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Improved PB transposon system and application thereof

A technology of transposon and purpose, applied in the field of improved PB transposon system, can solve the problems of low integration efficiency, limited loading capacity, and the transposition efficiency needs to be further improved, and achieve the effect of increasing the proportion and increasing the expression intensity

Pending Publication Date: 2020-05-29
SHANGHAI CELL THERAPY GRP CO LTD +1
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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
[0007] Another defect of the PB transposon system is that the length of the wild-type 5' and 3' inverted terminal repeats (Inverted Terminal Repeats, ITR) is more than 700bp, and the total length is about 1.5kb
Compared with the traditional binary PB transposition system, the transposition efficiency of this transposition system is significantly higher, but for the preparation of CAR-T cells, its transposition efficiency still needs to be further improved

Method used

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  • Improved PB transposon system and application thereof
  • Improved PB transposon system and application thereof
  • Improved PB transposon system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] The construction of embodiment 1 carrier

[0099] Construction of pNBC vector

[0100] Commissioned Shanghai Jereh Biotechnology Co., Ltd. to synthesize sequentially connected CMV promoter sequence (SEQ ID NO:1), PiggyBac transposase coding sequence (SEQ ID NO:2) containing c-myc nuclear localization signal, and PiggyBac transposon 5' terminal repeat (SEQ ID NO:3), synthetic polyA tailing signal sequence (SEQ ID NO:4), multiple cloning insertion site (SEQ ID NO:5), SV40 polyA tailing signal sequence (SEQ ID NO :6) and PiggyBac transposon 3' terminal repeat sequence (SEQ ID NO: 7), and PacI and AscI restriction sites were added at both ends, respectively, and loaded into pUC57 (purchased from Shanghai Jereh Biotech), and the resulting vector was named It is a pNBC vector, see the vector structure figure 1 .

[0101] Construction of pNBC(-) vector

[0102] The construction method is the same as that of pNBC, the difference is that it does not contain the synthetic pol...

Embodiment 2

[0119] Embodiment 2: Construction of the vector containing exogenous gene expression cassette

[0120] 1. Construction of pNBC vector containing EGFP expression cassette

[0121] (1) Entrust Shanghai Jereh Biotechnology Co., Ltd. to synthesize the sequence of the DTS EF1α promoter (sequence shown in SEQ ID NO: 8), and add Xba I and EcoR I restriction sites at both ends, and load it into Example 1 For each prepared vector, pNB338C, pNB338C(-), pNB338C-AD, pNB338C-D3 and pNB338C-D5 were respectively obtained.

[0122] (2) Entrust Shanghai Jereh Biotechnology Co., Ltd. to synthesize the coding sequence of EGFP (SEQ ID NO: 9), and add EcoR I and Sal I restriction sites at both ends, and load each vector obtained in (1) , the corresponding vectors were obtained, named pNB338C-EGFP, pNB338C-EGFP(-), pNB338C-EGFP-AD, pNB338C-EGFP-D3 and pNB338C-EGFP-D5, respectively.

[0123] 2. Construction of pNB338B-EGFP vector

[0124] Based on the pNB328-EGFP vector disclosed in CN105154473A,...

Embodiment 3

[0133] Example 3: Detection of Integration Efficiency of Vector Containing EGFP Expression Cassette in CHO Cells

[0134] Prepare 2×10 6 For CHO cells with vigorous metabolism, pNB338B-EGFP, pNB338C-EGFP, pNB338C-EGFP(-), pNB338C-EGFP-AD, pNB338C-EGFP-D3, pNB338C- EGFP-D5 and pNB338CFL-EGFP(-) were transfected into the nucleus (Nucleofector TM 2b program H-014), set at 37°C, 5% CO 2 Incubator culture. After the cells were confluent, they were subcultured at a ratio of 1:5. On the 13th day after transfection and after 3 passages, the proportion of EGFP-positive cells was detected by flow cytometry, and the CHO cells without plasmid transfection were used as the control of flow cytometry detection.

[0135] Dilute and passage at a ratio of 1:5, and the non-integrated plasmid will be lost quickly with cell division. After 3 passages, the green fluorescent positive cells can be considered to have stably integrated the green fluorescent expression frame. The efficiency of in...

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Abstract

The present invention relates to an improved PB transposon system and application thereof. Specifically, the present invention relates to a nucleic acid construct. The nucleic acid construct sequentially contains a promoter for controlling the expression of PiggyBac transposase, a PiggyBac transposase coding sequence, a terminal repeat at the 5'-end of the PiggyBac transposon, an optional polyA tailing signal sequence 1, a polyclonal insertion site, a polyA tailing signal sequence 2 and a terminal repeat at the 3'-end of the PiggyBac transposon. A recombinant vector constructed by using the nucleic acid construct provided by the invention has obviously improved integration efficiency.

Description

technical field [0001] The present invention relates to an improved PB transposon system and its application. 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 transfected into eukaryotic cells and integrated into the genome. The stable expression level of recombinant genes is generally 1-2 orders of magnitude lower than that of transient expression. (2) The expression level of the host cells remains stable even though the host cells have undergone multiple passages or conditional changes. [0003] In view of the fact that stable expression can maintain long-term continuous expression of exogenous genes with cell division, in ex vivo cell modification (ex vivo), such as transgenic chimeric antigen receptor T cell (Chimeric Antigen ReceptorT-CellImmunotherapy, CAR-T) treatment research is of great...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N5/10A61K48/00A61P35/00A61P35/02
CPCC12N15/63A61K48/005A61P35/00A61P35/02C12N2800/90C12N2510/00
Inventor 刘韬刘辉金华君王超何周何江川吴碧寒刘天怡钱其军
Owner SHANGHAI CELL THERAPY GRP CO LTD
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