Construction method and applications of targeting oncolytic-adenovirus carier Ad-TD-gene
A virus vector and construction method technology, applied in the fields of biotechnology and gene therapy, can solve the problems of increasing the loading of foreign genes and affecting the anti-tumor effect of oncolytic adenovirus, so as to achieve the effect of good curative effect and safety, and increase the specificity
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Embodiment 1
[0036] Example 1. Construction of tumor-targeting adenoviral vectors Ad-TD-gene, Ad-TD-hCCL21 and their control viral vector Ad-TD-RFP.
[0037] 1. Construction of tumor targeting adenoviral vector Ad-TD-gene
[0038] (1) First use the PCR method to obtain the gene fragments on both sides of the sequence E1A-CR2 to be transformed. The upstream sequence is called the left arm, and the downstream sequence is called the right arm. Use genetic engineering methods to place the left arm and the right arm in the same order as the viral genes Connect to the plasmid vector pSuperShuttle to build the E1A-CR2 shuttle vector;
[0039] (2) Transform the Ad5 adenovirus vector and the shuttle vector into BJ5183 bacteria at a ratio of 1:2 to 10 for homologous recombination; use PCR to identify positive recombinant bacteria, extract the recombinant plasmid, and transfect it into 293 cells to obtain E1A-CR2 deleted Ad5R-CR2 viral vector;
[0040] (3) Construct the E1B19K shuttle vector with t...
Embodiment 2
[0048] Example 2. The endogenous promoter of the tumor-targeting adenoviral vector Ad-TD-gene expresses the specific expression of the reporter gene RFP in the cell
[0049] 293 cells were inoculated into a six-well plate, and when the cells grew to 75%, Ad-TD-RFP was injected at 1×10 5 The dose of pt / cell was used to infect 293 cells. After culturing for 48 hours, the expression of red fluorescent protein was observed under a fluorescent microscope. The results are shown in the attached figure 2 shown.
Embodiment 3
[0050] Example 3, Detection of the Killing Ability of Tumor Targeting Adenovirus Vectors Ad-TD-gene and Ad-TD-hCCL21 to Common Human Tumor Cells
[0051] HCT116 (colorectal cancer) cells, PT45 (pancreatic cancer) cells, H460 (lung cancer) cells, A549 (lung cancer) cells, SUIT2 (pancreatic cancer) cells in the logarithmic growth phase were collected, counted, and seeded at 2000 cells / well in in a 96-well plate. After 12 hours, the cells were attached and the viruses dl1520, Ad-TD, Ad-TD-hCCL21 and Ad-TD-mCCL21 (mCCL21, mouse CCL21 gene) were diluted. According to the highest concentration 1×10 4 pt / cell was added to a 96-well plate, and each column was decreased by 10 times, and the 10th column was a blank control. Six days later, MTS method detection (Buttke TM, J Immunol Methods, 1993, 157 (1-2): 233-40), converted into EC50 (that is, the amount of virus required for each virus to kill 50% of tumor cells, the lower the EC50 , indicating that the virus killing ability is st...
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