Oncolytic virus construction body, oncolytic virus and application thereof
A technology of oncolytic virus and construct, applied in the direction of virus/bacteriophage, application, virus, etc.
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Embodiment 1
[0046] Embodiment 1 Materials and methods
[0047] Cell line African green monkey kidney fibroblasts (CV-1) were obtained from the American Germplasm Collection. CV-1 cells were cultured in DMEM supplemented with antibiotic-antimycotic solution (100 U / mL penicillin G, 250 ng / mL amphotericin B, 100 units / mL streptomycin) and 10% fetal bovine serum (FBS; Invitrogen Corporation) Base. The culture environment is 37°C, 5% CO 2 . Human 143TK-cells (ATCC) were cultured in basal medium supplemented with 0.015 mg / ml of 5-bromo-2'-deoxyuridine (BUdR), 10% fetal bovine serum and Earle's BSS.
[0048] Human melanoma cell A375, human pancreatic cancer cell Panc1, human brain tumor cell U87, human lung cancer cell H226, and human head and neck cancer cell ATCC TCP-1012 were obtained from ATCC. Normal cell lines, including human normal cells PDF (human primary dermal fibroblasts), MRC5 (normal human lung fibroblasts), IMR-90 (ATCC CCL-186, normal human lung fibroblasts) and BJ (ATCC CRL-...
Embodiment 2
[0055] Example 2 Construction of recombinant vaccinia oncolytic virus (VV-iPD1 / GMCSF) co-expressing human PD1 inhibitor and GM-CSF
[0056] The nucleotide sequence encoding human fusion protein (iPD1) (comprising human PD1 signal peptide sequence, PD1 extracellular region and human IgG Fc fragment) is connected with restriction enzyme cutting sites (Not1 and SalI). After synthesis, it was cloned into the vaccinia virus shuttle vector (pSEL-DsRed) (RFP: red fluorescent protein red fluorescent protein), thereby constructing the pVV-iPD1 shuttle vector (such as figure 1 shown). After the human GM-CSF gene was synthesized with restriction enzyme sites (XhoI and EcoRV), it was cloned into the pVV-iPD1 shuttle vector to construct the shuttle vector pVV-iPD1 / GMCSF. In the obtained shuttle vector pVV-iPD1 / GMCSF, the iPD1 fusion gene is under the control of the pSEL promoter, and the GM-CSF gene is under the regulation of the p7.5 promoter. The human GM-CSF gene can also be cloned in...
Embodiment 3
[0058] Example 3 Construction of recombinant vaccinia oncolytic virus VV-imPD1 / mGMCSF co-expressing mouse PD1 inhibitor and GM-CSF
[0059] Human GM-CSF is not functional in mice. Human PD1-Fc fusion protein also does not efficiently bind mouse PD-L1. In order to detect the anti-tumor immune response mediated by fusion proteins of GM-CSF and PD1-Fc (iPD1), the inventors constructed a series of recombinant TK-VGF-vaccinia viruses to express the corresponding mouse proteins. The mouse fusion protein gene includes mouse PD1 signal peptide gene, mouse PD1 extracellular region gene and mouse IgG2a Fc fragment gene. The mouse fusion protein gene is linked to a restriction enzyme site. After synthesis, it was cloned into the vaccinia virus shuttle vector pSEL-DsRed to construct pVV-imPDL1 / mGMCSF (results such as figure 2 shown). Mouse pVV-imPD1 or pVV-mGMCSF shuttle vectors were also constructed. Next, recombinant double-deleted VGF expressing mPD1-Fc(imPD1) fusion protein gene...
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