CN113549656A - Lentiviral vector expression system for polygene transformation - Google Patents

Abstract

The invention provides a lentivirus vector expression system for realizing multi-gene transformation by using a split single screening marker, which comprises the lentivirus vector expression system, a construction method thereof and application in multi-gene transformation. The lentivirus vector expression system can realize the transformation of a plurality of target genes by using a single screening marker, and can improve the packaging rate of the expression system and the expression rate of the target genes in a plurality of cells.

Description

Lentiviral vector expression system for polygene transformation

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a lentivirus vector expression system for realizing multi-gene transformation by using a split single screening marker, which comprises a lentivirus vector expression system, a construction method thereof and application in multi-gene transformation.

Background

Lentiviral vectors (Lentiviral vectors) are viral vector systems which are transformed on the basis of HIV-1 viruses and can efficiently transfer target genes into primary cells or cell lines of animals and humans. Lentiviral vector-mediated gene expression is sustained and stable because the inserted gene is integrated into the host cell genome and divides as the cell genome divides. In addition, lentiviruses have a wide host range, can infect dividing and non-dividing cells, can infect almost all types of cells, and are particularly suitable for cells with low transfection efficiency of plasmid vectors.

Most of the conventional lentivirus expression vectors are formed by connecting expression elements of double promoters in series, and have double screening genes of antibiotic resistance genes and fluorescent proteins, so that the lentivirus expression vectors are used for selecting engineering cells of target genotypes, but the selection is limited. Only a limited number of well characterized antibiotic resistance genes are available for eukaryotic cells, as well as spectra of only a limited number of fluorescent proteins that can be clearly distinguished by common equipment. Researchers often encounter problems with not enough selectable markers if they want to transfer multiple genes into a single cell. On the other hand, selection using multiple antibiotics simultaneously is often harsh on cells.

In 2006, the Ministry of Onchidium Shimadu invented a "cocktail" method consisting of four transcription factors including OCT4, SOX2, KLF4 and c-Myc, which was able to successfully reprogram terminally differentiated dermal fibroblasts into stem cells with differentiation pluripotency. The breakthrough invention breaks through the ethical limitation of using human embryonic stem cells in medicine, and greatly expands the application potential of stem cell technology in clinical medicine. However, when the four genes are simultaneously expressed in one lentiviral vector, the packaging efficiency of the lentivirus is lower than that of the four genes, and how to effectively package the target genes with wide application prospects and improve the expression rate of the genes is a problem to be solved urgently.

Disclosure of Invention

In order to solve the problems, the invention provides a lentivirus vector expression system, a construction method and an application thereof, which are used for selecting a plurality of genes under a screening condition, and specifically comprises the following steps:

in a first aspect of the invention, there is provided a lentiviral vector expression system comprising two vectors, wherein the first lentiviral vector comprises a N-Screening Marker-TS1 fragment, the N-Screening Marker-TS1 segment comprises a Marker gene 5' end nucleotide sequence (N-Screening Marker) for coding N end amino acid of the Screening Marker protein and a shearing joint 1 (TS 1), the second lentiviral vector comprises a TS2-C-Screening Marker segment, the TS2-C-Screening Marker fragment comprises a splicing linker 2(TS2) and a Marker gene 3' end nucleotide sequence (C-Screening Marker) for coding C end amino acid of a Screening Marker protein, the screening marker proteins in the two lentivirus vectors are the same, and the N-terminal amino acid and the C-terminal amino acid of the screening marker proteins have a screening marker function after being connected.

Preferably, the marker gene includes a selection gene and a reporter gene, preferably, the selection gene includes an antibiotic resistance gene, more preferably, the antibiotic resistance gene includes, but is not limited to, a neomycin phosphotransferase gene (npt), a kanamycin resistance gene (nptII), a puromycin resistance gene (Puro), a tetracycline resistance gene, a penicillin resistance gene, a streptomycin resistance gene, and the like; the reporter gene includes Chloramphenicol Acetyltransferase (CAT), beta-galactosidase gene (LacZ), dihydrofolate reductase gene, human growth hormone (hGH), secreted alkaline phosphatase (SEAP), Luciferase (Luciferase), fluorescent protein, etc.

In a specific embodiment of the present invention, in the above-mentioned lentiviral vector system, the selection marker is divided into two parts, i.e., N-terminal and C-terminal, based on the splicing cis-element of viral RNA, and only when two lentiviral vectors are introduced into the same cell and the corresponding N-terminal and C-terminal RNAs of the selection marker are transcribed, a complete selection marker RNA is formed under the action of the splicing cis-elements TS1 and TS2, and the selection marker protein is further expressed.

In a specific embodiment, the invention provides a lentivirus expression system, which comprises two lentivirus vectors, wherein the first lentivirus vector comprises a nucleotide sequence of pLent-EF1a-MCS-CMV-N-Screening Marker-TS1, and preferably, the nucleotide sequence of the first lentivirus vector is shown as SEQ ID NO. 1.

The second lentivirus expression vector comprises the nucleotide sequence of pLent-EF1a-MCS-CMV-TS2-C-Screening Marker, and preferably, the nucleotide sequence of the second lentivirus expression vector is shown as SEQ ID NO. 2.

Further preferably, wherein the nucleotide sequence information of the Kozak sequence is GCCACC; the screening marker gene is puromycin resistance gene (Puro), and the DNA sequence of the coding N-Puro is shown in SEQ ID NO. 3; the sequence of the Tans helicing 1 (TS 1) is shown in SEQ ID NO. 4; the sequence of the Tans spicing 2(TS2) is shown in SEQ ID NO. 5; the DNA sequence of the code C-Puro is shown in SEQ ID NO. 6. Preferably, the first lentiviral vector and the second lentiviral vector further comprise a plurality of genes of interest. More preferably, the plurality comprises at least two, such as 2, 3, 4, 5, 6 or more.

More preferably, the genes of interest include OCT4, SOX2, KLF4, c-Myc, NANOG and LIN 28.

Preferably, the gene of interest is inserted into a first lentiviral vector and a second lentiviral vector, respectively.

More preferably, the first lentiviral vector is pLent-EF1a-Oct4-P2A-Sox2-CMV-N-Puro-TS1, and the second lentiviral vector comprises pLent-EF1a-Klf4-P2A-Myc-CMV-TS 2-C-Puro.

Further preferably, the nucleotide sequence of Oct4-P2A-Sox2 is shown in SEQ ID: no.7 shows that the nucleotide sequence of Klf4-P2A-Myc is shown as SEQ ID No. 8.

In a second aspect of the present invention, there is provided a method for constructing the above lentivirus vector expression system, wherein the method comprises:

1) obtaining an N-Screening Marker-TS1 fragment and a TS2-C-Screening Marker fragment by a PCR technology;

2) respectively inserting the N-Screening Marker-TS1 fragment and the TS2-C-Screening Marker fragment obtained in the step 1) into a first lentiviral vector and a second lentiviral vector to obtain a vector 1 and a vector 2 which contain a Marker gene and a shearing linker.

In a specific embodiment, the vector 1 is an overexpression vector pLent-EF1a-MCS-CMV-N-Screening Marker-TS1, the vector 2 is an overexpression vector pLent-EF1a-MCS-CMV-TS2-C-Screening Marker, and the steps 1) and 2) of the construction method comprise:

s1: an N-Screening Marker-TS1 fragment is obtained by a PCR technology and is cloned into a Puro position in a pLent-EF1a-MCS-CMV-Puro vector to obtain a vector 1.

S2: the TS2-C-Screening Marker fragment is obtained by a PCR technology and is cloned into Puro in a pLent-EF1a-MCS-CMV-Puro vector to obtain a vector 2.

Further, the specific operation is as follows:

(1) design of primers

（2）PCR

Obtaining an N-Puro-TS1 fragment and a TS2-C-Puro fragment by a PCR technology, taking products after the PCR reaction is finished, carrying out electrophoretic separation detection on the products, and if the size of a target strip is correct, recycling the target fragment by utilizing and using a PCR purification recycling kit.

(3) Enzyme digestion:

(A) carrying out enzyme digestion on N-Puro-TS1 and TS2-C-Puro obtained by PCR;

(B) adding samples, mixing uniformly, placing at 37 ℃ for enzyme digestion for 2h, detecting the size of a band of a target enzyme digestion by using 1.5% agarose gel electrophoresis after the reaction is finished, and recovering a target fragment by using a gel recovery kit;

(C) carrying out enzyme digestion on the vector pLent-EF1a-MCS-CMV-Puro, wherein the enzyme digestion system is shown in the following table, and then carrying out gel recovery on the vector;

(D) connecting:

and connecting the recovered target gene fragment with the vector subjected to the same double enzyme digestion.

After mixing, the mixture is subjected to microcentrifugation and is connected for 1h at the temperature of 22 ℃.

(4) And (3) transformation:

transforming the connecting product into escherichia coli DH5 alpha competent cells, and coating the cells on an LB plate with corresponding resistance for screening;

(A) the pre-prepared DH5a was taken out from-80 ℃ and placed in an ice bath.

(B) After the DH5a competent cells were thawed, 5. mu.L of the ligation product was placed in 20. mu.L of DH5a competent cells, mixed well and allowed to stand in ice bath for 15 minutes.

(C) The centrifuge tube was placed in a 42 ℃ water bath for 40 seconds (without shaking the tube during the process), then quickly moved to an ice bath and allowed to stand for 2 minutes.

(D) Add 200. mu.L of sterile LB medium (without antibiotics) to the tubes, mix well and shake for 1 hour at 37 ℃ in a shaker at 220 rpm. The purpose is to express the relative resistance marker gene on the plasmid and recover the thallus.

(E) Spreading into solid culture medium plate with corresponding resistance;

(F) incubated overnight in a 37 ℃ incubator.

(5) Enzyme digestion verification;

(6) sequencing:

after single colony is picked and cultured, plasmids are extracted and are subjected to enzyme digestion identification positive cloning by EcoRI/MssI double enzyme digestion, and sequencing verification is carried out.

Preferably, the construction method further comprises: 3) inserting the target gene into the vector 1 and the vector 2 obtained in the step 2) respectively to obtain a vector 3 and a vector 4.

More preferably, the target genes are Oct4, Sox2, Klf4 and Myc.

Further preferably, the vector 3 and the vector 4 are respectively a pLent-EF1a-Oct4-P2A-Sox2-CMV-N-Puro-TS1 vector (P3) and a pLent-EF1a-Klf4-P2A-Myc-CMV-TS2-C-Puro vector (P4).

Wherein the nucleotide sequence of Oct4-P2A-Sox2 is shown as SEQ ID: no.7 shows that the nucleotide sequence of Klf4-P2A-Myc is shown as SEQ ID No. 8.

In a third aspect of the invention, there is provided a use of any one of the lentiviral vector expression vectors described above for transformation of multiple genes of interest.

Preferably, the plurality of genes of interest comprises at least two genes of interest, such as 2, 3, 4, 5, 6 or more genes of interest.

Preferably, the target gene comprises a combination of two or more of any of OCT4, SOX2, KLF4, c-Myc, NANOG and/or LIN 28.

The lentivirus vector expression system, the construction method and the application thereof have the advantages that:

1. the lentivirus vector expression system can realize the expression of a plurality of target genes, such as Oct4, Sox2, Klf4 and Myc genes, by using a single selection marker, and after cells are transfected by the lentivirus vector expression system, the survival rate of the lentivirus vector is consistent with that of a GFP-PURO vector by only using a Puro antibiotic resistance gene for selection.

2. The expression rate of the target protein can be improved by using the lentiviral vector expression system, for example, two CMV promoters respectively start Oct4-P2A-Sox2 and Klf4-P2A-Myc, and compared with the CMV promoters starting Oct4-P2A-Sox2-T2A-Klf4-P2A-Myc, the expression amount of each gene is obviously improved. After all, the promoter has a limited promoter phase rate, and the more distant a gene from the promoter, the lower the expression level.

3. The slow virus vector expression system can stably express target protein in a plurality of cells, such as HEK293, 293A cells and the like.

4. For example, two screening markers, PURO and Blasticidin, are usually required to screen the same cell twice, and only one screening marker is required to screen the same cell once by using the lentivirus vector expression system of the invention.

Drawings

FIG. 1: the detection result is pLent-EF1a-MCS-CMV-Puro-TS1 (P1) electrophoresis detection result, wherein M is a Marker schematic diagram, P1 is an actual Marker strip, and C is a detection carrier electrophoresis strip.

FIG. 2: the detection result is pLent-EF1a-MCS-CMV-TS2-C-Puro (P2) electrophoresis detection result, wherein M is a Marker schematic diagram, P2 is an actual Marker strip, and C is a detection carrier electrophoresis strip.

FIG. 3: the kit is a pLent-EF1a-Oct4-P2A-Sox2-CMV-N-Puro-TS1 (P3) electrophoresis detection result, wherein M is a Marker schematic diagram, P3 is an actual Marker strip, and C is a detection carrier electrophoresis strip.

FIG. 4: the detection result is pLent-EF1a-Klf4-P2A-Myc-CMV-TS2-C-Puro (P4) electrophoresis, wherein M is a Marker schematic diagram, P4 is an actual Marker band, and C is a detection carrier electrophoresis band.

FIG. 5: FIG. 5A shows the result of transfection after 48 hours₂White light results for O + PEI, FIG. 5B is H₂Green light result of O + PEI; FIG. 5C is the IPS-N-PURO + IPS-C-PURO + PEI white light result, FIG. 5D is the IPS-N-PURO + IPS-C-PURO + PEI green light result, FIG. 5E is the GFP-PURO + PEI white light result, and FIG. 5F is the GFP-PURO + PEI green light result.

FIG. 6: cell growth status after 72 hours for PURO screening. Wherein, FIG. 6A is H₂White light results for O + PEI, FIG. 6B is H₂Green light results for O + PEI, white light results for IPS-N-PURO + IPS-C-PURO + PE1 in FIG. 6C, green light results for IPS-N-PURO + IPS-C-PURO + PE1 in FIG. 6D, GFP-PURO + PEI white light results in FIG. 6E, and green light results for GFP-PURO + PEI in FIG. 6F

FIG. 7: the target protein mRNA is expressed copy number in HEK293 cells.

FIG. 8: as a result of infection of the 293A cells of the IPS-N-PURO-MOI 6+ IPS-C-PURO-MOI 6 group, panels A and B are the results before and after pressurization, respectively.

FIG. 9: results of transfection of IPS-N-PURO-MOI 12 group 293A cells, Panels A and B are pre-and post-compression results, respectively

FIG. 10: results of transfection of IPS-C-PURO-MOI 12 group 293A cells, Panels A and B are pre-and post-pressurization results, respectively

FIG. 11: as a result of transfection of LENTI-PURO-MOI 12 group 293A cells, graphs A and B are the results before and after pressurization, respectively

FIG. 12: as a result of transfection of 293A cells in the blank control group, panels A and B are the results before and after pressurization, respectively.

FIG. 13: is the expression copy number of the target protein mRNA in 293A cells.

Detailed Description

The present invention will be described in detail with reference to specific examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

In each of the following examples, the major equipment and materials were obtained from several companies as indicated below:

1. experimental Material

Table 1: experimental Material

2. Experimental equipment:

table 2: main equipment

3. Statistical analysis

The experimental data are expressed as Mean ± standard deviation (Mean ± SD). The two sets of data were statistically significant using unpaired t-test (Non-paired t test) with P < 0.05.

Example 1: construction of first and second Lentiviral vectors

Firstly, construction of vectors pLent-EF1a-MCS-CMV-N-Puro-TS1 and pLent-EF1a-MCS-CMV-TS2-C-Puro

1. And (3) designing a primer. Specific primers are shown in table 3:

table 3: PCR primer

2. And PCR is carried out to obtain an N-Puro-TS1 fragment and a TS2-C-Puro fragment.

Table 4: and (3) PCR system: (Unit: μ L)

Reaction procedure:

table 5: PCR procedure

After the PCR reaction is finished, 2 μ l of PCR product is separated and detected by 1.5% agarose gel electrophoresis, and if the size of the target strip is correct, the target fragment is recovered by utilizing and using a PCR purification recovery kit.

3. Enzyme digestion

(1) Carrying out enzyme digestion on N-Puro-TS1 and TS2-C-Puro obtained by PCR, and screening a gene enzyme digestion system as shown in Table 6:

table 6: screening of Gene digestion System

(2) Adding the sample, uniformly mixing, placing at 37 ℃ for enzyme digestion for 2h, detecting the size of a band of the enzyme digestion target by using 1.5% agarose gel electrophoresis after the reaction is finished, and recovering the target fragment by using a gel recovery kit.

(3) The vector pLent-EF1a-MCS-CMV-Puro was subjected to enzyme digestion in the system shown in Table 7, and then the vector was recovered by gel.

Table 7: vector digestion system

4. Connection of

The recovered screening gene fragment was ligated to the vector subjected to the same double enzyme digestion, in the system shown in Table 8:

table 8: connection system

After mixing, the mixture is subjected to microcentrifugation and is connected for 1h at the temperature of 22 ℃.

5. Transformation of

Transforming the connecting product into escherichia coli DH5 alpha competent cells, and coating the cells on an LB plate with corresponding resistance for screening;

(1) taking prepared DH5a from-80 deg.C, placing in ice bath;

(2) after the DH5a competent cells are thawed, 5 microliter of the ligation product is taken to be put into 20 microliter of DH5a competent cells, fully and uniformly mixed, and kept stand for 15 minutes in ice bath;

(3) putting the centrifuge tube into a 42 ℃ water bath kettle for 40 seconds (without shaking the centrifuge tube), then quickly moving the centrifuge tube into an ice bath, and standing for 2 minutes;

(4) adding 200 μ L of sterile LB medium (without antibiotic) into the centrifuge tube, mixing, placing in a shaker at 37 deg.C and 220rpm, and shaking for 1 hr; aims to express related resistance marker genes on plasmids and recover thalli;

(5) spreading into solid culture medium plate with corresponding resistance;

(6) incubated overnight in a 37 ℃ incubator.

6. Enzyme digestion verification: the reaction system is shown in Table 9, and the size of the cleaved band was checked by 1.5% agarose gel electrophoresis.

Table 9: enzyme digestion verification reaction system

The results are shown in FIGS. 1 and 2: wherein, FIG. 1 is a pLent-EF1a-MCS-CMV-Puro-TS1 (P1) electrophoresis detection result to obtain a 520bp fragment, which indicates that a target fragment with a 5 'end of a screening gene Puro is obtained, and FIG. 2 is a pLent-EF1a-MCS-CMV-TS2-C-Puro (P2) electrophoresis detection result to obtain a 320bp fragment, which indicates that a target fragment with a 3' end of the screening gene Puro is obtained.

7. Sequencing

After single colony is picked and cultured, plasmids are extracted and are subjected to enzyme digestion identification positive cloning by EcoRI/MssI double enzyme digestion, and sequencing verification is carried out.

Secondly, constructing a pLent-EF1a-Oct4-P2A-Sox2-CMV-N-Puro-TS1 vector and a pLent-EF1a-Klf4-P2A-Myc-CMV-TS2-C-Puro vector:

1. primers were designed, see table 10:

TABLE 10: PCR primer

2. PCR

Table 11: and (3) PCR system: (Unit: μ L)

Reaction procedure:

table 12: PCR reaction procedure

After the PCR reaction is finished, 2 mul of PCR products are separated and detected by 1.5% agarose gel electrophoresis, and if the size of the target strip is correct, the target fragment is recovered by utilizing and using a PCR purification recovery kit.

3. Enzyme digestion

(1) The Oct4-P2A-Sox2 obtained by PCR is subjected to enzyme digestion with Klf4-P2A-Myc, and the enzyme digestion system of the target gene is shown in Table 13:

table 13: enzyme digestion system of target gene

(2) Adding the sample, uniformly mixing, placing at 37 ℃ for enzyme digestion for 2h, detecting the size of a band of the enzyme digestion target by using 1.5% agarose gel electrophoresis after the reaction is finished, and recovering the target fragment by using a gel recovery kit.

(3) The vector pLent-EF1a-MCS-CMV-N-Puro-TS1 and pLent-EF1a-MCS-CMV-TS2-C-Puro are subjected to enzyme digestion, the enzyme digestion system is shown in Table 14, and then the vector is recovered by glue.

Table 14: vector digestion system

4. Connection of

The recovered target gene fragment was ligated to the vector subjected to the same double enzyme digestion, and the ligation system is shown in table 15:

table 15: connection system

After mixing, the mixture is subjected to microcentrifugation and is connected for 1h at the temperature of 22 ℃.

5. Transformation of

Transforming the connecting product into escherichia coli DH5 alpha competent cells, and coating the cells on an LB plate with corresponding resistance for screening;

(1) taking prepared DH5a from-80 deg.C, placing in ice bath;

(2) after the DH5a competent cells are thawed, 5 microliter of the ligation product is taken to be put into 20 microliter of DH5a competent cells, fully and uniformly mixed, and kept stand for 15 minutes in ice bath;

(3) putting the centrifuge tube into a 42 ℃ water bath kettle for 40 seconds (without shaking the centrifuge tube), then quickly moving the centrifuge tube into an ice bath, and standing for 2 minutes;

(4) add 200. mu.L of sterile LB medium (without antibiotics) to the tubes, mix well and shake for 1 hour at 37 ℃ in a shaker at 220 rpm. Aims to express related resistance marker genes on plasmids and recover thalli;

(5) spreading into solid culture medium plate with corresponding resistance;

(6) incubated overnight in a 37 ℃ incubator.

6. The reaction system is shown in Table 16:

table 16: verification system

The results are shown in FIG. 3 and FIG. 4, wherein FIG. 3 is the result of electrophoretic detection of pLent-EF1a-Oct4-P2A-Sox2-CMV-N-Puro-TS1 (P3), FIG. 4 is the result of electrophoretic detection of pLent-EF1a-Klf4-P2A-Myc-CMV-TS2-C-Puro (P4), and the results show that the objective plasmids are obtained, which are respectively 2132bp and 2879bp fragments.

7. Sequencing

After single colony is picked and cultured, plasmid is extracted and is subjected to enzyme digestion identification positive cloning by SgfI/MluI double enzyme digestion, and sequencing verification is carried out.

Example 2: transfection of the vector into cells, transcription of the target Gene

Co-transfecting the constructed pLent-EF1a-Oct4-P2A-Sox2-CMV-N-Puro-TS1 (P3) vector and the pLent-EF1a-Klf4-P2A-Myc-CMV-TS2-C-Puro (P4) vector into HEK293 cells, and screening the Puro for the survival rate of 72 hours and detecting the mRNA levels of the four genes

Firstly, co-transfecting HEK293 cells with constructed pLent-EF1a-Oct4-P2A-Sox2-CMV-N-Puro-TS1 vector and pLent-EF1a-Klf4-P2A-Myc-CMV-TS2-C-Puro vector, and verifying survival rate of Puro screening for 72 hours

1.1 HEK293 cells growing normally, and laying a 6-pore plate;

1.2 transfection experiments were performed according to Table 17:

1.2.1 cell culture: taking a 6-well plate cell culture dish, adding 2mL of cell culture dish containing 1-2 multiplied by 10⁵Cell culture broth, CO 37 ℃₂The culture density is 40% -60%, the density is too high, and cells are not selected after transfection.

1.2.2 transfection solution preparation of solution A: diluting 1. mu.g of plasmid DNA with serum-free medium; and B, liquid B: mu.l PEI was diluted with serum free medium; and (4) respectively and uniformly mixing the solution A and the solution B, and standing for 5 minutes.

1.2.3 absorbing the solution B, adding the solution B into the solution A, and gently and uniformly mixing. Left at room temperature for 25 minutes.

1.2.4 slowly adding the A/B compound into the culture solution, shaking up, and culturing at 37 ℃ in an incubator.

Table 17: transfection system

1.3 Pictures were taken 48 hours after transfection and exposure time 1 s. The results are shown in FIG. 5, and are all transfection results: wherein FIG. 5A is H₂White light results for O + PEI, FIG. 5B is H₂The green light of O + PEI shows the results, FIG. 5C shows the white light results of IPS-N-PURO + IPS-C-PURO + PEI, FIG. 5D shows the green light results of IPS-N-PURO + IPS-C-PURO + PEI, FIG. 5E shows the white light results of GFP-PURO + PEI, and FIG. 5F shows the green light results of GFP-PURO + PEI, showing that the transfection process is normal and the cells are all growing normally.

1.4 cells were passaged to 10cm dishes with the addition of puromycin, screened for 72h, observed and photographed:

1.5 Experimental results: the results are shown in FIG. 6, which is the cell growth status after screening. Wherein, FIG. 6A is H₂White light results for O + PEI, FIG. 6B is H₂Green light results for O + PEI, white light results for IPS-N-PURO + IPS-C-PURO + PE1 in FIG. 6C, green light results for IPS-N-PURO + IPS-C-PURO + PE1 in FIG. 6D, white light results for GFP-PURO + PEI in FIG. 6E, green light results for GFP-PURO + PEI in FIG. 6F, and results showing transfection of H + PEI after 72H puromycin screening₂All O + PEI cell groups died; the cell survival rate of the IPS-N-purO + IPS-C-purO + PE1 plasmid transfected group can reach 40%, and is basically consistent with that of a GFP-PURO plasmid transfected control group.

1.6 conclusion of the experiment: puromycin resistance was obtained by co-transfection of two vectors that split the puromycin selectable marker.

Secondly, after IPS-N-PURO + IPS-C-PURO cotransfection and PURO screening, mRNA level detection of four IPS related genes

2.1 collecting cell samples of the gfp-PURO + PEI 1 group and the IPS-N-purO + IPS-C-purO + PE12 group of the transfection experiment, extracting RNA, and detecting mRNA level after reverse transcription.

2.2 primer design, as in Table 18:

table 18: PCR primer

2.3 absolute quantitative PCR by Syber green method, 3 multiple wells were made during the qPCR process.

The amplification system is shown in Table 19:

table 19: PCR amplification system

Amplification conditions: 95 ℃ for 3min, 95 ℃ for 5sec, 60 ℃ for 15 sec, 72 ℃ for 15 sec. The number of cycles 39.

2.4 results of the experiment are shown in Table 20 and FIG. 12 (copy number of target protein mRNA expressed in HEK293 cells):

table 20: target protein mRNA expression copy number

2.5 conclusion of the experiment: two overexpression vectors of the split screening marker are used for co-expressing four target genes, and the four target genes are obviously overexpressed on the mRNA level.

Example 3: packaging lentivirus infected 293A cell screening stable transfer cell

3.1 packaging of lentivirus products:

3.1.1 spreading HEK293T cells in a 10cm dish, wherein the cell fusion degree reaches 85% -90% the next day;

3.1.2 preparing a transfection reagent according to the proportion, standing for 3min at room temperature after mixing, adding PEI, whirling and mixing uniformly, standing for 30min at room temperature, adding into a 10cm cell culture dish, and replacing a fresh culture medium 6 hours after transfection;

3.1.3 Observation of transfection efficiency and photography;

3.1.4 transfection 72 hours later collecting the supernatant culture medium, 0.45um filter membrane; adding the filtered supernatant culture medium into an ultrafiltration tube for ultracentrifugation at 25000rpm at 4 ℃ and 1.5hs;

3.1.5 Collection of virus.

3.2 detection of viral titres:

3.2.1 hour before infection in 24-well cell plates at 2.5X 10⁵Seeding HEK293 cells at cell/well density;

3.2.2 performing gradient dilution on the lentivirus, performing three gradients in total, then adding a group of lentivirus with fluorescence known TU, uniformly mixing, and adding into a 24-well plate paved with cells;

3.2.3 cells were collected after 64-68 hours of infection and genomic DNA was extracted;

3.2.4 qPCR assay

IPS-N-PURO:pLent-Ef1a-Oct4-Sox2-cmv-N-Puro -7.19E+08 IU/ml

IPS-C-PURO:pLent-Ef1a-Klf4-Myc-cmv-C-Puro -1.87E+09 IU/ml

3.3 infection protocol:

3.3.1 in 6-well cell plates at 1.0X 10 hours before infection⁶Seeding HEK293 cells at cell/well density;

3.3.2 grouped as in Table 19, MOI added the corresponding amount of lentivirus.

Grouping is shown in table 21:

table 21: grouping

3.4 selection of stably transfected cells: after 72 hours of infection, PURO was added for screening, and stable transfected cells were obtained in one week of screening. The results are shown in FIGS. 8-12, where:

FIG. 8 shows the results of transfection of the IPS-N-PURO-MOI 6+ IPS-C-PURO-MOI 6 group 293A cells, panels A and B being the results before and after pressurization, respectively; the result shows that the cells of the group are in a stable amplification state after being screened by puromycin, and a stable transgenic cell line is obtained.

FIG. 9 shows the results of transfection of IPS-N-PURO-MOI 12 group 293A cells, panels A and B are the results before and after pressurization, respectively; the results showed that the group of cells did not have puromycin resistance and all cells died.

FIG. 10 shows the results of transfection of IPS-C-PURO-MOI 12 group 293A cells, panels A and B are the results before and after pressurization, respectively; the results showed that the group of cells did not have puromycin resistance and all cells died.

FIG. 11 shows the results of transfection of LENTI-PURO-MOI 12 group 293A cells, panels A and B are the results before and after pressurization, respectively; the results showed that the group of cells did not have puromycin resistance and all cells died.

FIG. 12 shows the results of transfection in 293A cells of the blank control group, and FIGS. A and B show the results before and after pressurization, respectively; the results showed that the group of cells did not have puromycin resistance and all cells died.

3.5 quantitative determination of mRNA expression of each gene of IPS stable cell line, the results are shown in Table 22 and FIG. 13 (expressed copy number of target protein mRNA in 293 cell):

table 22: target protein mRNA expression copy number

The above results show that the four genes of interest can be stably expressed in 293A cells.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Sequence listing

<110> Shandong Weizhen Biotech Co., Ltd

<120> a lentiviral vector expression system for multigene transformation

<130> 1

<160> 28

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1095

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60

gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 120

atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 180

aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 240

catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 300

catggtgatg cggttttggc agtacaccaa tgggcgtgga tagcggtttg actcacgggg 360

atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 420

ggactttcca aaatgtcgta ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt 480

acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagaatt ttgtaatacg 540

actcactata gggcggccgg gaattcgcca ccatgaccga gtacaagccc acggtgcgcc 600

tcgccacccg cgacgacgtc ccccgggcag tacgcaccct cgccgccgcg ttcgccgact 660

accccgccac gcgccacacc gtcgatccag accgccacat cgagcgggtc accgagctgc 720

aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg 780

gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg 840

agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg 900

aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg 960

tctcgcccga ccaccaggta agtatcaagg ttacaagaca ggtttaagga gaccaataat 1020

aacttcgtat agcatacatt atacgaagtt atcggacagc tttaaataag cacagtagtt 1080

tgtccaagtt taaac 1095

<210> 2

<211> 897

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60

gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 120

atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 180

aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 240

catgacctta cgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 300

catggtgatg cggttttggc agtacaccaa tgggcgtgga tagcggtttg actcacgggg 360

atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 420

ggactttcca aaatgtcgta ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt 480

acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagaatt ttgtaatacg 540

actcactata gggcggccgg gaattcttgg acaaactact gtgcttattt aaagctataa 600

cttcgtatag catacattat acgaagttat ctcttgcgtt tctgataggc acctattggt 660

cttactgaca tccactttgc ctttctctcc acagggcaag ggtctgggca gcgccgtcgt 720

gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc 780

gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt 840

gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgag tttaaac 897

<210> 3

<211> 406

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc ccgggcagta 60

cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccagac 120

cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac 180

atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag 240

agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt 300

tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag 360

cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagg 406

<210> 4

<211> 109

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

taagtatcaa ggttacaaga caggtttaag gagaccaata ataacttcgt atagcataca 60

ttatacgaag ttatcggaca gctttaaata agcacagtag tttgtccaa 109

<210> 5

<211> 125

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ttggacaaac tactgtgctt atttaaagct ataacttcgt atagcataca ttatacgaag 60

ttatctcttg cgtttctgat aggcacctat tggtcttact gacatccact ttgcctttct 120

ctcca 125

<210> 6

<211> 195

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg 60

gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc 120

ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc 180

aagcccggtg cctga 195

<210> 7

<211> 2130

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gcgatcgcca ccatggcggg acacctggct tcggatttcg ccttctcgcc ccctccaggt 60

ggtggaggtg atgggccagg ggggccggag ccgggctggg ttgatcctcg gacctggcta 120

agcttccaag gccctcctgg agggccagga atcgggccgg gggttgggcc aggctctgag 180

gtgtggggga ttcccccatg ccccccgccg tatgagttct gtggggggat ggcgtactgt 240

gggccccagg ttggagtggg gctagtgccc caaggcggct tggagacctc tcagcctgag 300

ggcgaagcag gagtcggggt ggagagcaac tccgatgggg cctccccgga gccctgcacc 360

gtcacccctg gtgccgtgaa gctggagaag gagaagctgg agcaaaaccc ggaggagtcc 420

caggacatca aagctctgca gaaagaactc gagcaatttg ccaagctcct gaagcagaag 480

aggatcaccc tgggatatac acaggccgat gtggggctca ccctgggggt tctatttggg 540

aaggtattca gccaaacgac catctgccgc tttgaggctc tgcagcttag cttcaagaac 600

atgtgtaagc tgcggccctt gctgcagaag tgggtggagg aagctgacaa caatgaaaat 660

cttcaggaga tatgcaaagc agaaaccctc gtgcaggccc gaaagagaaa gcgaaccagt 720

atcgagaacc gagtgagagg caacctggag aatttgttcc tgcagtgccc gaaacccaca 780

ctgcagcaga tcagccacat cgcccagcag cttgggctcg agaaggatgt ggtccgagtg 840

tggttctgta accggcgcca gaagggcaag cgatcaagca gcgactatgc acaacgagag 900

gattttgagg ctgctgggtc tcctttctca gggggaccag tgtcctttcc tctggcccca 960

gggccccatt ttggtacccc aggctatggg agccctcact tcactgcact gtactcctcg 1020

gtccctttcc ctgaggggga agcctttccc cctgtctccg tcaccactct gggctctccc 1080

atgcattcaa acacgcgagg aagcggagct actaacttca gcctgctgaa gcaggctgga 1140

gacgtggagg agaaccctgg accttcgcgt gccaccatgt acaacatgat ggagacggag 1200

ctgaagccgc cgggcccgca gcaaacttcg gggggcggcg gcggcaactc caccgcggcg 1260

gcggccggcg gcaaccagaa aaacagcccg gaccgcgtca agcggcccat gaatgccttc 1320

atggtgtggt cccgcgggca gcggcgcaag atggcccagg agaaccccaa gatgcacaac 1380

tcggagatca gcaagcgcct gggcgccgag tggaaacttt tgtcggagac ggagaagcgg 1440

ccgttcatcg acgaggctaa gcggctgcga gcgctgcaca tgaaggagca cccggattat 1500

aaataccggc cccggcggaa aaccaagacg ctcatgaaga aggataagta cacgctgccc 1560

ggcgggctgc tggcccccgg cggcaatagc atggcgagcg gggtcggggt gggcgccggc 1620

ctgggcgcgg gcgtgaacca gcgcatggac agttacgcgc acatgaacgg ctggagcaac 1680

ggcagctaca gcatgatgca ggaccagctg ggctacccgc agcacccggg cctcaatgcg 1740

cacggcgcag cgcagatgca gcccatgcac cgctacgacg tgagcgccct gcagtacaac 1800

tccatgacca gctcgcagac ctacatgaac ggctcgccca cctacagcat gtcctactcg 1860

cagcagggca cccctggcat ggctcttggc tccatgggtt cggtggtcaa gtccgaggcc 1920

agctccagcc cccctgtggt tacctcttcc tcccactcca gggcgccctg ccaggccggg 1980

gacctccggg acatgatcag catgtatctc cccggcgccg aggtgccgga acccgccgcc 2040

cccagcagac ttcacatgtc ccagcactac cagagcggcc cggtgcccgg cacggccatt 2100

aacggcacac tgcccctctc acacatgtga 2130

<210> 8

<211> 2886

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gccaccatga ggcagccacc tggcgagtct gacatggctg tcagcgacgc gctgctccca 60

tctttctcca cgttcgcgtc tggcccggcg ggaagggaga agacactgcg tcaagcaggt 120

gccccgaata accgctggcg ggaggagctc tcccacatga agcgacttcc cccagtgctt 180

cccggccgcc cctatgacct ggcggcggcg accgtggcca cagacctgga gagcggcgga 240

gccggtgcgg cttgcggcgg tagcaacctg gcgcccctac ctcggagaga gaccgaggag 300

ttcaacgatc tcctggacct ggactttatt ctctccaatt cgctgaccca tcctccggag 360

tcagtggccg ccaccgtgtc ctcgtcagcg tcagcctcct cttcgtcgtc gccgtcgagc 420

agcggccctg ccagcgcgcc ctccacctgc agcttcacct atccgatccg ggccgggaac 480

gacccgggcg tggcgccggg cggcacgggc ggaggcctcc tctatggcag ggagtccgct 540

ccccctccga cggctccctt caacctggcg gacatcaacg acgtgagccc ctcgggcggc 600

ttcgtggccg agctcctgcg gccagaattg gacccggtgt acattccgcc gcagcagccg 660

cagccgccag gtggcgggct gatgggcaag ttcgtgctga aggcgtcgct gagcgcccct 720

ggcagcgagt acggcagccc gtcggtcatc agcgtcagca aaggcagccc tgacggcagc 780

cacccggtgg tggtggcgcc ctacaacggc gggccgccgc gcacgtgccc caagatcaag 840

caggaggcgg tctcttcgtg cacccacttg ggcgctggac cccctctcag caatggccac 900

cggccggctg cacacgactt ccccctgggg cggcagctcc ccagcaggac taccccgacc 960

ctgggtcttg aggaagtgct gagcagcagg gactgtcacc ctgccctgcc gcttcctccc 1020

ggcttccatc cccacccggg gcccaattac ccatccttcc tgcccgatca gatgcagccg 1080

caagtcccgc cgctccatta ccaagagctc atgccacccg gttcctgcat gccagaggag 1140

cccaagccaa agaggggaag acgatcgtgg ccccggaaaa ggaccgccac ccacacttgt 1200

gattacgcgg gctgcggcaa aacctacaca aagagttccc atctcaaggc acacctgcga 1260

acccacacag gtgagaaacc ttaccactgt gactgggacg gctgtggatg gaaattcgcc 1320

cgctcagatg aactgaccag gcactaccgt aaacacacgg ggcaccgccc gttccagtgc 1380

caaaaatgcg accgagcatt ttccaggtcg gaccacctcg ccttacacat gaagaggcat 1440

tttgctagcg gaagcggagc tactaacttc agcctgctga agcaggctgg agacgtggag 1500

gagaaccctg gacctgctag cctggatttt tttcgggtag tggaaaacca gcagcctccc 1560

gcgacgatgc ccctcaacgt tagcttcacc aacaggaact atgacctcga ctacgactcg 1620

gtgcagccgt atttctactg cgacgaggag gagaacttct accagcagca gcagcagagc 1680

gagctgcagc ccccggcgcc cagcgaggat atctggaaga aattcgagct gctgcccacc 1740

ccgcccctgt cccctagccg ccgctccggg ctctgctcgc cctcctacgt tgcggtcaca 1800

cccttctccc ttcggggaga caacgacggc ggtggcggga gcttctccac ggccgaccag 1860

ctggagatgg tgaccgagct gctgggagga gacatggtga accagagttt catctgcgac 1920

ccggacgacg agaccttcat caaaaacatc atcatccagg actgtatgtg gagcggcttc 1980

tcggccgccg ccaagctcgt ctcagagaag ctggcctcct accaggctgc gcgcaaagac 2040

agcggcagcc cgaaccccgc ccgcggccac agcgtctgct ccacctccag cttgtacctg 2100

caggatctga gcgccgccgc ctcagagtgc atcgacccct cggtggtctt cccctaccct 2160

ctcaacgaca gcagctcgcc caagtcctgc gcctcgcaag actccagcgc cttctctccg 2220

tcctcggatt ctctgctctc ctcgacggag tcctccccgc agggcagccc cgagcccctg 2280

gtgctccatg aggagacacc gcccaccacc agcagcgact ctgaggagga acaagaagat 2340

gaggaagaaa tcgatgttgt ttctgtggaa aagaggcagg ctcctggcaa aaggtcagag 2400

tctggatcac cttctgctgg aggccacagc aaacctcctc acagcccact ggtcctcaag 2460

aggtgccacg tctccacaca tcagcacaac tacgcagcgc ctccctccac tcggaaggac 2520

tatcctgctg ccaagagggt caagttggac agtgtcagag tcctgagaca gatcagcaac 2580

aaccgaaaat gcaccagccc caggtcctcg gacaccgagg agaatgtcaa gaggcgaaca 2640

cacaacgtct tggagcgcca gaggaggaac gagctaaaac ggagcttttt tgccctgcgt 2700

gaccagatcc cggagttgga aaacaatgaa aaggccccca aggtagttat ccttaaaaaa 2760

gccacagcat acatcctgtc cgtccaagca gaggagcaaa agctcatttc tgaagaggac 2820

ttgttgcgga aacgacgaga acagttgaaa cacaaacttg aacagctacg gaactcttgt 2880

gcgtaa 2886

<210> 9

<211> 86

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cgtataatgt atgctatacg aagttattat tggtctcctt aaacctgtct tgtaaccttg 60

atacttacct ggtggtcggg cgagac 86

<210> 10

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gcgtgtttaa acttggacaa actactgtgc ttatttaaag ctgtccgata acttcgtata 60

atgtatgcta tacg 74

<210> 11

<211> 87

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gttatctctt gcgtttctga taggcaccta ttggtcttac tgacatccac tttgcctttc 60

tctccacagg gcaagggtct gggcagc 87

<210> 12

<211> 89

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gcgtgaattc ttggacaaac tactgtgctt atttaaagct ataacttcgt atagcataca 60

ttatacgaag ttatctcttg cgtttctgg 89

<210> 13

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gcgtgtttaa actcaggcac cgggcttgcg g 31

<210> 14

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gcgtgaattc gccaccatga ccgagtacaa gcccac 36

<210> 15

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gagtgcgatc gccaccatgg cgggacacct ggct 34

<210> 16

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gcgtacgcgt tcacatgtgt gagaggggca gt 32

<210> 17

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gagtgcgatc gccaccatga ggcagccacc tg 32

<210> 18

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gcgtacgcgt ttacgcacaa gagttccgta g 31

<210> 19

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

catgtacgtt gctatccagg c 21

<210> 20

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ctccttaatg tcacgcacga t 21

<210> 21

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ggctcctggc aaaaggtca 19

<210> 22

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ctgcgtagtt gtgctgatgt 20

<210> 23

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

gccgagtgga aacttttgtc g 21

<210> 24

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ggcagcgtgt acttatcctt ct 22

<210> 25

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

ctgggttgat cctcggacct 20

<210> 26

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

ccatcggagt tgctctcca 19

<210> 27

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

accctgggtc ttgaggaagt 20

<210> 28

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

ggcatgagct cttggtaatg ga 22

Claims (10)

1. A lentiviral vector expression system, wherein the lentiviral vector expression system comprises two lentiviral vectors, wherein the first lentiviral vector comprises a N-Screening Marker-TS1 fragment, the N-Screening Marker-TS1 segment comprises a Marker gene 5' end nucleotide sequence (N-Screening Marker) for coding N end amino acid of the Screening Marker protein and a shearing joint 1 (TS 1), the second lentiviral vector comprises a TS2-C-Screening Marker segment, the TS2-C-Screening Marker fragment comprises a splicing linker 2(TS2) and a Marker gene 3' end nucleotide sequence (C-Screening Marker) for coding C end amino acid of a Screening Marker protein, the screening marker proteins in the two lentivirus vectors are the same, and the N-terminal amino acid and the C-terminal amino acid of the screening marker proteins have a screening marker function after being connected.

2. The lentiviral vector expression system of claim 1, wherein the marker gene comprises a selection gene and a reporter gene.

3. The lentiviral vector expression system of claim 1, wherein the first and second lentiviral vectors further comprise a plurality of genes of interest.

4. The lentiviral vector expression system of claim 3, wherein the gene of interest comprises OCT4, SOX2, KLF4, and/or c-Myc.

5. The lentiviral expression system of any one of claims 1-4, wherein the first lentiviral expression vector comprises the nucleotide sequence of pLent-EF1a-MCS-CMV-N-Screening Marker-TS1 and the second lentiviral expression vector comprises the nucleotide sequence of pLent-EF1a-MCS-CMV-TS2-C-Screening Marker.

6. The method of constructing the lentiviral vector expression system of claim 1, wherein the method comprises:

1) obtaining an N-Screening Marker-TS1 fragment and a TS2-C-Screening Marker fragment by a PCR technology;

2) respectively inserting the N-Screening Marker-TS1 fragment and the TS2-C-Screening Marker fragment obtained in the step 1) into a first lentiviral vector and a second lentiviral vector to obtain a vector 1 and a vector 2 which contain a Marker gene and a shearing linker.

7. The build method of claim 6, further comprising: 3) inserting the target gene into the vector 1 and the vector 2 obtained in the step 2) respectively to obtain a vector 3 and a vector 4.

8. Use of the lentiviral vector expression vector of any one of claims 1-5 for transformation of multiple genes of interest.

9. The use of claim 8, wherein said plurality of genes of interest comprises at least two genes of interest.

10. The use of claim 9, wherein the gene of interest comprises OCT4, SOX2, KLF4 and/or c-Myc.

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