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Human PD1 gene sgRNA with high knockout rate, plasmid containing sgRNA and T cell

A knockout rate and cell technology, applied in the field of genetic engineering, can solve problems such as no knockout efficiency, no verification of knockout efficiency, unknown knockout efficiency, etc.

Inactive Publication Date: 2021-09-24
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the CN103820454B patent provides 180 PD1 sgRNA sequences, it only gives the effects of 4 single sgRNAs and 4 double sgRNAs, and does not verify the knockout efficiency of the remaining sgRNAs
Although the CN107586777A patent also provides 131 sgRNAs, it only provides sgRNA activity for its effect, and does not involve specific knockout efficiency
[0005] CN105671083B patent only discloses the effect of one sgRNA sequence, and the knockout efficiency of other sgRNA sequences is unknown

Method used

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  • Human PD1 gene sgRNA with high knockout rate, plasmid containing sgRNA and T cell
  • Human PD1 gene sgRNA with high knockout rate, plasmid containing sgRNA and T cell
  • Human PD1 gene sgRNA with high knockout rate, plasmid containing sgRNA and T cell

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Effect test

Embodiment 1

[0039] Embodiment 1: for the construction of human PD1 gene Crispr / Cas9 carrier

[0040] 1. Screening for effective sgRNA targets targeting the human PD1 gene

[0041] According to the gene sequence of PD1, design an effective sgRNA target for PD1 gene.

[0042] Table 1 lists 8 effective sgRNA target sequences for the PD1 gene.

[0043] Table 1 sgRNA target sequence of target PD1 gene

[0044]

[0045] 2. Construction of recombinant px458 vector

[0046] The vector selected in this invention is the px458 vector (purchased from Addgene), and each sgRNA sequence in Table 1 was constructed into the px458 vector by conventional methods, and the names of the recombinant vectors are px458-PD1-sg1, px458-PD1-sg1, px458-PD1-sg2, px458-PD1-sg3, px458-PD1-sg4, px458-PD1-sg5, px458-PD1-sg6, px458-PD1-sg7, px458-PD1-sg8.

[0047] In the present invention, 8 recombinant plasmids are purified and concentrated by the cesium chloride method, so that the concentration reaches 1 µg / µL. ...

Embodiment 2

[0048] Example 2: Construction of 293T cells stably expressing PD1 gene

[0049]1. Construction of lentivirus expressing PD1 protein

[0050] The lentiviral vector selected in the present invention is CD513B (purchased from addgene, pCDH-CMV-Nluc-EF1α-

[0051] copRFP-T2A-Puro), the full-length sequence of the PD1 gene coding region (GenBank: AY238517.1) was entrusted to Beijing Biomed Gene Technology Co., Ltd. to synthesize the full-length sequence, and the PD1 gene and CD513B vector were double-digested with XbaI and EcoRI, respectively. Recover the digested product, use T4 DNA to connect the two to form a CD513B vector containing the PD1 gene, named CD513B-PD1, and perform sequencing verification. After correctness, extract the plasmid and remove endotoxin. The CD513B-PD1 plasmid extracted in the present invention The concentration is 600ng / µL.

[0052] 2. Use 293T cells to package lentivirus

[0053] 293T cells were revived and used for transfection after 3 passages; in...

Embodiment 3

[0058] Example 3: Determination of PD1 sgRNA knockout efficiency

[0059] 293T-PD1 cells were treated with 1×10 6 cells / well in a six-well plate and incubated overnight.

[0060] Take 4 μg of the 8 different constructed recombinant px458 vectors containing PD1 sgRNA, mix them with 10 μl FuGENE HD transfection reagent, add DMEM medium to 100 μl, let stand for 15 minutes to form a complex, and aspirate the medium in the six-well plate Drop, add the complex that has stood still, add DMEM medium to 2mL, place at 37°C, 5% CO 2 Culture in the incubator for 6h, replace the DMEM medium containing 10Vol%FBS and continue to culture for 48h, and observe the transfection under the fluorescence microscope (see figure 2 ), collect the cells in one well of each group, and use the FLAG flow cytometry antibody to measure the transfection efficiency; at the same time, the cells in the other two wells are passaged, and the cells in each group are collected 3 days after passage, and the PD1 fl...

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Abstract

The invention provides a human PD1 gene sgRNA with a high knockout rate. The sgRNA is used for knocking out a PD1 gene; the nucleotide sequence of the sgRNA is shown as SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 in a sequence table. The sgRNA target spot aiming at the human PD1 gene is transfected into a 293T cell for stably expressing PD1, and the knockout rate of the PD1 gene is 71.92-90.97%; the sgRNA target spot aiming at the human PD1 gene is transfected into the T cell, and the knockout rate of the PD1 gene is 58.26-72.67%; and the sgRNA target spot aiming at the human PD1 gene is transfected into the T cell, and a Crispr-PD 1-T cell is obtained, shows a remarkable specific cytotoxicity effect on a colon cancer cell line LoVo and shows effect-target ratio gradient dependence, namely the higher the effect-target ratio is, the stronger the cytotoxicity effect is.

Description

technical field [0001] The invention relates to a human PD1 gene sgRNA with a high knockout rate, a plasmid and a T cell containing the sgRNA, and belongs to the technical field of genetic engineering. Background technique [0002] PD-1 (programmed death receptor 1, programmed death 1) protein is a member of the immunoglobulin superfamily and can be expressed in activated T cells, B cells and myeloid cells, as well as CD4 - CD8 - thymocytes. PD-1 has two ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC), both of which are new members of the B7 family. PD-1 is an immunosuppressive receptor that interacts with its ligands PD-L1 and PD-L2 to transmit inhibitory signals and play a negative regulatory role in the immune response. The combination of PD-1 on T cells and PD-L1 / PD-L2 in tumor cells can inhibit activated T cells from attacking tumor cells, resulting in the inability of the immune system to play its full role and allowing tumor cells to escape. [0003] Crispr / Cas9 technolo...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N5/10A61K35/17A61P35/00
CPCC12N15/1138C12N5/0636C07K14/70521A61K35/17A61P35/00C12N2310/20C12N2510/00C12N15/85
Inventor 刘明录强邦明韩庆梅冯建海王立新张传鹏金海锋王亮许淼
Owner SHANDONG XINRUI BIOTECH CO LTD
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