Alphavirus replicon particle
一种甲病毒属、复制子的技术，应用在病毒、病毒肽、病毒/噬菌体等方向，能够解决缺少基因、基因疗法临床应用限制等问题
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Embodiment 1
[0189] Construction of VEEV Replicon Particles (VRP)
[0190] Schematic procedure such as figure 2 shown.
[0191] Establishment of VEEV replicon plasmid expressing luciferase
[0192] 1) Full-length VEEV TC83 nonstructural protein (nsp) 1, nsp2, nsp3 and nsp4 fragments were synthesized (ThermoFisher).
[0193] Similarly, the synthesis (IDT) of gblocks corresponding to the individual fragments of the replicon construct was as follows −
[0194] 2) VEEV gblock1 - ClaI-RSVp-5'UTR-nsp1 (bp1-470)-RsrII-ApaI-26Sp-sgRNA up to ATG of VEEV CA gene. This fragment has a 36 bp overlap with the pBR322 backbone plasmid at the 5' end. This is snippet #1.
[0195] 3) VEEV gblock2 - This fragment has a 64 bp overlap with VEEV gblock1 starting at the ApaI site until the ATG start codon. Next are the first 72 bases of Nano Luciferase ORF (Promega) and VEEV 3'UTR.
[0196] 4) Clone fragment #2 - VEEV gblock2, full-length VEEV 3' UTR and VEEV poly-A signal (A (n=55) ) were first assemble...
Embodiment 2
[0208] Determining the ability to package VEEV replicons into VEE viral replicon particles (VRPs)
[0209] Infection and luciferase assay
[0210] Schematic scheme such as Figure 8 shown.
[0211] 293T cells were seeded in complete DMEM in 96-well plates at a density of approximately 10,000 cells per well. Cells were infected with undiluted or 2-fold serial dilutions of harvested VRP at 37°C. 14 hours post infection (hpi), VRP was removed; cells were washed with PBS and fresh DMEM was added to the wells. Cells were further incubated and harvested for luciferase assay at 24, 48 and 72 hpi. Luciferase assays were performed by adding equal amounts of infected cells and the Nano-Glo Luciferase Assay System (Promega) to white bottom opaque 96-well plates (Costar). Luciferase activity was measured immediately using a Bio-Tek Synergy HTX microplate reader.
[0212] result
[0213] The result is as Figure 9 shown. Luciferase expression was confirmed in cells infected with V...
Embodiment 3
[0215] New VEEV replicon constructs
[0216] New VEEV TC83 replicon plasmid constructs were made by introducing a multiple cloning site (MCS) to enable the introduction of various "genes of interest". Schematic scheme such as Figure 10 shown. The construct is as Figure 11 and shown in SEQ ID NO: 6. exist Figure 11 In the construct of , the nucleotide encoding luciferase was introduced as the target gene. The promoter in the plasmid can be selected according to the cells to be infected.
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