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Endoglin nucleic acid aptamer based on CRISPR/Cas9 and application

A nucleic acid aptamer and aptamer technology, which is applied in the fields of genetic engineering, immunology and oncology, to achieve the effects of small molecular weight, solving the difficulty of obtaining, and being easy to synthesize and modify

Inactive Publication Date: 2019-04-05
GUANGXI MEDICAL UNIVERSITY
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Problems solved by technology

[0004] As a popular technology in recent years, CRISPR / Cas9 technology has been the object of research and application by many scholars. CRISPR / Cas9 technology is mostly used to knock out (knock out) or knock in (knock in) target genes in target cells Gene therapy, the use of this method to construct cell models for further biological research has yet to be developed

Method used

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  • Endoglin nucleic acid aptamer based on CRISPR/Cas9 and application
  • Endoglin nucleic acid aptamer based on CRISPR/Cas9 and application
  • Endoglin nucleic acid aptamer based on CRISPR/Cas9 and application

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Embodiment

[0029] A kind of Endoglin nucleic acid aptamer and application based on CRISPR / Cas9 of the present invention are realized in the following ways:

[0030] 1. Construction of 293T-CD105 cell line expressing human Endoglin gene

[0031] Using CRISPR / Cas9 technology to insert the pCAG-ENG(cDNA)-pA-PuroR gene into the hAAVS1 site of the human embryonic kidney epithelial cell line 293T cells to construct 293T-CD105 cells stably expressing human Endoglin, and construct an empty vector for transfection 293T cells were used as negative control cells.

[0032] Before screening, it is necessary to investigate the expression of Endoglin protein in target cells and control cells. In the present invention, human CD105-PE antibody is used to incubate target cell 293T-CD105 and control cell 293T, and the expression of Endoglin is identified by flow cytometry. The results are attached figure 1 As shown, the expression level of CD105 protein in 293T-CD105 cells transfected with human Endogli...

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Abstract

The invention discloses Endoglin nucleic acid aptamer based on CRISPR / Cas9 and application, and belongs to the technical field of genetic engineering, immunology and oncology. The Endoglin nucleic acid aptamer is any one or more sequences in nucleotide sequences shown in SEQ ID NO: 1-4. The endoglin nucleic acid aptamer is screened by a whole-cell subtraction Cell-SELEX technology, the molecular weight is small, the endoglin nucleic acid aptamer is easy to synthetize and modify, can recognize Endoglin in a high-specificity mode and can be bonded with Endoglin in a high-affinity mode, does notconduct feature bonding with other cells, is free of immunogenicity, is stable and easy to modify, and is convenient to synthetize and store. An Endoglin detection kit and reagent paper prepared by the Endoglin nucleic acid aptamer have high detection sensitivity to Endoglin, and are prone to optimization. The Endoglin nucleic acid aptamer is applied to assessment of tumor diagnosis and tumor prognosis and monitoring, and tumor diagnosis.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering, immunology and oncology, and in particular relates to a CRISPR / Cas9-based Endoglin nucleic acid aptamer and its application. Background technique [0002] Tumor vascular endothelial cells are the basis of tumor angiogenesis and play a very important role in tumor growth and metastasis. Early diagnosis and treatment of tumors targeting tumor vascular endothelial cells has become an inevitable trend in the development of tumor-related research. Endoglin is a transmembrane glycoprotein on the surface of tumor vascular endothelial cells, overexpressed in actively proliferating vascular endothelial cells, but not or weakly expressed in normal vascular endothelial cells. Therefore, Endoglin is recognized as a specific marker of tumor angiogenesis and neovascularization. [0003] Nucleic acid aptamers are a class of DNA or RNA sequences that can bind to target molecules with high specific...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/68
CPCC12N15/115C12N2310/16G01N33/68
Inventor 赵永祥张志勇薛志刚张坤钟莉娉蒋雅峰
Owner GUANGXI MEDICAL UNIVERSITY
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