Oncolytic vaccinia virus carrying tachypleus tridentatus lectin gene, and construction method and application thereof
A vaccinia virus, construction method technology, applied in the application, virus, genetic engineering and other directions, to achieve the effect of simplifying the construction technology route, high repeatability, and improving the level of replication
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Embodiment 1
[0036] Example 1: Construction and identification of oncolytic vaccinia virus oncoVV-TTL
[0037] 1. Insert the gene sequence (SEQ ID NO: 1) of Tachypleus tridentatus lectin (TTL) into pCB plasmid through Xba I and Bgl II sites to obtain pCB-TTL plasmid.
[0038] pCB-TTL plasmid map such as figure 1 shown. Among them, vTK-L and vTK-R, through homologous recombination with the thymidine kinase TK region of the wild-type virus, insert foreign genes into the TK region and cause TK deletion at the same time. In addition, the plasmid also carries the xanthine-guanine phoshporibosyl transferase (gpt) gene as a screening gene, and the gpt gene is derived from Escherichia coli, viruses or cells in the presence of mycophenolic acid (MPA) , because MPA can block the synthesis of guanine, so that the virus or cell can not synthesize nucleic acid normally and die. In the presence of the gpt gene, cells or viruses can use hypoxanthine (hypoxanthine) and xanthine (xanthine) to synthesize...
Embodiment 2
[0057] Example 2: In vitro inhibitory effect of oncoVV-TTL on various tumor cells detected by MTT method
[0058] In this experiment, liver cancer cells BEL-7404 and MHCC97-H, colorectal cancer cells HT-29 and glioma cells U251MG were selected, respectively, according to 5×10 3 Inoculate into a 96-well plate at a density of 96 wells, add 90 μL of cell culture medium to each well and culture overnight, add 2MOI or 10MOI oncoVV-TTL virus respectively, set up 6 replicate wells, the experimental control group is cells without virus, and the blank group is Culture medium without cells.
[0059] 37°C, 5% CO 2 For culture, three time gradients of 24h, 48h, and 72h were set, and 20 μL of MTT solution (5 mg / mL) was added to each well in the dark at the corresponding time point. The incubator was left to stand for 4 hours, and after absorbing the culture solution of each group, 150 μL of dimethyl sulfoxide was added to each well, and placed on a shaker for 10 minutes to fully dissolve...
Embodiment 3
[0062] Example 3: The replication level of oncolytic vaccinia virus oncoVV-TTL in tumor cells is significantly higher than that of control virus oncoVV
[0063] The tumor cells BEL-7404 or MHCC97-H were divided into 5×10 3 The density per well was inoculated into a 96-well plate, 90 μL of cell culture medium was added to each well and cultured overnight, 5 MOI of oncoVV-TTL virus or control virus oncoVV was added respectively, and more than 3 replicate wells were set for each time gradient. The cells were collected together with the culture medium, and the replication efficiency of oncoVV-TTL and the control virus oncoVV in the tumor cells was detected by the TCID50 method (half tissue culture infection dose).
[0064] Test results such as image 3 Shown: Compared with the control group, the replication efficiency of the experimental group oncoVV-TTL virus in tumor cells MHCC97-H and BEL-7404 was significantly higher than that of the control virus oncoVV.
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