A long-lived plasma cell that secretes PD-1 antibody and its preparation method and application
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A PD-1, plasma cell technology, applied in the field of biomedicine, can solve the problem of low efficiency of large fragment exogenous gene knock-in, and achieve the effect of improving the infection rate
Active Publication Date: 2022-08-05
SUN YAT SEN UNIV
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However, efficiency and safety are the focus of genetic engineering technology. The gene editing efficiency of existing gene editing methods on human primary B cells, especially the knock-in efficiency of large fragments of foreign genes, has always been very low.
So far, no relevant studies and reports have been seen
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Embodiment 1
[0057] Example 1 Preparation of long-lived plasma cells secreting PD-1 antibody
[0058] 1. Plasmid construction and B lymphocyte culture
[0059] (1) Construction of Cas9 / sgRNA expression lentiviral plasmid
[0060] Design sgRNA with Zhang Feng's laboratory sgRNA design tool ((https: / / zlab.bio / guide-design-resources), synthesize oligonucleotide primers, form oligonucleotide primer pairs through annealing reaction, and clone into BsmBI digestion The lentiCRISPR v2 (Addgene) plasmid.
[0063] Annealing reaction procedure: heating at 95°C for 5 minutes, heating at 72°C for 10 minutes, and cooling to room temperature naturally.
[0064] (2) Construction of homologous recombination template lentiviral plasmid
[0065] Human peripheral blood lymphocytes (PBMCs) were isolated, and genomic DNA was extracted with...
Embodiment 2
[0094] Example 2 sgRNA sequence screening
[0095] In order to determine the appropriate sgRNA sequence, three sgRNAs were designed near the terminator of the GAPDH 3'-UTR coding region, as shown in Table 1, and cloned into the lentiCRISPR v2 vector respectively. Construction of viral plasmids. Then HEK293T cells were transfected, and the genomic DNA was extracted after puromycin selection.
[0096] Table 1 Sequences of sgRNAs
[0097]
[0098] Detection of mutants by T7E1 digestion:
[0099] a. Collect cells to extract genomic DNA, use this as a template, and use PCR to amplify DNA fragments with mutation points (sgRNA target sequences);
[0100] b. Configure the following reaction system:
[0102] Heating at 95°C for 5 minutes, naturally cooling to room temperature, and performing annealing reaction;
[0103] c. Add 1 μl T7E1 enzyme for digestion, and incubate at...
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Abstract
The invention discloses a long-lived plasma cell that secretes PD-1 antibody, and a preparation method and application thereof. Specifically, the PD-1 antibody gene is site-specifically integrated into B cells to obtain genetically engineered B cells; the genetically engineered B cells are induced to become long-lived plasma cells that secrete PD-1 antibody. The long-lived plasma cells secreting PD-1 antibody prepared by the present invention not only have the typical phenotype and transcription characteristics of long-lived plasma cells, but also can continuously secrete a large amount of PD-1 antibody, which is expected to replace the tumor mediated by PD-1 antibody drugs Immunotherapy, and the present invention provides a new idea for adoptive cell immunotherapy of tumors. In addition, the present invention introduces antibody genes into B cells and induces long-lived plasma cells that can continuously secrete large amounts of antibodies, and the new idea therapy of returning the long-lived plasma cells to patients for treatment can not only be used for the treatment of various tumors, but also in various infections. It also has broad application prospects in sexually transmitted diseases such as AIDS and chronic viral hepatitis B, as well as in protein-deficiency diseases such as hemophilia.
Description
technical field [0001] The invention belongs to the technical field of biomedicine. More specifically, it relates to a long-lived plasma cell that secretes PD-1 antibody and its preparation method and application. Background technique [0002] Tumors are extremely difficult to treat because of their unclear pathogenesis and high mortality. At present, traditional treatment methods include surgical resection, radiotherapy, and chemotherapy. All traditional treatment methods have defects such as large toxic and side effects, unclear drug targets, and easy tumor recurrence. Immune checkpoint blockade therapy has significant anti-tumor effects. Among them, PD-1-targeted immune checkpoint blockade therapy has achieved major breakthroughs in the field of tumor immunotherapy, such as PD-1 blocking antibodies nivolumab and Mumumab. These antibodies inhibit the exhaustion of T cells by blocking the binding of PD-1 to its ligand PD-L1, thereby exerting anti-tumor efficacy. However...
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