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Cell strain capable of reducing replicability adenovirus production and construction method and application thereof

A construction method and cell line technology, applied in the field of cell lines and construction to reduce the production of replicable adenovirus, can solve the problem of reduction and achieve the effect of reducing off-target effects

Active Publication Date: 2018-01-26
CANSINO BIOLOGICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a cell line that reduces the production of replicable adenovirus, which solves the risk of producing replicable adenovirus RCA during the production of Ad5 recombinant adenovirus by existing cell lines

Method used

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  • Cell strain capable of reducing replicability adenovirus production and construction method and application thereof
  • Cell strain capable of reducing replicability adenovirus production and construction method and application thereof
  • Cell strain capable of reducing replicability adenovirus production and construction method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0053] Construction of Crispr / Cas9n system for HEK293 cell E1 gene ITR and E1 Promoter

[0054] First, according to the HEK293 genome sequence in NCBI, select the E1 gene ITR E1 Promoter as the target site to design sgRNA, and the target site sequence is as follows figure 1 As shown, the sgRNA sequence is shown in Table 1 above.

[0055] Secondly, the construction of PX462.V2.0-sgRNA containing specific sgRNA sequence:

[0056] (1) Design and synthesize sgRNA that recognizes E1 gene ITR and E1 Promoter;

[0057] (2) The synthesized sgRNA oligonucleotides are annealed in vitro;

[0058] (3) Digest PX462.V2.0 through the Bsa I site and connect with sgRNA, named PX462.V2.0-sgRNA.

[0059] Finally, a homology repair template plasmid was designed according to the target site sequence of the sgRNA.

Embodiment 2

[0061] cell transfection

[0062] 4 × 10 in a six-well plate 5 Inoculate HEK293 cells at a density of 70%-90% (about 18-20h) per well, and start transfection when they grow to a fusion rate of 70%-90% (about 18-20h). The concentration of PX462.V2.0-sgRNA1, PX462.V2.0-sgRNA2, PX462.V2.0-sgRNA3, PX462.V2.0-sgRNA4 is 20ng / μL, and the PGK Promoter repair template plasmid is 25ng / μL), Take 20 μL and transfect cells with 5 μL of Lipo2000 transfection reagent, add SCR7 non-homologous recombination inhibitor (final concentration: 0.01 mM) after 12 hours, and add Puromycin (final concentration: 3 μg / mL) after 36 hours for screening.

Embodiment 3

[0064] screening verification

[0065] use image 3 The method shown verifies whether the E1 gene in HEK293.CS cells has been completely replaced. The method is: extract the genome of the transformed cells, and if the ProF / AdR primer pair can amplify bands, it proves that the original sequence is replaced by PGK Promoter, such as YSF The / R primer pair can amplify the fragment, which proves that the original sequence still exists in the replaced cells, and the replacement is not complete. Only when the ProF / AdR primer pair can amplify the band but the YSF / R primer pair cannot amplify the band. It proved that ITR and E1A Promoter were completely replaced by PGK Promoter in HEK293 cells. If the sequencing result of the fragment amplified by the YZF / R primer pair is a pure PGK Promoter sequence, it further proves that the original sequence has been completely replaced.

[0066] The verification primers are shown in Table 2, and the sequencing results are shown in Figure 4 It ...

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Abstract

The invention provides a cell strain capable of reducing replicability adenovirus production and a construction method and an application thereof. The cell strain HEK 293.CS capable of reducing replicability adenovirus production is a safe adenovirus production cell line by knocking out of a gene fragment in HEK293 which is homologous with an Ad5 adenovirus E1 gene, and simultaneously providing template plasmid for replacing the gene fragment to a non-homologous sequence which is capable of stabilizing the E1 gene expression. Compared with un-reconstructed HEK293 cell strain, the growth capability and virus production capability of HEK 293.CS are not decreased, but detectable RCA is not produced; the cell strain can be used for large-scale cultivation of recombinant human adenovirus type 5, and the RCA production probability during medicine manufacture such as vaccines and antibodies is reduced.

Description

technical field [0001] The invention relates to the field of cell line biotechnology, in particular to a cell line for reducing the production of replicable adenovirus, a construction method and application. Background technique [0002] Crispr / Cas9 has been shown to cut any given DNA through RNA-mediated cleavage, and the cleavage of target genes by Crispr / Cas9 introduces DNA double-strand breaks (DSBs), and DSBs are cleaved by non-homologous end binding (non-homologous End joining, NHEJ) or homologous-directed repair (homologous-directed repairHDR) method for repair. NHEJ repair often causes frameshift mutations of the target gene at the site of Cas9 cleavage, introducing loss-of-function mutations. The HDR repair method can knock in genes under the guidance of exogenous sequences and introduce gain-of-function mutations. In addition, by using DSBs and providing donors at the same time, target sequences can be inserted or mutated in the genome. However, Crispr / Cas9 can ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/90C12N7/00C12R1/93
CPCC12N7/00C12N2710/10352C12N15/86C12N2710/10343C12N15/907C12N2310/20A61K39/235C12N5/0686
Inventor 朱涛崔海燕陈伟伟段磊李军强马金莘春林邵忠琦宇学峰毛慧华
Owner CANSINO BIOLOGICS INC
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