CN117947022A - Sequence for promoting expression of exogenous protein of T cell and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of genetic engineering, and particularly relates to a sequence for promoting the expression of exogenous proteins of T cells and application thereof, wherein a nucleotide sequence of SEQ ID NO.1 and SEQ ID NO.2 are taken as target gene fragments, and a carrier containing the sequence is constructed through amplification, enzyme digestion and connection, and the obtained carrier or sequence is applied to the T cells, so that the expression of the exogenous proteins of the T cells can be promoted, including the enhancement of the expression efficiency of green fluorescent protein in the T cells and the improvement of the expression efficiency of CAR genes in the T cells.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a sequence for promoting the expression of exogenous proteins of T cells and application thereof.
Background
Currently, the means for constructing T cells stably expressing the expression of exogenous proteins are transposon/transposase systems, mRNA electroporation and lentiviruses. Although transduction and expression of foreign genes in T cells can be achieved by lentiviruses, the transduction efficiency is often limited to a large extent by the size of the DNA, and decreases when the target expression DNA sequence is long. Considering that the number of T cells obtained in peripheral blood of a clinical patient is limited, the problem of low transduction efficiency cannot be solved from the supply depending on the number of T cells. Therefore, how to promote the expression of exogenous proteins of T cells, optimize a lentivirus-mediated transfection system, improve the transduction efficiency of lentivirus and ensure the expression efficiency is a key problem which needs to be solved clinically at present.
Disclosure of Invention
The invention provides a sequence for promoting the expression of exogenous proteins of T cells and application thereof.
The technical scheme of the invention is as follows:
The invention provides a sequence for promoting the expression of exogenous proteins of T cells, which is a nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.
The invention also provides a vector for promoting the expression of exogenous proteins of T cells, which contains the nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.
The vector is a nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2 inserted into the vector.
The vector is a nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2 inserted into a lentiviral vector, an adenovirus vector, a synthetic polymer nano-vector or a lipid nano-particle vector.
The vector is a slow virus vector Lenti-PGK-EGFP inserted with the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO. 2.
The invention also provides a construction method of the vector for promoting the expression of the exogenous protein of the T cell, which comprises the following steps:
Amplifying target gene fragments of SEQ ID NO.1 and SEQ ID NO.2 respectively, carrying out enzyme digestion on the vector, wherein enzyme digestion sites are EcoRI and ClaI, and connecting the digested vector and target gene fragments.
The invention also provides an application of the sequence or the vector constructed by the construction method in promoting the expression of exogenous proteins of T cells.
The sequences of the invention are located upstream of the exogenous protein.
The application of the invention comprises the application of the sequence or the vector in improving the expression efficiency of enhanced green fluorescent protein and CAR genes in T cells.
The exogenous proteins comprise enhanced green fluorescent protein, CAR gene, luciferase, caspase9 modified binding protein fused with fk506, histone H2B-enhanced green fluorescent protein, phosphorylated form YAP5SA of YAP1 protein, red fluorescent protein, angiotensin converting enzyme 2 and gap junction protein.
Advantageous effects
The SEQ ID NO.1 and SEQ ID NO.2 sequences provided by the invention can promote the expression of exogenous proteins of T cells, wherein the promotion effect of SEQ ID NO.1 is more obvious;
According to the invention, lentiviral vectors Lenti-PGK-EGFP are selected, and are subjected to EcoRI and ClaI digestion and then connected to obtain NO.1-EGFP and NO.2-EGFP inserted with SEQ ID NO.1 and SEQ ID NO.2 sequences respectively, so that an expression vector capable of promoting the expression of exogenous proteins of T cells is successfully obtained;
The sequences SEQ ID NO.1 and SEQ ID NO.2 are applied to the transfected 293T cells, so that the Enhanced Green Fluorescent Protein (EGFP) has higher transfection efficiency; the Enhanced Green Fluorescent Protein (EGFP) can be applied to an activated T cell and an expression vector NO.1-EGFP constructed by SEQ ID NO.1 sequence, and can be expressed with high efficiency and stability; in addition, in the activated T cells, the expression vector NO.1-CAR can improve the expression efficiency of the CAR genes in the T cells.
Drawings
FIG. 1 is a graph showing the fluorescence of viral titers of No.1-EGFP, no.2-EGFP, no.3-EGFP, no.4-EGFP,
FIG. 2 is a graph showing the comparison of the transfection efficiency of Enhanced Green Fluorescent Protein (EGFP) in 293T cells 72h after transfection of different expression vectors, wherein (a) is the result of detecting the FITC fluorescent channel by a flow cytometer, (b) is a bar graph of the result obtained in (a),
FIG. 3 is a graph showing comparison of expression efficiency and difference between the Enhancement of Green Fluorescent Protein (EGFP) on days 2 (a), 4 (b) and 6 (c) after the activation of T cells for 48h in a double sense,
FIG. 4 is a graph showing comparison of expression efficiency and a graph showing comparison of expression variability of G4S Linker on days 2 (a), 4 (b) and 6 (c) after activating T cells for 48h in a double sense.
Detailed Description
The following examples are intended to illustrate the invention, but not to limit it further.
The invention provides a sequence for promoting the expression of exogenous proteins of T cells and application thereof.
Designing 4 segments of sequences, namely SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively, wherein the nucleotide sequences are specifically shown as sequence 1, sequence 2, sequence 3 and sequence 4 in a sequence table respectively;
In order to verify the promotion effect of the 4-segment sequences on the expression of the T cell exogenous proteins, the corresponding expression vectors containing the sequences SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 are constructed, and the expression vectors contain Enhanced Green Fluorescent Protein (EGFP) which are respectively named as NO.1-EGFP, NO.2-EGFP, NO.3-EGFP and NO.4-EGFP. In order to further verify whether the 4-segment sequence has a promoting effect on the expression effect of long-chain proteins, the Enhanced Green Fluorescent Protein (EGFP) in the expression vectors NO.1-EGFP, NO.2-EGFP, NO.3-EGFP and NO.4-EGFP is replaced by a CAR gene respectively to obtain the expression vectors NO.1-CAR, NO.2-CAR, NO.3-CAR and NO.4-CAR.
1. Construction of expression vectors containing different sequences
(1) And respectively carrying out PCR amplification by taking SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 as target gene fragments. The amplified product was verified to be the desired insert, and the PCR amplified product was recovered. The PCR amplification conditions and the PCR program setting parameters are shown in Table 1 and Table 2 respectively:
TABLE 1PCR amplification conditions
TABLE 2PCR Programming parameters
(2) And (3) enzyme cutting: selecting lentiviral vector Lenti-PGK-EGFP, and performing enzyme digestion for 4-6h at 37 ℃ with EcoRI and ClaI as enzyme digestion sites. The specific cleavage conditions are shown in Table 3. Obtaining enzyme cutting carriers with different sticky ends, verifying enzyme cutting effect by electrophoresis, and performing electrophoresis recovery.
TABLE 3 cleavage conditions
(3) And (3) connection: and (3) connecting the recovered enzyme digestion vectors with target gene fragments of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively to obtain expression vectors NO.1-EGFP, NO.2-EGFP, NO.3-EGFP and NO.4-EGFP.
The specific connection process is as follows:
(3.1) 2 Xclone Buffer (Northenzan, clonExpress IIOne Step Cloning Kit) was used as a linking system in which the total volume of vector=1:1 (mass ratio) was not more than 6 uL. The PCR was performed at 50℃for 15min.
(3.2) Transforming 6uL of the ligation product of (1) into 20uL of STBL III competent cells thawed on ice, and ice-bathing for 30min; heat shock at 42 ℃ for 1min and ice bath for 2min; sterilizing the sterile coating rod by using an alcohol lamp outer flame, and recovering the room temperature;
(3.3) to (2) adding 80uL LB liquid medium, mixing and dripping on the corresponding resistance plate, using sterilized sterile coating rod to coat the resistance plate, inverting at 37 ℃ incubator overnight. Colonies were selected for colony PCR and verified by 1% agarose gel electrophoresis, demonstrating successful ligation of ligation products.
(4) Colony identification: the colonies selected were subjected to colony PCR under the conditions shown in Table 4. Positive clones were verified by electrophoresis. Positive clones were picked, 35ml of LB+35uL (1:1000) antibiotics were cultured overnight at 37℃with shaking at 220rpm, bacterial liquid was collected the next day, and plasmids were extracted. The upstream primer and the downstream primer used in colony PCR are all primers already known in the art.
TABLE 4 colony PCR conditions
(5) Extracting plasmids: 1. mixing 800uL of the collected bacterial liquid with 400uL of 30% glycerol in an EP tube, centrifuging at 7830rpm for 10min, discarding the supernatant, collecting the precipitate, and adding 500uLP to blow-suspend the precipitate; adding 500uL P2, slightly shaking for 10 times, and standing for 5min; adding 500uLP times, and oscillating for 5 times; passing the supernatant through a column, centrifuging at 12000rpm for 2min, and discarding the waste liquid; adding 600uL of rinsing liquid, passing through a column, centrifuging at 12000rpm for 2min, and discarding the waste liquid; adding 500uL of rinsing liquid, passing through a column, and centrifuging at 12000rpm for 2min; centrifuging at 12000rpm for 2min; the new 1.5ml EP tube was replaced, 60uL of eluent (heated in a metal bath at 75deg.C) was added, and the mixture was centrifuged at 12000rpm for 1min, and the elution was repeated once. The eluent is collected and stored, and double enzyme digestion verification and sequencing verification prove that the plasmid construction is correct.
2. Application of
2.1 Transfection of 293T cells
The extracted target plasmid is used for packaging slow viruses, namely, 4 slow viruses are obtained after slow viruses are respectively packaged on expression vectors NO.1-EGFP, NO.2-EGFP, NO.3-EGFP and NO.4-EGFP containing different sequences. In particular to a special-shaped ceramic tile,
(1) Lentivirus package
293T cells were plated in six well plates with 1.8M cells per well and transfected after overnight incubation. The transfection system is shown in Table 5. After 48h of transfection, the supernatant was collected and mixed with lentiviral concentrate reagents at a ratio of 5:1 at 4℃overnight, the pellet was collected by centrifugation and resuspended in PBS to give lentiviral concentrate which was stored at-80 ℃.
TABLE 5 configuration of transfection System
(2) Virus titer assay
To detect the expression Enhanced Green Fluorescent Protein (EGFP) virus titer, T cells were infected with four lentiviruses to the same titer. The virus titer is measured by a limiting dilution method, and the infection of the 293T cells by the lentivirus is detected initially. First, 293T cells in good growth state were digested and counted, diluted to 1X 10 5/mL, and added to a 96-well plate of 100. Mu.L/well, 10 gradients were prepared for each virus. Culturing in a 5% CO 2 incubator at 37deg.C. Next, 10 1.5mL EP tubes were prepared for each virus, and 10 dilutions (10 0～109) were made in the EP tubes in a 10-fold gradient, i.e., 90. Mu.L of DMEM complete medium per tube with 10uL of virus concentrate. Then, 10 dilutions of the virus were added to 96-well plates, and after further culturing, 293T cells infected for 72 hours were collected, photographed and recorded after virus infection, and the positive rate was analyzed by flow cytometry.
As shown in FIG. 1, the number of fluorescent cells in the field gradually decreased with increasing dilution. By taking a photograph of the fluorescent cells, a dilution group having ten or less fluorescent cells is selected, and the virus titer is calculated based on the specific values of the fluorescent cells and the corresponding dilution factors. The same titer was then used to infect T cells to ensure that 4 lentiviruses transduced T cells at an approximate level.
As shown in FIG. 2, compared with the existence of 293T cells in the control group, the transfection efficiencies of NO.3-EGFP, NO.2-EGFP, NO.1-EGFP and NO.4-EGFP in the 293T cells are 99.3%, 97.3%, 93.6% and 91.6% respectively, and the transfection efficiencies of different expression vectors are from high to low, namely NO.3-EGFP > NO.2-EGFP > NO.1-EGFP > NO.4-EGFP, which shows that the sequences of SEQ ID NO.1 and SEQ ID NO.2 can improve the transfection efficiency of Enhanced Green Fluorescent Protein (EGFP) in the 293T cells compared with SEQ ID NO. 4.
2.2 Transfection of T cells
1 Well of 24-well plate was coated with 500uLPBS and 20uL R factor (1:50), four degrees overnight; taking activated 48h T cells and counting; the volumes of T cells and virus added were calculated according to 1:1000 polyagglutination amine. Diluting the viruses until the titer is the same, discarding PBS and R factor coating liquid, taking an equal volume of the virus dilution liquid, slowly adding the virus dilution liquid into a pre-coating hole, centrifuging at 4000rpm for 1h at 4 ℃, centrifuging at 4000rpm for 1h at 32 ℃, and centrifuging a proper amount of cell suspension to the bottom of the plate. The 24-well plate 1 wells were coated with 500uLPBS and 20uL R factors. Four degrees overnight.
And (3) feel: in view of the difficulty in infecting T cells, multiple passes are required, i.e., the same titer, same volume of virus is added to the pre-coated wells and the same transfection procedure is performed. The cell suspension obtained by transfection was collected, slowly added to a 24-well plate containing virus, and centrifuged at 4000rpm at 32℃for 1 hour. After 48h T cells were re-activated, the infection ratio was recorded by flow cytometry on day 2, day 4, and day 6, respectively.
As shown in FIG. 3, the results of the measurement are (a), (b) and (c) the expression efficiencies of EGFP measured on days 2, 4 and 6, respectively. Compared with No.2-EGFP, no.3-EGFP and No.4-EGFP, the expression vectors No.1-EGFP have the highest expression efficiency, and stable expression is always carried out under the condition that the expression efficiency is not obviously reduced, which indicates that the SEQ ID No.1 sequence can improve the expression efficiency of Enhanced Green Fluorescent Protein (EGFP) in T cells; from (d), it is clear that the data columns from left to right in the expression ratio graphs measured on the 2 nd, 4 th and 6 th days are respectively groups containing SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, and the promotion effect of the Enhanced Green Fluorescent Protein (EGFP) in T cells is better and has obvious difference compared with that of SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 and the promotion effect of the SEQ ID NO.1 sequence. Therefore, the expression vector of NO.1 constructed by the SEQ ID NO.1 sequence has the highest expression efficiency in T cells.
2.3 Substitution with CAR Gene, transfected T cells
To verify whether the 4-segment sequence has an enhanced expression effect on the CAR gene, the Enhanced Green Fluorescent Protein (EGFP) in the vectors No.1-EGFP, no.2-EGFP, no.3-EGFP, no.4-EGFP was replaced with a CAR gene comprising SSI VH, SSI VL, and an amino acid sequence G4S Linker encoding a short peptide linking SSI VH and SSI VL, CD8 hinge region, CD8 transmembrane domain, 4-1BB target, CD3 Zeta target, resulting in expression vectors No.1-CAR, no.2-CAR, no.3-CAR, no.4-CAR, and the same experiment of transfected T cells was performed.
The expression of the CAR gene was reflected by detecting the expression efficiency of G4S Linker, and the detection results are shown in FIG. 4. And (a), (b) and (c) are respectively the expression efficiencies of the G4S Linker measured on the 2 nd day, the 4 th day and the 6 th day, compared with the expression efficiencies of the NO.3-CAR and the NO.4-CAR, the expression vectors NO.1-CAR and the NO.2-CAR respectively maintain higher expression efficiencies of the G4S Linker, wherein the expression vectors NO.1-CAR have the highest expression efficiency in promoting the G4SLinker in T cells, and the expression efficiencies are not obviously reduced, and are always and stably expressed, so that the SEQ ID NO.1 and SEQ ID NO.2 sequences can improve the expression efficiencies of the G4S Linker in the T cells, and further the SEQ ID NO.1 and SEQ ID NO.2 sequences can improve the expression efficiencies of the CAR genes in the T cells.
As shown in (d), the expression promoting effect of the sequences SEQ ID NO.3 and SEQ ID NO.4 and SEQ ID NO.1 and SEQ ID NO.2 is better and has obvious difference in improving the expression efficiency of the G4S Linker in T cells. The NO.1-CAR expression vector constructed by the SEQ ID NO.1 sequence has highest expression efficiency in promoting G4SLinker in T cells.
Claims (10)
1. A sequence for promoting the expression of exogenous proteins of T cells, which is characterized in that the sequence is a nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.
2. A vector for promoting expression of a T cell exogenous protein comprising the nucleotide sequence set forth in SEQ ID No.1 or SEQ ID No.2 of claim 1.
3. The vector according to claim 2, wherein the nucleotide sequence shown in SEQ ID No.1 or SEQ ID No.2 of claim 1 is inserted into the vector.
4. The vector according to claim 2, characterized in that the nucleotide sequence shown in SEQ ID No.1 or SEQ ID No.2 of claim 1 is inserted in a lentiviral vector, or an adenoviral vector, or a synthetic polymer nanocarrier, or a lipid nanoparticle vector.
5. The vector according to claim 4, wherein the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2 of claim 1 is inserted into a lentiviral vector Lenti-PGK-EGFP.
6. A method of constructing the vector for promoting expression of a T cell exogenous protein according to any one of claims 2 to 5, comprising:
Amplifying target gene fragments of SEQ ID NO.1 and SEQ ID NO.2 respectively, carrying out enzyme digestion on the vector, wherein enzyme digestion sites are EcoRI and ClaI, and connecting the digested vector and target gene fragments.
7. Use of a sequence according to claim 1 or a vector according to any one of claims 2 to 5 or a vector constructed by a construction method according to claim 6 for promoting expression of a T cell exogenous protein.
8. The use according to claim 7, wherein the sequence is located upstream of the exogenous protein.
9. The use of claim 7, wherein said sequence or said vector is used to increase the efficiency of expression of enhanced green fluorescent protein, CAR gene in T cells.
10. The use according to claim 7, wherein the exogenous proteins comprise enhanced green fluorescent protein, CAR gene, luciferase, caspase9 modified binding protein fused to fk506, histone H2B-enhanced green fluorescent protein, phosphorylated form YAP5SA of YAP1 protein, red fluorescent protein, angiotensin converting enzyme 2, gap junction protein.
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