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Humanized pd-l1 tumor cell line and its animal model and application

A technology of PD-L1 and tumor cells, applied in the field of biomedicine, can solve the problems of high cost and time-consuming humanization of mice, and achieve the effects of reducing economic expenditure, strong killing and proliferation ability

Active Publication Date: 2019-07-23
SUZHOU INST OF SYST MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This type of method plays a very important role in the preclinical drug evaluation process, but there are also many shortcomings. The first point is that the establishment of humanized mice is not only time-consuming but also relatively expensive

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  • Humanized pd-l1 tumor cell line and its animal model and application
  • Humanized pd-l1 tumor cell line and its animal model and application
  • Humanized pd-l1 tumor cell line and its animal model and application

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Embodiment Construction

[0047] Knockout of murine PD-L1 and expression of human PD-L1 in colon cancer cells MC-38.

[0048] Studies have shown that PD-L1 expressed on the surface of tumor cells in tumor tissue acts as an anti-tumor suppressor molecule 20 . This discovery prompted us to establish a more direct and economical animal model. The method of establishing the model is as follows: figure 1 Shown:

[0049] Step 1: Knock out mouse-derived PD-L1 (mPD-L1) through CRISPR-Cas9: According to CRISPR-Cas9 technology, first design a short-chain guide RNA (sgRNA) through the http: / / crispr.mit.edu website , the design sequence is as follows: 5'-GCTTGCGTTAGTGGTGTACT-3'. The synthesized DNA duplex was inserted into the sgRNA expression vector (FG-BB-U6-sgRNA) through BbsI. Then co-transfect MC-38 cells with the Cas9 expression plasmid (FG-hEF / HTLV-Cas9-PGK-Puro-WPRE), and select with puro for 48 hours after 48 hours.

[0050]Then expand the culture, obtain the knockout cell bank, extract the DNA and p...

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Abstract

The invention provides a humanized PD-L1 tumor cell line MC-38-hPD-L1, a builtanimal tumor model with the same and a method for constructing the humanized PD-L1 tumor cell line. The method particularly includes knocking out animal-origin PD-L1 by the aid of CRISPR-CAS9; carrying out amplification and cultivation to obtain knocked-out cell banks; extracting DNA (deoxyribonucleic acid) and carrying out PCR (polymerase chain reaction) amplification; recycling and cloning amplification products; carrying out over-expression on human-origin PD-L1 in MC-38 cell lines of mPD-L1 KO by the aid of lentivirus systems; packaging lentivirus and screening Puromycin to obtain the humanized MC-38 cell line of PD-L1. The humanized PD-L1 tumor cell line, the animal tumor model and the method have the advantages that as shown by results, high killing efficiency and multiplication capacity are obviously presented by tumor infiltration CD8 T lymphocytes after antibody treatment is carried out, tumor infiltration Treg cells can be obviously inhibited after antibody treatment is carried out, and accordingly the method is proved to be effective and feasible from the aspect of molecular mechanisms.

Description

technical field [0001] The invention relates to a humanized PD-L1 cell line for evaluating the curative effect of PD-L1 antibody, a mouse model with the cell line, and a preparation method of the cell line and the model, belonging to the technical field of biomedicine. Background technique [0002] In recent years, targeted therapy and immunotherapy have increasingly become new strategies for tumor treatment. Immunotherapy, which specifically blocks immune nodes, has gradually become the mainstream of treatment, and has achieved exciting curative effects. The way to specifically block the immune node is mainly to apply the monoclonal antibody that can directly block the immune node. To sum up, now there are mainly killer T lymphocyte antigen 4 (CTLA-4) 1,2 , Programmed cell death protein 1 (PD-1) 3-7 and programmed cell death ligand 1 (PD-L1) 8-12 , These several kinds of immunoregulatory molecules can provide negative control signals to T lymphocytes, thereby inhibiting...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/867C12N15/12A01K67/027A61K49/00A61K39/395A61P35/00
CPCA01K67/027A01K2227/105A01K2227/107A01K2267/0331A61K49/0008A61K2039/505C07K14/705
Inventor 秦晓峰黄安飞彭迪
Owner SUZHOU INST OF SYST MEDICINE