A non-viral transfection reagent and its preparation method and application

Abstract

The present invention belongs to the field of genetic engineering, and in particular to a transfection reagent and its preparation method and application, wherein the transfection reagent is composed of a cationic polymer with a final concentration of 10 to 50 ng / ml, a recombinant polypeptide with a final concentration of 0.1 to 20 μg / ml, and a cationic liposome with a final concentration of 5 to 80 ng / ml. The present invention can significantly improve the transfection efficiency of non-viral vectors with a single lysosomal escape effect, thereby improving the delivery efficiency of nucleic acids; in addition, the present invention adopts a molecular self-assembly method, which can conveniently prepare a transfection complex on a large scale, and the complex is degradable in vivo and has a high safety of use.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and in particular relates to a non-viral transfection reagent and a preparation method and application thereof. Background Art

[0002] Cell transfection technology refers to a method of incorporating exogenous DNA or RNA carrying a target gene into a host cell so that the cell acquires a new genetic marker. It is an important analytical tool for analyzing the expression function of genes, gene products, and proteins in cells. The main purpose of cell transfection is to study the function of genes, gene products, and recombinant proteins by enhancing or inhibiting the expression of specific genes in cells. Cell transfection technology has great application value in the field of genetic engineering, such as transfecting target genes into cells through gene therapy to treat diseases or improve patients' symptoms, and obtaining human tissue plasminogen activator through Chinese hamster ovary cells.

[0003] So far, the gene vectors developed mainly include viral vectors and non-viral vectors. The most commonly used non-viral transfection media are liposomes and cationic polymers. Their characteristics are similar to those of viruses and they can easily penetrate the cell membrane. Although viral vectors, such as retroviruses, adenoviruses, herpes viruses, and adeno-associated viruses, have high transfection efficiency, they have the risk of self-replication in the human body. Non-viral vectors have the advantages of easy large-scale preparation, low cost, no size limit for exogenous genes, low immunogenicity, and relative safety. Due to the limitations of viral media, cationic polymer transfection reagents are increasingly valued.

[0004] Cationic polymers can be used as a non-viral vector with the characteristics of cell-like structure and biomembrane. They can be degraded in vivo while protecting the biological activity of the gene fragments they carry. They are also easy to operate and have good reproducibility. They are gene transfection vectors with potential for clinical application. The transfection principle of cationic polymers is that the positively charged polymer forms a positively charged complex with the negatively charged phosphate group of nucleic acid, then interacts with the negatively charged proteoglycan on the cell surface and enters the cell through endocytosis. Commonly used cationic polymers include poly-L-lysine (PLL), polyethyleneimine (PEI), dendritic virus envelope glycoprotein polymers, chitosan, etc.

[0005] However, due to their extremely high cytotoxicity, they can cause problems such as blood cell coagulation during the in vivo transfection process. Existing cationic polymers cannot simultaneously meet the two basic requirements of safety, low toxicity, and efficient transfection. Therefore, their application is greatly limited.

[0006] Virus envelope glycoprotein B (gB) can mediate the virus invasion process by binding to receptors widely present on the surface of mammalian cells, such as heparin sulfateproglycan (HSPG) on various mammalian cells, and gB glycoprotein can function independently without relying on other viral envelope proteins. At the same time, gB can bind to host cells independently of HSPG, and gB can also bind to cell integrins (integrin) or epidermal growth factor receptor (EGFR) to promote the virus invasion process. Although the viral envelope glycoprotein peptide itself can quickly and efficiently enter the cell, its ability to bind and load siRNA or plasmid DNA is limited, and the transfection polypeptide alone cannot efficiently transport siRNA or plasmid DNA. Summary of the invention

[0007] In view of the safety risks of viral transfection, the inefficiency and cytotoxic side effects of cationic polymers, the present invention discloses a non-viral transfection reagent and a preparation method and application thereof. The non-viral transfection reagent can improve the transfection efficiency while reducing the toxic side effects of a single cationic polymer on cells.

[0008] The present invention is achieved through the following technical solutions:

[0009] A non-viral transfection reagent comprises: a cationic polymer with a final concentration of 10-50 ng / ml, a recombinant polypeptide with a final concentration of 0.1-20 μg / ml, and a cationic liposome with a final concentration of 5-80 ng / ml.

[0010] Furthermore, the non-viral transfection reagent includes: a cationic polymer with a final concentration of 20 ng / ml, a recombinant polypeptide with a final concentration of 8 μg / ml, and a cationic liposome with a final concentration of 40 ng / ml.

[0011] Furthermore, the cationic polymer of the non-viral transfection reagent is one or multiple cationic polymers in any ratio based on the proton sponge effect.

[0012] Preferably, the cationic polymer includes polyethyleneimine, polylysine, polyamidoamine or chitosan.

[0013] Furthermore, the recombinant polypeptide is a viral envelope glycoprotein; the viral envelope glycoprotein is a dendritic virus envelope glycoprotein, preferably a recombinant herpes virus envelope glycoprotein.

[0014] Preferably, the amino acid sequence of the recombinant gB polypeptide is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0015] Furthermore, the cationic liposomes are didodecyldimethylammonium bromide DDAB, amine cationic lipids including quaternary ammonium, primary amine, secondary amine and other amine salt liposomes, such as trimethyl-2,3-dioleyloxypropylammonium chloride DOTMA, dimethyl-2-hydroxyethyl-2,3-dioleyloxypropylammonium bromide DORIE, dimethyl-2-hydroxyethyl-2,3-dioleyloxypropylammonium bromide DORI and other quaternary ammonium salt liposomes.

[0016] Furthermore, the transfection reagent also includes a solvent; the solvent is HBS (pH 7.4).

[0017] A preparation method for the above-mentioned non-viral transfection comprises the following steps:

[0018] The cationic polymer, the recombinant polypeptide, the cationic liposome and the solvent are mixed to obtain a non-viral transfection preparation.

[0019] Furthermore, the preparation method of the non-viral transfection reagent comprises the following steps:

[0020] 1) preparing a cationic polymer solution;

[0021] 2) Preparation of recombinant polypeptides; Preparation of cationic liposome / recombinant polypeptide complexes;

[0022] 3) Preparation of transfection reagent.

[0023] Furthermore, the preparation of the cationic polymer solution in step 1) includes:

[0024] (1) Preparation of HBS (pH 7.4): Dissolve NaCl in ultrapure water, add HEPES, adjust pH, make up to volume, filter and store at 4°C until use;

[0025] (2) Dissolve the cationic polymer powder in the HBS (pH 7.4) prepared in step (1), shake evenly to prepare a PEI solution of the required concentration, filter, and store at 4°C for later use.

[0026] Furthermore, the preparation of the cationic liposome / viral envelope glycoprotein complex in step 2) includes: taking equal amounts of cationic liposomes and viral envelope glycoproteins and dissolving them in 1×HBS (pH 7.4), shaking them evenly, filtering them to obtain a cationic liposome / viral envelope glycoprotein complex solution, and storing them at 4° C. for future use.

[0027] A method for transfection using the non-viral transfection reagent, comprising:

[0028] (4) Take one tube, numbered as tube 1: add non-viral transfection reagent and serum-free medium to serum-free medium and incubate at room temperature for 5 min;

[0029] (5) Take another tube, numbered as tube 2, and add nucleic acid and Opti-MEM medium;

[0030] (6) Slowly add the solution in tube 1 to tube 2 at a rate of 1 ml per minute, and vortex to mix; let stand, and add to serum-free culture medium; after 12-16 hours, replace with complete nutrient medium (such as DMEM medium containing 10% serum); centrifuge and filter.

[0031] Furthermore, the serum-free culture medium includes Opti-MEM culture medium, DMEM, α-MEM, EMEM, 1640 and other basic culture medium,

[0032] Furthermore, the nucleic acid is one of plasmid DNA, gene fragment, double-stranded or single-stranded DNA fragment, cDNA, or a combination of multiple types in any ratio.

[0033] Beneficial effects of the present invention:

[0034] The transfection polypeptide of the present invention can be used as a cell transfection enhancer to assist cationic polymer-mediated cell transfection. When used in combination with a cationic polymer, the cationic polymer and the polypeptide are mainly bound together by charge action, and this binding is covalent binding and will not destroy the biological activity of the protein. However, this binding can mask the enzyme cleavage site and binding site of the protein, so that it cannot be recognized by the enzyme in the culture medium, thereby effectively protecting the polypeptide from degradation by the enzyme and improving the transfection efficiency.

[0035] The envelope glycoprotein B polypeptide provided by the present invention makes the DNA polymer adhere to the cell by invading or binding the structure when acting on the cell membrane, thereby improving the gene transfection efficiency and not causing cytotoxicity.

[0036] The viral envelope glycoprotein B polypeptide provided by the present invention has no infection potential of viral vectors, and thus has no safety issues of viral replication and infection, and has the potential to be applied to gene therapy.

[0037] The present invention can significantly improve the transfection efficiency of non-viral vectors with a single lysosomal escape effect, thereby improving the delivery efficiency of nucleic acids; in addition, the present invention adopts a molecular self-assembly method, which can conveniently prepare transfection complexes on a large scale, and the complexes are degradable in vivo and have high safety in use. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1The expression of GFP gene after Hela cells were transfected with cationic PEI polymer alone; 1(A) is the bright field image; 1(B) is the fluorescence image; 1(C) is the overlay image.

[0039] Figure 2 The expression of GFP gene after liposome transfection of Hela cells alone; 2(A) is the bright field image; 2(B) is the fluorescence image; 2(C) is the overlay image.

[0040] Figure 3 The expression of GFP gene after Hela cells were transfected with PEI and liposome composition; 3(A) is a bright field image; 3(B) is a fluorescence image; 3(C) is an overlay image.

[0041] Figure 4 The expression of GFP gene after Hela cells were transfected with PEI and polypeptide combination; 4(A) is a bright field image; 4(A) is a fluorescence image; 4(A) is an overlay image.

[0042] Figure 5 The expression of GFP gene after Hela cells were transfected with liposome and peptide combination; 5(A) is the bright field image; 5(A) is the fluorescence image; 5(A) is the overlay image.

[0043] Figure 6 The expression of GFP gene after Hela cells were transfected with the transfection reagent (cation, polypeptide and liposome composition) of the present invention; wherein 6(A) is a bright field image; 6(A) is a fluorescence image; 6(A) is an overlay image.

[0044] Figure 7 The figure shows the expression of GFP gene after Hela cells were transfected with commercially available Lipofectamine 2000.

[0045] Figure 8 The figures are comparisons of cell viability and OD value after transfection with each reagent 48h and 72h. Figure 8(A) is a comparison of cell viability; Figure 8(B) is a comparison of OD value.

[0046] Fig. 9 Bit Figures 1 to 7 Transfection efficiency profile of transfection experiments; 9-1 is PEI transfection ( Figure 1 ), the transfection efficiency was 2.1%; 9-2 was liposome transfection ( Figure 2 ), the transfection efficiency was 24.5%; 9-3 was 3. Cationic and liposome complex (composition 4, Figure 3 ) transfection efficiency was 38.8%; 9-4 was 3. Cations combined with polypeptides (composition 3, Figure 4) transfection efficiency is 30%; 9-5 is a combination of liposomes and peptides (composition 2, Figure 5 ), the transfection efficiency was 66.2%; 9-6 is the transfection reagent of the present invention (cation, polypeptide and liposome combination (composition 1, Figure 6 )) The transfection efficiency was 82.6%; 9-7 was Lipofectamine2000 ( Figure 7 ), the transfection efficiency was 60.5%. DETAILED DESCRIPTION

[0047] In order to better illustrate the problem to be solved by the technical solution of the present invention, the technical solution adopted and the beneficial effects achieved, it is now further described in conjunction with specific implementation methods. It is worth noting that the technical solution of the present invention includes but is not limited to the following implementation methods.

[0048] If no specific techniques or conditions are specified in the examples of the present invention, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained through commercial purchase or other channels.

[0049] Example 1 Preparation of the transfection reagent of the present invention

[0050] 1.1 Inducible expression and purification of cationic amphiphilic polypeptides

[0051] A recombinant peptide polypeptide of a class of viral envelope glycoproteins that fuses and binds to the host cell membrane, comprising the amino acid sequences shown below: IYNGWYAX and TXQMXTIYQ, and the polypeptide is a recombinant polypeptide secreted by cells.

[0052] In the above amino acid sequence, X is a replaceable acidic amino acid. In addition to Asp, Glu can also achieve the same effect. When X is Asp, the sequence of the gB recombinant polypeptide is shown in SEQ ID NO: 1; when X is Glu, the polypeptide sequence is shown in SEQ ID NO: 2.

[0053] When X is Asp, the recombinant polypeptide is obtained by the following method: synthesizing a coding gene (NCBI Gene ID: 3783680) related to host adhesion in the BALF4 gene of EBV virus, the sequence of which is as shown in SEQ ID NO: 3, connecting the recombinant polypeptide gB gene with the correct sequence to the eukaryotic secretory expression vector pSecTag2B by T4 DNA ligase, transforming into competent Escherichia coli, screening positive clones, and screening the correctly connected recombinant plasmid pSecTag2B-gB by enzyme digestion, and after linearization, the recombinant plasmid pSecTag2B-gB is transformed into HEK293T cells for expression, and the expression product is in the culture supernatant. After detection, the expression amount of the recombinant polypeptide gB of the present invention is 5 mg / mL to 12 mg / mL.

[0054] When X is Glu, the sequence is as shown in SEQ ID NO: 4, and other methods are the same as those when X is Asp.

[0055] 1.2 Preparation of cationic polymer

[0056] Preparation of 1×HBS (pH 7.4): Dissolve 8.76 g NaCl in 900 ml ultrapure water, add 20 ml 1 M (4.766 g) HEPES, adjust the pH to 7.4, make up to 1 L, filter (0.2 μm filter membrane) and store at 4°C for future use.

[0057] Weigh 50 mg of PEI powder and dissolve it in 50 ml of 1×HBS (pH 7.4), shake it evenly to make a 1.0 mg / ml PEI solution, filter it with a 0.2 μm filter membrane, and store it at 4°C for later use.

[0058] 1.3 Preparation of cationic liposome / recombinant gB polypeptide complex

[0059] Take 4 mg of commercial cationic liposome didodecyldimethylammonium bromide (DDAB), dissolve it in 10 mL of 1×HBS (pH 7.4), shake it evenly, filter it through a 0.2 μm filter membrane to obtain a 400 μg / ml liposome solution, dilute the liposome solution 10 times with 8 mg / ml of the recombinant gB polypeptide solution prepared in 1.1, prepare a cationic liposome / recombinant gB polypeptide complex solution containing 40 μg / ml cationic liposome and 8 mg / ml gB polypeptide, and store it at 4°C for use.

[0060] 1.4 Preparation of the transfection reagent of the present invention

[0061] The cationic polymer solution prepared in 1.2 and the complex prepared in 1.3 are mixed in a ratio of 1:50.

[0062] Example 2 Transfection using the transfection reagent of the present invention

[0063] Take two 1.5ml EP tubes, of which tube 1: 2μL of the transfection reagent of the present invention + 0.25mL Opti-MEM culture medium, room temperature for 5min; tube 2: 2ul 1ug plasmid (the plasmid is the vector pcDNA3.1(+) containing green fluorescent protein (GFP), which is convenient for observing the transfection efficiency of cells by fluorescence expression, pcDNA3.1(+) is purchased from addgene) + 0.25ml Opti-MEM culture medium; tube 1 is slowly added to tube 2 (1ml per minute), and then inverted to mix for 5 seconds. Let stand for 20min, and add to cells that have grown to 80% confluence. Change to DMEM culture medium containing 10% serum for 12-16h. Centrifuge at 1300rpm for 5min, and filter with a 0.45μm filter membrane.

[0064] Example 3 Transfection effect detection

[0065] Cationic polymer transfection efficiency and toxicity detection; PEI transfection efficiency and toxicity detection using different cells.

[0066] The positively charged amino groups in the structure of amino acid cationic liposomes can be completely compressed with the negatively charged phosphate groups on the plasmid through electrostatic adsorption to form a stable cationic liposome complex, which is compressed into dispersed particles by the cationic liposome. At this time, the primary structure has not changed, and it can also avoid degradation by enzymes and salts in the body. After preparing a complex suspension of amino acid cationic liposomes and PEI or viral envelope recombinant polypeptides in a certain ratio, it was observed that flocculation will occur regardless of the ratio of the liposomes. The polypeptide not only protects and promotes the penetration of plasmid DNA, but also reduces the toxicity of cationic liposomes. The polypeptide head rich in hydrophilic amino acids (M TRR V L S VVVLL AA L ACR LGA QT , where the underlined part is a hydrophilic amino acid), in a lower concentration aqueous solution, a hydrophilic barrier can be formed on the surface of the liposome, thereby increasing the stability of the liposome in the transfection reagent system and improving the properties of the liposome.

[0067] from Figure 1 ( Figure 9-1 )and Figure 2 ( Figure 9-2 ) It can be seen that the GFP gene expression in cells transfected with PEI and liposome is very low.

[0068] The cells were transfected with DNA plasmids. The transfection method of plasmid DNA was the same as in Example 2. The transfection reagents were compositions 1-4 in Table 1. The transfection results of each composition were observed. Figure 3-Figure 6 ( Figure 9-3 to 9-6).

[0069] Table 1 Composition and final concentration of each transfection reagent

[0070]

[0071] The expression of GFP gene in HeLa cells was shown in Figure 3. Figure 3-Figure 6 ( Figure 9-3 To 9-6). Figure 3-Figure 6 ( Figure 9-3 9-6), it can be seen that compared with cells transfected with PEI and liposome complexes, the cationic and liposome complexes (compositions 4, Figure 3 ), cation and polypeptide combination (composition 3, Figure 4 ), liposome and peptide combination (composition 2, Figure 5 ), the cation, polypeptide and liposome of the present invention are used in combination (composition 1, Figure 6 )、Lipofectamine2000( Figure 7 , Figure 9-7 ), these combined composite transient transfection reagents can efficiently deliver plasmid DNA into target cells and inhibit the expression of target genes, and the expression of GFP gene in cells is significantly enhanced. Among them, the cation, polypeptide and liposome of the present invention are combined ( Figure 6 , Figure 9-6 ) has the strongest effect and is superior to the commercially available Lipofectamine 2000 ( Figure 7 , Figure 9-7 ), that is, through the combined action of PEI, gB polypeptide and liposome, the transfection efficiency of GFP gene in HeLa cells was significantly improved, indicating that the plasmid DNA system delivered by PEI / polypeptide liposome complex can stably transfect HeLa cells.

[0072] Example 4 Cytotoxicity Experiment

[0073] 4000 cells / 100 μL cell suspension were inoculated in two 96-well plates. The culture plates were pre-cultured in an incubator (37°C, 5% CO2), and transfected with the compositions in Table 1, and then transfected for 48h and 72h, and the cell survival rate was determined by the CCK8 method. 20 μL CCK solution was added to each well, and the culture plates were incubated in the incubator for 4 hours, and the absorbance at 450nm was measured by an enzyme-labeled instrument. The results are shown in Figure 8 (8(A), 8(B)).

[0074] CCK8 staining method to measure cell number and viability

[0075] Experiments have shown that the vector is substantially non-cytotoxic, biodegradable, and has a high gene transfection efficiency: when a transfection experiment is conducted with cervical cancer cells Hela as target cells and GFP as target gene, the transfection efficiency is better than that of the commercially available cationic liposome Lipofectamine 2000. At the concentration with the highest transfection efficiency, the transfection efficiency of Lipofectamine 2000 is 60.5%, while the transfection efficiency of the present invention is 82.6%; at the concentration with the highest transfection efficiency, the cell survival rates of PEI and liposome after 48 hours of transfection are 71.59% and 78.47%, respectively, and the cytotoxicity is relatively strong. The commercially available Lipofectamine 3000 has a higher survival rate of 90.13%, while the survival rate of cells transfected by the cationic polymer gene transfection reagent of the present invention is 93.66%, and the toxicity is the lowest.

[0076] At present, the main strategies for non-viral vector lysosomal escape are proton sponge effect, membrane fusion effect, membrane perforation or membrane interference effect. The transfection complex in the present invention has two lysosomal escape effects: the proton sponge effect of cationic polymers and the membrane perforation or membrane interference effect of viral envelope glycoproteins. The combined use of these two effects can significantly improve the transfection efficiency of non-viral vectors with a single lysosomal escape effect, thereby improving the delivery efficiency of nucleic acids. The present invention adopts a molecular self-assembly method, which can conveniently prepare transfection complexes on a large scale, and the complex is degradable in vivo, not easy to accumulate in vivo, and has high safety in use.

[0077] The above experiments and experimental results show that the new transgenic complex formed by gB polypeptide, cationic polymer and nucleic acid can not only efficiently deliver DNA but also has low cytotoxicity.

[0078] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. Sequence Listing <110> Guangzhou Huiyuanyuan Pharmaceutical Technology Co., Ltd. <120> A non-viral transfection reagent and its preparation method and application <130> HPCNP201810060 <141> 2018-08-06 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 250 <212> PRT <213> Artificial Sequence <400> 1 Met Thr Arg Arg Arg Val Leu Ser Val Val Val Leu Leu Ala Ala Leu 1 5 10 15 Ala Cys Arg Leu Gly Ala Gln Thr Pro Glu Gln Pro Ala Pro Pro Ala 20 25 30 Thr Thr Val Gln Pro Thr Ala Thr Arg Gln Gln Thr Ser Phe Pro Phe 35 40 45 Arg Val Cys Glu Leu Ser Ser His Gly Asp Leu Phe Arg Phe Ser Ser 50 55 60 Asp Ile Gln Cys Pro Ser Phe Gly Thr Arg Glu Asn His Thr Glu Gly 65 70 75 80 Leu Leu Met Val Phe Lys Asp Asn Ile Ile Pro Tyr Ser Phe Lys Val 85 90 95 Arg Ser Tyr Thr Lys Ile Val Thr Asn Ile Leu Ile Tyr Asn Gly Trp 100 105 110 Tyr Ala Asp Ser Val Thr Asn Arg His Glu Glu Lys Phe Ser Val Asp 115 120 125 Ser Tyr Glu Thr Asp Gln Met Asp Thr Ile Tyr Gln Cys Tyr Asn Ala 130 135 140 Val Lys Met Thr Lys Asp Gly Leu Thr Arg Val Tyr Val Asp Arg Asp 145 150 155 160 Gly Val Asn Ile Thr Val Asn Leu Lys Pro Thr Gly Gly Leu Ala Asn 165 170 175 Gly Val Arg Arg Tyr Ala Ser Gln Thr Glu Leu Tyr Asp Ala Pro Gly 180 185 190 Trp Leu Ile Trp Thr Tyr Arg Thr Arg Thr Thr Val Asn Cys Leu Ile 195 200 205 Thr Asp Met Met Ala Lys Ser Asn Ser Pro Phe Asp Phe Phe Val Thr 210 215 220 Thr Thr Gly Gln Thr Val Glu Met Ser Pro Phe Tyr Asp Gly Lys Asn 225 230 235 240 Lys Glu Thr Phe His Glu Arg Ala Asp Ser 245 250 <210> 2 <211> 250 <212> PRT <213> Artificial Sequence <400> 2 Met Thr Arg Arg Arg Val Leu Ser Val Val Val Leu Leu Ala Ala Leu 1 5 10 15 Ala Cys Arg Leu Gly Ala Gln Thr Pro Glu Gln Pro Ala Pro Pro Ala 20 25 30 Thr Thr Val Gln Pro Thr Ala Thr Arg Gln Gln Thr Ser Phe Pro Phe 35 40 45 Arg Val Cys Glu Leu Ser Ser His Gly Asp Leu Phe Arg Phe Ser Ser 50 55 60 Asp Ile Gln Cys Pro Ser Phe Gly Thr Arg Glu Asn His Thr Glu Gly 65 70 75 80 Leu Leu Met Val Phe Lys Asp Asn Ile Ile Pro Tyr Ser Phe Lys Val 85 90 95 Arg Ser Tyr Thr Lys Ile Val Thr Asn Ile Leu Ile Tyr Asn Gly Trp 100 105 110 Tyr Ala Glu Ser Val Thr Asn Arg His Glu Glu Lys Phe Ser Val Asp 115 120 125 Ser Tyr Glu Thr Glu Gln Met Glu Thr Ile Tyr Gln Cys Tyr Asn Ala 130 135 140 Val Lys Met Thr Lys Asp Gly Leu Thr Arg Val Tyr Val Asp Arg Asp 145 150 155 160 Gly Val Asn Ile Thr Val Asn Leu Lys Pro Thr Gly Gly Leu Ala Asn 165 170 175 Gly Val Arg Arg Tyr Ala Ser Gln Thr Glu Leu Tyr Asp Ala Pro Gly 180 185 190 Trp Leu Ile Trp Thr Tyr Arg Thr Arg Thr Thr Val Asn Cys Leu Ile 195 200 205 Thr Asp Met Met Ala Lys Ser Asn Ser Pro Phe Asp Phe Phe Val Thr 210 215 220 Thr Thr Gly Gln Thr Val Glu Met Ser Pro Phe Tyr Asp Gly Lys Asn 225 230 235 240 Lys Glu Thr Phe His Glu Arg Ala Asp Ser 245 250 <210> 3 <211> 750 <212> DNA <213> Artificial Sequence <400> 3 atgactcggc gtagggtgct aagcgtggtc gtgctgctag ccgccctggc gtgccgtctc 60 ggtgcgcaga ccccagagca gcccgcaccc cccgccacca cggtgcagcc taccgccacg 120 cgtcagcaaa ccagctttcc tttccgagtc tgcgagctct ccagccacgg cgacctgttc 180 cgcttctcct cggacatcca gtgtccctcg tttggcacgc gggagaatca cacggagggc 240 ctgttgatgg tgtttaaaga caacattatt ccctactcgt ttaaggtccg ctcctacacc 300 aagatagtga ccaacattct catctacaat ggctggtacg cggactccgt gaccaaccgg 360 cacgaggaga agttctccgt tgacagctac gaaactgacc agatggatac catctaccag 420 tgctacaacg cggtcaagat gacaaaagat gggctgacgc gcgtgtatgt agaccgcgac 480 ggagttaaca tcaccgtcaa cctaaagccc accgggggcc tggccaacgg ggtgcgccgc 540 tacgccagcc agacggagct ctatgacgcc cccgggtggt tgatatggac ttacagaaca 600 agaactaccg tcaactgcct gataactgac atgatggcca agtccaacag ccccttcgac 660 ttctttgtga ccaccaccgg gcagactgtg gaaatgtccc ctttctatga cgggaaaaat 720 aaggaaacct tccatgagcg ggcagactcc 750 <210> 4 <211> 750 <212> DNA <213> Artificial Sequence <400> 4 atgactcggc gtagggtgct aagcgtggtc gtgctgctag ccgccctggc gtgccgtctc 60 ggtgcgcaga ccccagagca gcccgcaccc cccgccacca cggtgcagcc taccgccacg 120 cgagagcaaa ccagctttcc tttccgagtc tgcgagctct ccagccacgg cgacctgttc 180 cgcttctcct cggagatcca gtgtccctcg tttggcacgc gggagaatca cacggagggc 240 ctgttgatgg tgtttaaaga gaacattatt ccctactcgt ttaaggtccg ctcctacacc 300 aagatagtga ccaacattct catctacaat ggctggtacg cgggacccgt gaccaaccgg 360 cacgaggaga agttctccgt tgagagctac gaaactgagc agatggagac catctaccag 420 tgctacaacg cggtcaagat gacaaaagag gggctgacgc gcgtgtatgt agagcgcgag 480 ggagttaaca tcaccgtcaa cctaaagccc accgggggcc tggccaacgg ggtgcgccgc 540 tacgccagcc agacggagct ctatgaggcc cccgggtggt tgatatggac ttacagaaca 600 agaactaccg tcaactgcct gataactgag atgatggcca agtccaacag ccccttcgac 660 ttctttgtga ccaccaccgg gcagagtgtg gaaatgtccc ctttctatga cgggaaaaat 720 aaggaaacct tccatgagcg ggcagactcc 750

Claims

1. A non-viral transfection reagent, characterized in that include: A cationic polymer with a final concentration of 10 to 50 ng / ml, a recombinant polypeptide with a final concentration of 0.1 to 20 μg / ml, and a cationic liposome with a final concentration of 5 to 80 ng / ml; the recombinant polypeptide is a viral envelope glycoprotein; the viral envelope glycoprotein is a recombinant gB polypeptide; the amino acid sequence of the recombinant gB polypeptide is SEQ ID NO: 1; The cationic polymer is polyethyleneimine; The cationic liposome is didodecyl dimethyl ammonium bromide DDAB.

2. A method for preparing the non-viral transfection reagent according to claim 1, characterized in that: The following steps are involved: 1) preparing a cationic polymer solution; 2) Preparation of cationic liposome / viral envelope glycoprotein complex; 3) Preparation of transfection reagent.

3. The method for preparing the non-viral transfection reagent according to claim 2, characterized in that: The preparation operation of the cationic polymer solution in step 1) includes: (1) Preparation of HBS at pH 7.4: Dissolve NaCl in ultrapure water, add HEPES, adjust pH, make up to volume, filter and store at 4°C; (2) Dissolve the cationic polymer powder in the HBS prepared in step (1) with a pH of 7.4, shake evenly to prepare a polyethyleneimine solution of the required concentration, filter, and store at 4°C.

4. The method for preparing the non-viral transfection reagent according to claim 2, characterized in that: The preparation of the cationic liposome / virus envelope glycoprotein complex in step 2) includes: taking equal amounts of cationic liposomes and virus envelope glycoproteins, dissolving them in 1×HBS pH 7.4, shaking them evenly, filtering them to obtain a cationic liposome / recombinant polypeptide complex solution, and storing them at 4° C. for future use.

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