A double-copy human p53 gene recombinant adenovirus and its preparation method

A technology of p53 gene and recombinant adenovirus, which is applied in the field of biomedicine, can solve problems such as side effects, and achieve the effects of strong specificity, increased gene expression, and reduced virus usage

Active Publication Date: 2019-03-15
SINOSHENG SHENZHEN GENE IND DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • A double-copy human p53 gene recombinant adenovirus and its preparation method
  • A double-copy human p53 gene recombinant adenovirus and its preparation method
  • A double-copy human p53 gene recombinant adenovirus and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0042] Apply the golden gate cloning technology (goldengate cloning) that is used for one-time assembly of multiple molecular fragments in the new molecular cloning technology, design and synthesize the double-copy p53 gene expression cassette, that is, include the eukaryotic cell expression promoter (used in the embodiment of the present invention Developing reading frame (ORF) and polyadenylation tail (poly A) for cytomegalovirus (CMV promoter), p53 tumor suppressor gene: construction of human cytomegalovirus (CMV) virus early promoter--Kozak sequence-P53 Gene--3' end non-coding sequence polyadenylation tail (poly A) sequence-human cytomegalovirus (CMV) virus early promoter--Kozak sequence--P53 gene--3' end non-coding sequence polyadenylation Nucleotide tail (poly A) sequence.

[0043] The Golden Gate cloning technique utilizes type IIS restriction endonuclease, whose DNA...

Embodiment 2

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

[0054] Use a series of dilutions of purified virus (double-copy human p53 gene recombinant adenovirus) to infect monolayer HEK293 cells, and serially serially dilute the purified virus at a ratio of 1:10, and select 10 6 to 10 13Dilute virus (double-copy human p53 gene recombinant adenovirus) suspensions were added to dense single-layer HEK293 cell culture flasks to allow the virus to adsorb, and cultured at 37°C. After 1 hour, the virus suspension was sucked away, and then covered with a layer to melt agar, cultured at 37°C. At least 2 copies of each dilution gradient. After 24 hours, stain with neutral red and check the results. After the virus (recombinant adenovirus with double-copy human p53 gene) replicates in HEK293 cells, it can produce a limited infection focus, namely plaque. Live cells were stained with neutral red, and unstained plaques could be seen, and the virus conce...

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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, and in particular relates 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. The characteristics of malignant tumor cells are uncontrollable unlimited growth and destruction of local and metastatic tissue structures. The ultimate goal of tumor treatment is to completely kill or remove tumor tissue. 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. Gene therapy is a new anti-tumor treatment method developed at present, which directly targets the gene disorder of tumor cells and cures the root cause. [0003] P53 gene is the most important tumor suppressor gene. When the gene body of the cel...

Claims

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Application Information

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Patent Type & Authority Patents(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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