CN113073118B - Application of Tet-off expression control system in cascade amplification of promoter activity - Google Patents

Abstract

The invention discloses an application of a Tet-off expression regulation system in cascade amplification of promoter activity. The invention is based on the inventor's discovery that the Tet-off expression control system has the effect of cascade amplification of promoter activity, and the amplification effect of weak promoters is particularly obvious. The invention provides a method for constructing a high-efficiency expression stable cell strain by using a slow virus infection method. The method realizes short construction period, high efficiency, high expression and high stability of the cell strain, and provides reliable basis for realizing large-scale and high-level expression of the recombinant protein.

Description

Application of Tet-off expression control system in cascade amplification of promoter activity

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a Tet-off expression regulation system in cascade amplification of promoter activity.

Background

Expression of recombinant proteins by stable mammalian cell lines is an important technology in the biopharmaceutical field for the production of protein pharmaceuticals. However, construction of stable cell lines by conventional transfection is a time-consuming and labor-consuming process. In the traditional transfection technology, exogenous genes are randomly integrated in a host cell genome, so that obvious heterogeneity appears in gene copy number and integration sites, and the obtained engineering cell strain has low protein yield and unstable expression in a long-term culture process.

Lentiviruses belong to the retrovirus family, the name being derived from this virus for a latency period of up to several years, of which the most well known is human immunodeficiency virus (HIV-1). The HIV-1 virus has a diameter of about 120nm, contains two positive strand RNAs, can effectively enter the nucleus, and has high infection efficiency on cells in the dividing stage and cells in the non-dividing stage. The slow virus can integrate the carried gene into the genome of the cell after infecting the cell, and can continuously and stably express for a long time, and simultaneously can stably inherit along with cell division, so that the slow virus becomes an effective tool for introducing exogenous genes into the cell, and is also an effective stable cell strain construction means.

The lentiviral vector is a gene therapy vector obtained by modifying the HIV-1 based, and has the characteristics of higher titer, better biological safety, stronger capability of introducing exogenous fragments and the like by removing pathogenic genes and reducing the homology of helper plasmids and vector plasmids. In the existing mature lentivirus infection method construction protein expression cell strain technology, most of lentiviruses obtained by packing HEK 293T cells through adherence culture infect HEK 293T cells or other adherence culture cells; however, the use of adherent cells to express proteins has the following drawbacks: (1) The cell density is low, the expression capacity of the exogenous gene is limited, and the protein yield is low; (2) The culture medium with serum is required for culture, and risks such as pathogen pollution and the like exist; (3) The adherence culture mode is unfavorable for large-scale culture and industrialized production. Therefore, developing a stable expression cell line constructed by using lentivirus to infect suspension cells is a requirement for the industrial production of modern recombinant proteins. In the method for constructing the high-expression cell strain by using the slow virus infection method reported by AgataOberbek et al, the cell strain capable of stably expressing the fusion protein for a long time is obtained by using the slow virus to infect the suspension-cultured CHO DG44 cells. However, the cell strain obtained by the stable cell strain construction method has low yield of only 50-250 mg/L, and can not meet the requirement of the current generation of rapidly developed biopharmaceutical productivity.

Therefore, by optimizing the expression system of the lentiviral vector, the expression level of the exogenous protein is improved, and the method becomes a research hot spot.

Disclosure of Invention

The primary purpose of the invention is to overcome the defects and shortcomings of the prior art and provide the application of a Tet-off expression control system in cascade amplification of promoter activity.

Another object of the present invention is to provide a lentiviral recombinant expression vector comprising a Tet-off expression control system.

Still another object of the present invention is to provide the use of the above lentiviral recombinant vector and a method for constructing a stable cell line with high expression by lentiviral infection.

The aim of the invention is achieved by the following technical scheme: the application of the Tet-off expression regulation system in the cascade amplification of the promoter activity is based on the discovery that the Tet-off expression regulation system has the effect of cascade amplification of the promoter activity, and the amplification effect of the Tet-off expression regulation system is particularly obvious for a weak promoter; the specific application steps are as follows: the promoter is arranged at the upstream of a transcription activator tTA2 expression element in a Tet-off expression regulation system, so that the tTA2 gene is directly driven to express, and finally the expression quantity of the target protein is improved.

The promoter is preferably a weak promoter. The weak promoter is a promoter which has relatively weak binding force with RNA polymerase compared with a constitutive viral promoter such as CMV promoter and SV40 promoter.

The weak promoter is preferably derived from a host cell' S endogenous promoter, including but not limited to the CHO endogenous promoter S100a6 promoter, CIRP promoter, PDI promoter, etc.

A lentiviral recombinant expression vector comprises a lentiviral vector skeleton containing a Tet-off expression control system and a promoter A; the promoter is arranged upstream of the transcriptional activator tTA2 expression element and is used for directly driving the tTA2 gene to express.

The Tet-off expression control system comprises a transcriptional activator tTA2, a Tet response element TRE and the like.

The promoter A is preferably a weak promoter. The weak promoter is a constitutive viral promoter with high transcription activity, such as CMV promoter, SV40 promoter, etc., and has weak binding force with RNA polymerase.

The weak promoter is preferably derived from a host cell endogenous promoter, including but not limited to CHO endogenous promoters, such as the S100a6 promoter, CIRP promoter, PDI promoter, and the like.

The CIRP promoter is preferably at least one of CIRP-P867 with a nucleotide sequence shown as SEQ ID NO.1 and CIRP-P2588 with a nucleotide sequence shown as SEQ ID NO. 2.

The PDI promoter is preferably a PDI-P598 promoter with a nucleotide sequence shown in SEQ ID NO. 3.

The S100a6 promoter is preferably an S100a6-dS18 promoter with a nucleotide sequence shown in SEQ ID NO. 4.

The lentiviral vector backbone includes, but is not limited to: pTet-IRES-EGFP, pTet-Off, pTet-On, pLUT-Off and pLUT-Off-EGFP vectors.

The lentiviral vector is applied to preparation of a high-efficiency expression stable cell strain.

The high-efficiency expression stable cell strain is preferably obtained by a lentivirus infection method.

A method for constructing a high-efficiency expression stable cell strain by using a lentivirus infection method comprises the following steps:

(1) Cloning target protein genes into multiple cloning sites in the lentiviral recombinant expression vector to obtain a lentiviral vector capable of expressing target proteins;

(2) Carrying out slow virus packaging on slow virus vectors capable of expressing target proteins to obtain virus liquid;

(3) Infecting host cells with the obtained virus liquid to obtain a high-efficiency expression stable cell bank; and further screening the cell bank to obtain the cell strain with stable efficient expression.

The target protein gene in the step (1) includes, but is not limited to, RFP, GFP, mutant line thereof, luciferase, fc fusion protein, HSA fusion protein, monoclonal antibody.

The mutant strain of GFP is preferably EGFP.

The lentiviral vector capable of expressing the target protein described in step (2) is preferably a lentiviral vector obtained in high concentration and high purity by: transferring the lentiviral vector capable of expressing the target protein into competent cells of escherichia coli, copying and amplifying in the escherichia coli, and extracting plasmids to obtain the lentiviral vector with high concentration and high purity.

The escherichia coli competent cells include but are not limited to escherichia coli TOP10 competent cells and escherichia coli DH5 alpha competent cells.

The transformation method includes but is not limited to heat shock method, electric transformation method and the like.

The steps of lentivirus packaging are preferably as follows: mixing the lentiviral vector capable of expressing the target protein with a packaging plasmid to obtain a packaging system, and transfecting the packaging system into HEK 293T cells for culture; then changing fresh culture medium, continuously culturing to obtain virus liquid; more preferably as follows:

(21) 24 hours prior to co-transfection, HEK 293T cells to be transfected were passaged at appropriate densities;

(22) On the day of transfection, observing the growth condition of HEK 293T cells under a microscope, and carrying out a transfection experiment when the cell confluency is 70-90% and the condition is good;

(23) Preparing a DNA-transfection reagent complex according to the transfection reagent specification;

(24) Dropwise adding the DNA-transfection reagent complex into HEK 293T cells to be transfected, and continuing culturing;

(25) Changing fresh culture medium; the volume ratio of fresh culture medium to the culture medium during transfection is 3:10-3:12;

(26) And (4) after transfection for 46-48 hours, taking culture solution for centrifugation, and filtering the supernatant through a 0.45 mu m filter membrane to obtain the recombinant lentivirus suspension.

The passaged medium described in step (21) includes, but is not limited to, DMEM complete medium, DMEM GluMAX medium, advanced DMEM/F12 medium, etc.

The DMEM complete medium is DMEM medium containing FBS and L-glutamine.

The concentration of the FBS in the DMEM complete medium is preferably 10% by volume.

The concentration of the L-glutamine in the DMEM complete medium is preferably 2-4 mmol/L.

In the preparation of the DNA-transfection reagent complex in the step (24), the transfection reagent includes, but is not limited to, lipofectamine2000, lipofectamine3000, turbofect and other liposome transfection reagents.

The packaging system includes but is not limited to a second generation three plasmid packaging system and a third generation four plasmid packaging system.

When the packaging system is a second generation three-plasmid system, the lentiviral vector capable of expressing the target protein is proportioned with packaging plasmids pMD2.G and psPAX2 according to the mass ratio of 5:2.5-3:2-3; preferably, mixing is performed in a mass ratio of 5:3:3, 5:3:2 or 2:1:1.

When the packaging system is a third-generation four-plasmid system, the lentiviral vector capable of expressing the target protein is proportioned with packaging plasmids pMDLg-PRRE, pRSV-Rev and pMD2.G according to the mass ratio of 5:2-3:2-3:2.5-3; mixing is preferably performed in a mass ratio of 2:1:1:1, 5:3:3:3 or 5:2:2:3.

The conditions for the centrifugation in the step (26) are preferably 800rpm to 1000rpm for 5min to 8min.

Such transfection methods include, but are not limited to, electric shock transfection, liposome transfection, PEI transfection, and the like.

The fresh medium is preferably fresh DMEM medium.

The virus solution is preferably obtained for 46-48 hours after transfection.

The host cell described in step (3) is a mammalian cell, preferably a CHO cell, including but not limited to a CHO-S cell, a CHO-DG44 cell, a CHO-K1 cell, etc.

The step of infection described in step (3) is preferably as follows: taking lentivirus suspension subjected to centrifugation and filtration treatment, and infecting cells to be infected with proper density; after the virus liquid infects cells for 4 to 6 hours, fresh CD culture medium is added for continuous culture; changing fresh culture medium 46-48 hours after virus liquid infects cells; 4-5 days after virus liquid infects cells, detecting protein expression intensity to obtain a high-efficiency stable expression cell bank; more preferably as follows:

(31) Adding the centrifuged and filtered lentivirus suspension into the prepared host cells to be infected, gently mixing, and placing into a shaker at 130rpm with 8% CO ₂ Culturing at 37 ℃;

(32) 4-6 hours after virus infection, fresh CD culture medium is added, and the culture is continued;

(33) Changing fresh CD complete culture medium 46-48 hours after virus infection;

(34) Sampling and detecting the cell Viability (VCD) 4-5 days after lentivirus infection, observing the cell state by microscopic examination, detecting the protein concentration or the expression intensity of the reporter gene, and obtaining a cell pool (cell pool);

(35) The obtained high expression cell bank can be directly applied to downstream experiments such as protein expression or promoter activity research; alternatively, a monoclonal cell line having a high protein expression level may be selected by a monoclonal cell sorting technique.

The amount of lentiviral suspension described in step (31) is preferably between 0.2 and 0.6X10 per 1mL of lentiviral suspension ⁶ Individual CHO cells to be infected were counted.

The volume ratio of the lentiviral suspension to the CHO cells to be infected is preferably 1-2: 1, proportioning; more preferably 1:1 or 2:1 by volume.

The CHO cells to be infected include, but are not limited to, CHO-S cells, CHO-DG44 cells, CHO-K1 cells, and the like.

The medium described in step (32) is preferably a CD complete medium including, but not limited to, CD FortiCHO complete medium, CD OptiCHO complete medium, CD CHO complete medium,Expression Medium, etc.

The additional volume of fresh medium in step (32) is preferably 1.5 to 3 times the volume at the time of infection; more preferably 2.5 times.

Compared with the prior art, the invention has the following advantages and effects:

first, the present inventors have found that the Tet-off expression control system has a function of cascade amplifying the promoter activity, and is particularly remarkable in amplifying a weak promoter.

Secondly, the pLUT-off lentivirus recombinant vector modified by the invention is used as a skeleton, suspension cultured CHO cells are used as host cells, high-level expression of exogenous proteins including but not limited to monoclonal antibodies, fc fusion proteins, HSA fusion proteins and the like in the suspension cells can be realized, the protein expression quantity is more than 4 times of that of the prior reporting technology, the operation is simple, and the cell strain construction period is shorter. Compared with the traditional transfection technology, the invention has the following advantages: the lentiviral vector integrates a target protein gene into a transcription active region of a host cell genome through reverse transcriptase and integrase, so that the target protein is expressed in higher yield and has high expression stability; the construction process is simple, the steps are few, and the time consumption is short. Compared with the traditional lentivirus infection technology, the invention has the following advantages: (1) The protein expression host cell is a CHO cell in suspension culture. The suspension culture CHO cells are cultured by using a serum-free culture medium, so that adverse effects of serum on virus infection and risks of other microorganism pollution are avoided, and the suspension culture CHO cells can be applied to large-scale bioreactor culture; the suspension CHO cells have high growth speed and high cell density, can obtain the target protein with higher concentration in a shorter time, and are suitable for the later large-scale culture and the production of the target protein. (2) The virus infection efficiency is high, the positive rate of the infected suspension CHO cells is close to 100%, the virus infection efficiency can be directly applied to protein expression without resistance screening or reporter gene screening, and the construction period of cell strains is greatly shortened. (3) The yield of the cell strain stably expressed by the fusion protein constructed by the invention is more than 4 times that of the cell strain stably expressed by the fusion protein constructed by the prior art, and the long-term expression stability is good. The invention provides a novel and efficient technology for constructing a stable cell strain by infecting suspension CHO cells with replication-defective lentiviruses, realizes short cell strain construction period, high efficiency, high expression and high stability, and provides a reliable basis for realizing large-scale and high-level expression of recombinant proteins.

Drawings

FIG. 1 is a diagram of plasmid maps of recombinant lentiviral expression vectors pLUT-off-PEx-GOI and pLenti-PEx-GOI; PEx in A is a different promoter, x=1, 2,3,4, PE1 is CIRP-P2588 promoter, PE2 is CIRP-P867 promoter, PE3 is S100a6-dS18 promoter, PE4 is PDI-P598 promoter, and the same applies below; GOI is EGFP reporter gene or GLP-1-Fc fusion protein expression gene; in B, x=1, 2, goi is EGFP reporter.

FIG. 2 is a flow chart of the construction of cell lines by lentiviral infection.

FIG. 3 is a graph showing the results of comparison of expression intensities after transduction of the same recombinant expression vector into the same host cell by lentiviral infection and conventional transfection.

FIG. 4 is a graph showing the results of comparison of expression intensities of promoters containing a Tet-off expression control system and those not containing a Tet-off expression control system.

FIG. 5 is a graph showing the comparison of GFP expression intensities after 6d and 60d passages after resuscitating a pool of cells infected with pLUT-off-PEx-EGFP lentivirus.

FIG. 6 is a graph showing the results of the yield of a pLUT-off-PE3-GLP-1-Fc monoclonal cell strain cultured in a 15 mL-shake tube.

FIG. 7 is a graph showing the results of 250 mL-shake flask fermentation of a pLUT-off-PE3-GLP-1-Fc monoclonal cell strain.

FIG. 8 is a graph showing the results of comparison of yields after 6d of resuscitation and 60d of passage of a pLUT-off-PE3-GLP-1-Fc monoclonal cell line.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.

Example 1 construction of pLUT-off-PEx-EGFP lentiviral recombinant plasmid,

the EcoRI-EGFP-XhoI gene fragment was obtained by PCR amplification experiments using pLEGFP-C1 (ClonTech, cat#6084-1) plasmid as a template, and the PCR primers were SeqPF and SeqPR.

SeqPF:5’-ACCGGAATTCTCAGATCCGCTAGCGCTAC-3’；

SeqPR:5’-TGAGCTCGAGATCAGAGTCCGGACTTGTACAGC-3’。

EcoRI and XhoI double digestion is carried out on the EcoRI-EGFP-XhoI gene fragment and the pLUT-off plasmid (the sequence is shown as SEQ ID NO. 5), and the EcoRI-EGFP-XhoI gene fragment with the restriction enzyme cohesive end and the pLUT-off linearization vector are obtained through a glue recovery purification method. The pLUT-off linearization vector and EcoRI-EGFP-XhoI gene fragment are connected through T4DNA ligase, and the connection system and connection conditions are as follows: pLUT-off linearization vector 20ng, ecoRI-EGFP-XhoI fragment 200ng, T4 Buffer (T4 Buffer) 2. Mu. L, T4DNA ligase 1. Mu. L, ddH ₂ O was made up to 20. Mu.L. Connection conditions: the reaction was carried out at 16℃overnight. And (3) transforming the ligation product into competent cells of escherichia coli TOP10, selecting a monoclonal colony for culture, performing bacterial liquid PCR (polymerase chain reaction) screening positive monoclonal, selecting a monoclonal with correct target bands for bacterial liquid culture, extracting plasmids for enzyme digestion identification, and obtaining an intermediate plasmid pLUT-off-EGFP.

Different CHO endogenous promoters (hereinafter collectively PEx, x=1, 2,3,4, PE1 is CIRP-P2588 promoter, PE2 is CIRP-P867 promoter, PE3 is S100a6-dS18 promoter, PE4 is PDI-P598 promoter) were inserted into the pvt-off-EGFP vector through SpeI and XhoI cleavage sites, respectively, wherein the PDI-P598 promoter passed through SpeI and SalI cleavage sites, to obtain a pvt-off-PEx-EGFP recombinant plasmid (plasmid map is shown in fig. 1A). Wherein the PEx gene fragment is synthesized by gold SpA.

EXAMPLE 2 second Generation packaging System lentiviral packaging and infection thereof

The process is as shown in fig. 2:

1. plasmid extraction: the plasmid extraction kit with endotoxin elimination effect is used for extracting a large amount of lentivirus recombinant vectors pLUT-off-PEx-EGFP and packaging plasmids psPAX2 (Addgene) and pMD2.G (Addgene), and simultaneously extracting lentivirus positive control plasmids pLVX-IRES-ZsGreen1 (Clontech) to obtain high-concentration high-purity endotoxin-free plasmids required by virus packaging of a lentivirus second-generation packaging system.

2. Cell culture: HEK 293T (ATCC) was resuscitated and prepared for co-transfection virus packaging experiments after 3 passages in conventional subculture methods.

3. Lentivirus packaging was performed using a second generation packaging system:

(1) HEK 293T cells were incubated at 0.3X10 at 24 hours prior to co-transfection ⁵ The individual cells/mL, 10mL of DMEM complete medium (4 mM L-glutamine, 10% FBS, the same applies below) was inoculated into a 25T cell culture flask and cultured in a carbon dioxide incubator; on the day of transfection, cells were observed by microscopic examination, the cell state was good, the confluence was about 70%, and the method was applicable to transfection experiments. 10mL of fresh DMEM complete medium is replaced and returned to the incubator for use.

(2) 4.6 μg of pLUT-off-PEx-EGFP, 2.7 μg of psPAX2 and 2.7 μg of pMD2.G (mass ratio about 5:3:3) were added to an appropriate amount of DMEM basal medium (without FBS) and gently mixed; followed by the addition of 20. Mu.L of transfection reagent TurboFect ^TM Transfection Reagent (Invitrogen, cat#R0531) was gently mixed to a final volume of 1mL; the reaction was allowed to stand at room temperature for 20min to form a DNA-transfection reagent complex.

(3) Adding the DNA-transfection reagent complex solution into HEK 293T cells to be transfected, wherein the temperature is 37 ℃ and the concentration of CO is 5% ₂ Incubation in incubator.

(4) Meanwhile, a positive control group and a negative control group were set, respectively, the positive group was co-transfected with pLVX-IRES-ZsGreen1 plasmid (Clontech, cat# 632187) and the packaging vector in the above-described manner, and the negative control group was not transfected with DNA.

(5) After 28-32 hours of cultivation, the culture supernatant was gently removed, and 3mL of fresh DMEM basal medium was added to continue the cultivation.

(6) Culturing for 15-18 hr (after co-transfection for about 48 hr), collecting culture supernatant of virus packaging cell, centrifuging at 800rpm for 5min, and filtering the supernatant with 0.45 μm filter membrane to obtain slow virus solution for infection.

4. Lentiviral infection with EGFP and ZsGreen1 reporter genes:

(1) Prior to viral infection, CHO-S suspension cells (Invitrogen) were treated for density, viability and growth status. In this example, the density of CHO-S cells to be infected was 4.0X10 ⁶ The cell/mL has the activity of 99 percent and the diameter of 11.9 mu m, and has good observation state under the mirror, and can be used for virus infection.

(2) The CHO-S suspension cells to be infected are usedExpression Medium (Mirus, cat#MIR6200) (4 mM L-glutamine, 0.3% Poloxamer 188) dilutes the cell density to 0.5X10) ⁶ cells/mL and dispensed into 50 mL-shake tubes, 1mL per tube and labeled.

(3) Adding the slow virus solution filtered by 0.45 μm filter membrane into CHO-S cells to be infected, and infecting 0.5X10 with 2mL virus solution/tube ⁶ After gently mixing individual cells (1 mL), they were returned to the shaker and continued at 130rpm,8% CO ₂ Culturing at 37 ℃.

(4) 4 hours after virus infection, 5mL fresh was addedExpression Medium (4 mM L-glutamine, 0.3% Poloxamer 188) and further cultivation.

(5) 2 days after virus infection, centrifugation at 800rpm for 5min, removal of supernatant and addition of 8mL freshExpression Medium (4 mM L-glutamine in 0.3% Poloxamer 188) was resuspended and the experimental and negative control groups (NC) were supplemented with puromycin (puromycin) to a final concentration of 5. Mu.g/mL (5P) and the culture continued.

(6) 4 days after virus infection (2 days of 5P pressure culture), the viable cell density and cell viability were sampled and examined and the cell state was observed by microscopic examination, and the cell detection results are shown in Table 1 below. All cell banks were resuspended in 3mL fresh medium after centrifugation at 800 rpm. Finally, puromycin was added to a final concentration of 10. Mu.g/mL (10P) at 0.5M/mL,5mL passage, experimental and negative control groups (NC), and culture was continued.

TABLE 1 cell densities and cell viability of cell pools 4 days after lentiviral infection (2 days after 5P pressurization)

Cell bank	VCD(×10 6 Individual cells/mL)	Cell viability (%)
			NC	1.92	85
pLVX-IRES-ZsGreenl	2.2	90
			pLUT-off-PE1-EGFP	2.8	90
pLUT-off-PE2-EGFP	3.4	92
			pLUT-off-PE3-EGFP	2.5	91
pLUT-off-PE4-EGFP	3.2	90

Fluorescent expression intensities of different infected groups were observed under ultraviolet lamp: the pLUT-off-PE3-GFP > pLUT-off-PE1-GFP > pLVX-IRES-ZsGreen1 is more than or equal to pLUT-off-PE2-GFP > pLUT-off-PE4-GFP > NC.

(7) 6 days after virus infection (5P pressure culture for 2 days+10P pressure culture for 2 days), the viable cell density and cell viability were sampled and examined, and the cell state was observed by microscopic examination, and the cell detection results are shown in Table 2 below.

TABLE 2 cell densities and cell viability of cell pools after 6 days post lentiviral infection (5P 2 days and 10P 2 days)

Cell bank	VCD(×10 6 Individual cells/mL)	Cell viability (%)
			NC	0.28	20
pLVX-IRES-ZsGreen1	4.7	97
			pLUT-off-PE1-EGFP	3.5	96
pLUT-off-PE2-EGFP	3.4	95
			pLUT-off-PE3-EGFP	4.4	97
pLUT-off-PE4-EGFP	2.9	95

After two rounds of incremental pressure culture, the survival rate of cells of the group infected by the pLUT-off-PEx-EGFP series vector is not obviously reduced, and the growth is not obviously inhibited, which indicates that the proportion of cells containing exogenous target genes in the cell bank is close to 100%, namely, the positive rate is close to 100%.

EXAMPLE 3 third Generation packaging System lentiviral packaging and infection thereof

1. Plasmid extraction: the plasmid extraction kit with endotoxin elimination effect is used for extracting a large amount of lentivirus recombinant vectors pLUT-off-PEx-EGFP and packaging plasmids pMDLg-PRRE (Addgene), pRSV-Rev psPAX2 (Addgene) and pMD2.G (Adgene), and simultaneously extracting lentivirus positive control plasmids pLVX-IRES-ZsGreen1 to obtain high-concentration high-purity endotoxin-free plasmids required by the virus packaging of a lentivirus third-generation packaging system.

2. Cell culture: HEK 293T cells were resuscitated and passaged 3 times by conventional subculture methods and prepared for co-transfection virus packaging experiments.

3. Lentivirus packaging was performed using a third generation packaging system:

(1) HEK 293T cells were incubated at 0.3X10 at 24 hours prior to co-transfection ⁶ Each cell/mL, 10mL of DMEM complete medium was inoculated into a 25T cell culture flask, and cultured in a carbon dioxide incubator. On the day of transfection, cells were observed by microscopic examination, the cell state was good, the confluence was about 80%, and the method was applicable to transfection experiments. 10mL of fresh DMEM complete medium is replaced and returned to the incubator for use.

(2) Mu.g of pLUT-off-PEx-EGFP, 2. Mu.g of pMDLg-PRRE, 2. Mu.g of pRSV-Rev and 2. Mu.g of pMD2.G (mass ratio of 2:1:1:1) were added to DMEM basal medium (without FBS) and gently mixed; followed by the addition of 20. Mu.L of transfection reagent TurboFect ^TM Transfection Reagent, mixing gently to give a final volume of 1mL; standing at room temperature for reaction for 20min to form a DNA-transfection reagent complex;

(3) Adding the DNA-transfection reagent complex solution into HEK 293T cells to be transfected, at 37℃with 5% CO ₂ Incubation in incubator.

(4) Meanwhile, a positive control group and a negative control group are respectively arranged, the positive group is co-transfected with pLVX-IRES-ZsGreen1 plasmid and packaging vector by the method, and the negative control group is not transfected with DNA.

(5) After 28-32 hours of cultivation, the supernatant was gently removed, and 3mL of fresh DMEM basal medium was added to continue the cultivation.

(6) Culturing for 15-18 hours (about 48 hours of co-transfection), taking the culture supernatant of virus packaging cells, centrifuging at 800rpm for 5 minutes, and filtering the supernatant by a 0.45 mu m filter membrane to obtain the slow-record virus suspension for infection.

4. Lentiviral infection with EGFP and ZsGreen1 reporter genes:

(1) Before virus infection, density and activity detection and growth state observation are carried out on CHO-S suspension cells to be infected. In this example, the density of CHO-S cells to be infected was 5.2X10 ⁶ The cell/mL has the activity rate of 98 percent and the diameter of 11.2 mu m, and has good observation state under the mirror, and can be used for virus infection.

(2) Suspending CHO-S to be infectedCell useExpression Medium (4 mM L-glutamine, 0.3% Poloxamer 188) cell density was diluted to 0.5X10 ⁶ cells/mL and dispensed into 50 mL-shake tubes, 1mL per tube and labeled.

(3) Adding the slow virus solution filtered by 0.45 μm filter membrane into CHO-S cells to be infected, 1mL virus solution/tube, i.e. 1mL virus solution is infected with 0.5X10 ⁶ After gently mixing individual cells (1 mL), they were returned to the shaker and continued at 130rpm,8% CO ₂ Culturing at 37 ℃.

(4) 4 hours after virus infection, 5mL fresh was addedExpression Medium (4 mM L-glutamine, 0.3% Poloxamer 188) and further culturing

(5) 2 days after virus infection, centrifugation at 800rpm for 5min, removal of supernatant, addition of 5mL freshExpression Medium (4 mM L-glutamine in 0.3% Poloxamer 188) and the experimental and negative control groups (NC) were resuspended with puromycin to a final concentration of 5 μg/mL (5P) and the culture was continued.

(6) 4 days after virus infection (2 days of 5P pressure culture), the viable cell density and cell viability were sampled and examined and the cell state was observed by microscopic examination, and the cell detection results are shown in Table 3 below. All cell banks were resuspended in 3mL fresh medium after centrifugation at 800 rpm. Finally, the culture was continued with a final concentration of puromycin of 10. Mu.g/mL (10P) in the experimental group and negative control group (NC) at 0.5M/mL and 5mL passages.

TABLE 3 cell densities and cell viability of cell pools 4 days after lentiviral infection (2 days after 5P pressurization)

Cell bank	VCD(×10 6 Individual cells/mL)	Cell viability (%)
			NC	2.0	90
pLVX-IRES-ZsGreen1	5.2	96
			pLUT-off-PE1-EGFP	4.6	96
pLUT-off-PE2-EGFP	4.4	95
			pLUT-off-PE3-EGFP	5.5	97
pLUT-off-PE4-EGFP	3.8	96

Fluorescent expression intensities of different infected groups were observed under ultraviolet lamp: pLUT-off-PE3-GFP > pLUT-off-PE1-GFP

>pLVX-IRES-ZsGreen1≥pLUT-off-PE2-GFP>pLUT-off-PE4-GFP>>NC

Second generation packaging system to third generation packaging system fluorescence intensity comparison:

for pLVX-IRES-ZsGreen1 plasmid, the second generation > the third generation; for the pLUT-off-PEx-EGFP series vector, the third generation is > the second generation.

(7) 6 days after virus infection (5P pressure culture for 2 days+10P pressure culture for 2 days), the viable cell density and cell viability were sampled and examined, and the cell state was observed by microscopic examination, and the cell detection results are shown in Table 4 below.

TABLE 4 cell densities and cell viability of cell pools after 6 days post lentiviral infection (2 days 5P compression and 2 days 10P compression)

Cell bank	VCD(×10 6 Individual cells/mL)	Cell viability (%)
			NC	0.56	30
pLVX-IRES-ZsGreen1	6.0	98
			pLUT-off-PE1-EGFP	5.6	97
pLUT-off-PE2-EGFP	5.8	96
			pLUT-off-PE3-EGFP	6.1	98
pLUT-off-PE4-EGFP	4.8	96

After two rounds of incremental pressurization, the survival rate of cells of the group infected by the pLUT-off-PEx-EGFP series vector is not obviously reduced, and the growth is not obviously inhibited, which indicates that the proportion of cells containing exogenous target genes in the cell bank is close to 100%, namely, the positive rate is close to 100%.

The results of example 2 and example 3, taken together, demonstrate that lentiviral infection efficiencies approaching 100% can be achieved regardless of whether the virus is packaged in a second generation packaging system or a third generation packaging system.

EXAMPLE 4 comparison of the expression intensity of pLUT-off-PEx-EGFP cell bank with the expression intensity of the conventional transfection method

(1) Transient transfection of CHO-S cells with pLUT-off-PEx-EGFP recombinant plasmid with FreeStyle MAX Reagent (Gibco ^TM Cat # 16447100), the transfection procedure was performed following the instructions of the reagents.

(2) 48 hours after transient transfection, cells were taken for GFP detection, while lentiviral cell banks obtained in examples 2 or 3 (cultured for less than 6 days after resuscitation) were taken for GFP detection. The cell density was measured to be 1X 10 ⁶ The detection wavelength of each cell/mL is 480nm of emission wavelength and 520nm of absorption wavelength.

(3) The expression intensities of the same recombinant expression vector after transduction into the same host cell by different DNA delivery methods were compared. The results are shown in FIG. 3. The fluorescence expression intensity obtained by the slow virus infection method is 1.4-4.4 times of the fluorescence intensity of 48 hours after transient transfection, and the expression intensity of the vector with low expression intensity in the transient transfection process can be improved to more than 4 times by slow virus infection.

EXAMPLE 5 study of the effect of Tet-off promoter Cascade amplification

(1) PE1 (CIRP-P2588 promoter) and PE2 (CIRP-P867 promoter) with relatively weak promoter strength are taken and subcloned into lentiviral recombinant vector pliti-EGFP (the sequence is shown as SEQ ID NO. 6) through ClaI and EcoRI cleavage sites to be used as direct promoters to drive EGFP reporter gene expression, so that pLenti-PEx-GOI (shown as figure 1B) is obtained. Wherein the CIRP-P867 promoter and the CIRP-P2588 promoter are obtained by total gene synthesis of Kirschner company.

(2) Using the lentiviral infection method of example 2 or 3, the complete-PE 1-EGFP, complete-PE 2-EGFP, pLUT-off-PE1-EGFP, and pLUT-off-PE2-EGFP were transduced into CHO-S host cells, respectively, and expressed.

(3) GFP intensities were measured from a pool of lentivirus infected cells 4d after infection, and GFP expression intensities with and without Tet-off control systems were compared. The cell density was measured to be 1X 10 ⁶ Individual cells/mL, detection wavelength: the emission wavelength is 480nm and the absorption wavelength is 520nm.

(4) As a result, as shown in FIG. 4, PE2 was hardly active without the effect of the cascade amplification of the Tet-off promoter, whereas the PE2 promoter became a strong promoter with the effect of the cascade amplification of the Tet-off promoter, and the activity was improved by 91 times; PE1 with stronger promoter activity is also improved by 4.5 times under the amplification effect of Tet-off.

EXAMPLE 6pLUT-off-PEx-EGFP cell bank stable passage experiment

(1) The pLUT-off-PEx-EGFP cell bank was resuscitated with CD FortiCHO complete medium for cultivation.

(2) When the cell density is more than or equal to 2 multiplied by 10 ⁶ At 0.2X10 g/mL ⁶ The inoculation density of each cell/mL, the inoculation volume of 5mL and the complete medium of CD fortcho were subcultured.

(3) Culturing until 6 days, washing partial cells with PBS (pH 7.4, 0.01 mol/L), inoculating to black 96-well plate at appropriate cell density, detecting GFP fluorescence intensity with a multifunctional enzyme-labeled instrument under the conditions of emission wavelength 488nm and absorption wavelength 520nm, and recording as cell bank reporter gene expression intensity data at 6d of resuscitation. The remaining cells were at 0.2X10 ⁶ Subculturing was continued with 5mL per cell/mL.

(4) Cells were harvested 60 days after subculture and GFP intensity was again measured according to step (3).

(5) GFP detection results show that the GFP expression intensity of the cell bank constructed by a lentiviral infection method after 60 days of subculture is 94-100% of the GFP expression intensity after 6 days of resuscitative culture, and the stability of different cell banks is not significantly different. FIG. 5 shows cell density of 0.25X10 ⁶ And comparing the GFP expression intensities of the stable cell libraries before and after stable passage with each cell/mL, wherein the emission wavelength is 488nm, the absorption wavelength is 520nm.

EXAMPLE 7pLUT-off-PE3-GLP-1-Fc lentiviral packaging and infection thereof

1. Construction of pLUT-off-PE3-GLP-1-Fc lentiviral recombinant plasmid

The preparation of pLUT-off-PE3-GLP-1-Fc was described in example 1: GLP-1-Fc (Dulaglutide, trade name Trulicity) recombinant protein genes (the sequence is shown in SEQ ID NO.7 and delivered by pUC57-GLP-1-Fc recombinant vector) are synthesized by the company of Kirsrui, recombinant vectors pLUT-off-PE3-EGFP and pUC57-GLP-1-Fc are respectively digested by restriction enzymes EcoRI and XhoI, the pLUT-off-PE3 linearization vector and the GLP-1-Fc genes in the digested products are recovered by glue, and T4 ligase is connected with the linearization vector and the gene fragment to obtain the pLUT-off-PE3-GLP-1-Fc recombinant vector.

2. packaging and transfection of pLUT-off-PE3-GLP-1-Fc lentiviruses

1. Plasmid extraction: the plasmid extraction kit with endotoxin elimination effect is used for extracting a large amount of lentivirus recombinant protein expression vectors pLUT-off-PE3-GLP-1-Fc and packaging plasmids pMDLg-PRRE, pRSV-Rev and pMD2.G to obtain high-concentration high-purity endotoxin-free plasmids required by the virus packaging of a third-generation packaging system of lentiviruses.

2. Cell culture: HEK 293T cells were recovered and passaged 3 times by conventional subculture methods for use in cotransfection virus packaging experiments.

3. Lentivirus packaging was performed using the third generation packaging system virus packaging method, in the same manner as in example 3.

(1) HEK 293T cells were incubated at 0.3X10 at 24 hours prior to co-transfection ⁵ Complete culture of individual cells/mL, 10mL DMEMInoculating the culture medium into a 25T cell culture flask, and culturing in a carbon dioxide incubator; on the day of transfection, cells were observed by microscopic examination, the cell state was good, the confluence was about 80%, and the method was applicable to transfection experiments. 10mL of fresh DMEM complete medium is replaced and returned to the incubator for use.

(2) Add 4. Mu.g of pLUT-off-PE3-GLP-1-Fc, 2. Mu.g of pMDLg-PRRE, 2. Mu.g of pRSV-Rev and 2. Mu.g of pMD2.G (mass ratio 2:1:1:1) to DMEM basal medium (without FBS) and mix gently; followed by the addition of 20. Mu.L of transfection reagent TurboFect ^TM Transfection Reagent, mixing gently to give a final volume of 1mL; and standing at room temperature for reaction for 20min to form a DNA-transfection reagent complex.

(3) Adding the DNA-transfection reagent complex solution into HEK 293T cells to be transfected, wherein the temperature is 37 ℃ and the concentration of CO is 5% ₂ Incubation in incubator.

(4) Meanwhile, a positive control group and a negative control group were set, the positive group was co-transfected with pLVX-IRES-ZsGreen1 plasmid and packaging vector in the above manner, and the negative control group was not transfected with DNA.

(5) After 28 to 32 hours of cultivation, the culture supernatant was gently removed, and 3mL of fresh DMEM medium was added to continue the cultivation.

(6) Culturing for 15-18 hr (about 48 hr after co-transfection), collecting culture supernatant of virus packaging cells, centrifuging at 800rpm for 5min, and filtering the supernatant with 0.45 μm filter membrane to obtain slow virus solution for infection.

4. Lentivirus infection CHO-S cell containing GLP-1-Fc fusion protein gene

(1) Before virus infection, density and activity detection and growth state observation are carried out on CHO-S suspension cells to be infected. In this example, the density of CHO-S cells to be infected was 5.8X10 ⁶ The cell/mL has the activity of 99 percent and the diameter of 11.8 mu m, and has good observation state under the mirror, and can be used for virus infection.

(2) The density of CHO-S suspension cells to be infected was adjusted to 0.5X10 with CD fortifer complete medium ⁶ cells/mL and dispensed into 50 mL-shake tubes, 1mL per tube and labeled.

(3) Adding the slow virus solution filtered by 0.45 μm filter membrane into CHO-S cells to be infected, and treating 1mL of the virusVenom/tube, i.e. 1mL virus infection 0.5X10 ⁶ Individual cells (1 mL) were gently mixed and returned to the shaker for further culture.

(4) 4 hours after virus infection, 5mL of fresh CD FortiCHO complete medium is added, and the culture is continued.

(5) 2 days after virus infection (about 48 hours), centrifugation at 800rpm for 5min, supernatant removal, and resuspension with 5mL fresh CD fortcho complete medium, and cultivation continued.

(6) 4 days after viral infection, at 0.3X10 ⁶ The inoculation density of each cell/mL and the inoculation volume of 5mL are used for continuous culture for two days, and the cell bank is taken for limiting dilution.

EXAMPLE 8 limiting dilution method for sorting GLP-1-Fc monoclonal cell lines

(1) Preparation of cloning Medium containing 6mM L-Glutamine (per 100 mL): CD FortiCHO basal medium 97mL,200mM L-glutamine 3mL, prepared basal medium is preheated at 37 ℃ for standby.

(2) Cell bank sampling detects cell density, cell viability and observes growth status. Cells were finally diluted to 0.3 cells/40. Mu.L using a gradient dilution method with cloning medium, and the diluted cells were inoculated into 96-well plates, 40. Mu.L/well, i.e., 0.3 cells/well, using a row gun.

(3) Inoculated 96 well plates were placed in CO ₂ Incubator at 37℃with 5% CO ₂ And absolute resting culture under saturated humidity for 4 hours, the wells were observed under a microscope, wells containing only one cell were confirmed and labeled, and medium was added to 200. Mu.L. Repositioning cells to CO ₂ The incubator continues stationary culture.

(4) After 13 days of static culture, the growth of the monoclonal cells was observed under a microscope, wells with obvious cell proliferation were found and marked, and 50. Mu.L/well of CD fortcho complete medium was supplemented.

(5) After 17 days of stationary culture, the culture supernatant of the single cell colony was picked up and subjected to SDS-PAGE detection. According to SDS-PAGE detection result, selecting the monoclonal with obvious target band, transferring to 15 mL-shaking tube, and making amplification culture.

(6) The 15 mL-shake tube system is normally cultured for 5 days, the cell viability and the viable cell density are sampled and detected, the growth state of the cells is observed under a mirror, and the monoclonal cell culture supernatant with good growth state is selected for ELISA detection, and the ELISA detection result is shown in figure 6.

(7) According to ELISA detection results, monoclonal cells with higher yield are selected and transferred to a 50 mL-shake tube for expansion culture.

(8) Culturing for 4d in a 50 mL-shake tube system, sampling and detecting the cell viability, viable cell density and observing the growth state of the cells under a microscope. After centrifugation at 800rpm for 5min, the culture supernatant was taken for ELISA detection.

(9) According to ELISA detection results, 12 monoclonal cells with higher yield are selected and transferred to a 125 mL-shake flask, and 30mLCD11V complete culture medium is subjected to expansion culture; simultaneously inoculating the strain into a 30mLCD fortcho complete culture medium to culture and prepare for strain preservation, and obtaining a stable cell strain for efficiently expressing GLP-1-Fc recombinant protein.

Example 9 shake flask fermentation yield assessment

(1) After 3d of cell culture from 50 mL-shake flask transfer to 125 mL-shake flask expansion, the cells were incubated at 0.5X10 ⁶ Individual cells, 50mLCD11V complete medium was inoculated into 250 mL-shake flasks, 130rpm,8% CO ₂ Culturing at 37deg.C, and inoculating the culture medium with the same day as 0d.

(2) Starting from 3d of fermentation, 2mL per day were sampled for the following parameter measurements: viable Cell Density (VCD), cell viability (availability), cell Diameter (Diameter), pH, osmolality, lactate concentration and glucose concentration, while observing the cell state under a microscope.

(3) When the cell density is higher than 2.0X10 ⁷ At each cell/mL, the culture temperature was reduced to 33 ℃.

(4) When the glucose content in the culture medium is lower than 2g/L, adding glucose solution to 4g/L;

(5) The feed media PFF06 3%, 4%, 5% (V/V) were added to the fermentations 3d, 5d, 7d, 9d, 11d, 13d, respectively.

(6) Culture supernatants were taken at fermentation positions 9d, 11d, and 14d, respectively, and the effective concentration and purity of the target protein were measured by high performance liquid chromatography (HPSEC).

(7) At fermentation stage 14d or when the cell viability is below 80%, the fermentation experiment is ended and the fermentation supernatant is centrifuged for further purification.

(8) The shake flask fermentation yield of the monoclonal cell line in this example is shown in FIG. 7. In this example, the highest yield of shake flask fermentation of the cell line obtained by one time of lentiviral infection was 2g/L.

EXAMPLE 10 Stable passaging experiments with monoclonal cell lines

(1) 12 cell lines were picked and resuscitated with CD fortcho complete medium.

(2) When the cell density is greater than 2X 10 ⁶ Each cell/mL was passaged with CD fortcho complete medium at 0.2M/mL,5mL system.

(3) Culturing until the 6 th day, the cell state is good and the doubling time is stable, and inoculating is used for shake flask fermentation yield evaluation as yield data of cell strains at the initial stage of recovery.

(4) Shake flask fermentation yield evaluation experiments were performed again after 60 days of subculture.

(5) The yields after 6 days after resuscitating and 60 days after passaging of the stable cell line were compared as shown in FIG. 8. In this example, the yield of the cell line after 60 days subculture was 94% -100% of the yield of the freshly recovered cell line.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Sequence listing

<110> Pinghai Federal pharmaceutical Co., ltd

<120> use of Tet-off expression control System in Cascade amplifying promoter Activity

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 813

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CIRP-P867 promoter nucleotide sequence

<400> 1

gttgagggta ggaagtcgct gtcatctgtg tccccaacca cacacaccct cttgcgaagt 60

gaagaaattt ccagcatgcc attctggttg ggtggtccag actttggggt cctgtggcca 120

gggctaatgg agggccactg caggctagaa gtcagttgtt ttaggccata gaaccaccat 180

ttaaggtcag ctcagttgta tgacagtctt acatgtggtt ttgggatgat aaaagcagaa 240

gacatccctg tgtcgtgtcc cccccttgaa aatacctgtt ttccagacag ggtttctctg 300

tacctttgga gcttgtcctg gaactctata gcaggctggc ttcgtactca cagatccacc 360

tgcctctgcc tcccgagtgc tgggactaaa ggtgtgtgcc accaccaccc agtgtgtttg 420

ttcatttttg acatagagca tgtagcccag gctggtatca gacttgctgt gtagttgcaa 480

atggttttgg acttctgatc cttgtgcctc caatatccca aaggctagga gtgacaggat 540

tgtttggaaa cactctccca agttgttcca cagagtcctt tgtctcattg agtgactgga 600

tgaccggtgt gaccccgcac ttatggctca ggttccccag gaaggtcatg gattactttt 660

atatacaaaa gtagggaagg aaaggggcag gaagctttca gtgactgtca ccctagcact 720

aaagcttatg cagggcattt caagaccagt ctggcctata aagggaggtc atgtctcaaa 780

aactaaaaga ctgagtgtgc tatctggctt cag 813

<210> 2

<211> 821

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CIRP-P2588 promoter nucleotide sequence

<400> 2

ggatgtggtc aggggtgaag ccccgccaga accctccagt gaggggctga gggcatggtc 60

aggagtccct tcagatggca tgcttgaagc cctgggtgta tgcagaagag cagtgctctg 120

ccagctgtaa tgtgcacctg gttgggggtg agcagaaagc aggattcaaa tggcagttaa 180

gttatggagc aggttgagat ttgcaggcaa actgagttgg gttcaggaga gacgctgcag 240

caagtctgaa ggaggagtac tgactttgtg gttaatattg tgatgaaagt gttaggactg 300

atcttatgga atgtctgaga gccttgggtc cagagaatca ttaggagtca gttgttttgg 360

gagtcagctc agtggataac ggggcttgct gctaatcctg aatttgatcc taaggatgga 420

aggagagaca ccagaaaatt gccttctggc tttcacacac gcacttcagc taataaatgt 480

aatattaaaa agaaaagaaa aaaaaaagca gtgcgttgaa gctctttgtt cgccgccgca 540

gaccaaccgc gcggagagca acgaagtggg tcagctccgc ccctgaaccg cgctgtcacc 600

gccctccggc cgctaggggg cgggagaatg cggccggggc acatcccagc caatcaaaac 660

gctcagcgcc tccgcgcggg cagggacgaa gccggggata gtcccgcccc tccactgaag 720

cgcagctgcc gaatctggcg cgtcggattg gtcagctagg cgtagtgggc ggttctgggg 780

ggcgtgcccg aatgggtggg gtatatcagg cgggactcag c 821

<210> 3

<211> 595

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> PDI-P598 promoter nucleotide sequence

<400> 3

cagtacggaa acgcggtcca gtcagaatgc aacacgaggg gcttcggggg cgcaggcgca 60

gctccaccca ggggcccggg actccgcccc ctgccacgtt cgacgaagaa ccgcgcaggg 120

tgcgcgcatc tctcggccaa tcaggagccg ccgggccggg ccgtggccag cggcggcccc 180

tccgggcgtg gggaaggcgt gggaaaagtc gccagtcaca agtccagaaa gagaaagttc 240

gcctcggcca gccaatcaga ggctggggaa cgcggcgtgt gagcgcgcgc gcgcgcgctg 300

cgggccaatc ccgggcgagg acggcagggc ctccgggtcc ttccgggctc cgcggccccg 360

cctcgagtgg gtgtccagtc ctcgcgcgga gagggtgggc ctctcagcgc ctcggccaat 420

cagacggcgg ggcggcgcgc gtgcgcgcgg cggctggcgc gcgcggcgag ggggcggtgt 480

gggcgcgtcc ccggcccagg atttataaag gcgaggtccg gacccaggcg cgctctcgtc 540

gccttggctg tcccggcggc gccaacccaa ccgccccgcc cgctgccgac gtccg 595

<210> 4

<211> 222

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> S100a6-dS18 promoter nucleotide sequence

<400> 4

cctcatgcca ctcccaatcc gggacagtcc tggcagcaga ggcgtggaaa actgaggggg 60

ttgttggggt gtgttttgct agcctcaggc gccgggtggg gctcggggcg ggccggcact 120

ccttgggcgg gcctcccgga tgctagccgc tataaggcca gccggactgc gacacagtcc 180

atcccctcga ccactccttt ggctcttcgc tgtctacctg cc 222

<210> 5

<211> 9494

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> pLUT-off plasmid sequence

<400> 5

acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca 60

acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta 120

cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga 180

attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct 240

ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa 300

gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc 360

tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc 420

ccgaacaggg acttgaaagc gaaagggaaa ccagagctct ctcgacgcag gactcggctt 480

gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540

actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 600

attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta 660

aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 720

gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga 780

tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg 840

atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900

aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga 960

caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc 1020

acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc 1080

tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140

gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200

ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260

ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 1320

ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa 1380

atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440

ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500

acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560

ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620

agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680

tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740

tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcggtt 1800

aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat 1860

aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaat tcaaaatttt 1920

atcgatgcag gtccgaggtt ctagacgagt ttactcccta tcagtgatag agaacgatgt 1980

cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata 2040

gagaacgtat gtcgagttta ctccctatca gtgatagaga acgtatgtcg agtttatccc 2100

tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag aacgtatgtc 2160

gaggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg aaccgtcaga 2220

tcgcaccggt cagctagcac tgcagcgtct caagcttcag aattctatct agattctcga 2280

gacacgcgtg gcctccgcgc cgggttttgg cgcctcccgc gggcgccccc ctcctcacgg 2340

cgagcgctgc cacgtcagac gaagggcgca gcgagcgtcc tgatccttcc gcccggacgc 2400

tcaggacagc ggcccgctgc tcataagact cggccttaga accccagtat cagcagaagg 2460

acattttagg acgggacttg ggtgactcta gggcactggt tttctttcca gagagcggaa 2520

caggcgagga aaagtagtcc cttctcggcg attctgcgga gggatctccg tggggcggtg 2580

aacgccgatg attatataag gacgcgccgg gtgtggcaca gctagttccg tcgcagccgg 2640

gatttgggtc gcggttcttg tttgtggatc gctgtgatcg tcacttggtg agtagcgggc 2700

tgctgggctg gccggggctt tcgtggccgc cgggccgctc ggtgggacgg aagcgtgtgg 2760

agagaccgcc aagggctgta gtctgggtcc gcgagcaagg ttgccctgaa ctgggggttg 2820

gggggagcgc agcaaaatgg cggctgttcc cgagtcttga atggaagacg cttgtgaggc 2880

gggctgtgag gtcgttgaaa caaggtgggg ggcatggtgg gcggcaagaa cccaaggtct 2940

tgaggccttc gctaatgcgg gaaagctctt attcgggtga gatgggctgg ggcaccatct 3000

ggggaccctg acgtgaagtt tgtcactgac tggagaactc ggtttgtcgt ctgttgcggg 3060

ggcggcagtt atggcggtgc cgttgggcag tgcacccgta cctttgggag cgcgcgccct 3120

cgtcgtgtcg tgacgtcacc cgttctgttg gcttataatg cagggtgggg ccacctgccg 3180

gtaggtgtgc ggtaggcttt tctccgtcgc aggacgcagg gttcgggcct agggtaggct 3240

ctcctgaatc gacaggcgcc ggacctctgg tgaggggagg gataagtgag gcgtcagttt 3300

ctttggtcgg ttttatgtac ctatcttctt aagtagctga agctccggtt ttgaactatg 3360

cgctcggggt tggcgagtgt gttttgtgaa gttttttagg caccttttga aatgtaatca 3420

tttgggtcaa tatgtaattt tcagtgttag actagtaaat tgtccgctaa attctggccg 3480

tttttggctt ttttgttaga cgctagaaga tccataactt cgtatagtat acattatacg 3540

aagttatgcc accatgtcta gactggacaa gagcaaagtc ataaactctg ctctggaatt 3600

actcaatgaa gtcggtatcg aaggcctgac gacaaggaaa ctcgctcaaa agctgggagt 3660

tgagcagcct accctgtact ggcacgtgaa gaacaagcgg gccctgctcg atgccctggc 3720

aatcgagatg ctggacaggc atcataccca cttctgcccc ctggaaggcg agtcatggca 3780

agactttctg cggaacaacg ccaagtcatt ccgctgtgct ctcctctcac atcgcgacgg 3840

ggctaaagtg catctcggca cccgcccaac agagaaacag tacgaaaccc tggaaaatca 3900

gctcgcgttc ctgtgtcagc aaggcttctc cctggagaac gcactgtacg ctctgtccgc 3960

cgtgggccac tttacactgg gctgcgtatt ggaggatcag gagcatcaag tagcaaaaga 4020

ggaaagagag acacctacca ccgattctat gcccccactt ctgagacaag caattgagct 4080

gttcgaccat cagggagccg aacctgcctt ccttttcggc ctggaactaa tcatatgtgg 4140

cctggagaaa cagctaaagt gcgaaagcgg cgggccggcc gacgcccttg acgattttga 4200

cttagacatg ctcccagccg atgcccttga cgactttgac cttgatatgc tgcctgctga 4260

cgctcttgac gattttgacc ttgacatgct ccccgggtaa ataacttcgt atagtataca 4320

ttatacgaag ttatggatcc gcggccgcaa attccgcccc tctccctccc ccccccctaa 4380

cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc 4440

caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac 4500

gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt 4560

gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg 4620

caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata 4680

agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga 4740

aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt 4800

accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc 4860

gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac 4920

acgataatac catggccacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg 4980

tcccccgggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca 5040

ccgttgaccc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc 5100

gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct 5160

ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc tcgcgcatgg 5220

ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc 5280

accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg 5340

gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgctgggg 5400

tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct 5460

tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca 5520

agcccggtgc ctgagttcgc gtctggaacg tcgacaatca acctctggat tacaaaattt 5580

gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg 5640

ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt 5700

ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg 5760

tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc 5820

agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg 5880

cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt 5940

tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg tgttgccacc tggattctgc 6000

gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg 6060

gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga 6120

tctccctttg ggccgcctcc ccgcctggaa ttaattcgag ctcggtacct ttaagaccaa 6180

tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag 6240

ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt 6300

tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc 6360

aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta 6420

actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat 6480

gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga 6540

acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 6600

ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 6660

atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc 6720

cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga 6780

ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg 6840

cttttgcggg cccaaattcg taatcatggt catagctgtt tcctgtgtga aattgttatc 6900

cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct 6960

aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa 7020

acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 7080

ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 7140

gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 7200

caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 7260

tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 7320

gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 7380

ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 7440

cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 7500

tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 7560

tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 7620

cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 7680

agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga 7740

agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 7800

gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 7860

aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 7920

ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 7980

gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 8040

taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 8100

tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 8160

tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 8220

gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 8280

gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 8340

ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 8400

cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 8460

tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 8520

cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 8580

agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 8640

cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 8700

aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 8760

aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 8820

gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 8880

gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca 8940

tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 9000

ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata 9060

aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc 9120

tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca 9180

gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg 9240

cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat 9300

gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg 9360

aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg 9420

caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg 9480

ccagtgccaa gctg 9494

<210> 6

<211> 8107

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> plenti-EGFP lentiviral recombinant vector sequence

<400> 6

acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca 60

acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta 120

cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga 180

attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct 240

ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa 300

gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc 360

tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc 420

ccgaacaggg acttgaaagc gaaagggaaa ccagagctct ctcgacgcag gactcggctt 480

gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540

actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 600

attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta 660

aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 720

gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga 780

tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg 840

atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900

aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga 960

caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc 1020

acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc 1080

tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140

gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200

ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260

ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 1320

ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa 1380

atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440

ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500

acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560

ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620

agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680

tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740

tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcggtt 1800

aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat 1860

aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaat tcaaaatttt 1920

atcgatgcag gtccgaggtt ctagacgagt ttactcccta tcagtgatag agaacgatgt 1980

cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata 2040

gagaacgtat gtcgagttta ctccctatca gtgatagaga acgtatgtcg agtttatccc 2100

tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag aacgtatgtc 2160

gaggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg aaccgtcaga 2220

tcgcaccggt cagctagcac tgcagcgtct caagcttcag aattctcaga tccgctagcg 2280

ctaccggtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 2340

ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag 2400

ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 2460

gtgccctggc ccccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc 2520

ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg 2580

agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg 2640

agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca 2700

acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg 2760

acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca 2820

gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc 2880

tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc 2940

gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg 3000

agctgtacaa gtccggactc tgatctcgag ggggttgggg ttgcgccttt tccaaggcag 3060

ccctgggttt gcgcagggac gcggctgctc tgggcgtggt tccgggaaac gcagcggcgc 3120

cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc gtcacccgga tcttcgccgc 3180

tacccttgtg ggccccccgg cgacgcttcc tgctccgccc ctaagtcggg aaggttcctt 3240

gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca cgtctcacta gtaccctcgc 3300

agacggacag cgccagggag caatggcagc gcgccgaccg cgatgggctg tggccaatag 3360

cggctgctca gcagggcgcg ccgagagcag cggccgggaa ggggcggtgc gggaggcggg 3420

gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg tgttccgcat tctgcaagcc 3480

tccggagcgc acgtcggcag tcggctccct cgttgaccga atcaccgacc tctctcccca 3540

gggggatcca ccggagctta ccatgaccga gtacaagccc acggtgcgcc tcgccacccg 3600

cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac 3660

gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt 3720

cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt 3780

ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc 3840

gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct 3900

ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga 3960

ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg 4020

cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg 4080

gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat 4140

gacccgcaag cccggtgcct gagtcgacaa tcaacctctg gattacaaaa tttgtgaaag 4200

attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat 4260

gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc 4320

ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg 4380

cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct 4440

ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct 4500

tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg 4560

gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac 4620

gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct 4680

gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct 4740

ttgggccgcc tccccgcctg gaattaattc gagctcggta cctttaagac caatgactta 4800

caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat 4860

tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca 4920

gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag 4980

cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag 5040

atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct 5100

tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt 5160

tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc 5220

atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt 5280

ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc 5340

gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc 5400

tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc 5460

gggcccaaat tcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac 5520

aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt 5580

gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 5640

gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 5700

ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 5760

atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 5820

gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 5880

gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 5940

gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 6000

gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 6060

aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 6120

ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 6180

taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 6240

tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 6300

gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt 6360

taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 6420

tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 6480

tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 6540

ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 6600

taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 6660

tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 6720

cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 6780

gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 6840

cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 6900

ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 6960

aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 7020

atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 7080

tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 7140

gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 7200

aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 7260

acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 7320

ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 7380

tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 7440

aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 7500

catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 7560

atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 7620

aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag 7680

gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca 7740

catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc 7800

ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc 7860

agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag 7920

gagaaaatac cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg 7980

atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg 8040

attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtgc 8100

caagctg 8107

<210> 7

<211> 1083

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> GLP-1-Fc recombinant protein Gene sequence

<400> 7

gaattcgctg actagcgttt aaacttaagc ttagcgcaga ggcttggggc agccgagcgg 60

cagccaggcc ccggcccggg cctcggttcc agaagggaga ggagcccgcc aaggcgcgca 120

agagagcggg ctgcctcgca gtccgagccg gagagggagc gcgagccgcg ccggccccgg 180

acggcctccg aaaccatggg cgtgaaggtc ctgttcgccc tgatttgcat cgccgtcgca 240

gaggcacacg gcgagggcac cttcacctcc gacgtgtcct cctatctcga agagcaggcc 300

gccaaggaat tcatcgcctg gctggtgaag ggcggcggcg gtggtggtgg ctccggaggc 360

ggcggctctg gtggcggtgg cagcgctgag tccaaatatg gtcccccatg cccaccctgc 420

ccagcacctg aggccgccgg gggaccatca gtcttcctgt tccccccaaa acccaaggac 480

actctcatga tctcccggac ccctgaggtc acgtgcgtgg tggtggacgt gagccaggaa 540

gaccccgagg tccagttcaa ctggtacgtg gatggcgtgg aggtgcataa tgccaagaca 600

aagccgcggg aggagcagtt caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 660

caccaggact ggctgaacgg caaggagtac aagtgcaagg tctccaacaa aggcctcccg 720

tcctccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagagcc acaggtgtac 780

accctgcccc catcccagga ggagatgacc aagaaccagg tcagcctgac ctgcctggtc 840

aaaggcttct accccagcga catcgccgtg gagtgggaaa gcaatgggca gccggagaac 900

aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcagg 960

ctaaccgtgg acaagagcag gtggcaggag gggaatgtct tctcatgctc cgtgatgcat 1020

gaggctctgc acaaccacta cacacagaag agcctctccc tgtctctggg ttgataactc 1080

gag 1083

<210> 8

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> primer SeqPF

<400> 8

accggaattc tcagatccgc tagcgctac 29

<210> 9

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> primer SeqPR

<400> 9

tgagctcgag atcagagtcc ggacttgtac agc 33

Claims (3)

1. The application of a lentivirus recombinant expression vector in preparing a high-efficiency expression stable cell strain is characterized in that: the lentiviral recombinant expression vector comprises a lentiviral vector skeleton containing a Tet-off expression regulation system and a promoter A; the promoter A is arranged at the upstream of the tTA2 expression element and is used for directly driving the tTA2 to express;

the promoter A is a weak promoter, specifically an S100a6 promoter, a CIRP promoter or a PDI promoter;

the CIRP promoter is a CIRP-P867 promoter with a nucleotide sequence shown as SEQ ID NO.1 or a CIRP-P2588 promoter with a nucleotide sequence shown as SEQ ID NO. 2;

the PDI promoter is a PDI-P598 promoter with a nucleotide sequence shown as SEQ ID NO. 3;

the S100a6 promoter is an S100a6-dS18 promoter with a nucleotide sequence shown in SEQ ID NO. 4;

the slow virus vector skeleton is pLUT-off;

the nucleotide sequence of the pLUT-off is shown as SEQ ID NO. 5;

the cells are CHO cells.

2. A method for constructing a high-efficiency expression stable cell strain by using a lentivirus infection method is characterized by comprising the following steps:

(1) Cloning a target protein gene into a multiple cloning site in the lentiviral recombinant expression vector of claim 1 to obtain a lentiviral vector capable of expressing the target protein;

(2) Carrying out slow virus packaging on slow virus vectors capable of expressing target proteins to obtain virus liquid;

(3) Infecting host cells with the obtained virus liquid to obtain a high-efficiency expression stable cell bank; monoclonal screening is carried out on the cell bank to obtain a high-efficiency expression stable cell strain;

the target protein gene in the step (1) comprises RFP, GFP, EGFP, luciferase and GLP-1-Fc;

the nucleotide sequence of the GLP-1-Fc recombinant protein is shown as SEQ ID NO. 7.

3. The method for constructing a stable cell line with high expression by using lentiviral infection method according to claim 2, wherein:

the specific steps of lentiviral packaging described in step (2) are as follows: mixing the lentiviral vector in the step (1) with a packaging plasmid to obtain a packaging system; transfecting the packaging system into HEK 293T cells for culture; then changing fresh culture medium, continuously culturing to obtain virus liquid;

the host cell described in step (3) is a CHO cell;

the step (3) is specifically as follows: taking lentivirus suspension obtained through centrifugation and filtration treatment, and infecting host cells; after the virus liquid infects cells for 4 to 6 hours, fresh CD culture medium is added for continuous culture; changing fresh culture medium 46-48 hours after virus liquid infects cells; 4-5 days after virus liquid infects cells, detecting protein expression intensity to obtain a high-efficiency stable expression cell bank; and (3) carrying out monoclonal screening on the cell library to obtain the high-efficiency expression stable cell strain.