Liposome for transfecting DNA plasmids to CHO cells and preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to lipidosome for transfecting DNA plasmids to CHO cells and a preparation method and application of the lipidosome. The liposome comprises GM < 1-X >, DOPE, cholesterol and a folic acid derivative, wherein X is a hydrophilic joint. As a CHO transfection reagent, the liposome disclosed by the invention has relatively high transfection efficiency, is convenient to amplify and is controllable in cost. The domestic product market is reinforced, and more choices are provided for transfection reagents with determined chemical components in China.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a liposome for transfecting a DNA plasmid into a CHO cell, a preparation method and an application thereof. Background Art

[0002] With the mature development of biomedical technology, more and more targets have been discovered and used in the development of biopharmaceuticals, making the recombinant protein industry enter a period of vigorous development. The expression of recombinant proteins requires the support of high-expression hosts, vectors, and cell culture systems. Mammalian cell expression systems have attracted attention due to their unique advantages in processing, secretion, and glycosylation. Among the many expression systems, Chinese hamster ovary cells (CHO) have the characteristics of efficient amplification and expression of recombinant genes, accurate post-transcriptional modification functions, and stable integration of exogenous proteins. They can be cultured in high-density suspensions, can withstand high shear forces and osmotic pressures, and rarely secrete their own endogenous proteins, which facilitates the separation and purification of downstream products. They have become the most widely used mainstream biological product expression system.

[0003] In the CHO expression system, transient transfer system and stable transfer system are both frequently used tools. However, the application value of transient transfer system is limited due to the lack of efficient transfection reagents during its development. The steps for expressing recombinant protein biological products using the Chinese hamster ovary cell transient transfer expression system are as follows: 1) Design and synthesize exogenous genes to complete the construction of expression vectors; 2) Use appropriate transfection technology to deliver the vectors to CHO host cells; 3) Control key parameters to ensure the fermentation process; 4) Express the target protein through CHO cell fermentation; 5) Separate and purify the target protein. In the application of CHO cell transient transfer expression system, the state of CHO cells and the yield of protein are directly affected by the transfection reagents, which are ultimately reflected in the expression of recombinant proteins.

[0004] The development of culture media in CHO transient transfection systems has become mature, and domestic culture media manufacturers have successively launched chemically synthesized culture media products. However, the development of transfection reagents is very slow, which is mainly limited by the large differences in transfection of different cells. It is difficult to obtain a more efficient transfection formula through certain public ingredients. At the same time, in addition to affecting the transfection efficiency, the transfection reagent may also contain polymers, which is not conducive to cell adsorption and degradation, and thus not conducive to the purification of subsequent products. Therefore, the development of efficient transfection reagents to improve the controllability of protein expression and reduce production costs has a broad prospect for commercial application. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a transfection reagent that supports high-efficiency transfection of DNA plasmids in CHO cells, has a stable process and controllable costs, and provides convenience for the large-scale production of biological products.

[0006] The details are as follows:

[0007] Transfecting DNA plasmid into liposomes of CHO cells, wherein the liposomes contain GM1-X, DOPE, cholesterol and a folic acid derivative, wherein X is a hydrophilic linker, and the folic acid derivative has a structure of formula I,

[0008]

[0009] wherein R1 is selected from -O-R2 or -NH-R2;

[0010] The R2 is selected from C 2－30 alkyl,

[0011] R3 and R4 are each selected from C 1－20 Alkyl or

[0012] Said n=1, 2, 3..., preferably, said n=4-7.

[0013] Furthermore, the folic acid derivative is selected from the following structures:

[0014]

[0015]

[0016]

[0017] Further, the X is a hydrophilic cationic active linker;

[0018] Preferably, X is selected from arginine, vitamin A1, pyridoxamine, putrescine or cyclamenine;

[0019] The hydrophilic linker of the present invention is chemically connected to COO in GM1 (sialic acid side chain). - On the group.

[0020] Furthermore, the ratio of GM1-X, DOPE, cholesterol and folic acid derivative is: 0.5-4:1-5:0.2-1:0.05-0.5.

[0021] Further, the concentration of GM1-X is 0.5-1 mg / mL, the concentration of DOPE is 0.5-2 mg / mL, the concentration of cholesterol is 0.05-1 mg / mL and the concentration of folic acid derivative is 0.05-0.5 mg / mL

[0022] Furthermore, the liposome particle size Z-Ave=100-150nm.

[0023] Furthermore, the liposome surface charge ZP=50-70mV.

[0024] On the other hand, the present invention discloses a method for preparing the liposome, wherein the method is selected from a thin layer dispersion method, an ultrasonic dispersion method, a freeze drying method or a reverse evaporation method.

[0025] Preferably, the preparation method is selected from a thin layer dispersion method, and the preparation method comprises:

[0026] (1) GM1-X, DOPE, cholesterol and folic acid derivatives were mixed in chloroform;

[0027] (2) Vacuum rotary evaporation;

[0028] Preferably, the step (2) further comprises:

[0029] (3) Hydration after adding water;

[0030] (4) Ultrasonic treatment.

[0031] Preferably, after the preparation method is completed, filtration and storage can be selected.

[0032] Furthermore, the step (1) includes the synthesis process of GM1-X, specifically as follows: S1) synthesis of tetradecyl GM1-X; S2) synthesis of GM1-X.

[0033] In another aspect, the present invention discloses a transfection method, comprising:

[0034] (1) mixing the liposome with the plasmid;

[0035] (2) after mixing, mixing the mixture of liposomes and plasmids with cells for transfection;

[0036] Wherein, the cell is a CHO cell, and preferably the CHO cell is selected from expiCHO, CHO-S, CHO-K1 or CHO-K12F10 cells.

[0037] In another aspect, the present invention discloses a method for detecting transfection effect, comprising:

[0038] (1) mixing the liposome with a plasmid carrying fluorescence;

[0039] (2) after mixing, mixing the mixture of liposomes and plasmids with cells for transfection;

[0040] (3) After transfection is completed, observe the fluorescence to detect the transfection effect;

[0041] Preferably, the plasmid carrying fluorescence is an eGFP plasmid.

[0042] Beneficial Effects

[0043] 1. The liposome of the present invention can be used as a transfection reagent to improve CHO transfection efficiency, improve protein production efficiency, and reduce production costs, and has broad application prospects and value.

[0044] 2. The liposome of the present invention is used as a transfection reagent, and its formula does not contain polymers, which is conducive to cell adsorption and degradation, and to product purification. It can improve the stability of production batches and facilitate adjustments to project problems, thereby ensuring product quality and improving product controllability.

[0045] 3. The transfection reagent formula of the present invention is known, which is easy to scale up, can match cells for large-scale high-density culture, and is suitable for large-scale production. In addition, more ingredients can be added based on this basic formula to facilitate the development of more effective transfection reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a diagram of the transfection effect of different addition amounts of liposome 1;

[0047] Figure 2 is the transfection efficiency graph of liposome 1 to liposome 5;

[0048] Figure 3 The figure shows the transfection efficiency of liposome 5 in different CHO cells. DETAILED DESCRIPTION

[0049] Specifically, in order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein. Unless otherwise indicated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0051] As used herein, the terms "including," "comprising," "having," "may," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures.

[0052] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (comprising recombinant technology), microbiology, biochemistry and immunology, which are within the capabilities of those skilled in the art. This technology is fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, 1984); "Animal Cell Culture" (RI Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (DM Weir and CC Black, 1989). ell, ed.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0053] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0054] DOPE and cholesterol described in the following examples were from Sigma; chloroform was purchased from Sinopharm Group; CHO-S, expiCHO cells, ExpiCHO TMExpression Medium and ExpiFectamine CHOReagent were from Thermo; the cell line CHO-K12F10 was obtained by domestication and deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) with the accession number: CGMCC45325; CHO-K1 was from ECACC.

[0055] Example 1 Synthesis of GM1-X

[0056] 1. Synthesis of Tetradecyl GM1

[0057] GM1 (5g, 3.2mmol), pyridine (50mL), acetic anhydride (25mL, 245mmol) were added to a 250mL dry and clean eggplant-shaped bottle in sequence, and stirred to completely dissolve the solid; the reaction solution was cooled to 0℃ with an ice-water bath, and 4-dimethylaminopyridine (0.1g, 0.8mmol) was added to the reaction mixture, and stirred overnight at low temperature without removing the cooling bath. The reaction solution was poured into cold 500mL 2N hydrochloric acid, and stirred thoroughly. A white solid slowly precipitated, filtered, and the filter cake was washed with a small amount of water and then dried in vacuum at 45℃ to obtain an off-white solid (6.3g, 2.9mmol, yield: 90%). MS found: m / z 2135[(M+H) + ].

[0058] This embodiment provides a method for synthesizing tetradecaned GM1. In some specific practices, the tetradecaned GM1 can be purchased from outside.

[0059] 2. Synthesis of Tetradecyl GM1-X

[0060] Dissolve tetradecaned GM1 (500 mg, 0.23 mmol) in N,N-dimethylformamide (5 mL), add diisopropylethylamine (100 mg, 0.69 mmol), HOBt (50 mg, 0.35 mmol), EDCI (68 mg, 0.35 mmol) in sequence, stir at room temperature for 30 minutes under nitrogen protection, then add X; stir the above reaction mixture at room temperature overnight under nitrogen protection. Pour the reaction solution into 50 mL of cold water, stir well, dialyze the aqueous solution at room temperature for 2 days, and then freeze-dry to obtain a white solid.

[0061] Wherein, when X is arginine, vitamin A1, pyridoxamine, putrescine and cyclamen, the synthesis results are shown in Table 1.

[0062] Table 1 Tetradecyl GM1-X

[0063]

[0064] 3. Synthesis of GM1-X

[0065] Dissolve tetradecaned GM1-X in methanol (5 mL), add sodium tert-butoxide (10 mg, 0.1 mmol), and stir at room temperature overnight. Pour the reaction solution into 50 mL of cold water, adjust the pH to 2-3 with 2N hydrochloric acid; dialyze the aqueous solution at room temperature for 2 days and then freeze-dry to obtain a white solid.

[0066] Wherein, when X is arginine, vitamin A1, pyridoxamine, putrescine and cyclopentane, the synthesis results are shown in Table 2.

[0067] Table 2 GM1-X

[0068]

[0069]

[0070] Example 2 Synthesis of folic acid derivatives

[0071] 1. Folic acid derivative 1 (folate lauramide)

[0072] The structure is as follows:

[0073]

[0074] The synthesis steps are as follows:

[0075] Folic acid (200 mg, 0.45 mmol) was added to DMSO (20 mL) in batches and stirred at room temperature for 3 hours under nitrogen protection until the folic acid was completely dissolved. NHS (69 mg, 0.6 mmol) and DCC (124 mg, 0.6 mmol) were added in sequence. After the addition, the mixture was stirred at room temperature for 1 hour. A large amount of white solid precipitated. Dodecylamine (111 mg, 0.6 mmol) was dissolved in DMF (10 mL) and added dropwise to the above reaction solution; stirred at room temperature overnight. Filtered, the filtrate was concentrated under high vacuum at 40°C; the residue was purified by high pressure preparative liquid chromatography to obtain 56 mg of yellow solid (yield: 22%), MS found: m / z 567 [(M+H) + ].

[0076] 2. Folic acid derivative 2 (folate myristic acid amide)

[0077] The structure is as follows:

[0078]

[0079] The synthesis steps are as follows:

[0080] Based on 1, dodecylamine was replaced with tetradecylamine (128 mg, 0.6 mmol), and finally 73 mg of yellow solid (yield: 27%) was obtained. MS found: m / z 595 [(M+H) + ].

[0081] 3. Folic acid derivative 3 (folic acid palmitoylamide)

[0082] The structure is as follows:

[0083]

[0084] The synthesis steps are as follows:

[0085] Based on 1, dodecylamine was replaced with hexadecylamine (149 mg, 0.6 mmol), and finally 33 mg of yellow solid (yield: 12%) was obtained. MS found: m / z 623 [(M+H) + ].

[0086] 4. Folic acid derivative 4 (folate amide)

[0087] The structure is as follows:

[0088]

[0089] The synthesis steps are as follows:

[0090] Based on 1, dodecylamine was replaced with octadecylamine (161 mg, 0.6 mmol), and finally 47 mg of yellow solid (yield: 16%) was obtained. MS found: m / z 651 [(M+H) + ].

[0091] 5. Folic acid derivative 5 (folate eicosamide)

[0092] The structure is as follows:

[0093]

[0094] The synthesis steps are as follows:

[0095] Based on 1, dodecylamine was replaced with eicosylamine (178 mg, 0.6 mmol), and finally 11 mg of yellow solid (yield: 4%) was obtained. MS found: m / z 679 [(M+H) + ].

[0096] 6. Folic acid derivative 6 (folic acid tetraglycol monoamide)

[0097] The structure is as follows:

[0098]

[0099] The synthesis steps are as follows:

[0100] Based on 1, dodecylamine was replaced with 1-aminotetraglycol (115 mg, 0.6 mmol), and finally 80 mg of yellow solid (yield: 31%) was obtained. MS found: m / z 575 [(M+H) + ].

[0101] 7. Folic acid derivative 7 (folate pentaethylene glycol monoamide)

[0102] The structure is as follows:

[0103]

[0104] The synthesis steps are as follows:

[0105] Based on 1, dodecylamine was replaced with 1-aminopentaglycol (143 mg, 0.6 mmol), and finally 75 mg of yellow solid (yield: 27%) was obtained. MS found: m / z 619 [(M+H) + ].

[0106] 8. Folic acid derivative 8 (folic acid hexaethylene glycol monoamide)

[0107] The structure is as follows:

[0108]

[0109] The synthesis steps are as follows:

[0110] Based on 1, dodecylamine was replaced with 1-aminohexaglycol (169 mg, 0.6 mmol), and finally 75 mg of yellow solid (yield: 25%) was obtained. MS found: m / z 663 [(M+H) + ].

[0111] 9. Folic acid derivative 9 (folic acid heptaglycol monoamide)

[0112] The structure is as follows:

[0113]

[0114] The synthesis steps are as follows:

[0115] Based on 1, dodecylamine was replaced with 1-aminoheptaglycol (196 mg, 0.6 mmol), and finally 77 mg of yellow solid was obtained (yield: 24%), MS found: m / z 707 [(M+H) + ].

[0116] 10. Folic acid derivative 10 (folic acid-(2-dodecyloxy) dodecylamide)

[0117] The structure is as follows:

[0118]

[0119] The synthesis steps are as follows:

[0120] Based on 1, dodecylamine was replaced with folic acid-(2-dodecyloxy) dodecylamide, and finally 25 mg of yellow solid (yield: 8%) was obtained. MS found: m / z 751 [(M+H) + ].

[0121] 11. Folic acid derivative 11 (folic acid-(2-dodecyloxy) octadecylamide)

[0122] The structure is as follows:

[0123]

[0124] The synthesis steps are as follows:

[0125] Based on 1, dodecylamine was replaced with 2-dodecyloxyoctadecylamine (272 mg, 0.6 mmol), and finally 33 mg of yellow solid (yield: 9%) was obtained. MS found: m / z 835 [(M+H) + ].

[0126] 12. Folic acid derivative 12 (folic acid-(2-tetraethylene glycol) dodecylamide)

[0127] The structure is as follows:

[0128]

[0129] The synthesis steps are as follows:

[0130] Based on 1, dodecylamine was replaced with tetraglycol dodecylamine (226 mg, 0.6 mmol), and finally 45 mg of yellow solid (yield: 13%) was obtained. MS found: m / z 759 [(M+H) + ].

[0131] 13. Folic acid derivative 13 (folic acid-8-pentadecanamide)

[0132] The structure is as follows:

[0133]

[0134] The synthesis steps are as follows:

[0135] Based on 1, dodecylamine was replaced with 8-pentadecanamine (136 mg, 0.6 mmol), and finally 33 mg of yellow solid (yield: 12%) was obtained. MS found: m / z 609 [(M+H) + ].

[0136] 14. Folic acid derivative 14 (lauryl folate)

[0137] The structure is as follows:

[0138]

[0139] The synthesis steps are as follows:

[0140] Based on 1, dodecylamine was replaced with n-dodecanol (111 mg, 0.6 mmol), and finally 23 mg of yellow solid (yield: 9%) was obtained. MS found: m / z 568 [(M+H) + ].

[0141] 15. Folic acid derivatives 15

[0142] The structure is as follows:

[0143]

[0144] The synthesis steps are as follows:

[0145] Based on 1, dodecylamine was replaced with 8-pentadecanol (136 mg, 0.6 mmol), and finally 21 mg of yellow solid (yield: 7.6%) was obtained. MS found: m / z 610 [(M+H) + ]

[0146] Example 3 Preparation of liposomes by thin layer dispersion method

[0147] 1. Preparation: GM1-X stock solution 4mg / ml, solvent chloroform and methanol 5:1, DOPE stock solution 100mg / mL, solvent chloroform. Cholesterol 10mg / mL, solvent chloroform. Wash several 24 eggplant bottles and set aside.

[0148] 2. Add 4mg GM1-X, 3mg DOPE, 0.5mg cholesterol and 0.5mg folic acid derivative (folic acid tetraglycol monoamide) into a 24-mouth eggplant bottle, then add 15ml chloroform and mix well. Put it into a rotary evaporator to drain the solvent, the temperature is 40℃, the speed is 60rpm, and the time is 2 hours.

[0149] In some other embodiments, the added amounts of GM1-X, DOPE, cholesterol and folic acid derivative are 2.5 mg, 5 mg, 1 mg and 0.25 mg respectively;

[0150] In some other embodiments, the added amounts of GM1-X, DOPE, cholesterol and folic acid derivative are 20 mg, 25 mg, 5 mg and 2.5 mg respectively;

[0151] In some other embodiments, the added amounts of GM1-X, DOPE, cholesterol and folic acid derivative are 2.5 mg, 25 mg, 5 mg and 2.5 mg respectively;

[0152] In some other embodiments, the added amounts of GM1-X, DOPE, cholesterol and folic acid derivative are 20 mg, 25 mg, 5 mg and 0.25 mg respectively;

[0153] In some other embodiments, the added amounts of GM1-X, DOPE, cholesterol and folic acid derivative are 20 mg, 5 mg, 5 mg and 2.5 mg respectively;

[0154] In some other embodiments, the added amounts of GM1-X, DOPE, cholesterol and folic acid derivative are 20 mg, 25 mg, 1 mg and 2.5 mg respectively;

[0155] In some other embodiments, the added amounts of GM1-X, DOPE, cholesterol and folic acid derivative are 10 mg, 10 mg, 2 mg and 0.5 mg respectively;

[0156] In some other embodiments, the added amounts of GM1-X, DOPE, cholesterol and folic acid derivative are 5 mg, 4 mg, 1 mg and 1 mg respectively;

[0157] In some other embodiments, the added amounts of GM1-X, DOPE, cholesterol and folic acid derivative are 10 mg, 8 mg, 1 mg and 2 mg respectively.

[0158] 3. After the rotary steaming is completed, cover the lid and observe whether the thin layer is even and set aside.

[0159] 4. Add 5 ml of pure water and introduce it gently to avoid breaking the thin layer and affecting hydration.

[0160] 5. Place in an incubator at 50°C and 150 rpm for 2 hours, and observe whether the hydration is uniform to avoid lumpy particles.

[0161] 6. Place the ice in an ultrasonic crusher and perform ultrasonic treatment for 10 minutes. The parameters are designed as follows: 2% power, 5 seconds on, and 5 seconds off. Prevent local overheating in the ice from causing damage.

[0162] 7. After the ultrasound is completed, the liposomes are transparent and free of precipitation. Filter them at 0.22um and set aside.

[0163] In this example, X is arginine, vitamin A1, pyridoxamine, putrescine and cyclohexine, and the synthesized liposomes are named liposome 1, liposome 2, liposome 3, liposome 4 and liposome 5 in sequence.

[0164] In this embodiment, the folic acid derivative selected is folic acid derivative 14.

[0165] 8. A certain amount of the prepared liposomes was taken out for particle size and other characterization measurements. The results are shown in Table 3. It can be seen that the replacement of the hydrophilic linker has little effect on the performance of the liposomes.

[0166] Table 3 Liposome characterization

[0167] name Particle size (nm) Charge (mV) pH Liposome 1 137.1 64.6 3.12 Liposome 2 146 65.3 4.26 Liposome 3 147.1 61.6 3.45 Lipofectamine 4 149.5 63.2 3.56 Liposome 5 150.9 56.6 3.18

[0168] Example 4 Liposome transfection efficiency verification

[0169] 1. CHO Cell Preparation

[0170] (1) ExpiCHO, CHO-S, CHO-K1, and CHO-K12F10 cryopreserved cells were taken out of liquid nitrogen, and the cells were quickly lysed in a preheated water bath (within 1 min). The outer wall was wiped with 75% ethanol and then placed in a biosafety cabinet.

[0171] (2) Add 9 ml of preheated ExpiCHO TM Expression Medium into a 15 ml centrifuge tube, slowly add the cell freezing solution, and centrifuge at room temperature at 1000 rpm for 5 minutes.

[0172] (3) Discard the supernatant and add 8 ml ExpiCHO TM Resuspend the cells in Expression Medium, transfer to a culture dish, and place in an incubator at 37°C, 120 rpm, 8% CO2 for culture. Observe the cell status the next day.

[0173] (4) Pipette 20ul of cell suspension, add TypanBlue dye, let stand for 1min, then observe cell viability under a microscope and count the cells.

[0174] (5) The next day, record the activity and number in the same way.

[0175] (6) After 72 hours, cells can reach the stage of passage.

[0176] (7) After counting, 5x10 5 Cells / ml are passaged and after determining the total volume, the amount of cells to be added is calculated and introduced directly into new culture medium without centrifugation.

[0177] (8) 37°C, 180 rpm, 8% CO2 The cell density should not exceed 6x10 6 cells / ml, 2-3 days to meet

[0178] (9) Centrifuge at 1000 rpm at room temperature for 5 min to collect the cells for transfection.

[0179] (10) Adjust the cell density to 6x10 6 cells / ml, 2ml system was placed in a 6-well plate for later use.

[0180] 2. Transfection

[0181] This example investigates the transfection performance of liposomes 1 to 5.

[0182] (1) The amount of plasmid added was 2 ug, and the amount of liposome 5 added was 4 ul, 6 ul, and 8 ul in different gradients. Liposome 1 and plasmid were mixed and incubated for 5 min, and then added to 2 ml CHO cell culture medium for culture.

[0183] (2) Incubate at 37°C, 5% CO2 for 24 h and observe the number of fluorescent cells under a fluorescence microscope to determine the transfection efficiency. Figure 1 As shown, it can be seen that the transfection efficiency is highest when 6ul is added.

[0184] (3) Liposomes 2 to 5 were added in the same manner, and the amount with the highest transfection efficiency was selected. The results are as follows Figure 2 As shown, it can be seen that the hydrophilic heads selected in the present invention all have good transfection efficiency, among which cyclamen, namely liposome 5, has the highest transfection efficiency, close to 100%. The cyclamen hydrophilic linker was selected for subsequent experiments.

[0185] (4) Transfection efficiency of lipofectamine 5 in different CHO cells

[0186] The operation method is the same as expiCHO transfection, and the results are as follows Figure 3 As shown, it can be seen that liposome 5 can achieve a higher transfection efficiency in CHO.

[0187] Example 5 Verification of folic acid derivatives

[0188] In this example, a hydrophilic linker of cyclopentane was selected, CHO-K12F10 cells were used, and the operation method was the same as that in Example 4. The efficiency of transfection of CHO cells with different folic acid derivatives was investigated. The transfection results are shown in Table 4. It can be seen that the different folic acid derivatives listed in this example can achieve a high transfection efficiency, and the replacement of folic acid derivatives will not have a significant impact on the transfection efficiency.

[0189] Table 4 Transfection effect of different folic acid derivatives synthetic liposomes

[0190] Folic acid derivatives Transfection efficiency % Folic acid derivatives Transfection efficiency% 1 90 8 100 2 95 9 95 3 100 10 90 4 95 11 95 5 90 12 95 6 90 13 90 7 95 15 100

[0191] The above experimental results show that the transfection reagent of the present invention can achieve high-efficiency transfection in the CHO cell transient expression system, can support transfection under higher cell density, cell density, and cell survival rate, meet the needs of seed passage and amplification stages in the early stage of large-scale production, and has great application prospects. In particular, it has the same effect on expiCHO, CHO-S, CHO-K1 and CHO-K12F10 cells, and has obvious advantages in transfection efficiency and live cell density, which is conducive to antibody expression and production.

[0192] The culture medium of the present invention is a stable and balanced whole, and the components interact with each other to jointly promote DNA plasmid transfection into CHO cells, providing an efficient plasmid delivery environment for the growth of Chinese hamster ovary cells. The cationic liposomes of the present invention can promote the expression of functional proteins in Chinese hamster ovary cells and improve their performance.

[0193] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. Transfecting DNA plasmid into liposomes of CHO cells, characterized in that, The liposome comprises GM1-X, DOPE, cholesterol and a folic acid derivative, wherein X is a hydrophilic linker, and the folic acid derivative has a structure of formula I, wherein R1 is selected from -O-R2 or -NH-R2; The R2 is selected from C2-30 alkyl, R3 and R4 are each selected from C1-20 alkyl or Said n=1, 2, 3..., preferably, said n=4-7.

2. The liposome according to claim 1, wherein The folic acid derivative is selected from the following structures:

3. The liposome according to claim 1 or 2, wherein The X is a hydrophilic cationic active linker; preferably, the X is selected from arginine, vitamin A1, pyridoxamine, putrescine or cyclohexine.

4. The liposome according to any one of claims 1 to 3, characterized in that The ratio of GM1-X, DOPE, cholesterol and folic acid derivative is: 0.5-4:1-5:0.2-1:0.05-0.

5.

5. The liposome according to any one of claims 1 to 3, characterized in that The GM1-X concentration is 0.5-1 mg / mL, the DOPE concentration is 0.5-2 mg / mL, the cholesterol concentration is 0.05-1 mg / mL and the folic acid derivative concentration is 0.05-0.5 mg / mL.

6. The liposome according to any one of claims 1 to 3, characterized in that The liposome particle size Z-Ave=100-150nm; preferably, the liposome surface charge ZP=50-70mV.

7. A method for preparing the liposome according to any one of claims 1 to 6, characterized in that: The preparation method is selected from thin layer dispersion method, ultrasonic dispersion method, freeze drying method or reverse evaporation method.

8. A method for preparing liposomes as claimed in claim 7, characterized in that: The preparation method is selected from a thin layer dispersion method, and the preparation method comprises: (1) GM1-X, DOPE, cholesterol and folic acid derivatives were mixed in chloroform; (2) Vacuum rotary evaporation; Preferably, the step (2) further comprises: (3) Hydration after adding water; (4) Ultrasonic treatment; Preferably, the step (1) includes the synthesis process of GM1-X, specifically as follows: S1) synthesis of tetradecaned GM1-X; S2) synthesis of GM1-X.

9. A transfection method, characterized in that: include: (1) mixing the liposome according to any one of claims 1 to 6 with a plasmid; (2) after mixing, mixing the mixture of liposomes and plasmids with cells for transfection; Wherein, the cell is a CHO cell, and preferably the CHO cell is selected from expiCHO, CHO-S, CHO-K1 or CHO-K12F10 cells.

10. A method for detecting transfection effect, characterized in that: include: (1) mixing the liposome described in any one of claims 1 to 6 with a plasmid carrying fluorescence; (2) after mixing, mixing the mixture of liposomes and plasmids with cells for transfection; (3) After transfection is completed, observe the fluorescence to detect the transfection effect; Preferably, the plasmid carrying fluorescence is an eGFP plasmid.

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