CN114686576A - Method for detecting copy number of lentiviral vector of single CAR-T cell and application of method - Google Patents
Method for detecting copy number of lentiviral vector of single CAR-T cell and application of method Download PDFInfo
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Abstract
The invention provides a method for detecting the copy number of a lentiviral vector of a single CAR-T cell, which comprises the following steps: s1, designing qPCR primers and probes of beta-globin by taking the beta-globin as an internal reference gene, and establishing a standard curve 1 by utilizing a plasmid template of the beta-globin; s2, designing a primer and a probe of the target gene, and establishing a standard curve 2 by using a plasmid template of the target gene; s3, performing PCR amplification on the sample to be detected by respectively using the beta-globin and the qPCR primers of the target gene, calculating the beta-globin copy number of the sample to be detected from the standard curve 1, calculating the CAR copy number of the sample to be detected from the standard curve 2, and then calculating the copy number of the target gene relative to the cell number according to a formula. The invention takes beta-globin as an endogenous reference gene, can stably and effectively detect the copy number of the integrated viral vector integrated into the CAR-T cell genome, and can convert the copy number of the integrated viral vector into the average copy number of the viral vector in each CAR-T cell genome.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for detecting the copy number of a lentiviral vector of a single CAR-T cell and application thereof.
Background
CAR-T refers to a Chimeric Antigen Receptor (CAR) T cell, in which a Single chain variable region (Scfv) of an antibody capable of recognizing a certain tumor antigen and an intracellular signal region are coupled in vitro to form a chimeric protein, and the chimeric protein is transfected into a T cell of a patient cultured in vitro by a gene transduction method to express the Chimeric Antigen Receptor (CAR). This process is introduced by recombinant lentiviral vectors, i.e., foreign genes (chimeric antigen receptors, CARs) are introduced into host cells (T cells). Since lentiviral vectors are randomly inserted into the host genome during transduction, neither the copy number nor the site of insertion is fixed. When the exogenous gene is inserted with low copy number (1 or 2), the exogenous gene can be well expressed, and the expression of the exogenous gene with multiple copy number insertion is unstable and even the phenomenon of gene silencing occurs. Therefore, determining the copy number of the lentiviral vector in the CAR-T cell genome can provide a basis for the expression of the CAR-T cell target gene.
In the prior art, copy numbers mostly represent copies by taking DNA as a unit, so that the copy number in each cell cannot be accurately quantified, and further the influence of the single-cell copy number of the CAR on CAR-T cells cannot be evaluated.
Disclosure of Invention
In view of the above, the present invention provides a method for accurately quantifying the copy number of a lentiviral vector per cell.
The technical scheme of the invention is realized as follows: in a first aspect, the invention provides a method for detecting the lentiviral vector copy number of a single CAR-T cell, comprising the steps of:
s1, designing qPCR primers and probes of beta-globin by taking the beta-globin as an internal reference gene, and establishing a standard curve 1 by utilizing a plasmid template of the beta-globin;
s2, designing a primer and a probe of the target gene, and establishing a standard curve 2 by using a plasmid template of the target gene;
s3, performing PCR amplification on a sample to be detected by respectively using the beta-globin and the qPCR primers of the target gene, calculating the beta-globin copy number of the sample to be detected from the standard curve 1, calculating the CAR copy number of the sample to be detected from the standard curve 2, and then calculating the copy number of the target gene relative to the cell number according to a formula;
the copy number calculation formula of the target gene relative to the cell number is as follows:
on the basis of the technical scheme, preferably, the nucleotide sequence of the reference gene beta-globin is shown as SEQ ID NO. 1; the nucleotide sequence of the upstream primer of the reference gene beta-globin is shown as SEQ ID No.2, the nucleotide sequence of the downstream primer is shown as SEQ ID No.3, and the nucleotide sequence of the probe is shown as SEQ ID No. 4.
Based on the above technical solution, preferably, the target gene element is at least one selected from the group consisting of CD30scFv, WPRE, HIV-1 Ψ, and RRE.
On the basis of the above technical solution, preferably, the target gene is selected from CD30scFv and WPRE.
Based on the above technical scheme, preferably, the nucleotide sequence of the CD30scFv is shown as SEQ ID NO.5, the nucleotide sequence of the CD30scFv upstream primer is shown as SEQ ID NO.6, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.7, and the nucleotide sequence of the probe is shown as SEQ ID NO. 8.
On the basis of the technical scheme, preferably, the nucleotide sequence of the WPRE is shown as SEQ ID NO.9, the nucleotide sequence of the CD30scFv upstream primer is shown as SEQ ID NO.10, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.11, and the nucleotide sequence of the probe is shown as SEQ ID NO. 12.
On the basis of the technical scheme, preferably, the probes of the reference gene beta-globin, the target gene CD30scFv and the WPRE are labeled with fluorescent groups at the 5 'ends and are labeled with quenching groups FAM at the 3' ends; the fluorescent group is selected from one of VIC, FAM, TET, HEX, CY3, CY5, Texas Red, LC RED640 and LC RED 705.
Reaction system of PCR amplification: AceQ qPCR Probe Master Mix15.0. mu.L, PF Primer 20. mu.M 1.2. mu.L, PR Primer 20. mu.M 1.2. mu. L, Probe 20. mu.M 0.3. mu. L, Sample 5.0.0. mu. L, RNase-free Water 7.3. mu. L, Total 30. mu.L; reaction conditions for PCR amplification: 95 ℃ for 5 min; (95 ℃, 10 s; 60 ℃, 30 s; X45).
In a second aspect, the invention also provides the use of a method for detecting the copy number of a lentiviral vector of a single CAR-T cell, for detecting the copy number of a lentiviral vector integrated into the genome of each CAR-T cell on average.
In a third aspect, the invention also provides a method for detecting the copy number of the lentiviral vector of a single CAR-T cell, and application of the method in detecting the copy number of the CAR in a sample to be detected.
Compared with the prior art, the method for detecting the copy number of the lentiviral vector of the single CAR-T cell and the application thereof have the following beneficial effects:
(1) the invention takes beta-globin as an endogenous reference gene and CD30scFv and WPRE as gene elements, can stably and effectively detect the copy number of a virus vector integrated in the CAR-T cell genome, and can convert the copy number into the copy number of the virus vector integrated in each CAR-T cell genome on average.
(2) The beta-globin of the invention is a nuclear single copy gene, one cell expresses two copies of the beta-globin gene, and the beta-globin gene is stably expressed in all types of human cells.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram of the position of a beta-globin sequence and corresponding qPCR primers and probes in example II of the present invention
Figure 2 is a schematic diagram of the mCD30-01CAR sequence and the corresponding qPCR primer, probe positions in example two of the present invention;
FIG. 3 is a schematic diagram of a WPRE sequence and corresponding qPCR primers and probe positions in example two of the present invention;
FIG. 4 is a standard amplification curve of the working mCD30-01 plasmid in the sixth embodiment of the present invention;
FIG. 5 shows the standard amplification of mCD30-01 plasmid in the sixth embodiment of the present invention;
FIG. 6 is a working standard amplification curve of β -globin plasmid according to the sixth embodiment of the present invention;
FIG. 7 shows the working standard amplification of β -globin plasmid according to the sixth embodiment of the present invention;
FIG. 8 is a WPRE plasmid working standard amplification curve in the sixth embodiment of the present invention;
FIG. 9 shows the amplification of the WPRE plasmid working standard in the sixth embodiment of the present invention;
FIG. 10 is a flow chart of the method for detecting the copy number of the lentiviral vector of a single CAR-T cell of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The invention relates to a method for detecting the copy number of a lentiviral vector of a single CAR-T cell, which comprises the following steps:
first, two standard curves were established: firstly, establishing a standard curve 1 by using a beta-globin plasmid standard product with known copy number according to an endogenous reference gene beta-globin; secondly, a plasmid with a known initial copy number and a target gene (CAR gene) is used as a standard substance, and a standard curve 2 can be made; where the abscissa represents the Ct value and the ordinate represents the logarithm of the initial copy number.
Secondly, respectively using qPCR primers of beta-globin and a target gene (CAR gene) to carry out PCR reaction on the sample to be detected to obtain a Ct value of the sample about the beta-globin reaction, and calculating the copy number (copies/mu L) of the beta-globin of the sample from a standard curve 1; the Ct value of the sample for CAR was obtained, i.e.the copy number of CAR (copies/. mu.L) for this sample was calculated from the standard curve 2.
Finally, calculating the copy number of the target gene relative to the cell number according to a formula;
the copy number calculation formula of the target gene relative to the cell number is as follows:
beta-globin is a nuclear single copy gene, and one human cell expresses two copies of beta-globin, namely the cell mass is expressed as copy number of beta-globin/2 (cells/mu L). The copy number of the target gene relative to the cell number can be expressed as the ratio of the initial amount of the target gene to the initial amount of the cell number, i.e., copy number/cell.
The target gene is at least one selected from CD30scFv, WPRE, HIV-1 Ψ and RRE.
The nucleotide sequence of the internal reference gene beta-globin is shown as SEQ ID NO.1, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.2, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.3, and the nucleotide sequence of the probe is shown as SEQ ID NO. 4.
The nucleotide sequence of the CD30scFv is shown as SEQ ID NO.5, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.6, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.7, and the nucleotide sequence of the probe is shown as SEQ ID NO. 8.
The nucleotide sequence of the WPRE is shown as SEQ ID NO.9, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.10, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.11, and the nucleotide sequence of the probe is shown as SEQ ID NO. 12.
The 5 'ends of the beta-globin probe sequences, the CD30scFv probe sequences and the WPRE probe sequences are all marked with fluorescent groups, and the 3' ends are all marked with quenching groups FAM; the fluorescent group is selected from one of VIC, FAM, TET, HEX, CY3, CY5, Texas Red, LC RED640 and LC RED 705.
EXAMPLE 1 Standard plasmid construction and preparation of Standard
1.1. Selection of reference genes and target genes
The results of the normalization experiment using the reference genes, which are most commonly genes stably expressed in all cell types, must be constant in the expression level of the reference genes in all samples to be examined and not be changed by each treatment during the study as an appropriate normalization factor. Beta-globin is a nuclear single copy gene, one cell expresses two copies of the beta-globin gene, and the beta-globin gene is stably expressed in all types of human cells. In the present example, the results of the normalization experiment using β -globin gene as reference gene, the nucleotide sequence of β -globin is shown in SEQ id.1.
Vectors derived from HIV-1 lentiviruses have a number of cis-acting elements, such as cPPT and WPRE, which greatly affect the packaging efficiency of the lentivirus. The universal element WPRE (woodchuck hepatitis B virus post-transcriptional regulatory element) on the CAR vector is used as a target gene, and the expression quantity of the CAR gene can be monitored, so that the universal purpose is achieved; the CAR scFv is used as a target gene to detect, and the expression quantity of the CAR gene can be monitored to achieve the purpose of specifically recognizing the CAR. The nucleotide sequences of CD30scFV and WPRE are shown as SEQ ID.5 and SEQ ID.9, respectively.
1.2. Standard quality grain construction
Designing common primers at two ends of the beta-globin, CD30scFv and WPRE gene sequences by using SnapGene software, amplifying full-length target genes and reference gene DNA fragments, and purifying and recovering the full-length target genes and the reference gene DNA fragments by using a glue recovery kit. With reference to the pClone007 Versatile Simple Vector Kit specification, the DNA fragments of the full-length target gene and the reference gene are respectively connected with a T Vector (pClone007) to construct plasmid standards pClone-wpre, pClone-CD30 and pClone-beta-globin.
TABLE 1 plasmid Standard information
| Name (R) | Stock concentration (ng/. mu.L) | BaseNumber (bp) |
| β-globin | 27.11 | 2038 |
| mCD30-01 | 151.61 | 11879 |
| WPRE | 111.45 | 3283 |
Example 2 primer and Probe design
Good primer and probe design is one of the most important steps in real-time fluorescent quantitative PCR. Primer probe design is required to ensure the specificity of the primer probe to the target sequence, and not to form an internal secondary structure, and to avoid complementary hybridization between the 3' end of each primer itself or with other primers. Primers and probes were designed using PrimerExpress3.0 software, the design rules are shown in tables 2-4.
TABLE 2 Probe design principles
TABLE 3 primer design principles
TABLE 4 amplicons follow principles
| Index (es) | Amplicons |
| Length of | 70bp～150bp |
| Tm value | Maximum 85 DEG |
| GC content | |
| 40％～55％ | |
| Target sequence | To amplify AT-rich target sequences |
Primers and probes designed by the above design principles were searched in NCBI Primer Blast and RTPrimerDB databases, respectively, and their specificity was determined. Labeling of fluorophores on the probe. The fluorophores on the probes of the multiplex real-time fluorescent quantitative PCR can not be mutually interfered. The 5 'end of the probe is labeled FAM and the 3' end is labeled MGB. And (4) dissolving and subpackaging the primer probe. The primers and probe dry powder were dissolved to 100. mu.M with nucleic-Free water, used as a stock solution, and stored at-20 ℃. When in use, a small amount of the stock solution is diluted to 10 mu M as working concentration and stored at 4 ℃ for no more than 6 months. Note that the probe was stored in a brown 1.5mL centrifuge tube protected from light.
EXAMPLE 3 sample treatment
A method for detecting the copy number of a lentivirus vector adopts a detection reagent to carry out fluorescence quantitative PCR on a sample to be detected, and specifically comprises the following steps.
3.1 cell lysis
About 5 to 6X 106Adding 25U Cryonase (TM) Cold-active nucleic acid into the culture medium, and digesting at 4 ℃ for 1 h; centrifuging at room temperature at 500g for 5min, removing most of the culture medium by using a pipette, reserving about 100 μ L of the culture medium, and uniformly blowing the cells; 1mL of DNAZOL was added and gently whipped about 50 times (if not immediately extracted, it was stored temporarily at-20 ℃ C. and when extracted, it was removed from-20 ℃ C. and after it melted, it was gently whipped 10 times).
3.2 DNA precipitation
Adding 0.5mL of absolute ethyl alcohol into the cell homogenate, reversing the cell homogenate for 10 times, and standing at room temperature for 1-3 min to obtain white DNA precipitate; centrifuge at 18000g for 15min at 4 ℃.
3.3 DNA washing
Removing the supernatant, adding 1mL of 75% ethanol for washing, reversing the mixture from top to bottom for 5 times, and standing at room temperature for 1-3 min; centrifuging at 15000g at 4 deg.C for 5 min; the supernatant was removed (care was taken to suck the supernatant dry) and left to air dry the precipitate (the precipitate turned from white to transparent) by uncapping.
3.4 DNA solubilization
Adding a proper volume of 8mM NaOH solution (according to the size of the precipitate), and dissolving at 4 ℃ overnight; the pH was adjusted to 8.4 by addition of 0.1M HEPES. (calculated as 1mL of 8mM NaOH with 86. mu.L of 0.1M HEPES).
3.5 DNA concentration determination
The DNA concentration and A260nm/A230nm, A260nm/A280nm, etc. were measured by an ultramicro ultraviolet spectrophotometer Q5000.
EXAMPLE 4 dilution of plasmid standards
4.1 mCD30-01 plasmid working standard dilution
The concentration of the mCD30-01 plasmid working standard is 151.61 ng/. mu.L, and is converted into copy number by the following formula: plasmid copy number (copies/. mu.L) ═ 6.02X 1014X plasmid concentration (ng/u L)/(plasmid base number x 660), and the converted copy number of the mCD30-01 plasmid working standard is 1.16 x 1010copies/. mu.L. And taking the mCD30-01 plasmid working standard storage solution out of a refrigerator at the temperature of-20 ℃, melting at room temperature, flicking the bottom of the tube after completely melting, and quickly centrifuging for 10s in a short time. The dilution procedure for the working standard of plasmid mCD30-01 is shown in Table 5 below.
TABLE 5mCD30-01 plasmid working Standard formulations
| Pipe number | Volume of dilution | Concentration of |
| 1 | Standard |
1.16×109 |
| 2 | 10 μ L No.1 tube +90RNase-free Water | 1.16×108 |
| 3 | 10 μ L No.2 tube +90RNase-free Water | 1.16×107 |
| 4 | 10 μ L No.3 tube +90RNase-free Water | 1.16×106 |
| 5 | 10 μ L No.4 tube +90RNase-free Water | 1.16×105 |
| 6 | 10 μ L No.5 tube +90RNase-free Water | 1.16×104 |
| 7 | 10 μ L No.6 tube +90RNase-free Water | 1.16×103 |
| 8 | 10 μ L No.7 tube +90RNase-free Water | 1.16×102 |
According to the standard curve to be detected and the number of samples to be detected, the required reaction hole number is calculated, and 3 repeated holes/sample are made. The number of reaction wells (standard curve of 5 concentration gradients +1 no-template control NTC +1 control NEG with water as template + sample to be tested) × 3, 2 more reaction wells were added to the number of reaction wells calculated, the template was removed and CAR copy number detection main reaction mixture was prepared as shown in table 6.
TABLE 6 copy number detection reaction System
The main reaction system was dispensed into a transparent eight-way tube at 25. mu.L/well, and 1.16X 10 was added at 5. mu.L/well6、1.16×105、1.16×104、1.16×103、1.16×102mCD30-01 plasmid working standard and sample to be detected.
4.2 dilution of working standards for beta-globin plasmids
The concentration of the working standard product of the beta-globin plasmid is 27.11 ng/. mu.L, and is converted into copy number by the following formula: plasmid copy number (copies/. mu.L) ═ 6.02X 1014Xthe plasmid concentration (ng/. mu.L)/(plasmid base number. times.660), in exchangeThe copy number of the calculated beta-globin plasmid working standard product is 1.21 multiplied by 1010copies/. mu.L. And (3) taking out the working standard product storage solution of the beta-globin plasmid from a refrigerator at the temperature of-20 ℃, melting at room temperature, flicking the bottom of the tube after the working standard product storage solution is completely melted, and quickly centrifuging for 10s in a short time. The β -globin plasmid working standard dilution procedure is shown in table 7 below.
TABLE 7 formulation of working standards for beta-globin plasmids
| Pipe number | Dilution volume | Concentration of |
| 1 | Standard |
1.21×109 |
| 2 | 10 μ L No.1 tube +90RNase-free Water | 1.21×108 |
| 3 | 10 μ L No.2 tube +90RNase-free Water | 1.21×107 |
| 4 | 10 μ L No.3 tube +90RNase-free Water | 1.21×106 |
| 5 | 10 μ L No.4 tube +90RNase-free Water | 1.21×105 |
| 6 | 10 μ L No.5 tube +90RNase-free Water | 1.21×104 |
| 7 | 10 μ L No.6 tube +90RNase-free Water | 1.21×103 |
| 8 | 10 uL No.7 tube +90RNase-free Water | 1.21×102 |
According to the standard curve to be detected and the number of samples to be detected, the required reaction hole number is calculated, and 3 repeated holes/sample are made. The number of reaction wells (5 concentration gradient standard curve +1 no-template control NTC +1 water as template control NEG + sample to be tested) x 3, 2 more reaction wells were added to the calculated number of reaction wells, the template was removed and the beta-globin copy number detection master reaction mixture was prepared according to the reaction shown in Table 6 below.
TABLE 8 copy number detection reaction System
| Reaction system | Addition amount (μ L) |
| AceQ qPCR Probe Master Mix | 15.0 |
| F Primer(20μM) | 1.2 |
| R Primer(20μM) | 1.2 |
| Probe(20μM) | 0.3 |
| |
5 |
| RNase-free Water | 7.3 |
| |
30 |
The main reaction system was dispensed into a transparent eight-way tube at 25. mu.L/well, and 1.21X 10 was added at 5. mu.L/well6、1.21×105、1.21×104、1.21×103、1.21×102A copies/mu L beta-globin plasmid working standard and a sample to be detected.
4.3 dilution of the WPRE plasmid working Standard
The concentration of the WPRE plasmid working standard is 111.45 ng/. mu.L, and is converted into copy number by the following formula: plasmid copy number (copies/. mu.L) ═ 6.02X 1014X plasmid concentration (ng/uL)/(number of plasmid bases x 660), and the converted copy number of the WPRE plasmid working standard is 3.10 x 1010copies/. mu.L. And (3) taking out the working standard product storage solution of the beta-globin plasmid from a refrigerator at the temperature of-20 ℃, melting at room temperature, flicking the bottom of the tube after the working standard product storage solution is completely melted, and quickly centrifuging for 10s in a short time. The β -globin plasmid working standard dilution procedure is shown in table 9 below.
TABLE 9WPRE plasmid working Standard preparation
According to the standard curve to be detected and the number of samples to be detected, the required reaction hole number is calculated, and 3 repeated holes/sample are made. The number of reaction wells (standard curve of 5 concentration gradients +1 no-template control NTC +1 control NEG with water as template + sample to be tested) × 3, 2 more reaction wells were added to the number of reaction wells calculated, the template was removed, and the CAR copy number detection main reaction mixture was prepared.
TABLE 10 copy number detection reaction System
| Reaction system | Addition amount (μ L) |
| AceQ qPCR Probe Master Mix | 15.0 |
| F Primer(20μM) | 1.2 |
| R Primer(20μM) | 1.2 |
| Probe(20μM) | 0.3 |
| |
5 |
| RNase-free Water | 7.3 |
| |
30 |
The main reaction system was dispensed into a transparent eight-way tube at 25. mu.L/well, and 3.10X 10 was added at 5. mu.L/well6、3.10×105、3.10×104、3.10×103、3.10×102A copies/mu LWPRE plasmid working standard and a sample to be detected.
EXAMPLE 5 sample testing
The CFX ConnectTM Real-Time System fluorescent quantitative PCR instrument was opened and placed in the octal tube. The QPCR reaction program was created under the Protocol label as shown in table 11. 2 detection target targets are created under the Plate label and are respectively named as beta-globin, mCD30 and WPRE, and the reporter fluorescent group is selected as FAM. And running the program, selecting a saving path, and clicking for saving.
TABLE 11 qPCR reaction procedure
Example 6 analysis of results
6.1 establishment of mCD30-01 plasmid working standard curve
Establishment of mCD30-01 plasmid working standard curve and CAR-T cell gDNA sample CAR gene copy number detection 1.16 x 106～1.16×102The amplification curve of the copy/mu L serial dilution mCD30-01 plasmid working standard is in a standard S shape and has equal intervals. 1.16X 106The copies/. mu.L standard enters the exponential amplification phase at approximately 22 cycles, after which the standard is sequentially delayed by 3-4 cycles，1.16×102The copies/. mu.L enters the exponential amplification phase at approximately 35 cycles. The amplification curves of the negative control RNase-free Water and the blank control NTC are not raised, and the amplification curves are shown in FIG. 4. The standard curve is automatically given by the Ct value measured by the instrument, and the mCD30-01 plasmid working standard is 1.16 multiplied by 106～1.16×102A linear relation in the range of copies/. mu.L, a correlation coefficient R20.998, slope-3.241, and amplification efficiency E of 103.5%, the standard curve is shown in FIG. 5.
6.2 establishment of working Standard Curve of beta-globin plasmid
Establishment of beta-globin plasmid working standard curve and CAR-T cell gDNA sample CAR gene copy number detection 1.21 x 106～1.21×102Amplification curves of the working standard products of the copies/mu L serial dilution beta-globin plasmid are in a standard S type and are at equal intervals. 1.16X 106The copies/. mu.L standard enters the exponential amplification phase at approximately 20 cycles, after which the standard is sequentially delayed by 3 to 4 cycles, 1.16X 102The copies/. mu.L enters the exponential amplification phase at approximately 34 cycles. The amplification curves of the negative control RNase-free WaternEG and the blank control NTC were not raised, and the amplification curves are shown in FIG. 6. The standard curve is automatically given by the Ct value measured by the instrument, and the working standard product of the beta-globin plasmid is 1.16 multiplied by 106～1.16×102A linear relation in the range of copies/. mu.L, a correlation coefficient R2The slope was-3.354 at 0.998, and the amplification efficiency E was 98.7%, and the standard curve is shown in FIG. 7.
6.3 establishment of working Standard Curve for WPRE plasmid
Establishment of WPRE plasmid working standard curve and CAR-T cell gDNA sample CAR gene copy number detection 3.10 × 106～3.10×102The amplification curves of the copy/. mu.L serial dilution WPRE plasmid working standard are in a standard S shape and are equally spaced. 3.10X 106The copies/. mu.L standard enters the exponential amplification phase at approximately 22 cycles, after which the standard is sequentially delayed by 3 to 4 cycles, 1.16X 101The copies/. mu.L enters the exponential amplification phase at approximately 35 cycles. Amplification curves of negative control RNase-free Waterneg and blank control NTC have no raising head, and amplification is carried outThe increasing curve is shown in fig. 8. The standard curve is automatically given by the Ct value measured by the instrument, and the WPRE plasmid working standard is 3.10 multiplied by 106～3.10×102A linear relation in the range of copies/. mu.L, a correlation coefficient R2The slope was-3.406 at 0.999, and the amplification efficiency E was 96.6%, as shown in FIG. 9.
The verification of the mCD30-01 plasmid working standard curve, the beta-globin plasmid working standard curve and the WPRE plasmid working standard curve proves that the three plasmid working standard curves can be stably and normally amplified, and corresponding primers and probes can effectively and specifically identify target genes respectively.
Example 7 detection of clinical CAR T cell samples
And (3) detecting the CAR gene in the same sample according to the experimental method, and calculating the number of the lentivirus copy number in each CAR T cell by using three plasmid working standard curves. YZC0103202203002-02 is a CART cell sample, and TZC0103202203002-01 is a T cell sample. The three plasmid working standard curves are respectively shown in table 12, table 13 and table 14, and the gene detection data of the CAR T cell sample (YZC0103202203002-02) are respectively shown in table 15, table 16 and table 17; the final analysis results of the CAR T cell sample YZC0103202203002-02 and T cell sample TZC0103202203002-01 are shown in Table 18. The result shows that the data of the copy number of the lentiviral vector for detecting CART cells by using the primer combination of beta-globin and CD30scFv as specificity is 0.79 copies/cell, while the corresponding control T cells are 0 copies/cell, and the result shows that the detection combination can effectively and specifically detect the expression of the CAR gene; the data of the lentiviral vector copy number of the CAR T cells detected by using the WPRE and CD30scFv as specific primer combination are 0.82 copies/cell, while the corresponding control T cells are 0 copies/cell, and the results show that the detection combination can effectively and specifically detect the expression of the CAR gene. More importantly, the results of the two detection methods are almost consistent, which shows the feasibility of the detection method.
TABLE 12 detection data and calibration curves for beta-globin plasmid working standards
TABLE 13 mCD30-01 plasmid working Standard test data and Standard Curve
TABLE 14 WPRE plasmid working standards test data and calibration curves
TABLE 15 sample beta-globin Gene detection data
TABLE 16 sample CAR Gene detection data
TABLE 17 WPRE Gene detection data for samples
TABLE 18 analysis of the results
In conclusion, aiming at the requirement of CAR-T cytoplasm control, the invention analyzes the commonly used lentiviral vector sequences, and primer combinations of beta-globin and CD30scFv and primer combinations of WPRE and CD30scFv are developed and designed aiming at specific and universal primers respectively, and the primers can be effectively applied to the detection of the copy number of the lentiviral vector of a single cell. By using the digital PCR technology to match the primer and the probe, the copy number of the viral vector in the CAR-T cell genome can be accurately quantified, and the effectiveness and safety of CAR-T cell therapeutic products are guaranteed.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Wuhan university of science and technology
<120> a method for detecting the copy number of lentiviral vectors of a single CAR-T cell and uses thereof
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Claims (10)
1. A method for detecting the lentiviral vector copy number of a single CAR-T cell, wherein: the method comprises the following steps:
s1, designing qPCR primers and probes of beta-globin by taking the beta-globin as an internal reference gene, and establishing a standard curve 1 by utilizing a plasmid template of the beta-globin;
s2, designing a primer and a probe of a target gene, and establishing a standard curve 2 by using a plasmid template of the target gene;
s3, respectively using qPCR primers of beta-globin and a target gene to carry out PCR amplification on a sample to be detected, calculating the beta-globin copy number of the sample to be detected from a standard curve 1, calculating the CAR copy number of the sample to be detected from a standard curve 2, and then calculating the copy number of the target gene relative to the cell number according to a formula;
the copy number calculation formula of the target gene relative to the cell number is as follows:
2. a method for detecting the lentiviral vector copy number of a single CAR-T cell according to claim 1, wherein: the nucleotide sequence of the reference gene beta-globin is shown in SEQ ID NO. 1; the nucleotide sequence of the upstream primer of the reference gene beta-globin is shown as SEQ ID No.2, the nucleotide sequence of the downstream primer is shown as SEQ ID No.3, and the nucleotide sequence of the probe is shown as SEQ ID No. 4.
3. A method for detecting the lentiviral vector copy number of a single CAR-T cell according to claim 1, wherein: the target gene element is at least one of CD30scFv, WPRE, HIV-1 Ψ and RRE.
4. A method for detecting the lentiviral vector copy number of a single CAR-T cell according to claim 3, wherein: the gene element of interest is selected from the group consisting of CD30scFv and WPRE.
5. A method for detecting the lentiviral vector copy number of a single CAR-T cell according to claim 4, wherein: the nucleotide sequence of the CD30scFv is shown in SEQ ID NO.5, the nucleotide sequence of the CD30scFv upstream primer is shown in SEQ ID NO.6, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.7, and the nucleotide sequence of the probe is shown in SEQ ID NO. 8.
6. A method for detecting the lentiviral vector copy number of a single CAR-T cell according to claim 4, wherein: the nucleotide sequence of the WPRE is shown as SEQ ID NO.9, the nucleotide sequence of the CD30scFv upstream primer is shown as SEQ ID NO.10, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.11, and the nucleotide sequence of the probe is shown as SEQ ID NO. 12.
7. A method for detecting the lentiviral vector copy number of a single CAR-T cell according to claim 4, wherein: probes of the reference gene beta-globin, the target gene CD30scFv and the WPRE are all marked with fluorescent groups at the 5 'ends and are marked with quenching groups FAM at the 3' ends; the fluorescent group is selected from one of VIC, FAM, TET, HEX, CY3, CY5, Texas Red, LC RED640 and LC RED 705.
8. A method for detecting the lentiviral vector copy number of a single CAR-T cell according to claim 1, wherein: the reaction system of the PCR amplification comprises: AceQ qPCR Probe Master Mix15.0. mu.L, PF Primer 20. mu.M 1.2. mu.L, PR Primer 20. mu.M 1.2. mu. L, Probe 20. mu.M 0.3. mu. L, Sample 5.0.0. mu. L, RNase-free Water 7.3. mu. L, Total 30. mu.L; reaction conditions for PCR amplification: 95 ℃ for 5 min; (95 ℃, 10 s; 60 ℃, 30 s; X45).
9. Use of a method according to any one of claims 1 to 8 for detecting the copy number of a lentiviral vector of a single CAR-T cell, wherein: the lentiviral vector copy number is the number of lentiviral vector copies integrated into the average per CAR-T cell genome.
10. Use of a method according to any one of claims 1 to 8 for detecting the copy number of a lentiviral vector of a single CAR-T cell for detecting the copy number of a CAR in a test sample.
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