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293A cell strain for producing adenovirus as well as preparation and application thereof

An adenovirus and cell technology, applied in the field of cell engineering, can solve the problems of RCA and HDEP pollution, achieve the effect of preventing RCA and HDEP and improving safety

Active Publication Date: 2021-12-07
YUNZHOU BIOSCIENCES (GUANGZHOU) INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art, to provide a 293A cell strain for producing adenovirus and its preparation and application, which can solve the problem of RCA and HDEP contamination in the adenovirus production process

Method used

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  • 293A cell strain for producing adenovirus as well as preparation and application thereof
  • 293A cell strain for producing adenovirus as well as preparation and application thereof
  • 293A cell strain for producing adenovirus as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Obtaining of exogenous fragments

[0040]Gene fragments LoxM2 (nucleotide sequence shown in SEQ ID NO.1), HPRT intron (nucleotide sequence shown in SEQ ID NO.2) and hygro cassette (nucleotide sequence shown in SEQ ID NO.3) were synthesized respectively shown), the three gene fragments were ligated sequentially to obtain the exogenous fragment (the nucleotide sequence is shown in SEQ ID NO.5), and the gRNA recognition sequence (the nucleotide sequence is shown in SEQ ID NO. NO.4), to obtain an exogenous fragment containing the gRNA recognition sequence.

Embodiment 2

[0041] Example 2 Construction of recombinant plasmids

[0042] (1) Construct plasmid pDown-HBV ployA-HPRT intron-mPGK-Hygro-SV40 pA-gRNA2

[0043] The foreign fragment containing the gRNA recognition sequence obtained in Example 1 was cloned into the plasmid pDown-SapI-ccdB-Chl-SapI to obtain the recombinant plasmid pDown-HBV ployA-HPRT intron-mPGK-Hygro-SV40 pA-gRNA2, which Recombinant plasmid map such as figure 1 shown.

[0044] (2) Construction of plasmid carrying hCas9 and gRNA

[0045] Synthetic gene fragment hCas9 (nucleotide sequence shown in SEQ ID NO.6) and gRNA transcription frame (nucleotide sequence shown in SEQ ID NO.7), gRNA sequence is cloned into plasmid PX330-U6-BbsI-CBh -hSpCas9-CMV>EGFP / Puro obtained the recombinant plasmid pRP[CRISPR]-EGFP / Puro-hCas9-U6>{293A / Ad-3522nt}, the recombinant plasmid map is as follows figure 2 shown.

Embodiment 3

[0046] Example 3 Construction of VB-293A cell strain

[0047] Use 3×10 6 Cells were plated in a 6-well plate, and the confluence of the cells reached 80%-90% the next day. The two plasmids constructed in Example 2 were co-transfected into 293A cells (Thermo Fisher product number: R70507) with Lipofect amine3000 transfection reagent. According to the principle of NHEJ, hCas9 / gRNA will simultaneously recognize and cut the target site of the gene and the two target sites of the plasmid pDown-HBV ployA-HPRT intron-mPGK-Hygro-SV40 pA-gRNA2. The linearized exogenous fragment is inserted into the targeted site in the genome of the cell, resulting in targeted integration. The total amount of transfected plasmids is 2.5μg, plasmid pRP[CRISPR]-EGFP / Puro-hCas9-U6>{293A / Ad-3522nt}: 1.7μg; plasmid pDown-HBV ployA-HPRT intron-mPGK-Hygro-SV40 pA - gRNA2: 0.8 μg; Lipofectamine 3000: 7.5 μL. Change the solution every other day.

[0048] Passage the cells 48 hours after transfection to a 6...

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Abstract

The invention relates to the technical field of cell engineering, in particular to a 293A cell strain for producing adenovirus as well as preparation and application thereof. The cell provided by the invention is obtained by inserting a section of exogenous fragment containing LoxM2, HPRT intron and Hygro cassete into the position of an Ad5 gene in a 293A cell genome. The replicative adenovirus genome generated by the cell disclosed by the invention cannot form complete adenovirus particles due to the fact that the genome exceeds the packaging limit of the adenovirus particles, so that the problem of RCA and HDEP pollution in the adenovirus production process can be solved.

Description

technical field [0001] The invention relates to the technical field of cell engineering, in particular to a 293A cell strain for producing adenovirus and its preparation and application. Background technique [0002] Replication-defective adenoviruses have been widely used in gene medicine, vaccine preparation, and oncolytic adenoviruses. [0003] Most of the currently used replication-deficient adenoviruses are serotype 5 adenoviruses that have deleted the E1 and E3 regions. Since the E1 region is an essential region for adenovirus replication, replication-defective adenoviruses must proliferate in complementing cell lines containing the E1 region. The most widely used replication-deficient adenovirus packaging cell is 293 cells, which contain 1-4344 bp sequence in Ad5 genome, including 5' inverted repeat (ITR), packaging signal, E1a, E1b gene and pIX coding sequence. However, the replication-defective adenovirus has an overlapping region with the adenovirus genome fragme...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N7/01C12N15/85C12N15/65C12R1/93
CPCC12N5/0686C12N7/00C12N15/85C12N15/65C12N2510/02C12N2710/10052C12N2800/30C12N2800/107
Inventor 施金秀陈咏罗燕蒙伟能蓝田
Owner YUNZHOU BIOSCIENCES (GUANGZHOU) INC
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