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Kit for detecting foreign plasmid residues based on resistance gene and use method of kit

A resistance gene and kit technology, applied in the field of detection kits for exogenous plasmid residues, can solve the problems of limited infection efficiency, tumorigenic risk, long cycle, etc., and achieve good repeatability, good specificity, and high sensitivity Effect

Pending Publication Date: 2020-09-08
SHANGHAI CELL THERAPY GRP CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, CAR-T immunotherapy generally uses lentivirus, mRNA or plasmid transfection to transfect CAR constructs into T cells to direct them to surface-exposed tumor-associated antigens (TAA), such as Chinese patent 201610327646. The CAR-T technology disclosed a CAR-T transgenic vector targeting CD30 replication-defective recombinant lentivirus, but most of the lentivirus infection methods modify the gene insertion site is not clear, the infection efficiency is limited, and the whole process is complicated and difficult. The operation is long and has a potential risk of tumorigenesis
However, we use the plasmid vector to introduce the CAR system (CN 20150638974.7), although the transduction efficiency is greatly improved and the transduction cycle is shortened, but some plasmids will remain in the T cells, which may have an unknown impact on the subsequent therapeutic effect

Method used

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  • Kit for detecting foreign plasmid residues based on resistance gene and use method of kit
  • Kit for detecting foreign plasmid residues based on resistance gene and use method of kit
  • Kit for detecting foreign plasmid residues based on resistance gene and use method of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Example 1: Screening of primer pairs for specific amplification of Kana resistance gene

[0097] 1. Design multiple sets of primer pairs based on Kana resistance gene and commission Shanghai Jereh Biotechnology Co., Ltd. to synthesize. The sequence of the primer pair is shown in Table 1 below.

[0098] Table 1: Sequences of primer pairs 1-4

[0099]

[0100]

[0101] The sequence of the Kana resistance gene is shown in SEQ ID NO:9.

[0102] 2. The specificity and sensitivity screening of primers

[0103] Samples, reagents, instruments

[0104] Instrument: LightCycle480 fluorescent quantitative PCR detector (Roche)

[0105] Sample: Shanghai Jierui Biotechnology Co., Ltd. was commissioned to synthesize CD19CAR exogenous gene (including CD8 signal peptide, scFv, CD8 transmembrane region, CD8 hinge region, 4-1BB and CD3B). The sequence is shown in SEQ ID NO: 14 and is included in it. Introduce a polyclonal restriction site (BglII-XbaI-EcoRI-BamHI) upstream, insert a restriction site ...

Embodiment 2

[0125] Example 2 According to the preferred primer pairs, design probes to detect amplification efficiency

[0126] 1. Design the probes for the amplification of Kana resistance gene and the amplification primers and probes for the internal reference Actin gene. The sequence is shown in Table 5, and the synthesis is commissioned by Shanghai Jereh Biotechnology Co., Ltd. The nucleotides with capital letters in the probe A sequence are locked nucleic acid modified nucleotides.

[0127] Table 5: Primers and probe sequences

[0128]

[0129] Samples, reagents, instruments

[0130] Instrument: LightCycle480 fluorescent quantitative PCR detector (Roche)

[0131] Sample: Plasmid pNB328-CD19CAR (CD19CAR)

[0132] Reagents: Genome extraction kit (Takara), Taqman Master Mix reagent (ABI).

[0133] Take the aforementioned plasmid pNB328-CD19CAR (CD19CAR) as a template, the preferred primer pair 4 and probe A combination; configure the reaction solution according to the reaction system of the fluore...

Embodiment 3

[0141] Example 3: Detection of the residual amount of resistance genes in CAR-T cells

[0142] Instrument: LightCycle480 fluorescent quantitative PCR detector (Roche),

[0143] Samples: 4 CD19CAR-T samples. CD19CAR-T cells were constructed as follows: Peripheral blood mononuclear cells (PBMCs) were isolated and obtained by conventional methods in the field. The PBMCs were adhered and cultured for 2-4 hours, among which the non-adherent suspended cells were the initial T cells. The suspended cells were collected in a 15ml centrifuge tube, centrifuged at 1200rpm for 3min, discarded the supernatant, added physiological saline, centrifuged at 1200rpm for 3min, discarded the physiological Saline, and repeat this step; take a 1.5ml centrifuge tube, add 5 heart tubes, 6 Cells were centrifuged at 1200rpm for 3min, the supernatant was discarded, and the electroporation kit (purchased from Lonza) was added. A total of 100μl of electroporation reagent and 4μg of pNB328-CD19CAR plasmid were ad...

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Abstract

The invention provides a combination of multiple primer pairs. The combination comprises a primer pair 5 and any one, two, three or four selected from primer pairs 1, 2, 3 and 4. The invention also provides a probe, a probe combination and a kit containing the primer pair combination and / or the probe or the probe combination. The invention also provides a method for determining a copy number of CAR plasmid and a method for controlling the residue of the CAR plasmid. The primer pair combination, the probe or the probe combination or the kit provided by the invention is simple and convenient tooperate, is good in specificity, high in sensitivity, high in reaction efficiency and good in reaction system repeatability, and can be used for quantitative detection of the copy number of the CAR plasmid containing the kana resistance gene or quantitative detection of the residual level of the CAR plasmid containing the kana resistance gene in peripheral blood of a patient treated by CAR-T celltransfusion.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a detection kit based on resistance gene-based foreign plasmid residues and a method of use thereof. Background technique [0002] CAR-T cell technology is through genetic recombination of single-chain antibodies (scFv) that recognize tumor-associated antigens and intracellular signal domain "immunoreceptor tyrosine activation motif (ITAM)" in vitro to generate recombinant plasmids, and then Transfection of natural T cells in vitro, purified and expanded T cells express chimeric antigen receptor (CAR), T cells expressing CAR are called CAR-T cells. CAR-T cells can bind tumor antigens in an antigen-dependent, non-MHC-restricted manner, and activate and activate specific tumor-killing effects. At present, CAR immunotherapy has become a new adoptive immunotherapy technology, which has obvious therapeutic effects on many solid tumors, hematological tumors, especially B-cell lympho...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6851
CPCC12Q1/6851C12Q2531/113C12Q2537/16C12Q2545/114C12Q2563/107
Inventor 郝方元金华君张喜艳刘相岑钱其军
Owner SHANGHAI CELL THERAPY GRP CO LTD
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