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Anti-GCC nucleic acid, preparation method thereof, immune cell having the nucleic acid, and application thereof

An immune cell and nucleic acid technology, applied in the field of genes, can solve the problems of less than 50% survival rate, recurrence and distant metastasis of colorectal cancer patients, and achieve the effect of improving clinical effectiveness.

Inactive Publication Date: 2018-12-07
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although surgery is an effective treatment, nearly 1 / 3 of colorectal cancer patients still have recurrence and distant metastasis after surgery, and the 5-year survival rate is less than 50%.

Method used

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  • Anti-GCC nucleic acid, preparation method thereof, immune cell having the nucleic acid, and application thereof
  • Anti-GCC nucleic acid, preparation method thereof, immune cell having the nucleic acid, and application thereof
  • Anti-GCC nucleic acid, preparation method thereof, immune cell having the nucleic acid, and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Anti-GCC nucleic acid comprising at least a Leader nucleotide sequence, a GCC single-chain antibody nucleic acid artificial sequence, a CD8 hinge region nucleotide sequence, a transmembrane-stimulatory domain nucleotide sequence, and a CD3ζ signaling region nucleotide sequence , the self-cleaving peptide T2A nucleotide sequence, the chemokine IL7 nucleotide sequence and the chemokine CCL19 nucleotide sequence. Wherein, the transmembrane-stimulatory domain nucleotide sequence is selected from CD8, CD27, CD28, CD137 (ie 4-1BB), CD134 (ie OX40), all or part of DNA fragments of ICOS molecules.

[0050] In this embodiment, the nucleic acid of described anti-GCC, it comprises CD8 Leader nucleic acid artificial sequence (SEQ ID NO.2), GCC single-chain antibody nucleic acid artificial sequence (SEQ ID NO. 3), CD8 hinge region nucleic acid artificial sequence (SEQ ID NO. 4), CD8 transmembrane region nucleic acid artificial sequence (SEQ ID NO. 5), 4-1BB costimulatory region nucl...

Embodiment 2

[0052] The preparation method of the nucleic acid for the treatment of GCC, comprises the steps:

[0053] (1) According to the artificial sequence of Leader nucleic acid, artificial nucleic acid sequence of GCC single-chain antibody, artificial nucleic acid sequence of CD8 hinge region, artificial nucleic acid sequence of CD8 transmembrane region, artificial nucleic acid sequence of 4-1BB costimulatory region, artificial nucleic acid sequence of CD3ζ signaling region , Synthesize the entire expression frame of the self-cleaving peptide T2A nucleic acid artificial sequence, the IL7 nucleic acid artificial sequence, the self-cleaving peptide T2A nucleic acid artificial sequence, and the CCL19 nucleic acid artificial sequence and insert it into the standard vector pUC to obtain pUC-GCC-CAR;

[0054] (2) Double-enzyme digestion of pUC-GCC-CAR, cut off the agar part of the GCC-CAR DNA fragment by agarose gel electrophoresis, treated with DNA extraction kit sol solution, passed throu...

Embodiment 3

[0059] The plasmid containing the gene described in Example 2, the plasmid is a plasmid obtained by inserting the fusion gene fragment GCC-CAR DNA into the lentiviral expression vector pLent-C-GFP, named 7×19pLent-GCC-CAR. The plasmid was prepared by the following preparation method: the above-purified GCC-CAR DNA fragment and the linearized pLent-C-GFP DNA fragment were ligated in the following system: 10×buffer: 1 μl; T4 ligase: 1 μl; GCC-CAR DNA fragment: 4 μl; linearized pLent-C-GFP DNA fragment: 4 μl, formed by ligation.

[0060] In the present embodiment, this plasmid is prepared by the following method:

[0061] According to the artificial sequence of Leader nucleic acid, artificial nucleic acid sequence of GCC single chain antibody, artificial nucleic acid sequence of CD8 hinge region, artificial nucleic acid sequence of CD8 transmembrane region, artificial nucleic acid sequence of 4-1BB costimulatory region, artificial nucleic acid sequence of CD3ζ signal transduction...

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Abstract

An anti-GCC nucleic acid, a preparation method thereof, an immune cell having the nucleic acid, and an application thereof are provided. The anti-GCC nucleic acid at least includes: a Leader nucleotide sequence, a GCC single chain antibody nucleotide artificial sequence, a CD8 hinge region nucleotide sequence, a transmembrane-stimulation structural domain nucleotide sequence, a CD3[zeta] signal transduction region nucleotide sequence, a self-lytic peptide T2A nucleotide sequence, a chemotactic factor IL7 nucleotide sequence and a chemotactic factor CCL19 nucleotide sequence. The anti-GCC nucleic acid can improve the pertinence of the immune cell and reduce the damage on non-tumor cells, thereby improving effectiveness and safety.

Description

technical field [0001] The present invention relates to the field of gene technology, in particular to an anti-GCC nucleic acid, a preparation method thereof, an immune cell having the nucleic acid and applications thereof. Background technique [0002] Colorectal cancer is one of the common digestive tract malignant tumors, accounting for 10% of all tumors in the world, and it ranks third in the incidence of malignant tumors in the world. Although surgery is an effective treatment method, nearly one-third of colorectal cancer patients still experience recurrence and distant metastasis after surgery, and the 5-year survival rate is less than 50%. At present, relevant studies have also shown that the incidence of colorectal disease is increasing year by year and becoming younger, which seriously affects human health. Therefore, early detection and early treatment are the keys to improve the curative effect, prognosis and survival rate of colorectal cancer. [0003] In the t...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/10C12N5/10A61K35/17A61P35/00
CPCA61P35/00A61K35/17C12N5/0636C12N5/0646C07K14/522C07K14/523C07K16/00C07K2319/02C07K2319/03C12N2510/00A61K39/4611A61K39/0011A61K39/4644A61K39/4631C07K14/7051
Inventor 刘明录王立新卢永灿马洪华王亮刘敏韩国英冯建海
Owner SHANDONG XINRUI BIOTECH CO LTD
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