Micro-annullus DNA recombinant plasmid containing recombinant chimeric antigen receptor gene expression box, micro-annullus DNA containing expression box and application

A chimeric antigen receptor and gene expression cassette technology, which is applied in the field of biotechnology and can solve the problems of fatal immune response, high cost of viral vectors, insertion and mutation of foreign genes, etc.

Inactive Publication Date: 2015-04-29
SHENZHEN INST OF ADVANCED TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, viral vectors have a high transfection rate in vivo, but the cost of preparing viral vectors is high, and viral vectors have the risk of insertion mutation and immunogenicity, which will bring the risk of cancer induction and may trigger fatal immune responses in patients
Traditional plasmid vectors are safer than viral vectors, but have a lower transfection rate than viral vectors, and foreign genes can only be expressed transiently. For stable and long-l

Method used

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  • Micro-annullus DNA recombinant plasmid containing recombinant chimeric antigen receptor gene expression box, micro-annullus DNA containing expression box and application
  • Micro-annullus DNA recombinant plasmid containing recombinant chimeric antigen receptor gene expression box, micro-annullus DNA containing expression box and application
  • Micro-annullus DNA recombinant plasmid containing recombinant chimeric antigen receptor gene expression box, micro-annullus DNA containing expression box and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0248] This embodiment provides a method for constructing a minicircle DNA recombinant parent plasmid p2ФC31.CAR containing a recombinant chimeric antigen receptor gene expression cassette, which includes the following steps:

[0249] (1) This example provides a method for constructing a pUC57.CAR recombinant vector containing a recombinant chimeric antigen receptor (CAR) gene

[0250] (1a) Synthesize the sequence of HindⅢ restriction site-CAR gene sequence (SEQ ID NO:16-SEQ ID NO:8-SEQ ID NO:9-SEQ ID NO:10-SEQ ID NO:11-SEQ ID NO:12-SEQ ID NO:13-SEQ ID NO:14)-stop codon (TAG)-EcoRI restriction site nucleotide sequence, wherein, SEQ ID NO:8- SEQ ID NO:9-SEQ ID NO:10-SEQ ID NO:11-SEQ ID NO:12-SEQ ID NO:13-SEQ ID NO:14 corresponding to the CD8 signal peptide encoding CAR-tumor cell epitope Binding site – hinge region – transmembrane region of type I transmembrane protein – activates the intracellular signal transduction region of immune effector cells:

[0251] The SEQ ID NO: 16 encod...

Embodiment 2

[0263] This embodiment combines as Image 6 The shown construction flow chart of minicircle DNA containing recombinant chimeric antigen receptor gene expression cassette provides a method for preparing minicircle DNA containing recombinant chimeric antigen receptor gene expression cassette, including the following steps:

[0264] (1a) Transform the p2ФC31.CAR plasmid obtained in Example 1 into TOP10 and culture it overnight;

[0265] (1b) Suspend the bacteria in the culture broth obtained in step (1a) in a fresh induction solution containing 1% arabinose, incubate at 32°C with shaking (250 times / min) for 2 hours to induce the expression of recombinant enzyme ΦC31, and mediate Form the bacterial plasmid (pBackbone) DNA loop containing the attL site and the minicircle DNA containing the attR site;

[0266] (1c) Then the incubation temperature was increased to 37°C, and the endonuclease I-Sce1 was induced to cut the built-in I-Sce1 restriction site on pBackbone. The chain-shaped pBackbo...

Embodiment 3

[0268] This embodiment provides a method for constructing a minicircle DNA recombinant parent plasmid pMC.CAR containing a recombinant chimeric antigen receptor gene expression cassette, which includes the following steps:

[0269] (1) Refer to the step of Example 1 (1) Construction of pUC57.CAR recombinant vector containing recombinant chimeric antigen receptor (CAR) gene

[0270] (2) Refer to the step (2) of Example 1 to construct the pEF1a.bpA.CAR recombinant expression vector containing the recombinant chimeric antigen receptor gene expression cassette

[0271] (3) Such as Figure 8 As shown, this example provides a method for constructing a pMC.CAR minicircle DNA recombinant parent plasmid containing a recombinant chimeric antigen receptor gene expression cassette

[0272] (3a) Culture the positive clones selected in step (1c), and purify the pEF1a.bpA.CAR recombinant expression vector with a plasmid purification kit (OMEGA).

[0273] (3b) Double digestion of pMC.BESPX vector and p...

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Abstract

The invention provides a micro-annullus DNA recombinant plasmid containing a recombinant chimeric antigen receptor gene expression box and a preparation method of the micro-annullus DNA recombinant plasmid. The micro-annullus DNA recombinant plasmid can be used for implementing site-specific recombination in host bacteria, eliminating a prokaryotic plasmid part and forming micro-annullus DNA containing the recombinant chimeric antigen receptor gene expression box. The invention also provides the micro-annullus DNA containing the recombinant chimeric antigen receptor gene expression box as well as the preparation method and application of the micro-annullus DNA; the micro-annullus DNA can be used for stably and lastingly expressing a recombinant chimeric antigen receptor gene in a host cell and is an efficient and safe gene treatment vector; moreover, the invention also provides the host cell containing the micro-annullus DNA as well as the preparation method and application of the host cell; besides, the invention also provides a recombinant chimeric antigen receptor as well as the preparation method and application thereof.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to a minicircle DNA recombinant parent plasmid containing a recombinant chimeric antigen receptor gene expression cassette, a construction method and application thereof, and a microcircle DNA recombinant parent plasmid containing a recombinant chimeric antigen receptor gene expression cassette Minicircle DNA and its construction method and application, a host cell with microcircle DNA containing a recombinant chimeric antigen receptor gene expression cassette and its application, and a recombinant chimeric antigen receptor and its expression method and application. Background technique [0002] Clinically, recombinant chimeric antigen receptors have been used to treat various diseases, including cancer and autoimmune diseases. However, the current recombinant chimeric antigen receptors are generally produced in vitro and then injected into the body for treatment. This method has man...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/85A61K48/00A61P35/00C07K19/00
Inventor 陈志英马飞何成宜
Owner SHENZHEN INST OF ADVANCED TECH
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