CN117230014A - Method for constructing CHO cell strain with high expression of protein difficult to secrete - Google Patents
Method for constructing CHO cell strain with high expression of protein difficult to secrete Download PDFInfo
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Abstract
The invention relates to the technical field of cell strains, and discloses a method for constructing a CHO cell strain with high expression of protein difficult to secrete, and constructing plasmids to obtain plvx-CMV-SRP14-IRES-Puro; simultaneously transferring two packaging plasmids psPAX2 and pMD2.G of plvx-CMV-SRP14-IRES-Puro and lentivirus into 293T cells, concentrating and purifying the obtained packaged lentivirus, infecting CHO cells, constructing a polyclonal CHO-SRP14 cell strain without a medicine sieve, screening with 20ug/ml puromycin until all cells of control CHO die (trypan blue staining method), obtaining a positive CHO-SRP14 cell strain group, and carrying out monoclonal selection: the monoclonal CHO-SRP14 cell strain is obtained by selecting monoclonal from the polyclonal CHO-SRP14 cells by an infinite dilution method and culturing.
Description
Technical Field
The invention relates to the technical field of cell lines, in particular to a method for constructing a CHO cell line with high expression of proteins difficult to secrete.
Background
Culturing mammalian cells to produce recombinant proteins in a highly efficient secretory manner is an ideal way to obtain recombinant proteins. CHO cells have become the first choice for mammalian cells because they function similar to human post-translationally modified proteins. However, the desire to continue to express certain proteins, such as immunoglobulins, at high levels remains a difficulty because continued expression results in reduced yields, possibly in the inability of the cell to handle the synthesis or processing of certain heterologous proteins, thereby causing cellular stress and cytotoxic effects.
Mammalian cells secrete proteins in a complex way involving the transfer of polypeptides from the cytoplasm to the lumen of the Endoplasmic Reticulum (ER) where they fold and assemble. The first step in protein secretion relies on a signal peptide, a specific sequence at the amino terminus of a polypeptide, that mediates association of the translation ribosome with Signal Recognition Particles (SRPs). The association of SRPs results in the cessation of translation of ribosomes and interfaces with the SRP Receptor (SR) on the endoplasmic reticulum membrane. Translation continues as the nascent polypeptide enters the endoplasmic reticulum lumen via the translocation channel. When the SRP complex is improperly functioning, the signal peptide is not removed, inclusion bodies precipitated in the endoplasmic reticulum accumulate, and low secretion levels result. For example, patent publication number CN111304243a, "construction method of CHO cell line for efficiently expressing exogenous protein, CHO cell line constructed and application thereof", discloses an expression plasmid for constructing the signal peptide sequence and exogenous protein gene, then transfecting CHO cells, and gradually screening monoclonal cells stably expressing exogenous protein, which cannot allow efficient processing and secretion of exogenous protein in CHO cells.
Disclosure of Invention
(one) solving the technical problems
Aiming at the defects of the prior art, the invention provides a method for constructing a CHO cell strain with high expression of protein difficult to secrete, which has the effects of high secretion and high expression.
(II) technical scheme
(1) Inserting the base sequence of the gene synthesized SRP14 into a polyclonal enzyme cutting site of a plasmid plvx-CMV-MCS-IRES-Puro to obtain an expression plasmid vector plvx-CMV-SRP14-IRES-Puro;
(2) Detecting the expression level of the secretory protein of the expression plasmid, then carrying out slow virus packaging on the expression plasmid, carrying out virus infection removal and culture on CHO cells in a logarithmic phase by using the virus after measuring the titer of the virus, changing the liquid after 2 days of culture, and culturing for 2-4d by using a new culture medium to obtain a polyclonal CHO-SRP14 cell strain without a medicine sieve;
(3) Culturing the polyclonal CHO-SRP14 cell strain without medicine sieve to logarithmic phase, counting living cells with trypan blue, passaging the cells, centrifuging, culturing with complete culture medium containing 20/ml puromycin until the non-infected virus CHO dies completely, and culturing the stable polyclonal CHO-SRP14 cell strain to logarithmic phase to obtain monoclonal CHO-SRP14 cell strain.
Further, the step of lentiviral packaging of step (2) of claim 1, comprising:
(1) The cells were spread the day before packaging
When the confluence rate of 293T cells reaches 80-90%, filtering supernatant, adding phosphate buffer solution for cleaning, adding trypsin, uniformly distributing in the whole culture dish by rotation, standing for 1-2min, blowing for 5-10 times by a pipetting gun, diluting cell suspension by using a culture medium, adding 10-15uL into a blood cell counting plate, and diluting cells to 0.35-0.45X10 6 Inoculating the obtained cell suspension to another dish, shaking uniformly, and then placing into an incubator for culturing;
(2) Viral supernatant
When the cell confluence rate reaches 70% -80% after 24-36h passage, carrying out transfection experiment, adding the cells into a complete culture medium under the same condition for 1-2h before transfection, respectively diluting plasmids and polyethyleneimine transfection reagent with the culture medium, standing for 5-10min, adding a polyethyleneimine transfection reagent tube into the plasmid tube, shaking and mixing for 15-20s, standing for 10-20min, adding the mixture into a culture dish, placing the culture dish into an incubator for culture, changing liquid after 16-18h, adding an equal amount of toxigenic culture medium, placing the cell supernatant into a centrifuge tube after 48-56h, centrifuging for 3-5min, and taking a supernatant filtering membrane;
(3) Virus concentration
The virus supernatant was transferred to a concentration tube and centrifuged for 10-15min at 3-6deg.C.
Further, the method of claim 1, wherein the disease is a disease of step (2)Conditions at the time of toxic infection of CHO cells are: 0.65 to 0.75X10 6 cells/ml,24 well plate, 500-550 ul/well.
Further, the conditions for culturing the polyclonal CHO-SRP14 cell strain without the drug screening in the step (3) to the logarithmic growth phase are as follows: the cell density is regulated to be 0.65 to 0.75X10 6 cells/ml, puromycin with a final concentration of 20ug/ml was added to a six-well plate at a speed of 150-180rmp at 37℃and 5% CO 2 Culturing for 2-3d.
Further, the method for culturing the stable polyclonal CHO-SRP14 cell strain in the step (3) to the logarithmic growth phase comprises the following steps: 500xg of relative 5 centrifugal force for 3-5min, re-suspending with new culture medium after centrifugation, performing cell count dilution to 0.45-0.55cell/120 ul/hole, plating with a 96-well plate, removing polyclonal holes after culturing for 4-5d, culturing for 7-8d, adding culture solution, culturing for 14-16d, and sucking out cells with a cell confluence degree of 40% -50% of the bottom area in the 96-well plate, culturing to a 24-well plate, and shaking plate culturing.
Further, the phosphate buffer salt solution of step (1) of claim 1 comprises Na 2 HPO 4 、KH 2 PO 4 Is a kind of the above-mentioned materials.
(III) beneficial technical effects
Synthesizing SRP14 gene of CHO by gene and constructing plasmid to obtain plvx-CMV-SRP14-IRES-Puro; co-transferring the constructed plasmid and the secretory protein difficult to express into CHO cells, and detecting the expression level of the secretory protein by taking the independent secretory protein as a control; simultaneously transferring two packaging plasmids psPAX2 and pMD2.G of plvx-CMV-SRP14-IRES-Puro and lentivirus into 293T cells to form packaged lentivirus; concentrating and purifying the slow virus, and then infecting CHO cells to construct a polyclonal CHO-SRP14 cell strain without a medicine sieve; then screening the medicine, screening with puromycin until all cells of the control CHO die (trypan blue staining method), and obtaining a positive CHO-SRP14 cell strain; the polyclonal CHO-SRP14 cells and the CHO cells are respectively transfected with the same secretory protein, and the expression quantity of the secretory protein is compared; monoclonal selection: selecting a monoclonal from the polyclonal CHO-SRP14 cells by an infinite dilution method, and culturing to obtain a monoclonal CHO-SRP14 cell strain; and respectively transfecting the secreted proteins by the monoclonal cell strains, and comparing the expression quantity of the secreted proteins to obtain the monoclonal cell strain with the highest expression quantity.
Drawings
FIG. 1 shows the expression levels of recombinant proteins produced by CHO-SRP14 cells and CHO cells.
FIG. 2 is a graph of viral titers.
FIG. 3 shows the expression level of recombinant proteins produced by CHO-SRP14 monoclonal cell line.
Detailed Description
Example 1
(1) Inserting the base sequence of the gene synthesized SRP14 into a polyclonal enzyme cutting site of a plasmid plvx-CMV-MCS-IRES-Puro to obtain an expression plasmid vector plvx-CMV-SRP14-IRES-Puro;
(2) Detecting the expression level of secretory protein, packaging with slow virus, measuring the titer of virus, and infecting CHO cells cultured to logarithmic phase with virus to obtain cell density of 0.65X10 6 culturing cells/ml, 24-well plate and 500 ul/well with new culture medium for 2d to obtain cell strain of polyclonal CHO-SRP14 without drug sieve;
(3) The cell line of polyclonal CHO-SRP14 without drug screening was cultured to the logarithmic growth phase with a cell density of 0.65X10 6 cells/ml, puromycin at a final concentration of 20ug/ml was added to a six-well plate at a speed of 150rmp at 37℃and 5% CO 2 Culturing for 2d, counting living cells by trypan blue, passaging the cells, centrifuging, replacing liquid, continuously culturing by using a complete culture medium containing 20ug/ml puromycin until the CHO not infected by virus is dead, culturing the obtained stable polyclonal CHO-SRP14 cell strain to logarithmic phase, culturing for 3min under the relative centrifugal force of 500xg, re-suspending by using a new culture medium after centrifuging, diluting the cell count to 0.45cell/120 ul/hole, plating by using a 96-well plate, removing the polyclonal hole after culturing for 4d, adding the culture solution after culturing for 7d, culturing for 14d, wherein the cell confluency is 50% of the bottom area in the 96-well plate, sucking out the cells, culturing to the 24-well plate, and culturing by shaking to obtain the monoclonal CHO-SRP14 cell strain.
The step of lentivirus packaging includes:
(1) The cells were spread the day before packaging
When the 293T cell confluence rate reached 80%, naH was added to the filtered supernatant 2 PO 4 /2HPO 4 Washing with buffer solution, adding trypsin, rotating uniformly in the whole culture dish, standing for 1min, blowing with a pipetting gun for 5 times, diluting cell suspension with culture medium, adding 10uL into a blood cell counting plate, and diluting cells to 0.35X10 6 Inoculating the obtained cell suspension to another dish, shaking uniformly, and then placing into an incubator for culturing;
(2) Viral supernatant
When the cell confluence rate reaches 70% after 24 hours of passage, carrying out transfection experiment, adding the cells into a complete culture medium under the same condition for 1 hour before transfection, respectively diluting plasmids and polyethyleneimine transfection reagents with the culture medium, standing for 5 minutes, adding a polyethyleneimine transfection reagent tube into the plasmid tube, shaking and mixing for 15 seconds, standing for 10 minutes, adding the mixture into a culture dish, placing the culture dish into an incubator for culture, changing liquid after 16-18 hours, adding an equal amount of toxigenic culture medium, placing the culture dish into the incubator again, placing cell supernatant into a centrifuge tube after 48 hours, centrifuging for 3 minutes, and taking a supernatant filtering membrane.
(3) Virus concentration
The virus supernatant was transferred to a concentration tube and centrifuged for 10min at 3 ℃.
Example 2
(1) Inserting the base sequence of the gene synthesized SRP14 into a polyclonal enzyme cutting site of a plasmid plvx-CMV-MCS-IRES-Puro to obtain an expression plasmid vector plvx-CMV-SRP14-IRES-Puro;
(2) Detecting the expression level of secretory protein, packaging with slow virus, infecting CHO cells with virus according to the titer of virus, culturing to logarithmic phase, and culturing at cell density of 0.75X10 6 The cells/ml,24 pore plates and 550 ul/pore are replaced again, and a new culture medium is used for culturing for 4d, so that a polyclonal CHO-SRP14 cell strain without a medicine sieve is obtained;
(3) Culturing cell lines of polyclonal CHO-SRP14 without drug screeningTo logarithmic growth phase, cell density was 0.75X10 6 cells/ml, puromycin at a final concentration of 20ug/ml was added to a six-well plate at a rotation speed of 180rmp at 37℃and 5% CO 2 Culturing for 3d, counting living cells by trypan blue, passaging the cells, centrifuging, replacing liquid, continuously culturing by using a complete culture medium containing 20ug/ml puromycin until the CHO not infected by virus is dead, culturing the obtained stable polyclonal CHO-SRP14 cell strain to logarithmic phase, culturing for 5min under the relative centrifugal force of 500xg, re-suspending by using a new culture medium after centrifuging, diluting the cell count to 0.55cell/120 ul/hole, plating by using a 96-well plate, removing the polyclonal hole after culturing for 5d, adding the culture solution after culturing for 8d, culturing for 16d, wherein the cell confluency is 60% of the bottom area in the 96-well plate, sucking out the cells, culturing to the 24-well plate, and culturing by shaking to obtain the monoclonal CHO-SRP14 cell strain.
The step of lentivirus packaging includes:
(1) The cells were spread the day before packaging
When the 293T cell confluence rate reached 90%, naH was added to the filtered supernatant 2 PO 4 /2HPO 4 Washing with buffer solution, adding trypsin, rotating uniformly distributed in the whole culture dish, standing for 2min, blowing with a pipetting gun for 10 times, diluting cell suspension with culture medium, adding 15uL into a blood cell counting plate, and diluting cells to 0.45X10 6 Inoculating the obtained cell suspension to another dish, shaking uniformly, and then placing into an incubator for culturing;
(2) Viral supernatant
And (3) when the cell confluence rate reaches 80% after 36h passage, carrying out transfection experiments, adding the cells into a complete culture medium under the same conditions for 2h before transfection, respectively diluting plasmids and polyethyleneimine transfection reagents with the culture medium, standing for 10min, adding a polyethyleneimine transfection reagent tube into the plasmid tube, shaking and mixing uniformly for 20s, standing for 20min, adding the mixture into a culture dish, placing the culture dish into an incubator for culturing, changing the liquid after 16-18h, adding an equivalent amount of toxigenic culture medium, placing the culture dish into the incubator again, placing cell supernatant into a centrifuge tube after 56h, centrifuging for 5min, and taking a supernatant filtering membrane.
(3) Virus concentration
The virus supernatant was transferred to a concentration tube and centrifuged for 15min at 6 ℃.
Example 3
(1) Inserting the base sequence of the gene synthesized SRP14 into a polyclonal enzyme cutting site of a plasmid plvx-CMV-MCS-IRES-Puro to obtain an expression plasmid vector plvx-CMV-SRP14-IRES-Puro;
(2) Detecting the expression level of secretory protein, packaging with slow virus, infecting CHO cells with virus according to the titer of virus, culturing to logarithmic phase, and culturing at cell density of 0.7X10 6 The cells/ml,24 pore plates and 520 ul/pore are replaced again, and a new culture medium is used for culturing for 3d, so that a polyclonal CHO-SRP14 cell strain without a medicine sieve is obtained;
(3) The cell line of polyclonal CHO-SRP14 without drug screening was cultured to the logarithmic growth phase with a cell density of 0.7X10 6 cells/ml, puromycin at a final concentration of 20ug/ml was added to a six-well plate at a rotation speed of 160rmp at 37℃and 5% CO 2 Culturing for 1.5d, counting living cells by trypan blue, passaging the cells, centrifuging, replacing liquid, continuously culturing by using a complete culture medium containing 20ug/ml puromycin until the CHO not infected by viruses is dead, culturing the obtained stable polyclonal CHO-SRP14 cell strain to logarithmic phase, culturing to obtain a monoclonal CHO-SRP14 cell strain by using a new culture medium after centrifugation for 4min under the relative centrifugal force of 500xg, performing cell counting dilution to 0.5cell/120 ul/hole, plating by using a 96-well plate, removing the polyclonal hole after culturing for 4.5d, adding the culture liquid after culturing for 7.5d, culturing for 15d, sucking out the cells, culturing to the 24-well plate, and culturing by shaking plate to obtain the monoclonal CHO-SRP14 cell strain.
The step of lentivirus packaging includes:
(1) The cells were spread the day before packaging
When the 293T cell confluency rate reaches 95%, naH is added into the filtered supernatant 2 PO 4 /2HPO 4 Washing with buffer solution, adding trypsin, rotating and uniformly distributing in the whole culture dish, standing for 1.5min, and transferringThe cell suspension was diluted with medium by blowing 8 times with a liquid gun, 12uL was added to a blood cell counting plate, and the cells were diluted to 0.4X10 6 Inoculating the obtained cell suspension to another dish, shaking uniformly, and then placing into an incubator for culturing;
(2) Viral supernatant
When the cell confluence rate reaches 75% after 30 hours of passage, carrying out transfection experiment, adding the cells into a complete culture medium under the same condition for 1.5 hours before transfection, respectively diluting plasmids and polyethyleneimine transfection reagents with the culture medium after preparation, standing for 8 minutes, adding the polyethyleneimine transfection reagent tube into the plasmid tube, shaking and mixing for 18 seconds, standing for 15 minutes, adding the mixture into a culture dish, placing the culture dish into an incubator for culture, changing liquid after 16-18 hours, adding an equal amount of toxigenic culture medium, placing the culture dish into the incubator again, placing cell supernatant into a centrifuge tube after 52 hours, centrifuging for 4 minutes, and taking a supernatant filtering membrane.
(3) Virus concentration
The virus supernatant was transferred to a concentration tube and centrifuged for 12min at 5 ℃.
Example 4
(1) Inserting the base sequence of the gene synthesized SRP14 into a polyclonal enzyme cutting site of a plasmid plvx-CMV-MCS-IRES-Puro to obtain an expression plasmid vector plvx-CMV-SRP14-IRES-Puro;
(2) Detecting the expression level of secretory protein, packaging with slow virus, infecting CHO cells with virus according to the titer of virus, culturing to logarithmic phase, and culturing at cell density of 0.7X10 6 The cells/ml,24 pore plates and 520 ul/pore are replaced again, and a new culture medium is used for culturing for 3d, so that a polyclonal CHO-SRP14 cell strain without a medicine sieve is obtained;
(3) Culturing the cell strain of polyclonal CHO-SRP14 without drug sieve to logarithmic phase with cell density of 0.75X106 cells/ml, adding 20ug/ml puromycin into six-well plate at rotation speed of 180rmp at 37deg.C and 5% CO 2 Culturing for 3d, counting living cells with trypan blue, passaging cells, centrifuging, replacing liquid, culturing with complete medium containing 24ug/ml puromycin until the CHO not infected by virus is dead, and stabilizingCulturing the fixed polyclonal CHO-SRP14 cell strain to logarithmic phase, under the relative centrifugal force of 500xg for 5min, re-suspending with new culture medium, cell count dilution to 0.55cell/120 ul/hole, 96-well plate plating, 5d culturing, eliminating polyclonal hole, 8d culturing, adding culture liquid, 16d culturing, cell confluency 60% of the bottom area in 96-well plate, sucking out the cells, culturing to 24-well plate, and shaking plate culturing to obtain monoclonal CHO-SRP14 cell strain.
The step of lentivirus packaging includes:
(1) The cells were spread the day before packaging
When the confluence rate of 293T cells reaches 95%, adding phosphate buffer solution into the filtered supernatant for cleaning, adding trypsin, uniformly distributing in the whole culture dish by rotation, standing for 1.5min, blowing 8 times by a liquid-transfering gun, diluting the cell suspension by a culture medium, adding 12uL into a blood cell counting plate, and diluting the cells to 0.4X10 6 Inoculating the obtained cell suspension to another dish, shaking uniformly, and then placing into an incubator for culturing;
(2) Viral supernatant
When the cell confluence rate reaches 75% after 30 hours of passage, carrying out transfection experiment, adding the cells into a complete culture medium under the same condition for 1.5 hours before transfection, respectively diluting plasmids and polyethyleneimine transfection reagents with the culture medium after preparation, standing for 8 minutes, adding the polyethyleneimine transfection reagent tube into the plasmid tube, shaking and mixing for 18 seconds, standing for 15 minutes, adding the mixture into a culture dish, placing the culture dish into an incubator for culture, changing liquid after 16-18 hours, adding an equal amount of toxigenic culture medium, placing the culture dish into the incubator again, placing cell supernatant into a centrifuge tube after 52 hours, centrifuging for 4 minutes, and taking a supernatant filtering membrane.
(3) Virus concentration
The virus supernatant was transferred to a concentration tube and centrifuged for 12min at 5 ℃.
Comparative example 1
(1) Detecting the expression level of secretion protein by plvx-CMV-MCS-IRES-Puro expression plasmid, packaging with slow virus, infecting CHO cells with virus according to virus titer, culturing to logarithmic phase, culturing in fine phaseCell density of 0.7X10 6 The cells/ml,24 pore plates and 520 ul/pore are replaced again, and a new culture medium is used for culturing for 3d, so that a polyclonal CHO-SRP14 cell strain without a medicine sieve is obtained;
(2) Culturing the cell strain of polyclonal CHO-SRP14 without drug sieve to logarithmic phase with cell density of 0.75X106 cells/ml, adding 20ug/ml puromycin into six-well plate at rotation speed of 180rmp at 37deg.C and 5% CO 2 Culturing for 3d, counting living cells by trypan blue, passaging the cells, centrifuging, replacing liquid, continuously culturing by using a complete culture medium containing 2ug/ml puromycin until the CHO not infected by virus is dead completely, culturing the obtained stable polyclonal CHO-SRP14 cell strain to logarithmic phase, culturing for 5min under the relative centrifugal force of 500xg, re-suspending by using a new culture medium after centrifuging, diluting the cell count to 0.55cell/120 ul/hole, plating by using a 96-well plate, removing the polyclonal hole after culturing for 5d, adding the culture solution after culturing for 8d, culturing for 16d, wherein the cell confluency is 60% of the bottom area in the 96-well plate, sucking out the cells, culturing to the 24-well plate, and culturing by shaking to obtain the monoclonal CHO-SRP14 cell strain.
The step of lentivirus packaging includes:
(1) The cells were spread the day before packaging
When the confluence rate of 293T cells reaches 95%, adding phosphate buffer solution into the filtered supernatant for cleaning, adding trypsin, uniformly distributing in the whole culture dish by rotation, standing for 1.5min, blowing 8 times by a liquid-transfering gun, diluting the cell suspension by a culture medium, adding 12uL into a blood cell counting plate, and diluting the cells to 0.4X10 6 Inoculating the obtained cell suspension to another dish, shaking uniformly, and then placing into an incubator for culturing;
(2) Viral supernatant
When the cell confluence rate reaches 75% after 30 hours of passage, carrying out transfection experiment, adding the cells into a complete culture medium under the same condition for 1.5 hours before transfection, respectively diluting plasmids and polyethyleneimine transfection reagents with the culture medium after preparation, standing for 8 minutes, adding the polyethyleneimine transfection reagent tube into the plasmid tube, shaking and mixing for 18 seconds, standing for 15 minutes, adding the mixture into a culture dish, placing the culture dish into an incubator for culture, changing liquid after 16-18 hours, adding an equal amount of toxigenic culture medium, placing the culture dish into the incubator again, placing cell supernatant into a centrifuge tube after 52 hours, centrifuging for 4 minutes, and taking a supernatant filtering membrane.
(3) Virus concentration
The virus supernatant was transferred to a concentration tube and centrifuged for 12min at 5 ℃.
Table 1 transfection liquid formulation
(1) 293T cells were plated the day before infection at a cell density of 0.2X10 6 cells/mL,500 ul/well, plated on 24 well plates. The confluency of cells on the day of infection was approximately 20-40%.
(2) Respectively adding 10ul of concentrated lentivirus into the holes, and slowly shaking and uniformly mixing.
(3) The genome of the cells was extracted 48h after infection.
(4) By qPCR detection, lentivirus titers were obtained.
TABLE 2 genome dilution 10-fold, ligand (ul)
| qPCR taq mix | PrimerF | PrimerR | Genome (genome) | H 2 O |
| 10 | 0.8 | 0.8 | 2 | 6.4 |
The rpp gene is taken as an internal reference gene, the WPRE gene carried by the slow virus is measured, and the titer of the virus is calculated relatively quantitatively. The virus titer calculation method is as follows: titer (Transducing Units per mL, TU/ml): TU mL-1= (2 (Ct 1-Ct 2). Times.Nx2.times.1000)/V
CT1 = inner reference primer amplification CT value CT2 = WPRE primer amplification CT value
N=number of cells at infection (approximately equal to 2 x 105 cells)
V = volume of virus solution at addition (ul) in combination with figure 2
Viral titer = 2.24e+8
FIG. 1 shows the method for testing the expression level of recombinant protein produced by CHO-SRP 14: CHO-SRP14 cells and CHO were cultured at 7X 10 6 cell/ml density inoculation of 30ml in 150ml shake flask, transfection of recombinant protein expression plasmid, sixth day after transfection of cell supernatant, purification of protein, detection of protein expression, comparison of SRP14 over-expressed CHO cells and CHO cells to recombinant protein production.
FIG. 3 shows the method for testing the expression level of recombinant proteins produced by CHO-SRP14 monoclonal cell strain: monoclonal CHO-SRP14 (1-200) cells and CHO were cultured at 7X 10 6 10ml of cells/ml are inoculated into 50ml shaking tubes, plasmids expressing recombinant proteins are transfected, cell supernatants are collected on the sixth day after transfection, proteins are purified, and the expression amount of the proteins is detected.
Claims (6)
1. A method for constructing a CHO cell strain with high expression of a protein difficult to secrete is characterized by comprising the following steps: the method comprises the following steps:
(1) Inserting the base sequence of the gene synthesized SRP14 into a polyclonal enzyme cutting site of a plasmid plvx-CMV-MCS-IRES-Puro to obtain an expression plasmid vector plvx-CMV-SRP14-IRES-Puro;
(2) Detecting the expression level of the secretory protein of the expression plasmid, then carrying out slow virus packaging on the expression plasmid, carrying out virus infection removal and culture on CHO cells in a logarithmic phase by using the virus after measuring the titer of the virus, changing the liquid after 2 days of culture, and culturing for 2-4d by using a new culture medium to obtain a polyclonal CHO-SRP14 cell strain without a medicine sieve;
(3) Culturing the polyclonal CHO-SRP14 cell strain without medicine sieve to logarithmic phase, counting living cells with trypan blue, passaging the cells, centrifuging, culturing with complete culture medium containing 20ug/ml puromycin until the non-infected virus CHO dies completely, and culturing the stable polyclonal CHO-SRP14 cell strain to logarithmic phase to obtain monoclonal CHO-SRP14 cell strain.
2. The method for constructing a CHO cell line highly expressing a difficult-to-secrete protein of claim 1, wherein: the step (2) of lentiviral packaging comprises the steps of:
(1) The cells were spread the day before packaging
When the confluence rate of 293T cells reaches 80-90%, filtering supernatant, adding phosphate buffer solution for cleaning, adding trypsin, uniformly distributing in the whole culture dish by rotation, standing for 1-2min, blowing for 5-10 times by a pipetting gun, diluting cell suspension by using a culture medium, adding 10-15uL into a blood cell counting plate, and diluting cells to 0.35-0.45X10 6 Inoculating the obtained cell suspension to another dish, shaking uniformly, and then placing into an incubator for culturing;
(2) Viral supernatant
When the cell confluence rate reaches 70% -80% after 24-36h passage, carrying out transfection experiment, adding the cells into a complete culture medium under the same condition for 1-2h before transfection, respectively diluting plasmids and polyethyleneimine transfection reagent with the culture medium, standing for 5-10min, adding a polyethyleneimine transfection reagent tube into the plasmid tube, shaking and mixing for 15-20s, standing for 10-20min, adding the mixture into a culture dish, placing the culture dish into an incubator for culture, changing liquid after 16-18h, adding an equal amount of toxigenic culture medium, placing the cell supernatant into a centrifuge tube after 48-56h, centrifuging for 3-5min, and taking a supernatant filtering membrane;
(3) Virus concentration
The virus supernatant was transferred to a concentration tube and centrifuged for 10-15min at 3-6deg.C.
3. The method for constructing a CHO cell line highly expressing a difficult-to-secrete protein of claim 1, wherein: the conditions for the infection of CHO cells with viruses in step (2) are: 0.65 to 0.75X10 6 cells/ml,24 well plate, 500-550 ul/well.
4. The method for constructing a CHO cell line highly expressing a difficult-to-secrete protein of claim 1, wherein: the conditions for culturing the cell strain of the polyclonal CHO-SRP14 without the medicine screen in the step (3) to the logarithmic growth phase are as follows: the cell density is regulated to be 0.65 to 0.75X10 6 cells/ml, puromycin with a final concentration of 20ug/ml was added to a six-well plate at a speed of 150-180rmp at 37℃and 5% CO 2 Culturing for 2-3d.
5. The method for constructing a CHO cell line highly expressing a difficult-to-secrete protein of claim 1, wherein: the method for culturing the stable polyclonal CHO-SRP14 cell strain in the step (3) to the logarithmic growth phase comprises the following steps: the relative centrifugal force of 500xg is 3-5min, new culture medium is used for resuspension after centrifugation, cell count dilution is carried out to 0.45-0.55cell/120 ul/hole, 96-well plates are used for plating, polyclonal holes are removed after 4-5d of culture, culture solution is added after 7-8d of culture, after 14-16d of culture, the cell confluence is 40% -50% of the bottom area in the 96-well plates, cells are sucked out and cultured to 24-well plates, and shaking plates are used for culture.
6. The method for constructing a CHO cell line highly expressing a difficult-to-secrete protein of claim 2, wherein: the phosphate buffer salt solution of the step (1) comprises Na 2 HPO 4 、KH 2 PO 4 Is a kind of the above-mentioned materials.
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