CN111909931A - Fluorescent probe, primer pair, fluorescent quantitative PCR kit and detection method - Google Patents

Fluorescent probe, primer pair, fluorescent quantitative PCR kit and detection method Download PDF

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CN111909931A
CN111909931A CN201910380678.XA CN201910380678A CN111909931A CN 111909931 A CN111909931 A CN 111909931A CN 201910380678 A CN201910380678 A CN 201910380678A CN 111909931 A CN111909931 A CN 111909931A
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唐超
张宏玲
李浩莎
任异菲
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Shenzhen Bindebio Technology Co ltd
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Abstract

The invention provides a fluorescent probe, which comprises a single-stranded nucleotide fragment, wherein the 5 'end of the single-stranded nucleotide fragment is marked with a fluorescent reporter group, the 3' end of the nucleotide fragment is marked with a fluorescent quenching group, and the single-stranded nucleotide fragment comprises a nucleotide sequence shown in SEQ ID NO: 1. The fluorescent probe has the advantages of high specificity and high sensitivity, and can be used for rapidly and quantitatively analyzing the copy number of the slow virus containing the WPRE element, particularly the copy number of the slow virus in CAR-T mediated by the slow virus gene containing the WPRE element. The invention also provides a primer pair, a fluorescent quantitative PCR kit and a detection method.

Description

Fluorescent probe, primer pair, fluorescent quantitative PCR kit and detection method
Technical Field
The invention relates to the field of medical biology, in particular to a fluorescent probe, a primer pair, a fluorescent quantitative PCR kit and a detection method.
Background
Immune cell therapy is the only method which has the possibility of completely eliminating cancer cells in the prior art, and is considered as a treatment means with the most development prospect in the comprehensive treatment mode of tumors in the twenty-first century. Compared with the traditional treatment means, the immune cell treatment technology has the great advantages of strong specificity and almost no toxic or side effect in the aspect of treating malignant tumors, overcomes the defects of the traditional operation, radiotherapy and chemotherapy, and becomes the fourth means of treating tumors. Among them, Chimeric Antigen Receptor T cell (CAR-T) technology has received extensive attention and research as one of the current latest immune cell therapy technologies.
At present, most of CAR-T is prepared by adopting a lentivirus transfection method to perform gene transduction so as to enable the CAR-T to express a Chimeric Antigen Receptor (CAR) by virtue of the advantages of safer integration sites of lentivirus vectors, larger transgene load, smaller gene toxicity and the like. However, because the quality control of CAR-T is very strict, there are still great challenges in the industrial application of CAR-T. The method has important significance for the quality control of CAR-T, for example, the method has guiding significance in the aspects of testing the quality control of a lentiviral vector transfected with CAR gene or the rate of genetically modified CAR-T positive cells and the like. However, the traditional detection method mainly determines the positive rate of the CAR-T after transfection by detecting the amount of protein expressed by a target gene on a T cell by flow cytometry at the protein level; or qualitatively by detecting the CAR gene, etc. The detection methods have the defects of low detection specificity, low sensitivity, complexity, time and repeatability and the like, and have strict requirements on operating environment.
Disclosure of Invention
In view of this, the invention provides a fluorescent probe, a primer pair, a fluorescent quantitative PCR kit and a detection method. The fluorescent probe has the advantages of high specificity and high sensitivity, and can be used for rapidly and quantitatively analyzing the copy number of the slow virus containing the WPRE element, particularly the copy number of the slow virus in CAR-T mediated by the slow virus gene containing the WPRE element.
In a first aspect, the present invention provides a fluorescent probe, which comprises a single-stranded nucleotide fragment, wherein a fluorescent reporter group is labeled at the 5 'end of the single-stranded nucleotide fragment, a fluorescent quencher group is labeled at the 3' end of the nucleotide fragment, and the single-stranded nucleotide fragment comprises the nucleotide sequence shown in SEQ ID NO: 1.
In the present invention, the fluorescent probe is used for specifically detecting WPRE (Woodchick hepatitis virus post-translational regulatory element) elements. The WPRE element is a viral post-transcriptional regulatory element that enhances viral-mediated transgene expression levels. Optionally, the WPRE element comprises a sequence as set forth in SEQ ID NO: 4.
In the invention, when the fluorescence reporter group and the fluorescence quenching group respectively marked at two ends of the fluorescence probe are close to each other, a fluorescence signal emitted by the excited fluorescence reporter group is absorbed by the fluorescence quenching group. When the target gene fragment of the WPRE element is amplified, the fluorescence reporter group can be specifically identified with the target gene fragment of the WPRE element, and after the fluorescence reporter group is separated from the fluorescence quenching group, the fluorescence reporter group emits a fluorescence signal, so that the purpose of detecting the copy number of the target gene fragment can be achieved by detecting the fluorescence signal. Wherein, each time the target gene segment is copied, one fluorescent probe corresponds to the target gene segment, so that the fluorescent signal accumulation is completely synchronous with the target gene segment amplification product.
Alternatively, the fluorescent probes may be used, but are not limited to, for specifically detecting WPRE element copy number in a variety of objects, including plasmids, vectors, viruses, genomes of bacteria, or genomes of cells. For example, the fluorescent probes are used to detect WPRE elements in recombinant lentiviruses or CAR-T cells containing WPRE elements.
Optionally, the fluorescent reporter comprises one or more of carboxyfluorescein (FAM), carboxytetramethylrhodamine (TAMRA), 2, 7-dimethyl-4, 5-dichloro-6-carboxyfluorescein (JOE), and hexachloro-6-methylfluorescein (HEX); the fluorescence quenching group comprises one or more of carboxytetramethylrhodamine (TAMRA), 4- [ (2-chloro-4-nitro-phenyl) -azo ] -aniline (Eclipse) and Black Hole Quencher (BHQ). Wherein the carboxyfluorescein (FAM) comprises 5-carboxyfluorescein (5-FAM) or 6-carboxyfluorescein (6-FAM). The Black Hole Quencher (BHQ) comprises a black hole quencher 1(BHQ-1), a black hole quencher 2(BHQ-2) or a black hole quencher (BHQ-3).
Alternatively, the fluorescent reporter group may also include, but is not limited to, one or more of tetrachloro-6-carboxyfluorescein (TET), anthocyanidin dyes (Cyanines dyes), carboxy-X-Rhodamine (ROX), Texas Red dye (Texas Red), Fluorescein Isothiocyanate (FITC), and Acridine orange (Acridine orange). Alternatively, the anthocyanidin dye can be used but does not include fluorescent dyes with different emission wavelengths, such as Cy3, Cy5 or Cy5.5. The carboxyl-X-rhodamine comprises 5-carboxyl-X-rhodamine (5-ROX) or 6-carboxyl-X-rhodamine (6-ROX).
Alternatively, the fluorescence quenching group may also include, but is not limited to, 4- (4 '-oxanilino) azobenzene benzoic acid (DABCYL) or 4- (N, N-dimethylamino) azobenzene-4' -sulfonic acid chloride (DABSYL).
The fluorescent probe can increase the detection sensitivity to a certain degree by selecting the matched fluorescent reporter group and the matched fluorescent quenching group or the fluorescent reporter group with low background fluorescence.
In a second aspect, the present invention provides a primer pair, comprising a forward primer and a reverse primer, wherein the forward primer comprises the nucleotide sequence shown in SEQ ID NO: 2, and the reverse primer comprises a nucleotide sequence shown as SEQ ID NO: 3.
In the invention, the single-stranded nucleotide fragment comprises one or more of polydeoxyribonucleotide, polyribonucleotide and other types of polynucleotides; for example, purine or pyrimidine bases in other types of polynucleotides are modified to pyrimidine or purine bases. The source of the single-stranded nucleotide fragment of the present invention is conventional in the art, and for example, the source of the single-stranded nucleotide fragment may include a synthetic method or a method in which the single-stranded nucleotide fragment is obtained by extraction from a naturally occurring genome. Wherein the artificial synthesis method of the single-stranded nucleotide fragment comprises the following steps: one or more of a phosphoric acid triester method, a phosphoric acid diester method, a diethyl phosphoric acid amide method and a solid-phase carrier method.
In the invention, the reaction efficiency of the primer pair is high, and the WPRE element can be rapidly and specifically detected; the WPRE element can be continuously amplified during PCR amplification reaction, and the size of the amplified WPRE element is 127 bp.
In a third aspect, the present invention also provides a fluorescent quantitative PCR kit, comprising the fluorescent probe according to the first aspect of the present invention and/or the primer pair according to the second aspect of the present invention.
In particular, the fluorescent quantitative PCR kit may, but is not limited to, comprise a fluorescent probe as described in the first aspect of the invention. Or the fluorescent quantitative PCR kit may, but is not limited to, comprise a primer pair as described in the second aspect of the invention. Or the fluorescent quantitative PCR kit may, but is not limited to, comprise the fluorescent probe according to the first aspect of the invention and the primer pair according to the second aspect of the invention.
Optionally, when the fluorescent quantitative PCR kit comprises the fluorescent probe according to the first aspect of the invention, the fluorescent quantitative PCR kit further comprises other PCR primer pairs for amplification of WPRE elements.
Optionally, when the fluorescent quantitative PCR kit comprises a primer pair according to the second aspect of the invention, the fluorescent quantitative PCR kit may further comprise other fluorescent probes.
Optionally, the fluorescent quantitative PCR kit further comprises a reaction buffer, a positive control and a negative control.
Alternatively, the reaction buffer may also include, but is not limited to, a PCR buffer. The reaction buffer may include, but is not limited to, one or more of salt ions, enzymes, or a stable pH buffer system necessary for the PCR amplification reaction. The reaction buffer may be, but is not limited to, an existing reaction buffer. For example, the reaction buffer contains dNTP, magnesium chloride, DNA polymerase, and the like. Alternatively, the dNTP may be a dNTP conventional in the art, including dATP, dGTP, dTTP, and dCTP. The DNA polymerase includes a thermostable DNA polymerase.
Optionally, the positive control contains the WPRE element.
Alternatively, the negative control may include, but is not limited to, DEPC water. The DEPC water is MiliQ pure water which is treated by DEPC (diethyl pyrocarbonate) and sterilized by high temperature and high pressure, and is colorless liquid; contains no impurity RNA, DNA and protein.
In a fourth aspect, the present invention also provides a fluorescent quantitative PCR detection method, comprising:
extracting DNA of a sample to be detected, wherein the sample to be detected comprises target cells transfected by a first recombinant gene transfer vector containing a WPRE element, and carrying out quantitative PCR amplification reaction on the DNA of the sample to be detected; in the quantitative PCR amplification reaction, the fluorescent probe according to the first aspect of the invention and/or the primer pair according to the second aspect of the invention are/is used;
and after the reaction is finished, detecting whether the DNA of the sample to be detected contains the WPRE element or not, and detecting the copy number of the WPRE element in the sample to be detected.
Optionally, after the reaction is finished, by recording the Ct value of the sample to be detected, detecting whether the DNA of the sample to be detected contains the WPRE element, and detecting the copy number of the WPRE element in the sample to be detected.
In the present invention, the Ct value is the corresponding amplification Cycle (Cycle Threshold) when the fluorescence signal of the amplification product reaches a set fluorescence Threshold during the fluorescent quantitative PCR amplification process.
Optionally, the first recombinant gene delivery vector further comprises a target gene, and the target cell transfected by the first recombinant gene delivery vector is prepared by a process comprising:
(1) packaging the first recombinant gene transfer vector and transfecting host cells to obtain recombinant viruses;
(2) transfecting a target cell with the recombinant virus to introduce the WPRE element and the target gene into the genome of the target cell; the target cells include one or more of tumor cells, stem cells, and immune cells.
Optionally, the immune cell comprises a T lymphocyte or an NK cell. Preferably, the immune cells comprise CD3 positive T lymphocytes.
Optionally, the first recombinant gene delivery vector is a viral vector, including at least one of a lentiviral vector, a retroviral vector, and an adenoviral vector. Preferably, the first recombinant gene delivery vector is a lentiviral vector. The first recombinant gene delivery vector is obtained by gene modification on the basis of a gene delivery vector. Wherein, the gene delivery vector can be but is not limited to pWPXLD plasmid vector, pLEX-MCS vector, pSico vector and pCgpV vector.
Optionally, the first recombinant gene delivery vector is co-transfected with an envelope plasmid and a packaging plasmid to obtain a recombinant virus.
In one embodiment of the present invention, the first recombinant gene delivery vector may be a pwxld recombinant plasmid. Adopting a pWPXLD plasmid vector, and when the pWPXLD plasmid vector does not contain a WPRE element, inserting the WPRE element and a target gene fragment into the pWPXLD plasmid vector by a genetic engineering method to obtain the pWPXLD recombinant plasmid. In this case, the envelope plasmid may be pmd2.g and the packaging plasmid may be psPAX 2. The envelope plasmid pmd2.g encodes a vesicular stomatitis virus glycoprotein capsid that aids in the adhesion of the recombinant lentivirus to the cell membrane and maintains the infectivity of the recombinant lentivirus.
The pWPXLD recombinant plasmid is a manually modified lentivirus vector, and can be packaged and transfected into a host cell to obtain a recombinant lentivirus which has only one-time infection capacity and high infection efficiency, and the recombinant lentivirus is a virus (RCL) without replication capacity, is very safe and can be widely used for gene mediation of a target cell. Compared with other gene operation technologies such as transfection, electrotransformation, retrovirus and adenovirus, the pWPXLD recombinant plasmid adopted by the invention has more outstanding advantages. The pWPXLD recombinant plasmid is packaged and transfected to a host cell to obtain the recombinant lentivirus, and the WPRE element and the target gene fragment can be efficiently integrated on the genome of a target cell.
The recombinant lentiviruses of the present invention are typically packaged using transient transfection or using cell lines. Human cell lines that can be used as packaging cells upon transient transfection include, for example, 293 cells, 293T cells, 293FT cells, 293LTV cells, 293EBNA cells, and other clones isolated from 293 cells; SW480 cells, u87MG cells, HOS cells, C8166 cells, MT-4 cells, Molt-4 cells, HeLa cells, HT1080 cells, TE671 cells, and the like. Monkey-derived cell lines, for example, COS1 cells, COS7 cells, CV-1 cells, BMT10 cells, and the like can also be used. Furthermore, commonly used calcium phosphate and PEI transfection reagents, as well as some transfection reagents such as Lipofectamine2000, FuGENE and S93fectin, are also commonly used.
Alternatively, the host cell may comprise HEK293T cells, 293T cells, 293FT cells, SW480 cells, u87MG cells, HOS cells, COS1 cells or COS7 cells.
Further, optionally, the host cell is a HEK293T cell.
Optionally, in the first recombinant gene delivery vector, the WPRE element is located downstream of the target gene; the target gene includes a chimeric antigen receptor gene that targets tumor cells.
In the present invention, the WPRE element is a non-coding gene and is not involved in transcription and translation, but the WPRE element can replicate together with the genome in the target cell. For example, when a recombinant lentivirus containing a WPRE element introduces the WPRE element into the genome of a target cell, the copy number of the WPRE element can be considered a lentivirus copy number, which can be used to reflect the success or failure of lentivirus transfection. And, if the target gene is introduced into the genome of the target cell together with the WPRE element, the copy number of the target gene will correspond to the copy number of the WPRE element; the copy number of the target gene can be counted by detecting the copy number of the WPRE element.
Alternatively, the target gene may also be, but is not limited to, other functional genes, for example the other functional genes may be gene fragments for effecting gene silencing, gene knockout or gene interference. Or the other functional gene may be a gene expressing a cell surface receptor protein.
For example, when the target cell is a CD3 positive T lymphocyte and the target gene is a CAR gene, the test sample is a CAR-T cell that can target the antigen of interest. Further, the gene encoding the CAR gene may include, but is not limited to, a gene encoding a signal peptide, a gene encoding a single chain antibody, a gene encoding an extracellular hinge region, a gene encoding a transmembrane region, and a gene encoding an intracellular signal region, which are sequentially linked from the 5 'end to the 3' end. For example, the CAR gene can be a chimeric antigen receptor gene targeting CD19, CD22, CD33, BCMA, or the like, resulting in a CAR-T targeting CD19, CD22, CD33, or BCMA.
Alternatively, when the target cell is a tumor cell, the target gene may be a gene fragment for gene silencing. Or when the target cell is a stem cell, the target gene can be a gene segment for gene knockout, and the target cell can be used for researching stem cell differentiation and other problems.
Alternatively, the CD3 positive T lymphocytes are isolated from human peripheral blood mononuclear cells. The human-derived peripheral blood mononuclear cells are derived from autologous venous blood, autologous bone marrow, umbilical cord blood, placental blood and the like. Further optionally, the source is fresh peripheral blood or bone marrow collected after one month of surgery or one month of chemotherapy for the cancer patient.
Specifically, the process for obtaining the CD3 positive T lymphocyte is as follows: adding CD3/CD28 immunomagnetic beads into peripheral blood mononuclear cells according to a certain proportion, incubating for a period of time, putting a magnet for screening to obtain CD3 positive T lymphocytes coated by the immunomagnetic beads, and removing the magnetic beads to obtain CD3 positive T lymphocytes.
Optionally, detecting the copy number of the WPRE element in the sample to be tested, including drawing a standard curve, includes the following steps:
providing a plurality of standards of different concentration gradients, said standards being a second recombinant gene delivery vector comprising said WPRE element;
performing a quantitative PCR amplification reaction on the WPRE element in each standard; and after the reaction is finished, drawing a standard curve according to the Ct value corresponding to the copy number of the WPRE element in the standard substance with different concentration gradients.
Optionally, when a standard curve is drawn, the reaction conditions of the quantitative PCR amplification reaction are the same as those when the sample to be detected is detected.
Optionally, the second recombinant gene delivery vector is a viral vector, including at least one of a lentiviral vector, a retroviral vector, and an adenoviral vector. Preferably, the second recombinant gene delivery vector is a lentiviral vector. The second recombinant gene delivery vector is obtained by gene modification on the basis of the gene delivery vector. Wherein, the gene delivery vector can be but is not limited to pWPXLD plasmid vector, pLEX-MCS vector, pSico vector and pCgpV vector. Further optionally, the second recombinant gene delivery vector contains a WPRE element and no target gene.
Alternatively, the positive control may be obtained by diluting the standard with different dilution times.
Alternatively, the standard curve may be, but is not limited to, the Ct value of the standard measured as the abscissa and the log of the WPRE element copy number of the standard at each concentration as the ordinate.
In the invention, the Ct value measured after the sample to be measured is detected can obtain the original copy number of the sample according to a standard curve. The detection method can greatly improve the authenticity and the accuracy of quantitative analysis data of the copy number of the lentivirus in a sample to be detected, and improve the quantitative analysis efficiency of the fluorescent quantitative PCR detection method.
Optionally, the procedure for quantitative PCR amplification reaction comprises: culturing at 45-55 deg.C for 2-5 min; pre-denaturation at 95-98 deg.C for 1-2 min; denaturation at 94-98 deg.C for 10-30 s, annealing and extension at 55-65 deg.C for 1-3 min, and 35-45 cycles.
Further, optionally, the procedure for quantitative PCR amplification reaction comprises: culturing at 45-55 deg.C for 2 min; pre-denaturation at 95-98 deg.C for 2 min; denaturation at 94-98 deg.C for 15 seconds, annealing and extension at 55-65 deg.C for 1 minute, for 35-45 cycles.
The fluorescent quantitative PCR detection method provided by the invention has the advantages of high sensitivity, strong specificity, good repeatability, accurate quantification, high speed and the like, and can become an important tool for quantitative analysis of a lentivirus expression vector, quantitative analysis of the copy number of lentiviruses in CAR-T and determination of the relationship between the number of CAR-T cells and the required virus amount.
The lower detection limit of the fluorescent quantitative PCR detection method can reach 1.7 copies/mu L of DNA. The lower limit value of the detection is far lower than that of the traditional fluorescent quantitative PCR detection method, so that the fluorescent quantitative PCR detection method has higher sensitivity and stronger specificity.
In a fifth aspect, the invention provides the use of a fluorescent probe according to the first aspect, a primer pair according to the second aspect or a fluorescent quantitative PCR assay according to the fourth aspect for the detection of the lentiviral copy number of CAR-T. Because CAR-T has important prospect in preparing medicaments for preventing, diagnosing and treating malignant tumors; the application has important significance for quantitative detection of the lentiviral copy number in CAR-T in the industrial and clinical application processes of CAR-T, and is beneficial to research on the relationship between CAR-T cell positive rate and lentivirus transfection titer.
The invention has the beneficial effects that:
(1) the fluorescent probe has high specificity, and when the fluorescent reporter group and the quenching group at two ends are very close, the fluorescent quenching efficiency is high; WPRE elements can be detected with high specificity.
(2) The primer pair can specifically identify the nucleotide sequence of the WPRE element and quickly amplify the WPRE element in a PCR amplification reaction.
(3) The fluorescent quantitative PCR kit provided by the invention can be used for quantitatively detecting the copy number of the slow virus containing the WPRE element or the copy number of the slow virus WPRE element in CAR-T mediated by a gene of a slow virus transfection method, and has the advantages of high sensitivity, strong specificity, good repeatability, accurate quantification, high speed and the like.
(4) The fluorescent quantitative PCR detection method provided by the invention is simple and practical, and can be used as an important tool for quantitatively detecting the copy number of the lentivirus WPRE element in CAR-T mediated by a lentivirus transfection method gene, and determining the lentivirus titer and the relation between the CAR-T cell number and the required virus amount; provides an important detection means for stability of CAR-T preparation production process and release standard after batch production.
Drawings
FIG. 1 is a plasmid map of a lentiviral recombinant plasmid provided in one embodiment of the present invention.
FIG. 2 is a standard curve of the fluorescence quantitative PCR detection method according to an embodiment of the present invention.
FIG. 3 is a fluorescent quantitative PCR amplification plot of a standard curve provided in accordance with an embodiment of the present invention.
Figure 4 is a flow cytometric assay of CAR-T provided by an embodiment of the present invention.
Detailed Description
While the following is a description of the preferred embodiments of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, all chemical reagents used in the following examples are commercially available reagents. Wherein, the PCR tube used in the fluorescent quantitative PCR detection method is a Real-time PCR tube purchased from BIOptics company, and the product number is: b60109; Real-timePCR tube cover, available from BIOptics, Cat #: b57801; the Probe qPCR Mix, purchased from TOYOBO, Cat #: QPS-101.
Example one
A method of making a chimeric antigen receptor T cell (CAR-T), comprising the steps of:
(1) construction of pWPXLD-CAR recombinant plasmid
Inserting a Chimeric Antigen Receptor (CAR) gene fragment between BamH1 and EcoR1 cleavage sites downstream of a eukaryotic promoter in a pWPXLD plasmid vector, the WPRE element being downstream of the CAR gene fragment. Then transferred into escherichia coli competent cell DH5 alpha, and positive clone PCR identification and sequencing identification are carried out. The size and the sequence of the fragment which accords with the target are identified through PCR product gel electrophoresis detection and sequencing, and the pWPXLD-CAR recombinant plasmid containing the WPRE element and the CAR target gene shown in figure 1 is successfully constructed.
(2) Recombinant lentivirus construction
The pWPXLD-CAR recombinant plasmid, the packaging plasmid psPAX2 and the envelope plasmid pMD2G are co-transfected into a cultured HEK293T cell. Collecting virus-containing supernatant in 48h, filtering with 0.45 μm filter membrane, and storing in an ultra-low temperature refrigerator at-80 deg.C; harvesting virus-containing supernatants for the second 72h, filtering with 0.45 μm filter membrane, mixing with the virus supernatants harvested for the 48h, adding into an ultracentrifuge tube, placing into a Beckman ultracentrifuge one by one, setting the centrifugation parameters to be 25000rpm, the centrifugation time to be 2h, and controlling the centrifugation temperature to be 4 ℃; after the centrifugation is finished, removing the supernatant, removing the liquid remained on the tube wall as much as possible, adding a virus preservation solution, and lightly and repeatedly blowing and resuspending; after fully dissolving, centrifuging at high speed 10000rpm for 5min, taking supernatant to measure titer by a fluorescence method, and measuring the virus according to 100 mu L, 2 multiplied by 108Subpackaging each/mL, and storing in an ultra-low temperature refrigerator at-80 ℃ to obtain the recombinant lentivirus.
(3) Preparation of chimeric antigen receptor T cells
a) Isolation of PBMC (peripheral blood mononuclear cells)
PBMC is derived from autologous venous blood, autologous bone marrow, umbilical cord blood, placental blood, etc. Preferably fresh peripheral blood or bone marrow taken from cancer patients after one month of surgery and one month of chemotherapy.
Drawing blood from a patient and sending the blood to a blood separation chamber; collecting peripheral blood mononuclear cells, and taking intermediate layer cells after Ficoll centrifugal separation; PBMC were obtained after PBS wash.
b) Separation of antigen specific T lymphocyte by immunomagnetic bead method
Taking the PBMC, adding a serum-free basal culture medium to prepare a cell suspension; adding CD3/CD28 immunomagnetic beads according to the ratio of the magnetic beads to the cells being 3:1, and incubating for 1-2h at room temperature; screening the cells incubated with the magnetic beads by using a magnet; after washing with PBS and removal of immunomagnetic beads, CD 3-positive T lymphocytes were obtained.
c) Preparation of antigen-specific T lymphocytes by lentivirus transfection method
And (3) adding the recombinant lentivirus with the virus titer corresponding to the number of the CD3 positive cells into the CD3 positive T lymphocytes obtained by the immunomagnetic bead separation method for culture.
On the 3 rd day of the culture, cell counting and medium exchange were performed to adjust the cell concentration to 1X 106Inoculating and culturing the seeds per mL; on the 5 th day of culture, the state of cells was observed, and if the cell density increased, the cell concentration was diluted to 1X 106And (4) detecting the activity of the cells per mL, and continuing to culture. Expanding and culturing to 9-11 days, and collecting cells to obtain chimeric antigen receptor T cells (CAR-T).
(4) And (3) detecting the CAR expression rate of the obtained CAR-T by flow cytometry, extracting DNA (deoxyribonucleic acid), and detecting the copy number of the WPRE element gene of the lentivirus by using a fluorescent probe and a fluorescent quantitative PCR (polymerase chain reaction) detection method.
The primer pair used comprises a forward primer and a reverse primer:
a forward primer: 5'-GTTGTCGGGGAAGCTGACGT-3'
Reverse primer: 5'-CAGGCCGCGGGAAGGAAGGTC-3'
The primer pair can specifically recognize the nucleotide sequence of the WPRE element, and the size of the amplified target fragment is 127 bp.
Wherein, the fluorescent probe is: 5 '-FAM-CCACCTGGATTCTGCGCGGGA-BHQ-1-3'
The reaction process comprises the following steps: 500nmol of each of the forward primer and the directional primer is taken, 250nmol of the fluorescent Probe is taken, 10 mu L of reaction buffer solution Probe qPCR Mix and 2 mu L of extracted DNA template are taken, and ultrapure water is added to regulate the reaction system to 20 mu L; the reaction conditions of the fluorescent quantitative PCR are as follows: culturing at 50 deg.C for 2 min; pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 1min, for 40 cycles.
The lower limit of the detection of the fluorescent quantitative PCR detection method can reach 1.7 copies/mu L DNA.
In order to evaluate the detection effect of the fluorescent quantitative PCR detection method described in the present invention, the following effect examples were performed.
Effects of the embodiment
1. Evaluation of relationship between lentivirus WPRE element gene copy number and CAR-T cell positivity in production-successful CAR-T
Respectively taking 2 × 106Cell number CAR-T cells prepared by the method described in example 1 and untransfected T lymphocytes (negative control) were subjected to DNA extraction (QIAamp DNABlood Mini Kit, QIAGEN, cat # 51106), concentration was measured, and then subjected to fluorescent quantitative PCR detection.
(a) Establishing a standard curve: the standard pWPXLD plasmid vector containing WPRE element was used as a standard (purchased from Addgene, Inc.), and its concentration was determined to be 20.35ng/uL, and converted to copy number/. mu.L as: 1775000000 copies/. mu.L, 10 concentration gradient dilutions were made, the lowest concentration gradient being 1.775 copies/. mu.L; the 10 concentration gradients and their corresponding log values for the standard are shown in table 1 below:
table 1: 10 concentration gradients of standard substance and corresponding log values thereof
Plasmid concentration (copies/. mu.L) Corresponding logarithmic value
1775000000 9.249198357
177500000 8.249198357
17750000 7.249198357
1775000 6.249198357
177500 5.249198357
17750 4.249198357
1775 3.249198357
177.5 2.249198357
17.75 1.249198357
1.775 0.249198357
The reaction system (20. mu.L) for fluorescent quantitative PCR was configured as follows: 500nmol of each of forward primer and reverse primer, 250nmol of fluorescent Probe, 10 muL of reaction buffer solution Probe qPCR Mix, 2 muL of each concentration gradient standard product DNA, and performing constant volume by using DEPC water;
the procedure of the fluorescent quantitative PCR amplification reaction is as follows: culturing at 50 deg.C for 2 min; pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 1min for 40 cycles; each sample was subjected to 3 replicate wells.
After the reaction is finished, data are recorded, referring to table 2, the corresponding logarithm values of the concentration of 10 concentration gradients are measured as ordinate, the Ct value is measured as abscissa, and a standard curve y ═ 0.3005x +12.277 is drawn, (R ═ 0.3005x +12.27720.998), see figures 2 and 3 together.
Table 2: data table corresponding to horizontal and vertical coordinates of standard curve
Figure BDA0002053269380000131
(b) Detection evaluation of sample to be tested
Configuring different gradient infection groups for the recombinant lentiviruses prepared by the preparation method of the embodiment 1, and adopting the recombinant lentiviruses with different volumes to infect the groups, wherein the volumes of the recombinant lentiviruses are respectively as follows: 0 mu L, 5 mu L, 10 mu L and 20 mu L, transfecting the number of CD3 positive cells to obtain CAR-T, culturing, and taking a part of CAR-T cells to perform flow cytometry detection; and (3) taking another part of CAR-T cells, extracting total DNA of the cells, and then carrying out fluorescence quantitative PCR detection, wherein the detection result of flow cytometry is shown in figure 4, wherein ACP-A +' refers to the staining positive rate of antibody APC (allophycocyanin), and can reflect the positive rate of CAR-T. Where CAR-T was flow tested for positive rates in figure 4, the corresponding CAR positive rates were 0 (negative control), 8.13%, 26.4% and 49.9% when transfected with 0, 5, 10 and 20 μ L virus, respectively.
The experimental group in the fluorescent quantitative PCR detection method process specifically comprises the following steps: 1. DEPC water is used as a negative reference substance; 2. pWPXLD vector plasmid is used as a positive control (20.35 ng/. mu.L of the standard pWPXLD vector plasmid is diluted 1000 times to obtain the positive control with the concentration of 2.035 pg/. mu.L); 3. the sample 1 to be tested is CAR-T cells after 0 mu L of recombinant lentivirus transfection is added; 4. the sample 2 to be tested is CAR-T cells after 5 mu L of recombinant lentivirus transfection is added; 5. the sample 2 to be detected is CAR-T cells after 10 mu L of recombinant lentivirus transfection is added; 6. the sample 4 to be tested is CAR-T cells after 20. mu.L of recombinant lentivirus transfection is added.
According to the prepared standard curve: when y is-0.3005 x +12.277, the lentivirus WPRE gene copy number, i.e. lentivirus copy number, of each experimental group can be rapidly and quantitatively determined, as shown in table 3.
Table 3: fluorescence quantitative PCR detection data table
Figure BDA0002053269380000141
The lentivirus copy number of each sample to be tested, which is measured by the fluorescent quantitative PCR detection method of the invention, is compared with the data of the positive rate of the flow detection CAR-T, see Table 4. As can be seen in Table 4, the lentivirus copy number of each sample to be detected measured by the fluorescent quantitative PCR detection method of the present invention is gradually increased; the percent CAR positivity was also a linear relationship with a gradient increase when transfected with 0. mu.L, 5. mu.L, 10. mu.L and 20. mu.L of virus. The fold relation between the lentivirus copy numbers of the samples to be detected, which is measured by the fluorescent quantitative PCR detection method, corresponds to the fold relation of the CAR positive rate of the samples to be detected, which is measured by flow cytometry. Therefore, the virus copy number measured by the fluorescent quantitative PCR detection method is positively correlated with the CAR-T flow-type positive rate.
Table 4: comparison of fluorescence quantitative PCR detection data with flow cytometry detection data
Figure BDA0002053269380000142
However, in the existing method, a great error exists in the detection process of detecting the CAR positive rate by adopting the flow cytometry, and the error can be reduced as much as possible by introducing a CAR antibody with excellent specificity; and when different CAR-T cell positive rates are detected, a corresponding antibody needs to be found for each target point, so that the detection cost is huge. In addition, flow cytometry is also highly required for detection of objects, for example, only living cells can be detected. Compared with the traditional method, the fluorescent quantitative PCR detection method can specifically detect the WPRE element in the sample to be detected, and can efficiently and accurately detect the corresponding copy number no matter how the detection target changes. On one hand, the fluorescent quantitative PCR detection method can be used for rapidly and quantitatively analyzing the copy number of the lentivirus WPRE gene in CAR-T cells, the process is easy to operate, the accuracy of a test result is high, the detection line is low, and the repeatability is high; in another aspect, the assay can be used to determine the relationship between CAR-T cell positivity rate and the amount of lentivirus required. The detection method of the invention can also more accurately determine the virus titer and the virus infection efficiency.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
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ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgca 588

Claims (10)

1. A fluorescent probe is characterized by comprising a single-stranded nucleotide fragment, wherein the 5 'end of the single-stranded nucleotide fragment is marked with a fluorescent reporter group, the 3' end of the nucleotide fragment is marked with a fluorescent quenching group, and the single-stranded nucleotide fragment comprises a nucleotide sequence shown as SEQ ID NO: 1.
2. The fluorescent probe of claim 1, wherein the fluorescent reporter group comprises one or more of carboxytetramethylrhodamine, carboxyfluorescein, 2, 7-dimethyl-4, 5-dichloro-6-carboxyfluorescein, and hexachloro-6-methylfluorescein; the fluorescence quenching group comprises one or more of 4- [ (2-chloro-4-nitro-phenyl) -azo ] -aniline and a black hole quenching agent.
3. A primer pair, comprising a forward primer and a reverse primer, wherein the forward primer comprises the sequence set forth in SEQ ID NO: 2, and the reverse primer comprises a nucleotide sequence shown as SEQ ID NO: 3.
4. A fluorescent quantitative PCR kit comprising the fluorescent probe according to any one of claims 1 to 2 and/or the primer pair according to claim 3.
5. The quantitative fluorescence PCR kit of claim 4, further comprising a reaction buffer, a positive control, and a negative control.
6. A fluorescent quantitative PCR detection method is characterized by comprising the following steps:
extracting DNA of a sample to be detected, wherein the sample to be detected comprises target cells transfected by a first recombinant gene transfer vector containing a WPRE element, and carrying out quantitative PCR amplification reaction on the DNA of the sample to be detected; in the quantitative PCR amplification reaction, the fluorescent probe according to claim 1 or 2 and/or the primer pair according to claim 3 are/is used;
and after the reaction is finished, detecting whether the DNA of the sample to be detected contains the WPRE element or not, and detecting the copy number of the WPRE element in the sample to be detected.
7. The fluorescent quantitative PCR assay of claim 6 wherein the first recombinant gene delivery vector further comprises a target gene and the target cells transfected with the first recombinant gene delivery vector are prepared by:
(1) packaging the first recombinant gene transfer vector and transfecting host cells to obtain recombinant viruses;
(2) transfecting a target cell with the recombinant virus to introduce the WPRE element and the target gene into the genome of the target cell; the target cells include one or more of tumor cells, stem cells, and immune cells.
8. The fluorescent quantitative PCR assay of claim 7 wherein in the first recombinant gene delivery vector, the WPRE element is located downstream of the target gene; the target gene includes a chimeric antigen receptor gene that targets tumor cells.
9. The fluorescent quantitative PCR detection method of claim 6, wherein detecting the copy number of the WPRE element in the test sample comprises plotting a standard curve, comprising the steps of:
providing a plurality of standards of different concentration gradients, said standards being a second recombinant gene delivery vector comprising said WPRE element;
performing a quantitative PCR amplification reaction on the WPRE element in each standard; and after the reaction is finished, drawing a standard curve according to the Ct value corresponding to the copy number of the WPRE element in the standard substance with different concentration gradients.
10. Use of a fluorescent probe according to any of claims 1-2, a primer pair according to claim 3 or a fluorescent quantitative PCR detection method according to any of claims 6-9 for the detection of lentiviral copy number in CAR-T.
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