Preparation method of PD-1 (programmed death 1) gene deficiency type T-lymphocyte preparation

A technology for lymphocytes and gene defects, applied in the field of molecular biology, can solve the problems of complex antibody preparation and purification process, expensive antibody drugs, and high manufacturing costs, and achieve the effects of increasing speed, ensuring survival rate, and simplifying production procedures

Active Publication Date: 2017-12-15
百福生命科学研究(珠海横琴)股份有限公司
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Problems solved by technology

However, there is currently no PD-1 antibody in China, and PD-1 antibody therapeutic drugs are still under development and have not yet entered clinical trials
In addition, the preparation and purification proce

Method used

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  • Preparation method of PD-1 (programmed death 1) gene deficiency type T-lymphocyte preparation
  • Preparation method of PD-1 (programmed death 1) gene deficiency type T-lymphocyte preparation
  • Preparation method of PD-1 (programmed death 1) gene deficiency type T-lymphocyte preparation

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Experimental program
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Effect test

Embodiment 1

[0046] (1) Construction of PD-1 knockout vector

[0047] ① Design of gRNA target sequence

[0048] Obtain the full gene sequence of PD-1 (EF064716.1) from NCBI, design the gRNA target sequence based on the CRISPR-DO website, and then select two suitable target sequences (each 20bp in length) according to the set parameters, and then send them to Shanghai Sangong Bioengineering Co., Ltd. synthesized two complementary target sequences (adding sticky ends after BbsI digestion).

[0049] For example: Target1: F 5'-ATAGTGTGAGGAGTGGATAGGCCA-3'

[0050] R 5'-AAATTGGCCTATCCACTCCTCCACA-3'

[0051] Target2: F 5'-ATAGAGGGCCCGGCGCAATGACAG-3'

[0052] R 5'-AAATCTGTCATTGCGCCGGGCCCT-3'

[0053] ② Knockout vector construction

[0054] The pU6gRNA-CMV-Cas9-GFP expression vector was digested with BbsI, and after recovery, it was ligated with the oligo double strand formed by the target1 sequence to construct a PD-1 gene knockout vector containing a gRNA ( figure 1 ).

[0055] Using the p...

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Abstract

The invention belongs to the technical field of molecular biology and particularly relates to a preparation method of a PD-1 (programmed death 1) gene deficiency type T-lymphocyte preparation. The preparation method comprises steps of construction of a PD-1 knockout vector, separation and activation of T-lymphocytes, electrotransfection of the T-lymphocytes, identification by T7E1 restriction enzyme digestion, flow cytometer screening and sequencing analysis. By means production of the gene deficiency type immune cell preparation, on one hand, the cell production routine is simplified, and on the other hand, the preparation obtaining speed is increased.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a preparation method of a PD-1 gene-deficient T lymphocyte preparation. Background technique [0002] The CAR-T technology in cellular immunotherapy (T lymphocytes are modified by genetic engineering) is currently the most effective tumor cell therapy. It has been developed to the fourth generation by continuously increasing the number of intracellular signaling molecules of CAR elements. Compared with traditional non-specific DC cell therapy, it has the advantages of strong specificity, significant curative effect, and low side effects, and has a significant curative effect in blood cancer. However, cellular immunotherapy has its disadvantages. The fundamental reason is that the PD-1 gene in T lymphocytes, PD-1 (Programmed Death 1) programmed death receptor-1: is an important immunosuppressive molecule. One mechanism by which cancer cells evade immune kill...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10
CPCC12N5/0636C12N15/85C12N2510/00
Inventor 王清路
Owner 百福生命科学研究(珠海横琴)股份有限公司
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