Transfection reagent for CHO cell DNA transfection and preparation method thereof

Abstract

The invention relates to a transfection reagent for CHO (Chinese hamster ovary) cell DNA (deoxyribonucleic acid) transfection, and the formula of the transfection reagent comprises N-(2, 3-dioleoyloxy-1-propyl) trimethylamine methyl sulfate, polyethyleneimine, N-methyl pyrrolidone and pure water. The invention also relates to a preparation method of the transfection reagent, which comprises the following steps: S1, mixing 150-250g / L of N-methyl pyrrolidone and pure water to 1L to obtain a solution; and S2, when the temperature of the solution is reduced to 30 DEG C or below, sequentially adding 800 to 1200 mg / L of N-(2, 3-dioleoyloxy-1-propyl) trimethylamine methyl sulfate and 400 to 600 mg / L of polyethyleneimine, and uniformly mixing, so as to obtain the transfection reagent. According to the invention, the permeability of transfected DNA to cell membranes is improved, so that the efficiency of PEI and DNA compounds entering cells is improved; meanwhile, according to the scheme, the dosage of the PEI is relatively reduced, on one hand, the toxicity of the PEI to cells is reduced, and on the other hand, the compound of the PEI and the transfection DNA can efficiently release the transfection DNA in the cells, so that the overall transfection efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of transfection reagents, and particularly relates to a transfection reagent for DNA transfection of CHO cells and a preparation method thereof. Background Art

[0002] Chinese Hamster Ovary (CHO) cells are the most widely used host cells for producing antibodies and protein drugs. More than 70% of the commercially available therapeutic protein drugs are produced by CHO cells. Compared with other mammalian cell expression systems, CHO cells have the advantages of post-translational modification similar to humans, clear historical background, rapid growth, and high expression level.

[0003] In order to obtain antibodies or proteins in a short time, transient transfection technology is widely used, including physical methods (such as electroporation), chemical methods, and biological methods (such as Sendai virus). Chemical methods have become the most important transient transfection methods due to their convenience and low cost. Chemical transfection methods mainly include calcium phosphate co-precipitation method and cationic polymers. The calcium phosphate co-precipitation method on CHO cells has strong toxicity and low transfection efficiency, and is basically not used.

[0004] Polyethyleneimine (PEI) is a widely studied cationic polymer that can enter cells by interacting with cell surface proteoglycans and carry exogenous plasmids into cells (as Figure 1 shown). PEI is divided into linear and branched forms. The transfection efficiency of PEI on CHO cells is relatively low, usually only about 40%.

[0005] In the prior art, patent CN 119913209 A discloses a composition for delivering nucleic acids into cells, which is prepared by mixing solution A and solution B. Solution A is an aqueous liquid containing a cationic polymer and a water-soluble calcium salt, and the cationic polymer contained in solution A is preferably polyethyleneimine; solution B is a solution containing a cationic lipid, a neutral lipid, and an optional ethanol solution. By introducing calcium ions and cationic polymers simultaneously, the knockdown efficiency of transfected siRNA can be improved more than that by introducing calcium ions alone, but the calcium salt will increase the cost and the difficulty of post-treatment.

[0006] Therefore, it is necessary to provide a new transfection reagent to solve the above problems. Summary of the Invention

[0007] The main object of the present invention is to provide a transfection reagent for DNA transfection of CHO cells, which can significantly improve the transient transfection efficiency of CHO, thereby increasing the expression level of proteins.

[0008] The present invention achieves the above object through the following technical solutions: A transfection reagent for DNA transfection of CHO cells, the formula of which includes: N-(2,3-dioleyloxy-1-propyl) trimethylammonium methyl sulfate, polyethyleneimine, N-methylpyrrolidone and pure water.

[0009] Specifically, the content of N-(2,3-dioleyloxy-1-propyl) trimethylammonium methyl sulfate in the formula is 800-1200 mg / L.

[0010] Specifically, the content of the polyethyleneimine in the formula is 400-600 mg / L.

[0011] Specifically, the molecular weight of the polyethyleneimine used is 40 kd.

[0012] Specifically, the content of N-methylpyrrolidone in the formula is 150-250 g / L.

[0013] Another main object of the present invention is to provide a preparation method, by which the above transfection reagent for DNA transfection of CHO cells can be obtained.

[0014] The present invention achieves the above object through the following technical solutions: A preparation method of a transfection reagent, the steps of which include:

[0015] S1. First, mix 150-250 g / L of N-methylpyrrolidone and pure water to a volume of 1 L to obtain a solution;

[0016] S2. Wait until the temperature of the solution drops below 30°C, and then sequentially add 800-1200 mg / L of N-(2,3-dioleyloxy-1-propyl) trimethylammonium methyl sulfate and 400-600 mg / L of polyethyleneimine. After mixing evenly, the transfection reagent is obtained.

[0017] The beneficial effects of the technical solutions of the present invention are:

[0018] The present invention improves the permeability of the transfected DNA to the cell membrane by adding N-methylpyrrolidone and N-(2,3-dioleyloxy-1-propyl) trimethylammonium methyl sulfate, thereby enhancing the efficiency of the entry of the PEI and DNA complex into the cell; at the same time, this solution relatively reduces the dosage of PEI. On the one hand, it reduces the toxicity of PEI to cells, and on the other hand, it enables the complex of PEI and transfected DNA to efficiently release the transfected DNA in the cell, thus improving the overall efficiency of transfection. Description of the Drawings

[0019] Figure 1 It is a transfection efficiency diagram of the transfection reagent of the present invention and a conventional PEI reagent;

[0020] Figure 2This is the graph of the antibody expression results for each group in Examples 1-4 and Control Examples 1-4 of the present invention. Detailed implementation mode

[0021] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following will describe the specific implementation mode of the present invention in detail in combination with specific embodiments and drawings. Many specific details are set forth in the following description to fully understand the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific implementations disclosed below.

[0022] Example:

[0023] A transfection reagent formulation for DNA transfection of CHO cells, which comprises the following steps:

[0024] S1. First, mix 150-250 g / L of N-methylpyrrolidone and purified water to a volume of 1 L to obtain a solution.

[0025] A large amount of heat is generated when N-methylpyrrolidone and pure water are mixed. Therefore, it is necessary to wait for the heat generated after mixing to dissipate before proceeding to the next step.

[0026] The N-methylpyrrolidone is purchased from Aladdin Biology, with the product number M103246 and a purity of ≥99%.

[0027] S2. When the temperature of the solution drops below 30°C, sequentially add 800-1200 mg / L of N-(2,3-dioleyloxy-1-propyl) trimethylammonium methyl sulfate and 400-600 mg / L of polyethyleneimine, and after mixing evenly, obtain the transfection reagent.

[0028] The N-(2,3-dioleyloxy-1-propyl) trimethylammonium methyl sulfate is purchased from Aladdin Biology, with the product number D292990 and a purity of ≥98%.

[0029] The polyethyleneimine is purchased from Polyscience, product number: 24765, with a molecular weight of 40 kD.

[0030] During the transfection process, the transfection DNA and the transfection reagent need to form a complex to help the DNA penetrate the cell membrane of CHO cells, and then the DNA reacts with the intracellular DNA. Therefore, the transfection efficiency is affected by the two speeds of the transfection DNA entering the cell membrane and the release of the transfection DNA.

[0031] If the PEI content of the transfection reagent is too high, although it can help the previous DNA enter the cell membrane, the complex structure is too compact, and it is difficult to release the transfected DNA in the cell, which will lead to the inhibition of transfection efficiency and affect the expression.

[0032] Both N-methylpyrrolidone and N-(2,3-dioleyloxy-1-propyl)trimethylammonium methyl sulfate are organic solvents and do not use calcium salts. They are easy to handle, but can improve the permeability of the transfected DNA to the cell membrane, thereby enhancing the efficiency of the PEI-DNA complex entering the cell. At the same time, this scheme relatively reduces the dosage of PEI. On the one hand, it reduces the toxicity of PEI to cells, and on the other hand, it enables the PEI-transfected DNA complex to efficiently release the transfected DNA in the cell, thus improving the overall transfection efficiency.

[0033] To verify the transfection effect of this formulation on CHO cells, multiple examples and control examples were set up for experimental verification. The situations of each example and control example are shown in Table 1, and the numbers in Table 1 correspond to Figure 2 the abscissa numbers in the upper part.

[0034] Table 1

[0035]

[0036]

[0037] Unless otherwise specified, the reagents and instruments used in the above examples are all conventional selections in the art. For the experimental methods without specific conditions noted in the examples, they are usually carried out according to conventional conditions, such as the conditions described in the literature, books, or the methods recommended by the kit manufacturers. The unit mM represents mmol / L, and M represents mol / L.

[0038] Materials used in the experiment:

[0039] The culture medium was provided by Shanghai Youyi Biotechnology Co., Ltd. and was a culture medium that could culture CHO cells.

[0040] The cells were cultured in a 125 mL shake flask with an air-permeable cap (Zhejiang Baidi Technology, product number FL125). The shake flask was cultured in a shaker at 37 °C and 5% CO2 for 5 days, and the shaker speed was 120 rpm - 130 rpm.

[0041] After culturing for 48 h, the expression of green fluorescent protein of the transfection reagent of the present invention and the conventional PEI reagent was detected.

[0042] After culturing for 96 h, the antibody expression of Examples 1 - 4 and Control Examples 1 - 4 was detected.

[0043] The specific statistical test methods are as follows:

[0044] Determination of antibody expression level: It was determined by HPLC method (Agilent, model 1100): The HPLC system was equilibrated with 0.1 M phosphate buffer at a flow rate of 2 mL / min for 15 min until the baseline was stable, 50 μL of sample was injected, and elution was carried out at a flow rate of 2 mL / min.

[0045] Detection of green fluorescent protein: Microscope.

[0046] The detection results are as Figures 1 to 2 shown. Among them:

[0047] Figure 1 shows the expression of fluorescent protein of the transfection reagent of the present invention and the conventional PEI reagent;

[0048] Figure 2 shows the antibody expression results of each group of Examples 1-4 and Control Examples 1-4 ( Figure 2 the ordinate in which represents the molecular weight).

[0049] According to Figures 1 to 2 the results shown, the following conclusions can be drawn:

[0050] (1) More fluorescent protein is expressed and the fluorescence is stronger, indicating that the transfection reagent of the present invention can significantly improve the transfection efficiency compared with the conventional PEI reagent;

[0051] (2) The color blocks at the main molecular weights of Examples 1-4 are generally darker than those of Control Examples 1-4, indicating that the transfection reagent of the present invention can significantly increase the antibody expression level compared with the conventional PEI reagent.

[0052] The above are only some embodiments of the present invention. For those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A transfection reagent for CHO cell DNA transfection, characterized in that, Its formulation includes: N-(2,3-dioleyloxy-1-propyl)trimethylammonium methyl sulfate, polyethyleneimine, N-methylpyrrolidone and pure water.

2. A transfection reagent for CHO cell DNA transfection according to claim 1, characterized in that: The content of N-(2,3-dioleyloxy-1-propyl)trimethylammonium methyl sulfate in the formulation is 800 - 1200 mg / L.

3. A transfection reagent for CHO cell DNA transfection according to claim 1, characterized in that: The content of polyethyleneimine in the formulation is 400 - 600 mg / L.

4. A transfection reagent for CHO cell DNA transfection according to claim 1, characterized in that: The molecular weight of the used polyethyleneimine is 40 kd.

5. A transfection reagent for CHO cell DNA transfection according to claim 1, characterized in that: The content of N-methylpyrrolidone in the formulation is 150 - 250 g / L.

6. A preparation method of any one of the transfection reagents of claims 1 to 5, characterized in that the steps Including: S1. First, mix 150 - 250 g / L of N-methylpyrrolidone and pure water to a volume of 1 L to obtain a solution. S2. Wait until the temperature of the solution drops below 30 °C, then sequentially add 800 - 1200 mg / L of N-(2,3-dioleyloxy-1-propyl)trimethylammonium methyl sulfate and 400 - 600 mg / L of polyethyleneimine. After mixing evenly, the transfection reagent is obtained.

Images