WO2020224394A1 - 检测慢病毒的荧光探针、引物对、荧光定量pcr试剂盒及检测方法 - Google Patents

检测慢病毒的荧光探针、引物对、荧光定量pcr试剂盒及检测方法 Download PDF

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WO2020224394A1
WO2020224394A1 PCT/CN2020/084752 CN2020084752W WO2020224394A1 WO 2020224394 A1 WO2020224394 A1 WO 2020224394A1 CN 2020084752 W CN2020084752 W CN 2020084752W WO 2020224394 A1 WO2020224394 A1 WO 2020224394A1
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quantitative pcr
fluorescent
detection method
cells
wpre element
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French (fr)
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唐超
张宏玲
李浩莎
任异菲
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深圳宾德生物技术有限公司
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

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  • 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.
  • Immune cell therapy is the only method in the current technology that can completely eliminate cancer cells, and is considered to be the most promising treatment method in the comprehensive tumor treatment model in the 21st century. Compared with traditional treatment methods, immune cell therapy technology has great advantages in the treatment of malignant tumors with strong specificity and almost no toxic side effects. It makes up for the disadvantages of traditional surgery, radiotherapy and chemotherapy and becomes the fourth type of tumor treatment. means. Among them, Chimeric Antigen Receptor-Modified T Cells (CAR-T) technology, as one of the latest immune cell therapy technologies, has received extensive attention and research.
  • CAR-T Chimeric Antigen Receptor-Modified T Cells
  • CAR-T preparations use lentiviral transfection methods for gene transduction to make them Express chimeric antigen receptor (CAR).
  • CAR-T chimeric antigen receptor
  • due to the very strict quality control of CAR-T there are still huge challenges in the industrial application of CAR-T.
  • how to quickly and quantitatively detect the lentiviral copy number in CAR-T gene-mediated by the lentiviral transfection method plays an important role in the quality control of CAR-T. For example, it is used to test the lentiviral vector transfected with CAR gene. Quality control or the rate of genetically modified CAR-T positive cells have guiding significance.
  • the present invention provides a fluorescent probe, primer pair, fluorescent quantitative PCR kit and detection method.
  • the fluorescent probe has the advantages of high specificity and high sensitivity.
  • the fluorescent probe can be used for rapid and quantitative analysis of the copy number of lentiviruses containing WPRE elements, especially those mediated by lentiviral genes containing WPRE elements. Lentivirus copy number in CAR-T.
  • the present invention provides a fluorescent probe comprising a single-stranded nucleotide fragment, the 5'end of the single-stranded nucleotide fragment is labeled with a fluorescent reporter group, and the nuclear The 3'end of the nucleotide fragment is labeled with a fluorescence quenching group, and the single-stranded nucleotide fragment includes the nucleotide sequence shown in SEQ ID NO:1.
  • the fluorescent probe is used to specifically detect WPRE (Woodchuck hepatitis virus post-transcriptional regulatory element) elements.
  • the WPRE element is a viral post-transcriptional regulatory element, which can enhance virus-mediated transgene expression levels.
  • the WPRE element includes a nucleotide sequence as shown in SEQ ID NO:4.
  • the fluorescent reporter group and the fluorescence quenching group labeled at both ends of the fluorescent probe are close to each other, the fluorescent signal emitted by the excited fluorescent reporter group will be absorbed by the fluorescence quenching group .
  • the fluorescent reporter group can specifically recognize the target gene fragment of the WPRE element.
  • the fluorescent reporter group After the fluorescent reporter group is separated from the fluorescence quenching group, the fluorescent reporter group The cluster emits a fluorescent signal, so that the purpose of detecting the copy number of the target gene fragment can be achieved by detecting the fluorescent signal.
  • every time the target gene fragment is copied there is a fluorescent probe corresponding to it, so the fluorescence signal accumulation is completely synchronized with the target gene fragment amplified product.
  • the fluorescent probe can be, but not limited to, used to specifically detect the copy number of WPRE elements in a variety of subjects, including plasmids, vectors, viruses, bacterial genomes, or cell genomes.
  • the fluorescent probe is used to detect WPRE elements in recombinant lentiviruses containing WPRE elements or CAR-T cells.
  • the fluorescent reporter group includes carboxyfluorescein (FAM), carboxytetramethylrhodamine (TAMRA), 2,7-dimethyl-4,5-dichloro-6-carboxyfluorescein (JOE ) And one or more of hexachloro-6-methylfluorescein (HEX);
  • the fluorescence quenching group includes carboxytetramethylrhodamine (TAMRA), 4-[(2-chloro-4- One or more of nitro-phenyl)-azo]-aniline (Eclipse) and black hole quencher (BHQ).
  • the carboxyfluorescein (FAM) includes 5-carboxyfluorescein (5-FAM) or 6-carboxyfluorescein (6-FAM).
  • the black hole quencher (BHQ) includes black hole quencher 1 (BHQ-1), black hole quencher 2 (BHQ-2) or black hole quencher (BHQ-3).
  • the fluorescent reporter group may also include, but is not limited to, tetrachloro-6-carboxyfluorescein (TET), cyanine dyes (Cyanines dyes), carboxy-X-rhodamine (carboxy-X-rhodamine, One or more of ROX, Texas Red, Fluorescein Isothiocyanate (FITC), and Acridine Orange (Acridine Orange).
  • TET tetrachloro-6-carboxyfluorescein
  • Cyanines dyes Cyanines dyes
  • carboxy-X-rhodamine carboxy-X-rhodamine
  • FITC Fluorescein Isothiocyanate
  • Acridine Orange Acridine Orange
  • the anthocyanin dye may but does not include a variety of fluorescent dyes with different emission wavelengths, such as Cy3, Cy5, or Cy5.5.
  • the carboxy-X-rhodamine includes 5-carboxy-X-rhodamine (5-ROX
  • the fluorescence quenching group may also include, but is not limited to, 4-(4'-oxalkylaminophenylazo)benzoic acid (DABCYL) or 4-(N,N-dimethylamino)azo Benzene-4'-sulfonic acid chloride (DABSYL).
  • DBCYL 4-(4'-oxalkylaminophenylazo)benzoic acid
  • DABSYL 4-(N,N-dimethylamino)azo Benzene-4'-sulfonic acid chloride
  • the fluorescent probe of the present invention can increase the detection sensitivity to a certain extent by selecting matching fluorescent reporter groups and fluorescence quenching groups, or fluorescent reporter groups with low background fluorescence.
  • the present invention provides a primer pair, the primer pair includes a forward primer and a reverse primer, wherein the forward primer includes the nucleotide sequence shown in SEQ ID NO: 2, the The reverse primer includes the nucleotide sequence shown in SEQ ID NO: 3.
  • the single-stranded nucleotide fragments include one or more of polydeoxyribonucleotides, polyribonucleotides and other types of polynucleotides; for example, in other types of polynucleotides
  • the purine or pyrimidine base of the modified pyrimidine or purine base is a conventional source in the field.
  • the source of the single-stranded nucleotide fragment may include artificial synthesis or extraction from a naturally-occurring genome to obtain the single-stranded core.
  • the artificial synthesis method of the single-stranded nucleotide fragment includes one or more of the phosphotriester method, the phosphodiester method, the diethyl phosphate amide method and the solid phase carrier method.
  • the primer pair has high reaction efficiency and can quickly and specifically detect the WPRE element; during the PCR amplification reaction, the WPRE element can be continuously amplified, and the molecular weight of the amplified WPRE element is 127 bp .
  • 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.
  • the fluorescent quantitative PCR kit may include, but is not limited to, the fluorescent probe as described in the first aspect of the present invention.
  • the fluorescent quantitative PCR kit may but is not limited to include the primer pair as described in the second aspect of the present invention.
  • the fluorescent quantitative PCR kit may include, but is not limited to, the fluorescent probe described in the first aspect of the present invention and the primer pair described in the second aspect of the present invention.
  • the fluorescent quantitative PCR kit when the fluorescent quantitative PCR kit includes the fluorescent probe as described in the first aspect of the present invention, the fluorescent quantitative PCR kit further includes other PCR primer pairs for amplification of WPRE elements.
  • the fluorescent quantitative PCR kit when the fluorescent quantitative PCR kit includes the primer pair as described in the second aspect of the present invention, the fluorescent quantitative PCR kit may further include other fluorescent probes.
  • the fluorescent quantitative PCR kit further includes a reaction buffer, a positive control substance and a negative control substance.
  • the reaction buffer can also include but is not limited to PCR buffer.
  • the reaction buffer may, but is not limited to, contain one or more of salt ions, enzymes or a stable pH buffer system necessary for the PCR amplification reaction.
  • the reaction buffer can be, but is not limited to, an existing reaction buffer.
  • the reaction buffer contains dNTP, magnesium chloride, DNA polymerase and the like.
  • the dNTP may be a conventional dNTP in the art, including dATP, dGTP, dTTP, and dCTP.
  • the DNA polymerase includes a thermostable DNA polymerase.
  • the positive control substance contains the WPRE element.
  • the negative control substance may include, but is not limited to, DEPC water.
  • DEPC water is MiliQ pure water treated with DEPC (diethyl pyrocarbonate) and sterilized by high temperature and high pressure, a colorless liquid; it does not contain impurity RNA, DNA and protein.
  • the present invention also provides a fluorescent quantitative PCR detection method, including:
  • Extract the DNA of the sample to be tested, the sample to be tested includes target cells transfected with the first recombinant gene delivery vector containing the WPRE element, and perform a quantitative PCR amplification reaction on the DNA of the sample to be tested; the quantitative PCR amplification In the amplification reaction, 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 is used;
  • the reaction by recording the Ct value of the test sample, detect whether the DNA of the test sample contains the WPRE element, and detect the copy number of the WPRE element in the test sample .
  • the Ct value refers to the number of cycles (Cycle Threshold) corresponding to when the fluorescence signal of the amplified product reaches the set fluorescence threshold during the fluorescence quantitative PCR amplification process.
  • the first recombinant gene delivery vector further contains a target gene
  • the preparation process of the target cell transfected by the first recombinant gene delivery vector includes:
  • the target cell includes one or more of tumor cells, stem cells and immune cells kind.
  • the immune cells include T lymphocytes or NK cells.
  • the immune cells include CD3-positive T lymphocytes.
  • 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.
  • the first recombinant gene delivery vector is a lentiviral vector.
  • the first recombinant gene delivery vector is obtained by genetic modification on the basis of the gene delivery vector.
  • the gene delivery vector can be, but not limited to, pWPXLD plasmid vector, pLEX-MCS vector, pSico vector and pCgpV vector.
  • the first recombinant gene delivery vector is co-transfected with the envelope plasmid and the packaging plasmid into the host cell to obtain a recombinant virus.
  • the first recombinant gene delivery vector may be a pWPXLD recombinant plasmid.
  • the pWPXLD plasmid vector when used, when the pWPXLD plasmid vector does not contain the WPRE element, the WPRE element and the target gene fragment are inserted into the pWPXLD plasmid vector by the genetic engineering method to obtain the pWPXLD recombinant plasmid.
  • the envelope plasmid may be PMD2.G, and the packaging plasmid may be psPAX2.
  • the envelope plasmid PMD2.G encodes the vesicular stomatitis virus glycoprotein capsid, which assists the recombinant lentivirus to adhere to the cell membrane and maintains the infectivity of the recombinant lentivirus.
  • the pWPXLD recombinant plasmid of the present invention is an artificially modified lentiviral vector, which can be packaged and transfected into host cells to obtain a recombinant lentivirus with only one infection ability and high infection efficiency.
  • the recombinant lentivirus is a virus without replication ability. (RCL) is very safe and can be widely used for gene mediation of target cells. Compared with other gene manipulation technologies such as transfection, electroporation, retrovirus, adenovirus, etc., the use of pWPXLD recombinant plasmid in the present invention has more prominent advantages.
  • the pWPXLD recombinant plasmid is packaged and transfected into host cells to obtain a recombinant lentivirus that can efficiently integrate WPRE elements and target gene fragments into the genome of the target cell.
  • the packaging process of the recombinant lentivirus of the present invention usually adopts transient transfection or cell line packaging.
  • Human cell lines that can be used as packaging cells during transient transfection such as 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, etc.
  • Cell lines derived from monkeys such as COS1 cells, COS7 cells, CV-1 cells, and BMT10 cells, can also be used.
  • the commonly used calcium phosphate and PEI transfection reagents, as well as some transfection reagents such as Lipofectamine2000, FuGENE and S93fectin, are also frequently used.
  • the host cells may include HEK293T cells, 293 cells, 293T cells, 293FT cells, SW480 cells, u87MG cells, HOS cells, COS1 cells or COS7 cells.
  • the host cell is a HEK293T cell.
  • the WPRE element is located downstream of the target gene; the target gene includes a chimeric antigen receptor gene targeting tumor cells.
  • the WPRE element is a non-coding gene and does not participate in transcription and translation, but the WPRE element can replicate with the genome in the target cell.
  • the copy number of the WPRE element can be regarded as the copy number of the lentivirus, and the copy number of the WPRE element can be used to reflect Whether the transfection of lentivirus is successful or not.
  • the copy number of the target gene will correspond to the copy number of the WPRE element; the copy number of the WPRE element can be detected, and the copy number of the target gene can be counted. .
  • the target gene may also be, but is not limited to, other functional genes.
  • the other functional genes may be gene fragments used to achieve gene silencing, gene knockout, or gene interference.
  • the other functional gene may be a gene expressing a cell surface receptor protein.
  • the sample to be tested is a CAR-T cell that can target the target antigen.
  • the encoding gene of the CAR gene may include, but is not limited to, the encoding gene of the signal peptide, the encoding gene of the single-chain antibody, the encoding gene of the extracellular hinge region, the transmembrane encoding gene, the encoding gene of the single-chain antibody, the encoding gene of the extracellular hinge region, the encoding gene of the signal peptide sequentially connected from the 5'end to the 3'end, but not limited to Region coding gene, intracellular signal region coding gene.
  • the CAR gene can be a chimeric antigen receptor gene targeting CD19, CD22, CD33, or BCMA to obtain CAR-T targeting CD19, CD22, CD33 or BCMA.
  • the target gene when the target cell is a tumor cell, the target gene may be a gene fragment used for gene silencing. Or when the target cell is a stem cell, the target gene can be a gene fragment used for gene knockout, and the target cell can be used to study stem cell differentiation and other issues.
  • the CD3 positive T lymphocytes are isolated from human peripheral blood mononuclear cells.
  • the human peripheral blood mononuclear cells are derived from autologous venous blood, autologous bone marrow, umbilical cord blood, placental blood and the like. Further optionally, it is derived from fresh peripheral blood or bone marrow collected from a cancer patient one month after surgery and one month after radiotherapy and chemotherapy.
  • CD3 positive T lymphocytes are obtained.
  • CD3/CD28 immunomagnetic beads are added to the peripheral blood mononuclear cells in a certain proportion, and after incubation for a period of time, they are placed in a magnet for screening to obtain immunomagnetic bead coating After removing the magnetic beads, CD3 positive T lymphocytes can be obtained.
  • detecting the copy number of the WPRE element in the sample to be tested includes the following steps:
  • the standard products being a second recombinant gene delivery vector containing the WPRE element
  • a quantitative PCR amplification reaction is performed on the WPRE element in each of the standard products; after the reaction is completed, a standard curve is drawn according to the Ct value corresponding to the copy number of the WPRE element in the standard products with different concentration gradients.
  • the reaction conditions of the quantitative PCR amplification reaction are the same as those when the test sample is detected.
  • 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.
  • the second recombinant gene delivery vector is a lentiviral vector.
  • the second recombinant gene delivery vector is obtained by genetic modification on the basis of the gene delivery vector.
  • the gene delivery vector can be, but not limited to, pWPXLD plasmid vector, pLEX-MCS vector, pSico vector and pCgpV vector.
  • the second recombinant gene delivery vector contains a WPRE element and does not contain a target gene.
  • the positive control substance may be obtained from the standard substance through different dilution multiples.
  • the standard curve can be, but not limited to, the measured Ct value of the standard product as the abscissa, and the log value of the WPRE element copy number of the standard product of each concentration as the ordinate.
  • the Ct value of the sample to be tested can be obtained according to the standard curve to obtain its original copy number.
  • the detection method of the present invention can greatly improve the authenticity and accuracy of the quantitative analysis data of the copy number of the lentivirus in the sample to be tested, and improve the quantitative analysis efficiency of the fluorescent quantitative PCR detection method.
  • the procedure of the quantitative PCR amplification reaction includes: incubating at 45-55°C for 2-5 min; pre-denaturing at 95-98°C for 1-2 min; denaturing at 94-98°C for 10-30 seconds, and annealing extension at 55-65°C 1-3 minutes, a total of 35-45 cycles.
  • the procedure of the quantitative PCR amplification reaction includes: incubating at 45-55°C for 2 min; pre-denaturing at 95-98°C for 2 min; denaturing at 94-98°C for 15 seconds, and annealing and extension at 55-65°C for 1 minute, A total of 35-45 cycles.
  • the fluorescent quantitative PCR detection method provided by the present invention has many advantages such as high sensitivity, strong specificity, good repeatability, accurate quantification, fast speed, etc., and can be used for quantitative analysis of lentiviral expression vector, quantitative analysis of lentiviral copy number in CAR-T, And an important tool to determine the relationship between the number of CAR-T cells and the amount of virus required.
  • the detection limit of the fluorescence quantitative PCR detection method of the present invention can reach 1.7 copies/ ⁇ L of DNA.
  • the detection lower limit is far lower than the traditional fluorescent quantitative PCR detection method, therefore, the fluorescent quantitative PCR detection method of the present invention has higher sensitivity and stronger specificity.
  • the present invention provides a fluorescent probe as described in the first aspect, a primer pair as described in the second aspect, or a fluorescent quantitative PCR detection method as described in the fourth aspect in CAR-T lentivirus Application of copy number detection. Because CAR-T has important prospects in the preparation of drugs for the prevention, diagnosis and treatment of malignant tumors; this application has great potential for quantitative detection of the copy number of lentivirus in CAR-T during the industrialization and clinical application of CAR-T. Significantly, it is helpful to study the relationship between the positive rate of CAR-T cells and the titer of lentivirus transfection.
  • the fluorescent probe of the present invention has high specificity, and when the fluorescent reporter group and the quenching group at both ends are very close, the fluorescence quenching efficiency is high; the WPRE element can be detected with high specificity.
  • the primer pair of the present invention can specifically recognize the nucleotide sequence of the WPRE element, and rapidly amplify the WPRE element in the PCR amplification reaction.
  • the fluorescent quantitative PCR kit provided by the present invention can quantitatively detect the copy number of lentivirus containing WPRE element or the copy number of lentiviral WPRE element in CAR-T gene-mediated by the lentiviral transfection method, and has High sensitivity, strong specificity, good repeatability, accurate quantification, fast speed and many other advantages.
  • the fluorescent quantitative PCR detection method provided by the present invention is simple and practical, and can quantitatively detect the copy number of the lentiviral WPRE element in CAR-T gene-mediated by the lentiviral transfection method to determine the lentivirus titer, and An important tool for the relationship between the number of CAR-T cells and the amount of virus required; it provides an important detection method for the stability of the CAR-T preparation and production process and the release standards after mass production.
  • Figure 1 is a plasmid map of a lentiviral recombinant plasmid provided by an embodiment of the present invention.
  • Figure 2 is a standard curve of a fluorescent quantitative PCR detection method provided by an embodiment of the present invention.
  • Fig. 3 is a fluorescence quantitative PCR amplification diagram of a standard curve provided by an embodiment of the present invention.
  • Figure 4 is a flow cytometric detection diagram of CAR-T provided by an embodiment of the present invention.
  • the chemical reagents used in the following examples are all commercially available reagents.
  • the PCR tube used in the fluorescent quantitative PCR detection method is Real-time PCR tube, purchased from BIOplastics company, item number: B60109; Real-time PCR tube cover, purchased from BIOplastics company, item number: B57801; the Probe qPCR Mix, purchased from TOYOBO company, article number: QPS-101.
  • a method for preparing chimeric antigen receptor T cells includes the following steps:
  • the chimeric antigen receptor (CAR) gene fragment was inserted between the BamH1 and EcoR1 restriction sites downstream of the eukaryotic promoter in the pWPXLD plasmid vector, and the WPRE element was located downstream of the CAR gene fragment. Then it was transferred into E. coli competent cells DH5 ⁇ , and the positive clones were identified by PCR and sequencing. After PCR product gel electrophoresis detection and sequencing to identify the size and sequence of the target fragment, the pWPXLD-CAR recombinant plasmid containing the WPRE element and the CAR target gene as shown in Figure 1 was successfully constructed.
  • CAR chimeric antigen receptor
  • the pWPXLD-CAR recombinant plasmid, packaging plasmid psPAX2 and envelope plasmid pMD2G were co-transfected into cultured HEK293T cells.
  • the virus-containing supernatant was harvested at 48h, filtered through a 0.45 ⁇ m filter, and stored in an ultra-low temperature refrigerator at -80°C; at 72h, the virus-containing supernatant was harvested for the second time, filtered with a 0.45 ⁇ m filter, and combined with the virus supernatant harvested at 48h Add them to the ultracentrifuge tube together, put them into the Beckman ultracentrifuge one by one, set the centrifugation parameters to 25000rpm, the centrifugation time to 2h, and the centrifugal temperature to be controlled at 4°C; after centrifugation, discard the supernatant and try to remove the residue on the tube wall Add the virus preservation solution, gently pipetting and resuspend; after fully dissolving,
  • PBMC peripheral blood mononuclear cells
  • PBMC comes from autologous venous blood, autologous bone marrow, umbilical cord blood and placental blood. It is best from fresh peripheral blood or bone marrow collected from cancer patients one month after surgery and one month after radiotherapy and chemotherapy.
  • the patient's blood is drawn and sent to the blood separation chamber; the peripheral blood mononuclear cells are collected, Ficoll centrifugal separation, and the middle layer cells are collected; after washing with PBS, PBMCs are obtained.
  • PBMC blood pressure
  • serum-free basal medium to prepare a cell suspension
  • CD3/CD28 immunomagnetic beads according to the ratio of magnetic beads to cells of 3:1, and incubate for 1-2h at room temperature; incubate with a magnet pair
  • the cells of the magnetic beads are screened; after washing with PBS and removing the immunomagnetic beads, CD3 positive T lymphocytes are obtained.
  • the CD3 positive T lymphocytes obtained by the immunomagnetic bead separation method are taken, and the recombinant lentivirus with the virus titer corresponding to the number of CD3 positive cells is added for culture.
  • CAR-T chimeric antigen receptor T cells
  • the CAR-T will be obtained, and the CAR expression rate will be detected by flow cytometry.
  • DNA will be extracted, and the copy number of the lentiviral WPRE element gene will be detected using fluorescent probes and fluorescent quantitative PCR detection methods.
  • primer pairs used include forward primer and reverse primer:
  • the primer pair can specifically recognize the nucleotide sequence of the WPRE element, and the size of the amplified target fragment is 127 bp.
  • the fluorescent probe is: 5’-FAM-CCACCTGGATTCTGCGCGGGA-BHQ-1-3’
  • the reaction process includes: 500nmol of forward primer and directional primer, 250nmol of fluorescent probe, 10 ⁇ L of reaction buffer Probe qPCR Mix, 2 ⁇ L of extracted DNA template, add ultrapure water to adjust the reaction system to 20 ⁇ L; reaction conditions for fluorescence quantitative PCR Incubate at 50°C for 2min; 95°C for pre-denaturation for 2min; 95°C for 15s and 60°C for annealing for 1min, totaling 40 cycles.
  • the detection limit of the above-mentioned fluorescence quantitative PCR detection method is 1.7 copies/ ⁇ L DNA.
  • Plasmid concentration (copies/ ⁇ L) Corresponding logarithmic value 1775000000 9.249198357 177500000 8.249198357 17750000 7.249198357 1775000 6.249198357 177500 5.249198357 17,750 4.249198357 1775 3.249198357 177.5 2.249198357 17.75 1.249198357 1.775 0.249198357
  • the reaction system (20 ⁇ L) configured with fluorescence quantitative PCR is: forward primer and reverse primer 500nmol each, fluorescent probe 250nmol, reaction buffer Probe qPCR Mix 10 ⁇ L, each concentration gradient standard DNA 2 ⁇ L, constant volume with DEPC water;
  • the procedure of the fluorescence quantitative PCR amplification reaction is: incubating at 50°C for 2 minutes; 95°C for pre-denaturation for 2 minutes; 95°C for denaturation for 15 seconds, 60°C for annealing for 1 minute, a total of 40 cycles; each sample carries out 3 repeat holes.
  • the recombinant lentivirus prepared by the preparation method described in implementation 1 was configured with different gradient infection groups, and the recombinant lentivirus infection groups of different volumes were used.
  • the volumes of the recombinant lentivirus were: 0 ⁇ L, 5 ⁇ L, 10 ⁇ L and 20 ⁇ L.
  • the number of CD3 positive cells was measured.
  • CAR-T is obtained by transfection. After culturing, a part of CAR-T cells is taken for flow cytometry; another part of CAR-T cells is extracted for total cell DNA and then subjected to fluorescence quantitative PCR detection.
  • the experimental group in the process of the fluorescence quantitative PCR detection method is specifically: 1. DEPC water is a negative control; 2. pWPXLD vector plasmid is a positive control (20.35ng/ ⁇ L standard pWPXLD vector plasmid is diluted 1000 times to obtain the concentration The positive control substance is 2.035pg/ ⁇ L); 3.
  • the test sample 1 is the CAR-T cell transfected with 0 ⁇ L recombinant lentivirus; 4.
  • the test sample 2 is the CAR-T cell transfected with 5 ⁇ L recombinant lentivirus. CAR-T cells; 5.
  • Test sample 2 is CAR-T cell transfected with 10 ⁇ L recombinant lentivirus; 6.
  • Test sample 4 is CAR-T cell transfected with 20 ⁇ L recombinant lentivirus.
  • the copy number of the lentiviral WPRE gene of each experimental group that is, the copy number of the lentivirus, can be quickly and quantitatively measured, as shown in Table 3.
  • the comparison of the lentiviral copy number of each sample to be tested by the fluorescence quantitative PCR detection method of the present invention and the positive rate of CAR-T in flow detection is shown in Table 4. It can be seen from Table 4 that the copy number of the lentivirus of each sample to be tested measured by the fluorescence quantitative PCR detection method of the present invention gradually increases; and when 0 ⁇ L, 5 ⁇ L, 10 ⁇ L and 20 ⁇ L are used for transfection, the percentage of CAR positive rate is also A linear relationship with a rising gradient.
  • the multiple relationship between the copy numbers of the lentivirus of each test sample measured by the fluorescence quantitative PCR detection method of the present invention corresponds to the multiple relationship of the CAR positive rate of each test sample measured by flow cytometry. Therefore, the number of virus copies measured by the fluorescent quantitative PCR detection method of this application is positively correlated with the positive rate of CAR-T flow cytometry.
  • the fluorescent quantitative PCR detection method of the present invention can specifically detect the WPRE element in the sample to be tested, and can efficiently and accurately detect the corresponding copy number regardless of the change of the detection target.
  • the fluorescent quantitative PCR detection method of the present invention can be used to quickly and quantitatively analyze the copy number of the lentiviral WPRE gene in CAR-T cells, the process is easy to operate, the test result has high accuracy, low detection line, and high repeatability;
  • the detection method can be used to determine the relationship between the positive rate of CAR-T cells and the amount of lentivirus required.
  • the detection method of the present invention can also more accurately determine the virus titer and virus infection efficiency.

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Abstract

本发明提供一种检测慢病毒的荧光探针，其针对WPRE元件，所述探针包括如SEQ ID NO：1所示的核苷酸序列，5'端标记有荧光报告基团，3'端标记有荧光淬灭基团。本发明还提供检测慢病毒的引物对、荧光定量PCR试剂盒及检测方法。

Description

[根据细则37.2由ISA制定的发明名称]　检测慢病毒的荧光探针、引物对、荧光定量PCR试剂盒及检测方法
本申请要求了2019年05月08日提交中国专利局的，申请号201910380678.X，发明名称为“一种荧光探针、引物对、荧光定量PCR试剂盒及检测方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及医学生物领域，特别涉及一种荧光探针、引物对、荧光定量PCR试剂盒及检测方法。
背景技术
免疫细胞治疗是现有科技中唯一有可能彻底清除癌细胞的方法，被认为是二十一世纪肿瘤综合治疗模式中最有发展前途的一种治疗手段。与传统治疗手段相比，免疫细胞治疗技术在治疗恶性肿瘤方面，具有特异性强、几乎无毒副作用的巨大优势，它弥补了传统的手术、放疗和化疗的弊端，成为肿瘤治疗的第四种手段。其中，嵌合抗原受体T细胞(Chimeric Antigen Receptor–Modified T Cells，CAR-T)技术作为当前最新的免疫细胞治疗技术之一，受到广泛的关注和研究。
目前，凭借慢病毒载体(lentiviral vector)的整合位点更安全，转基因负荷量更大和基因毒性更小等优势，大部分CAR-T的制备均采用慢病毒转染方法进行基因转导以使其表达嵌合抗原受体(CAR)。然而，由于CAR-T的质量控制非常严格，现在CAR-T产业化应用过程中仍然面临巨大挑战。其中，如何快速定量检测经慢病毒转染方法基因介导的CAR-T中的慢病毒拷贝数对于CAR-T的质量控制就具有重要作用，例如其在检验转染CAR基因的慢病毒载体的质量控制或基因修饰的CAR-T阳性细胞率等方面均具有指导意义。但是，传统的检 测方法主要通过在蛋白水平上，通过流式细胞术检测目的基因在T细胞上表达的蛋白量来确定CAR-T转染后的阳性率；或者通过检测CAR基因来定性检测等。这些检测方法普遍存在检特异性低、灵敏度低、复杂费时和重复性差等缺点，且对操作环境要求严格。
发明内容
有鉴于此，本发明提供了一种荧光探针、引物对、荧光定量PCR试剂盒及检测方法。其中，所述荧光探针具有特异性高和灵敏度高的优点，该荧光探针可以用于快速、定量分析含有WPRE元件的慢病毒拷贝数，特别是经含有WPRE元件的慢病毒基因介导的CAR-T中的慢病毒拷贝数。
第一方面，本发明提供了一种荧光探针，所述荧光探针包括一段单链核苷酸片段，所述单链核苷酸片段的5’端标记有荧光报告基团，所述核苷酸片段的3’端标记有荧光淬灭基团，所述单链核苷酸片段包括如SEQ ID NO：1所示的核苷酸序列。
本发明中，所述荧光探针用于特异性检测WPRE(Woodchuck hepatitis virus post-transcriptional regulatory element)元件。所述WPRE元件为一病毒的转录后调控元件，可以增强病毒介导的转基因表达水平。可选地，所述WPRE元件包括如SEQ ID NO：4所示的核苷酸序列。
本发明中，所述荧光探针两端分别标记的荧光报告基团和荧光淬灭基团在相互靠近时，被激发后的荧光报告基团所发射的荧光信号会被荧光淬灭基团吸收。但当WPRE元件的目的基因片段扩增时，所述荧光报告基团可以与WPRE元件的目的基因片段特异性识别，所述荧光报告基团与所述荧光淬灭基团分离后，荧光报告基团发射荧光信号，从而通过检测所述荧光信号可以达到检测目的基因片段的拷贝数的目的。其中，目的基因片段每复制一次，就有一个荧光 探针与之对应，所以荧光信号累积与目的基因片段扩增产物完全同步。
可选地，所述荧光探针可以但不限于用于特异性检测多种对象中的WPRE元件拷贝数，所述对象包括质粒、载体、病毒、细菌的基因组或细胞的基因组。例如，所述荧光探针用于检测含有WPRE元件的重组慢病毒或CAR-T细胞中的WPRE元件。
可选地，所述荧光报告基团包括羧基荧光素(FAM)、羧基四甲基罗丹明(TAMRA)、2,7-二甲基-4,5-二氯-6-羧基荧光素(JOE)和六氯-6-甲基荧光素(HEX)中的一种或多种；所述荧光淬灭基团包括羧基四甲基罗丹明(TAMRA)、4-[(2-氯-4-硝基-苯基)-偶氮基]-苯胺(Eclipse)和黑洞淬灭剂(black hole quencher，BHQ)中的一种或多种。其中，所述羧基荧光素(FAM)包括5-羧基荧光素(5-FAM)或6-羧基荧光素(6-FAM)。所述黑洞淬灭剂(BHQ)包括黑洞淬灭剂1(BHQ-1)、黑洞淬灭剂2(BHQ-2)或黑洞淬灭剂(BHQ-3)。
可选地，所述荧光报告基团还可以但不限于包括四氯-6-羧基荧光素(TET)、花青素染料(Cyanines dyes)、羧基-X-罗丹明(carboxy-X-rhodamine，ROX)、德克萨斯红染料(Texas Red)、异硫氰酸荧光素(FITC)和吖啶橙(Acridine orange)中的一种或多种。可选地，所述花青素染料可以但不包括Cy3、Cy5或Cy5.5等多种不同发射波长的荧光染料。所述羧基-X-罗丹明包括5-羧基-X-罗丹明(5-ROX)或6-羧基-X-罗丹明(6-ROX)。
可选地，所述荧光淬灭基团还可以但不限于包括4-(4’-恶烷氨基苯偶氮)苯甲酸(DABCYL)或4-(N,N-二甲基氨基)偶氮苯-4’-磺酸氯(DABSYL)。
本发明所述荧光探针通过选择匹配的荧光报告基团和荧光淬灭基团，或是背景荧光低的荧光报告基团可以一定程度的增加检测的灵敏度。
第二方面，本发明提供了一种引物对，所述引物对包括正向引物和反向引 物，其中，所述正向引物包括如SEQ ID NO：2所示的核苷酸序列，所述反向引物包括如SEQ ID NO：3所示的核苷酸序列。
本发明中，所述单链核苷酸片段包括多聚脱氧核糖核苷酸、多聚核糖核苷酸和其他类型的多核苷酸中的一种或几种；例如其他类型的多核苷酸中的嘌呤或嘧啶碱基经过修饰后的嘧啶或嘌呤碱基。本发明所述的单链核苷酸片段来源为本领域常规来源，例如，所述单链核苷酸片段的来源可以包括人工合成方法或从天然存在的基因组中提取获得所述的单链核苷酸片段。其中所述单链核苷酸片段的人工合成方法包括：磷酸三酯法、磷酸二酯法、二乙基磷酸酰胺法和固相载体法中的一种或几种。
本发明中，所述引物对的反应效率高，可以快速、特异性检测WPRE元件；在PCR扩增反应时，可以不断扩增所述WPRE元件，所述扩增的WPRE元件的分子量大小为127bp。
第三方面，本发明还提供了一种荧光定量PCR试剂盒，包括如本发明第一方面所述的荧光探针和/或如本发明第二方面所述的引物对。
具体地，所述荧光定量PCR试剂盒可以但不限于包括如本发明第一方面所述的荧光探针。或所述荧光定量PCR试剂盒可以但不限于包括如本发明第二方面所述的引物对。或所述荧光定量PCR试剂盒可以但不限于包括本发明第一方面所述的荧光探针和如本发明第二方面所述的引物对。
可选地，当所述荧光定量PCR试剂盒包括如本发明第一方面所述的荧光探针时，所述荧光定量PCR试剂盒还包括其他用于WPRE元件扩增的PCR引物对。
可选地，当所述荧光定量PCR试剂盒包括如本发明第二方面所述的引物对时，所述荧光定量PCR试剂盒还可以包括其他荧光探针。
可选地，所述荧光定量PCR试剂盒还包括反应缓冲液、阳性对照品和阴性对 照品。
可选地，所述反应缓冲液还可以但不限于包括PCR缓冲液。所述反应缓冲液中可以但不限于包含有PCR扩增反应必要的盐离子、酶或稳定的pH缓冲体系中的一种或多种。所述反应缓冲液可以但不限于为现有反应缓冲液。例如，所述反应缓冲液中含有dNTP、氯化镁和DNA聚合酶等。可选地，所述dNTP可以为本领域常规的dNTP，包括dATP、dGTP、dTTP和dCTP。所述DNA聚合酶包括热稳定DNA聚合酶。
可选地，所述阳性对照品中含有所述WPRE元件。
可选地，所述阴性对照品可以但不限于包括DEPC水。所述DEPC水是用DEPC(diethyl pyrocarbonate，焦碳酸二乙酯)处理过并经高温高压灭菌的MiliQ纯水，无色液体；不含杂质RNA、DNA和蛋白质。
第四方面，本发明还提供了一种荧光定量PCR检测方法，包括：
提取待测样本的DNA，所述待测样本包括经含有WPRE元件的第一重组基因传递载体转染的靶细胞，对所述待测样本的DNA进行定量PCR扩增反应；所述定量PCR扩增反应中，使用本发明第一方面所述的荧光探针和/或如本发明第二方面所述的引物对；
反应结束后，检测所述待测样本的DNA是否含有所述WPRE元件，以及检测所述待测样本中的所述WPRE元件的拷贝数。
可选地，反应结束后，通过记录所述待测样本的Ct值，检测所述待测样本的DNA是否含有所述WPRE元件，以及检测所述待测样本中的所述WPRE元件的拷贝数。
本发明中，所述Ct值是指在荧光定量PCR扩增过程中，扩增产物的荧光信号达到设定的荧光阈值时的所对应的扩增循环数(Cycle Threshold)。
可选地，所述第一重组基因传递载体还含有靶基因，经所述第一重组基因传递载体转染的所述靶细胞的制备过程包括：
(1)将所述第一重组基因传递载体进行包装并转染宿主细胞，得到重组病毒；
(2)将所述重组病毒转染靶细胞，以导入所述WPRE元件和所述靶基因至所述靶细胞的基因组；所述靶细胞包括肿瘤细胞、干细胞和免疫细胞中的一种或多种。
可选地，所述免疫细胞包括T淋巴细胞或NK细胞。优选地，所述免疫细胞包括CD3阳性T淋巴细胞。
可选地，所述第一重组基因传递载体为病毒载体，包括慢病毒载体、逆转录病毒载体和腺病毒载体中的至少一种。优选地，所述第一重组基因传递载体为慢病毒载体。所述第一重组基因传递载体是通过在基因传递载体基础上经基因改造后的得到。其中，所述基因传递载体可以但不限于为pWPXLD质粒载体、pLEX-MCS载体、pSico载体和pCgpV载体。
可选地，将所述第一重组基因传递载体与包膜质粒、包装质粒共转染宿主细胞，得到重组病毒。
在本发明一实施方式中，所述第一重组基因传递载体可以为pWPXLD重组质粒。采用pWPXLD质粒载体，当所述pWPXLD质粒载体不含有WPRE元件时，通过基因工程方法将WPRE元件和靶基因片段插入至所述pWPXLD质粒载体上得到上述pWPXLD重组质粒。此时，所述包膜质粒可以为PMD2.G，所述包装质粒可以为psPAX2。所述包膜质粒PMD2.G编码水疱性口炎病毒糖蛋白衣壳，所述水疱性口炎病毒糖蛋白衣壳协助重组慢病毒向细胞膜粘附，并保持重组慢病毒的感染性。
本发明所述pWPXLD重组质粒为经人工改造后的慢病毒载体，可经包装和转染宿主细胞后得到重组慢病毒仅具有一次感染能力且感染效率高，该重组慢病毒是无复制能力的病毒(RCL)，十分安全，可广泛用于靶细胞的基因介导。相比于转染、电转、逆转录病毒、腺病毒等其他基因操作技术，本发明采用pWPXLD重组质粒具有更突出的优势。由所述pWPXLD重组质粒经包装并转染宿主细胞，得到重组慢病毒可以高效地将WPRE元件和靶基因片段均整合至靶细胞的基因组上。
本发明所述重组慢病毒的包装过程通常采用瞬时转染或采用细胞系包装。瞬时转染时可以用作包装细胞使用的人类细胞株，例如包括293细胞、293T细胞、293FT细胞、293LTV细胞、293EBNA细胞及其他的从293细胞分离的克隆；SW480细胞、u87MG细胞、HOS细胞、C8166细胞、MT-4细胞、Molt-4细胞、HeLa细胞、HT1080细胞、TE671细胞等。也可以采用来源于猴子的细胞株，例如，COS1细胞、COS7细胞、CV-1细胞、BMT10细胞等。而且，通常采用的磷酸钙和PEI转染试剂，还有一些转染试剂如Lipofectamine2000、FuGENE和S93fectin也被经常使用。
可选地，所述宿主细胞可以包括HEK293T细胞、293细胞、293T细胞、293FT细胞、SW480细胞、u87MG细胞、HOS细胞、COS1细胞或COS7细胞。
进一步地，可选地，所述宿主细胞为HEK293T细胞。
可选地，所述第一重组基因传递载体中，所述WPRE元件位于所述靶基因的下游；所述靶基因包括靶向肿瘤细胞的嵌合抗原受体基因。
本发明中，所述WPRE元件为非编码基因，不参与转录和翻译，但所述WPRE元件可以与靶细胞内的基因组一起复制。例如，当含WPRE元件的重组慢病毒将所述WPRE元件导入至靶细胞的基因组时，所述WPRE元件的拷贝数就可以认为 是慢病毒拷贝数，所述WPRE元件的拷贝数可以用于反映慢病毒的转染成功与否。并且，如果靶基因与WPRE元件一并导入至靶细胞的基因组，那么靶基因的拷贝数会与WPRE元件的拷贝数相对应；可以通过检测WPRE元件的拷贝数，可以对靶基因拷贝数进行统计。
可选地，所述靶基因还可以但不限于为其他功能性基因，例如所述其他功能性基因可以为用于实现基因沉默、基因敲除或基因干扰的基因片段。或所述其他功能性基因可以为表达细胞表面受体蛋白的基因。
例如，当靶细胞为CD3阳性T淋巴细胞时，所述靶基因为CAR基因时，所述待测样本为可以靶向目的抗原的CAR-T细胞。进一步地，所述CAR基因的编码基因可以但不限于包括从5’端到3’端顺次连接的信号肽的编码基因、单链抗体的编码基因、胞外铰链区的编码基因、跨膜区的编码基因、胞内信号区的编码基因。例如所述CAR基因可为靶向CD19、CD22、CD33或BCMA等的嵌合抗原受体基因，得到靶向CD19、CD22、CD33或BCMA的CAR-T。
可选地，当所述靶细胞为肿瘤细胞时，所述靶基因可以为用于基因沉默的基因片段。或当所述靶细胞为干细胞时，所述靶基因可以为用于基因敲除的基因片段，所述靶细胞可以用于研究干细胞分化等问题。
可选地，所述CD3阳性T淋巴细胞是从人源外周血单个核细胞中分离获得。所述人源外周血单个核细胞来源于自体静脉血、自体骨髓、脐带血和胎盘血等。进一步可选地，来源于癌症患者手术一个月后、放化疗一个月后采集的新鲜外周血或骨髓。
具体地，所述CD3阳性T淋巴细胞的获得过程如下：向外周血单个核细胞中按一定比例加入CD3/CD28免疫磁珠，孵育一段时间后，放入磁铁进行筛选，得到免疫磁珠包被的CD3阳性T淋巴细胞，去除磁珠后，获得CD3阳性T淋巴细胞。
可选地，检测所述待测样本中的所述WPRE元件的拷贝数，包括绘制标准曲线，包括以下步骤：
提供多个不同浓度梯度的标准品，所述标准品为含有所述WPRE元件的第二重组基因传递载体；
对各个所述标准品中的所述WPRE元件进行定量PCR扩增反应；反应完成后，根据所述不同浓度梯度的标准品中的所述WPRE元件的拷贝数对应的Ct值绘制得到标准曲线。
可选地，绘制标准曲线时，所述定量PCR扩增反应的反应条件与检测所述待测样本时的一致。
可选地，所述第二重组基因传递载体为病毒载体，包括慢病毒载体、逆转录病毒载体和腺病毒载体中的至少一种。优选地，所述第二重组基因传递载体为慢病毒载体。所述第二重组基因传递载体是通过在基因传递载体基础上经基因改造后的得到。其中，所述基因传递载体可以但不限于为pWPXLD质粒载体、pLEX-MCS载体、pSico载体和pCgpV载体。进一步地，可选地，所述第二重组基因传递载体含有WPRE元件，不含有靶基因。
可选地，所述阳性对照品可以为所述标准品经不同稀释倍数得到的。
可选地，所述标准曲线可以但不限于以测得所述标准品的Ct值为横坐标，以所述各个浓度的标准品的有WPRE元件拷贝数的对数值为纵坐标。
本发明中，所述待测样本经检测后测得的Ct值可以根据标准曲线获得其原始拷贝数。本发明所述检测方法，可以大大提高待测样本中的慢病毒拷贝数的定量分析数据的真实性和准确性，提高所述荧光定量PCR检测方法的定量分析效率。
可选地，所述定量PCR扩增反应的程序包括：45-55℃培育2-5min；95-98℃ 预变性1-2min；94-98℃变性10-30秒，55-65℃退火延伸1-3分钟，共35-45个循环。
进一步地，可选地，所述定量PCR扩增反应的程序包括：45-55℃培育2min；95-98℃预变性2min；94-98℃变性15秒，55-65℃退火延伸1分钟，共35-45个循环。
本发明提供的荧光定量PCR检测方法以灵敏度高、特异性强、重复性好、定量准确、速度快等诸多优点，可以成为慢病毒表达载体定量分析、CAR-T中慢病毒拷贝数定量分析、以及确定CAR-T细胞数量与所需病毒量的关系的重要工具。
本发明所述荧光定量PCR检测方法的检测下限可达到1.7copies/μL的DNA。所述检测下限值远远低于传统的荧光定量PCR检测方法，因此，本发明所述荧光定量PCR检测方法具有更高的灵敏性，以及更强的特异性。
第五方面，本发明提供了一种如第一方面所述的荧光探针、如第二方面所述的引物对或如第四方面所述的荧光定量PCR检测方法在CAR-T的慢病毒拷贝数检测方面的应用。由于CAR-T在制备预防、诊断和治疗恶性肿瘤的药物中的具有重要前景；该应用在CAR-T在产业化和临床应用的过程中，对CAR-T中慢病毒拷贝数的定量检测具有重要意义，其有利于研究CAR-T细胞阳性率与慢病毒转染滴度之间的关系。
本发明的有益效果：
(1)本发明所述荧光探针特异性高，且两端的荧光报告基团和淬灭基团非常靠近时，荧光淬灭效率高；可高特异性检测WPRE元件。
(2)本发明所述引物对可以特异性识别WPRE元件的核苷酸序列，并在PCR扩增反应中快速扩增WPRE元件。
(3)本发明提供的荧光定量PCR试剂盒能够对含WPRE元件的慢病毒拷贝数或经慢病毒转染方法基因介导的CAR-T中的慢病毒WPRE元件的拷贝数进 行定量检测，具有灵敏度高、特异性强、重复性好、定量准确、速度快等诸多优点。
(4)本发明提供的荧光定量PCR检测方法简单实用，可以对经慢病毒转染方法基因介导的CAR-T中的慢病毒WPRE元件的拷贝数进行定量检测，确定慢病毒滴度，以及CAR-T细胞数量与所需病毒量的关系的重要工具；为CAR-T制备生产工艺的稳定性、批量生产后的放行标准提供了重要检测手段。
附图说明
图1为本发明一实施例提供的慢病毒重组质粒的质粒图谱。
图2为本发明一实施例提供的荧光定量PCR检测方法的标准曲线。
图3为本发明一实施例提供的标准曲线的荧光定量PCR扩增图。
图4为本发明一实施例提供的CAR-T的流式细胞术检测图。
具体实施方式
以下所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明的保护范围。
若无特别说明，下面实施例使用的化学试剂均为市售试剂。其中，荧光定量PCR检测方法中使用PCR管为Real-time PCR管，采购自BIOplastics公司，货号：B60109；Real-timePCR管盖，采购自BIOplastics公司，货号：B57801；所述Probe qPCR Mix，采购自TOYOBO公司，货号：QPS-101。
实施例一
一种嵌合抗原受体T细胞(CAR-T)的制备方法，包括以下步骤：
(1)构建pWPXLD-CAR重组质粒
将嵌合抗原受体(CAR)基因片段插入到pWPXLD质粒载体中真核启动子 下游的BamH1和EcoR1酶切位点之间，所述WPRE元件位于CAR基因片段的下游。然后转入大肠杆菌感受态细胞DH5α，进行阳性克隆PCR鉴定和测序鉴定。经过PCR产物凝胶电泳检测和测序鉴定符合目的片段大小和序列，成功构建如图1所示的含有WPRE元件和CAR靶基因的pWPXLD-CAR重组质粒。
(2)重组慢病毒构建
将pWPXLD-CAR重组质粒、包装质粒psPAX2、包膜质粒pMD2G三者共转染入培养好的HEK293T细胞。第48h收获含病毒的上清，经0.45μm滤膜过滤，-80℃超低温冰箱中保存；第72h二次收获含病毒的上清，0.45μm滤膜过滤，与第48h收获的病毒上清合并一起加入超速离心管中，逐一放入至Beckman超速离心机内，设置离心参数为25000rpm，离心时间为2h，离心温度控制在4℃；离心结束后，弃去上清，尽量去除残留在管壁上的液体，加入病毒保存液，轻轻反复吹打重悬；经充分溶解后，高速离心10000rpm，离心5min后，取上清荧光法测定滴度，病毒按照100μL，2×10 8个/mL分装，保存于-80℃超低温冰箱，得到重组慢病毒。
(3)嵌合抗原受体T细胞的制备
a)PBMC(外周血单个核细胞)的分离
PBMC来源于自体静脉血、自体骨髓、脐带血和胎盘血等。最好是来源于癌症患者手术一个月后、放化疗一个月后采集的新鲜外周血或骨髓。
抽取病人血液，送样至血液分离室；采集外周血单个核细胞，Ficoll离心分离后取中间层细胞；经PBS洗涤后，得到PBMC。
b)免疫磁珠法分离抗原特异性T淋巴细胞
取上述PBMC，加入不含血清的基础培养基，配成细胞悬液；按磁珠与细胞的比例为3:1，加入CD3/CD28免疫磁珠，室温孵1-2h；采用磁铁对孵育好磁珠的 细胞进行筛选；PBS洗涤，去除免疫磁珠后，得到CD3阳性T淋巴细胞。
c)慢病毒转染法制备抗原特异性T淋巴细胞
取上述经过免疫磁珠分离法得到的CD3阳性T淋巴细胞，加入与CD3阳性细胞数相应的病毒滴度的所述重组慢病毒进行培养。
培养的第3天，进行细胞计数和换液，调整细胞浓度为1×10 6个/mL，接种，培养；培养的第5天，观察细胞状态，如果细胞密度增大，则稀释细胞浓度为1×10 6个/mL，检测细胞活性，继续培养。扩增培养到第9-11天，收集细胞，得到嵌合抗原受体T细胞(CAR-T)。
(4)将得到CAR-T，进行流式细胞术检测其CAR表达率，同时提取DNA，使用荧光探针、荧光定量PCR检测方法检测慢病毒WPRE元件基因拷贝数。
其中，使用的引物对，包括正向引物和反向引物：
正向引物：5’-GTTGTCGGGGAAGCTGACGT-3’
反向引物：5’-CAGGCCGCGGGAAGGAAGGTC-3’
所述引物对可以特异性识别WPRE元件的核苷酸序列，扩增目的片段大小为127bp。
其中，荧光探针为：5’-FAM-CCACCTGGATTCTGCGCGGGA-BHQ-1-3’
反应过程包括：正向引物和方向引物各取500nmol，荧光探针取250nmol，10μL反应缓冲液Probe qPCR Mix，2μL提取的DNA模板，加超纯水调节至20μL反应体系；荧光定量PCR的反应条件为：50℃培育2min；95℃预变性2min；95℃变性15s，60℃退火1min，共40个循环。
上述荧光定量PCR检测方法的检测下限为可达到1.7copies/μL DNA。
为了评估本发明所描述的荧光定量PCR检测方法的检测效果，进行如下效果实施例。
效果实施例
1、评估制备成功的CAR-T中慢病毒WPRE元件基因拷贝数与CAR-T细胞的阳性率之间的关系
分别取2×10 6细胞数的经过实施例1所述的方法制备的CAR-T细胞与未经转染的T淋巴细胞(阴性对照)，进行DNA提取(QIAamp DNA Blood Mini Kit，QIAGEN公司，货号51106)，测量浓度后，进行荧光定量PCR检测。
(a)建立标准曲线：将含有WPRE元件的标准pWPXLD质粒载体作为标准品(采购自Addgene公司)，检测其浓度为20.35ng/uL，将此浓度换算成拷贝数/μL为：1775000000copies/μL，进行10个浓度梯度稀释，最低浓度梯度为1.775copies/μL；所述标准品的10个浓度梯度及其相对应的对数值如下表1所示：
表1：标准品的10个浓度梯度及其对应的对数值
质粒浓度(copies/μL) 相对应的对数值
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
配置荧光定量PCR的反应体系(20μL)为：正向引物、反向引物各500nmol，荧光探针250nmol，反应缓冲液Probe qPCR Mix 10μL，各个浓度梯度标准品DNA 2μL，用DEPC水进行定容；
所述荧光定量PCR扩增反应的程序为：50℃培育2min；95℃预变性2min；95℃变性15s，60℃退火1min，共40个循环；每个样品进行3个重复孔。
反应结束后，记录数据，参见表2，将测得的10个浓度梯度的浓度相应对 数值为纵坐标，以测得的Ct值为横坐标，绘制得到标准曲线y＝-0.3005x+12.277，(R 2＝0.998)，具体一并参见图2和图3。
表2：标准曲线的横、纵坐标相应数据表
Figure PCTCN2020084752-appb-000001
(b)待测样本的检测评估
将实施1所述制备方法制备的重组慢病毒，配置不同梯度感染组，采用不同体积的重组慢病毒感染组，重组慢病毒体积分别为：0μL、5μL、10μL和20μL，对CD3阳性细胞数进行转染得到CAR-T，经过培养后，取一部分CAR-T细胞进行流式细胞术检测；取另一部分CAR-T细胞先进行细胞总DNA提取，然后进行荧光定量PCR检测，其中，流式细胞术的检测结果如图4所示，其中“APC-A+”是指抗体APC(allophycocyanin，异丙氰苷)染色阳性率，可以反映出CAR-T的阳性率。其中，图4中流式检测CAR-T的阳性率，在使用0μL、5μL、10μL和20μL病毒转染时，对应的CAR阳性率百分比分别为0(阴性对照)、8.13％、26.4％和49.9％。
所述荧光定量PCR检测方法过程中的实验组具体为：1、DEPC水为阴性对照品；2、pWPXLD载体质粒为阳性对照品(将20.35ng/μL标准品pWPXLD载体质粒稀释1000倍后得到浓度为2.035pg/μL的所述阳性对照品)；3、待测样本1为加入0μL重组慢病毒转染后的CAR-T细胞；4、待测样本2为加入5μL重 组慢病毒转染后的CAR-T细胞；5、待测样本2为加入10μL重组慢病毒转染后的CAR-T细胞；6、待测样本4为加入20μL重组慢病毒转染后的CAR-T细胞。
根据制备的标准曲线：y＝-0.3005x+12.277，可以得快速、定量测得各个实验组的慢病毒WPRE基因拷贝数，即慢病毒拷贝数，参见表3所示。
表3：荧光定量PCR检测数据表
Figure PCTCN2020084752-appb-000002
将由本发明所述荧光定量PCR检测方法测得的各个待测样本的慢病毒拷贝数与流式检测CAR-T的阳性率的数据对比，参见表4。表4中可以看出，本发明荧光定量PCR检测方法测得的各个待测样本的慢病毒拷贝数是逐渐增加的；而采用0μL、5μL、10μL和20μL病毒转染时，CAR阳性率百分比也是一个梯度上升的线性关系。本发明荧光定量PCR检测方法测得的各个待测样本的慢病毒拷贝数之间的倍数关系与流式细胞术测得的各个待测样本的CAR阳性率的倍数关系对应。因此，本申请荧光定量PCR检测方法测得的病毒拷贝数与CAR-T流式阳性率正相关。
表4：荧光定量PCR检测数据与流式细胞检测数据对比
Figure PCTCN2020084752-appb-000003
但是由于现有方法中，采用流式细胞术检测CAR阳性率的检测过程中存在 很大误差，需要引入特异性非常出色的CAR抗体采可以尽量降低误差；并且当对于不同的CAR-T细胞阳性率的检测时，每一种靶点，均需找一种相应的抗体，造成检测成本巨大。此外，流式细胞术对于检测对象的要求也非常高，例如只能检测活细胞等。相比于传统方法，本发明所述荧光定量PCR检测方法可以特异性检测的待测样本中的WPRE元件，无论检测靶点如何变化，都可以高效、精准地检测其对应的拷贝数。本发明所述荧光定量PCR检测方法一方面可以用于快速定量分析CAR-T细胞中慢病毒WPRE基因拷贝数，过程易操作，测试结果准确度高、检测线低、可重复性高；另一方面，所述检测方法可以用于确定CAR-T细胞阳性率与所需慢病毒量之间的关系。本发明所述检测方法也可以更加准确地测定病毒滴度和病毒感染效率。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (13)

  1. 一种荧光探针，其特征在于，所述荧光探针包括一段单链核苷酸片段，所述单链核苷酸片段的5’端标记有荧光报告基团，所述核苷酸片段的3’端标记有荧光淬灭基团，所述单链核苷酸片段包括如SEQ ID NO：1所示的核苷酸序列。
  2. 如权利要求1所述的荧光探针，其特征在于，所述荧光报告基团包括羧基四甲基罗丹明、羧基荧光素、2,7-二甲基-4,5-二氯-6-羧基荧光素和六氯-6-甲基荧光素中的一种或多种；所述荧光淬灭基团包括4-[(2-氯-4-硝基-苯基)-偶氮基]-苯胺和黑洞淬灭剂中的一种或多种。
  3. 如权利要求1所述的荧光探针，其特征在于，所述荧光报告基团为羧基四甲基罗丹明，所述荧光淬灭基团为黑洞淬灭剂。
  4. 一种引物对，其特征在于，所述引物对包括正向引物和反向引物，其中，所述正向引物包括如SEQ ID NO：2所示的核苷酸序列，所述反向引物包括如SEQ ID NO：3所示的核苷酸序列。
  5. 一种荧光定量PCR试剂盒，其特征在于，包括如权利要求1-3任意一项所述的荧光探针和/或如权利要求4所述的引物对。
  6. 如权利要求5所述的荧光定量PCR试剂盒，其特征在于，所述荧光定量PCR试剂盒还包括反应缓冲液、阳性对照品和阴性对照品。
  7. 一种荧光定量PCR检测方法，其特征在于，包括：
    提取待测样本的DNA，所述待测样本包括经含有WPRE元件的第一重组基因传递载体转染的靶细胞，对所述待测样本的DNA进行定量PCR扩增反应；所述定量PCR扩增反应中，使用如权利要求1或2所述的荧光探针和/或如权利要求4所述的引物对；
    反应结束后，检测所述待测样本的DNA是否含有所述WPRE元件，以及检测所述待测样本中的所述WPRE元件的拷贝数。
  8. 如权利要求7所述的荧光定量PCR检测方法，其特征在于，所述第一重组基因传递载体还含有靶基因，经所述第一重组基因传递载体转染的所述靶细胞的制备过程包括：
    (1)将所述第一重组基因传递载体进行包装并转染宿主细胞，得到重组病毒；
    (2)将所述重组病毒转染靶细胞，以导入所述WPRE元件和所述靶基因至所述靶细胞的基因组；所述靶细胞包括肿瘤细胞、干细胞和免疫细胞中的一种或多种。
  9. 如权利要求8所述的荧光定量PCR检测方法，其特征在于，所述第一重组基因传递载体中，所述WPRE元件位于所述靶基因的下游；所述靶基因包括靶向肿瘤细胞的嵌合抗原受体基因。
  10. 如权利要求8所述的荧光定量PCR检测方法，其特征在于，所述定量PCR扩增反应的程序包括：45-55℃培育2-5min；95-98℃预变性1-2min；94-98℃变性10-30秒，55-65℃退火延伸1-3分钟，共35-45个循环。
  11. 如权利要求7所述的荧光定量PCR检测方法，其特征在于，检测所述待测样本中的所述WPRE元件的拷贝数，包括绘制标准曲线，包括以下步骤：
    提供多个不同浓度梯度的标准品，所述标准品为含有所述WPRE元件的第二重组基因传递载体；
    对各个所述标准品中的所述WPRE元件进行定量PCR扩增反应；反应完成后，根据所述不同浓度梯度的标准品中的所述WPRE元件的拷贝数对应的Ct值绘制得到标准曲线。
  12. 如权利要求7所述的荧光定量PCR检测方法，其特征在于，所述荧光定量PCR检测方法的检测下限为1.7copies/μL的DNA。
  13. 一种如权利要求1-3任一项所述的荧光探针、如权利要求4所述的引物对或如权利要求7-12任一项所述的荧光定量PCR检测方法在CAR-T中的慢病毒拷贝数检测方面的应用。
PCT/CN2020/084752 2019-05-08 2020-04-14 检测慢病毒的荧光探针、引物对、荧光定量pcr试剂盒及检测方法 WO2020224394A1 (zh)

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