Application of NKG2A in detection of human NK cells in mouse humanized tumor model

A NK cell and tumor technology, applied in the biological field, can solve the problems of inability to apply human cell tissue distribution, inability to distinguish human cells and human tumor cells, research and other problems, and achieve low cost, reduce toxic side effects, and reduce uncertainty. sexual effect

Pending Publication Date: 2022-03-22
安徽中盛溯源生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some DNA sequences that distinguish humans from different animals have been reported in the prior art, they cannot be applied to the study of the tissue distribution of human-derived cells without gene editing in animals with human-derived tumors, because these human-specific DNA sequences cannot distinguish between treatments. Human cells and human tumor cells

Method used

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  • Application of NKG2A in detection of human NK cells in mouse humanized tumor model
  • Application of NKG2A in detection of human NK cells in mouse humanized tumor model
  • Application of NKG2A in detection of human NK cells in mouse humanized tumor model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Screening of Candidate Genes

[0049] 1. Screen candidate genes: Search the expression profiles of NK cells and MOLM13 cells through the NCBI official website, and screen a series of genes that are highly expressed in human NK cells, but not expressed in MOLM13 cells (human tumor cells) or expressed in very few cells.

[0050] The expression levels of candidate specific genes CD2, NKG2A, and NKp46 in NK cells and MOLM13 cells were detected.

[0051] First, use Tiangen RNA extraction kit (RNAprep Pure Cell / Bacteria Kit, #DP403) to extract total mRNA from NK cells and MOLM13 cell samples; according to the amount of 2ug RNA, use Vazyme RNA reverse transcription kit (HiScript II Q RT SuperMix for Qprc, #R223-01) reverse-transcribed mRNA into cDNA, and finally followed the Transgege qPCR kit ( Top Green qPCR SuperMix, #AQ131), the expression of CD2, NKG2A, and NKp46 genes in NK cells and MOLM13 cells was detected by RT-qPCR detection method, and the reaction system is show...

Embodiment 2

[0063] The expression of the obtained genes NKG2A and NKp46 in mouse tissues, MOLM13 cells and NK cells will be screened.

[0064] Mouse tissue preparation: prepare mortar and liquid nitrogen in advance; take out the spleen of the fresh test mouse and put it directly in the mortar poured with liquid nitrogen, add liquid nitrogen while grinding, and keep the liquid nitrogen always present; grind the well Divide the spleen tissue powder into two 1.5ml EB tubes; immediately add 1ml Trizol to one tube, and 700μl lysate from the DP430 kit to the other tube, fully vortex and lyse, 5min (two RNA extraction methods), two The extraction method is shown in Table 4.

[0065] Table 4 Two RNA extraction methods from mouse tissue

[0066] sample method of extraction Elution volume Elution concentration (ng / μl) 260 / 280 260 / 230 1 / 2 mouse spleen powder DP430 30μl 1239.4 2.14 2.02 1 / 2 mouse spleen powder Trizol 50μl 1304 2.05 2.05

[0067] Then RN...

Embodiment 3

[0074] Plasmid standard, cell standard preparation and detection limit determination

[0075] 1. Preparation of plasmid standard

[0076] In this example, the carrier plasmid is pUC57; pUC57, as a commonly used carrier plasmid, does not specifically bind to NKG2A, NKp46 and other gene primers, making the detection more accurate. The plasmid DNA containing NKG2A and NKp46 marker gene structure was prepared by vector plasmid as a standard product. The structure of the standard product is as follows: figure 1 shown. The amplified sequences of the three pairs of primers for plasmid synthesis are shown in Table 6.

[0077] Table 6 Plasmid Synthesis 3 pairs of primers

[0078]

[0079]

[0080] 将三对引物扩增序列首尾相连形成融合基因(SEQ ID NO:19,AGCTCCATTTTAGCAACTGAACAGGAAATAACCTATGCGGAATTAAACCTTCAAAAAGCTTCTCAGGATTTTCAAGGGAATGACAAAACCTATCACTGCAAAGATTTACCATCAGCTCCAGAGAAGCTCATTGTTGGGATCCTGGGAATTATCTGTCTTATCTTAATGGCCTCTGTGGTAACGATAGTTGTGGACCCGAAGTGATCTCGGGAGAGAAGGTGACCTTCTACTGCCGTCTAGACACTGCAACA...

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Abstract

The invention provides application of NKG2A in detection of human NK cells in a mouse humanized tumor model, and belongs to the technical field of biology. The invention relates to an application of NKG2A in detection of human NK cells in a mouse humanized tumor model. The NKG2A fragment is adopted as a detection target, human NK cells and human tumor cells can be distinguished in a mouse tumor-bearing tumor model, and the NKG2A fragment has the characteristics of short detection time, high detection efficiency and high detection sensitivity, is beneficial to research on non-clinical pharmacokinetics, particularly animal experiments, exploration of in-vivo dynamic and prediction of a treatment scheme in the cell treatment research and development process, and has a wide application prospect. The method is very helpful for reducing the uncertainty of cell therapy, increasing the curative effect and reducing the toxic and side effects, and has important clinical guiding significance.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the application of NKG2A in detecting human NK cells in mouse human tumor models. Background technique [0002] NK cells generally refer to natural killer cells (natural killer cells, NK), which are important immune cells in the body. Perforin and tumor necrosis factor, destroy target cells. NK cells are not only related to anti-tumor, anti-viral infection and immune regulation, but also participate in the occurrence of hypersensitivity and autoimmune diseases in some cases, and can recognize target cells and killing mediators. Common NK cells include NK cells derived from peripheral blood, NK cells derived from cord blood, and NK cells differentiated from hPSCs. [0003] NK cells do not express genetically rearranged antigen-specific receptors, but instead rely on receptors encoded by a series of germline genes. Activating receptors (NKp46, NKp44, NKp30, NKG2D, etc.) ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/12G01N33/68
CPCC12Q1/6888C12Q1/686G01N33/6872G01N33/56972C12Q2600/158G01N2333/70503C12Q2563/107C12Q2521/107
Inventor 高雅丽俞君英张颖焦璐琰
Owner 安徽中盛溯源生物科技有限公司
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