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Oncolytic adenovirus construction method and application

An oncolytic adenovirus and construction method technology, applied in the field of oncolytic adenovirus construction, can solve the problem that the killing effect of animal tumor cells has not been studied in depth, and achieve the effect of inhibiting the occurrence of tumors

Inactive Publication Date: 2018-08-31
复百澳(苏州)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the killing effect of endotoxin (crystal protein, Cry gene) on animal tumor cells has not been deeply studied

Method used

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  • Oncolytic adenovirus construction method and application
  • Oncolytic adenovirus construction method and application
  • Oncolytic adenovirus construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Nucleotide sequence of effector gene CRY for whole gene synthesis

[0029] The CRY plasmid was obtained by synthesizing the gene sequence of the effector gene CRY through the whole gene synthesis of Suzhou Synbio Biotechnology Co., Ltd.

Embodiment 2

[0030] Example 2: Construction of oncolytic adenoviral plasmids

[0031] (1) Enzyme digestion system:

[0032]

[0033]

[0034] (2) Agarose gel electrophoresis and gel return to the target product

[0035] Use 1% Agrose, 120V constant voltage electrophoresis for 30-40min, cut out the single target fragment under ultraviolet irradiation and put it into a clean centrifuge tube for gel recovery and purification.

[0036] (3) Ligate the recovered carrier product and the enzyme-cut gel back product (1h at room temperature)

[0037]

[0038] (4) Connection product conversion

[0039] 1) Take 50ul of competent cells (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.) and put them on ice to melt, take 5ul of the connection product and mix with competent cells;

[0040] 2) Ice bath for 30 minutes, heat shock in 42°C water bath for 90 seconds, and continue ice bath for 2 minutes;

[0041] 3) Add 1ml non-resistant SOB liquid medium to the mixture, shake the ...

Embodiment 3

[0044] Example 3: Ligation product identification

[0045] After plasmid digestion and identification, the recombinant plasmid after transfection was consistent with the size of the target gene fragment (results such as figure 1 shown), the amplified product was 1017bp, which proved that the recombinant plasmid was successfully constructed.

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Abstract

The invention discloses an oncolytic adenovirus construction method and application, the method comprises the following steps: step one: the gene sequence of the effect gene CRY is synthesized by thewhole gene of the Suzhou Synbio Technologies Co., Ltd, and the CRY plasmids are obtained; step two: the oncolytic adenovirus vector gene after ExcoR I and Xba I double enzyme digestion and the CRY plasmids are constructed into recombined plasmids; step three: the recombined plasmids obtained in the step two and auxiliary plasmids are jointly transferred to the cells for virus packing, and after lesions occur to the cells, the oncolytic adenovirus is obtained. The oncolytic adenovirus construction method can not only be used for preparing disease treatment drugs for mice breast cancer (not onlylimited to breast cancer, but also including lung cancer, liver cancer and the like), but also can directly kill tumor cells and can induce more cells to apoptosis under the action of the treatment protein, and accordingly, the tumor is effectively inhibited.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a construction method and application of an oncolytic adenovirus. Background technique [0002] Oncolytic virus is a kind of virus that preferentially infects and kills tumor cells. In the early stage, some tumor cells are specifically infected and destroyed by oncolytic viruses. Subsequently, oncolytic viruses replicate and proliferate in tumor cells, releasing new infectious virus particles to infect and destroy other tumor cells. Oncolytic viruses exert oncolytic effects by directly lysing tumor cells or stimulating the host to produce an anti-tumor immune response. [0003] Oncolytic adenovirus is the most commonly used oncolytic virus at present. There are many strategies to enhance the tumor targeting of oncolytic adenovirus, including functional genes involved in the regulation of cell cycle nodes in the adenovirus genome (such as E1A or E1B) to mutate or (and) ...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N7/01A61K48/00A61P35/00C12R1/93
CPCA61K48/0025A61K48/0058A61P35/00C12N7/00C12N15/86C12N2710/10343C12N2710/10352
Inventor 刘骏周春艳金丽肖倩谢伟建刘清波
Owner 复百澳(苏州)生物科技有限公司
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