Synthetic chimeric vaccinia virus
A vaccinia virus, virus technology, applied in the direction of viruses, antiviral agents, virus antigen components, etc., can solve the problems of no scrape-free area status regulations and guidelines, prion contamination, etc.
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Embodiment 1
[0162] Example 1. Selection and Design of Overlapping Fragments of Viral Genomes
[0163] Synthetic chimeric VACV ACAM2000 containing VACV WR strain hairpin and duplex sequences (scVACV ACAM2000-WR DUP / HP)
[0164] The design of the scVACV genome was based on the previously described genome sequence of VACV ACAM2000 [GenBank accession number AY313847] (Osborne JD et al. Vaccine. 2007;25(52):8807-32). The genome was divided into nine overlapping segments (Fig. 1). These fragments were designed such that they shared at least 1.0 kbp of overlapping sequence (ie, homology) with each adjacent fragment to provide sites where homologous recombination would drive full-length genome assembly (Table 1). These overlapping sequences provide sufficient homology for accurate recombination between co-transfected fragments (Yao XD, Evans DH. Journal of Virology. 2003;77(13):7281-90).
[0165] Table 1: VACV ACAM2000 genomic fragments used in this study. The size and sequence within the VACV...
Embodiment 2
[0176] Ligation of VACV WR F and S-terminal hairpin loops to VACV ACAM2000 right and left ITR fragments
[0177] Synthesize the same 70bp repeat fragment as VACV WR strain ( Figure 2C ; SEQ ID NO: 10). SapI and NheI restriction sites were included at the 5' and 3' ends of the 70 bp tandem repeat to facilitate ligation to the VACV WR hairpin sequence and the VACVACAM2000 right and left ITR fragments, respectively. Before the VACV WR terminal hairpin loop can be ligated onto the 70bp tandem repeat, the loop must be extended by an additional 58bp using a duplex sequence synthesized by IDT technology ( Figure 3A ). This is due to the extra sequence immediately downstream of the concatemer resolution site, before the first 70 bp repeat found in VACV strain WR. The duplex sequence is produced by synthesizing two single-stranded DNA molecules that, when annealed together, will produce a 5'-TGT overhang at the 5' end and a 5'-GGT overhang at the 3' end end of the duplex DNA ( ...
Embodiment 3
[0182] Example 3. Preparation of VACV ACAM2000 overlapping DNA fragments
[0183] Each VACV ACAM2000 overlapping DNA fragment in Table 1 was cloned into the plasmid provided by GeneArt using the restriction enzyme I-SceI. Before transfecting these synthetic DNA fragments into BGMK cells, the plasmid was digested with I-SceI and the product was run on a gel to confirm that the DNA fragments were successfully linearized ( Figure 5). After digestion at 37°C for 2 hours, the reaction was subsequently heat inactivated at 65°C. Samples were kept on ice or at 4°C until formation of terminal hairpins / duplexes / 70bp tandem repeats / ITR fragments (as described above).
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