CN116425857B - Glycosylation-free modified IL-15 and preparation method thereof - Google Patents
Glycosylation-free modified IL-15 and preparation method thereof Download PDFInfo
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- C07K14/52—Cytokines; Lymphokines; Interferons
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Abstract
The invention relates to the technical field of IL-15 preparation, in particular to an IL-15 modified by glycosylation and a preparation method thereof. The glycosylation-free modified IL-15 is characterized in that 3N-glycosylation sites in an IL-15 sequence are mutated, glycosylation modification can not be generated even if 293 cells are used for expression after mutation, the biological activity of the glycosylation-free modified IL-15 is 2-3 times higher than that of glycosylation-modified proteins, and the glycosylation-free modified IL-15 has the advantages of no animal source component, low endotoxin content, high biological activity and the like when being used for NK cell culture.
Description
Technical Field
The invention relates to the technical field of IL-15 preparation, in particular to an IL-15 modified by glycosylation and a preparation method thereof.
Background
IL-15, interleukin-15, is composed of 114 amino acids and is produced by monocytes and macrophages, and it can stimulate and activate B cells, induce NK cell differentiation, and chemotaxis T lymphocytes. Besides, IL-15 can exert a wide variety of roles in various tissues and cells outside the immune system, and it has been demonstrated that IL-15 can promote lymphocyte and NK cell proliferation and enhance its biological activity, and can also transform peripheral blood and spleen lymphocytes into LAK cells with high tumoricidal effect in vitro. IL-15 functions similarly to IL-2 but IL-2 causes T cells to excessively differentiate, forming aged T cells that have a weak killing capacity, and may induce activated T cell apoptosis, whereas IL-15 has an advantage in immune cell therapy in that it does not cause activated T cell apoptosis, and therefore IL-15 is often an important component in immune cell culture media. It has been reported that IL-15 itself has 3 glycosylation sites, and the physiological activity after glycosylation modification is lower than that of non-glycosylation modified IL-15 expressed by prokaryotes, but the prokaryotic expressed IL-15 has the following problems: endotoxin is lipopolysaccharide in cell wall of gram negative bacteria, and its toxic component is lipid A, which can cause fever, microcirculation disturbance, endotoxin shock and disseminated intravascular coagulation. The introduction of endotoxin is difficult to avoid when using prokaryotic cell expression purification. Accordingly, those skilled in the art have been working on a purification method for expression of non-glycosylation modified IL-15, which can obtain IL-15 expression with high purity, high activity, low endotoxin and meeting the requirements of GMP standards and clinical use standards.
Disclosure of Invention
Aiming at the problems and the defects existing in the prior art, the invention aims to provide the glycosylation-free modified IL-15 and the preparation method thereof, which can ensure high activity rate when being used for NK cell culture, can enable NK cells to be rapidly activated and greatly amplified, and is easier to ensure the quality and the bioactivity compared with prokaryotic expression IL-15, thereby being beneficial to controlling the quality of cell medicines.
In order to solve the technical problems, the invention provides the following technical scheme:
an aglycosylation-free modified IL-15, the amino acid mutation site of said IL-15 being N119D, N D and N160D, the signal peptide of said IL-15 being altered to an IgG heavy chain signal peptide.
Preferably, the amino acid sequence of IL-15 is shown in SEQ ID NO: 4.
Nucleotide sequences encoding the above IL-15.
Preferably, a Kozak sequence is added to the front end.
Preferably, the nucleotide sequence of IL-15 is shown in SEQ ID NO: shown at 5.
The preparation method of the IL-15 uses a transfection reagent PEI to transfect a target protein plasmid capable of expressing the IL-15 into 293 cells.
Preferably, the carrier used by the target protein plasmid is UCOE ® An expression vector.
Preferably, the final concentration of the target protein plasmid is 1.0. Mu.g/mL and the final concentration of the transfection reagent PEI is 3. Mu.g/mL.
Compared with the prior art, the invention has the following beneficial effects: the glycosylation-free modified IL-15 is characterized in that 3N-glycosylation sites in an IL-15 sequence are mutated, glycosylation modification can not be generated even if 293 cells are used for expression after mutation, the biological activity of the glycosylation-free modified IL-15 is 2-3 times higher than that of glycosylation-modified proteins, and the glycosylation-free modified IL-15 has the advantages of no animal-derived components, low endotoxin content, high biological activity and the like when being used for NK cell culture.
The non-glycosylation modified IL-15 has obvious promotion effect on the purity, amplification efficiency and killing potential of NK cells, can reduce the residual risk of host residual protein/nucleic acid possibly existing in prokaryotic expression by using human 293 cell expression, and adopts UCOE ® The expression vector can obtain continuous, stable and high-level expression of the target gene, makes the following mutations N119D, N D and N160D on the natural sequence of IL-15, and converts the original signal peptide (1-48) of the protein into the heavy chain signal peptide of the IgG antibody, thereby improving the secretion efficiency of the protein.
The action mechanism of the invention is as follows: such risks can be reduced by using prokaryotic expression where there may be residues of host residual protein/nucleic acid and 293 cell expression of human origin.
The potential 3 glycosylation site residues of IL-15 were mutated to render them incapable of glycosylation modification. Then, gene synthesis is carried out according to the designed sequence, and the gene synthesis is constructed to UCOE ® The expression vector contains a ubiquitin-like opening element, and the gene can effectively prevent gene silencing, and the target gene can be continuously and stably expressed at a high level no matter where the gene is integrated into chromatin.
The natural sequence of IL-15 is mutated into N119D, N D and N160D as follows, and the original signal peptide (1-48) of the protein is converted into the heavy chain signal peptide of the IgG antibody, so that the secretion efficiency of the protein can be improved.
The plasmid was transfected into 293 cells using PEI transfection, IL-15 in the supernatant was collected after cultivation and purified using ion exchange chromatography, after which the final protein purity was checked by SDS-PAGE and its effect on its viability and activity when used in NK cell culture was examined. Compared with commercial IL-15, the glycosylation-free modified IL-15 has obvious promotion effect on the purity, amplification efficiency and killing potential of NK cells.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is a diagram showing the result of SDS-PAGE of the protein according to example 1 of the present invention;
FIG. 2 is a chart showing the gray scale analysis of SDS-PAGE detection protein according to example 1 of the present invention;
FIG. 3 is a graph of total cells obtained from IL-15 of example 2 of the present invention;
FIG. 4 is a bar graph of the proportion of NK cells harvested by D14 in example 2 of the present invention;
FIG. 5 is a graph showing NK cell killing activity of example 2 of the present invention;
FIG. 6 is a graph of total cells obtained from IL-15 of example 3 of the invention;
FIG. 7 is a bar graph of the proportion of NK cells harvested by D14 in example 3 of the present invention;
FIG. 8 is a graph showing NK cell killing activity of example 3 of the present invention;
FIG. 9 is a graph of total cells obtained from IL-15 of example 4 of the present invention;
FIG. 10 is a bar graph of the proportion of NK cells harvested by D14 in example 4 of the present invention;
FIG. 11 is a graph showing NK cell killing activity of example 4 of the present invention;
FIG. 12 is an SDS-PAGE gel of the detection protein according to example 1 of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
And (3) a step of: experimental materials:
Opti-MEM Medium (Thermofiser Siemens, cat. No. A4124802), HEK 293 cells (Thermofiser Siemens, cat. No. A14527), transfection reagent PEI (Sigma, cat. No. 919012), UCOE ® Expression vector (Merck Merck, cat. No. 5048670001), anion exchange packing (Cytiva Situo, cat. No. 17531610), IL-15 gene synthesis, vector construction and plasmid extraction services were performed by Nanjin Kirschner Biotechnology, monoclonal CD16 antibody (Biolegend Barceix, cat. No. 302002), CD3 antibody (Biolegend Barceix, cat. No. 300439), CD56 antibody (Biolegend Barceix, cat. No. 362508), K562 cell line (ATCC (American type culture collection library), cat. No. CCL-243), RPMI-1640 medium (Prime, cat. No. PM 150110), CCK-8 reagent (MedChemexpress (Shanghai Hao's) and cat. No. HY-K0301), SCGM NK cell medium (CellGenix (Sadolis), cat. No. 20802-0500), well KBM581 lymphocyte serum-free medium (Corning (kaning) No. 88-243), CD-5-K-5, CD-15G-15 (GmbH-35), and CD-15-35 (GmbH-15).
And II: the experimental method comprises the following steps:
(1) Design of non-glycosylation modified IL-15 amino acid sequence and construction of expression vector:
the potential 3 glycosylation site residues of IL-15 were mutated to render them incapable of glycosylation modification. Then, gene synthesis is carried out according to the designed sequence, and the gene synthesis is constructed to UCOE ® The expression vector contains a ubiquitin-like opening element, and the gene can effectively prevent gene silencing, and the target gene can be continuously and stably expressed at a high level no matter where the gene is integrated into chromatin.
The natural sequence of IL-15 was mutated N119D, N D and N160D as follows, and the original signal peptide of the protein (amino acids 1-48 of the natural sequence of IL-15) was converted into the heavy chain signal peptide of the IgG antibody to increase the secretion efficiency of the protein.
IL-15 natural amino acid sequence is shown as SEQ ID NO:1 is shown as follows:
MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS；
the amino acid sequence of IL-15 after mutation is shown as SEQ ID NO:2 is shown as follows:
MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILADNSLSSNGNVTESGCKECEELEEKDIKEFLQSFVHIVQMFIDTS；
the amino acid sequence of the heavy chain signal peptide of the IgG antibody is shown as SEQ ID NO:3, shown in the following:
MEWSWVFLFFLSVTTGVHS；
the amino acid sequence of the IL-15 mutation of the original signal peptide of the protein converted into the heavy chain signal peptide of the IgG antibody is shown as SEQ ID NO:4, as follows:
MEWSWVFLFFLSVTTGVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILADNSLSSNGNVTESGCKECEELEEKDIKEFLQSFVHIVQMFIDTS；
the nucleotide sequence of the Kozak+IgG heavy chain signal peptide+IL-15 after mutation is shown in SEQ ID NO:5, as follows: 5'-gccaccatggaatggagctgggtgttcctgttctttctgtccgtgaccacaggcgtgcattctaactgggtgaatgtaataagtgatttgaaaaaaattgaagatcttattcaatctatgcatattgatgctactttatatacggaaagtgatgttcaccccagttgcaaagtaacagcaatgaagtgctttctcttggagttacaagttatttcacttgagtccggagatgcaagtattcatgatacagtagaaaatctgatcatcctagcagataacagtttgtcttctaatgggaatgtaacagaatctggatgcaaagaatgtgaggaactggaggaaaaagatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatcgatacttcttga-3', wherein the 1 st to 6 th nucleotide sequence is a Kozak sequence and the 7 th to 63 th nucleotide sequence is an IgG heavy chain signal peptide nucleotide sequence.
(2) HEK 293 cell subculture: suspension of HEK 293 cells at 0.6X10 6 The density of each living cell/mL is inoculated in a 125mL shaking flask, the culture system is 30 mL HEK 293 cell culture medium, and the incubator is set at 37 ℃ and 8% CO 2 The rotation speed was 95rpm (50 mm diameter-varying bed) or 125rpm (19 mm diameter-varying bed), and samples were taken every 72 hours, counted and passaged.
(3) Transient protein method: transfecting HEK 293 cells which are passaged for 3 times and have the activity rate of more than 90%, and adjusting the cell density to 2 multiplied by 10 before transfection 6 Each living cell/mL. Transfection testThe PEI agent is dissolved by injection water, the concentration is 1mg/mL, the PEI agent is used after being sterilized and filtered by a 0.22 mu M membrane, and the plasmid solution with known concentration is obtained by amplifying and purifying by outsourcing service after the target protein plasmid is obtained. And respectively adding target protein plasmid and transfection reagent PEI into 1200 mu L of Opti-MEM culture medium or sterile PBS for dilution, standing for 5 minutes at room temperature after mixing, slowly adding transfection reagent PEI diluent into the dilution containing target protein plasmid, mixing, incubating for 10-20 minutes at room temperature, adding the mixture of the two into suspension HEK 293 cell culture solution, wherein the final concentration of target protein plasmid is 1.0 mu g/mL, and the final concentration of transfection reagent PEI is 3 mu g/mL. Finally, transferring the cell culture mixture into an incubator for culture without changing liquid, and harvesting the protein after 72 hours. Protein production in the supernatant was detected by SDS-PAGE.
(4) Ion exchange chromatography: the protein purifier pump head was placed in buffers QA and QB and then first pumped, after which 3 column volumes of QB were used to rinse the anion exchange column, wash off the possibly remaining impurities on the column, and then 5 column volumes of QA were used to equilibrate the anion exchange column. The a pump was then placed into the collected supernatant protein sample to begin loading until the protein was fully bound to the ion exchange column, and care was taken to observe the a280 absorbance value and conductivity value of the uv detector during loading to ensure that the protein was bound to the ion exchange column. After loading, the pump A is placed in QA again, and the residual protein in the pump A is completely flushed into the ion exchange column, the absorption value of the ultraviolet detector A280 is kept stable, and then the target protein is ready to be eluted from the ion exchange column.
(5) Activity detection and NK cell culture: performing activity identification on the purified IL-15 according to a general rule 3524 recombinant human interleukin-2 biological activity assay in pharmacopoeia 2020 edition; NK cell culture and cell activity detection were performed according to the industry general methods. CTLL-2 cells were cultured in complete medium at 37℃in 5% CO 2 Culturing under conditions sufficient to collect CTLL-2 cells by centrifugation, washing 3 times with RPMI-1640 culture solution, and re-suspending in basic culture solution to give a culture solution containing 6.0X10 per 1ml 5 Cell suspension of individual cells at 37℃in 5% CO 2 And (5) standby under the condition. Adding standard solutionAnd a sample solution in 96-well cell culture plate, 50. Mu.L of cell suspension was added to each well, and the mixture was subjected to 5% CO at 37 ℃ 2 Culturing for 18-24 hours under the condition; then 20. Mu.L of MTT solution was added to each well, and the mixture was heated to 37℃with 5% CO 2 After culturing for 4-6 hours under the condition, 150 mu L of lysate is added into each hole, and the temperature is 37 ℃ and the concentration of CO is 5% 2 And preserving heat for 18-24 hours under the condition. All of the above operations are performed under aseptic conditions. Mixing the liquid in the cell plate, placing the liquid in an enzyme-labeled instrument, taking 630nm as a reference wavelength, measuring absorbance at the wavelength of 570nm, and recording the measurement result; fresh PBMCs were isolated from human whole blood using density gradient centrifugation for activated expansion of NK cells, and the isolated autologous plasma was kept for NK cell culture after inactivation at 56 ℃ for 30 min. On day 0, the isolated PBMC cell suspension was transferred to a prepared monoclonal CD16 antibody coated T75 flask and cell density was adjusted to 2X 10 using SCGM NK medium 6 Inoculating cells per mL, adding commercial IL-15-1 with a final concentration of 500IU/mL or commercial IL-15-2 with the same concentration or IL-15 obtained by the purification method of the invention with the same concentration, 10% heat-inactivated autologous plasma, placing into 37 ℃ and 5% CO 2 Cell activation was performed in the incubator. Transferring cells into new T75 culture flask 5 days after activation, adding NK culture medium containing IL-15 and 10% heat-inactivated autologous plasma, testing cell viability and density every 2-3 days, and maintaining cell density at 1-1.5X10 6 NK cells were harvested and examined on day 14 of culture at each/mL.
(6) NK cell viability and proliferation efficiency detection: total cell proliferation curve data were obtained by sampling and counting on days 0,5,7,9, 11, 14 of culture, respectively.
(7) NK cell identification: cell immunophenotyping assays were performed on day 8 and day 14 of cell culture. About 1.0X10 respectively 6 The individual cells were centrifuged at 2000rpm for 5 minutes, the supernatant removed and resuspended in PBS, and the fluorescently labeled CD3 antibody and CD56 antibody were used to incubate the PBS-resuspended cells under the following conditions: at 4℃for 15 min, then washed 2 times with 1mL of PBS, the supernatant discarded, and resuspended in 0.5 mL of PBS. And detecting and analyzing the treated cells by using a flow cytometer.
(8) NK fineCell killing activity assay (CCK 8 method): taking K562 cell strain with vigorous growth as target cell, washing with RPMI-1640 culture solution for 2 times, and adjusting cell density to 1×10 6 /ml. 50 μl of each well was plated in a 96-well plate. The NK cells cultured until day 14 were collected and the cell density was adjusted to 1X 10 6 Per mL, 2X 10 6 Per mL, 5X 10 6 Per mL, 1X 10 7 50 mu L of each/mL is taken and added into a 96-well plate, so that the effective target ratios are respectively as follows: 1:1, 2:1, 5:1, 10:1, into four groups, respectively experimental group (K562 + NK), control group (NK + RPMI-1640), maximum release group (K562 + RPMI-1640) and blank group (RPMI-1640), wherein the control group does not contain K562 cells, and the obtained absorbance is the value of K562 cell 0 killing; the maximum release group was subjected to K562 cell lysis, and the absorbance obtained was a value of 100% killing; the blank is medium, no cells are contained, and the absorbance obtained is the background value. Each group had 3 duplicate wells. After the inoculated cells are placed in an incubator for co-culture for 12 hours, 10 mu LCCK-8 reagent is added into each hole, and the cells are uniformly shaken for continuous culture for 3 hours. The enzyme label instrument detects the light absorption value at the wavelength of 450nm, and the killing rate is calculated according to the calculation mode: 1- [ experimental group (OD value) -control group (OD value) -blank group (OD value) ]/[ maximum release group (OD value) -blank group (OD value) ]. Times.100.
The present invention will be specifically described with reference to the following examples.
Example 1: expression purification of the aglycosylated modified rhIL-15 protein was performed entirely according to the above experimental procedure.
The potential three N-glycosylation residues N119, N143 and N160 in the human IL-15 sequence (SEQ ID No. 1) are mutated into aspartic acid (SEQ ID No. 2), the original signal peptide is changed into IgG heavy chain signal peptide (SEQ ID No. 3), and the sequence of Kozak is added to construct the UCOE ® In an expression vector.
The experimental results are shown in FIGS. 1-2: plasmid transfection experiments were performed as described above, and the proteins in the supernatant were collected and purified using ion exchange chromatography after 72 hours of transfection. IL-15 has a theoretical isoelectric point of 4.4, and when the pH of the environment in which the protein is positioned is greater than 4.4, the protein is negatively charged and can be combined with anion exchange with positive chargeAnd (3) a chromatographic column, wherein after the protein is combined to the anion exchange chromatographic column, the salt concentration is increased so as to elute the target protein from the chromatographic column, and finally, the non-glycosylation modified IL-15 with higher purity is obtained. As shown in FIG. 12, the molecular weight of the protein obtained by SDS-PAGE was about 12kDa, which is consistent with the theoretical molecular weight; the purity of the gray level analysis is more than 95 percent; IL-15 bioactivity is about 4.3X10 7 IU/mg, meets GMP requirements.
Example 2: to ensure consistent experimental conditions, the activity of IL-15 of the invention and two commercial IL-15 were tested simultaneously using the same pharmacopoeia method.
The activity of the IL-15 obtained by the invention is as follows: 4.3X10 7 IU/mg, commercial IL-15-1 Activity is 2.6X10 7 IU/mg, commercial IL-15-2 Activity is 2.1X10 7 In this example, the final concentration of each IL-15 addition was adjusted to be 500IU/mL based on the results of activity detection. Under the same conditions, the IL-15 provided by the invention has advantages of NK cell amplification, NK proportion and killing property, because the removal of glycosylation modification improves the physiological activity of IL-15 on NK activation and amplification.
The experimental results are shown in fig. 3: the final total cell expansion obtained by adding the IL-15 of the invention is slightly higher than that of the commercial IL-15-1 and the commercial IL-15-2.
The experimental results are shown in fig. 4: the addition of the IL-15 can increase the NK cell proportion (namely the proportion of the obtained D14 harvested CD3-CD56+ cells) by at least 10 percent, so that the NK cell proportion is obviously increased.
The experimental results are shown in fig. 5: the tumor cell killing activity of the IL-15 is higher than that of the control commercial IL-15 under the conditions that the effective target ratio is 1:1, 2:1 and 5:1.
Example 3: other experimental conditions were unchanged, and only the NK cell medium was replaced with corning KBM581 NK medium.
The experimental results are shown in fig. 6: the final total cell expansion obtained by adding the IL-15 of the invention is slightly higher than that of the commercial IL-15-1 and the commercial IL-15-2.
The experimental results are shown in fig. 7: the addition of the IL-15 can increase the NK cell proportion (namely the proportion of the obtained D14 harvested CD3-CD56+ cells) by at least 10 percent, so that the NK cell proportion is obviously increased.
The experimental results are shown in fig. 8: the tumor cell killing activity of the IL-15 is higher than that of the control commercial IL-15 under the conditions that the effective target ratio is 1:1, 2:1 and 5:1.
Example 4: other experimental conditions are unchanged, and only the NK cell culture medium is replaced by the Bao Ri Yi NK cell serum-free culture medium.
The experimental results are shown in fig. 9: the final total cell expansion obtained by adding the IL-15 of the invention is slightly higher than that of the commercial IL-15-1 and the commercial IL-15-2.
The experimental results are shown in fig. 10: the addition of the IL-15 can increase the NK cell proportion (namely the proportion of the obtained D14 harvested CD3-CD56+ cells) by at least 10 percent, so that the NK cell proportion is obviously increased.
The experimental results are shown in fig. 11: the tumor cell killing activity of the IL-15 is higher than that of the control commercial IL-15 under the conditions that the effective target ratio is 1:1, 2:1 and 5:1.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. An aglycosylation modified IL-15, wherein the amino acid mutation site of said IL-15 is N119D, N D and N160D, and the signal peptide of said IL-15 is changed to an IgG heavy chain signal peptide;
the front end of the nucleotide sequence of the IL-15 is added with a Kozak sequence;
the nucleotide sequence of the IL-15 is shown as SEQ ID NO: shown at 5.
2. The aglycosylated modified IL-15 according to claim 1, wherein the amino acid sequence of said IL-15 is as set forth in SEQ ID NO: 4.
3. The method for preparing IL-15 according to claim 1, wherein the target protein plasmid capable of expressing the IL-15 is transfected into 293 cells using the transfection reagent PEI.
4. The method for preparing IL-15 according to claim 3, wherein the vector used in the target protein plasmid is UCOE ® An expression vector.
5. The method for producing IL-15 according to claim 4, wherein the final concentration of the target protein plasmid is 1.0. Mu.g/mL and the final concentration of the transfection reagent PEI is 3. Mu.g/mL.
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