WO2022253000A1 - Use of cd4＋t cell line in lentiviral infectious titer detection and lentiviral infectious titer detection method - Google Patents

Use of cd4＋t cell line in lentiviral infectious titer detection and lentiviral infectious titer detection method Download PDF

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WO2022253000A1
WO2022253000A1 PCT/CN2022/093906 CN2022093906W WO2022253000A1 WO 2022253000 A1 WO2022253000 A1 WO 2022253000A1 CN 2022093906 W CN2022093906 W CN 2022093906W WO 2022253000 A1 WO2022253000 A1 WO 2022253000A1
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cells
lentivirus
cell line
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tested
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吴昊泉
高旭
吕朋
丁彦甫
李昕然
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康霖生物科技（杭州）有限公司
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Definitions

  • the invention relates to the field of genetic engineering, in particular to the use and method of CD4+T cell lines in the detection of lentivirus infection titer.
  • Recombinant lentivirus gene therapy vectors are currently one of the most widely used gene therapy vectors. Most recombinant lentiviruses are developed on the basis of HIV-1 (human immunodeficiency virus type I). With a wide range of hosts, extremely low immunogenicity and excellent infection efficiency, it can effectively integrate the expression framework of the target gene into the genome of dividing and non-dividing cells.
  • HIV-1 human immunodeficiency virus type I
  • the third-generation pseudo-enveloped lentiviral vector with VSVG as the membrane protein can effectively integrate the expression framework of the target gene into a wide range of cell genomes including division and non-division
  • the high-efficiency expression of the target gene without being affected by cell division makes it possible for gene therapy drugs using lentivirus as a carrier to achieve the goal of one-time treatment and life-long effectiveness in clinical practice. At present, it has been successfully applied to a number of listed gene and cell therapy drugs and gene therapy drugs under clinical research, and has great commercial value.
  • the integration of the expression framework of the target gene into the genome of the target cell by the lentiviral vector is a prerequisite for its continuous lifelong high-efficiency expression of the target gene. Integrating the expression framework copy of the target gene into the cell genome in the human body is the ultimate goal of lentiviral vector gene therapy drug administration, that is, the target copy number integrated into the cell genome is the real effective dose of the lentiviral vector gene therapy drug .
  • the ideal drug administration of gene therapy should achieve the integration of the target gene in the target cell population just in line with the copy number of the dose.
  • the dose of a drug in all drug research is not only related to the efficacy of the drug, but also closely related to the toxicity and safety of the drug.
  • the dose is the most important indicator in drug application. Precisely controlled dose-related indicators.
  • lentiviral vector gene therapy drugs are integrated into the cell genome after one application, and drug-related toxicity and safety will continue irreversibly for life.
  • the importance of drug dosage that is, the control of the copy number of the target gene, is far more important than the metabolic pathway of conventional drugs.
  • the lentiviral vector that is, the infection titer.
  • the crude products, semi-finished products, intermediate products, eluted substances, raw materials, and final product preparations of lentiviral vectors are all guided by the detection of infectious titers.
  • Various levels of lentiviral vectors The first important indicator of the application.
  • the current mainstream method for detection of lentiviral vector infection titers is to detect the infection titer of the target cells by culturing adherent 293T cells
  • the method includes pre-inoculation of 293T cells; after the completion of cell attachment, the lentiviral vector sample to be tested is used to infect the adhered 293T cells; after a period of time, the number of copies of the target gene integrated into the cell genome and the cell endogenous
  • the ratio of gene copy numbers was used to calculate the infection titer of the lentiviral vector.
  • the lentiviral vector infection titer detection method inoculated with adherent 293T cells has some defects as follows:
  • lentiviral particles interact with the non-cultured surface of cells, but cannot interact with the cultured surface, which reduces the probability of virus infection of cells and affects the accurate measurement of lentiviral vector infection titer.
  • HIV-1 virus mainly infects CD4+ T cells in the blood in vivo.
  • the lentiviral gene therapy vector with VSV-G envelope has a wide host range
  • 293T cells are derived from human embryonic kidney epithelial cells and are not specific HIV-infected host cells are therefore likely not one of the most sensitive cell lines for infection with lentiviral gene therapy vectors.
  • the purpose of the present invention is to provide the use and method of CD4+T cell lines in the detection of lentivirus infection titer, so as to solve the problems in the prior art.
  • the present invention firstly provides the use of CD4+T cell line in the detection of lentivirus infection titer.
  • the CD4+T cell lines include MT4 cells, C8166 cells or Jurkat cells.
  • the Jurkat is selected from Jurkat-E6 cells.
  • the present invention also provides a detection method for the infection titer of the lentivirus, the detection method comprising the following steps: inoculating the lentivirus to be tested into the cell suspension of the CD4+ T cell line, and continuing to cultivate the lentivirus to be tested after being inoculated CD4+T cell line, detect the infection titer of the lentivirus to be tested after the culture is completed.
  • the lentivirus to be tested is diluted and then inoculated into the cell suspension of the CD4+ T cell line.
  • the CD4+T cell line is selected from MT4 cells, C8166 cells or Jurkat cells.
  • the use of the CD4+ T cell line of the present invention in the detection of lentivirus infection titer has the following beneficial effects:
  • MT4 and other blood-derived CD4+ leukemia T cell lines are significantly more sensitive to lentivirus infection than 293T cell lines derived from human embryonic kidney epithelial cells.
  • the 293T cell line is 5-10 times higher, that is, closer to the real infection titer of the lentivirus to be tested, and can be used as a detection cell line for accurately measuring the lentivirus infection titer.
  • the infection operation can be carried out immediately after the cells are inoculated, which is convenient and precise to control the precise number of cells when the infection occurs, and can also shorten the operation period of the lentiviral vector infection titer detection.
  • the infection titer detection technology based on suspension cells is convenient for high-throughput large-scale detection. Compared with adherent cells, it is also more conducive to automatic workstation operation, reducing manual operation errors and interference.
  • the scope of application includes the quality detection of lentiviral gene therapy vector infection titer biological activity indicators of various scales.
  • Figure 1-1 shows a schematic diagram of the lentiviral vector construct pCCL-sin-EF1 ⁇ -WPRE-EGFP of the present invention.
  • 1-2 are schematic diagrams showing the lentiviral vector construct pCCL-sin-PGK-WPRE-EGFP of the present invention.
  • Figure 2-1 shows the trend graph of the biological titer of PGK-EGFP lentivirus with the number of culture days when 293T is used as the detection cell line.
  • Figure 2-2 shows the trend graph of the biological titer of EF1a-EGFP lentivirus with the number of days of culture when 293T is used as the detection cell line.
  • Figure 3-1 shows the trend of the infection efficiency of PGK-EGFP lentivirus with the number of days of culture when MT4 is used as the detection cell line.
  • Figure 3-2 shows the trend of the infection efficiency of EF1 ⁇ -EGFP lentivirus with the number of days of culture when MT4 is used as the detection cell line.
  • the method is more convenient to operate, and easy to expand the throughput.
  • the present invention firstly provides the use of CD4+T cell line in the detection of lentivirus infection titer.
  • the CD4+T cell line is a suspension cell line of human origin. Using this sensitive human suspension cell line, the biological activity of the recombinant lentivirus, especially the infectious titer, can be accurately detected. Compared with the widely used detection cell line derived from traditional adherent 293T, it can obtain the real biological activity index of lentivirus, which can be used to accurately guide the application of recombinant lentivirus in vitro and in vivo gene therapy.
  • the scope of application includes Qualitative and quantitative quality detection of lentiviruses of all sizes.
  • the CD4+ T cell line includes MT4 cells, C8166 cells, Jurkat cells.
  • the MT4 cells are human blood-derived CD4+ acute lymphoblastic leukemia T cell lines.
  • the MT4 cells are cells derived from ECACC number 08081402 of the European Accredited Cell Culture Collection.
  • the C8166 cells are cells from the European Accredited Cell Culture Collection ECACC number 88051601.
  • the Jurkat is selected from Jurkat-E6 cells.
  • the Jurkat-E6 cells are a clone of the Jurkat-FHCRC cell line (a derivative of the Jurkat cell line).
  • the Jurkat-E6 cells are TIB-152 cells numbered by the American Standard Biological Collection (ATCC).
  • 293T cells, K562 cells, MT4 cells, C8166 cells and Jurkat cells were used to detect the infection titer of lentivirus.
  • the infection titer of the virus is higher than that of suspension cell lines such as K562 and 293T.
  • suspension cell lines such as K562 and 293T.
  • Direct titer also known as physical titer, is obtained by strictly measuring the actual number of virus particles, usually expressed as the number of virus particles/mL, that is, VP/mL.
  • Infectious titer also known as functional titer, indicates how much virus actually infects target cells. Functional titer can be expressed in transfection unit/mL, ie TU/mL, plaque/mL, ie pfu/mL, or infection unit/mL, ie ifu/mL. The method of expression depends on the viral vector. Infectious titers are a more accurate reflection of the biological activity of viral vectors than direct titers because it is possible to determine how much virus has the potential to actually enter target cells.
  • the present invention also provides a detection method for lentivirus infection titer, said detection method comprising the following steps: inoculating the lentivirus to be tested into the cell suspension of the CD4+T cell line, continuing to cultivate the CD4+T cell line, culturing After the end, detect the infection titer of the lentivirus to be tested.
  • the lentivirus to be tested can be any lentivirus obtained by lentiviral packaging, for example, it can be a lentivirus carrying a fluorescent reporter group, or it can be a lentivirus carrying any group or not.
  • the examples in this application use lentiviruses carrying fluorescent reporter groups for experiments.
  • the lentivirus to be tested is diluted and then inoculated into the cell suspension of the CD4+ T cell line.
  • the dilution ratio of the lentivirus to be tested is, for example, 5-4000 times.
  • the lentivirus to be tested can be diluted with the culture medium of the CD4+ T cell line.
  • the number of cells of the CD4+T cell line in the cell suspension of the CD4+T cell line is 4.00E+04-1.60E+05 per well. In a preferred embodiment, the number of cells of the CD4+T cell line in the cell suspension of the CD4+T cell line is 1.00E+05-1.60E+05 per well.
  • the culture period is continued for 2-6 days.
  • the volume of the cell suspension of the CD4+ T cell line is 30-200 ⁇ l per well.
  • the multi-well plate is a 96-well plate.
  • conversion can be made based on the data of the above-mentioned 96-well cell culture plates.
  • the infection titer of the lentivirus to be tested can be detected by flow cytometry or fluorescent quantitative PCR.
  • the steps when using flow cytometry are as follows: use flow cytometry to detect the virus positive rate of the CD4+T cell line after the culture, and then calculate the infection titer of the lentivirus to be tested.
  • the positive rate of the virus refers to the percentage of the CD4+T cell line infected by the lentivirus to be tested in the total number of cells.
  • the expression percentage of the fluorescent reporter gene GFP (GFP%) is used to represent the positive rate of the virus.
  • the calculation method of detecting the infection titer of the lentivirus to be tested by flow cytometry is as follows: after cell flow cytometry analysis, the data between 5-30% of the fluorescence percentage is substituted into the following formula for calculation:
  • the infection titer values obtained after substituting different fluorescence percentages into the formula are different, the value that appears multiple times is selected as the infection titer; if multiple values appear multiple times, the largest value among the values that appear multiple times is selected as the waiting Measure the infectious titer of lentivirus.
  • fluorescent quantitative PCR For lentiviruses that do not carry fluorescent reporter genes, use fluorescent quantitative PCR to detect the infection titer of the lentiviruses to be tested.
  • the method of fluorescent quantitative PCR is well known to those skilled in the art. For example: extract the total DNA of cells, run quantitative PCR with the total DNA as a template, calculate the lentivirus to be tested according to the ratio of the copy number of the target gene integrated into the cell genome in the cell to the copy number of the endogenous gene in the cell according to the result of quantitative PCR infection titer.
  • the CD4+ T cell line is selected from MT4 cells, C8166 cells or Jurkat cells.
  • the detection method of the lentivirus infection titer may also include other steps or details well known in the art, for example, when the lentivirus to be tested is inoculated into the cell suspension of the CD4+ T cell line, it can be The ratio of virus co-infection reagents is well known to those skilled in the art.
  • the culture conditions and culture medium of the CD4+ T cell line are based on the conditions and culture medium recommended by the cell culture center from which the cells originate, and those skilled in the art can also obtain the information on the basis of professional knowledge or various laboratory conditions. Adjust the culture conditions and culture medium.
  • the reagents used in the detection method are all commercially available reagents unless otherwise specified.
  • multiple lentiviruses carrying the GFP reporter gene were obtained by transiently transfecting adherent 293T cells, and the purified lentiviruses were used to inoculate different infection titer detection cell lines in different ways. After the cells were harvested at different detection time points, the expression of the reporter gene was detected by flow cytometry.
  • the differences in the detection of lentivirus infection titer between 293T cell lines and CD4+ T cell lines derived from multiple blood sources were compared, and the number, method, time, cell culture volume, and cell inoculation amount were analyzed in detail. Effects of virus vector infection ratio, infection time and other factors on lentivirus infection titer.
  • Embodiment 1 The acquisition of the lentiviral vector construct carrying the GFP reporter gene
  • the lentiviral vector skeleton used in the present invention is from the third-generation lentiviral vector skeleton prepared by Kanglin Biotechnology (Hangzhou) Co., Ltd.
  • the lentiviral vector contains chimeric LTR promoter, HIV-1 packaging signal ( ⁇ ), central polypurine region (cPPT), Rev response element (RRE), polypurine fragment (PPT), self-inactivating long terminal repeat sequence , woodchuck hepatitis B virus post-transcriptional regulatory element (WPRE), SV40 virus polyadenylation signal (SV40 pA signal), SV40 virus replication initiation site (SV40 ori).
  • FIG. 1-1 The schematic diagrams of the lentiviral vector constructs pCCL-sin-EF1 ⁇ -WPRE-EGFP and pCCL-sin-PGK-WPRE-EGFP carrying the GFP fluorescent protein reporter gene constructed by the aforementioned lentiviral vector are shown in Figure 1-1 and Figure 1- 2, the two can obtain EF1a-EGFP lentivirus and PGK-EGFP lentivirus respectively.
  • the above lentiviral vector construct (pCCL-sin-EF1 ⁇ -WPRE-EGFP, pCCL-sin-PGK-WPRE-EGFP), packaging plasmid (pKL-Kan-GagPol; pKL-Kan-Rev) and envelope plasmid (pKL -Kan-Vsvg) were simultaneously co-transfected into 293T cells at a ratio of 4:2.6:1:1, and the lentivirus was packaged in the 293T cell line.
  • the transfection method is the transient transfection of eukaryotic cells mediated by PEI cationic polymer
  • the PEI cationic polymer is the PEI-Max transfection reagent purchased from Polysciences (purchased from Polysciences, catalog number: 24765-1), and the transfection operation refers to the production Standardized operation recommended by the manufacturer.
  • the lentivirus transfected cell culture supernatant was harvested. First, centrifuge at 4000 rpm for 5 minutes at room temperature on a bench-top swinging bucket machine to remove cell debris.
  • the conventional purification method removes various impurities produced in the virus production process, and the prepared virus suspension is subpackaged and frozen at -80 degrees for later use.
  • the samples are respectively labeled: the sample to be tested EF1 ⁇ -EGFP and the sample to be tested PGK– EGFP.
  • 293T cells (ATCC-CCL-243) derived from human embryonic kidney epithelial cells were purchased from the American Standard Biological Collection;
  • MT4 Human blood-derived CD4+ acute lymphoblastic leukemia T cell line——MT4 (European Accredited Cell Culture Collection Center ECACC-08081402) was purchased from Shanghai Suer Biotechnology Co., Ltd.;
  • C8166 European Certified Cell Culture Collection Center ECACC-88051601 was purchased from Nanjing Kebai Biotechnology Co., Ltd.;
  • Jurkat-E6 (ATCC-TIB-152, American Standard Biological Collection) was purchased from American Standard Biological Collection;
  • Each cell was cultured in accordance with the culture conditions recommended by the cell culture center, after stable passage, it was frozen and stored in liquid nitrogen for future use.
  • the specific steps of the flow cytometry method are as follows: collect part of the cells at the same time, resuspend the cells with the flow cytometry buffer (phosphate buffered saline PBS; 2% fetal bovine serum FBS), and send them to the flow cytometer for detection.
  • the instrument is (Agilent Technologies (China) Co., Ltd. NovoCyte Flow Cytometer Systems).
  • the percentage (GFP%) of the positive cells expressing the fluorescent reporter gene GFP in the total number of CD4+T cells infected by the lentivirus to be tested represents the positive rate of the virus.
  • the 293T detection cells were inoculated in a 48-well cell culture plate at a density of 5.00E+04 cells per well, and the inoculated cell culture volume was 200 ⁇ l/well, and 12 cells were inoculated.
  • the virus to be tested was diluted to a volume of 100 ⁇ l with medium as a diluent to infect the cells, and the infection titers of the sample to be tested EF1 ⁇ -EGFP and the sample to be tested PGK–EGFP were detected by flow cytometry.
  • the test sample EF1 ⁇ -EGFP was diluted 80 times and the test sample PGK-EGFP was diluted 10,000 times.
  • the culture time was 3-6 days after infection.
  • the data are as follows:
  • the trend charts of the infection titers of the tested viruses with the number of culture days are shown in Figures 2-1 and 2-2, respectively.
  • the PGK-EGFP infection titer of the sample to be tested tended to be stable after 3-6 days of culture; the EF1 ⁇ -EGFP of the sample to be tested reached the highest value after 3 days, and then gradually stabilized, with a slight downward trend.
  • the 293T test cells were inoculated in 48-well cell culture plates at different densities, and the inoculated cell culture volume was 200 ⁇ l/well. After 12 hours of inoculating the cells, the virus to be tested was diluted to a volume of 100 ⁇ l with the medium as the diluent, and then the cells were infected. , the test sample EF1 ⁇ -EGFP was diluted 80 times, and the infection titer of the test sample PGK-EGFP was detected by flow cytometry.
  • the impact data of the number of inoculated cells on the virus infection efficiency is shown in Table 2. When the number of inoculated cells is 1.5E+05, the measured virus infection titer tends to be stable.
  • the above test data show that the highest infectious titer of PGK-EGFP in the sample to be tested is 2.12E+07 infectious units per milliliter (transduction units, TU/ml); the highest infectious titer of EF1 ⁇ -EGFP in the sample to be tested is 1.45E+09 infectious units per milliliter (transduction units, TU/ml).
  • Example 4 Infection Titer Detection Conditions Influence on the Detection System in which the Suspension MT4 Detection Cell Line is the Target Cell
  • the MT4 detection cells were inoculated in a 96-well cell culture plate at a density of 1.00E+05 cells per well, and the cell culture volume was 50 ⁇ l/well, and the virus to be tested was Infect the cells after diluting to a volume of 20 ⁇ l with the medium as a diluent, and detect the infection titers of the test sample EF1 ⁇ -EGFP and the test sample PGK-EGFP by flow cytometry, wherein the test sample EF1 ⁇ -EGFP is diluted 80 times and the test sample PGK-EGFP was diluted 10,000 times, and the culture time was 3-6 days after infection.
  • the data are as follows:
  • Figures 3-1 and 3-2 show the trend of the infection efficiency of the tested samples PGK-EGFP and EF1 ⁇ -EGFP with the number of days of culture. It can be seen that both tend to be stable on the 4th to 6th day.
  • the MT4 detection cells were seeded in 96-well cell culture plates at different cell densities, and the cell culture volume was 50 ⁇ l/well.
  • the virus to be tested was diluted to a volume of 20 ⁇ l with the medium as a diluent, and then the cells were infected.
  • the sample to be tested was EF1 ⁇ – EGFP was diluted 80 times and the test sample PGK–EGFP was diluted 10,000 times, and the culture time was 3 days after infection.
  • the infection titers of the test sample EF1 ⁇ -EGFP and the test sample PGK–EGFP were detected by flow cytometry, and inoculated
  • Table 4 The data of the effect of cell number on virus infection efficiency are shown in Table 4.
  • the cell culture volume is 30 ⁇ l-150 ⁇ l/well, and dilute the virus to be tested to volume with the medium as the diluent
  • the test sample EF1 ⁇ -EGFP was diluted 200 times and the test sample PGK-EGFP was diluted 10000 times, and the culture time was 3 days after infection.
  • the test sample EF1 ⁇ -EGFP and The infection titer of the sample PGK-EGFP to be tested, and the data of the influence of different cell culture volumes on the virus infection efficiency are shown in Table 5.
  • the above infection titer detection data show that when MT4 is used as the detection cell line: the highest infection titer of PGK-EGFP in the test sample is 1.03E+08 infection units per milliliter (transduction units, TU/ml); the test sample EF1 ⁇ -EGFP The highest infectious titer was 1.29E+10 infectious units per milliliter (transduction units, TU/ml). They were 4.86 times and 8.9 times higher than the infection titers measured when 293T was used as the detection cell line, respectively.
  • Example 2 Using the same detection method as described in Example 2, use suspension cell lines such as MT4, C8166, Jurkat-E6, K562 as detection target cells, and inoculate them in 96-well cell culture plates at a cell density of 1.00E+05 cells/well , the cell culture volume is 30 ⁇ l/well, the virus to be tested is diluted to a volume of 20 ⁇ l with the medium as the diluent, and then the cells are infected. The culture time is 3 days after infection to detect the sample PGK-EGFP to be tested, and the data are shown in Table 6-1 to 6-4 shown.
  • suspension cell lines such as MT4, C8166, Jurkat-E6, K562 as detection target cells, and inoculate them in 96-well cell culture plates at a cell density of 1.00E+05 cells/well , the cell culture volume is 30 ⁇ l/well, the virus to be tested is diluted to a volume of 20 ⁇ l with the medium as the diluent, and then the
  • the detection results of 293T cells can refer to the detection results obtained under the best experimental conditions of 293T in Example 3, that is, the highest infection titer of PGK-EGFP in the sample to be tested is 2.12E+07TU/ml, it can be seen that the same virus can be used for The titer detected by 293T cells is much lower than the titer detected by CD4+T cells in this example.
  • Table 6-1 MT4 is used as the detection target cell
  • the above infection titer detection data show that when MT4 is used as the detection cell line: the infection titer of PGK-EGFP in the sample to be tested is higher than that of the target cells detected by suspension cell lines such as C8166, Jurkat-E6, and K562, and it is unexpectedly found that MT4 is the preferred slow cell line.
  • Virus infection titer assay cell lines show that when MT4 is used as the detection cell line: the infection titer of PGK-EGFP in the sample to be tested is higher than that of the target cells detected by suspension cell lines such as C8166, Jurkat-E6, and K562, and it is unexpectedly found that MT4 is the preferred slow cell line.
  • Virus infection titer assay cell lines show that when MT4 is used as the detection cell line: the infection titer of PGK-EGFP in the sample to be tested is higher than that of the target cells detected by suspension cell lines such as C8166, Jurkat-E6, and K562, and it

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Abstract

Provided are a use of a CD4＋T cell line in lentiviral infectious titer detection and a lentiviral infectious titer detection method. The CD4＋T cell line comprises MT4 cells, C8166 cells or Jurkat cells, and Jurkat is selected from Jurkat-E6 cells. The lentiviral infectious titer detection method comprises the following steps: inoculating a lentivirus to be tested into a cell suspension of a CD4+T cell line; continuing to culture the CD4+T cell line; and detecting the infectious titer of said lentivirus after the culture is finished.

Description

CD4+T细胞系在慢病毒感染滴度检测中的用途及方法Use and method of CD4+T cell line in detection of lentivirus infection titer 技术领域technical field
本发明涉及基因工程领域，特别是涉及CD4+T细胞系在慢病毒感染滴度检测中的用途及方法。The invention relates to the field of genetic engineering, in particular to the use and method of CD4+T cell lines in the detection of lentivirus infection titer.
背景技术Background technique
重组慢病毒(Lentivirus)基因治疗载体是目前应用最为广泛的基因治疗载体之一，大多数重组慢病毒是以HIV-1(人类免疫缺陷I型病毒)为基础发展而来。具有广泛的宿主范围、极低的免疫原性和优异的感染效率，能够有效的将目的基因的表达框架整合到包括分裂期及非分裂期细胞的基因组中。基于HIV的复制缺陷型的自我失活型，以VSVG为膜蛋白的第三代假包膜慢病毒载体能够有效的将目的基因的表达框架整合到包括分裂期及非分裂期的广泛的细胞基因组中，并且不受到细胞分裂影响的高效表达目的基因，使得以慢病毒为载体的基因治疗药物在临床上有可能达到一次治疗、终身有效的目标。目前已经被成功应用到多个上市基因及细胞治疗药物和临床在研基因治疗药物中，具有极大的商业化价值。Recombinant lentivirus gene therapy vectors are currently one of the most widely used gene therapy vectors. Most recombinant lentiviruses are developed on the basis of HIV-1 (human immunodeficiency virus type I). With a wide range of hosts, extremely low immunogenicity and excellent infection efficiency, it can effectively integrate the expression framework of the target gene into the genome of dividing and non-dividing cells. Based on HIV's replication-deficient self-inactivation type, the third-generation pseudo-enveloped lentiviral vector with VSVG as the membrane protein can effectively integrate the expression framework of the target gene into a wide range of cell genomes including division and non-division In addition, the high-efficiency expression of the target gene without being affected by cell division makes it possible for gene therapy drugs using lentivirus as a carrier to achieve the goal of one-time treatment and life-long effectiveness in clinical practice. At present, it has been successfully applied to a number of listed gene and cell therapy drugs and gene therapy drugs under clinical research, and has great commercial value.
慢病毒载体将目的基因的表达框架整合到目的细胞基因组中是其持续终身高效表达目的基因的前提条件。将目的基因的表达框架拷贝整合到人体内细胞基因组中是慢病毒载体基因治疗药物给药的最终目的，即整合到细胞基因组中的目的拷贝数，即为慢病毒载体基因治疗药物的真实有效剂量。The integration of the expression framework of the target gene into the genome of the target cell by the lentiviral vector is a prerequisite for its continuous lifelong high-efficiency expression of the target gene. Integrating the expression framework copy of the target gene into the cell genome in the human body is the ultimate goal of lentiviral vector gene therapy drug administration, that is, the target copy number integrated into the cell genome is the real effective dose of the lentiviral vector gene therapy drug .
理想的基因治疗药物给药应该达到目的基因在目的细胞群体中整合正好符合给药剂量的拷贝数。所有药物研究中药物的剂量不仅与药效相关，更与药物带来的毒性及安全性息息相关，剂量是药物应用中最为重要的指标，整合的基因表达框架拷贝数是基因治疗药物研发中必须要精确控制的剂量相关指标。事实上，慢病毒载体基因治疗药物为一次应用即整合进细胞基因组，药物相关毒性及安全性将不可逆的持续终身。药物剂量，即目的基因拷贝数控制的重要性，更远胜于常规药物的代谢途径。The ideal drug administration of gene therapy should achieve the integration of the target gene in the target cell population just in line with the copy number of the dose. The dose of a drug in all drug research is not only related to the efficacy of the drug, but also closely related to the toxicity and safety of the drug. The dose is the most important indicator in drug application. Precisely controlled dose-related indicators. In fact, lentiviral vector gene therapy drugs are integrated into the cell genome after one application, and drug-related toxicity and safety will continue irreversibly for life. The importance of drug dosage, that is, the control of the copy number of the target gene, is far more important than the metabolic pathway of conventional drugs.
要达到在目的细胞群体中整合正好符合给药剂量的目的基因拷贝数，就必须对慢病毒载体的生物学活性，即感染滴度进行准确的测量及检测。在各种离体及在体基因治疗应用中，慢病毒载体的粗制品、半成品、中间产物、洗脱物质、原料药、终产品制剂等均以感染滴度检测为指导慢病毒载体各种层次应用的第一重要指标。In order to achieve the integration of the target gene copy number that just meets the dosage in the target cell population, it is necessary to accurately measure and detect the biological activity of the lentiviral vector, that is, the infection titer. In various in vitro and in vivo gene therapy applications, the crude products, semi-finished products, intermediate products, eluted substances, raw materials, and final product preparations of lentiviral vectors are all guided by the detection of infectious titers. Various levels of lentiviral vectors The first important indicator of the application.
绝大部分商业化的慢病毒基因治疗载体采用具有广泛宿主范围的VSV-G包膜，慢病毒载体感染滴度检测当前的主流方法是以培养的贴壁293T细胞为目的细胞的感染滴度检测方法， 包括预先接种293T细胞；待细胞贴壁完成后以待测慢病毒载体样本感染贴壁后293T细胞；通过检测一段时间后293T细胞内整合进细胞基因组的目的基因拷贝数与细胞内源性基因拷贝数的比值，推算出该慢病毒载体的感染滴度。但接种贴壁293T细胞的慢病毒载体感染滴度检测方法有如下的一些缺陷：Most commercialized lentiviral gene therapy vectors use the VSV-G envelope with a wide host range. The current mainstream method for detection of lentiviral vector infection titers is to detect the infection titer of the target cells by culturing adherent 293T cells The method includes pre-inoculation of 293T cells; after the completion of cell attachment, the lentiviral vector sample to be tested is used to infect the adhered 293T cells; after a period of time, the number of copies of the target gene integrated into the cell genome and the cell endogenous The ratio of gene copy numbers was used to calculate the infection titer of the lentiviral vector. However, the lentiviral vector infection titer detection method inoculated with adherent 293T cells has some defects as follows:
1)感染滴度检测过程中，慢病毒颗粒与细胞的非培养表面相互作用，但是并不能与培养表面相互作用，减少了病毒感染细胞的几率，影响了慢病毒载体感染滴度的准确测量。1) During the infection titer detection process, lentiviral particles interact with the non-cultured surface of cells, but cannot interact with the cultured surface, which reduces the probability of virus infection of cells and affects the accurate measurement of lentiviral vector infection titer.
2)预先接种贴壁的293T细胞在贴壁的过程中伴随着细胞的增殖，感染发生时的精确细胞数量，较难以通过常规的悬浮细胞技术方法准确控制。2) Pre-seeded 293T cells adhered to the wall are accompanied by cell proliferation during the process of adhesion, and the precise number of cells at the time of infection is difficult to accurately control through conventional suspension cell technology.
3)细胞在贴壁的过程增加了慢病毒载体感染滴度检测的操作周期。3) The process of cell attachment increases the operating cycle of lentiviral vector infection titer detection.
4)HIV-1病毒在体内主要感染血液中的CD4+T细胞，虽然采用VSV-G包膜的慢病毒基因治疗载体具有广泛的宿主范围，但293T细胞来源于人胚胎肾上皮细胞，并非特异HIV病毒感染的宿主细胞，所以有可能不是对慢病毒基因治疗载体感染最敏感的细胞系之一。4) HIV-1 virus mainly infects CD4+ T cells in the blood in vivo. Although the lentiviral gene therapy vector with VSV-G envelope has a wide host range, 293T cells are derived from human embryonic kidney epithelial cells and are not specific HIV-infected host cells are therefore likely not one of the most sensitive cell lines for infection with lentiviral gene therapy vectors.
发明内容Contents of the invention
鉴于以上所述现有技术的缺点，本发明的目的在于提供CD4+T细胞系在慢病毒感染滴度检测中的用途及方法，用于解决现有技术中的问题。In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide the use and method of CD4+T cell lines in the detection of lentivirus infection titer, so as to solve the problems in the prior art.
为实现上述目的及其他相关目的，本发明首先提供CD4+T细胞系在慢病毒感染滴度检测中的用途。To achieve the above purpose and other related purposes, the present invention firstly provides the use of CD4+T cell line in the detection of lentivirus infection titer.
所述CD4+T细胞系包括MT4细胞、C8166细胞或Jurkat细胞。The CD4+T cell lines include MT4 cells, C8166 cells or Jurkat cells.
所述Jurkat选自Jurkat-E6细胞。The Jurkat is selected from Jurkat-E6 cells.
本发明还提供一种慢病毒感染滴度的检测方法，所述检测方法包括如下步骤：将待测慢病毒接种到CD4+T细胞系的细胞悬液中，继续培养接种待测慢病毒后的CD4+T细胞系，培养结束后检测所述待测慢病毒的感染滴度。The present invention also provides a detection method for the infection titer of the lentivirus, the detection method comprising the following steps: inoculating the lentivirus to be tested into the cell suspension of the CD4+ T cell line, and continuing to cultivate the lentivirus to be tested after being inoculated CD4+T cell line, detect the infection titer of the lentivirus to be tested after the culture is completed.
优选的，将待测慢病毒稀释后再接种到CD4+T细胞系的细胞悬液中。Preferably, the lentivirus to be tested is diluted and then inoculated into the cell suspension of the CD4+ T cell line.
优选的，所述CD4+T细胞系选自MT4细胞、C8166细胞或Jurkat细胞。Preferably, the CD4+T cell line is selected from MT4 cells, C8166 cells or Jurkat cells.
如上所述，本发明的CD4+T细胞系在慢病毒感染滴度检测中的用途，具有以下有益效果：As mentioned above, the use of the CD4+ T cell line of the present invention in the detection of lentivirus infection titer has the following beneficial effects:
1.MT4等血液来源的CD4+白血病T细胞系对慢病毒感染的敏感性显著高于来源于人胚胎肾上皮细胞的293T细胞系，其中MT4细胞系对同一慢病毒载体感染滴度的检测值比293T细胞系高5-10倍，即更接近于待测慢病毒的真实感染滴度，可以用作精确测量慢病毒感染滴 度的检测细胞系。1. MT4 and other blood-derived CD4+ leukemia T cell lines are significantly more sensitive to lentivirus infection than 293T cell lines derived from human embryonic kidney epithelial cells. The 293T cell line is 5-10 times higher, that is, closer to the real infection titer of the lentivirus to be tested, and can be used as a detection cell line for accurately measuring the lentivirus infection titer.
2.接种细胞后可以立即进行感染操作，方便精确控制感染发生时的精确细胞数量，同时还可以缩短慢病毒载体感染滴度检测的操作周期。2. The infection operation can be carried out immediately after the cells are inoculated, which is convenient and precise to control the precise number of cells when the infection occurs, and can also shorten the operation period of the lentiviral vector infection titer detection.
3.基于悬浮细胞的感染滴度检测技术，方便高通量大规模检测，相对于贴壁细胞也更有利于全自动工作站操作，减少人工操作误差和干扰。3. The infection titer detection technology based on suspension cells is convenient for high-throughput large-scale detection. Compared with adherent cells, it is also more conducive to automatic workstation operation, reducing manual operation errors and interference.
4.应用范围包括各种规模的慢病毒基因治疗载体感染滴度生物学活性指标的质量检测。4. The scope of application includes the quality detection of lentiviral gene therapy vector infection titer biological activity indicators of various scales.
附图说明Description of drawings
图1-1显示为本发明的慢病毒载体构建体pCCL-sin-EF1α-WPRE-EGFP的示意图。Figure 1-1 shows a schematic diagram of the lentiviral vector construct pCCL-sin-EF1α-WPRE-EGFP of the present invention.
图1-2显示为本发明的慢病毒载体构建体pCCL-sin-PGK-WPRE-EGFP的示意图。1-2 are schematic diagrams showing the lentiviral vector construct pCCL-sin-PGK-WPRE-EGFP of the present invention.
图2-1显示为293T作为检测细胞系时，PGK-EGFP慢病毒的生物滴度随培养天数的变化趋势图。Figure 2-1 shows the trend graph of the biological titer of PGK-EGFP lentivirus with the number of culture days when 293T is used as the detection cell line.
图2-2显示为293T作为检测细胞系时，EF1a-EGFP慢病毒的生物滴度随培养天数的变化趋势图。Figure 2-2 shows the trend graph of the biological titer of EF1a-EGFP lentivirus with the number of days of culture when 293T is used as the detection cell line.
图3-1显示为MT4作为检测细胞系时，PGK-EGFP慢病毒的感染效率随培养天数的变化趋势。Figure 3-1 shows the trend of the infection efficiency of PGK-EGFP lentivirus with the number of days of culture when MT4 is used as the detection cell line.
图3-2显示为MT4作为检测细胞系时，EF1α-EGFP慢病毒的感染效率随培养天数的变化趋势。Figure 3-2 shows the trend of the infection efficiency of EF1α-EGFP lentivirus with the number of days of culture when MT4 is used as the detection cell line.
具体实施方式Detailed ways
为了获得更接近真实的慢病毒感染滴度生物学活性指标，以用于精确指导重组慢病毒的离体及在体基因治疗的最佳剂量应用；为了使慢病毒感染滴度检测数据更加准确、稳定可靠，方法更加方便操作，易于通量放大，本发明首先提供CD4+T细胞系在慢病毒感染滴度检测中的用途。In order to obtain a biological activity index closer to the real lentivirus infection titer, which can be used to accurately guide the optimal dose application of recombinant lentivirus in vitro and in vivo gene therapy; in order to make the detection data of lentivirus infection titer more accurate, Stable and reliable, the method is more convenient to operate, and easy to expand the throughput. The present invention firstly provides the use of CD4+T cell line in the detection of lentivirus infection titer.
所述CD4+T细胞系为人源的悬浮细胞系。利用这种敏感的人源悬浮细胞系，对重组慢病毒的生物学活性尤其是感染滴度进行精确检测。与广泛应用的传统贴壁293T来源的检测细胞系相比，能获得慢病毒的更接近真实生物学活性指标，能用于精确指导重组慢病毒的离体及在体基因治疗应用，应用范围包括各种规模的慢病毒的定性、定量质量检测。The CD4+T cell line is a suspension cell line of human origin. Using this sensitive human suspension cell line, the biological activity of the recombinant lentivirus, especially the infectious titer, can be accurately detected. Compared with the widely used detection cell line derived from traditional adherent 293T, it can obtain the real biological activity index of lentivirus, which can be used to accurately guide the application of recombinant lentivirus in vitro and in vivo gene therapy. The scope of application includes Qualitative and quantitative quality detection of lentiviruses of all sizes.
在一种实施方式中，所述CD4+T细胞系包括MT4细胞、C8166细胞、Jurkat细胞。In one embodiment, the CD4+ T cell line includes MT4 cells, C8166 cells, Jurkat cells.
所述MT4细胞是人血液来源的CD4+急性淋巴母细胞白血病T细胞系。在一种实施方式中，所述MT4细胞为源自欧洲认证细胞培养物收藏中心ECACC编号为08081402的细胞。The MT4 cells are human blood-derived CD4+ acute lymphoblastic leukemia T cell lines. In one embodiment, the MT4 cells are cells derived from ECACC number 08081402 of the European Accredited Cell Culture Collection.
在一种实施方式中，所述C8166细胞为来源于欧洲认证细胞培养物收藏中心ECACC编号为88051601的细胞。In one embodiment, the C8166 cells are cells from the European Accredited Cell Culture Collection ECACC number 88051601.
在一种实施方式中，所述Jurkat选自Jurkat-E6细胞。所述Jurkat-E6细胞是Jurkat-FHCRC细胞株(Jurkat细胞株的衍生)的一个克隆。在一种实施方式中，所述Jurkat-E6细胞为美国标准生物品收藏中心ATCC编号为TIB-152的细胞。In one embodiment, the Jurkat is selected from Jurkat-E6 cells. The Jurkat-E6 cells are a clone of the Jurkat-FHCRC cell line (a derivative of the Jurkat cell line). In one embodiment, the Jurkat-E6 cells are TIB-152 cells numbered by the American Standard Biological Collection (ATCC).
同时用293T细胞、K562细胞、MT4细胞、C8166细胞、Jurkat细胞检测慢病毒的感染滴度，本申请的发明人意外的发现，以MT4细胞、C8166细胞、Jurkat细胞为检测细胞系时待测慢病毒的感染滴度高于K562、293T等悬浮细胞系。更意外的是，以MT4为检测细胞系时待测慢病毒的感染滴度最高，即高于C8166、Jurkat-E6、K562、293T等悬浮细胞系，因此，MT4细胞可首选作为慢病毒感染滴度检测细胞系。At the same time, 293T cells, K562 cells, MT4 cells, C8166 cells and Jurkat cells were used to detect the infection titer of lentivirus. The infection titer of the virus is higher than that of suspension cell lines such as K562 and 293T. What is even more surprising is that when MT4 is used as the detection cell line, the infection titer of the tested lentivirus is the highest, which is higher than that of suspension cell lines such as C8166, Jurkat-E6, K562, and 293T. Therefore, MT4 cells can be the first choice for lentivirus infection titers. To test the cell line.
病毒滴度主要有两种表示方式：There are two main ways to express the virus titer:
1.直接滴度，也称为物理滴度，通过严格测定病毒的实际颗粒数量得到，通常以病毒颗粒数量/mL表示，即VP/mL。1. Direct titer, also known as physical titer, is obtained by strictly measuring the actual number of virus particles, usually expressed as the number of virus particles/mL, that is, VP/mL.
2.感染滴度，也称为功能滴度，表示有多少病毒实际感染了靶细胞。功能滴度可采用转染单位/mL表示，即TU/mL，也可能用蚀斑/mL表示，即pfu/mL，或者以感染单元/mL表示，即ifu/mL。表示方法取决于病毒载体。感染滴度比直接滴度更准确的反映病毒载体的生物活性，因为可以测定有多少病毒具有实际进入靶细胞的潜力。2. Infectious titer, also known as functional titer, indicates how much virus actually infects target cells. Functional titer can be expressed in transfection unit/mL, ie TU/mL, plaque/mL, ie pfu/mL, or infection unit/mL, ie ifu/mL. The method of expression depends on the viral vector. Infectious titers are a more accurate reflection of the biological activity of viral vectors than direct titers because it is possible to determine how much virus has the potential to actually enter target cells.
本发明还提供一种慢病毒感染滴度的检测方法，所述检测方法包括如下步骤：将待测慢病毒接种到CD4+T细胞系的细胞悬液中，继续培养CD4+T细胞系，培养结束后检测所述待测慢病毒的感染滴度。The present invention also provides a detection method for lentivirus infection titer, said detection method comprising the following steps: inoculating the lentivirus to be tested into the cell suspension of the CD4+T cell line, continuing to cultivate the CD4+T cell line, culturing After the end, detect the infection titer of the lentivirus to be tested.
所述待测慢病毒可以是任意经过慢病毒包装的方法得到的慢病毒，例如可以是携带荧光报告基团的慢病毒，也可以是携带或不携带任何基团的慢病毒。为简化实验过程，本申请中的实施例以携带荧光报告基团的慢病毒进行实验。The lentivirus to be tested can be any lentivirus obtained by lentiviral packaging, for example, it can be a lentivirus carrying a fluorescent reporter group, or it can be a lentivirus carrying any group or not. In order to simplify the experimental process, the examples in this application use lentiviruses carrying fluorescent reporter groups for experiments.
在一种实施方式中，将待测慢病毒稀释后再接种到CD4+T细胞系的细胞悬液中。本发明中对待测慢病毒的稀释比例不做具体限制。待测慢病毒的稀释比例例如为5～4000倍。在一种实施方式中，可以用CD4+T细胞系的培养基稀释待测慢病毒。In one embodiment, the lentivirus to be tested is diluted and then inoculated into the cell suspension of the CD4+ T cell line. In the present invention, there is no specific limitation on the dilution ratio of the lentivirus to be tested. The dilution ratio of the lentivirus to be tested is, for example, 5-4000 times. In one embodiment, the lentivirus to be tested can be diluted with the culture medium of the CD4+ T cell line.
在一种实施方式中，在多孔细胞培养板中进行实验时，CD4+T细胞系的细胞悬液中CD4+T细胞系的细胞数量为每孔4.00E+04～1.60E+05个。在一较佳实施方式中，CD4+T细胞系的细胞悬液中CD4+T细胞系的细胞数量为每孔1.00E+05～1.60E+05个。In one embodiment, when the experiment is performed in a multi-well cell culture plate, the number of cells of the CD4+T cell line in the cell suspension of the CD4+T cell line is 4.00E+04-1.60E+05 per well. In a preferred embodiment, the number of cells of the CD4+T cell line in the cell suspension of the CD4+T cell line is 1.00E+05-1.60E+05 per well.
在一种实施方式中，接种待测慢病毒后，继续培养时间为2～6天。In one embodiment, after the lentivirus to be tested is inoculated, the culture period is continued for 2-6 days.
在一种实施方式中，在多孔细胞培养板中进行实验时，CD4+T细胞系的细胞悬液的体积为每孔30～200μl。In one embodiment, when the experiment is performed in a multi-well cell culture plate, the volume of the cell suspension of the CD4+ T cell line is 30-200 μl per well.
在一种实施方式中，所述多孔板为96孔板。利用其他规格的细胞培养板时可根据上述96孔细胞培养板的数据进行换算。In one embodiment, the multi-well plate is a 96-well plate. When using cell culture plates of other specifications, conversion can be made based on the data of the above-mentioned 96-well cell culture plates.
培养结束后可以通过流式细胞术或荧光定量PCR检测待测慢病毒的感染滴度。采用流式细胞术时的步骤如下：用流式细胞仪检测培养结束后CD4+T细胞系的病毒阳性率，进而计算得到所述待测慢病毒的感染滴度。After the culture, the infection titer of the lentivirus to be tested can be detected by flow cytometry or fluorescent quantitative PCR. The steps when using flow cytometry are as follows: use flow cytometry to detect the virus positive rate of the CD4+T cell line after the culture, and then calculate the infection titer of the lentivirus to be tested.
所述病毒阳性率是指被所述待测慢病毒感染的CD4+T细胞系占细胞总数的百分比。在本发明的实施例中，用荧光报告基因GFP表达百分率(GFP％)表示病毒阳性率。The positive rate of the virus refers to the percentage of the CD4+T cell line infected by the lentivirus to be tested in the total number of cells. In the embodiment of the present invention, the expression percentage of the fluorescent reporter gene GFP (GFP%) is used to represent the positive rate of the virus.
采用流式细胞术检测待测慢病毒的感染滴度的计算方法如下：细胞流式分析后，将荧光百分比5-30％之间的数据代入以下公式计算：The calculation method of detecting the infection titer of the lentivirus to be tested by flow cytometry is as follows: after cell flow cytometry analysis, the data between 5-30% of the fluorescence percentage is substituted into the following formula for calculation:
滴度(TU/ml)＝细胞数*荧光百分比*10 3/病毒原液体积(μl) Titer (TU/ml) = cell number * fluorescence percentage * 10 3 / virus stock solution volume (μl)
若不同荧光百分比代入公式后得到的感染滴度数值不同，则选择多次出现的值作为感染滴度，若有多个数值多次出现，则选择多次出现的数值中最大的一个数值作为待测慢病毒的感染滴度。If the infection titer values obtained after substituting different fluorescence percentages into the formula are different, the value that appears multiple times is selected as the infection titer; if multiple values appear multiple times, the largest value among the values that appear multiple times is selected as the waiting Measure the infectious titer of lentivirus.
对于不携带荧光报告基因的慢病毒，则采用荧光定量PCR检测待测慢病毒的感染滴度。荧光定量PCR的方法为本领域技术人员所熟知的。例如：提取细胞总DNA，以总DNA为模板运行定量PCR，根据定量PCR的结果以细胞内整合进细胞基因组的目的基因拷贝数与细胞内源性基因拷贝数的比值，推算出待测慢病毒的感染滴度。For lentiviruses that do not carry fluorescent reporter genes, use fluorescent quantitative PCR to detect the infection titer of the lentiviruses to be tested. The method of fluorescent quantitative PCR is well known to those skilled in the art. For example: extract the total DNA of cells, run quantitative PCR with the total DNA as a template, calculate the lentivirus to be tested according to the ratio of the copy number of the target gene integrated into the cell genome in the cell to the copy number of the endogenous gene in the cell according to the result of quantitative PCR infection titer.
在一种实施方式中，所述CD4+T细胞系选自MT4细胞、C8166细胞或Jurkat细胞。In one embodiment, the CD4+ T cell line is selected from MT4 cells, C8166 cells or Jurkat cells.
在一种实施方式中，所述慢病毒感染滴度的检测方法中还可以包括其他本领域熟知的步骤或细节，例如将待测慢病毒接种到CD4+T细胞系的细胞悬液中时可以加入病毒助感染试剂，其所占比例也是本领域人员所熟知的。再例如，所述CD4+T细胞系的培养条件和培养基均为根据细胞所来源的细胞培养中心推荐的条件和培养基，本领域技术人员也可以在专业认知或各实验室条件的基础上进行调整培养条件和培养基。所述检测方法中使用的试剂若无特别说明均为市售试剂。In one embodiment, the detection method of the lentivirus infection titer may also include other steps or details well known in the art, for example, when the lentivirus to be tested is inoculated into the cell suspension of the CD4+ T cell line, it can be The ratio of virus co-infection reagents is well known to those skilled in the art. For another example, the culture conditions and culture medium of the CD4+ T cell line are based on the conditions and culture medium recommended by the cell culture center from which the cells originate, and those skilled in the art can also obtain the information on the basis of professional knowledge or various laboratory conditions. Adjust the culture conditions and culture medium. The reagents used in the detection method are all commercially available reagents unless otherwise specified.
本发明实施例通过瞬时转染贴壁293T细胞的方法，获得携带GFP报告基因的多个慢病毒，并使用纯化后的慢病毒，以不同的方式接种到不同的感染滴度检测细胞系，在不同的检测时间点收获细胞后，以流式细胞仪的方法检测报告基因的表达。本发明实施例按照上述流程比较了293T细胞系，以及多个血液来源的CD4+T细胞系在慢病毒感染滴度检测方面的差 异，详细分析了细胞接种数量、方式、时间、细胞培养体积、病毒载体感染比率、感染时间等因素对慢病毒感染滴度的影响。In the embodiment of the present invention, multiple lentiviruses carrying the GFP reporter gene were obtained by transiently transfecting adherent 293T cells, and the purified lentiviruses were used to inoculate different infection titer detection cell lines in different ways. After the cells were harvested at different detection time points, the expression of the reporter gene was detected by flow cytometry. In the embodiment of the present invention, according to the above process, the differences in the detection of lentivirus infection titer between 293T cell lines and CD4+ T cell lines derived from multiple blood sources were compared, and the number, method, time, cell culture volume, and cell inoculation amount were analyzed in detail. Effects of virus vector infection ratio, infection time and other factors on lentivirus infection titer.
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention.
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are to describe specific specific embodiments, It is not intended to limit the protection scope of the present invention; in the description and claims of the present invention, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural forms.
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to those skilled in the art's grasp of the prior art and the description of the present invention, the methods, equipment, and materials described in the embodiments of the present invention can also be used Any methods, apparatus and materials of the prior art similar or equivalent to the practice of the present invention.
实施例1携带GFP报告基因的慢病毒载体构建体的获得 Embodiment 1 The acquisition of the lentiviral vector construct carrying the GFP reporter gene
本发明中使用的慢病毒载体骨架来源于康霖生物科技(杭州)有限公司自备第3代慢病毒载体骨架。该慢病毒载体包含嵌合型LTR启动子、HIV-1包装信号(ψ)、中心聚嘌呤区(cPPT)、Rev应答元件(RRE)、多嘌呤片段(PPT)、自失活长末端重复序列、土拨鼠乙肝病毒转录后调控元件(WPRE)、SV40病毒的多腺苷酸化信号(SV40 pA signal)、SV40病毒的复制起始位点(SV40 ori)。利用前述慢病毒载体构建的携带GFP荧光蛋白报告基因的慢病毒载体构建体pCCL-sin-EF1α-WPRE-EGFP和pCCL-sin-PGK-WPRE-EGFP的示意图分别如图1-1和图1-2所示，二者可分别获得EF1a-EGFP慢病毒和PGK-EGFP慢病毒。The lentiviral vector skeleton used in the present invention is from the third-generation lentiviral vector skeleton prepared by Kanglin Biotechnology (Hangzhou) Co., Ltd. The lentiviral vector contains chimeric LTR promoter, HIV-1 packaging signal (ψ), central polypurine region (cPPT), Rev response element (RRE), polypurine fragment (PPT), self-inactivating long terminal repeat sequence , woodchuck hepatitis B virus post-transcriptional regulatory element (WPRE), SV40 virus polyadenylation signal (SV40 pA signal), SV40 virus replication initiation site (SV40 ori). The schematic diagrams of the lentiviral vector constructs pCCL-sin-EF1α-WPRE-EGFP and pCCL-sin-PGK-WPRE-EGFP carrying the GFP fluorescent protein reporter gene constructed by the aforementioned lentiviral vector are shown in Figure 1-1 and Figure 1- 2, the two can obtain EF1a-EGFP lentivirus and PGK-EGFP lentivirus respectively.
将上述慢病毒载体构建体(pCCL-sin-EF1α-WPRE-EGFP，pCCL-sin-PGK-WPRE-EGFP)、包装质粒(pKL-Kan-GagPol；pKL-Kan-Rev)和包膜质粒(pKL-Kan-Vsvg)按照4:2.6:1:1比例同时共转染293T细胞，在该293T细胞系中进行慢病毒的包装。转染方法为PEI阳离子聚合物介导的真核细胞瞬时转染，PEI阳离子聚合物为购自Polysciences的PEI-Max转染试剂(购自Polysciences，货号：24765-1)，转染操作参照生产商推荐标准化操作进行。转染完成48小时后，收获慢病毒(转染细胞培养上清)，首先在台式吊桶式机上，室温4000rpm离心5分钟去除细胞碎片，再以顺序步骤的切向流、分子筛、离子交换树脂的常规纯化方法去除病毒生产 过程中产生的各种杂质，并将制备好的病毒重悬液分装冻存于-80度备用，分别标记样本为：待测样本EF1α-EGFP和待测样本PGK–EGFP。The above lentiviral vector construct (pCCL-sin-EF1α-WPRE-EGFP, pCCL-sin-PGK-WPRE-EGFP), packaging plasmid (pKL-Kan-GagPol; pKL-Kan-Rev) and envelope plasmid (pKL -Kan-Vsvg) were simultaneously co-transfected into 293T cells at a ratio of 4:2.6:1:1, and the lentivirus was packaged in the 293T cell line. The transfection method is the transient transfection of eukaryotic cells mediated by PEI cationic polymer, the PEI cationic polymer is the PEI-Max transfection reagent purchased from Polysciences (purchased from Polysciences, catalog number: 24765-1), and the transfection operation refers to the production Standardized operation recommended by the manufacturer. 48 hours after the transfection was completed, the lentivirus (transfected cell culture supernatant) was harvested. First, centrifuge at 4000 rpm for 5 minutes at room temperature on a bench-top swinging bucket machine to remove cell debris. The conventional purification method removes various impurities produced in the virus production process, and the prepared virus suspension is subpackaged and frozen at -80 degrees for later use. The samples are respectively labeled: the sample to be tested EF1α-EGFP and the sample to be tested PGK– EGFP.
实施例2感染滴度检测方法Embodiment 2 infection titer detection method
感染滴度检测细胞系的获得：Infection titer detection cell line acquisition:
人胚胎肾上皮细胞来源的293T细胞(ATCC-CCL-243)购自美国标准生物品收藏中心；293T cells (ATCC-CCL-243) derived from human embryonic kidney epithelial cells were purchased from the American Standard Biological Collection;
人血液来源的CD4+急性淋巴母细胞白血病T细胞系——MT4(欧洲认证细胞培养物收藏中心ECACC-08081402)购自上海素尔生物科技有限公司；Human blood-derived CD4+ acute lymphoblastic leukemia T cell line——MT4 (European Accredited Cell Culture Collection Center ECACC-08081402) was purchased from Shanghai Suer Biotechnology Co., Ltd.;
C8166(欧洲认证细胞培养物收藏中心ECACC-88051601)购自南京科佰生物科技有限公司；C8166 (European Certified Cell Culture Collection Center ECACC-88051601) was purchased from Nanjing Kebai Biotechnology Co., Ltd.;
Jurkat-E6(美国标准生物品收藏中心ATCC-TIB-152)购自美国标准生物品收藏中心；Jurkat-E6 (ATCC-TIB-152, American Standard Biological Collection) was purchased from American Standard Biological Collection;
各细胞培养按照细胞培养中心推荐的培养条件下，经稳定传代后冻存以液氮保存备用。Each cell was cultured in accordance with the culture conditions recommended by the cell culture center, after stable passage, it was frozen and stored in liquid nitrogen for future use.
感染滴度检测方法：Infection titer detection method:
将待测慢病毒样本以不同稀释比例稀释到室温的新鲜细胞培养基中，再以不同体积接种于预铺在96孔或48孔细胞培养板中的各种检测细胞系。经过一定时间的培养后，通过常规离心的方法收集细胞，通过本领域熟知的方法，以基于GFP信号的流式细胞术数据计算出慢病毒初始收获液感染滴度。流式细胞术法具体步骤如下：同时收集部分细胞，以流式细胞缓冲液(磷酸盐缓冲液PBS；2％胎牛血清FBS)重悬细胞后，送入流式细胞仪运行检测，仪器为(安捷伦科技(中国)有限公司NovoCyte Flow Cytometer Systems)。Dilute the lentivirus samples to be tested into fresh cell culture medium at room temperature at different dilution ratios, and inoculate various detection cell lines pre-plated in 96-well or 48-well cell culture plates with different volumes. After culturing for a certain period of time, the cells were collected by conventional centrifugation, and the infection titer of the initial lentivirus harvest solution was calculated based on the flow cytometry data of the GFP signal by a method well known in the art. The specific steps of the flow cytometry method are as follows: collect part of the cells at the same time, resuspend the cells with the flow cytometry buffer (phosphate buffered saline PBS; 2% fetal bovine serum FBS), and send them to the flow cytometer for detection. The instrument is (Agilent Technologies (China) Co., Ltd. NovoCyte Flow Cytometer Systems).
以荧光报告基因GFP表达阳性细胞占待测慢病毒感染的CD4+T细胞总数的百分比(GFP％)表示病毒阳性率，待测慢病毒的感染滴度的计算方法如下：细胞流式分析后，将荧光百分比5-30％之间的数据代入以下公式计算：滴度(TU/ml)＝细胞数*荧光百分比*10 3/病毒原液体积(μl)。 The percentage (GFP%) of the positive cells expressing the fluorescent reporter gene GFP in the total number of CD4+T cells infected by the lentivirus to be tested represents the positive rate of the virus. The calculation method of the infection titer of the lentivirus to be tested is as follows: after the cell flow analysis, Substitute the data between 5-30% fluorescence percentage into the following formula for calculation: titer (TU/ml)=cell number*fluorescence percentage*10 3 /virus stock solution volume (μl).
实施例3感染滴度检测条件对293T检测细胞系为靶细胞的检测系统的影响Example 3 Infection Titer Detection Conditions Influence on 293T Detection Cell Line as Target Cell Detection System
培养时间的影响Effect of incubation time
以实施例2所述的相同检测方法，将293T检测细胞以每孔细胞数量为5.00E+04个细胞的密度接种于48孔细胞培养板中，接种细胞培养体积为200μl/孔，接种细胞12小时后，将待测病毒以培养基为稀释液稀释到体积为100μl后感染细胞，以流式细胞术的方法检测待测样本EF1α-EGFP和待测样本PGK–EGFP的感染滴度，其中待测样本EF1α–EGFP稀释80倍和待测样本PGK–EGFP稀释10000倍，培养时间为感染后3-6天，数据如下：With the same detection method as described in Example 2, the 293T detection cells were inoculated in a 48-well cell culture plate at a density of 5.00E+04 cells per well, and the inoculated cell culture volume was 200 μl/well, and 12 cells were inoculated. Hours later, the virus to be tested was diluted to a volume of 100 μl with medium as a diluent to infect the cells, and the infection titers of the sample to be tested EF1α-EGFP and the sample to be tested PGK–EGFP were detected by flow cytometry. The test sample EF1α-EGFP was diluted 80 times and the test sample PGK-EGFP was diluted 10,000 times. The culture time was 3-6 days after infection. The data are as follows:
表1Table 1
Figure PCTCN2022093906-appb-000001
Figure PCTCN2022093906-appb-000001
待测病毒感染滴度随培养天数的变化趋势图分别如图2-1和2-2所示。待测样本PGK-EGFP感染滴度培养3-6天趋于稳定；待测样本EF1α-EGFP 3天达到最高值，后逐渐趋于稳定，有略微下降趋势。The trend charts of the infection titers of the tested viruses with the number of culture days are shown in Figures 2-1 and 2-2, respectively. The PGK-EGFP infection titer of the sample to be tested tended to be stable after 3-6 days of culture; the EF1α-EGFP of the sample to be tested reached the highest value after 3 days, and then gradually stabilized, with a slight downward trend.
接种细胞数量的影响The effect of the number of seeded cells
将293T检测细胞以不同的密度接种于48孔细胞培养板中，接种细胞培养体积为200μl/孔，接种细胞12小时后，将待测病毒以培养基为稀释液稀释到体积为100μl后感染细胞，待测样本EF1α–EGFP稀释80倍，以流式细胞术的方法检测待测样本PGK–EGFP的感染滴度。接种细胞数量对病毒感染效率的影响数据如表2所示，当接种细胞数量在1.5E+05时，测得病毒感染滴度趋于稳定。The 293T test cells were inoculated in 48-well cell culture plates at different densities, and the inoculated cell culture volume was 200 μl/well. After 12 hours of inoculating the cells, the virus to be tested was diluted to a volume of 100 μl with the medium as the diluent, and then the cells were infected. , the test sample EF1α-EGFP was diluted 80 times, and the infection titer of the test sample PGK-EGFP was detected by flow cytometry. The impact data of the number of inoculated cells on the virus infection efficiency is shown in Table 2. When the number of inoculated cells is 1.5E+05, the measured virus infection titer tends to be stable.
表2Table 2
Figure PCTCN2022093906-appb-000002
Figure PCTCN2022093906-appb-000002
以上检测数据显示：待测样本PGK-EGFP感染滴度最高为2.12E+07感染单位每毫升(transduction units,TU/ml)；待测样本EF1α-EGFP感染滴度最高为1.45E+09感染单位每毫升(transduction units,TU/ml)。The above test data show that the highest infectious titer of PGK-EGFP in the sample to be tested is 2.12E+07 infectious units per milliliter (transduction units, TU/ml); the highest infectious titer of EF1α-EGFP in the sample to be tested is 1.45E+09 infectious units per milliliter (transduction units, TU/ml).
实施例4感染滴度检测条件对悬浮MT4检测细胞系为靶细胞的检测系统的影响Example 4 Infection Titer Detection Conditions Influence on the Detection System in which the Suspension MT4 Detection Cell Line is the Target Cell
培养时间的影响Effect of incubation time
以实施例2所述的相同检测方法，将MT4检测细胞以每孔细胞数量为1.00E+05个细胞的密度接种于96孔细胞培养板中，细胞培养体积为50μl/孔，将待测病毒以培养基为稀释液稀释到体积为20μl后感染细胞，以流式细胞术的方法检测待测样本EF1α-EGFP和待测样本PGK–EGFP的感染滴度，其中待测样本EF1α–EGFP稀释80倍和待测样本PGK–EGFP稀释10000倍，培养时间为感染后3-6天，数据如下：With the same detection method described in Example 2, the MT4 detection cells were inoculated in a 96-well cell culture plate at a density of 1.00E+05 cells per well, and the cell culture volume was 50 μl/well, and the virus to be tested was Infect the cells after diluting to a volume of 20 μl with the medium as a diluent, and detect the infection titers of the test sample EF1α-EGFP and the test sample PGK-EGFP by flow cytometry, wherein the test sample EF1α-EGFP is diluted 80 times and the test sample PGK-EGFP was diluted 10,000 times, and the culture time was 3-6 days after infection. The data are as follows:
表3table 3
Figure PCTCN2022093906-appb-000003
Figure PCTCN2022093906-appb-000003
Figure PCTCN2022093906-appb-000004
Figure PCTCN2022093906-appb-000004
待测样本PGK-EGFP和EF1α-EGFP的感染效率随培养天数的变化趋势如图3-1和3-2所示，可见二者均在第4-6天趋于稳定。Figures 3-1 and 3-2 show the trend of the infection efficiency of the tested samples PGK-EGFP and EF1α-EGFP with the number of days of culture. It can be seen that both tend to be stable on the 4th to 6th day.
接种细胞数量的影响The effect of the number of seeded cells
将MT4检测细胞以不同细胞密度接种于96孔细胞培养板中，细胞培养体积为50μl/孔，将待测病毒以培养基为稀释液稀释到体积为20μl后感染细胞，其中待测样本EF1α–EGFP稀释80倍和待测样本PGK–EGFP稀释10000倍，培养时间为感染后3天，以流式细胞术的方法检测待测样本EF1α-EGFP和待测样本PGK–EGFP的感染滴度，接种细胞数量对病毒感染效率的影响数据如表4所示，当接种细胞数量在1.40E+05时，测得两种慢病毒的感染滴度均达到最高值，继续增大接种细胞数量后，两种慢病毒的感染滴度均稍有下降。The MT4 detection cells were seeded in 96-well cell culture plates at different cell densities, and the cell culture volume was 50 μl/well. The virus to be tested was diluted to a volume of 20 μl with the medium as a diluent, and then the cells were infected. The sample to be tested was EF1α– EGFP was diluted 80 times and the test sample PGK–EGFP was diluted 10,000 times, and the culture time was 3 days after infection. The infection titers of the test sample EF1α-EGFP and the test sample PGK–EGFP were detected by flow cytometry, and inoculated The data of the effect of cell number on virus infection efficiency are shown in Table 4. When the number of inoculated cells was 1.40E+05, the infection titers of the two lentiviruses reached the highest values. After continuing to increase the number of inoculated cells, both The infection titers of all lentiviruses decreased slightly.
表4Table 4
Figure PCTCN2022093906-appb-000005
Figure PCTCN2022093906-appb-000005
Figure PCTCN2022093906-appb-000006
Figure PCTCN2022093906-appb-000006
细胞培养体积的影响Effect of Cell Culture Volume
将MT4检测细胞以1.00E+05个细胞/孔的细胞密度接种于96孔细胞培养板中，细胞培养体积为30μl-150μl/孔不等，将待测病毒以培养基为稀释液稀释到体积为20μl后感染细胞，其中待测样本EF1α–EGFP稀释200倍和待测样本PGK–EGFP稀释10000倍，培养时间为感染后3天，以流式细胞术的方法检测待测样本EF1α-EGFP和待测样本PGK–EGFP的感染滴度，不同的细胞培养体积对病毒感染效率的影响数据如表5所示，当细胞数量、病毒数量、感染后培养时间一定时，细胞培养体积越小，慢病毒的滴度越高。(50μl为铺满96孔培养板底部最小体积)Inoculate the MT4 detection cells in a 96-well cell culture plate at a cell density of 1.00E+05 cells/well, the cell culture volume is 30μl-150μl/well, and dilute the virus to be tested to volume with the medium as the diluent After 20 μl of infected cells, the test sample EF1α-EGFP was diluted 200 times and the test sample PGK-EGFP was diluted 10000 times, and the culture time was 3 days after infection. The test sample EF1α-EGFP and The infection titer of the sample PGK-EGFP to be tested, and the data of the influence of different cell culture volumes on the virus infection efficiency are shown in Table 5. When the number of cells, the number of viruses, and the culture time after infection are constant, the smaller the cell culture volume, the slower The higher the titer of the virus. (50μl is the minimum volume to cover the bottom of a 96-well culture plate)
表5table 5
Figure PCTCN2022093906-appb-000007
Figure PCTCN2022093906-appb-000007
Figure PCTCN2022093906-appb-000008
Figure PCTCN2022093906-appb-000008
Figure PCTCN2022093906-appb-000009
Figure PCTCN2022093906-appb-000009
以上感染滴度检测数据显示，以MT4为检测细胞系时：待测样本PGK-EGFP感染滴度最高为1.03E+08感染单位每毫升(transduction units,TU/ml)；待测样本EF1α-EGFP感染滴度最高为1.29E+10感染单位每毫升(transduction units,TU/ml)。分别比以293T为检测细胞系时测 的感染滴度高4.86倍和8.9倍。The above infection titer detection data show that when MT4 is used as the detection cell line: the highest infection titer of PGK-EGFP in the test sample is 1.03E+08 infection units per milliliter (transduction units, TU/ml); the test sample EF1α-EGFP The highest infectious titer was 1.29E+10 infectious units per milliliter (transduction units, TU/ml). They were 4.86 times and 8.9 times higher than the infection titers measured when 293T was used as the detection cell line, respectively.
实施例5多个CD4+T细胞检测细胞系的数据比较Example 5 Data comparison of multiple CD4+ T cell detection cell lines
以实施例2所述的相同检测方法，以MT4、C8166、Jurkat-E6、K562等悬浮细胞系为检测靶细胞，以1.00E+05个细胞/孔的细胞密度接种于96孔细胞培养板中，细胞培养体积为30μl/孔，将待测病毒以培养基为稀释液稀释到体积为20μl后感染细胞，培养时间为感染后3天检测待测样本PGK–EGFP，数据如表6-1至6-4所示。由于CD4+T细胞为悬浮细胞，293T细胞为贴壁细胞，贴壁细胞无法与悬浮细胞在相同的实验条件下进行对比，因此本实施例中未考察相同条件下293T细胞与CD4+T细胞的检测结果，293T细胞的检测结果可参考实施例3中293T的最佳实验条件得出的检测结果，即待测样本PGK-EGFP感染滴度最高为2.12E+07TU/ml，可见相同的病毒用293T细胞检测得到的滴度远低于本实施例中用CD4+T细胞检测得到的滴度。Using the same detection method as described in Example 2, use suspension cell lines such as MT4, C8166, Jurkat-E6, K562 as detection target cells, and inoculate them in 96-well cell culture plates at a cell density of 1.00E+05 cells/well , the cell culture volume is 30 μl/well, the virus to be tested is diluted to a volume of 20 μl with the medium as the diluent, and then the cells are infected. The culture time is 3 days after infection to detect the sample PGK-EGFP to be tested, and the data are shown in Table 6-1 to 6-4 shown. Since CD4+T cells are suspension cells and 293T cells are adherent cells, adherent cells cannot be compared with suspension cells under the same experimental conditions, so the comparison between 293T cells and CD4+T cells under the same conditions was not investigated in this example. Detection results, the detection results of 293T cells can refer to the detection results obtained under the best experimental conditions of 293T in Example 3, that is, the highest infection titer of PGK-EGFP in the sample to be tested is 2.12E+07TU/ml, it can be seen that the same virus can be used for The titer detected by 293T cells is much lower than the titer detected by CD4+T cells in this example.
表6-1以MT4为检测靶细胞Table 6-1 MT4 is used as the detection target cell
Figure PCTCN2022093906-appb-000010
Figure PCTCN2022093906-appb-000010
表6-2以C8166为检测靶细胞Table 6-2 Taking C8166 as the detection target cell
Figure PCTCN2022093906-appb-000011
Figure PCTCN2022093906-appb-000011
Figure PCTCN2022093906-appb-000012
Figure PCTCN2022093906-appb-000012
表6-3以Jurkat-E6为检测靶细胞Table 6-3 Take Jurkat-E6 as the detection target cell
Figure PCTCN2022093906-appb-000013
Figure PCTCN2022093906-appb-000013
表6-4以K562为检测靶细胞Table 6-4 Take K562 as the detection target cell
Figure PCTCN2022093906-appb-000014
Figure PCTCN2022093906-appb-000014
以上感染滴度检测数据显示，以MT4为检测细胞系时：待测样本PGK-EGFP感染滴度高于C8166、Jurkat-E6、K562等悬浮细胞系检测靶细胞，意外的发现MT4为优先的慢病毒感染滴度检测细胞系。The above infection titer detection data show that when MT4 is used as the detection cell line: the infection titer of PGK-EGFP in the sample to be tested is higher than that of the target cells detected by suspension cell lines such as C8166, Jurkat-E6, and K562, and it is unexpectedly found that MT4 is the preferred slow cell line. Virus infection titer assay cell lines.
以上的实施例是为了说明本发明公开的实施方案，并不能理解为对本发明的限制。此外，本文所列出的各种修改以及发明中方法的变化，在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述，但应当理解，本发明不应仅限于这些具体实施例。事实上，各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。The above examples are intended to illustrate the disclosed embodiments of the present invention, and should not be construed as limiting the present invention. In addition, various modifications set forth herein, as well as changes in the method of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in connection with various specific preferred embodiments of the invention, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as mentioned above which are obvious to those skilled in the art to obtain the invention should be included in the scope of the present invention.

Claims (10)

  1. CD4+T细胞系在慢病毒感染滴度检测中的用途。Use of CD4+ T cell line in detection of lentivirus infection titer.
  2. 根据权利要求1所述的用途，其特征在于，所述CD4+T细胞系包括MT4细胞、C8166细胞、Jurkat细胞。The use according to claim 1, characterized in that the CD4+ T cell line comprises MT4 cells, C8166 cells, and Jurkat cells.
  3. 根据权利要求2所述的用途，其特征在于，所述Jurkat选自Jurkat-E6细胞。The use according to claim 2, wherein the Jurkat is selected from Jurkat-E6 cells.
  4. 一种慢病毒感染滴度的检测方法，其特征在于，所述检测方法包括如下步骤：将待测慢病毒接种到CD4+T细胞系的细胞悬液中，继续培养接种待测慢病毒后的CD4+T细胞系，培养结束后检测所述待测慢病毒的感染滴度。A detection method for lentivirus infection titer, characterized in that the detection method comprises the following steps: inoculating the lentivirus to be tested into the cell suspension of CD4+ T cell line, and continuing to cultivate the lentivirus to be tested after being inoculated CD4+T cell line, detect the infection titer of the lentivirus to be tested after the culture is completed.
  5. 根据权利要求4所述的检测方法，其特征在于，将待测慢病毒稀释后再接种到CD4+T细胞系的细胞悬液中。The detection method according to claim 4, characterized in that the lentivirus to be tested is diluted and then inoculated into the cell suspension of the CD4+ T cell line.
  6. 根据权利要求4所述的检测方法，其特征在于，在多孔细胞培养板中进行实验时，CD4+T细胞系的细胞悬液中CD4+T细胞系的细胞数量为每孔4.00E+04～1.60E+05个；和/或，在多孔细胞培养板中进行实验时，CD4+T细胞系的细胞悬液的体积每孔为30～200μl。The detection method according to claim 4, characterized in that, when the experiment is carried out in a multi-well cell culture plate, the number of cells of the CD4+T cell line in the cell suspension of the CD4+T cell line is 4.00E+04 per well. 1. 60E+05 cells; and/or, when the experiment is performed in a multi-well cell culture plate, the volume of the cell suspension of the CD4+ T cell line is 30-200 μl per well.
  7. 根据权利要求4所述的检测方法，其特征在于，接种待测慢病毒后，继续培养时间为2～6天。The detection method according to claim 4, characterized in that, after the lentivirus to be tested is inoculated, the continuous culture time is 2 to 6 days.
  8. 根据权利要求4所述的检测方法，其特征在于，培养结束后通过流式细胞术或荧光定量PCR检测待测慢病毒的感染滴度。The detection method according to claim 4, characterized in that the infection titer of the lentivirus to be tested is detected by flow cytometry or fluorescent quantitative PCR after the culture is completed.
  9. 根据权利要求8所述的检测方法，其特征在于，通过流式细胞术检测时，待测慢病毒的感染滴度按照以下方法计算：将荧光百分比5-30％之间的数据代入以下公式：感染滴度(TU/ml)＝细胞数*荧光百分比*10 3/病毒原液体积(μl)，若不同荧光百分比代入公式后得到的感染滴度数值不同，则选择多次出现的值作为感染滴度，若有多个数值多次出现，则选择多次出现的数值中最大的一个数值作为待测慢病毒的感染滴度。 The detection method according to claim 8, characterized in that, when detected by flow cytometry, the infection titer of the lentivirus to be tested is calculated according to the following method: Substituting the data between 5-30% of the fluorescence percentage into the following formula: Infection titer (TU/ml) = number of cells * fluorescence percentage * 10 3 / volume of virus stock solution (μl), if the values of infection titers obtained after substituting different fluorescence percentages into the formula are different, select the value that appears multiple times as the infection titer If multiple values appear multiple times, select the largest value among the values that appear multiple times as the infection titer of the lentivirus to be tested.
  10. 根据权利要求4所述的检测方法，其特征在于，所述CD4+T细胞系选自MT4细胞、C8166细胞或Jurkat细胞。The detection method according to claim 4, wherein the CD4+ T cell line is selected from MT4 cells, C8166 cells or Jurkat cells.
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