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A self-assembling polypeptide, its preparation method, its nucleic acid complex and its application

A self-assembly and complex technology, applied in the field of cell biology, can solve problems such as low transfection efficiency and nucleic acid instability

Active Publication Date: 2019-07-19
天津天诚新药评价有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Therefore, the purpose of the invention of the present invention is to provide a kind of nucleic acid transfection carrier reagent for the low transfection efficiency of current transfection reagents and the unstable nucleic acid of transfection, thereby improving the stability of nucleic acid and making transfection in serum can also be performed in the presence of

Method used

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  • A self-assembling polypeptide, its preparation method, its nucleic acid complex and its application
  • A self-assembling polypeptide, its preparation method, its nucleic acid complex and its application
  • A self-assembling polypeptide, its preparation method, its nucleic acid complex and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Example 1 Preparation of self-assembling polypeptide 1 (SEQ ID NO.2)

[0104] During the preparation of self-assembling polypeptide 1, the reagents used are commercially available as follows:

[0105]

[0106]

[0107] The synthesis of the self-assembled peptide 1 was carried out using a CEM liberitity peptide synthesizer according to the instructions of the manufacturer of the instrument.

[0108] 1) Self-assembly of chloromethyl polystyrene resin as an insoluble solid phase.

[0109] Selected resin particle size: 100~200mesh, loading capacity: 0.10~0.34

[0110] 2) Covalently link amino acids whose amino groups are protected by blocking groups (G) to solid-phase self-assembly.

[0111] The selected amino acids are: Fmoc-Tyr(tBu)-OH, Fmoc-Ile-OH Fmoc-leu(tBu)-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Arg(pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp-OH, Fmoc-Cys(Trt)-OH, Fmoc- Gln(Trt)-OH, Fmoc-Asn(trt)-OH, Fmoc-Trp(tBu)-OH, ...

Embodiment 2

[0117] Example 2 Synthesis of Self-Assembling Polypeptide 2 (SEQ ID NO: 3)

[0118] The synthetic method of self-assembling polypeptide 2 is the same as that of self-assembling polypeptide 1 (SEQ ID NO) in Example 1 ： 2) The synthetic method is the same, except that each amino acid connected is:

[0119] The first amino acid: Ala

[0120] The second amino acid: Lys

[0121] The third amino acid: Arg

[0122] The fourth amino acid: Leu

[0123] Fifth Amino Acid: Arg

[0124] The sixth amino acid: Lys

[0125] The seventh amino acid: Arg

[0126] The eighth amino acid: Trp

[0127] The ninth amino acid: Trp

[0128] The tenth amino acid: Leu

[0129] The eleventh amino acid: Ser

[0130] Twelfth amino acid: Lys

[0131] Thirteenth amino acid: Trp

[0132] Fourteenth amino acid: Trp

[0133] The fifteenth amino acid: Leu

[0134] The sixteenth amino acid: Lys

[0135] Seventeenth amino acid: Trp

[0136] Eighteenth amino acid: Trp

[0137] The nineteenth amino ...

Embodiment 3

[0143] Example 3 BOC synthesis of self-assembling polypeptide 3 (SEQ ID NO: 4)

[0144] During the preparation of self-assembling polypeptide 3, the reagents used are commercially available as follows:

[0145]

[0146] The synthesis of peptide 3 was carried out by Activo-P14 semi-automatic peptide synthesizer, and the synthesis steps were carried out according to the instructions of the instrument manufacturer.

[0147] The protected amino acids required for the peptide synthesis process are: Arg(Mts), Arg(NO2), Arg(Tos), Asp(OBzl), Asp(OcHx), Cys(pMeBzl), Cys(Acm), Cys(pMeOBzl), Glu(OBzl), Glu(OcHx), His(Bom), His(Dnp), His(Tos), His(Z), Lys(2-Cl-Z), Ser(Bzl), Thr(Bzl), Trp (For) and Tyr (2-Br-Z).

[0148] Experiment specific steps:

[0149] 1) Resin selection: PAM resin, use as little N,N-dimethylformamide or dichloromethane as possible to swell the resin

[0150] 2) Removal of N-terminal BOC: Before HF cleavage, the N-terminal Boc protecting group must be removed ...

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Abstract

The present invention provides a self-assembled polypeptide for transfecting nucleic acid into cells, a preparation method of the self-assembled polypeptide, a self-assembled polypeptide and nucleic acid complex, and a method for transfecting a target nucleic acid into cells. According to the present invention, the self-assembled polypeptide having special physical and chemical properties and DNA or RNA molecules form the complex so as to effectively improve the stability of the nucleic acid; with the carrier, cell transfection can be performed in the presence of serum, and the transfection time can be prolonged to several days; and when the self-assembled polypeptide is used to carry out cell transfection, the deliberate serum removing is not required, such that the toxicity of the reagent on cells during the transfection is substantially reduced.

Description

technical field [0001] The invention relates to the field of cell biology, in particular to a reagent used in basic research in the field of cell biology. Specifically, the present invention relates to a self-assembling polypeptide, its preparation method, its nucleic acid complex and its use. Background technique [0002] The process of inserting DNA or RNA fragments into somatic cells or cell lines is cell transfection technology. Cell transfection technology is divided into chemical method and physical method. The physical method is electroporation, which can be used under certain conditions, but it is easy to cause high cytotoxicity. The chemical method is developing rapidly because of its simple operation and low price. [0003] Chemical methods usually include liposome transfection (currently the most widely used transfection method, in many literatures, if no chemical method is specifically indicated, it refers to liposome method), calcium phosphate method, and DEAE-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C07K1/08C07K1/04C12N15/87
Inventor 王玉丽郑学敏龚珉徐为人汤立达
Owner 天津天诚新药评价有限公司