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Kit for detecting replicable lentivirus (RCL) and application thereof

A lentivirus, replication technology, applied in applications, viruses, viral peptides, etc., can solve the problems of no inhibition test, low quantitative sensitivity, low consistency, etc., to eliminate false positive results, reduce cytotoxicity, and safety. high effect

Pending Publication Date: 2021-12-14
康美润源(北京)生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the existing methods have the following problems: 1. The positive control of the current RCL detection method has no protein label, and the quantitative sensitivity is low
2. The positive control strains used abroad are not highly consistent with the sequence of the transfer vector in the third-generation packaging system, and there are system differences, which can easily lead to false negatives
3. There is no dose-related inhibition test of the test product, which is prone to false negatives

Method used

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  • Kit for detecting replicable lentivirus (RCL) and application thereof
  • Kit for detecting replicable lentivirus (RCL) and application thereof
  • Kit for detecting replicable lentivirus (RCL) and application thereof

Examples

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Effect test

Embodiment 1

[0065] Example 1 Design and construction of conditionally replicable positive attenuated strain recombinant expression gene NL-TAA plasmid and virus packaging

[0066] HIV NL4 strain gene expression plasmid pUC19-NL4-EGFP (sequence is SEQ ID NO.1) (Wuhan Institute of Virology, Chinese Academy of Sciences). Based on the existing lentiviral vectors to study the gene elements of lentiviral strains of the same origin, design and add stop codons in the genes Vif, VPR, VPU, and Nef to weaken the expression of HIV NL4 strain genes and construct conditions for expressing HIV NL4 The plasmid of replicable attenuated strain, the specific construction process is as follows:

[0067] a. Transform HIV NL4 strain recombinant expression vector pUC19-NL4-EGFP into Escherichia coli competent strain DH-5a, the specific steps are as follows 1-7:

[0068] 1. After 30 minutes of ultraviolet irradiation on the ultra-clean workbench, turn on the fan and run for 5 minutes.

[0069] 2. Take out the ...

Embodiment 2

[0125] Example 2 Positive control, research on setting conditions of inhibition control and complete RCL detection method

[0126] (1) Experiment overview

[0127] ① Use 3 concentrations of NL4-3 HIV-EGFP-ATT virus (1TCID50, 5TCID50, 10TCID50) to infect C8166 cells respectively, do 3 repetitions for each concentration, and explore the amount of NL4-3 HIV-EGFP-ATT virus added in the positive control group .

[0128] ② Two concentrations of NL4-3 HIV-EGFP-ATT virus (5TCID50, 10TCID50) were used to infect C8166 cells respectively, and each test product (cell bank cells producing lentivirus, terminal cells producing lentivirus, Lentiviral supernatant, lentiviral products and final released chimeric antigen receptor cells, final released chimeric antigen receptor cell supernatant, the addition amount of each test product is based on the addition of each test product in the RCL test amount of research results), each concentration was repeated three times to explore the amount of v...

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Abstract

The invention relates to a kit for detecting a replicable lentivirus (RCL) and an application thereof. The kit comprises a recombinant expression vector pUC19-NL4-3HIV-EGFP-ATT or a recombinant bacterium NL4-3HIV-EGFP-ATT, and the recombinant bacterium NL4-3HIV-EGFP-ATT contains the recombinant expression vector pUC19-NL4-3HIV-EGFP-ATT. Compared with the existing method, the kit disclosed by the invention has the advantages that the occurrence of false positive results can be avoided; more detection points are used, the detection result is more reliable, and the safety is higher.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for detecting replicable lentivirus and its application. Background technique [0002] Chimeric Antigen Receptor T cell Immunotherapy (Chimeric Antigen Receptor T cell Immunotherapy), referred to as CAR-T technology. The structure of CAR (chimeric antigen receptor) includes extracellular single-chain antibody region, hinge region, transmembrane region and intracellular signal peptide region. A single-chain antibody (scFv) is composed of the light chain (VL) and heavy chain (VH) variable region fragments of a monoclonal antibody that specifically recognizes an antigen; the hinge region is generally an immunoglobulin sequence, such as CD8α or TCRβ Chain, which has the activity of deforming and stretching, provides more space for deforming and approaching the antigen for single-chain antibodies; the transmembrane region is generally composed of homologous or heterologous dimer me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/65C12N15/64C12N15/49C12N15/47C12N1/21C12N7/01C12N7/04C12Q1/70C12Q1/6851C12R1/19C12R1/93
CPCC12N15/85C12N15/65C07K14/005C12N7/00C12Q1/04C12Q1/702C12Q1/6851C12N2800/107C12N2740/16322C12N2760/20222C12N2740/15022C12N2740/15052C12N2740/15062G01N2333/155C12Q2531/113C12Q2545/114C12Q2563/107
Inventor 岳云强王悦鲁敬芳
Owner 康美润源(北京)生物技术有限公司
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