Recombinant adenovirus for expressing targetedly proliferating recombinant adenovirus for expressing proapoptosis gene and use thereof
A recombinant adenovirus and gene technology, applied in gene therapy, virus/bacteriophage, recombinant DNA technology, etc., can solve the problems that performance needs to be further enhanced
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Embodiment 1
[0065] Embodiment 1, the preparation of recombinant adenovirus
[0066] 1. Construction and identification of pClon15-PDCD5
[0067] figure 1 It is a flowchart of the construction of pClon15-PDCD5.
[0068] 1. Construction of pClon15-PDCD5
[0069] After pClon15 was digested with EcoRI (37°C water bath for 6h), electrophoresed on 1.2% agarose gel, a 3396bp linearized band of pClon15 was recovered with a gel extraction kit (QIAquick Gel Extraction).
[0070] pDC316-PDCD5 was digested with EcoR I (37°C water bath for 6h), electrophoresed on a 1.2% agarose gel, and the 423bp PDCD5 gene was recovered with a gel extraction kit (QIAquick Gel Extraction). Figure 5 . Figure 5 Middle, 1: PDCD5 gene; M: marker.
[0071] Ligate 2ul pClon15 linearized band and 8ul PDCD5 gene in 10ul Solution I at 12°C for 12h, transform the ligation product into DH5α Escherichia coli competent cells, spread agar plates (containing AMP), and culture in a biochemical incubator at 37°C for 10-12h . ...
Embodiment 2
[0152] Example 2, the expression level of PDCD5 after SG611-PDCD5 infected K562 cells
[0153] Real-time quantitative RT-PCR was used to detect the expression level of PDCD5 after different MOIs of SG611-PDCD5 infected K562 cells (Shanghai Kunken Biochemical Co., Ltd.; China Cell Bank Collection Cell Center Cell Catalog: QK10269). K562 cells in the logarithmic growth phase were taken, and 8×10 5 Each well was inoculated in a six-well plate, and the following four treatments were performed respectively, and three duplicate holes were set up for each treatment:
[0154] Treatment 1 to treatment 3: infection with SG611-PDCD5, SG611-EGFP or Ad-PDCD5 at a dose of MOI=1 respectively; treatment 4 (control): use an equal amount of normal saline as a control.
[0155] After infection, half of the cells were harvested at the same time every day, and total RNA was routinely extracted and reverse-transcribed into cDNA. Real-time quantitative RT-PCR was completed using TaqMan technology ...
Embodiment 3
[0157] Embodiment 3, flow cytometry detects the infection efficiency of SG611-EGFP to K562 cells
[0158] Take K562 cells in the logarithmic growth phase and adjust the cell concentration to 2×10 5 / ml, take 4ml (8×10 5 Cells) / well were inoculated in a six-well plate, and the following four treatments were performed respectively, and three duplicate holes were set up for each treatment:
[0159] Treatment 1 to Treatment 3: Infect SG611-EGFP at doses of MOI=1, 5, and 10 respectively; Treatment 4 (control): Use an equal amount of normal saline as a control.
[0160] After 24, 48, 72, 96, and 120 hours after infection, the cells were collected once, and the cells in each group were collected in triplicate wells and mixed. After washing twice with PBS, they were detected by a flow cytometer (FACSort type, Becton Dickinson, USA) and analyzed. Calculate the percentage of positive cells, the excitation light is 488nm, and the detection light is 530nm.
[0161] The result is as F...
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