Immuno-oncolytic virus combination drug for enhancing systemic immune response and its application
An oncolytic virus, enhancement system technology, used in drug combinations, anti-tumor drugs, resistance to vector-borne diseases, etc.
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Embodiment 1
[0051] Example 1: Attenuating mutation of wild-type virus: including the amino acid point mutation of the envelope protein or the mutation at the 3' end of the non-coding region, neuro-attenuated flaviviruses can be produced. The present inventors modified the infectious WNV cDNA and substituted five amino acids related to neurotoxicity in the WNV envelope protein. At the same time, the nucleotide sequence of the 3'-terminal stem-loop part of the dengue type 2 was replaced by the wild-type WNV 3'-terminal stem-loop sequence, and there were one or more mutations in the nucleotides in the secondary structure of the WNV 3'-terminal stem-loop. The engineered WNV exhibits attenuation characteristics. In the experiment of sensitive 3-week-old mice, subcutaneous injection of 3'-terminal mutant WNV (MutE) did not cause mouse death and neurological disease. Intracerebral injection of WNV envelope mutants (WN / Env5 and WNmutE-Env5) in 2-day-old mice showed a 1000-fold reduction in neuro...
Embodiment 2
[0052] Example 2: Construction of Attenuated Membrane Hybrid Flavivirus (ZIKA / WNV): The inventors linked the entire WNV genome to the pBR322 plasmid vector containing the CMV promoter with a cDNA structure. The Zika virus (ZIKA) envelope gene fragment was synthesized by PCR. The two ends of the fragment have restriction enzyme sites. After being cut with restriction enzymes, this fragment is ligated into the same site of the WNV cDNA cut by the same enzyme. The recombinant plasmid was transformed into Escherichia coli cells, which were propagated in vitro and extracted and purified. 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 cells. These viral RNAs replicated and multiplied, resulting in a hybrid flavivirus (ZIKA / WNV) with the Zika virus envelope based on the WNV RNA genome. Animal experimental data showed that hybrid flaviviru...
Embodiment 3
[0056] Example 3: The attenuated WNV and hybrid ZIKA / WNV flaviviruses obtained in Example 1 and Example 2, and the WNV wild virus strain were 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 mouse-derived T cell coactivator gene fragment or GFP gene fragment, these fragments are respectively connected in the attenuated WNV and hybrid ZIKA / WNV cDNA cut by the same enzyme, and the inserted site is the same as the above-mentioned cDNA. The construction of yellow fever virus (YF) 17D / GFP was the same. The cloned recombinant plasmids produced infectious virus in Vero cells, GFP expression was shown by fluorescence microscopy, and flavivirus production was detected by immunofluorescence. Lung or cervical cancer tumor cells were infected with these recombinant flaviviruses and three days later the cells were observed dying from oncolytic virus infection. Co...
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