阅读说明：本技术 多巯基肽自组装及其基因载体应用 (Self-assembly of multi-sulfhydryl peptide and application of gene vector thereof ) 是由 曹美文 王瑜 丁榛 玄甲明 刘祯 张子瑾 赵琳杰 孙路 王生杰 夏永清 于 2020-06-08 设计创作，主要内容包括：本发明提出一种多巯基肽与DNA共组装及其在基因载体中应用的方法,属于生物材料领域,该载体具有较低的细胞毒性,可成功的诱导基因缩合,能够保护基因不被酶所降解,并且获得了不错的转染效率。该技术方案包括将多肽分子施于缓冲液中,再将所述多肽与基因分子溶液在预定条件下充分混合制备得到,即多肽/基因分子共组装体溶液。本发明能够应用于基因转运过程中。(The invention provides a method for co-assembling multi-sulfhydryl peptide and DNA and applying the multi-sulfhydryl peptide and the DNA in a gene carrier, which belongs to the field of biological materials. The technical scheme comprises the steps of applying polypeptide molecules into a buffer solution, and fully mixing the polypeptide and a gene molecule solution under a preset condition to prepare the polypeptide/gene molecule co-assembly solution. The invention can be applied to the gene transfer process.)

1. The self-assembly of multi-sulfhydryl peptide and the application of gene carrier thereof are characterized in that polypeptide molecules are dissolved in buffer solution, and then the polypeptide solution and the gene molecule solution are fully mixed under preset conditions, so that the polypeptide molecules and DNA molecules are co-assembled, and the co-assembly can be used for the application of gene carrier.

2. The polymercapto peptide and DNA co-assembly of claim 1, wherein the seven polypeptide molecules used have the molecular formula Nap-FF-GPLGLAG (CK)_n)_mC (abbreviated as (CK)_n)_mC，n＝2,3,5，m＝2,3,4)，Nap-FF-GPLGLAG(CR_n)_mC (abbreviated as (CR)_n)_mC,n＝2,3,5，m＝2,3,4)，Nap-FF-GPLGLAGCK₁₁C。

3. The polymercapto peptide of claim 2, wherein the step of dissolving the polypeptide molecule in a buffer comprises: prepared at a concentration of 50. mu.M-1.5 mM (CK)₂)₄C、(CK₃)₃C、(CK₅)₂C、CK₁₁C、(CR₂)₄C、(CR₃)₃C、(CR₅)₂And C, dissolving the polypeptide molecule solution in a Tris buffer solution with the pH value of 7.4, and standing for more than 3 days, wherein the polypeptide can be self-assembled into a specific nano structure.

4. The polymercapto peptide of claim 3, wherein the polypeptide molecules self-assemble into specific nanostructures, e.g., fibrous structures, short rod-like structures, beaded structures.

5. The genetic vector of claim i wherein the step of formulating the polymercapto peptide/DNA co-assembly solution comprises: a50. mu.M-1.5 mM polypeptide solution was prepared and dissolved in 50mM Tris buffer at pH 7.4.

6. The genetic vector of claim l, wherein the step of formulating the solution of polypeptide/gene molecule co-assembly comprises: preparing a pDNA solution with the concentration of 50 mug/mL, and dissolving the pDNA solution in a buffer solution with the pH of 7.4 to obtain a gene molecule solution; and fully mixing the polypeptide solution and the gene molecule solution according to preset conditions to obtain a polypeptide/gene molecule co-assembly solution.

7. The gene transfer vector according to claim 7, wherein the predetermined conditions are: the charge ratio of the polypeptide molecules to the gene molecules is 0-50.0.

8. Use of a gene delivery vector according to any one of claims 1 to 8 for gene transfection, comprising: and co-culturing the polypeptide/gene co-assembly solution and the cells, changing the culture solution into a DMEM medium containing 10% serum after culturing for 4-6 hours, and continuously culturing for 24-48 hours to realize effective transfection of genes in the cells.

9. The use according to claim 9, wherein said preferred cells are 293E cells.

Technical Field

The invention belongs to the field of biological materials, and particularly relates to a gene vector and a using method thereof.

Background

Gene therapy is a promising therapeutic strategy, mainly by introducing genes with specific functions into cells for the purpose of disease treatment. After 20 years of intensive research, it is rapidly becoming an important strategy for treating various genetic diseases, and plays a very critical role in treating some life-threatening diseases, such as cancer, cardiovascular diseases, acquired immunodeficiency syndrome, certain autoimmune diseases and the like.

Gene therapy is the use of nucleic acids to repair, replace or modulate genes to prevent or treat disease, and the therapeutic approach of gene therapy determines its ability to fundamentally address the occurrence of disease. In recent years, gene therapy techniques have been developed rapidly, and a great deal of research has been devoted to the application of gene transfection techniques to specific clinical applications, but the progress has been slow, mainly because safe and effective gene delivery vectors have not been developed.

There are many ways of gene therapy, but the process of introducing therapeutic genes into target cells is not well known. However, most of gene drugs are in linear extended conformation in solution, have poor stability in a human body and are easily degraded by various enzymes, and meanwhile, because the gene drugs have negative charges and can generate electrostatic repulsion with anions on the outer layer of a cell membrane, the characteristics also provide requirements for a gene carrier, the gene carrier is an efficient DNA agglomeration reagent, and the gene carrier can agglomerate DNA into particles with small volume, so that DNA molecules can easily enter cells through endocytosis or endocytosis.

Self-assembling peptides would be an excellent choice for the development of next generation biomaterials as well as future genetic drug delivery materials, which can produce a well-defined series of nanostructures. Compared with other delivery carriers, the self-assembly peptide nano structure has various advantages, such as high biocompatibility of peptide molecules, adjustable and controllable structure of the peptide molecules, strong designability and the like. More positive charges can be introduced into multiple molecules through reasonable design, so that the multiple molecules have strong DNA binding capacity. Cysteine (Cys) can be introduced, and due to the introduction of a redox-sensitive thiol group, peptides may form inter-peptide disulfide bonds capable of stabilizing a DNA carrier complex, thereby playing a role in improving a gene delivery effect. In addition, changes in the charge type, charge spacing, and hydrophobicity of the molecules can also have an effect on the transfection efficiency of the gene.

Disclosure of Invention

The invention provides a self-assembly of multi-sulfhydryl peptide and an application method of a gene vector thereof, belonging to the field of biological materials.

In order to achieve the above object, the present invention provides a gene vector, wherein a polypeptide molecule is dissolved in a buffer solution, and then the buffer solution and a gene molecule solution are sufficiently mixed under a predetermined condition, such that the polypeptide molecule and a DNA molecule are co-assembled, thereby preparing a polypeptide/gene molecule co-assembly solution.

Preferably, the buffer used in the above embodiment is Tris buffer with a concentration of 50mM and a pH of 7.4. The polypeptide is dissolved in the buffer solution, the positive charges carried by the polypeptide and the DNA with negative charges are utilized to carry out co-assembly, and Cys in the molecule plays a role in stabilizing an assembly structure, so that the DNA is condensed into particles with small volume, and the gene molecule is transported.

Preferably, the series of polypeptide molecules has the following sequence:

the following polypeptide molecules were designed: Nap-FF-GPLGLAG (CK)_n)_mC (abbreviated as (CK)_n)_mC,n＝2,3,5，m＝2,3,4)，Nap-FF-GPLGLAG(CR_n)_mC (abbreviated as (CR)_n)_mC,n＝2,3,5，m＝2,3,4)，Nap-FF-GPLGLAGCK₁₁C

Preferably, the seven polythiol peptides are composed of essentially three moieties, the first of which is a Nap-Phe-Phe (Nap-FF) self-assembly motif that facilitates peptide self-assembly in aqueous solution by providing pi-pi interactions and hydrophobic interactions. The second part is a-PLGLA-fragment which can be specifically cleaved by matrix metalloproteinases in order to obtain enzyme-responsive self-assembly behavior. The third part is a hydrophilic segment.

Preferably, the hydrophilic fragment of the seven peptides consists of: (CK)₂)₄C、(CK₃)₃C、(CK₅)₂C、CK₁₁The hydrophilic segment of the C-four peptide consists of lysine (Lys) and Cys, (CR)₂)₄C、(CR₃)₃C、(CR₅)₂The hydrophilic segment of the C-strip peptide consists of arginine (Arg) and Cys, wherein the Cys plays a role in stabilizing the structure of an assembly body, and the charge property, the number and the distribution of the molecule are adjusted by adjusting the number and the position of Lys (Arg) and Cys residues in the segment, so that the self-assembly behavior of the molecule and the co-assembly behavior with DNA are regulated.

Preferably, the step of self-assembling the polypeptide molecules into specific nanostructures in a buffer comprises:

preparing 50 μ M-1.5mM polypeptide molecule solution, dissolving in pH 7.4 buffer solution, and standing for more than 3 days.

Preferably, the polypeptide molecule can self-assemble into a specific structure, such as (CK)₂)₄C is assembled to form a short rod-shaped structure, the length of the short rod-shaped structure is about 40-60nm, and the diameter of the short rod-shaped structure is about 7.5 nm. (CK)₃)₃C forms a similar bead structure, and the diameter of the bead is about 7 nm. CK (CK)₁₁C, forming a long fiber-shaped structure after assembly, wherein the diameter of the long fiber-shaped structure is about 8 nm. (CR)₃)₃C、(CR₅)₂C an assembly of two polypeptides, both forming a short-fiber-like structure, (CR)₃)₃The length of the fiber formed by C is about 200-300nm, (CR)₅)₂The length of the fiber formed by C is mostly about 150nm, and the fiber diameter of the two peptides is about 9.5 nm. In addition (CK)₅)₂C、(CR₂)₄C does not form an ordered assembly structure.

As an advantage, the preparation steps of the polypeptide/gene molecule co-assembly solution comprise:

preparing DNA solution with the concentration of 50 mug/mL, and dissolving the DNA solution in Tris buffer solution with the pH value of 7.4 to obtain gene molecule solution.

And fully mixing the polypeptide solution and the gene molecule solution according to preset conditions to obtain a polypeptide/gene molecule co-assembly solution.

In the technical scheme, the gene molecule takes a green fluorescent protein reporter gene pEGFP-N2 as a model.

Preferably, the predetermined condition is: the charge ratio of the polypeptide molecule to the DNA is 20.

In the above technical scheme, when the charge ratio of the polypeptide molecule to the DNA molecule is above 5, effective condensation of the DNA molecule can be induced, and the DNA molecule is protected from enzyme digestion degradation: when the ratio of the polypeptide molecules to the DNA molecules is 5-20, the DNA molecules can be converted from an extended conformation to a highly compressed condensed conformation, and gene transfection can be effectively mediated.

The invention also provides a use method of the gene vector according to any one technical scheme, which comprises the following steps:

co-culturing the polypeptide/gene molecule co-assembly solution and cells, updating the culture solution into a DMEM culture medium containing 10% serum after 6-10 hours, and continuing to culture for 24-48 hours to realize effective transfection of genes in the cells.

In the technical scheme, the cells are inoculated in a 96-well plate, and the density of the inoculated cell suspension is 1 × 10⁵cells/mL, cultured in a cell incubator for 24 hours after seeding. The complete medium was removed from each well and renewed with 100. mu.L of the medium, after which 50. mu.L of the peptide/DNA mixed solution was injected into each well and after 4-6 hours of co-culture with the cells, the original medium was replaced with 150. mu.L of fresh medium containing fetal bovine serum and the culture was continued for 24-48 hours.

Preferably, the cell is a 293E cell.

In the above technical scheme, when the ratio of polypeptide molecules to DNA molecules is 20, pEGFP-N2 plasmid is transfected into 293E cellsOptimal effect, (CK)₂)₄C、(CK₃)₃C、(CK₅)₂C、CK₁₁C、(CR₂)₄C、(CR₃)₃C、(CR₅)₂The C seven peptides all achieve higher transfection efficiency, which is respectively 29.67%, 49.49%, 11.39%, 37.73%, 54.49%, 48.74% and 48.98%.

Preferably, the polypeptide molecule is not so toxic to the cell at the charge ratio that provides the best transfection efficiency of the gene, and the cell can still maintain a good morphology.

Compared with the prior art, the invention has the advantages and positive effects that

(1) The hydrophilic segments of the seven polypeptide molecules provided by the invention are all composed of Cys, the Cys plays a role in stabilizing the structure of an assembly body, and the charge property, the number and the distribution of the molecules are adjusted by adjusting the number and the positions of Lys (Arg) and Cys residues in the segments, so that the self-assembly behavior of the molecules and the co-assembly behavior of the molecules and DNA are regulated and controlled.

(2) The gene vector provided by the invention has simple synthesis process, the obtained vector has lower cytotoxicity, the vector can successfully induce DNA condensation, and can protect the DNA condensation from being degraded by enzyme, and good transfection efficiency is obtained.

Drawings

FIG. 1 shows a transmission electron microscope characterization of an aggregate structure of a polypeptide in a Tris buffer according to an embodiment of the present invention, where (a): (CK)₂)₄C，(b)：(CK₃)₃C，(c)：(CK₅)₂C，(d)：CK₁₁C，(e)：(CR₂)₄C，(f)：(CR₃)₃C，(g)：(CR₅)₂C。

FIG. 2 is a graph showing the relative fluorescence intensity as a function of charge ratio for EB-replacement experiments according to the present invention.

FIG. 3 is a gel electrophoresis of the polypeptide/DNA complex of the present invention with HindIII and EcoRI enzymes at different charge ratios, (a) (CK)₂)₄C，(b):(CK₃)₃C，(c):(CK₅)₂C，(d)CK₁₁C，(e):(CR₂)₄C，(f):(CR₂)₄C，(g):7-(CR₅)₂C, lane 1: pDNA, lanes 2-7: fixed DNA concentration, polypeptide/DNA complexes containing varying amounts of polypeptide

FIG. 4 shows the gene transfection efficiency of 293E cells transfected with the polypeptide/pEGFP-N2 characterized by flow cytometry as provided in the examples of the present invention.

Detailed Description

In order to more clearly and specifically describe the method for co-assembling the polymercapto peptide and the DNA and the application thereof in the gene vector, which are provided by the embodiments of the present invention, the following description will be given with reference to specific embodiments.