DNA aptamer targeted PD-L1 extracellular fragment

A technology of PD-L1 and extracellular fragments, applied in the field of bioengineering, can solve the problems of short duration of clinical effects and achieve rapid screening, tight binding, and high specificity

Pending Publication Date: 2021-11-19
优葆优保健康科技(宁波)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, passive immunotherapy through the production of anti-PD-L1/PD antibodies in vitro and then injected into the patient's body can temporarily activate the spontaneously induced immune cells-cytotoxic T lymphocy

Method used

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  • DNA aptamer targeted PD-L1 extracellular fragment
  • DNA aptamer targeted PD-L1 extracellular fragment
  • DNA aptamer targeted PD-L1 extracellular fragment

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specific Embodiment

[0099] S1. Cell preparation: 95% of living cells adhere to the wall, 90% coverage, 5 million to 10 million cells (100mm×20mm culture dish). Washing Buffer twice.

[0100] S2. DNA library: Dissolve 20ul of 0.5mM (10nmol) DNA library into 350ul Binding Buffer, mix well, treat at 95°C for 5min, and quickly cool on ice. Keep on ice until ready to use. If not used immediately, store at -20°C, dissolve on ice when used and keep on ice for future use.

[0101] S3. Binding of DNA library to positively selected cell lines: Prepare DNA library, dissolve in 500ul BindingBuffer, bind cells with 1000ul Binding Buffer, and combine on a shaker at 50rpm for 1 hour.

[0102] S4. Eluting unbound non-specific DNA: washing the cells with washing buffer three times.

[0103] S5. Elution of specifically bound DNA: add 1 ml of DNAse-free pure water to the washed cells, scrape the cells, and transfer them into a 1.5 ml centrifuge tube. Heat at 95°C for 10 minutes and centrifuge at 13,100 g for 5 ...

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Abstract

The invention discloses a DNA aptamer of a targeted PD-L1 extracellular fragment, the aptamer is a nucleic acid aptamer PD-L1-Aptamer screened by adopting a Whole-Cell SELEX and a WCS technical screening method, a negative selection cell line PDL-1-KO cancer cell line constructed in the early stage is utilized, and the PD-L1-Aptamer with high specificity and tight combination is screened out through circulation for 20-30 rounds, and the invention relates to the technical field of bioengineering. According to the DNA aptamer of the targeted PD-L1 extracellular fragment, PDL-1 protein expressed on the surface of a cell is taken as a target spot, a corresponding nucleic acid aptamer (PD-L1-Aptamer) is screened out by adopting a whole-cell SELEX technology, and the screened nucleic acid aptamer is subjected to functional research, so that the application of the DNA aptamer in early diagnosis and treatment is developed, in the early stage, a PDL1 gene knockout tumor cell line is constructed by utilizing a crisp-9 technology, the tumor cell line can be used as a very key negative control cell line in the process of screening nucleic acid aptamers, and the purpose of rapidly screening target molecules is achieved by screening PD-L1 positive cells and negative cells and performing bidirectional screening.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a DNA aptamer targeting the extracellular fragment of PD-L1. Background technique [0002] Cancer is a disease in which cells proliferate uncontrollably due to genetic mutations in cells. It has become a major threat to human health and one of the main causes of human death. Cancer cases and deaths are rising rapidly around the world, with nearly half of new cancer cases occurring in Asia, most of them in China. Traditional tumor treatment methods mainly include surgery, radiotherapy and chemotherapy, but these methods have great limitations. For cancer cells that have undergone proximal invasion or distant metastasis, the recurrence rate of tumor metastasis after surgical resection is high . Radiotherapy and chemotherapy can cause serious damage to the body's own normal cells, especially the hematopoietic system and immune system. In recent years, targeted drug therap...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12N15/10
CPCC12N15/115C12N15/1048C12N2310/16C12N2330/31C12Q2531/113
Inventor 黄必胜胡咏武
Owner 优葆优保健康科技(宁波)有限公司
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