Preparation method and application of recombinant Sendai virus for human cell reprogramming

A technology of Sendai virus and human cells, applied in the field of preparation of recombinant Sendai virus, can solve the problems of easy introduction of other active viruses into packaged viruses, integration of viral genomes, and low reprogramming efficiency

Active Publication Date: 2021-05-11
北京默赛尔生物科技有限责任公司
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Problems solved by technology

[0004] The purpose of the present invention is to provide a preparation method of recombinant Sendai virus for human cell reprogramming, aiming to solve the existing method of cell reprogramming to induce iPS cells, the reprogramming efficiency is low, the virus genome is integrated, and the purity of the packaged virus is low. The problem of easy introduction of other active viruses in

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  • Preparation method and application of recombinant Sendai virus for human cell reprogramming
  • Preparation method and application of recombinant Sendai virus for human cell reprogramming
  • Preparation method and application of recombinant Sendai virus for human cell reprogramming

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[0032] On the one hand, the embodiment of the present invention provides a method for preparing a recombinant Sendai virus for human cell reprogramming, comprising the following steps:

[0033] S01. Provide pBluescript II SK(+) plasmid and T7 terminator, and use infusion fusion technology to prepare pBluescript II SK(+)T7terminator plasmid; after the pBluescript II SK(+)T7 terminator plasmid is digested with Kpn1 and Not1, Insert the P gene, L gene and NP gene respectively to obtain the T7-P / L / NP plasmid (T7-P / L / NP plasmid means three kinds of plasmids, which are respectively T7-P plasmid, T7-L plasmid and T7-NP plasmid ); after the T7-P / L / NP plasmid is cut into a linear plasmid using Kpn1 enzyme, it is fused with the IRES fragment to prepare the viral helper plasmid T7-IRES-P / L / NP (T7-IRES-P / L / NP represents three kinds of viral helper plasmids, respectively T7-IRES-P virus helper plasmid, T7-IRES-L virus helper plasmid and T7-IRES-NP virus helper plasmid);

[0034] S02. Inser...

Embodiment 1

[0086] A method for reprogramming human skin fibroblasts into iPS cells, comprising the following steps:

[0087] S11. Using the pBluescript II SK (+) plasmid as a template, using the first primer set to amplify to obtain a pBluescript II SK (+) plasmid with a fragment length of 2961bp, wherein the first primer set includes:

[0088] First upstream primer: CAGCTTTTGTTCCCTTTAGTGAGGG

[0089] First downstream primer: GAGCTCCACCGCGGTGGC.

[0090] Using pET-28a(+) as a template, the second primer set was used to amplify to obtain the T7 terminator with a fragment length of 159 bp. The second primer set includes:

[0091] Second upstream primer: ACCGCGGTGGAGCTCctgctaacaaagcccgaaagg

[0092] Second downstream primer: AGGGAACAAAAAGCTGatccggatatagttcctcc.

[0093] According to the operation of the VAZYME C115-ClonExpress Ultra One Step Cloning Kit kit, the pBluescript II SK(+) plasmid and the T7 terminator were subjected to infusion fusion to prepare the pBluescript II SK(+)T7term...

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Abstract

The invention provides a method for preparing recombinant Sendai virus for human cell reprogramming, comprising: preparing three auxiliary plasmids containing T7 terminator, P gene, L gene, NP gene and IRES fragment, and containing T7 promoter , hammerhead enzyme sequence, hepatitis B ribozyme sequence, pUC57-Simple plasmid of T7 terminator; digest the pUC57-Simple plasmid and connect the full-2 plasmid digestion product to obtain the main backbone plasmid of Sendai virus; provide BSR- T7 / 5 cells were cultured using DMEM serum-free complete medium; the medium was replaced with Opti-MEM medium before transfection, and the viral helper plasmid T7-IRES-P / L / NP and the Sendai virus main backbone plasmid were added ; Add polyethylenimine to lipo3000 as a transfection reagent for transfection; proliferate and culture the transfected cell bodies, and collect the recombinant Sendai virus.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of a recombinant Sendai virus used for human cell reprogramming. Background technique [0002] Sendai virus (SeV), also known as type 1 parainfluenza virus or HVJ (Hemagglutinating Virus of Japan, Japanese hemagglutination virus), is a negative-sense single-stranded single-stranded RNA virus of the Paramyxoviridae family. Sendai virus has the following characteristics, which make the Sendai virus vector have a strong advantage in the process of introducing foreign genes: (1). In the life cycle of Sendai virus, the viral protein and genome are in the cytoplasm, and the viral genome is not separated from the cell. Genome integration; (2). Sendai virus can infect the whole life cycle of cells, and can infect most cells with extremely high efficiency, including cell types that are difficult for lentivirus to infect; 3. Expression of foreig...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/86C12N7/01C12N5/10
CPCC12N5/0696C12N7/00C12N15/86C12N2510/00C12N2760/18843C12N2760/18852C12N2800/107
Inventor 不公告发明人
Owner 北京默赛尔生物科技有限责任公司
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