Immuneoncolytic viruscombination drug for enhancing systemic immune response and application thereof
An oncolytic virus, enhancement system technology, used in anti-tumor drugs, drug combinations, resistance to vector-borne diseases, etc.
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Embodiment 1
[0051] Example 1: Attenuating mutation of wild-type virus: Including amino acid point mutation of envelope protein or 3' terminal mutation of non-coding region, neuroattenuated flavivirus can be produced. The present inventor modified infectious WNV cDNA, and substituted 5 amino acids related to neurotoxicity of WNV envelope protein. At the same time, the nucleotide sequence of the dengue type 2 3' terminal stem-loop part was replaced by the wild-type WNV 3' terminal stem-loop sequence, and one or more mutations were generated in the nucleotide secondary structure of the WNV 3' terminal stem-loop. The engineered WNV exhibited attenuated characteristics. In the experiment of sensitive 3-week-old mice, subcutaneous injection of 3' terminal mutant WNV (MutE) did not cause death or neurological disease in mice. Intracerebral injection of WNV envelope protein mutants (WN / Env5 and WNmutE-Env5) into young mice at day 2 showed a 1000-fold reduction in neurotoxicity (Yu, 2008. Vaccine...
Embodiment 2
[0052] Example 2: Construction of attenuated cell membrane hybrid flavivirus (ZIKA / WNV): The inventors linked the entire WNV genome to the pBR322 plasmid vector containing the CMV promoter in a cDNA structure. Zika virus (ZIKA) envelope gene fragment was synthesized by PCR. Both ends of the fragment have restriction enzyme sites. After cutting with a restriction enzyme, this fragment is ligated to the same site of the WNV cDNA cut with the same enzyme. Transform the recombinant plasmid into Escherichia coli cells, and allow it to proliferate, extract and purify in vitro. The purified recombinant plasmid is transfected into animal or mosquito cells cultured in vitro, and the recombinant plasmid containing the CMV promoter transcribes infectious viral RNA in the cell. These viral RNAs reproduced and reproduced to produce a hybrid flavivirus (ZIKA / WNV) with a Zika virus envelope based on the WNV RNA genome. Animal experiment data showed that the hybrid flavivirus with Zika viru...
Embodiment 3
[0056] Example 3: The attenuated WNV obtained in Example 1 and Example 2, the hybrid ZIKA / WNV flavivirus, and the WNV wild strain are further genetically modified by conventional genetic engineering methods to make them into vectors carrying foreign gene fragments , the method is: PCR synthesis of each human or murine T cell coactivator gene fragment or GFP gene fragment, these fragments are respectively connected to the attenuated WNV and hybrid ZIKA / WNV cDNA cut by the same enzyme, and the insertion site is the same as the above-mentioned Yellow fever virus (YF) 17D / GFP was constructed identically. Cloned recombinant plasmids produced infectious virus in Vero cells, fluorescence microscopy revealed GFP expression, and flavivirus production was detected by immunofluorescence. Lung or cervical cancer tumor cells were infected with these recombinant flaviviruses, and cells were observed dying from oncolytic virus infection after three days. These flaviviruses containing exogen...
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