Method for electrotransfecting CHOZN cell lines

Abstract

The invention discloses a method for electrotransfecting a CHOZN cell line. The electrotransformation conditions used in the method are based on the following electrotransformation conditions: the cell number is 5E6, the plasmid amount is 30-50 [mu] g, the voltage is 300 V, and the capacitance is 950 [mu] F; and one or more of parameters such as capacitance, the cell number, the voltage and the like are adjusted. The method for electrotransfecting the CHOZN cell line has the advantages of both electrotransfection efficiency and cell viability, high repeatability and the like.

Description

Technical Field

[0001] The present application relates to the technical field of plasmid transfection in the construction of engineered cell lines, and specifically to a method for electroporation of CHOZN cell lines. Background Art

[0002] In addition to electric field parameters, cell factors, and the choice of electrotransfection reagents, the size of the plasmid is also an important factor affecting the electrotransfection efficiency in electrofection experiments. Generally speaking, the larger the molecular weight of the plasmid, the lower its electrotransfection efficiency will be. However, for many multi-expression frame double-antibody plasmids, modified site-specific integration plasmids, and gene-modified plasmids that prevent gene silencing on the market, the plasmid size is generally above 10kb, and their electrotransfection efficiency is generally lower than that of ordinary plasmids.

[0003] There are also many electroporation buffers on the market for engineering cell line screening. However, many buffers, such as the Mirus electroporation kit, recommend the electroporation condition range and cell number for conventional suspension cells and adherent cells, and list the appropriate electroporation condition range for different cells with different electroporators, but there are no clear and specific electroporation parameters for suspended CHOZN host cells.

[0004] MERCK has recommended electroporation conditions for CHOZN host cells (5×10 cells per electroporation). 6 The recommended amount of DNA is 30-50μg, and the electroporation parameters are 300 V, 950μF, exponential decay pulse). When operated correctly, a transfection efficiency of 60-80% can be achieved. However, these are electroporation parameters for pcGS3.2 plasmids. The size of pcGS3.2 plasmids (conventional recombinant antibodies) is about 10kb. For large plasmids with a molecular weight of about 15kb, there is not much reference data for electroporation conditions, and it still needs to be explored again.

[0005] Therefore, there is an urgent need in the art to develop a new electroporation method for CHOZN cell lines targeting large plasmids. Summary of the invention

[0006] Based on this, it is necessary to provide at least a method for electroporating CHOZN cell lines.

[0007] In the first aspect of the present application, a method for electroporating a CHOZN cell line with a plasmid is provided, wherein the electroporation conditions used are based on the following electroporation conditions:

[0008] The cell number was 5E6, the amount of plasmid was 30 μg to 50 μg, the voltage was 300 V, and the capacitance was 950 μF;

[0009] One or more of the following adjustments from 1) to 3) have been made:

[0010] 1) Capacitance increases or decreases;

[0011] 2) Increased cell number; and,

[0012] 3) Voltage reduction.

[0013] In some embodiments, the adjusting comprises increasing capacitance.

[0014] In some embodiments, the capacitance is increased to greater than 950 μF.

[0015] In some embodiments, the capacitance is increased to 1000 μF to 1100 μF.

[0016] In some embodiments, the adjusting further comprises one or more of: 2) increasing the number of cells and 3) decreasing the voltage.

[0017] In some embodiments, the cell number is increased to 5.5E6 ~11E6.

[0018] In some embodiments, the cell number is increased to 7.5E6-10E6.

[0019] In some embodiments, the voltage is reduced to 280 V to 290 V.

[0020] In some embodiments, the modulation includes an increase in cell number and a decrease in voltage.

[0021] In some embodiments, the cell number is 7.5E6-10E6, the voltage is 280 V-290 V, and the capacitance is 900 μF-1100 μF.

[0022] In some embodiments, the method satisfies one or more of the conditions i) to ii):

[0023] i) the length of the plasmid is greater than 10 kb; optionally, the length of the plasmid is 14.5 kb to 16 kb; and,

[0024] ii) The viability of the CHOZN cell line is >90%.

[0025] In some embodiments, the method comprises the steps of:

[0026] Centrifuge the electroporated cells to prepare cell pellets;

[0027] Resuspend the cell pellet using an electroporation buffer to prepare a cell suspension;

[0028] The cell suspension and the plasmid are mixed to prepare a plasmid / cell mixture, and electroporation is performed according to the adjusted electroporation conditions.

[0029] In some embodiments, the electroporation buffer is from a Mirus electroporation kit.

[0030] In some embodiments, the volume of the plasmid in the plasmid / cell mixture accounts for less than 10%.

[0031] The method for the CHOZN cell line used for large plasmid electroporation in the present application has the advantages of taking into account both electroporation efficiency and cell viability (for example, the 24h transfection efficiency can reach 72.3%, and the 24h viability can reach 63.11%), as well as high reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0033] Figure 1 Schematic diagram of the plasmid used in one embodiment of the present application.

[0034] Figure 2 These are the results of 4 groups of cells detected under a fluorescence microscope in one embodiment of the present application. DETAILED DESCRIPTION

[0035] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0037] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and the like, when used in other ways to mean "one or more", are also understood in the same way unless otherwise specified.

[0038] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0039] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0040] In this application, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent different technical solutions are related in terms of the content covered, but should not be understood as a limitation on the previous technical solution, nor can it be understood as a limitation on the protection scope of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0041] In this application, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. Unless otherwise specified, the descriptions of "optionally include", "optionally contain", etc. in this application, taking "optionally include" as an example, mean "may include or not include".

[0042] The terms "contain", "include" and "include" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0043] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0044] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form new technical solutions.

[0045] In the present application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0046] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0047] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0048] Unless otherwise specified, the term "CHOZN cell line" in this application refers to a cell line created in the art using Sigma's CompoZr zinc finger nuclease (ZFN) patented technology, which is genetically modified from parental CHO-K1 cells. In one embodiment of this application, the CHOZN cell line is derived from MERCK.

[0049] In the present application, "electroporation" is also referred to as "electroporation", which is a method of cell transfection. The principle is to use pulsed current to make holes in the cell membrane to introduce exogenous molecules into the cell.

[0050] In one aspect of the present application, a method for electroporating a CHOZN cell line with a plasmid is provided, wherein the electroporation conditions used are based on the following electroporation conditions:

[0051] The cell number was 5E6, the amount of plasmid was 30 μg to 50 μg, the voltage was 300 V, and the capacitance was 950 μF;

[0052] One or more of the parameters such as capacitance, number of cells and voltage are adjusted.

[0053] In some embodiments, one or more of the following adjustments 1) to 3) are made:

[0054] 1) Capacitance increases or decreases;

[0055] 2) Increased cell number; and,

[0056] 3) Voltage reduction.

[0057] The adjustment may be any one of 1) to 3), or a combination of any two of them, or may include 1) to 3) at the same time.

[0058] In some embodiments, the method includes or only increases the capacitance. For example, the capacitance is increased to greater than 950 μF as described above. In some embodiments, the capacitance is increased to 1000 μF to 1100 μF. In some embodiments, the capacitance is increased to 1010 μF, 1020 μF, 1030 μF, 1040 μF, 1050 μF, 1060 μF, 1070 μF, 1080 μF, 1090 μF, 1100 μF, or a range or value between any two values.

[0059] Without wishing to be bound by any theory, it has been found that when the capacitance is increased as described above, at least the 24h transfection efficiency and / or the 24h viability can be significantly increased.

[0060] In some embodiments, the adjustment further comprises: one or more of an increase in cell number and a decrease in voltage. For example, the adjustment comprises an increase in capacitance and an increase in cell number, or the adjustment comprises an increase in capacitance and a decrease in voltage, or the adjustment comprises an increase in capacitance, an increase in cell number, and a decrease in voltage.

[0061] In some embodiments, the cell number can be increased to 5.5E6 to 11E6, for example. In some embodiments, the cell number is increased to 5.5E6, 6E6, 6.5E6, 7E6, 7.5E6, 8E6, 8.5E6, 9E6, 9.5E6, 10E6, 10.5E6, 11E6, or a range or value between any two values. In some embodiments, the cell number is increased to 7.5E6 to 10E6.

[0062] In some embodiments, the voltage can be reduced to 280 V to 290 V. In some embodiments, the voltage is reduced to 280 V, 281 V, 282 V, 283 V, 284 V, 285 V, 286 V, 287 V, 288 V, 289 V, 290 V, or a range or value between any two values.

[0063] In some embodiments, the modulation in the method comprises a decrease in capacitance, a decrease in voltage, and an increase in cell number.

[0064] In this adjustment, the cell number can be increased to 5.5E6 to 11E6, for example. In some embodiments, the cell number is increased to 5.5E6, 6E6, 6.5E6, 7E6, 7.5E6, 8E6, 8.5E6, 9E6, 9.5E6, 10E6, 10.5E6, 11E6, or a range or value between any two values. In some embodiments, the cell number is increased to 7.5E6 to 10E6.

[0065] In this adjustment, the voltage may be reduced, for example, to 280 V to 290 V. In some embodiments, the voltage is reduced.

[0066] In some embodiments, the adjustment comprises an increase in cell number and a decrease in voltage to 280 V, 281 V, 282 V, 283 V, 284 V, 285 V, 286 V, 287 V, 288 V, 289 V, 290 V, or a range or value between any two values.

[0067] In this adjustment, the capacitance may be reduced to less than 950 μF. In some embodiments, the capacitance is reduced to less than 950 μF, but greater than 900 μF. In some embodiments, the capacitance is reduced to 949 μF, 948 μF, 947 μF, 946 μF, 945 μF, 944 μF, 943 μF, 942 μF, 941 μF, 940 μF, 939 μF, 938 μF, 937 μF, 936 μF, 935 μF, 934 μF, 933 μF, 932 μF, 931 μF, 930 μF, 929 μF, 928 μF, 927 μF, 926 μF, 925 μF, 924 μF, 923μF, 922 μF, 921 μF, 920 μF, 919 μF, 918 μF, 917 μF, 916 μF, 915 μF, 914 μF, 913 μF, 912μF, 911 μF, 910 μF, 909 μF, 908 μF, 907 μF, 906 μF, 905 μF, 904 μF, 903 μF, 902 μF, 901 μF, 900 μF, or a range or value between any two values.

[0068] In some embodiments, the cell number is 7.5E6-10E6, the voltage is 280 V-290 V, and the capacitance is 900 μF-1100 μF.

[0069] In some embodiments, the length of the plasmid in the method is greater than 10 kb, for example, greater than 10 kb, greater than 11 kb, greater than 12 kb, greater than 13 kb, greater than 14 kb, greater than 15 kb, greater than 16 kb, greater than 17 kb, greater than 18 kb, greater than 19 kb, greater than 20 kb, or even greater.

[0070] In some embodiments, the length of the plasmid is 14.5 kb to 16 kb. In some embodiments, the length of the plasmid is 14.5 kb, 14.6 kb, 14.7 kb, 14.8 kb, 14.9 kb, 15 kb, 15.1 kb, 15.2 kb, 15.3 kb, 15.4 kb, 15.5 kb, 15.6 kb, 15.7 kb, 15.8 kb, 15.9 kb, 16 kb, or a range or value between any two values.

[0071] The viability of the CHOZN cell line can be conventional in the art. In some embodiments, the viability of the CHOZN cell line is >90%.

[0072] Without wishing to be bound by any theory, it is believed that below 90%, the electroporation efficiency of cells will be significantly reduced.

[0073] The method for measuring the viability can be a conventional method in the art. In some embodiments, the viability is measured using a cell counter. The viability percentage is directly obtained from the cell counter without a calculation formula.

[0074] The steps of the method described in this application may be conventional steps in the art.

[0075] In some embodiments, the method comprises the steps of:

[0076] Centrifuge the electroporated cells to prepare cell pellets;

[0077] Resuspend the cell pellet using an electroporation buffer to prepare a cell suspension;

[0078] The cell suspension and the plasmid are mixed to prepare a plasmid / cell mixture, and electroporation is performed according to the adjusted electroporation conditions.

[0079] The method of preparing cell pellet by centrifugation can be conventional in the art. In some embodiments, the centrifugation condition is 1000 rpm, 5 minutes.

[0080] In some embodiments, the electroporation buffer is from a Mirus electroporation kit.

[0081] In some embodiments, the volume of the plasmid in the plasmid / cell mixture accounts for less than 10%.

[0082] In some embodiments, the instrument used to perform electroporation is a Bio-Rad electroporator.

[0083] Some examples are provided below.

[0084] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0085] Example 1 Screening for a suitable electroporation buffer

[0086] According to the survey results, three electroporation buffers were selected: Gene Pulser Electroporation buffer (Bio-Rad, 1652677), Medium (EX-CELL ®CD CHO Fusion (Sigma, 14365C-1000) + 6mM Glutamax-1 (Gibco, 35050-061)), Ingenio ® Electroporation Solution (Mirus, MIR50114) was used to electroporate CHOZN host cells (MERCK) using a Bio-rad, Gene pulser X cell system for the model large plasmid pCR-eGFP-HC molecule.

[0087] In this experiment, a CHOZN cell line was revived from WCB and cultured twice with complete growth medium (EX-CELL ® CD CHOFusion + 6mM Glutamax-1). The cells were then passaged to 0.5E6 / mL in 40mL and electroporated the next day. On the day of electroporation, a cell counter (Countstar, Altair) was used to count the cell density and viability. The minimum viability must be >90% for electroporation. The appropriate volume of cells required for transfection (5E6 cells / transfection) was calculated, the required volume of cell culture medium was taken out, centrifuged at 1000rpm for 5 minutes, and the cell pellet was resuspended in each electroporation buffer (the amount of electroporation buffer and the adjusted cell density refer to the instructions of each kit. When Medium is used as buffer, the cell density is adjusted to 6.25E6 / mL, and 800μL of cell suspension is taken for electroporation). The pre-cooled electroporation cup was taken out of the refrigerator and placed on ice to cool. When electroporating, the corresponding cell suspension (a total of ~5E6 cells) and the required amount of DNA (pCR-eGFP-HC plasmid, see the plasmid map) were taken out. Figure 1 ) in a sterile microcentrifuge tube (the volume of DNA in a single transfection should not exceed 10% of the total volume); transfer the DNA / cell mixture to a cooled electroporation cup and perform electroporation according to the set electroporation parameters; the electroporation program settings for 4 groups are as shown in Table 1, and group 1 is the control group for electroporation of empty plasmid (H2O). Transfer the electroporated cells to a T-25 cm 2 Resuspend the cells in a culture flask (try not to transfer white blood cell debris). Incubate the T-25 cm2 flask at 37°C and 5% CO2 for 24 (± 4) hours.

[0088] Table 1 Electroporation conditions and parameters of the four groups of cells

[0089]

[0090] After 24 hours of transfection, the results were observed under a fluorescence microscope. Figure 2As shown in the figure, under the same electroporation conditions, the fluorescence intensity of the electroporation system using the Mirus electroporation kit is significantly higher than that of the electroporation buffer of Medium and Bio-Rad.

[0091] After observation, the cells were gently blown to make a cell suspension, and samples were taken to measure the cell viability and FACS electroporation efficiency of the cell suspension. The test results are shown in Table 2 below. 24 hours after electroporation, under the same electroporation conditions, the FACS positive rate was 58.22% using the Mirus electroporation kit, which was significantly better than the Medium group (26.56%) and the Bio-Rad kit group (23.82%). The cell viability was 68.78%. This kit was subsequently selected to continue the electroporation parameter optimization.

[0092] Table 2 Cell viability and transfection efficiency of four groups of cells

[0093]

[0094] Example 2 Optimizing the electroporation procedure for large plasmids

[0095] Based on the excellent electroporation efficiency of the Mirus electroporation kit, the electroporation conditions were further optimized to take into account both electroporation efficiency and cell viability after electroporation. The cell number, plasmid addition amount, voltage and capacitance of the CHOZN cell suspension were used as factors, and cell electroporation efficiency and cell viability were used as response variables. A 4-factor 4-level orthogonal experiment was performed to explore the optimal electroporation conditions.

[0096] The specific experimental steps are as described in Example 1. The orthogonal experimental design scheme and experimental results are shown in Table 3 below. Group 17 is the current condition control, and Group 18 is the electroporated empty plasmid (H2O) control group.

[0097] The experimental results showed that after optimization, the electroporation efficiency of three groups was better than the current conditions (5E6 / 50μg / 300V / 950μF), namely 8 (5E6 / 50μg / 300V / 1000μF), 12 (7.5E6 / 50μg / 290V / 900μF), and 16 (10E6 / 50μg / 280V / 1100μF). Among them, the electroporation efficiency of group 8 was significantly better than the current conditions (72.3% vs 57.9%), and there was no significant difference in cell viability (63.1% VS 66.2%). The electroporation efficiency of groups 12 and 16 was not much different from the current conditions (60%, 59.7% VS 57.9%), and the cell viability was significantly better than the current conditions (76.43%, 76.3% VS 66.17%).

[0098] Table 3 4-factor 4-level orthogonal experimental design and results

[0099]

[0100] Example 3 Repeatability verification of the optimized procedure

[0101] The results of the three groups of electroporation schemes obtained in Example 2 were repeated and verified. The specific experimental steps were as described in Example 1. The specific test results are shown in Table 4 below. Group 17 is the control for the current conditions, and Group 18 is the control group for the electroporation of empty plasmid (H2O). The results in Table 4 show that the electroporation efficiency of the three groups of electroporation schemes that are better than the current conditions is not much different between the first and second electroporations, which are 72.3% VS 67%, 60% VS 60.2% and 59.7% VS 55.8%, respectively. The transfection efficiency is stable, and the cell viability is not much different for the two times. The 8 (5E6 / 50μg / 300V / 1000μF) with the highest transfection efficiency was selected as the preferred transfection program, and 12 (7.5E6 / 50μg / 290V / 900μF) and 16 (10E6 / 50μg / 280V / 1100μF) were selected as alternative programs.

[0102] Table 4 Cell viability and transfection efficiency of 6 groups of repeated electroporation procedures

[0103]

[0104] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0105] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A method for electroporating a CHOZN cell line using a plasmid, characterized in that: The electroporation conditions used are based on the following electroporation conditions: The cell number was 5E6, the amount of plasmid was 30 μg to 50 μg, the voltage was 300 V, and the capacitance was 950 μF; One or more of the following adjustments from 1) to 3) have been made: 1) Capacitance increases or decreases; 2) Increased cell number; and, 3) Voltage reduction.

2. The method according to claim 1, characterized in that The adjustment includes increasing the capacitance; optionally, the capacitance is increased to greater than 950 μF; optionally, the capacitance is increased to 1000 μF~1100 μF.

3. The method according to claim 2, characterized in that The adjustments also include: One or more of 2) increased cell number and 3) decreased voltage.

4. The method according to any one of claims 1 to 3, characterized in that The cell number increased to 5.5E6 ~11E6.

5. The method according to claim 4, characterized in that The cell number increased to 7.5E6~10E6.

6. The method according to any one of claims 1 to 3, characterized in that The voltage is reduced to 280 V~290 V.

7. The method according to claim 1, characterized in that The adjustment includes increasing the number of cells and decreasing the voltage; Optionally, the cell number is increased to 7.5E~10E6; Optionally, the voltage is reduced to 280 V~290 V.

8. The method according to claim 7, characterized in that The cell number is 7.5E6~10E6, the voltage is 280 V~290 V, and the capacitance is 900 μF~1100 μF.

9. The method according to any one of claims 1 to 8, characterized in that The method satisfies one or more of the conditions i) to ii): i) the length of the plasmid is greater than 10 kb; optionally, the length of the plasmid is 14.5 kb to 16 kb; and, ii) The viability of the CHOZN cell line is >90%.

10. The method according to any one of claims 1 to 9, characterized in that: It includes the following steps: Centrifuge the electroporated cells to prepare cell pellets; Resuspend the cell pellet using an electroporation buffer to prepare a cell suspension; The cell suspension and the plasmid are mixed to prepare a plasmid / cell mixture, and electroporation is performed according to the adjusted electroporation conditions; Optionally, the electrotransfer buffer is from a Mirus electrotransfer kit; Optionally, in the plasmid / cell mixture, the volume proportion of the plasmid is less than 10%.

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