust the pH of the solution to 4.5-5.5, add 0.23 grams of EDC to the HA solution, stir and dissolve, adjust the pH value of the solution with HCl or NaOH solution, and make It is stable at pH 4.5-5.5, and then slowly add the pre-prepared chitosan solution to it, and stir and react at 37°C for 5-7 days; adjust the pH...

Embodiment 3

[0042] The preparation of embodiment 3 carrier and plasmid DNA complex nanoparticle

[0043] Dissolve the above-mentioned refined chitosan-grafted hyaluronic acid targeting gene carrier with 5 mmol / L pH5.5 acetic acid-sodium acetate buffer solution to prepare a solution of 0.1-10 mg / ml, and use a disposable needle-type Filter (0.22 microns) to filter the solution before use. Plasmid DNA (approximately 130 kb in size) was dissolved in 4.3 mmol / L sodium sulfate solution to a concentration of 100 μg / ml as determined by UV. Before compounding, preheat the carrier and DNA solution to 55°C respectively, then mix on a vortex mixer for 1 minute according to the volume ratio of carrier and plasmid DNA at 1:1, and let stand at room temperature for 0.5 to 1 hour, and the carrier and plasmid DNA pass through Electrostatic self-assembly into nanocomposite particles.

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Abstract

This invention provides a kind of preparation method of gene vector which made chitosan as matrix, and made haluronic acid as target molecule. The method is: at 37DEG C, use hydoscine N butyl bromide degrade natural haluronic acid to the desired length, after dislysis and purification then freezing and drying, then according to the specific quality proportion, at 37DEG C coupled with four kinds of chitosans with different molecular weight catalysed by the EDC, and formed different target chitosan gene vector with four kind of different molecular weight. Dissolve the obtained gene vector with 5 mmol/l pH 5.5 buffer solution of acid acetic and sodium acetate to get 0.1 - 10 mg/ml solution, after the 0.22 mm membrane filter, composite with the reporter gene with FP to form a series of composition with quality proportion of vector and plasmid DNA. Using it to transfect Insect cell lines Sf9, can achieve the same or better transfection efficiency with the commercial transfection agent liposome. The effect of using this vector transfect silkworm is excellent. The best transfection effeciency is over 50%. This vector is safe and innocuity, cheap and easy to use.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a non-viral gene carrier targeting cells containing hyaluronic acid receptors. The invention also relates to the preparation method and application of the non-viral gene carrier. Background technique [0002] Professor Tan Jiazhen, a famous geneticist, once pointed out with great foresight: "The medical revolution in the 21st century will depend on the success of gene therapy research." Cells are used to correct gene defects or play a therapeutic role, so as to achieve the purpose of biomedical treatment of diseases. It consists of three basic elements: target gene, gene delivery carrier and target cell. With the completion of a more accurate human genome map and the discovery of various new genes, the acquisition of target genes will have a more solid scientific basis; and the development of cell biology, molecular biology and genetics and...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/87C12N15/65C12N15/89C12N15/85
Inventor 张伟李静周芬付铜权袁建军程时远闫翠娥
Owner HUBEI UNIV
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