Transposase for efficiently mediating exogenous gene integration, and use thereof

A transposase and human-derived technology, applied in the field of molecular biology, can solve problems such as limited loading capacity, low integration efficiency, and complicated preparation process of recombinant virus particles

Active Publication Date: 2016-04-13
SHANGHAI CELL THERAPY RES INST +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(2) Although the host cells have undergone multiple passages or conditional changes, the expression level remains stable
[0004] In order to achieve stable expression of exogenous genes in host cells, commonly used vector systems include: 1. Retrovirus system: can effectively infect host cells and mediate the efficient integration of exogenous gene expression cassettes into the genome, but its loading capacity is limited , and the preparation process of recombinant virus particles is complex
2. Eukaryotic expression plasmid system: The preparation process is relatively simple, but it is inserted into the host genome through random DNA recombination, and the integration efficiency is extremely low
3. Transposon system: the plasmid system is used, the preparation process is relatively simple, and the foreign gene is integrated into the genome by transposase, and the integration efficiency is relatively low
However, HIV-TAT is a virus-derived protein, which will increase the immunogenicity of the fusion protein and is not suitable for in vivo applications

Method used

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  • Transposase for efficiently mediating exogenous gene integration, and use thereof
  • Transposase for efficiently mediating exogenous gene integration, and use thereof
  • Transposase for efficiently mediating exogenous gene integration, and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Embodiment 1: Construction of pNB vector

[0121] According to the sequence of PiggyBac transposon 5' terminal repeat (SEQ ID NO: 1), polyclonal insertion site (SEQ ID NO: 2), polyA tailing signal sequence (SEQ ID NO: 3), PiggyBac transposon 3' terminal repeat (SEQ ID NO :4), PiggyBac transposase coding sequence (SEQIDNO:5) containing c-myc nuclear localization signal, CMV promoter sequence (SEQIDNO:6), spliced ​​into a long sequence (SEQIDNO:7), wherein contains c-myc The PiggyBac transposase coding sequence of the nuclear localization signal and the reverse complement of the CMV promoter sequence sequence (reverse complement here means that the expression frame of the exogenous gene is in the opposite direction to the expression frame of the PB gene, so the PiggyBac transposase coding sequence is displayed. Sequence, the reverse complementary sequence of the CMV promoter sequence), commissioned by Shanghai Jereh Biotechnology Co., Ltd. to synthesize, and added AscI ...

Embodiment 2

[0122] Embodiment 2: Construction of the pNB vector containing exogenous gene expression cassette

[0123] 1. According to the sequence of the EF1α promoter, entrust Shanghai Jereh Biotechnology Co., Ltd. to synthesize it, add XbaI and EcoRI restriction sites at both ends, load the pNB vector prepared in the previous example 1, and name it pNB328 vector.

[0124] The EF1α promoter sequence is shown in SEQ ID NO:8.

[0125] 2. According to the coding sequence of EGFP, entrust Shanghai Jereh Biotechnology Co., Ltd. to synthesize it, and add EcoRI and SalI restriction sites at both ends, load it into pNB328 vector, and name it pNB328-EGFP vector.

[0126] The EGFP coding sequence is shown in SEQ ID NO:9.

[0127] 3. According to the Luc luciferase coding sequence, entrust Shanghai Jereh Biotechnology Co., Ltd. to synthesize it, and add EcoRI and SalI restriction sites at both ends, load it into pNB328 vector, and name it pNB328-Luc vector.

[0128] The coding sequence of Luc ...

Embodiment 3

[0131] Example 3: Construction of PB vector and analysis of nuclear localization

[0132] The inventors used the method of immunofluorescence hybridization to detect the change of protein subcellular localization before and after artificially increasing the nuclear localization signal of PB transposase. Since there is no PB transposase antibody on the market, the inventors fused a His tag in front of PB, and used the His tag antibody to detect the subcellular localization of the His-PB fusion protein.

[0133] Specific steps are as follows:

[0134] 1. Construction of pcDNA3.1-cPB vector

[0135] According to the human c-myc nuclear localization signal coding sequence, 6×His tag coding sequence, and PB transposase coding sequence, a coding sequence of myc-his-PB fusion protein (SEQ ID NO: 17) was spliced ​​into a piece of myc-his-PB fusion protein coding sequence, commissioned by Shanghai Jereh Biotechnology Co., Ltd. Co., Ltd. synthesized the entire sequence, and added Hi...

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Abstract

The invention belongs to the field of molecular biology, and relates to a transposase for efficiently mediating exogenous gene integration, and a use thereof. The transposase is a fusion protein, and fuses a human c-myc pyrenoid positioning signal, and the human c-myc pyrenoid positioning signal can effectively guide aggregation of the transposase in a nucleus. The transposase is loaded in a transposon integration system, can mediate efficient integration of the exogenous gene in host cells, and can efficiently and stably express.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a transposase that efficiently mediates the integration of exogenous genes and its application. The transposase is an artificial transposase and a fusion protein. Specifically, it relates to a PiggyBac transposase fused with the nuclear localization signal of human c-myc protein, and the transposase can mediate the high-efficiency integration of exogenous exogenous genes in host cells, and high-efficiency and stable expression. The present invention also relates to a nucleic acid construct, a recombinant vector and a recombinant cell containing the nucleic acid construct, and their uses, such as the use of the transposase in transgenic T cell therapy. Background technique [0002] The expression forms of exogenous genes in host cells can be divided into transient expression and stable expression, among which stable expression refers to: (1) expression after exogenous genes are tra...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N9/12C12N15/62C12N15/85C12N5/10A61K48/00A61P35/00
Inventor 钱其军金华君李林芳吕赛群左明辉王颖吴红平吴孟超
Owner SHANGHAI CELL THERAPY RES INST
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