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
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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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