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TCR gene targeted to CD19, preparation method, plasmid with TCR gene, kit and application

A kit and gene technology, applied in the field of genes, can solve the problems of low experimental success rate, cumbersome analysis operation, long process and so on.

Active Publication Date: 2018-04-17
上海兴瑞一达生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned technologies have problems such as many steps, long process, cumbersome analysis operations, and low success rate of experiments.

Method used

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  • TCR gene targeted to CD19, preparation method, plasmid with TCR gene, kit and application
  • TCR gene targeted to CD19, preparation method, plasmid with TCR gene, kit and application
  • TCR gene targeted to CD19, preparation method, plasmid with TCR gene, kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] A safe and efficient TCR gene targeting CD19, the nucleic acid sequence of the gene is SEQ ID NO.1. Such as figure 1 As shown, the TCR gene is composed of CD8leader, CD19scFv, TRBC, T2A, CD8leader, myc-tag, and TRAC in series; wherein, the nucleic acid artificial sequence of the CD8leader is SEQ ID NO.2; the nucleic acid artificial sequence of the CD19scFv It is SEQ ID NO.3; the TRBC nucleic acid artificial sequence is SEQ ID NO.4; the self-cleaving polypeptide T2A nucleic acid artificial sequence is SEQ ID NO.5; the myc-tag nucleic acid artificial sequence is SEQ ID NO.6 ; The artificial sequence of the TRAC nucleic acid is SEQID NO.7.

Embodiment 2

[0071]The preparation method of the TCR gene comprises the following steps: according to the guide CD8leader nucleic acid artificial sequence, CD19scFv nucleic acid artificial sequence, TRBC nucleic acid artificial sequence, self-cleavage polypeptide T2A nucleic acid artificial sequence, myc-tag nucleic acid artificial sequence, TRAC nucleic acid artificial sequence entrusted Sangon Bioengineering (Shanghai) Co., Ltd. synthesized the entire expression cassette and inserted it into the standard vector pUC, so it was named pUC-Anti-CD19-TCR; pUC-Anti-CD19-TCR was subjected to Fast DigestAsiSI (purchased from ThermoFisher) and Fast Digest NotI (purchased from ThermoFisher Company) double enzyme digestion, 37°C, digestion 20min, 100μl digestion system: 10×buffer: 10μl; DNA 6μg; AsiSI enzyme: 3μl; NotI enzyme: 3μl; deionized water to make up the volume ; Utilize agar gel electrophoresis to excise the agar part containing the Anti-CD19-TCR DNA fragment, and place it in a centrifuge t...

Embodiment 3

[0073] The fusion gene fragment CD8leader-CD19scFv-TRBC-T2A-CD8leader-myc-tag-TRAC was inserted into the lentiviral expression vector pLent-C-GFP.

[0074] According to the guide CD8leader nucleic acid artificial sequence, CD19scFv nucleic acid artificial sequence, TRBC nucleic acid artificial sequence, self-cleavage polypeptide T2A nucleic acid artificial sequence, myc-tag nucleic acid artificial sequence, and TRAC nucleic acid artificial sequence, we commissioned Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize them Its entire expression cassette was inserted into the standard vector pUC, so it was named pUC-Anti-CD19-TCR, and the pUC-Anti-CD19-TCR and pLent-C-GFP vectors were carried out to Fast Digest AsiSI (purchased from ThermoFisher Company) and Fast Digest NotI (purchased from ThermoFisher Company) double enzyme digestion, 37 ° C, digestion 20min. 100μl enzyme digestion system: 10×buffer: 10μl; DNA 6μg; AsiSI enzyme: 3μl; NotI enzyme: 3μl; deionized water to ma...

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Abstract

The invention discloses a safe and efficient TCR gene targeted to CD19. The TCR gene is characterized in that a nucleotide sequence of the TCR gene is SEQ ID NO.1, and the TCR gene is formed by CD8leader, CD19scFv, TRBC, T2A, CD8leader, myc-tag and TRAC which are sequentially connected in series. The invention also discloses a preparation method of the TCR gene, a plasmid with the TCR gene, a kitand an application. The TCR gene disclosed by the invention has the advantages that clinical effectiveness and safety of a TCR technology are improved, and fussy experimental operations are also avoided.

Description

technical field [0001] The invention relates to the field of gene technology, in particular to a safe and high-efficiency TCR gene targeting CD19, a preparation method, a plasmid with the gene, and applications. Background technique [0002] Leukemia classification and prognosis stratification are complex, and its treatment has become a difficult problem. CD19 is a specific surface protein of normal and malignant B lymphocytes, which plays an important role in the development, proliferation and differentiation of B cells as well as malignant transformation. Due to the specificity of CD19 expression in B lymphocytes and the widespread expression of malignant tumors, it has become a potential molecular target for immunotherapy of B lymphocyte malignant tumors. Chimeric antigen receptor T cells targeting CD19 (CD19-CAR-T) can achieve a complete remission rate of over 90% in the treatment of refractory relapsed acute lymphoblastic leukemia in children and adults, and a complete...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/10C12N15/867C12N15/66A61K35/17A61P35/02
CPCA61K35/17C07K14/7051C07K14/70517C07K16/2803C07K2317/622C07K2319/41C12N15/62C12N15/66C12N15/86C12N2740/15043
Inventor 刘明录王立新韩国英万磊韩庆梅王亮卢永灿刘敏
Owner 上海兴瑞一达生物科技有限公司
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