Novel polypeptide for assisting PEI transfection of cells and application of novel polypeptide

Abstract

The invention relates to the technical field of biology correlation, in particular to a novel polypeptide for assisting PEI transfection of cells and application of the novel polypeptide, the polypeptide is named as LN, and the amino acid sequence of the polypeptide is KKLLALALHLAGGPKKKRKV. The invention belongs to the technical field of in-vitro cell gene transfection, and particularly relates to a cell transfection delivery compound and a preparation method and application thereof. The cell transfection delivery compound is prepared from the following raw materials: polyethyleneimine, a target gene DNA (deoxyribonucleic acid) fragment and synthesized polypeptide, and the polypeptide can penetrate through cell membranes of various types of cells to enter the cells. According to the cell transfection transfer compound, by introducing the polypeptide, the cell uptake ability is enhanced, the cytotoxicity caused by PEI is effectively buffered, and the experimental repeatability and operation efficiency of transfection are improved by the cell transfection transfer compound.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a novel polypeptide that assists in PEI transfection of cells and its applications. Background Technology

[0002] Gene delivery is a key technology in molecular biology and gene therapy research, aiming to efficiently deliver exogenous nucleic acids into target cells for expression. Polyethyleneimine (PEI), a traditional non-viral vector, is considered one of the most representative cationic polymer vectors due to its high cation density, excellent DNA compression ability, and "proton sponge effect," which effectively promotes gene escape from endosomes. However, while high molecular weight PEIs exhibit high transfection efficiency, they also possess strong cytotoxicity; while low molecular weight PEIs, although showing better biocompatibility, suffer from insufficient nucleic acid compression and intracellular release capabilities, leading to a significant decrease in transfection efficiency. Therefore, developing a substance that can synergize with PEI is of significant theoretical and practical value for constructing efficient and low-toxicity non-viral gene delivery systems.

[0003] Cell-penetrating peptides (CPPs) with their strong transmembrane delivery capability and nuclear localization peptides (NLSs) with their nuclear localization function are considered potential solutions to the above problems. CPPs and NLSs can be covalently or non-covalently bound to nucleic acid molecules to carry them into cells. To date, various CPPs and NLSs have been discovered, including those from natural sources as well as those synthesized artificially, such as simple polylysine and polyarginine structures. However, existing CPP and NLS systems still have several shortcomings: (1) Most classic CPPs are directly derived from natural protein fragments, making it difficult to optimize their sequence or modify their function; (2) Low efficiency of endocytosis and endosome escape: Most CPPs enter cells via energy-based endocytosis, and are easily transported to endosomes / lysosomes and degraded, resulting in low delivery efficiency; (3) Simple CPPs or NLSs have limited functions. Although CPPs can penetrate cells, they lack nuclear localization function, while NLSs have nuclear localization function but cannot effectively penetrate cells. Summary of the Invention

[0004] The purpose of this invention is to provide a novel polypeptide that assists in PEI transfection of cells and its application, in order to solve the problems of limited transfection efficiency and high cytotoxicity of PEI in non-viral gene transfection systems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A polypeptide that can enhance the transfection efficiency of polyethyleneimine (PEI) and reduce toxicity, the polypeptide is named LN and has the amino acid sequence SEQ NO: 1: KKLLALALHLLAGGPKKKRKV.

[0007] A cell transfection delivery complex, the raw materials for which are prepared include PEI, a target gene DNA fragment, and an LN polypeptide, wherein the DNA is the target gene, and the amino acid sequence of the LN polypeptide is shown in SEQ NO: 1.

[0008] Preferably, in the raw materials for preparing the cell transfection and delivery complex, the mass ratio of PEI, target gene DNA fragment, and LN polypeptide is (2-3):1:(0.5-1).

[0009] More preferably, in the raw materials for preparing the above-mentioned cell transfection and delivery complex, the mass ratio of PEI, target gene DNA fragment, and LN polypeptide is 2.5:1:0.5.

[0010] A method for preparing a cell transfection delivery complex, comprising: mixing PEI, a target gene DNA fragment, and an LN polypeptide.

[0011] A mixing reaction is performed to obtain a cell transfection and delivery complex; wherein the mixing reaction method is selected from any of the following:

[0012] (a1) Mix the raw materials in the preparation raw materials together for a reaction;

[0013] (b1) First, mix and react two of the raw materials in the preparation raw materials, and then add a third raw material to mix and react.

[0014] Preferably, the above preparation method includes:

[0015] (1) PEI and LN peptides were mixed and reacted in buffer to obtain a binary complex reaction solution;

[0016] (2) The binary complex and the target gene DNA dilution solution are mixed and reacted to obtain the cell transfection and delivery complex.

[0017] Preferably, in the above preparation method,

[0018] (a2) The buffer is selected from one or more combinations of PBS solution, 0.9% NaCl aqueous solution, water, DPBS solution, Opti-MEM solution, DMEM and complete culture medium; wherein the complete culture medium contains 10% fetal bovine serum, 1% penicillin-dextrin antibody and the remainder DMEM; and / or

[0019] (b2) The buffer volume in steps (1) and (2) is 10 μL to 100 μL.

[0020] Preferably, the above preparation method satisfies one or more of the following conditions:

[0021] (a3) In step (1), the reaction includes: reacting at 20℃~30℃ for 15min~25min;

[0022] (b3) In step (2), the reaction includes: reacting at 20℃~30℃ for 5min~15min.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] 1. Significantly Improved Transfection Efficiency: This invention significantly enhances the cell membrane permeability and endocytosis capacity of the complex by introducing a novel LN polypeptide into the PEI / DNA system. Fluorescence microscopy and flow cytometry analysis showed that the proportion of fluorescent protein (mcherry) positive cells in the LN / PEI / DNA system was approximately 20-30% higher than the control group without LN, demonstrating good stability and versatility in various cell lines.

[0025] 2. Significantly Reduced Cytotoxicity: The introduction of LN peptide in this invention enhances cellular uptake while effectively buffering the positive charge stress effect caused by PEI, significantly reducing cytotoxicity. CCK8 assay showed that the cell inhibition rate within 24 hours post-transfection was reduced by approximately 25% compared to the traditional PEI / DNA complex system.

[0026] 3. Simple operation and stable system: The preparation and transfection process of the complex in this invention is simple, requiring no additional solution changes or washing steps. The system has high stability and good reproducibility, and can be directly used in cell experiments. Compared with traditional liposome or PEI systems, this method significantly simplifies the transfection process and saves operation time.

[0027] 4. Wide range of applications: The LN-enhanced PEI / DNA complex system constructed in this invention can be widely used for gene transfection in various mammalian cell types, especially for efficient transfection of adherent cells. This system has significant application prospects in fields such as gene engineering vector delivery, gene function research, cell model construction, and small molecule drug delivery. Attached Figure Description

[0028] Figure 1 Flow cytometry patterns and transfection efficiency statistics are used to compare the results of transfecting HEK293T cells with the preparation of Example 1 (PEI / DNA / LN polypeptide ternary complex) and Comparative Example 1 (PEI / DNA binary complex).

[0029] Figure 2Flow cytometry and statistical graphs showing the effect of different component mixing order (PEI, DNA, LN peptide) on transfection efficiency in Preparation Example 2.

[0030] Figure 3 Flow cytometry and statistical graphs of different PEI and LN peptide pre-incubation times (first step reaction) in Preparation Example 3.

[0031] Figure 4 Flow cytometry plots and statistical graphs of DNA ligation incubation time (second step reaction) for different PEI / LN binary complexes in preparation Example 3.

[0032] Figure 5 Flow cytometry and statistical plots were prepared for the complexes using different buffer systems in Example 4.

[0033] Figure 6 Flow cytometry and statistical graphs were prepared for the complexes prepared using different volume buffer systems in Example 5.

[0034] Figure 7 Flow cytometry and statistical graphs of HEK 293T cells transfected with different PEI dosages when the LN addition amount was 0 in Preparation Example 6.

[0035] Figure 8 Flow cytometry plots and statistical graphs of HEK 293T cells transfected with different PEI dosages when the LN addition amount was 0.5 μg in Preparation Example 6.

[0036] Figure 9 Flow cytometry and statistical graphs of HEK 293T cells transfected with different PEI dosages when the LN addition amount was 1 μg in Preparation Example 6.

[0037] Figure 10 Flow cytometry plots and statistical graphs of HEK 293T cells transfected with different PEI dosages when the LN addition amount was 1.5 μg in Preparation Example 6.

[0038] Figure 11 Flow cytometry and statistical graphs of HEK 293T cells transfected with different PEI dosages when the LN addition amount was 2 μg in Preparation Example 6.

[0039] Figure 12 The flow cytometry results and statistical graphs of HEK 293T cells transfected with a fixed PEI dosage (2.5 μg) and varying LN dosage in Example 6 are shown.

[0040] Figure 13 Flow cytometry and statistical graphs showing the effect of different media exchange times (0–48 h) on transfection efficiency in Preparation Example 7.

[0041] Figure 14 Preparation and application example 1: Transfection complex transfected HeLa cells and HEK 293T cells followed by detection of CCK8 cell inhibition rate.

[0042] Figure 15 Flow cytometry and statistical graphs of different sized plasmids transfected in HEK 293T cells in Application Example 2.

[0043] Figure 16 Flow cytometry and statistical graphs of multiple plasmids (such as eGFP, mCherry, and BFP) co-transfected in HEK 293T cells in Application Example 3.

[0044] Figure 17 The fluorescence intensity changes in HeLa and HEK 293T cells after adding the LN-enhanced complex in Application Example 4 include dynamic curves of total fluorescence intensity and average fluorescence intensity.

[0045] Figure 18 Preparation and application example 5: Cell confluence after transfection of HeLa cells and HEK 293T cells with the transfection complex. Detailed Implementation

[0046] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0048] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0049] In a first aspect, embodiments of the present invention provide a cell transfection delivery complex, the raw materials for which include PEI, a target gene DNA fragment, and an LN polypeptide, wherein the DNA is the target gene, and the amino acid sequence of the LN polypeptide is shown in SEQ NO: 1.

[0050] It should be noted that the introduction of LN peptide in this invention significantly reduces cytotoxicity. CCK8 assays showed that, under the same transfection conditions, cells treated with the method of this invention exhibited significantly lower cytotoxicity than those treated with the traditional PEI / DNA binary transfection complex, with a relative reduction in cell inhibition rate of approximately 25%. This indicates that the introduction of LN peptide in this invention effectively buffers PEI-induced cytotoxicity while enhancing cellular uptake.

[0051] It should also be noted that the amino acid sequence of the LN polypeptide is shown in SEQ NO: 1, and the specific sequence is: KKLLALALHLLAGGPKKKRKV.

[0052] In some specific examples, the mass ratio of PEI, target gene DNA fragment, and LN polypeptide in the raw materials for preparing the above-mentioned cell transfection and delivery complex is (2-3):1:(0-1).

[0053] In some specific examples, the mass ratio of PEI, target gene DNA fragment, and LN polypeptide in the raw materials for preparing the above-mentioned cell transfection and delivery complex is 2.5:1:0.5.

[0054] It should be noted that the cell transfection delivery complex prepared with different mass ratios of PEI, target gene DNA fragment, and LN polypeptide has a certain impact on the transfection efficiency. In this invention, the preferred ratio is (2-3):1:(0-1), and the more preferred ratio is 2.5:1:0.5. The cell transfection delivery complex prepared with the preferred mass ratio has a higher transfection efficiency.

[0055] Secondly, embodiments of the present invention provide a method for preparing the cell transfection and delivery complex of the present invention, the method comprising: mixing PEI, a target gene DNA fragment, and an LN polypeptide to obtain the cell transfection and delivery complex; wherein the mixing reaction method is selected from any of the following:

[0056] (a1) Mix the raw materials in the preparation raw materials together for a reaction;

[0057] (b1) First, mix and react two of the raw materials in the preparation raw materials, and then add a third raw material to mix and react.

[0058] Preferably, the above preparation method includes:

[0059] (1) PEI and LN peptides were mixed and reacted in buffer to obtain a binary complex reaction solution;

[0060] (2) The binary complex and the target gene DNA dilution solution are mixed and reacted to obtain the cell transfection and delivery complex.

[0061] Preferably, in the above preparation method,

[0062] (a2) The buffer is selected from one or more combinations of PBS solution, 0.9% NaCl aqueous solution, water, DPBS solution, Opti-MEM solution, DMEM and complete culture medium; wherein the complete culture medium contains 10% fetal bovine serum, 1% penicillin antibody and the remainder DMEM;

[0063] (b2) The buffer volume in steps (1) and (2) is 10 μL to 100 μL.

[0064] Preferably, the above preparation method satisfies one or more of the following conditions:

[0065] (a3) In step (1), the reaction includes: reacting at 20℃~30℃ for 15min~25min;

[0066] (b3) In step (2), the reaction includes: reacting at 20℃~30℃ for 5min~15min.

[0067] In another aspect, the present invention provides a method for DNA transfection of cells, the method comprising: adding the above-mentioned cell transfection delivery complex or the cell transfection delivery complex prepared by the above preparation method to the culture medium of cells to be transfected for incubation.

[0068] (a1) Mix the raw materials in the preparation raw materials together for a reaction;

[0069] (b1) First, mix and react two of the raw materials in the preparation raw materials, and then add a third raw material to mix and react.

[0070] It should be noted that the preparation method of the cell transfection delivery complex in this invention is well known in the art, and it is preferred to prepare it according to the above preparation method.

[0071] In some specific examples, the above preparation method includes:

[0072] (1) PEI and LN peptides were mixed and reacted in buffer to obtain a binary complex reaction solution;

[0073] (2) The binary complex and the target gene DNA dilution solution are mixed and reacted to obtain the cell transfection and delivery complex.

[0074] It should be noted that, in this invention, the preferred method for preparing the cell transfection delivery complex is to mix PEI and LN peptides in a buffer solution according to the above preparation method to obtain a binary complex reaction solution, and then mix it with the target gene dilution solution to obtain the cell transfection delivery complex. The transfection efficiency of the cell transfection delivery complex prepared by this method is higher.

[0075] In some specific examples, in the above preparation method,

[0076] (a2) The buffer is selected from one or more combinations of PBS solution, 0.9% NaCl aqueous solution, water, DPBS solution, Opti-MEM solution, DMEM solution or DMEM culture medium; wherein the DMEM culture medium contains 10% fetal bovine serum, 1% penicillin antibody and the remainder is complete DMEM culture medium;

[0077] (b2) The buffer volume in steps (1) and (2) is 10 μL to 100 μL.

[0078] It should be noted that the buffer solution used in this invention is known in the art. The above-mentioned buffer solution is preferred, and DPBS is more preferred. The transfection efficiency of the cell transfection delivery complex prepared using DPBS as a buffer solution is higher than that of other buffer solutions. In addition, the total volume of the buffer solution in steps (1) and (2) can be 10 μL to 100 μL, for example, 10 μL, 20 μL, 30 μL, 40 μL or 100 μL, preferably 30 μL. The transfection efficiency of the cell transfection delivery complex prepared with this volume is higher.

[0079] In some specific examples, the above preparation method satisfies one or more of the following conditions:

[0080] (a3) In step (1), the reaction includes: reacting at 20℃~30℃ for 15min~25min;

[0081] (b3) In step (2), the reaction includes: reacting at 20℃~30℃ for 5min~15min.

[0082] Thirdly, embodiments of the present invention provide a method for DNA transfection of cells, the method comprising: adding the above-mentioned cell transfection delivery complex or the cell transfection delivery complex prepared by the above-mentioned preparation method to the culture medium of cells to be transfected for incubation.

[0083] It should be noted that the cell transfection delivery complex provided by this invention can be directly added to the cell culture system during co-incubation with cells without the need to change the culture medium or perform a washing step; after transfection, it can be directly incubated in the original culture system until subsequent detection or processing steps, compared to Lipofectamine. TM Compared with the existing PEI / DNA binary complex transfection system, the 2000 transfection reagent eliminates the experimental procedure of changing the medium shortly after transfection, reduces human interference, and improves experimental repeatability and operational efficiency.

[0084] In some specific examples, the above method satisfies one or more of the following conditions:

[0085] (a4) The incubation conditions include: incubation at 35℃-40℃ and 4%-6% carbon dioxide concentration for 40h-50h. Specifically, the incubation conditions for transfection in this invention are known in the art. Preferably, incubation is at 35℃-40℃ and 4%-6% carbon dioxide concentration for 40h-50h. More preferably, the incubation time is 48h. This incubation time can achieve higher transfection efficiency in a shorter time.

[0086] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0087] Preparation Example 1

[0088] (1) Take 1 μL of plasmid DNA with a concentration of 1 μg / μL (pcDNA 3.1 mcherry purchased from Fenghui Biotechnology Cat: FH1737) and dilute it into 50 μL of PBS solution (gibco product number 10010023, potassium dihydrogen phosphate (KH2PO4) 1 mM, disodium hydrogen phosphate heptahydrate (Na2HPO4 and 3 mM sodium chloride (NaCl) 155 mM, the same below) and gently vortex to obtain DNA dilution solution; take 2 μL of LN polypeptide with a concentration of 1 μg / μL and dilute it into 25 μL of PBS solution and gently vortex to obtain LN polypeptide dilution solution; add LN polypeptide dilution solution to DNA dilution solution and gently vortex to obtain LN polypeptide / DNA binary complex.

[0089] (2) Take 2 μL of PEI (linearized polyethyleneimine with a molecular weight of 40 kDa) with a concentration of 1 μg / μL and dilute it into 50 μL of PBS solution. Vortex the solution to obtain the PEI dilution. Add the PEI dilution to the LN polypeptide / DNA binary complex and vortex the solution gently. React at 25℃ for 10 min to obtain the PEI / DNA / LN polypeptide ternary complex.

[0090] Preparation Example 2

[0091] Preparation Example 2 is largely the same as Preparation Example 1, except that the order of adding DNA, LN and PEI in Preparation Example 2 is different from that in Preparation Example 1. Otherwise, they are the same as in Preparation Example 1, and different transfection complexes are prepared. The order of adding DNA, LN and PEI in Preparation Example 2 is shown in Table 1 below (where "+" represents the order of addition, " / " represents disordered mixing, and the interpretation of "+" and " / " is only applicable to the description of Preparation Example 2. For example, the order of addition in Preparation Example 1 can be labeled as: DNA / PEI+LN).

[0092] Preparation Example 3

[0093] Incubation time:

[0094] Preparation Example 3 is largely the same as Preparation Example 2 (component addition order: PEI / LN + DNA), except that the incubation time in Preparation Example 3 differs from that in Preparation Example 2, resulting in different transfection complexes. In Preparation Example 3, the incubation time refers to the time (in minutes) for mixing the two solutions. Preparation Example 3 involves two steps, with the incubation times set for each step according to the following conditions.

[0095] (1) Set the incubation time for step 2 to 10 min, and the time for connecting PEI / LN in step 1 is shown in Table 2.

[0096]

[0097] (2) Set the incubation time for step 1 to 10 min, and the DNA ligation time for step 2 as shown in Table 3.

[0098]

[0099] Preparation Example 4

[0100] Preparation Example 4 is largely the same as Preparation Example 3 (incubation time: 20+10, unit: min), except that different buffer solutions are used to replace the PBS solution in Preparation Example 3 in Preparation Example 4. Otherwise, the same as Preparation Example 3 is used to prepare different transfection complexes. The buffer solutions used in Preparation Example 4 are shown in Table 4 below.

[0101]

[0102] Preparation Example 5

[0103] Preparation Example 5 is largely the same as Preparation Example 4 (buffer is DPBS). The difference is that the volume of DPBS in steps (1) to (3) of Preparation Example 5 is shown in Table 5 below. The other steps are the same as in Preparation Example 4, and different transfection complexes are prepared.

[0104]

[0105] Preparation Example 6

[0106] Preparation Example 6 is largely the same as Preparation Example 5 (buffer volume is 30 μL), except that the amounts of DNA, LN, and PEI added in Preparation Example 6 are different from those in Preparation Example 5. Other aspects are the same as in Preparation Example 5, resulting in different transfection complexes. In Preparation Example 6, the amount of DNA used for fixation is 1 μg (concentration 1 μg / μL), and the amounts of LN and PEI (concentration of LN and PEI is 1 μg / μL) are set as follows:

[0107] (1) The amount of DNA was set to 1 μg, the amount of LN was set to 0 μg, and the amount of PEI was set as shown in Table 6 below;

[0108]

[0109] (2) The amount of DNA used is set to 1 μg, the amount of LN is set to 0.5 μg, and the amount of PEI is set as shown in Table 7 below;

[0110]

[0111] (3) The amount of DNA, LN and PEI is set to 1 μg and 1 μg respectively, as shown in Table 8 below;

[0112]

[0113] (4) The amount of DNA used is set to 1 μg, the amount of LN is set to 1.5 μg, and the amount of PEI is set as shown in Table 9 below;

[0114]

[0115] (5) The amount of DNA used is set to 1 μg, the amount of LN is set to 2 μg, and the amount of PEI is set as shown in Table 10 below;

[0116]

[0117] Preparation Example 7

[0118] Preparation Example 7 is largely the same as Preparation Example 6, except that the mass ratio of DNA, LN, and PEI in Preparation Example 7 is set at 2.5:1:0.5 (all concentrations are 1 μg / μL), resulting in different transfection complexes. In Preparation Example 7, the transfection time referred to in this invention is the time interval (in hours, h) between the addition of the PEI / DNA / LN complex to the cells and the first medium change. The time gradients are shown in Table 11: 0 h, 1 h, 2 h, 3 h, 6 h, 12 h, 24 h, and 48 h.

[0119]

[0120] Preparation of Comparative Example 1

[0121] Take 1 μl of plasmid DNA with a concentration of 1 μg / μl and 2 μl of PEI with a concentration of 1 μg / μl, and dilute them separately into 50 μl of PBS solution. Gently vortex to mix them to obtain DNA dilution and PEI dilution respectively. Add PEI dilution to DNA dilution and incubate at room temperature for 10 min to allow PEI and DNA to self assemble and form a binary nanocomposite.

[0122] Preparation Example 1: Transfection Efficiency Test

[0123] The transfection complexes prepared in Example 1 and Comparative Example 1 were added to HEK 293T cell culture plates with a cell growth density of 70%, respectively. The plates were gently shaken to mix, and then incubated in an incubator with a CO2 concentration of 5% and a temperature of 37°C for 48 hours. After the culture, the cells were photographed using a fluorescence microscope. After the photographs, the culture medium from each well was collected and stored for later use. 100 μL of 0.25% trypsin was added to each well, and the cells were allowed to digest for 2 minutes. Then, two times the volume of complete culture medium was added to terminate the digestion reaction. The cells were gently pipetted into the wells to completely detach the cells. The detached cell suspension was then aspirated into the previously stored original culture medium and mixed thoroughly. The cell mixture was centrifuged at 800 rpm for 5 minutes, and the supernatant was discarded after centrifugation. The cells were resuspended in 300 μL of flow cytometry buffer in the centrifuge tube, and then flow cytometry was performed for detection. The transfection efficiency was expressed as the proportion of cells expressing fluorescent protein to the total number of cells.

[0124] The test results are shown below:

[0125] The test results of the transfection complexes prepared in Example 1 and Comparative Example 1 are as follows: Figure 1 As shown in Table 12;

[0126]

[0127] The test results of the transfection complex prepared in Example 2 are as follows: Figure 2 As shown in Table 13;

[0128]

[0129] The test results of the transfection complex prepared in Example 3 are as follows: Figure 3 Figure 4 As shown in Tables 14 and 15;

[0130]

[0131] The test results of the transfection complex prepared in Example 4 are as follows: Figure 5 As shown in Table 16;

[0132]

[0133] The test results of the transfection complex prepared in Example 5 are as follows: Figure 6 And as shown in Table 17;

[0134]

[0135] The test results of the transfection complexes prepared with different amounts of LN peptide in Preparation Example 6 are shown in Figures 7-12 and Tables 18-22;

[0136]

[0137]

[0138]

[0139]

[0140]

[0141] The test results of the transfection complex prepared in Example 7 are as follows: Figure 13 As shown in Table 23.

[0142]

[0143] Application Example 1

[0144] The PEI / DNA / LN polypeptide complex prepared in Example 7 and the PEI / DNA complex prepared in Comparative Example 1 were added to 96-well cell culture plates containing HEK 293T and HeLa cells at a cell growth density of 70%. The cell culture plates were gently shaken to mix, and transfection was performed at 37°C and 5% CO2 concentration. CCK8 assay was performed after 24 hours, and the results are as follows: Figure 14 As shown.

[0145] Application Example 2

[0146] The method for preparing the PEI / DNA / LN polypeptide complex obtained in Example 7 involved experiments using plasmids of different sizes, as shown in the table. The PEI / DNA / LN complexes prepared with different plasmids were added to HEK 293T cell culture plates with a cell growth density of 70%. The plates were gently shaken to mix, and then transfected at 37°C with 5% carbon dioxide. Transfection efficiency was tested after 48 hours. The transfection efficiencies corresponding to different plasmid sizes are shown in the table. Figure 15 And as shown in Table 25 below.

[0147] Application Example 3

[0148] The method for preparing the PEI / DNA / LN polypeptide complex obtained in Example 7 was tested using 2-3 different plasmids, as shown in the table. The PEI / DNA / LN complexes prepared with different plasmids were added to HEK 293T cell culture plates with a cell growth density of 70%. The plates were gently shaken to mix, and then transfected at 37°C with 5% carbon dioxide. Transfection efficiency was tested after 48 hours. The transfection efficiencies corresponding to different time points are shown in the table. Figure 16 And as shown in Table 26.

[0149]

[0150] Application Example 4

[0151] The PEI / DNA / LN peptide complex and PEI / DNA complex prepared in Preparation Example 7 and Comparative Example 1 were added to HEK 293T cells and HeLa cells at a cell growth density of 70%. The cell culture plates were gently shaken to mix, and then placed in a high-content cell imaging system. The internal temperature of the instrument was 37°C, and 5% carbon dioxide was used. Each group was set to three replicates. The instrument was set to take pictures once per hour, with nine pictures taken per well. The changes in the average fluorescence intensity and total fluorescence intensity of the cells at different time points within 48 hours were calculated as follows. Figure 17 As shown.

[0152] Application Example 5

[0153] The PEI / DNA / LN polypeptide complex prepared in Example 7 and the PEI / DNA complex prepared in Comparative Example 1 were added to 24-well cell culture plates containing HEK 293T and HeLa cells at a cell growth density of 70%. The plates were placed in a high-content imaging system with three replicates per group and nine fields of view per well. Images were taken hourly to record cell confluence over 48 hours. Results Figure 18 As shown.

[0154] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A polypeptide capable of enhancing polyethyleneimine (PEI) transfection efficiency and reducing toxicity, characterized in that: The polypeptide is named LN and its amino acid sequence is SEQ NO: 1: KKLLALALHLLAGGPKKKRKV.

2. A cell transfection delivery complex, characterized in that: The raw materials for its preparation include PEI, target gene DNA fragment, and LN polypeptide. The DNA is the target gene, and the amino acid sequence of the LN polypeptide is shown in SEQ NO:

1.

3. The cell transfection delivery complex according to claim 2, characterized in that: In the preparation of the raw materials, the mass ratio of PEI, target gene DNA fragment, and LN polypeptide is (2-3):1:(0.5-1).

4. The cell transfection delivery complex according to claim 3, characterized in that: In the preparation materials, the mass ratio of PEI, target gene DNA fragment, and LN polypeptide is 2.5:1:0.

5.

5. A method for preparing a cell transfection delivery complex as described in any one of claims 2 to 4, characterized in that: The method includes: mixing PEI, target gene DNA fragment, and LN polypeptide to obtain a cell transfection delivery complex; The mixed reaction method is selected from any of the following: (a1) Mix all the raw materials used in the preparation of the raw materials together for the reaction; (b1) First, mix two raw materials in the preparation raw materials and react them, and then add another raw material and mix them together.

6. The method for preparing the cell transfection delivery complex according to claim 5, characterized in that, The method includes: (1) PEI and LN peptides were mixed and reacted in buffer to obtain a binary complex reaction solution; (2) Add the binary complex to the DNA dilution solution and mix in the buffer to obtain the cell transfection delivery complex.

7. The method for preparing the cell transfection delivery complex according to claim 6, characterized in that: The buffer solution is selected from one or more combinations of PBS solution, 0.9% NaCl aqueous solution, water, DPBS solution, Opti-MEM solution, DMEM solution or DMEM culture medium, wherein the DMEM culture medium contains 10% fetal bovine serum, 1% penicillin antibody and the remainder is complete DMEM culture medium, and in steps (1) and (2), the volume of the buffer solution is 10 μL to 100 μL.

8. The method for preparing the cell transfection delivery complex according to claim 6, characterized in that, The method satisfies one or more of the following conditions: (a) In step (1), the reaction includes: reacting at 20℃~30℃ for 15min~25min; (b) In step (2), the reaction includes: reacting at 20℃~30℃ for 5min~15min.

9. A method for transfecting cells with DNA, characterized in that, The method includes: adding the cell transfection delivery complex according to any one of claims 2 to 4 or the cell transfection delivery complex prepared by any one of the preparation methods according to claims 5 to 8 into the culture medium of cells to be transfected for incubation.

10. The use of the LN polypeptide as described in claim 1 in the preparation of gene delivery reagents or kits for enhancing cell transfection efficiency and reducing cytotoxicity.

Images