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 syst

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

Example Embodiment

[0120] Example 1: Construction of pNB vector

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

Example Embodiment

[0122] Example 2: Construction of pNB vector containing foreign gene expression cassette

[0123] 1. According to the EF1α promoter sequence, commissioned by Shanghai Jereh Biotechnology Co., Ltd. to synthesize, and add XbaI and EcoRI restriction sites at the two ends respectively, and load the pNB vector prepared in Example 1 above, named as 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, and add EcoRI and SalI restriction sites at both ends respectively, and load it into pNB328 vector, named pNB328-EGFP vector.

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

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

[0128] The Luc luciferase codi...

Example Embodiment

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

[0132] The present inventors used the method of immunofluorescence hybridization to detect the changes in the protein subcellular localization of the PB transposase before and after artificially increasing the nuclear localization signal. Since there is currently no PB transposase antibody on the market, the inventors fused a His tag in front of the PB, and used His tag antibodies 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 human c-myc nuclear localization signal coding sequence, 6×His tag coding sequence, PB transposase coding sequence, spliced ​​into a myc-his-PB fusion protein coding sequence (SEQ ID NO: 17), commissioned by Shanghai Jereh Biotechnology The entire sequence was synthesized by Co., Ltd., and HindIII and ClaI restriction sites were added at both ends, and lo...

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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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