Polypeptide, polypeptide-siRNA (ribonucleic acid) induction coassembly and application thereof

A co-assembly and multi-purpose technology, applied in the field of bioengineering, can solve the problems of high toxicity, cumbersome preparation and purification, and poor effect, and achieve the effect of simple synthesis, cumbersome preparation and purification, and increased positive charge

Active Publication Date: 2018-07-06
PEKING UNIV SHENZHEN GRADUATE SCHOOL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] Aiming at the above-mentioned technical problems existing in siRNA carriers in the prior art, the present invention provides a polypeptide, a polypeptide-siRNA inducible co-assembly and its use, and the polypeptide, polypeptide-siRNA inducible co-assembly and its use To solve the technical problems of cumbersome preparation and purification of siRNA carriers in the prior art, high toxicity and poor effect

Method used

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  • Polypeptide, polypeptide-siRNA (ribonucleic acid) induction coassembly and application thereof
  • Polypeptide, polypeptide-siRNA (ribonucleic acid) induction coassembly and application thereof
  • Polypeptide, polypeptide-siRNA (ribonucleic acid) induction coassembly and application thereof

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Embodiment 1

[0039] Preparation and separation and purification steps of the polypeptide of embodiment 1:

[0040] According to the standard -Fmoc solid-phase peptide synthesis technology, MBHA resin is used to synthesize the peptide on the resin (taking the peptide Wpc as an example):

[0041]

[0042] The specific operation steps are:

[0043] (1) Polypeptide solid-phase synthesis: Weigh Rink amide MBHA resin into a peptide tube, add N,N-dimethylformamide (DMF), and swell with nitrogen gas for 40 minutes. Add 50% (v / v) morpholine in N,N-dimethylformamide (DMF) solution, blow nitrogen gas for 40 min, and remove the Fmoc protecting group. Wash the resin 3 times with DMF and DCM alternately, each time for 1min, mix the prepared Fmoc-AA-OH (5eq, 0.4M, DMF) solution, 6-chlorobenzotriazole-1,1,3,3- Tetramethyluronium hexafluorophosphate (HCTU) (4.9eq, 0.38M, DMF) solution and N,N-diisopropylethylamine (DIPEA) (7.0eq) were mixed and added to the resin and blown with nitrogen for 2h. The r...

Embodiment 2

[0047] Example 2 Polypeptides form co-assembled polypeptide-siRNA nanoparticles under the induction of siRNA (or other nucleic acid molecules)

[0048] Agarose gel retardation electrophoresis was used to determine the loading degree of peptides on siRNA. The amount of siRNA was 0.4 μg. The aqueous solution of peptides and siRNA were incubated at room temperature for 5 minutes to form stable peptide-siRNA co-assembled nanoparticles, the final 10 μg The polypeptide can be fully loaded with 0.4μg siRNA. The concentration of polypeptide was determined from the absorbance of Trp at 280 nm. ( figure 1 )

[0049] The co-assembled nanoparticles formed by the peptide and siRNA were characterized by atomic force microscopy (AFM), which proved that the peptide-siRNA formed regular and uniform nanoparticles. ( figure 2 )

[0050] The co-assembled nanoparticles formed by the peptide and siRNA were characterized by scanning electron microscopy (SEM), which proved that the peptide-siRN...

Embodiment 3

[0053] Example 3 Polypeptide-siRNA co-assembled nanoparticles can be used as a simple and low-toxic transfection reagent to effectively transfect siRNA into various cell lines and trigger RNAi effects

[0054] In order to verify whether the polypeptide-siRNA co-assembled nanoparticles can be used as siRNA carriers to carry siRNA, the most intuitive way is to use flow cytometry and laser confocal microscopy imaging to measure and observe the fluorescence of transfected cells in real time. Taking HeLa cells as a model and using flow cytometry, the polypeptide and fluorescently labeled (FAM) siRNA were incubated at room temperature for 5 minutes according to the ratio determined by agarose gel electrophoresis, and then added to HeLa cells pre-incubated with OPTi medium. After continuing to culture in the incubator for 4 hours, the cells were washed and digested, and the fluorescence intensity of the HeLa cells was detected by flow cytometry. It can be clearly seen that the fluore...

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Abstract

The invention provides polypeptide. The structural formula of the polypeptide is as shown below (the structural formula is shown in the description). The invention further provides a polypeptide-siRNAinduction coassembly which is composed of polypeptide and siRNA. The invention further provides application of the polypeptide-siRNA induction coassembly to cell carrying. The invention further provides application of the polypeptide-siRNA induction coassembly to preparation of drugs for treating cervical cancer. After polypeptide and target siRNA are mixed and hatched for 5 minutes at the room temperature, stable and uniform nano particles can be formed. A cytotoxicity test proves that the coassembly nano particles have high biocompatibility, and the cytotoxicity and hemolysis toxicity of the coassembly nano particles are both low. A cell proliferation resistant test further verifies that the polypeptide-siRNA nano particles can effectively block a cervical cancer cell line HeLa in the G2 stage, so that growth of the cervical cancer line HeLa is hindered.

Description

technical field [0001] The invention belongs to the field of bioengineering and relates to a polypeptide, specifically a polypeptide, a polypeptide-siRNA inducible co-assembly and its application. Background technique [0002] RNAi technology is one of the most widely used biological technologies, due to the highly specific and efficient expression regulation of specific gene mRNA by RNAi technology. Therefore, RNAi technology is widely used in many fields such as functional genomics, genetics, gene therapy, and viral disease treatment. However, siRNA is easily degraded by RNase, which exists widely, both in vivo and in vitro, and siRNA cannot pass through the cell membrane alone to trigger RNAi effects. Therefore, RNAi technology is inseparable from the help of highly efficient and low-toxic siRNA carriers. [0003] Commonly used siRNA transfection vectors include viral vectors, liposomes, polymers, inorganic nanoparticles, and polypeptide biological vectors. Viral vector...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C12N15/87A61K31/7088A61K47/62A61K47/69A61P35/00
CPCA61K31/7088C07K7/06C12N15/87
Inventor 李子刚尹丰李文君王冬园
Owner PEKING UNIV SHENZHEN GRADUATE SCHOOL
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