CN112175993B - Construction method and application of single-copy cell strain for stably and highly expressed protein - Google Patents

Abstract

The invention provides a construction method of a single copy cell strain of a stable high expression protein and application thereof, wherein the method is based on 293T cells, can be quickly and accurately inserted, and can stably express a target gene at a high level. The method establishes a 293T cell strain which is stable and high-efficiency in expression and can replace target genes by combining random insertion with a high-throughput screening strategy. Based on this cell line, we successfully expressed the adenosine A2A receptor protein (ADORA 2A) belonging to one of the GPCR families. By this method, repeated cassette recombination replacement can be performed, and the antibody or other protein gene to be expressed or optimized can be inserted into the cell gene cassette quickly and accurately without destroying the integrity of the gene cassette.

Description

Construction method and application of single-copy cell strain for stably and highly expressed protein

Technical Field

The invention relates to the technical field of biology, in particular to a construction method of a single copy cell strain for stably and highly expressing protein and application thereof.

Background

Antibody drug research has been vigorously developed in recent years, and the number of monoclonal antibodies entering advanced clinical studies has rapidly increased every year since 2010. One of the key technologies driving the development of therapeutic antibodies is the affinity maturation of antibodies in vitro. Technologies currently widely used for in vitro antibody affinity optimization include ribosome display, phage display, yeast display, bacterial display, and mammalian cell display technologies. Since antibodies screened by mammalian cell display technology are more closely related to human native proteins in terms of post-translational modifications of the protein and structure, the technology has received increasing attention. In the mammalian cell display technology, constructing a cell line with replaceable target genes and stably and efficiently expressed is an important link of the technology. Two strategies can be adopted for establishing a stable and high-expression cell strain: firstly, obtaining an ideal cell strain from a large number of cells in which target genes are randomly inserted into a genome through high-throughput screening; secondly, the target gene is integrated to an ideal preset site on the genome in a fixed-point insertion mode to obtain cell clone. There have been many studies reporting on the first strategy, while the second strategy has not been reported.

In order to continuously improve mammalian cell display systems and increase the utility of mammalian systems in improving the affinity, stability or other properties of antibodies or other proteins, several solutions have been proposed in the prior art: 1. constructing a stably expressed single copy gene display cell strain; 2. increasing the transcription and expression level of the target gene; 3. screening AID mutants with higher mutagenesis activity; 4. transient transfection of AID avoids a reduction in its mutagenic activity. However, low or no expression was observed in CHO cells, which are most commonly used in mammalian display systems, presumably because: the post-translational modification of the protein in CHO cells is different from that of human protein, and the different modifications can cause the difference of post-translational modification and stability. In addition, there are cytological functional studies of human proteins that need to be performed in human cells. In the prior art, no technical scheme and technical suggestion for constructing 293T cells which stably and efficiently express and can replace target genes by using a random screening strategy are provided.

Disclosure of Invention

The invention provides a construction method of a single copy cell strain of a stable high expression protein, which is based on 293T cells, can be quickly and accurately inserted and can stably express a target gene at a high level;

further, the method comprises the following steps:

(1) Randomly integrating an anti-TNF alpha antibody expression plasmid containing a replaceable gene cassette into the genome of a 293T cell, and performing flow sorting and enrichment to obtain the 293T-anti-TNF cell stably expressing an anti-TNF alpha single-chain antibody at a high level;

(2) Transfect pFRT-EGFP-LoxP (pCDFGL) to Replace plasmid and Replace anti-TNF alpha single-chain antibody gene displayed on the surface of a cell membrane (first round of recombination replacement, namely Replace 1), cells which are successfully replaced express GFP so as to emit green fluorescence, and cells which only express GFP are enriched through multi-round flow sorting (named as 293T-GFP);

(3) Replacing the GFP gene in the cells with pFRT-RFP-LoxP (pFRL) plasmid (second round of recombination replacement, namely Replace 2), and enriching the cells expressing only RFP (named 293T-RFP) by multi-round flow sorting;

(4) Screening excellent single cell clone of stable high expression protein by using a 96-pore plate single cell sorting screen;

(5) After excellent cloning is obtained, based on the replaceability of a cell strain inserted gene frame, a replacement plasmid pFRT-Ab-LoxP and the like carrying a foreign gene are accurately inserted into the gene frame so as to stably express a target protein for subsequent research (a third round of recombination replacement, namely Replace 3).

Further, hygromysin B antibiotic with the concentration of 100 mu g/mL is selected and used for screening 293T in the screening process;

further, in step (1), the expression plasmid pCDNA3.1-hygro-SV40-DHFR-SV40polyA-FRT-TNF-Loxp (pCD) was transfected into 293T cells seeded the previous day. The following day, hygromycin B antibiotic at a concentration of 100. Mu.g/mL was added for one week for selection, and then cultured in media without hygromycin antibiotic for a period of time to allow stable growth. 293T negative cells which are not successfully transfected with plasmids die gradually under antibiotic screening because of not having hyglomycin B resistance, cells which survive after pressurization have a large amount of anti-TNF alpha single-chain antibody molecules expressed on the cell membranes of the cells due to the successfully transfected plasmids, and after stable culture for a period of time, cell populations which stably express the antibody molecules at high levels are sorted out by a flow cytometer and named as 293T-anti-TNF cells (such as gate T in figure 1A) for subsequent multiple rounds of recombination replacement to increase the probability of obtaining single-copy cell strains.

Further, in step (2), the pFRT-EGFP-LoxP (pCDFGL) replacement plasmid and recombinase plasmid pF2AC (expressing Flp and Cre double recombinases) are co-transformed into 293T-anti-TNF cells, and the GFP gene is used for replacing the anti-TNF alpha antibody gene in the cells under the combined action of Flp and Cre enzymes. The replacement successful cell transcribes and expresses the GFP protein, so that the fluorescent light emits green; flow sorting of GFP fluorescence-expressing cells alone (gate G in FIG. 2B), designated 293T-GFP; and (3) carrying out two-round flow sorting on the sorted 293T-GFP cells after carrying out long-time amplification culture under the antibiotic-free pressure, and enriching 0.5% of the cells with the strongest GFP fluorescence in each round of sorting until a fluorescence signal of a marked anti-TNF alpha antibody cannot be detected, so as to obtain a dominant cell population with higher protein expression level and more stable and uniform expression.

Further, in step (3), the pFRT-RFP-LoxP (pFRL) replacement plasmid and the recombinase plasmid pF2AC are co-transformed into 293T-GFP cells, and the RFP gene replaces GFP in the cells under the combined action of Flp and Cre enzyme. The replacement of successful cells transcribes and expresses the RFP protein so that the cells fluoresce spontaneously in red; flow sorting enriched RFP-only fluorescent cells (gate R in fig. 3B), named 293T-RFP; and (3) carrying out two-round flow sorting on the sorted 293T-RFP cells after long-time amplification culture under the antibiotic-free pressure, and enriching 0.5% of the cells with the strongest RFP fluorescence in each round of sorting until no GFP fluorescence signal is detected.

Further, in step (4), after enriching the strongest RFP fluorescence signal by two rounds of flow sorting, single cell sorting was performed in 96-well plates, each well sorting only one cell, and 3 96-well plates were divided. After the cells in the 96-well plate grow for a week in culture, the wells are observed one by one under a microscope to ensure that a plurality of cells grow in a cloning manner, or the wells are not sorted into cells, or the growth state of the monoclonal cells is not good, and the monoclonal cell strains with good growth state are kept alive for numbering. After the monoclonal cell strains are cultured and amplified, the uniformity and the stability of each monoclonal expression RFP signal are detected in a flow mode, and a plurality of monoclonal cell strains which are ranked at the top are selected from the detected signals for subsequent experiments.

Further, in step (5), the monoclonal cell line obtained in the above step was co-transfected with the pFRT-foreign protein-loxP replacement plasmid and the pF2AC recombinase plasmid, and RFP in the cells was replaced with a foreign gene by the action of recombinase. 293T monoclonal cells with foreign proteins displayed on the surfaces of the flow sorting cells.

Preferably, the foreign protein is an ADORA2A protein.

Advantageous effects

In the research, a stable and high-efficiency expression monoclonal cell strain capable of replacing target genes is obtained by combining site-specific insertion with a high-throughput screening strategy. The unique application of the cell lines obtained using the method of the present application compared to cell lines obtained earlier in the art is: (1) Can be used as a vector for researching the functions of human proteins (researching the cell biological functions of expressed proteins); (2) Studying that some human proteins can only be expressed in human cells, for example, some human GPCRs cannot be successfully expressed in CHO cells and can be expressed in human cells; (3) The human cell line expression full-length antibody is closer to a human body self antibody in structure and modification, and is more suitable for development of therapeutic antibodies. (4) Repeated cassette recombination substitutions can be made, and the antibody or other protein gene to be expressed or optimized can be inserted into the cellular gene cassette quickly and accurately without disrupting the integrity of the gene cassette.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for the embodiments will be briefly described below.

FIG. 1 is a schematic diagram of the flow chart of detecting the antibody display level and constructing cell lines; (A) As shown, cell populations stably and highly expressing antibody genes in gate T were sorted out. (B) And (3) constructing a single copy cell strain of the stable high expression protein. Firstly, transferring an expression plasmid containing an anti-TNF alpha single-chain antibody (expressed as TNF) into 293T cells, randomly integrating the expression plasmid into a cell genome, and carrying out flow sorting on the cells (named as 293T-anti-TNF) which stably and highly express the anti-TNF alpha; then, pFRT-EGFP-LoxP (pCDFGL) exchange plasmids are used for replacing an anti-TNF alpha single-chain antibody gene (Replace 1) displayed on the surface of a cell membrane, and the cells which only express GFP (named 293T-GFP) are enriched by flow sorting; then pFRT-RFP-LoxP (pFRL) replaces plasmid to Replace GFP gene (Replace 2) in the cells, and the cells which only express RFP are enriched by flow sorting (named as 293T-RFP); then, performing single cell sorting on a 96-pore plate to screen excellent single cell clone of the stable high-expression protein; after obtaining an excellent clone, the antibody plasmid pFRT-Ab-LoxP was inserted into the gene frame accurately to express the target protein (Replace 3) stably.

Fig. 2 is a schematic diagram of the first round of reassembly and GFP sorting, wherein (a) the schematic diagram of the first round of reassembly: after 293T-anti-TNF cells co-transform pCDFGL and pF2AC plasmids, under the action of Flp and Cre recombinase, anti-TNF alpha antibody gene (Ab for short) is replaced by GFP gene, and the cells transcribe and express GFP protein, so that the cells generate green fluorescence. (B) One group of GFP positive cells contains only GFP fluorescence, the other group contains double fluorescence signals of GFP and a labeled anti-TNF alpha antibody, and the cells (gate G) only containing GFP fluorescence are enriched through flow sorting and named as 293T-GFP.

Fig. 3 is a schematic diagram of the second round of reconfiguration and RFP sorting, wherein, a) the schematic diagram of the second round of reconfiguration: after 293T-GFP cells co-transform pCDFGL and pF2AC plasmids, under the action of Flp and Cre double recombinases, GFP gene is replaced by RFP gene, and cells are transformed to express RFP protein, so that cells generate red fluorescence. (B) RFP positive cells one group contains only RFP fluorescence, the other group contains RFP and GFP double fluorescence signal, flow sorting enrichment RFP fluorescence only cells (gate R), named 293T-RFP.

FIG. 4 is a schematic diagram of single cell sorting and detection in a 96-well plate.

FIG. 5 is a graph showing the detection of RFP expression levels of monoclonal cells in a 96-well plate, in which 292T cells in the upper left corner are used as negative controls (controls), the horizontal axis represents the RFP protein expression level, and the vertical axis represents the number of cells. The finer the peak of the histogram represents the more stable and uniform the RFP expression. Among them, clones S39 and S38 showed the highest (100%) expression level of RFP and the most uniform expression, and 3 additional times (S78, S16 and S7).

FIG. 6 shows flow sorting and detection of ADORA2A, in which (A) cells are divided into two parts, most of which are mainly displaying RFP protein, and only a few of which successfully replace RFP and display ADORA2A on the cell surface (as shown in gate A), and the cells in the sorting gate A are enriched. (B) flow-detecting the sorting result of gate A: the anti-FLAG-APC flow antibody was used to label the cell-expressed FLAG protein, and as shown on the abscissa, the majority of cells with APC signal detected (77%) was observed, indicating that the 293T-S39 monoclonal cells displaying ADORA2A protein on the cell surface reached 77%.

Detailed Description

In order to make the technical solutions of the present invention better understood, those skilled in the art will now describe the present invention in further detail with reference to the accompanying drawings.

Example 1 construction of 293T cell-based protein high expression System and optimization thereof

Transient expression plasmid mainly used for expressing anti-TNF alpha single-chain antibody in experiment

pCDNA3.1-hygro-SV40-DHFR-SV40polyA-FRT-TNF-Loxp (pCD for short), double recombinase expression plasmid (pF 2 AC) and pFRT-EGFP-LoxP (pCDFGL) and pFRT-RFP-LoxP (pFRL) replacement plasmids for two rounds of recombination replacement (Chen C, li N, ZHao Y, et al, coupling recombination-mediated cassette exchange with fluidic affinity for antibiotic affinity purification, 2016,113 (1): 39-51).

Used in experimentsThe cell line is 293T cell (human embryonic kidney epithelial cell line) and various subcellular clones constructed on the basis of the cell line: DMEM medium (Invitrogen, CA) containing 10% calf serum (Hyclone) and 100U/mL diabody (penicillin and streptomycin) was used at 37 ℃ with 5% CO ₂ Adherent culture in an incubator.

In the present study, the plasmid transfected 293T cells all adopt polyethyleneimine (PEI for short) transfection reagent, and the specific transfection process is as follows:

1. the 293T cells were trypsinized the day before transfection and counted in a 10cm dish using 200 ten thousand cells of the common double-resistant-free DMDM medium to achieve approximately 80% confluence on the next day of transfection;

2. mu.g of the plasmid to be transfected and 40. Mu.L of PEI were mixed with 1200. Mu.L of serum-free medium Opti-MEM (Gibco) equilibrated at 37 ℃ in advance, and the mixture was gently mixed by vortexing and left at room temperature for 25 minutes, each for a short period of 5 minutes.

3. The old medium in the plate is sucked away, the Opti-MEM is gently rinsed twice, the mixture of the plasmid to be transfected and the PEI is slowly added into each plate, the Opti-MEM is supplemented to 5 mL/plate, and the plate is replaced by the ordinary double-antibody-free DMEM medium after 5 to 6 hours.

4. The next day after transfection, the cells were expanded and screened by adding the corresponding antibiotics as required.

5. Detecting and sorting by a flow cytometer, wherein a flow antibody for marking the display level of the anti-TNF alpha single-chain antibody is PE-conjugated anti-HA antibody (Abcam), and the dilution ratio is 1; the flow antibody that marks the display level of ADORA2A protein is APC-conjugated anti-FLAG antibody (Abcam), 1; the plasmids pFRT-EGFP-LoxP (pCDFGL) and pFRT-RFP-LoxP (pFRL) can perform autofluorescence after substitution expression, and respectively are Green Fluorescence (GFP) and Red Fluorescence (RFP). The labeled cells were detected or sorted using a FACS AriaIII (BD) flow cytometer. Flow sorting enriched cells stably expressing anti-TNF alpha (named 293T-anti-TNF).

Optimization of transfection conditions

1.1 concentration optimization of hygromycin (hyglomycin B antibiotic) screening

First, to find a suitable concentration for hygromycin (hygromycin B antibiotic) screening, we set up the following experiment: meanwhile, 293T cells with the density of 1 ten thousand in 5 plates are planted, the culture volume is 4mL, hygromysin B antibiotics with the concentration of 0, 50, 100, 200 and 250 mu g/mL are respectively added in the next day, the growth state of the cells is observed and recorded every day, and the cells are continuously cultured for 7 days. 293T cells placed under 100. Mu.g/mL antibiotic selection conditions are exemplified: the growth state of the cells on the first day is good, and the adherence is complete; from the second day, the growth rate of the cells is slower than that of the normally cultured cells (0 mu g/mL), the cells gradually fall off to form spherical floating death, and the cell density is less than that of the normally cultured cells (0 mu g/mL); only about 15% of the cells were observed to survive on day 5, with intact adherent walls; 5% of the cell density survived on day 6; on day 7, no intact adherent cells were observed in the dish, all falling off as spheres and losing luster. In contrast, about 10% of cells survived by 7 days after the addition of cells with antibiotic concentration less than 100. Mu.g/mL; the cells with the antibiotic concentration higher than 100 mug/mL are obviously floated and shed on the 2 nd day, more than 90% of the cells float and die on the 4 th day, and no viable cells are observed on the 5 th day; normal cultured cells (0. Mu.g/mL) reached full cell density after 7 days. In view of the above, we selected hygromysin B antibiotic at a concentration of 100. Mu.g/mL to screen 293T.

1.2 optimization of pressure screening strategies

Subsequently, we transfected pCDNA3.1-hygro-SV40-DHFR-SV40polyA-FRT-TNF-Loxp (pCD for short) expression plasmid into 293T cells seeded the day before. The following day, hygromycin B antibiotic at a concentration of 100. Mu.g/mL was added for one week for selection, and then cultured in media without hygromycin antibiotic for a period of time to allow stable growth. 293T negative cells which are not successfully transfected with plasmids die gradually under antibiotic selection because they do not have hygromysin B resistance, cells which survive after pressurization are sorted out by a flow cytometer from cell populations which stably express antibody molecules at high levels after a period of stable culture due to the expression of a large amount of anti-TNF alpha single-chain antibody molecules on the cell membranes of the cells which are successfully transfected with plasmids, and the cell populations are named as 293T-anti-TNF cells (such as gate T in figure 1A) and are used for subsequent multiple rounds of recombination replacement to increase the probability of obtaining single-copy cell strains.

Example 2 first round reassembly

A pFRT-EGFP-LoxP (pCDFGL) replacement plasmid and a recombinase plasmid pF2AC (expressing Flp and Cre double recombinases) are cotransformed into 293T-anti-TNF cells, and under the combined action of Flp and Cre enzymes, a GFP gene is used for replacing an anti-TNF alpha antibody gene in the cells. The cells that were successful in the replacement transcriptionally expressed the GFP protein, and thus spontaneously fluoresced green, as shown in fig. 2A. The first round of reconstruction replacement is detected in a streaming mode, and the result shows that: the ordinate represents the display level of anti-TNF α antibody and the abscissa represents the replacement level of GFP protein. After replacement, the GFP positive cells are divided into two groups, one group only contains GFP fluorescence, and the other group contains double fluorescence signals of GFP and marked anti-TNF alpha antibody (which indicates that some cells contain a plurality of replacement sites, and if the anti-TNF alpha antibody gene of some sites is not replaced by the GFP gene, the anti-TNF alpha antibody and GFP protein can be simultaneously expressed in the cells). Cells expressing only GFP fluorescence (gate G in FIG. 2B) were flow sorted and named 293T-GFP.

And (3) carrying out two-round flow sorting on the sorted 293T-GFP cells after carrying out long-time amplification culture under the antibiotic-free pressure, and enriching 0.5% of the cells with the strongest GFP fluorescence in each round of sorting until a fluorescence signal of a marked anti-TNF alpha antibody cannot be detected, so as to obtain a dominant cell population with higher protein expression level and more stable and uniform expression.

Example 3 second round of reassembly

The plasmid pFRT-RFP-LoxP (pFRL) and the plasmid pF2AC as recombinase were co-transfected into 293T-GFP cells in the same manner as in example 2, and the RFP gene was substituted for GFP in the cells by the combined action of Flp and Cre enzymes. Transcription of RFP protein by the cells with successful replacement resulted in spontaneous red fluorescence by the cells, as shown in fig. 3A. And (3) detecting a second round of recombination replacement by streaming, wherein the result shows that: the abscissa represents the expression level of GFP protein and the ordinate represents the level of RFP replacement. RFP positive cells were separated into two clusters, one containing only RFP fluorescence and the other containing dual fluorescence of GFP and RFP, and the cells that only RFP fluorescence were enriched by flow sorting (gate R in FIG. 3B), named 293T-RFP.

In order to obtain a dominant cell population with stronger protein expression capability and more stable expression, two rounds of flow sorting are carried out on the 293T-RFP cells after long-time amplification culture under antibiotic-free pressure, and 0.5% of the cells with the strongest RFP fluorescence are enriched in each round of sorting until no GFP fluorescence signal is detected.

Example 4 Single cell sorting and detection of 96-well plates

After enrichment of the strongest RFP fluorescent signal by two rounds of flow sorting, single cell sorting in 96-well plates was performed. The abscissa represents the expression level of RFP and the ordinate represents the residual GFP signal. Cells expressing the strongest RFP fluorescence (fig. 4, gate R) were sorted into 96-well plates, with only one cell sorted per well, for a total of 3 96-well plates. After the cells in the 96-well plate are cultured and grown for one week, the wells are observed one by one under a microscope, and the wells with a plurality of cell clones growing, or the wells without sorted cells, or the growth state of the monoclonal cells is not good are excluded, and finally 86 monoclonal cell strains with good growth state are remained to survive (the numbers are from S1 to S86).

After the 86 monoclonal cell strains are cultured and amplified, the uniformity and stability of the RFP expression signal of each monoclonal are detected by flow, and the proportion of cells which express RFP at high level and are expressed more uniformly is found to be most. We picked the top 5 monoclonal cell lines from the pool and the results are shown in fig. 5. 292T cells in the upper left corner of FIG. 5 served as negative controls (Control), the abscissa represents the expression level of RFP protein, and the ordinate represents the number of cells. The finer the peak of the histogram represents the more stable and uniform the RFP expression. Among them, clones S39 and S38 showed the highest (100%) and most uniform expression of RFP, and 3 clones (S78, S16 and S7) were selected. Since the growth rate of the S39 cell strain from the 96-well plate culture to the 6-well plate is faster than that of the S38 clone, the 293T-S39 clone selected was selected for subsequent experiments.

Example 5 construction of stably expressing GPCR cells

The pFRT-ADORA2A-loxP replacement plasmid (constructed in the laboratory, see Chen C, li N, ZHao Y, et al. Coupling recombinant enzyme-mediated cassette exchange with a host hypermutation for the antibody affinity purification in CHO cells [ J ]. Biotechnology & Bioengineering,2016,113 (1): 39-51.) and pF2AC recombinase plasmid were co-transformed into 293T-S39 monoclonal cell strain, and the RFP in the cells was replaced by the ADORA2A gene under the action of the recombinase. Among them, ADORA2A is an adenosine a2A receptor, and the protein encoded by this gene is one of several receptor subtypes of adenosine, and is also a member of the G protein-coupled receptor family (GPCR). Its activity is mediated by G protein, and through activating adenylate cyclase, it induces the synthesis of cAMP in cell and participates in the physiological change of cell and the occurrence of many diseases. The reason we chose ADORA2A as a model protein expression is that: it is not expressed in CHO-puro cell lines (Chen C, li N, ZHao Y, et al, coupling recombinant-mediated cassette exchange with therapeutic hypermutation for antibody affinity mapping in CHO cells [ J ]. Biotechnology & Bioengineering,2016,113 (1): 39-51.) constructed earlier in this laboratory. The ADORA2A gene is a recombinant gene (SP-ADORA 2A-FLAG-TM) containing a signal peptide, a FLAG tag and a transmembrane sequence, which is expressed by transcription and then shows GPCR protein on the cell surface under the action of the signal peptide and the transmembrane sequence, and the expression level of ADORA2A protein can be directly reflected by labeling the cell with anti-FLAG-tag flow antibody (anti-FLAG-APC, 1. As shown in fig. 6A, the ordinate represents the expression level of RFP protein and the abscissa represents the replacement level of ADORA 2A. The cells are divided into three parts, most of the cells mainly display RFP, and only a few cells successfully replace RFP gene to display ADORA2A (gate A) on the cell surface. Flow sorting of display cells in gate a, flow detection of sorting results for ADORA2A protein expressing cells after expanded culture, as shown in figure 6B: the ordinate represents the expression level of RFP and the abscissa represents the display level of ADORA2A protein expressed by the cells after successful replacement of RFP. The anti-FLAG-APC flow antibody is used for marking the FLAG tag protein expressed by the cells, the majority of the cells with APC signals is detected (77%), representing that the 293T-S39 monoclonal cells with ADORA2A protein displayed on the cell surface reach 77%, and the 293T-S39 cell strain screened by the inventor successfully displays the adenosine A2A receptor protein (ADORA 2A) belonging to one GPCR family.

The results of the above experiments show that the human GPCR gene ADORA2A can be rapidly and accurately inserted into the gene frame of 293T-S39 cells, and the expression of ADORA2A protein reaches 77% after flow sorting. The inability to express the gene using CHO-puro cells, however, demonstrates the value of 293T-S39 cells obtained by screening to contain alternative gene cassettes for expression of human GPCRs.

All publications mentioned in this specification are herein incorporated by reference in their entirety. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the disclosure as shown in the specific embodiments without departing from the spirit or scope of the disclosure as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Although the disclosure has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present disclosure as defined by the appended claims.

Claims (3)

1. A construction method of a single copy cell strain of a stable high expression protein is characterized in that the method is based on 293T cells, can be inserted rapidly and accurately, and can express a target gene at a stable high level; the method comprises the following steps:

(1) Randomly integrating an anti-TNF alpha antibody expression plasmid containing a replaceable gene cassette into the genome of a 293T cell, and carrying out flow sorting and enrichment to obtain the 293T-anti-TNF cell stably expressing an anti-TNF alpha single-chain antibody at a high level;

(2) Transfect pFRT-EGFP-LoxP and Replace plasmid pCDFGL and Replace the anti-TNF alpha single-chain antibody gene displayed on the surface of the cell membrane, the first round of recombination replacement is Replace, namely Replace 1; cells successfully replaced express GFP to emit green fluorescence, and the cells only expressing GFP are enriched through multi-round flow sorting and named as 293T-GFP;

(3) pFRT-RFP-LoxP is used for replacing plasmid pFRL to Replace GFP gene in cells, and the second round of recombination replacement is Replace, namely Replace2; enriching cells only expressing RFP by multi-round flow sorting, and naming as 293T-RFP;

(4) Screening out excellent single cell clone of stable high expression protein by using 96-pore plate single cell sorting;

(5) After excellent cloning is obtained, based on the replaceability of a cell strain inserted into a gene frame, a replacement plasmid pFRT-Ab-LoxP and the like carrying a foreign gene are accurately inserted into the gene frame so as to stably express a target protein for follow-up research, and the third round of recombination replacement is Replace, namely Replace 3;

in step (1), pCDNA3.1-hygro-SV40-DHFR-SV40polyA-FRT-TNF-Loxp expression plasmid pCD is transfected into 293T cells of the previous day; adding hygromycin B antibiotic with the concentration of 100 mu g/mL for screening for one week on the next day, and then culturing in a culture medium without hygromycin antibiotic for a period of time to ensure that the hygromycin B antibiotic can grow stably; 293T negative cells which are not successfully transfected with plasmids die gradually under antibiotic screening because of not having hyglomysin B resistance, cells which survive after pressurization have expression of a large amount of anti-TNF alpha single-chain antibody molecules on cell membranes of the cells due to successful transfection of plasmids, and cell populations which stably express the antibody molecules at high levels are sorted out through a flow cytometer after stable culture for a period of time, and are named as 293T-anti-TNF cells for subsequent multi-round recombination replacement to increase the probability of obtaining single-copy cell strains;

in the step (2), 293T-anti-TNF cells are cotransformed by pFRT-EGFP-LoxP replacing plasmid pCDFGL and recombinase plasmid pF2AC, and under the combined action of Flp and Cre enzyme, GFP gene replaces anti-TNF alpha antibody gene in the cells; the replacement successful cell transcribes and expresses the GFP protein, so that the fluorescent light emits green; flow sorting of cells expressing only GFP fluorescence, named 293T-GFP; carrying out two-round flow sorting on the sorted 293T-GFP cells after long-time amplification culture under the pressure without antibiotics, and enriching 0.5% of cells with strongest GFP fluorescence in each round of sorting until a fluorescence signal of a marked anti-TNF alpha antibody cannot be detected so as to obtain a dominant cell population with higher protein expression level and more stable and uniform expression;

in the step (3), co-transforming 293T-GFP cells with pFRT-RFP-LoxP replacement plasmid pFRL and recombinase plasmid pF2AC, and replacing GFP in the cells with RFP genes under the combined action of Flp and Cre enzyme; the replacement successful cell transcribes and expresses the RFP protein so that the cell spontaneously fluoresces in red; enriching cells only with RFP fluorescence by flow sorting, and naming as 293T-RFP; carrying out two-round flow sorting on the sorted 293T-RFP cells after long-time amplification culture under the antibiotic-free pressure, and enriching 0.5% of cells with the strongest RFP fluorescence in each round of sorting until GFP fluorescence signals cannot be detected; in the step (4), after the strongest RFP fluorescence signals are enriched by two-round flow sorting, 96-pore plate single cell sorting is carried out, each pore only sorts one cell, and 3 96-pore plates are divided in total; after the cells in the 96-well plate grow for a week, observing one by one under a microscope to enable a plurality of cells in the holes to grow in a clonal manner, or to exclude the holes without sorted cells or the monoclonal cells with poor growth state, and remaining monoclonal cell strains with good growth state to survive and carry out numbering; after the monoclonal cell strains are cultured and amplified, detecting the uniformity and stability of each monoclonal expression RFP signal in a flow mode, and selecting a plurality of monoclonal cell strains with the top rank from the signals for subsequent experiments;

in the step (5), co-transferring the pFRT-foreign protein-loxP replacing plasmid and the pF2AC recombinase plasmid into the monoclonal cell strain obtained in the step, and replacing RFP in the cells by foreign genes under the action of recombinase; 293T monoclonal cells with foreign proteins displayed on the surfaces of flow sorting cells;

the foreign protein is ADORA2A protein, and the ADORA2A protein is a protein encoded by a recombinant gene SP-ADORA2A-FLAGTM which comprises a signal peptide, a FLAG tag and a transmembrane sequence.

2. The single-copy cell line of the stably and highly expressed protein prepared and selected according to the method of claim 1, which is the single-copy cell obtained in step (4) of claim 1.

3. The use of the single-copy cell line of claim 2 for expressing recombinant proteins.

Images