CN114990163A

CN114990163A - Lentiviral vector for stem cell gene modification and construction method and application thereof

Info

Publication number: CN114990163A
Application number: CN202210342741.2A
Authority: CN
Inventors: 夏玉龙; 吴金芸; 叶华衍; 孙毅; 林靖
Original assignee: Zhonghaixia Fujian Cell Biotechnology Co ltd
Current assignee: Zhonghaixia Fujian Cell Biotechnology Co ltd
Priority date: 2022-03-31
Filing date: 2022-03-31
Publication date: 2022-09-02

Abstract

The invention discloses a lentiviral vector for stem cell gene modification and a construction method and application thereof. The vector can be used for genetic modification of stem cells and endows the stem cells with stronger immunoregulation capability. Meanwhile, the expression of the reporter gene RFP is beneficial to the pharmacokinetics research before clinic, and lays the theoretical foundation of the early stage for the development of clinical tests.

Description

Lentiviral vector for stem cell gene modification and construction method and application thereof

Technical Field

The invention relates to a lentivirus vector and preparation and application thereof, belonging to the technical field of biology.

Background

Stem cells are a class of cells that have not been fully differentiated, have self-renewal, high proliferation and differentiation potential. Stem cells can be classified into three types, i.e., pluripotent stem cells, multipotent stem cells and unipotent stem cells, according to the size of differentiation potential; stem cells can be classified into two types, embryonic stem cells and adult stem cells, depending on the developmental stage. Stem cell therapy refers to the process of applying stem cells from human autologous or allogeneic sources to transfuse (implant) into a human body after in vitro operation and acting on disease treatment. The stem cells have great development potential and clinical application value in the fields of disease treatment, tissue repair, anti-aging, beauty treatment and the like, and become one of the important directions in the field of life science in recent years. Although the stem cells have a wide clinical application prospect, the clinical application of the stem cells also faces some challenges, for example, how to effectively improve the treatment effect of the stem cell treatment is the key point for further popularization and application of the stem cell treatment.

It is a common technical method at present to enhance the expression of specific proteins in cells by using a genetic modification method so as to directionally endow a certain cell with specific functions. And has been widely used in the field of disease treatment, for example, chimeric antigen receptor T cells, which utilize recombinant lentiviruses to transduce foreign gene expression, thereby conferring T cells with the ability to recognize specific tumor antigens, thereby targeted killing of tumor cells. Therefore, the importance of gene modification and the safety of gene modification have been widely recognized by the scientific community, and the relationship between human health, environment, agricultural development, economy and politics has been intensively studied and gradually advanced into the lives of people. Stem cells have immunomodulatory effects and stem cell therapy has important research value in a number of tissue inflammatory diseases, such as osteoarthritis. However, it is one of the directions of research on stem cell therapy to safely and effectively genetically modify stem cells to improve their therapeutic effects on osteoarthritis.

Disclosure of Invention

The invention discloses a lentiviral vector pLL3.7-EF1a-MCS-PGK-RFP for stem cell gene modification, a construction method thereof and application thereof in preparation of osteoarthritis drugs. The invention also establishes a method for detecting the virus titer by the flow cytometry, and provides a detection standard for clinical application. The invention provides a lentiviral vector pLL3.7-EF1a-MCS-PGK-RFP for stem cell gene modification, the nucleotide sequence and map of which are shown in figure 1:

the invention also discloses a preparation method of the lentiviral vector pLL3.7-EF1a-MCS-PGK-RFP, which comprises the following steps:

s1: synthesis of EF1-MCS sequences

The sequence of the pLL3.7 vector (the sequence of the pLL.7 vector is shown as SEQ ID NO. 1) is analyzed by using SnapGene software to obtain a corresponding map (shown in figure 1), and a single enzyme cutting site XbaI (located at 7170bp) and a single enzyme cutting site Pci I (1390bp) are found to be capable of cutting a CMV + EGHFP element (a sequence with the length of 1867bp is cut out together) on the initial vector, and the size of the vector after cutting is 5780 bp. The synthesized EF1a-MCS-PGK-RFP sequence is added with Xba I or Pci I sequence at two ends. The total length of the synthetic gene is 1646bp (shown in SEQ ID NO. 2). The sequence of the synthesized EF1a-MCS-PGK-RFP gene containing XbaI and PciI enzyme cutting sites is shown as SEQ ID NO. 3.

S2：

The pLL3.7 vector (purchased from Shanghai Yang Sheng) and the EF1a-MCS-PGK-RFP sequence containing the restriction enzyme sites Xba I and PciI are subjected to double enzyme digestion by restriction enzymes Xba I and PciI, and the target products after enzyme digestion are respectively recovered.

The invention also discloses a using method of the lentivirus vector pLL3.7-EF1a-MCS-PGK-RFP, which comprises the following steps:

step 1: cloning a target gene into a pLL3.7-EF1a-MCS-PGK-RFP vector to obtain a recombinant lentivirus vector;

step 2: co-transfecting the recombinant lentiviral vector obtained in the step 1, psPAX2 and pMD2.0G into a host cell;

and step 3: culturing a host cell, wherein the host cell is a 293T cell;

and 4, step 4: and separating to obtain the recombinant lentivirus.

The invention also discloses a recombinant lentiviral vector, which is prepared by the following method: the neutralizing antibody sequences of the immunomodulatory cytokine genes or anti-inflammatory cytokines are cloned into the polyclonal site of a vector such as pLenti-EF 1-MCS-PGK-RFP. Wherein the immunoregulatory cytokine gene is selected from the group consisting of human TGF-beta, IL-10, IL-4, and the like, or any combination thereof. Neutralizing antibodies against inflammatory cytokines include anti-human IL-6, TNF-a, IL-1, and the like.

The invention has the following beneficial effects: the lentiviral vector for stem cell gene modification can be used for stem cell gene modification and endows stem cells with stronger immunoregulatory capacity. Meanwhile, the expression report gene RFP is helpful for the pharmacokinetics research before clinic, and lays the theoretical foundation of the early stage for the development of clinical tests.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is a map of the pLL3.7 plasmid vector;

FIG. 2 shows a diagram of double-restriction electrophoresis of pLL3.7-EF1a-MCS-PGK-RFP recombinant plasmid;

FIG. 3 pLL3.7-EF1a-MCS-PGK-RFP plasmid map;

FIG. 4 double digestion of pLL3.7-EF1a-IL-10-PGK-RFP recombinant lentiviral vector plasmid;

FIG. 5 pLL3.7-EF1-IL-10-PGK-RFP recombinant lentiviral vector plasmid map;

FIG. 6 fluorescent microscope observation of cells expressing red fluorescence;

FIG. 7 ELISA detects IL-10 secretion levels.

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

Examples

The preparation method of the lentivirus vector pLL3.7-EF1a-MCS-PGK-RFP comprises the following steps:

step 1: synthesis of EF1-MCS sequence (Suzhou Jinwei Zhi Biotechnology Co., Ltd.)

The sequence of the pLL3.7 vector (pLL.7 vector sequence is as follows) is analyzed by using SnapGene software to obtain a corresponding map (as shown in figure 1), and a single enzyme cutting site XbaI (located at 7170bp) and a single enzyme cutting site Pci I (1390bp) are found to be capable of cutting off a CMV + EGHFP element (a sequence with the length of 1867bp is cut off together) on the initial vector, and the size of the vector after cutting off is 5780 bp. The synthesized EF1a-MCS-PGK-RFP sequence is added with Xba I or Pci I sequence at two ends. The total length of the synthetic gene is 1646bp (the sequence is shown below).

The sequence of the pLL3.7 plasmid vector is shown in SEQ ID NO. 1.

The EF1a-MCS-PGK-RFP gene sequence containing XbaI and PciI enzyme cutting sites and formed by Jinzhi Biotechnology Limited, Suzhou is shown as SEQ ID NO. 3.

And 2, step:

the pLL3.7 vector (purchased from Shanghai Kangshihibul) and the EF1a-MCS-PGK-RFP sequence containing the enzyme cutting sites Xba I and PciI are subjected to double enzyme cutting by using restriction enzymes Xba I and Pci I, and target products after enzyme cutting are respectively recovered;

the specific method comprises the following steps:

1) enzyme digestion

pLL3.7 plasmid or EF1a-MCS-PGK-RFP gene 1ug, 1. mu.L restriction enzyme Xba I (NEB, R0145S) and 1. mu.L restriction enzyme Pci I (NEB, R0655S), 5ul were added

Buffer (NEB), and finally double distilled water was added to make the whole reaction volume 50. mu.L. The sample is placed at 37 ℃ for reaction for 1 hour;

2) linearized plasmid recovery

Required reagent

1 × TAE: 4.84g Tris (sollabio, # T8060-100g), 0.744g Na2EDTA.2H2O (Shanghai test, #10009717), 1.142mL glacial acetic acid (national drug, #10000218) were fully dissolved in appropriate amount of ddH2O, and the volume was determined to 1L for use.

Agarose (Biowest, BY-R0100)

Agarose gel recovery kit: common agarose gel DNA recovery kit (Tiangen, # DP209-02)

DNA loading: 6x DNA loading (gold overall, # GH101)

Experimental procedure

A. 1% agarose gel preparation

Weighing 0.5g of agarose, adding 50mL of 1 XTAE solution, uniformly mixing, boiling in a microwave oven for 2min, and adding into a gel tank for condensation;

B. agarose gel electrophoresis

Adding DNA loading to 1x of linearized plasmid and target fragment samples, adding into sample wells, and performing 150V electrophoresis for 15 min;

C. linearized plasmid and target band cutting gel

Cutting the agarose gel containing the target band under an ultraviolet gel cutting instrument and transferring the agarose gel into a new 1.5mL centrifuge tube;

D. destination strip recovery

The target fragment was recovered according to the procedure of the agarose gel recovery kit, and stored at-20 ℃ after the concentration was determined.

3) Connection of

Required reagent

Connecting the kit: minerva Super Fusion Cloning Kit (Suzhou Yuheng, # M2026-50T)

Experimental procedure

A. Reaction system

The following ligation reaction system was prepared:

*: adding the target gene segment recovered in the step 2) and the linearized vector into a reaction system according to a molar ratio of 2: 1.

B. Connection of

Mixing, and reacting at 37 deg.C for 30min

C. Storage of

After the reaction is finished, the ligation product containing the recombinant lentivirus plasmid pLL3.7-EF1a-MCS-PGK-RFP is obtained and is used immediately or stored at-20 ℃ for later use.

3) Transformation of

Required reagent

DH5 alpha competent cell (Kangshenghe, # KTSM101L)

LB liquid medium: 10G tryptone (Sigma-Aldrich, # T9410-250G), 5G yeast extract (Sigma-Aldrich, # T9410-250G), 10G NaCl (Hu test, #10019308) were added to ddH ₂ Adjusting pH to 7.0 after O is dissolved, fixing volume to 1L, sterilizing at 121 deg.C under high pressure for 15min, and cooling to room temperature

LB solid medium: 10G tryptone (Sigma-Aldrich, # T9410-250G), 5G yeast extract (Sigma-Aldrich, # T9410-250G), 10G NaCl (Hu test, #10019308), adjusting pH to 7.0, adding 10G agar powder (avadin, # A109143-5kg), making a volume of 1L, autoclaving at 121 deg.C for 15min, and cooling to room temperature

Antibiotics: weighing 1g of corresponding antibiotic powder (sigma), dissolving in 20mL of double distilled water, sterilizing at 0.22 μm, filtering, washing hair, sterilizing, packaging, and storing at-20 deg.C

Experimental procedure

a. Taking a piece of DH5 alpha competence (100 mu L) to be placed on ice for unfreezing, sucking a proper amount of the ligation product obtained in the step 2) after the competence cells are unfrozen, adding the ligation product into the competence cells in the molten state, slightly blowing and beating for several times, uniformly mixing, and placing on ice for 20 min;

b. after the ice bath is finished, placing the mixture in a water bath kettle, thermally shocking the mixture for 90s at 42 ℃, and immediately placing the mixture on ice for 2 min;

c. adding 500 μ L of LB liquid culture medium without antibody into the mixture, resuscitating at 37 deg.C and 200rpm for 1 h;

d. sucking a proper amount of recovered bacteria liquid and coating the bacteria liquid on an LB solid culture medium containing 100 mu g/mL of corresponding antibiotics;

e.37 ℃ in an incubator, inverting the culture overnight to show that a monoclonal colony appears on an LB solid culture medium;

4) plasmid extraction

Required reagent

LB liquid medium: 10G tryptone (Sigma-Aldrich, # T9410-250G), 5G yeast extract (Sigma-Aldrich, #70161-500G), 10G NaCl (Shanghai test, #10019308) were dissolved in ddH2O, adjusted to pH 7.0, adjusted to 1L, autoclaved at 121 ℃ for 15min, and cooled to room temperature

Plasmid mini-extraction kit: omega plasmid petite kit (Omega, # D6943-01-100T)

Antibiotics: 1g of the corresponding antibiotic powder (sigma) was weighed and dissolved well in 20mL ddH ₂ O, 0.22 mu m sterilizing, filtering, washing hair, sterilizing, subpackaging and storing at-20 ℃.

Experimental procedure

a) Picking a monoclonal grown in the LB solid medium in the step 3), adding 5mL of LB liquid medium containing 100 mu g/mL of corresponding antibiotics, and culturing overnight (12-16h) at 37 ℃ by a bacterial culture shaker at 200 rpm; (ii) a

b) Centrifuging 1.5-5 ml of bacterial liquid at room temperature of 10000 Xg for 1min

c) The supernatant was removed, 250. mu.l of solution I (containing RNase A) was added, and the cells were shaken by a vortex shaker until they were completely suspended.

d) Adding 250 mu l of solution II, and gently inverting the centrifuge tube for 4-6 times to obtain a clear lysate. Incubation for 2min at room temperature, vigorous mixing will shear the chromosomal DNA, reducing plasmid purity. (storage solution II should screw bottle cap)

e) Add 350. mu.l of solution III, mix gently by inversion several times until white flocculent precipitate appears, centrifuge at room temperature 10000 Xg for 10min.

f) The supernatant was aspirated with special care and transferred to a clean adsorption column equipped with 2ml centrifuge tubes. It is ensured that there are no aspiration deposits and cell debris. Centrifuging at room temperature 10000 Xg for 1min until the lysate completely passes through the absorption column

g) Discarding the filtrate, adding 500. mu.l Buffer HB, centrifuging at 10000 Xg for 1min, cleaning the absorption column, removing residual protein to ensure the purity of DNA.

h) If the following steps have low requirements on plasmid purity, such as enzyme digestion and other screening methods, the step can be omitted

i) Discarding the filtrate, washing the absorption column with 750. mu.l Wash Buffer diluted with 100% ethanol, centrifuging at 10000 Xg for 1min to note: the Wash Buffer concentrate must be diluted with pure ethanol before use, as indicated by the fact that if the label is frozen, it must be brought to room temperature before use.

j) This step is optional: then 750 mul Wash Buffer was added to clean the absorption column

k) The column must be centrifuged at 10000 Xg for 1min to ensure that the ethanol is removed, which can affect the next steps.

l) the column is placed in a clean 1.5ml centrifuge tube and 50-100. mu.l (depending on the desired final concentration) of sterile deionized water or TE buffer is added to the filter and centrifuged at 10000 Xg for 5 min.

m) measuring the concentration, and storing in a refrigerator at the temperature of 20 ℃ below zero for later use.

5) Enzyme digestion verification

Taking 1 μ g of the pLL3.7-EF1a-MCS-PGK-RFP plasmid recombinant plasmid extracted in the step 4), adding 1 μ L of restriction enzyme Pci I (NEB, R0655S) and 1 μ L of restriction enzyme XbaI (NEB, R0145S), and 5 μ L

Buffer (NEB), and finally double distilled water was added to make the whole reaction volume 50. mu.L. The sample was left to react at 37 ℃ for 1 hour. Subsequently, the band of interest was ligated as determined by 1% agarose gel electrophoresis, which showed that a band of about 1800bp was cut. The double digestion results show that the pLL3.7-EF1a-MCS-PGK-RFP recombinant lentiviral vector plasmid is successfully constructed (as shown in FIG. 2).

The sequencing result and the EF1a-MCS-PGK-RFP synthesized sequence Blast are completely correct. Thus, the Pre-Lenti-EF1-MCS vector is successfully constructed. The full-length sequence of pLL3.7-EF1a-MCS-PGK-RFP is shown in SEQ ID NO.2, the vector map is shown in figure 3, and the length is as follows: 7420 bp.

The full-length sequence of Lenti-EF1a-MCS-PGK-RFP is shown in SEQ ID NO. 3.

and 3, step 3: culturing a host cell, wherein the host cell is a 293T cell;

and 4, step 4: and separating to obtain the recombinant lentivirus.

The invention also discloses a recombinant lentiviral vector, which is prepared by the following method: the neutralizing antibody sequence of the immunomodulatory cytokine gene or anti-inflammatory cytokine is cloned into the polyclonal site of a vector such as pLenti-EF 1-MCS-PGK-RFP. Wherein the immunoregulatory cytokine gene is selected from the group consisting of human TGF-beta, IL-10, IL-4, and the like, or any combination thereof. Neutralizing antibodies against inflammatory cytokines include anti-human IL-6, TNF-a, IL-1, and the like.

Construction of expression human IL-10 Lentiviral vector pLL3.7-EF1-IL-10-PGK-RFP

IL-10 Gene Synthesis

1) Obtaining a CDS sequence of IL-10(Homo sapiens) from NCBI, analyzing an IL-10 gene sequence by using SnapGene software, and finding that single enzyme cutting sites EcoRI (a recognition sequence GAATTC) and BamHI (a recognition sequence GGATCC) are suitable for gene cloning and can be matched with a multiple cloning site of a pLL3.7-EF1-MCS-PGK-RFP vector; designing an IL-10 gene sequence containing an enzyme cutting site and a Kzake sequence as follows, and sending the gene sequence to Suzhou Jinwei Zhi Biotech limited for gene synthesis;

the gene sequence of IL-10 containing enzyme cutting site and Klake sequence is shown in SEQ ID NO. 4.

2.2 enzyme digestion

Double-enzyme cutting pLL3.7-EF1-MCS-PGK-RFP carrier and the IL-10 gene containing enzyme cutting sites BamH I and EcoR I by using restriction endonucleases BamH I and EcoR I, respectively recovering the target products after enzyme cutting; the enzyme digestion reaction system and the reaction conditions are constructed as the pLL3.7-EF1-MCS-PGK-RFP vector.

2.3 the product after digestion was recovered and the same procedure as that for the construction of pLL3.7-EF1-MCS-PGK-RFP vector was carried out using the Biomiga gel recovery kit (cat # DC-3511-01).

2.4 ligation, the procedure was identical to that of the construction of pLL3.7-EF1-MCS-PGK-RFP vector.

2.5 transformation, plating, and constructing the pLL3.7-EF1-MCS-PGK-RFP vector as before.

2.6 selecting clone, shaking bacteria, and constructing the vector by the same operation as the pLL3.7-EF1-MCS-PGK-RFP vector.

2.7 plasmid extraction, the procedure was the same as that of the pLL3.7-EF1-MCS-PGK-RFP vector construction, using the endotoxin-free plasmid extraction kit from Biomiga (cat # PD 1220-02).

2.8 enzyme digestion identification

Extraction step 2.7 extraction of recombinant plasmid 1. mu.g, adding 1. mu.L of restriction enzyme BamH I (NEB, R0136S) and 1. mu.L of restriction enzyme EcoR I (NEB, R0101S), 5. mu.L

Buffer (NEB), and finally double distilled water was added to make the whole reaction volume 50. mu.L. The sample was left to react at 37 ℃ for 1 hour. Subsequent identification by 1% agarose gel electrophoresis revealed that the band of interest was ligated by cutting an approximately 543bp band. The double digestion result shows that the pLL3.7-EF1a-MCS-PGK-RFP recombinant lentiviral vector plasmid is successfully constructed (as shown in FIG. 4). For further confirmation, sequencing verification was performed and the sequencing results were aligned with the IL-10 gene and were completely correct. Thus, the IL-10 lentiviral vector pLL3.7-EF1-IL-10-PGK-RFP was obtained (vector map is shown in FIG. 5).

1：pLL3.7-EF1-IL-10-PGK-RFP

2: the pLL3.7-EF1-IL-10-PGK-RFP plasmid is subjected to double enzyme digestion by BamH I and EcoR I

III, pLL3.7-EF1-IL-10-PGK-RFP lentivirus package

3.1 Lentiviral packaging follows conventional methods, roughly as follows:

day 1 (afternoon): cell seeding

293T cells at 5X10 ⁵ The density of individual cells/well was seeded in 6-well culture plates containing 2mL of lentivirus packaging medium. Cells were incubated overnight at 37 ℃ under 5% CO2 to ensure that cell densities reached 60-70% confluence during transfection.

Day 2 (morning): transfection

All plasmids were diluted to 1ug/ul with Opti-MEM;

1) tube A: 250ul of serum-free Opti-MEM medium diluted 7ul of Lipofectamine 3000 was vortexed for 10 s.

2) And a tube B: dilution of 4ug of plasmid pLL3.7-EF1-IL-10-PGK-RFP in 250ul serum-free Opti-MEM medium: 2ul, psPAX 2: 1.2ul, pMD2G:0.8ul) and 6ul of P3000 reagent were vortexed for 10 s.

3) Preparation of liposome-DNA complexes: transfer the tube contents to tube B and mix well and incubate for 10min at room temperature.

Prior to addition of the complexes, 1mL of medium was removed from each well to give a total volume of 1mL per well.

4) 500uL of liposome-DNA complex was added to each well, and the liquid was carefully added against the walls of the wells to avoid damaging the cells. The plate was gently agitated to distribute it evenly.

5) The plates were incubated at 37 ℃ with 5% CO2 for 8 hours.

6) After 8 hours of transfection, the plate medium in each well was replaced. The medium containing the liposome-DNA complexes was carefully aspirated from each well and the aspirated medium was treated with 10% bleaching solution before disposal. Replace with 2mL of pre-warmed lentiviral packaging medium.

7) The plates were returned to the incubator and incubated overnight at 37 ℃ under 5% CO 2.

Day 3: collecting the first batch of viruses

1) 48 hours after transfection, 2mL of cell supernatant (containing packaged recombinant lentivirus lenti-IL-10-RFP) was collected from each well, packed into 15mL conical tubes and stored at 4 ℃.

2) The collected medium was replaced with 2mL of pre-warmed lentiviral packaging medium.

3) The plates were incubated overnight at 37 ℃ with 5% CO 2.

Day 4: collecting a second batch of viruses

1) About 72 hours after transfection, 2mL of cell supernatant was collected from each well and mixed with the first collected supernatant to make the total volume of collected supernatant 4 mL.

2) Cell debris was removed by centrifugation at 2,000rpm for 10 minutes at room temperature. The supernatant was collected and transferred, and the cell pellet was discarded.

3) And (3) virus concentration: the collected supernatant was transferred to a sterile centrifuge tube according to a Lenti-X Concentrator: the supernatant was added to a Lenti-X Concentrator at a ratio of 1:3 and incubated overnight at 4 ℃.

5) Centrifuge at 1500g for 60min at 4 ℃ and discard the supernatant.

6) Adding DMEM basic culture medium to resuspend virus precipitate, subpackaging at 50 ul/tube, preserving at-80 ℃ in a refrigerator for standby, and limiting freeze-thaw times of the subpackaged virus so as to maintain virus titer.

3.2. And (4) determining the virus titer.

a. 293T cells according to 2X10 ⁵ A 24-hole plate is paved on each hole; b. adding 293T cells according to three gradient virus concentrated solutions of 0.01ul, 0.1ul and 1 ul; c. detecting the RFP positive cell proportion by flow cytometry after 48 hours; d. calculate Titer Titer ═ according to the formula (2X 10) ⁵ x RFP positivity rate)/volume of added virus x1000 TU/ml.

Fourth, infection of mesenchymal Stem cells

Material

Human umbilical cord mesenchymal stem cells (Saiko biology, Cat number HUXUC)

Human umbilical cord mesenchymal stem cell complete medium (Youkang biology, cat # NC0103)

4.1 recovery and culture of human umbilical mesenchymal stem cells

a) A37 ℃ water bath was prepared.

b) Human umbilical cord mesenchymal stem cell complete medium was prepared and incubated to 37 ℃.

c) 9mL of human umbilical cord mesenchymal stem cell complete medium was added to a 15mL centrifuge tube.

d) The cryopreserved human umbilical cord mesenchymal stem cells are taken out of the liquid nitrogen tank and immediately placed into a refrigerator at the temperature of minus 80 ℃ (the aim is to slightly volatilize the liquid nitrogen entering the cryopreserved pipe).

e) Standing at-80 deg.C for 2-3min, taking out the frozen cells, quickly placing the frozen tube into 37 deg.C warm water, and quickly shaking to melt the contents in the tube as soon as possible. Carefully observing, and taking out the frozen tube after the contents of the frozen tube are completely melted.

f) The outer wall of the freezing storage pipe orifice is disinfected by 70% -75% alcohol.

g) The cryopreservation tube was opened in a clean bench and the cell cryopreservation suspension was transferred with a pipette into a 15mL centrifuge tube containing 9mL complete medium. During this process, it is desirable to avoid the generation of bubbles as much as possible.

h) To reduce cell loss, 1mL of complete medium was added to the vial, gently pipetted, and the 1mL of cell suspension was aspirated into the centrifuge tube using a pipette, and the cells in the centrifuge tube were gently pipetted and gently and pipetted into the tube.

i) The cell suspension was centrifuged for 5min at 250g (corresponding to 1134rpm for Eppendorf 5810R centrifuge). The supernatant was removed as much as possible and 1-2mL of complete medium (preheated to 37 ℃) was added to the cell pellet and gently pipetted evenly.

j) The cells were all inoculated into 1T 25 flask or culture vessel of equivalent bottom area, and sufficient complete medium was added. The cell culture vessel was gently shaken to evenly distribute the cells.

k) Culturing in an incubator at 37 deg.C and 5% CO2 with saturated humidity.

l) the next day after recovery, the recovered cells were replaced with fresh human umbilical mesenchymal stem cell complete medium (pre-warmed to 37 ℃).

m), cells were replaced with fresh complete medium every two days until the cells reached 80% confluence.

4.2 infection of human umbilical cord blood mesenchymal Stem cells by recombinant lentiviruses

a) The day before the experiment, the mesenchymal stem cells with the confluence degree reaching 80 percent in the step 1m) are treated according to the specification of 1x10 ⁵ Inoculating into 6-well plate containing mesenchymal stem cell complete culture medium;

b) the cells reached 70-90% full, and the recombinant lentivirus obtained in step three was added in an amount of MOI ═ 5 (meaning viral particle number: mesenchymal stem cell number is 5:1), the mesenchymal stem cells are infected, polybrene is added during infection to enable the final concentration to reach 6 mug/mL, and the control hole is not infected with virus and is not added with polybrene;

c) after 72h infection, the expression of RFP was observed by fluorescence microscopy, and the level of IL-10 secretion was measured by collecting cell culture supernatants. The results showed that almost all cells expressed RFP fluorescence (fig. 6), and the level of IL-10 secretion was extremely significant compared to the control group (fig. 7).

The above results fully show that successfully constructing plasmid vector for expressing immunoregulation can be used for gene modification of stem cells and endow stem cells with stronger immunoregulation capability. Meanwhile, the expression of the reporter gene RFP is beneficial to the preclinical pharmacokinetic research and lays the early theoretical foundation for the development of clinical tests.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will understand that various changes, modifications and substitutions can be made without departing from the spirit and scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Mediterranean gorges (Fujian) cell Biotechnology Ltd

<120> lentiviral vector for stem cell gene modification, and construction method and application thereof

<141> 2022-03-31

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ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac 420

ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc 480

aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc 540

gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc 600

gctaccggtc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat 660

cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga 720

gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc 780

cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta 840

ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca 900

ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt 960

cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg 1020

caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc 1080

cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg 1140

cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct 1200

gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa 1260

gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga 1320

cgagctgtac aagtaggaat tcgtcgaggg acctaataac ttcgtatagc atacattata 1380

cgaagttata catgtttaag ggttccggtt ccactaggta caattcgata tcaagcttat 1440

cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt 1500

tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc 1560

ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga 1620

gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc 1680

cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct 1740

ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg 1800

gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct 1860

gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc 1920

cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg 1980

tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata 2040

ccgtcgacct cgatcgagac ctagaaaaac atggagcaat cacaagtagc aatacagcag 2100

ctaccaatgc tgattgtgcc tggctagaag cacaagagga ggaggaggtg ggttttccag 2160

tcacacctca ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact 2220

ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga caagatatcc 2280

ttgatctgtg gatctaccac acacaaggct acttccctga ttggcagaac tacacaccag 2340

ggccagggat cagatatcca ctgacctttg gatggtgcta caagctagta ccagttgagc 2400

aagagaaggt agaagaagcc aatgaaggag agaacacccg cttgttacac cctgtgagcc 2460

tgcatgggat ggatgacccg gagagagaag tattagagtg gaggtttgac agccgcctag 2520

catttcatca catggcccga gagctgcatc cggactgtac tgggtctctc tggttagacc 2580

agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa 2640

gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga 2700

gatccctcag acccttttag tcagtgtgga aaatctctag cagcatgtga gcaaaaggcc 2760

agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 2820

cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 2880

tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 2940

tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 3000

gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 3060

acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 3120

acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 3180

cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 3240

gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 3300

gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 3360

agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 3420

ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 3480

ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 3540

atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 3600

tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 3660

gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 3720

ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 3780

caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 3840

cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 3900

cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 3960

cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 4020

agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 4080

tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 4140

agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 4200

atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 4260

ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 4320

cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 4380

caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 4440

attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 4500

agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg 4560

acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc 4620

cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc 4680

gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct 4740

tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg 4800

attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat 4860

ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 4920

ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 4980

ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 5040

tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 5100

tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 5160

cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga 5220

ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 5280

aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg 5340

taggcgtgta cggtgggagg tctatataag cagcgcgttt tgcctgtact gggtctctct 5400

ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc 5460

ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg 5520

gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc 5580

gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct 5640

tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt 5700

gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag 5760

aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt 5820

aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt 5880

agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg 5940

atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag 6000

gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag 6060

taagaccacc gcacagcaag cggccggccg ctgatcttca gacctggagg aggagatatg 6120

agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 6180

gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 6240

ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 6300

acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 6360

ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 6420

ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 6480

tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 6540

aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 6600

aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 6660

aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 6720

acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 6780

agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 6840

tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 6900

gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 6960

gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 7020

tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 7080

agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 7140

agatccagtt tggttagtac cgggcccgct ctagagatcc gacgcgccat ctctaggccc 7200

gcgccggccc cctcgcacag acttgtggga gaagctcggc tactcccctg ccccggttaa 7260

tttgcatata atatttccta gtaactatag aggcttaatg tgcgataaaa gacagataat 7320

ctgttctttt taatactagc tacattttac atgataggct tggatttcta taagagatac 7380

aaatactaaa ttattatttt aaaaaacagc acaaaaggaa actcacccta actgtaaagt 7440

aattgtgtgt tttgagacta taaatatccc ttggagaaaa gccttgttaa cgcgcggtga 7500

ccctcgaggt cgacggtatc gataagctcg cttcacgaga ttccagcagg tcgagggacc 7560

taataacttc gtatagcata cattatacga agttatatta agggttccaa gcttaagcgg 7620

ccgcgtggat aaccgtatta ccgccat 7647

<210> 2

<211> 1646

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tctagacagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagc 60

tgaagcttcg aggggctcgc atctctcctt cacgcgcccg ccgccctacc tgaggccgcc 120

atccacgccg gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt 180

ccgccgtcta ggtaagttta aagctcaggt cgagaccggg cctttgtccg gcgctccctt 240

ggagcctacc tagactcagc cggctctcca cgctttgcct gaccctgctt gctcaactct 300

acgtctttgt ttcgttttct gttctgcgcc gttacagatc caagctgtga ccggggttta 360

gtgaaccgtc agatccgcca ccgctagcgc taatgtacaa gacgcgtcgc gtttcgaacc 420

cgggcccgga tcctgcgaat tctgcctaga taattctacc gggtagggga ggcgcttttc 480

ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg ctacacaagt 540

ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccggc tccgttcttt 600

ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc ccccgccccg 660

cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag tctcgtgcag 720

atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg ccaatagcag 780

ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc 840

aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc 900

acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc gggcctttcg 960

acatggcctc ctccgaggac gtcatcaagg agttcatgcg cttcaaggtg cgcatggagg 1020

gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg 1080

gcacccagac cgccaagctg aaggtgacca agggcggccc cctgcccttc gcctgggaca 1140

tcctgtcccc tcagttccag tacggctcca aggcctacgt gaagcacccc gccgacatcc 1200

ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg 1260

aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct 1320

acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga 1380

ccatgggctg ggaggcctcc accgagcgga tgtaccccga ggacggcgcc ctgaagggcg 1440

agatcaagat gaggctgaag ctgaaggacg gcggccacta cgacgccgag gtcaagacca 1500

cctacatggc caagaagccc gtgcagctgc ccggcgccta caagaccgac atcaagctgg 1560

acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgagcgc gccgagggcc 1620

gccactccac cggcgcctaa acatgt 1646

<210> 3

<211> 7420

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ttaagggttc cggttccact aggtacaatt cgatatcaag cttatcgata atcaacctct 60

ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct 120

atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat 180

tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt 240

caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat 300

tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc 360

ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga 420

caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc 480

cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga 540

ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc 600

tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgatc 660

gagacctaga aaaacatgga gcaatcacaa gtagcaatac agcagctacc aatgctgatt 720

gtgcctggct agaagcacaa gaggaggagg aggtgggttt tccagtcaca cctcaggtac 780

ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg 840

ggggactgga agggctaatt cactcccaac gaagacaaga tatccttgat ctgtggatct 900

accacacaca aggctacttc cctgattggc agaactacac accagggcca gggatcagat 960

atccactgac ctttggatgg tgctacaagc tagtaccagt tgagcaagag aaggtagaag 1020

aagccaatga aggagagaac acccgcttgt tacaccctgt gagcctgcat gggatggatg 1080

acccggagag agaagtatta gagtggaggt ttgacagccg cctagcattt catcacatgg 1140

cccgagagct gcatccggac tgtactgggt ctctctggtt agaccagatc tgagcctggg 1200

agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc 1260

ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct 1320

tttagtcagt gtggaaaatc tctagcagca tgtgagcaaa aggccagcaa aaggccagga 1380

accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 1440

acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 1500

cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 1560

acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 1620

atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 1680

agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 1740

acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 1800

gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 1860

gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 1920

gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 1980

gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 2040

acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 2100

tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 2160

ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 2220

catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 2280

ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 2340

caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 2400

ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 2460

tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 2520

cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 2580

aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 2640

tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 2700

gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 2760

cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 2820

aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 2880

tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 2940

tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 3000

gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 3060

atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 3120

taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc 3180

tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 3240

gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc 3300

tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt 3360

tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta 3420

ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac 3480

ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc 3540

aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt 3600

ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac 3660

gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac 3720

cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt 3780

gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc 3840

aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt 3900

tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg 3960

ggaggtctat ataagcagcg cgttttgcct gtactgggtc tctctggtta gaccagatct 4020

gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc 4080

cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc 4140

tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa 4200

agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac 4260

ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta 4320

gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg 4380

ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg 4440

ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg 4500

ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag 4560

atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga 4620

caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca 4680

gcaagcggcc ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga 4740

agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag 4800

gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt 4860

gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag 4920

gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag 4980

gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc 5040

ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga 5100

aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa 5160

cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc 5220

ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta 5280

ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg 5340

tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct 5400

gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac 5460

ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga 5520

gacagagaca gatccattcg attagtgaac ggatcggcac tgcgtgcgcc aattctgcag 5580

acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc 5640

aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca 5700

aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc cagtttggtt 5760

agtaccgggc ccgctctaga cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc 5820

cgccagaaca cagctgaagc ttcgaggggc tcgcatctct ccttcacgcg cccgccgccc 5880

tacctgaggc cgccatccac gccggttgag tcgcgttctg ccgcctcccg cctgtggtgc 5940

ctcctgaact gcgtccgccg tctaggtaag tttaaagctc aggtcgagac cgggcctttg 6000

tccggcgctc ccttggagcc tacctagact cagccggctc tccacgcttt gcctgaccct 6060

gcttgctcaa ctctacgtct ttgtttcgtt ttctgttctg cgccgttaca gatccaagct 6120

gtgaccgggg tttagtgaac cgtcagatcc gccaccgcta gcgctaatgt acaagacgcg 6180

tcgcgtttcg aacccgggcc cggatcctgc gaattctgcc tagataattc taccgggtag 6240

gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg ctgggcactt 6300

ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt aggcgccaac 6360

cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta gtcaggaagt 6420

tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca 6480

ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc ctttggggca 6540

gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga aggggtgggt 6600

ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg tcctccggag 6660

gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc tcttcctcat 6720

ctccgggcct ttcgacatgg cctcctccga ggacgtcatc aaggagttca tgcgcttcaa 6780

ggtgcgcatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg 6840

ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc 6900

cttcgcctgg gacatcctgt cccctcagtt ccagtacggc tccaaggcct acgtgaagca 6960

ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg 7020

cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga 7080

cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt 7140

aatgcagaag aagaccatgg gctgggaggc ctccaccgag cggatgtacc ccgaggacgg 7200

cgccctgaag ggcgagatca agatgaggct gaagctgaag gacggcggcc actacgacgc 7260

cgaggtcaag accacctaca tggccaagaa gcccgtgcag ctgcccggcg cctacaagac 7320

cgacatcaag ctggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga 7380

gcgcgccgag ggccgccact ccaccggcgc ctaaacatgt 7420

<210> 4

<211> 555

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggatccgcca ccatgcacag ctcagcactg ctctgttgcc tggtcctcct gactggggtg 60

agggccagcc caggccaggg cacccagtct gagaacagct gcacccactt cccaggcaac 120

ctgcctaaca tgcttcgaga tctccgagat gccttcagca gagtgaagac tttctttcaa 180

atgaaggatc agctggacaa cttgttgtta aaggagtcct tgctggagga ctttaagggt 240

tacctgggtt gccaagcctt gtctgagatg atccagtttt acctggagga ggtgatgccc 300

caagctgaga accaagaccc agacatcaag gcgcatgtga actccctggg ggagaacctg 360

aagaccctca ggctgaggct acggcgctgt catcgatttc ttccctgtga aaacaagagc 420

aaggccgtgg agcaggtgaa gaatgccttt aataagctcc aagagaaagg catctacaaa 480

gccatgagtg agtttgacat cttcatcaac tacatagaag cctacatgac aatgaagata 540

cgaaactgag aattc 555

Claims

1. The nucleotide sequence of the lentiviral vector for the stem cell gene modification is shown as SEQ ID NO. 2.

2. Use of the lentiviral vector of claim 1 for genetic modification of a stem cell.

3. The method of constructing a lentiviral vector according to claim 1, comprising the steps of: selecting a single enzyme cutting site XbaI and a single enzyme cutting site Pci I to cut a CMV + EGHFP element on the initial vector, wherein the size of the vector after enzyme cutting is 5780bp, the synthesized EF1a-MCS-PGK-RFP sequence is added with the sequence of XbaI or Pci I at two ends respectively.

4. Use of the lentiviral vector of claim 1, comprising the steps of:

step 1: cloning a target gene into a vector to obtain a recombinant lentivirus vector;

step 2: co-transfecting the recombinant lentiviral vector with psPAX2 and pMD2.0G to a host cell;

and step 3: culturing host cells

And 4, step 4: and separating to obtain the recombinant lentivirus.

5. A recombinant lentiviral vector is prepared by the following method: cloning of neutralizing antibody sequences of immunomodulatory cytokine genes or anti-inflammatory cytokines into the polyclonal site of the lentiviral vector for stem cell genetic modification of claim 1.

6. The recombinant lentiviral vector of claim 5, wherein the immunomodulatory cytokine gene is selected from any one or more of human TGF- β, IL-10, or IL-4; the neutralizing anti-inflammatory cytokine antibody includes any one or more of anti-human IL-6, TNF-a or IL-1.