Longevity plasma cell capable of secreting PD-1 antibody as well as preparation method and application of longevity plasma cell
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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 compliance, improving infection rate and broad application prospects
Active Publication Date: 2020-08-18
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] Use the sgRNA design tool of Zhang Feng Laboratory ((https: / / zlab.bio / guide-design-resources) to design sgRNA, synthesize oligonucleotide primers, form oligonucleotide primer pairs through annealing reaction, and clone into BsmBI digestion 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 w...
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 adjacent to the 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 after puromycin selection, genomic DNA was extracted.
[0096] Table 1 sgRNAs sequences
[0097]
[0098] Detection of mutants using T7E1 enzyme digestion method:
[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 sequence);
[0100] b. Configure the following reaction system:
[0102] Heating at 95°C for 5 minutes, naturally cooling to room temperature, and annealing reaction;
[0103] c. Add 1 μl T7E1 enzyme for digestion, ...
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Abstract
The invention discloses a longevity plasma cell capable of secreting a PD-1 antibody as well as a preparation method and application of the longevity plasma cell. The method specifically comprises thefollowing steps: integrating a PD-1 antibody gene into a B cell at a fixed point to obtain a genetically engineered B cell; and inducing the genetically engineered B cell into a longevity plasma cellsecreting a PD-1 antibody. The prepared longevity plasma cell capable of secreting the PD-1 antibody has typical phenotype and transcription characteristics of the longevity plasma cell; in addition,a large number of PD-1 antibodies can be continuously secreted, PD-1 antibody drug-mediated tumor immunotherapy is expected to be replaced, Meanwhile, a new thought is provided for tumor adoptive cellular immunotherapy. Moreover, the antibody gene is introduced into B cells to be induced into long-life plasma cells capable of continuously secreting a large number of antibodies; the new thought therapy for treating the long-life plasma cell transfused patient not only can be used for treating various tumors, but also has a wide application prospect in various infectious diseases such as AIDS (acquired immune deficiency syndrome) and chronic viral hepatitis B, and protein-deficient 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 secreting PD-1 antibody and its preparation method and application. Background technique [0002] Because of its unclear pathogenesis and high mortality rate, tumors are extremely difficult to treat. At present, the traditional treatment methods include surgical resection, radiotherapy, and chemical drug treatment. The traditional treatment methods all have defects such as high toxicity and side effects, unclear drug targets, and easy tumor recurrence. Immune checkpoint blockade therapy has a significant anti-tumor effect. Among them, immune checkpoint blockade therapy targeting PD-1 has made a major breakthrough in the field of tumor immunotherapy, such as the PD-1 blocking antibody nivolumab and Pai Monoclonal antibody. These antibodies inhibit the exhaustion of T cells by blocking the binding of PD-1 and its ligand PD-L1, thereby...
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