Double-copy human p53 gene recombinant adenovirus and preparation method thereof

A p53 gene, recombinant adenovirus technology, applied in the field of biomedicine, can solve problems such as side effects, achieve the effects of strong specificity, increased efficacy and safety, and increased gene expression

Active Publication Date: 2016-07-13
SINOSHENG SHENZHEN GENE IND DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Traditional tumor treatment methods include three main methods of surgery, chemotherapy and radiotherapy and some adjuvant therapies, all of which have significant side effects and limitations

Method used

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  • Double-copy human p53 gene recombinant adenovirus and preparation method thereof
  • Double-copy human p53 gene recombinant adenovirus and preparation method thereof
  • Double-copy human p53 gene recombinant adenovirus and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] 1. Construction of a double-copy human p53 tumor suppressor gene eukaryotic expression cassette:

[0042] Applying the golden gate cloning technology (goldengatecloning), which is used to assemble multi-molecular fragments at one time in the novel molecular cloning technology, design and synthesize a double-copy p53 gene expression structure box, that is, including the eukaryotic cell expression promoter (the one used in the embodiment of the present invention is Cytomegalovirus CMV promoter), p53 tumor suppressor gene development reading frame (ORF) and polyadenylation tail (polyA): constructed into human cytomegalovirus (CMV) virus early promoter--Kozak sequence-P53 gene- - 3' non-coding sequence polyadenylation tail (polyA) sequence - human cytomegalovirus (CMV) virus early promoter - Kozak sequence - P53 gene - 3' non-coding sequence polyadenylation tail (polyA) sequence.

[0043] The Golden Gate cloning technology uses type ⅡS restriction endonuclease, its DNA rec...

Embodiment 2

[0053] (1) Double plaque formation experiment and determination of virus particle content

[0054] Monolayer HEK293 cells were infected with a series of dilutions of purified virus (double-copy human p53 gene recombinant adenovirus), and the purified virus was serially serially diluted at a ratio of 1:10, and 10 were selected. 6 to 10 13 The diluted virus (double-copy human p53 gene recombinant adenovirus) suspensions were added to dense monolayer HEK293 cell culture flasks to allow virus adsorption, cultured at 37°C, aspirated after 1 hour, and then covered with a layer of thawing agar at 37°C. At least 2 copies of each dilution gradient. Neutral red staining was performed 24 hours later to check the results. After the virus (double-copy human p53 gene recombinant adenovirus) replicated in HEK293 cells, it could produce a localized infection foci, namely plaques. Viable cells were stained with neutral red, and unstained plaques were seen. The virus concentration and titer...

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Abstract

The invention discloses double-copy human p53 gene recombinant adenovirus and a preparation method thereof. A commercialized 5-type recombinant replication-deficient adenovirus construction system (AdEasy) is inserted into a double-copy human p53 tumor inhibition gene eukaryotic expression box as shown in SEQ ID NO.1 to construct a p53 tumor inhibition gene recombinant adenovirus expression carrier system, and recombinant replication-deficient adenovirus granules for expressing double-copy human p53 tumor inhibition gene are further obtained. Experiment shows that after being injected by tumor cells, the double-copy human p53 gene recombinant adenovirus can efficiently express p53 tumor inhibition genes carried by the virus. As the double-copy human p53 tumor inhibition gene eukaryotic expression box is integrated, the p53 tumor inhibition gene expression amount can be greatly increased, meanwhile the virus amount can be reduced, and a relatively good gene treatment effect can be achieved. The double-copy human p53 gene recombinant adenovirus is good in specificity and wide in spectrum, directly aims at gene mutation of tumor cells, and can be applied to malignant tumor of various tissue types at the early stage, the middle stage and the late stage.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to a double-copy human p53 gene recombinant adenovirus and a preparation method thereof. Background technique [0002] Normal tissue cells develop into malignant tumor cells due to long-term accumulation of gene mutations, and malignant tumor cells are characterized by uncontrollable unlimited growth and destruction of local and metastatic tissue structures. The ultimate goal of treating a tumor is to completely kill or remove the tumor tissue. Traditional tumor treatment methods include surgery, chemotherapy and radiotherapy as the three main methods and some adjuvant therapies, all of which have significant side effects and limitations. Gene therapy is a new anti-tumor treatment method currently developed, which directly targets the genetic disorder of tumor cells and cures the root cause. [0003] The p53 gene is the most important tumor suppressor gene. When the cell genome is dama...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N7/01A61K38/17A61K48/00A61P35/00
CPCA61K38/1758A61K48/005C12N7/00C12N15/86C12N2710/10021C12N2710/10043Y02A50/30
Inventor 高贵光炜
Owner SINOSHENG SHENZHEN GENE IND DEV CO LTD
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